**The Molecular Frame of Pancreatic Carcinogenesis**

Elisabeth Heßmann, Sandra Baumgart, Nai ming Chen, Shiv Singh, Garima Singh, Alex König, Albrecht Neeße and Volker Ellenrieder

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57422

## **1. Introduction**

Annually, approximately 43,140 people are diagnosed (incidence 10-12/100000) with pancre‐ atic ductal adenocarcinoma (PDAC) in the United States and the mortality rate of 36800 almost equals this number [1]. PDAC ranks fourth on the list of cancer-related causes of death and despite extensive scientific and clinical effort, the prognosis of this exceptionally lethal disease has not improved significantly over the past decades [2]. Surgical resection, for which only a minority (less than 20%) of the patients qualify due to late diagnosis in advanced stages, is currently the only chance of cure, improving 5-year survival rates from <4% if left untreated to 20-30% after resection [3]. Unresectable tumors are characterized by early invasion and metastases as well as by an extreme chemoresistance. Despite subtle progress over the years in terms of therapeutic strategies in many malignancies, no major conventional treatment options have come forward from numerous clinical trials in pancreatic cancer.

Considering its bad prognosis much effort was put into revealing the hidden secrets of pancreatic cancer that explain the severity of this disease. Among the different fields of tumor biology in pancreatic cancer research, this chapter will focus on the morphological and molecular features that cause and accompany pancreatic carcinogenesis.

## **2. Morphological features of pancreatic carcinogenesis**

Although there was little improvement in pancreatic cancer treatment during the past decades, much effort has been made in understanding the pathogenesis of pancreatic cancer. In contrast to its rapid progress after diagnosis, recent published data clearly show that the clonal

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

evolution of the earliest alterations in cancer initiating cells towards frankly invasive and metastasized PDAC takes at least more than a decade [4, 5]. This creates an important window of opportunity for early detection and much effort is put into attempts to map the molecular and morphological changes resulting in pancreatic cancer formation.

instance, the BRCA2 protein is involved in DNA damage repair, especially after occurrence of interstrand brakes [13]. Germline BRCA2 gene mutations are responsible for approximately 10% of familial pancreatic cancers [14]. Mismatch repair family (MMR) genes target base substitution mismatches as well as intersection deletions that arise as a result of errors occurring regularly during replication. Alterations in these mismatch repair genes lead to genetic instability and make the genome vulnerable for additional, more severe genetic

The Molecular Frame of Pancreatic Carcinogenesis

http://dx.doi.org/10.5772/57422

5

One of the most important and best studied proteins involved in DNA damage repair is the tumor suppressor protein p53. p53 is responsible for the cellular response to genotoxic stress as it mediates apoptosis and cell cycle arrest [16]. p53 is frequently disrupted in many human

Cell cycle regulation and progression is affected in virtually all transformed pancreatic cells. Enhanced activation of genes promoting G1/S-phase transition or loss of cell cycle inhibitors results in uncontrolled cell division which facilitates tumor progression and unrestrained

In normal pancreatic tissue, cells are anchored to each other and their surroundings via multiple connections. A decrease in these interactions can allow cells to detach from their surroundings and allows transformation, migration and metastasis. As such, cell to cell

The other pathways discovered by Jones and colleagues which proofed to be frequently affected by genetic alterations in pancreatic cancer are signaling cascades that can be divided into three groups: embryonic signaling pathways, MAPKinase signaling pathways and TGFßsignaling pathways [9]. The transforming growth factor ß (TGFß) pathway has been linked to PDAC for many years. TGFß signaling is involved in a wide range of cellular processes including differentiation, proliferation, apoptosis and angiogenesis [20]. As discussed in detail later in this chapter, TGFß signaling functions as a double-edged sword as it comprises tumor-

All MAPK signaling pathways consist of the same basic kinase components. Stimulation of an upstream MAP2K kinase by growth factors, stress or other extracellular signals leads to phosphorylation of one of the terminal MAPK: extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK) or p38 [8]. These signaling cascades result in the activation of

One growth factor receptor responsible for many signaling events in early carcinogenesis is the Epidermal Growth Factor Receptor (EGFR). EGFR is located in the cell membrane and is activated by binding of its specific ligands, including epidermal growth factor (EGF) and Transforming Growth Factor alpha (TGFα) [6, 21]. Upon activation, EGFR undergoes dimeri‐ zation, thus stimulating its intrinsic intracellular protein-tyrosine kinase activity resulting in autophosphorylation of several tyrosine residues in the C-terminal region of the receptor. This autophosphorylation elicits activation of numerous downstream kinases and signal transduc‐ tion cascades that modulate cancer associated phenotypes as cell proliferation, migration and adhesion [6]. Recent work has proven a high impact of EGFR signaling on induction of

malignancies and the tumor suppressor is lost in 50-75% of PDACs [17].

adhesion and interaction plays an important role in carcinogenesis [18, 19].

alterations [15].

tumor growth [1].

suppressive as well as oncogenic qualities.

multiple oncogenic cellular functions.

The current model of pancreatic carcinogenesis describes a stepwise process from healthy acinar cells to frank pancreatic adenocarcinoma: Recent lineage-tracing studies have shown that acinar cells harboring molecular alterations are induced to transdifferentiate, generating duct-like cells through a process known as acinar-to-ductal metaplasia (ADM) [6]. ADM lesions then convert to precancerous pancreatic intraepithelial neoplasia (PanIN) that progress to PDAC over time [7]. PanIN lesions are found in the smaller pancreatic ducts and are classified in four grades based on the degree of dysplasia reflected in the cytonuclear atypia and architectural change of the epithelial cell: PanIN-1A, -1B, -2 and -3 [7]. The lowest grade PanIN lesions can be flat (-1A) or papillary (-1B), but are characterized by absence of nuclear atypia and retained nuclear polarity. PanIN-2 lesions show micropapillary features with evidence of nuclear atypia and infrequent mitoses. PanIN-3 lesions demonstrate all hallmarks of cancer, including a widespread loss of polarity, nuclear atypia and frequent mitoses and are considered as *Carcinoma in situ* [1, 8]. Yet, the lesion is confined within the basement membrane and no invasive growth is present. The increasing grades of dysplasia in the various PanIN lesions manifest the morphological steps of tumor progression that precede invasive PDAC. These consecutive steps of tumor progression are accompanied by a cumulative occurrence of molecular alterations.

#### **3. Molecular characteristics of pancreatic carcinogenesis**

#### **3.1. Genetic alterations in pancreatic carcinogenesis**

For many decades pancreatic cancer was described as an exclusively genetic disease. In 2008 Jones and colleagues discovered 1561 somatic gene mutations within more than 20000 analyzed genes, yielding an average rate of 63 genetic abnormalities per pancreatic cancer, emphasizing the extreme complexity of this disease [9]. These genetic alterations can be clustered in 12 partially overlapping signaling pathways (compare Fig. 1). Five of the pathways comprise specific cellular functions: apoptosis, DNA-damage repair, G1/S phase cell cycle progression, cell-cell adhesion and invasion.

Apoptosis or programmed cell death, plays an essential role in carcinogenesis since resistance to apoptosis is a key factor of the survival of a cancer cell [1]. In PDAC, genes implicated in the apoptosis pathway (Bcl2, Mcl-1, p53, NF-kB among others) were found altered in all tumors studied and many reports document impaired apoptotic signaling in this disease [10, 11]. For example, a high fraction of apoptotic cells has been correlated with longer overall survival as well as absence of nodal involvement [12]. Moreover, resistance to chemotherapeutics is mostly a result of defective apoptosis pathways.

DNA damage control genes code for proteins that repair any damage that occurs in the cell during its lifespan and thus are responsible for safeguarding the integrity of DNA [1]. For instance, the BRCA2 protein is involved in DNA damage repair, especially after occurrence of interstrand brakes [13]. Germline BRCA2 gene mutations are responsible for approximately 10% of familial pancreatic cancers [14]. Mismatch repair family (MMR) genes target base substitution mismatches as well as intersection deletions that arise as a result of errors occurring regularly during replication. Alterations in these mismatch repair genes lead to genetic instability and make the genome vulnerable for additional, more severe genetic alterations [15].

evolution of the earliest alterations in cancer initiating cells towards frankly invasive and metastasized PDAC takes at least more than a decade [4, 5]. This creates an important window of opportunity for early detection and much effort is put into attempts to map the molecular

4 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

The current model of pancreatic carcinogenesis describes a stepwise process from healthy acinar cells to frank pancreatic adenocarcinoma: Recent lineage-tracing studies have shown that acinar cells harboring molecular alterations are induced to transdifferentiate, generating duct-like cells through a process known as acinar-to-ductal metaplasia (ADM) [6]. ADM lesions then convert to precancerous pancreatic intraepithelial neoplasia (PanIN) that progress to PDAC over time [7]. PanIN lesions are found in the smaller pancreatic ducts and are classified in four grades based on the degree of dysplasia reflected in the cytonuclear atypia and architectural change of the epithelial cell: PanIN-1A, -1B, -2 and -3 [7]. The lowest grade PanIN lesions can be flat (-1A) or papillary (-1B), but are characterized by absence of nuclear atypia and retained nuclear polarity. PanIN-2 lesions show micropapillary features with evidence of nuclear atypia and infrequent mitoses. PanIN-3 lesions demonstrate all hallmarks of cancer, including a widespread loss of polarity, nuclear atypia and frequent mitoses and are considered as *Carcinoma in situ* [1, 8]. Yet, the lesion is confined within the basement membrane and no invasive growth is present. The increasing grades of dysplasia in the various PanIN lesions manifest the morphological steps of tumor progression that precede invasive PDAC. These consecutive steps of tumor progression are accompanied by a cumulative occurrence of

and morphological changes resulting in pancreatic cancer formation.

**3. Molecular characteristics of pancreatic carcinogenesis**

For many decades pancreatic cancer was described as an exclusively genetic disease. In 2008 Jones and colleagues discovered 1561 somatic gene mutations within more than 20000 analyzed genes, yielding an average rate of 63 genetic abnormalities per pancreatic cancer, emphasizing the extreme complexity of this disease [9]. These genetic alterations can be clustered in 12 partially overlapping signaling pathways (compare Fig. 1). Five of the pathways comprise specific cellular functions: apoptosis, DNA-damage repair, G1/S phase cell cycle

Apoptosis or programmed cell death, plays an essential role in carcinogenesis since resistance to apoptosis is a key factor of the survival of a cancer cell [1]. In PDAC, genes implicated in the apoptosis pathway (Bcl2, Mcl-1, p53, NF-kB among others) were found altered in all tumors studied and many reports document impaired apoptotic signaling in this disease [10, 11]. For example, a high fraction of apoptotic cells has been correlated with longer overall survival as well as absence of nodal involvement [12]. Moreover, resistance to chemotherapeutics is mostly

DNA damage control genes code for proteins that repair any damage that occurs in the cell during its lifespan and thus are responsible for safeguarding the integrity of DNA [1]. For

**3.1. Genetic alterations in pancreatic carcinogenesis**

progression, cell-cell adhesion and invasion.

a result of defective apoptosis pathways.

molecular alterations.

One of the most important and best studied proteins involved in DNA damage repair is the tumor suppressor protein p53. p53 is responsible for the cellular response to genotoxic stress as it mediates apoptosis and cell cycle arrest [16]. p53 is frequently disrupted in many human malignancies and the tumor suppressor is lost in 50-75% of PDACs [17].

Cell cycle regulation and progression is affected in virtually all transformed pancreatic cells. Enhanced activation of genes promoting G1/S-phase transition or loss of cell cycle inhibitors results in uncontrolled cell division which facilitates tumor progression and unrestrained tumor growth [1].

In normal pancreatic tissue, cells are anchored to each other and their surroundings via multiple connections. A decrease in these interactions can allow cells to detach from their surroundings and allows transformation, migration and metastasis. As such, cell to cell adhesion and interaction plays an important role in carcinogenesis [18, 19].

The other pathways discovered by Jones and colleagues which proofed to be frequently affected by genetic alterations in pancreatic cancer are signaling cascades that can be divided into three groups: embryonic signaling pathways, MAPKinase signaling pathways and TGFßsignaling pathways [9]. The transforming growth factor ß (TGFß) pathway has been linked to PDAC for many years. TGFß signaling is involved in a wide range of cellular processes including differentiation, proliferation, apoptosis and angiogenesis [20]. As discussed in detail later in this chapter, TGFß signaling functions as a double-edged sword as it comprises tumorsuppressive as well as oncogenic qualities.

All MAPK signaling pathways consist of the same basic kinase components. Stimulation of an upstream MAP2K kinase by growth factors, stress or other extracellular signals leads to phosphorylation of one of the terminal MAPK: extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK) or p38 [8]. These signaling cascades result in the activation of multiple oncogenic cellular functions.

One growth factor receptor responsible for many signaling events in early carcinogenesis is the Epidermal Growth Factor Receptor (EGFR). EGFR is located in the cell membrane and is activated by binding of its specific ligands, including epidermal growth factor (EGF) and Transforming Growth Factor alpha (TGFα) [6, 21]. Upon activation, EGFR undergoes dimeri‐ zation, thus stimulating its intrinsic intracellular protein-tyrosine kinase activity resulting in autophosphorylation of several tyrosine residues in the C-terminal region of the receptor. This autophosphorylation elicits activation of numerous downstream kinases and signal transduc‐ tion cascades that modulate cancer associated phenotypes as cell proliferation, migration and adhesion [6]. Recent work has proven a high impact of EGFR signaling on induction of pancreatic metaplasia, and overexpression of the receptor already occurs in early pancreatic precursor lesions [6, 21]. The relevance of EGFR dependent signal cascades was emphasized by a therapeutic beneficial effect of the EGFR inhibitor Erlotinib in a subgroup of pancreatic cancer patients [22].

Conceptually, these data suggest that pancreatic cancer is substantially a disease of pathways. But research into these pathways rendered clearly that these cascades must ultimately engage the function of epigenetic regulators to influence gene expression in a heritable manner. Thus studies into epigenetics in pancreatic cancer demonstrate a logical extension to the genetic

The Molecular Frame of Pancreatic Carcinogenesis

http://dx.doi.org/10.5772/57422

7

Conceptually, these data suggest that pancreatic cancer is substantially a disease of pathways. But research into these pathways rendered clearly that these cascades must ultimately engage the function of epigenetic regulators to influence gene expression in a heritable manner. Thus studies into epigenetics in pancreatic cancer demonstrate a logical extension

Figure 1. The commonly altered signaling pathways in PDAC accompanied by affected genes from these pathways (adopted from [1]).

**Figure 1. The commonly altered signaling pathways in PDAC accompanied by affected genes from these path‐**

Epigenetics are defined as any heritable genomic mechanism unrelated to changes in the DNA sequence [32]. Epigenetic modifications are involved in normal cellular development and maintenance, but they are also responsible for deregulation of gene expression, resulting in diseased cellular phenotypes. Most notably, deregulation of epigenetic mechanisms can contribute to cancer development [33-38]. The past years have witnessed an explosive increase in our knowledge about epigenetic features in pancreatic carcinogenesis. Several well-known epigenetic mechanisms are active in pancreatic cancer, sub-divided into DNA methylation, histone modification and microRNAs, all of them affecting the cell by induction or suppression of gene expression [39-42]. For instance, the introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in the transforming pancreatic epithelial cell that play a role in the neoplastic process [43]. Hypermethylation of promoter

Epigenetics are defined as any heritable genomic mechanism unrelated to changes in the DNA sequence [32]. Epigenetic modifications are involved in normal cellular development and maintenance, but they are also responsible for deregulation of gene expression, resulting in diseased cellular phenotypes. Most notably, deregulation of epigenetic mechanisms can contribute to cancer development [33-38]. The past years have witnessed an explosive increase in our knowledge about epigenetic features in pancreatic carcinogenesis. Several well-known epigenetic mechanisms are active in pancreatic cancer, sub-divided into DNA methylation, histone modification and microRNAs, all of them affecting the cell by induction or suppression of gene expression [39-42]. For instance, the introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in the transforming pancreatic epithelial cell that play a role in the neoplastic process [43]. Hypermethylation of promoter cytosine-phospho-guanine (CpG) islands is closely linked to gene silencing and loss of tumor suppressor function in many cancer entities [44]. Since the first detailed analysis of DNA hypermethylation in pancreatic cancer was reported in 1997 by Schutte et al., many tumor-suppressor or cancer-related genes that undergo aberrant methyl‐ ation during pancreatic cancer development have been identified, including APC, RUNX3,

By influencing the structure of chromatin, in addition to DNA methylation, posttranslational modifications of histone tail residues highly affect the transcriptional activity of genes. While acetylation of histones is primarily associated with transcriptional activation, methylation of histones can lead to both, activation and repression, depending on the modified residue [46,

Similar to Notch-signaling, the hedgehog pathway belongs to the developmental programs of pancreatogenesis. The hedgehog gene was originally identified in Drosophila when a large-scale screening for mutations revealed an altered segmentation pattern of larvae, resulting in a short, fat larva covered in a "lawn" of denticles resembling a hedgehog [27]. Early in development, around embryonic day 8.5-9.0, the hedgehog ligands Indian Hedgehog (Ihh) and Sonic hedgehog (Shh) are expressed throughout the endodermal epithelium of the primitive gut but are noticeably absent in the developed organ [28]. Sonic hedgehog signaling is reactivated in the case of pancreatic regeneration, for example in response to inflammation-associated pancreatic injury [29]. Through inappropriate activation of these pathways, chronic injury might contribute to misdirection of tissue repair, ultimately resulting in neoplasia. Aberrant expression of members of the hedgehog-pathway in chronic pancreatitis and pancreatic carcinogenesis was first noted by Kayed and colleagues [30]. Subsequent research proved that the ligand Shh is expressed aberrantly in pancreatic cancer and its precursor lesions and that Shh functions as a mediator of cancer initiation and growth [31]. Mice with transgenic misexpression of Shh in the pancreatic endoderm develop lesions resembling PanIN, and hedgehog inhibition induces apoptosis and blockes proliferation in pancreatic cancer cells *in vivo* and *in vitro* [31]. Thus, hedgehog signaling can be

Hedgehog-, Notch- and Wnt-signaling cascades [9]. Several studies have shown upregulation of these pathways during pancreatic carcinogenesis and in invasive pancreatic cancer and their inhibition results in decreased tumor proliferation and enhanced apoptosis [1]. For instance, activation of the Notch signaling pathway is involved in cell proliferation and angiogenesis in a variety of human cancers, including pancreatic cancer [23]. Notch signaling is initiated when Notch ligand binds to its receptor between adjacent cells. Upon activation, Notch is cleaved and releases the Notch intracellular domain (NICD) via a cascade of proteolytic enzymes including ʏ-secretase. Finally, NICD translocates into the nucleus and activates its target genes such as Hes-1, Hey-1, Cyclin D1 and cMyc [24]. Additional to its growth promoting functions accumulating evidence shows a molecular link between Notch and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [25]. During the EMT process, epithelial cells gain a mesenchymal phenotype accompanied by the cumulative expression of the mesenchymal markers Vimentin, Slug, Snail and ZEB1 and reduced expression of the epithelial marker E-cadherin. EMT-type cells harbor an increased migratory and invasive capacity resulting in invasion and spread of tumor cells even during early carcinogenesis [26]. Inhibition of Notch-signaling leads to

paradigm of this malignant disease.

to the genetic paradigm of this malignant disease.

**ways** (adopted from [1]).

described as an early and late mediator of pancreatic ductal adenocarcinoma.

**Signaling pathway Affected genes**  Apoptosis p53, NF-ƙB, PI3K/Akt DNA damage repair p53, BRCA2, MMR-genes G1/S transition p16Ink4a, p14arf, p15Ink4b, Cyclin D Regulation of invasion TGFß, Integrin signaling Embryonic signaling Notch, Hedgehog, Wnt

MAPK signaling Erk, Jnk, p38 TGFß signaling TGFß, Smad-proteins

**4. Epigenetic mechanisms in pancreatic carcinogenesis**

**4. Epigenetic mechanisms in pancreatic carcinogenesis** 

SOCS-1, p16Ink4a, Cyclin D2 and CHD13 [44, 45].

reduction of EMT resulting in a better clinical outcome [25].

Since embryogenesis shares many characteristics with carcinogenesis, not surprisingly many embryonic pathways are involved in tumor development. The three embryonic pathways operative in pancreatic carcinogenesis are the Hedgehog-, Notch- and Wnt-signaling cascades [9]. Several studies have shown upregulation of these pathways during pancreatic carcino‐ genesis and in invasive pancreatic cancer and their inhibition results in decreased tumor proliferation and enhanced apoptosis [1]. For instance, activation of the Notch signaling pathway is involved in cell proliferation and angiogenesis in a variety of human cancers, including pancreatic cancer [23]. Notch signaling is initiated when Notch ligand binds to its receptor between adjacent cells. Upon activation, Notch is cleaved and releases the Notch intracellular domain (NICD) via a cascade of proteolytic enzymes including Y-secretase. Finally, NICD translocates into the nucleus and activates its target genes such as Hes-1, Hey-1, Cyclin D1 and cMyc [24]. Additional to its growth promoting functions accumulating evidence shows a molecular link between Notch and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [25]. During the EMT process, epithelial cells gain a mesenchymal phenotype accompanied by the cumulative expression of the mesenchymal markers Vimentin, Slug, Snail and ZEB1 and reduced expression of the epithelial marker E-cadherin. EMT-type cells harbor an increased migratory and invasive capacity resulting in invasion and spread of tumor cells even during early carcinogenesis [26]. Inhibition of Notch-signaling leads to reduction of EMT resulting in a better clinical outcome [25].

Similar to Notch-signaling, the hedgehog pathway belongs to the developmental programs of pancreatogenesis. The hedgehog gene was originally identified in Drosophila when a largescale screening for mutations revealed an altered segmentation pattern of larvae, resulting in a short, fat larva covered in a "lawn" of denticles resembling a hedgehog [27]. Early in development, around embryonic day 8.5-9.0, the hedgehog ligands Indian Hedgehog (Ihh) and Sonic hedgehog (Shh) are expressed throughout the endodermal epithelium of the primitive gut but are noticeably absent in the developed organ [28]. Sonic hedgehog signaling is reactivated in the case of pancreatic regeneration, for example in response to inflammationassociated pancreatic injury [29]. Through inappropriate activation of these pathways, chronic injury might contribute to misdirection of tissue repair, ultimately resulting in neoplasia. Aberrant expression of members of the hedgehog-pathway in chronic pancreatitis and pancreatic carcinogenesis was first noted by Kayed and colleagues [30]. Subsequent research proved that the ligand Shh is expressed aberrantly in pancreatic cancer and its precursor lesions and that Shh functions as a mediator of cancer initiation and growth [31]. Mice with transgenic misexpression of Shh in the pancreatic endoderm develop lesions resembling PanIN, and hedgehog inhibition induces apoptosis and blockes proliferation in pancreatic cancer cells *in vivo* and *in vitro* [31]. Thus, hedgehog signaling can be described as an early and late mediator of pancreatic ductal adenocarcinoma.

Conceptually, these data suggest that pancreatic cancer is substantially a disease of pathways. But research into these pathways rendered clearly that these cascades must ultimately engage the function of epigenetic regulators to influence gene expression in a heritable manner. Thus studies into epigenetics in pancreatic cancer demonstrate a logical extension to the genetic paradigm of this malignant disease. described as an early and late mediator of pancreatic ductal adenocarcinoma. Conceptually, these data suggest that pancreatic cancer is substantially a disease of pathways. But research into these pathways rendered clearly that these cascades must ultimately engage the function of epigenetic regulators to influence gene expression in a heritable manner. Thus studies into epigenetics in pancreatic cancer demonstrate a logical extension

apoptosis and blockes proliferation in pancreatic cancer cells *in vivo* and *in vitro* [31]. Thus, hedgehog signaling can be

Hedgehog-, Notch- and Wnt-signaling cascades [9]. Several studies have shown upregulation of these pathways during pancreatic carcinogenesis and in invasive pancreatic cancer and their inhibition results in decreased tumor proliferation and enhanced apoptosis [1]. For instance, activation of the Notch signaling pathway is involved in cell proliferation and angiogenesis in a variety of human cancers, including pancreatic cancer [23]. Notch signaling is initiated when Notch ligand binds to its receptor between adjacent cells. Upon activation, Notch is cleaved and releases the Notch intracellular domain (NICD) via a cascade of proteolytic enzymes including ʏ-secretase. Finally, NICD translocates into the nucleus and activates its target genes such as Hes-1, Hey-1, Cyclin D1 and cMyc [24]. Additional to its growth promoting functions accumulating evidence shows a molecular link between Notch and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [25]. During the EMT process, epithelial cells gain a mesenchymal phenotype accompanied by the cumulative expression of the mesenchymal markers Vimentin, Slug, Snail and ZEB1 and reduced expression of the epithelial marker E-cadherin. EMT-type cells harbor an increased migratory and invasive capacity resulting in invasion and spread of tumor cells even during early carcinogenesis [26]. Inhibition of Notch-signaling leads to

Similar to Notch-signaling, the hedgehog pathway belongs to the developmental programs of pancreatogenesis. The hedgehog gene was originally identified in Drosophila when a large-scale screening for mutations revealed an altered segmentation pattern of larvae, resulting in a short, fat larva covered in a "lawn" of denticles resembling a hedgehog [27]. Early in development, around embryonic day 8.5-9.0, the hedgehog ligands Indian Hedgehog (Ihh) and Sonic hedgehog (Shh) are expressed throughout the endodermal epithelium of the primitive gut but are noticeably absent in the developed organ [28]. Sonic hedgehog signaling is reactivated in the case of pancreatic regeneration, for example in response to inflammation-associated pancreatic injury [29]. Through inappropriate activation of these pathways, chronic injury might contribute to misdirection of tissue repair, ultimately resulting in neoplasia. Aberrant expression of members of the hedgehog-pathway in chronic pancreatitis and pancreatic carcinogenesis was first noted by Kayed and colleagues [30]. Subsequent research proved that the ligand Shh is expressed aberrantly in pancreatic cancer and its

reduction of EMT resulting in a better clinical outcome [25].

to the genetic paradigm of this malignant disease.


Figure 1. The commonly altered signaling pathways in PDAC accompanied by affected genes from these pathways (adopted from [1]). **Figure 1. The commonly altered signaling pathways in PDAC accompanied by affected genes from these path‐ ways** (adopted from [1]).

deregulation of gene expression, resulting in diseased cellular phenotypes. Most notably, deregulation of epigenetic

#### Epigenetics are defined as any heritable genomic mechanism unrelated to changes in the DNA sequence [32]. Epigenetic modifications are involved in normal cellular development and maintenance, but they are also responsible for **4. Epigenetic mechanisms in pancreatic carcinogenesis**

**4. Epigenetic mechanisms in pancreatic carcinogenesis** 

pancreatic metaplasia, and overexpression of the receptor already occurs in early pancreatic precursor lesions [6, 21]. The relevance of EGFR dependent signal cascades was emphasized by a therapeutic beneficial effect of the EGFR inhibitor Erlotinib in a subgroup of pancreatic

6 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Since embryogenesis shares many characteristics with carcinogenesis, not surprisingly many embryonic pathways are involved in tumor development. The three embryonic pathways operative in pancreatic carcinogenesis are the Hedgehog-, Notch- and Wnt-signaling cascades [9]. Several studies have shown upregulation of these pathways during pancreatic carcino‐ genesis and in invasive pancreatic cancer and their inhibition results in decreased tumor proliferation and enhanced apoptosis [1]. For instance, activation of the Notch signaling pathway is involved in cell proliferation and angiogenesis in a variety of human cancers, including pancreatic cancer [23]. Notch signaling is initiated when Notch ligand binds to its receptor between adjacent cells. Upon activation, Notch is cleaved and releases the Notch intracellular domain (NICD) via a cascade of proteolytic enzymes including Y-secretase. Finally, NICD translocates into the nucleus and activates its target genes such as Hes-1, Hey-1, Cyclin D1 and cMyc [24]. Additional to its growth promoting functions accumulating evidence shows a molecular link between Notch and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [25]. During the EMT process, epithelial cells gain a mesenchymal phenotype accompanied by the cumulative expression of the mesenchymal markers Vimentin, Slug, Snail and ZEB1 and reduced expression of the epithelial marker E-cadherin. EMT-type cells harbor an increased migratory and invasive capacity resulting in invasion and spread of tumor cells even during early carcinogenesis [26]. Inhibition of Notch-signaling leads to reduction of EMT

Similar to Notch-signaling, the hedgehog pathway belongs to the developmental programs of pancreatogenesis. The hedgehog gene was originally identified in Drosophila when a largescale screening for mutations revealed an altered segmentation pattern of larvae, resulting in a short, fat larva covered in a "lawn" of denticles resembling a hedgehog [27]. Early in development, around embryonic day 8.5-9.0, the hedgehog ligands Indian Hedgehog (Ihh) and Sonic hedgehog (Shh) are expressed throughout the endodermal epithelium of the primitive gut but are noticeably absent in the developed organ [28]. Sonic hedgehog signaling is reactivated in the case of pancreatic regeneration, for example in response to inflammationassociated pancreatic injury [29]. Through inappropriate activation of these pathways, chronic injury might contribute to misdirection of tissue repair, ultimately resulting in neoplasia. Aberrant expression of members of the hedgehog-pathway in chronic pancreatitis and pancreatic carcinogenesis was first noted by Kayed and colleagues [30]. Subsequent research proved that the ligand Shh is expressed aberrantly in pancreatic cancer and its precursor lesions and that Shh functions as a mediator of cancer initiation and growth [31]. Mice with transgenic misexpression of Shh in the pancreatic endoderm develop lesions resembling PanIN, and hedgehog inhibition induces apoptosis and blockes proliferation in pancreatic cancer cells *in vivo* and *in vitro* [31]. Thus, hedgehog signaling can be described as an early and

cancer patients [22].

resulting in a better clinical outcome [25].

late mediator of pancreatic ductal adenocarcinoma.

mechanisms can contribute to cancer development [33-38]. The past years have witnessed an explosive increase in our knowledge about epigenetic features in pancreatic carcinogenesis. Several well-known epigenetic mechanisms are active in pancreatic cancer, sub-divided into DNA methylation, histone modification and microRNAs, all of them affecting the cell by induction or suppression of gene expression [39-42]. For instance, the introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in the transforming pancreatic epithelial cell that play a role in the neoplastic process [43]. Hypermethylation of promoter Epigenetics are defined as any heritable genomic mechanism unrelated to changes in the DNA sequence [32]. Epigenetic modifications are involved in normal cellular development and maintenance, but they are also responsible for deregulation of gene expression, resulting in diseased cellular phenotypes. Most notably, deregulation of epigenetic mechanisms can contribute to cancer development [33-38]. The past years have witnessed an explosive increase in our knowledge about epigenetic features in pancreatic carcinogenesis. Several well-known epigenetic mechanisms are active in pancreatic cancer, sub-divided into DNA methylation, histone modification and microRNAs, all of them affecting the cell by induction or suppression of gene expression [39-42]. For instance, the introduction of genome-wide screening techniques has accelerated the discovery of a growing list of genes with abnormal methylation patterns in the transforming pancreatic epithelial cell that play a role in the neoplastic process [43]. Hypermethylation of promoter cytosine-phospho-guanine (CpG) islands is closely linked to gene silencing and loss of tumor suppressor function in many cancer entities [44]. Since the first detailed analysis of DNA hypermethylation in pancreatic cancer was reported in 1997 by Schutte et al., many tumor-suppressor or cancer-related genes that undergo aberrant methyl‐ ation during pancreatic cancer development have been identified, including APC, RUNX3, SOCS-1, p16Ink4a, Cyclin D2 and CHD13 [44, 45].

> By influencing the structure of chromatin, in addition to DNA methylation, posttranslational modifications of histone tail residues highly affect the transcriptional activity of genes. While acetylation of histones is primarily associated with transcriptional activation, methylation of histones can lead to both, activation and repression, depending on the modified residue [46,

47]. For instance, Polycomb proteins, which are known for their crucial role in induction of repressive histone modifications, embody oncogenic properties in many human cancers. Polycomb proteins can be divided into two functional biochemical categories, Polycomb repressive complexes (PRC) 1 and 2. While members of the PRC 2 complex initiate gene repression by catalysation of H3K27 trimethylation, proteins belonging to PRC1 maintain the repressive state [48, 49]. Under physiological conditions, the activity of Polycomb proteins is crucial in development as well as in maintenance and proliferation of pluripotent progenitor cells in a variety of tissues. Overexpression of these proteins may promote tumorigenesis by fostering a self-renewing population of cells [50, 51]. Indeed, overexpression of Polycomb proteins is responsible for malignant progression and poor prognosis in breast [52], bladder [53] and prostate [54] cancer and shows strong association with hallmarks of cancer, including induced cellular proliferation [55], angiogenesis [56], survival [57] and migration [58]. Enhancer of Zeste Homolog 2 (EZH2) is the only PRC2 protein member thus far studied in pancreatic cancer. Strong nuclear accumulation of EZH2 was found in 55% of well differenti‐ ated tumors and 98% of poorly differentiated samples in a comprehensive immunohistochem‐ ical analysis of PDACs, indicating a significant correlation between EZH2 expression and dedifferentiation in pancreatic cancer [59]. Additionally, EZH2 overexpression participates in epithelial-to-mesenchymal transition (EMT) and invasion through repression of epithelial proteins like E-cadherin [60].

**5. Impact of Kras activation on pancreatic carcinogenesis**

on growth factor stimulation [67].

immunity [69, 70].

The mutation of Kras belongs to the earliest events in pancreatic carcinogenesis. Kras proteins comprise a family of signal-transducing GTPases that mediate a wide variety of cellular functions including proliferation, differentiation and survival and are frequently mutated in human cancers [66]. Although Kras is a GTPase, its intrinsic activity is inefficient and requires GTPase activating proteins to promote GTP hydrolysis and attenuate downstream signaling [1]. Oncogenic mutation of Kras (KrasG12D) is generally accepted to represent the initial key event in pancreatic carcinogenesis and found in virtually all invasively growing pancreatic tumors [7]. Due to its prominent role in pancreatic carcinogenesis Kras is considered to be an attractive therapeutic target of PDAC-treatment, but specific biochemical properties of the protein have made this an elusive goal [67]. Activating Kras point mutations at codon 12 (from GGT to GAT or GTT and more rarely CGT) result in substitution of glycine with aspartate, valine or arginine. Oncogenic Kras mutations lock the protein in its GTP-bound form thus permitting its constitutive interaction with and activation of multiple effectors, independent

The activation of Kras engaged effector pathways, like the RAF-mitogen-activated kinase (MAPK)-cascade, phosphoinositide-3-kinase- (PI3K) signaling and the Ral GDS pathway results in stimulation of proliferation, invasion, metastases and survival thus enabling pancreatic cancer progression [3]. Given the aforementioned limitations in Kras inhibition, these downstream targets may provide alternative effective points of therapeutic intervention

The impact of constitutive Kras activation is not limited on the epithelial cell but also partici‐ pates in the modulation of the tumor environment. One hallmark of PDAC is an extensive stromal remodeling, the most prominent features of which are the recruitment of inflammatory and mesenchymal cells as well as fibrotic replacement of pancreatic parenchyma [68]. Recent studies revealed that even early stages of PanIN development are associated with a stromal reaction, which is characterized by a robust desmoplastic response and recruitment of immune cells. Subclasses of these immune cells, immature myeloid cells, suppress infiltrating T cells and thus establish an immune privilege in the tumor microenvironment promoting pancreatic carcinogenesis [69, 70]. Mechanistically, constitutive activation of Kras in pancreatic ductal cells triggers the production of the cytokine GM-CSF, which, in turn, promotes the expansion

Due to its high biological relevance for pancreatic carcinogenesis, a genetically engi‐ neered mouse model (GEMM) with pancreas specific Kras mutation was created, allow‐ ing detailed investigations of morphological as well as molecular features of this disease [71]. This transgenic mouse model bares a mutation of the endogenous murine Kras gene specifically in pancreatic progenitor cells by crossing mice with a conditionally activated Kras allel (LSL-KrasG12D) to transgenic strains that express Cre recombinase in pancreatic lineages (PdxCre or p48Cre). These "KC" mice develop low and high grade PanIN lesions recapitulating pancreatic carcinogenesis in the human situation but only slowly progress

T-cell-driven-antitumor

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and thus are the focus of ongoing studies in pancreatic specific systems.

of immunosuppressive myeloid cells, leading to the evasion of CD8+

The third group among the epigenetic players in pancreatic carcinogenesis comprises the MicroRNA (miRNA) family, a class of small non-protein coding RNAs which participate in post-transcriptional control of gene expression in eukaryotic organisms [61]. In the last years, advanced global screening technologies have enabled large scale analyses of miRNA profiles in diverse tissue samples, indicating that miRNAs can function as either onco‐ genes or tumor suppressors in the development of various cancer types, including pancreatic cancer [62, 63]. The analysis of miRNA expression patterns has let to complete‐ ly novel insights into pancreatic cancer biology. Specific miRNAs, such as the miR-200 family, miR-34a and miR-155 are involved in PDAC-biology by regulating genes associat‐ ed with metastasis and cell stemness [64, 65].

The era of epigenetics in pancreatic cancer has emerged strongly within the last years and deepened our understanding of pancreatic cancer biology. One of the most important charac‐ teristics of epigenetic mechanisms which clearly demarcates them from genetics is their reversibility. This feature provides new targets for novel therapeutic interventions in pancre‐ atic cancer and other epithelial tumors.

The manifold genetic and epigenetic events observed in pancreatic carcinogenesis mirror the complexity of this malignancy and lead to the assumption that targeting one molecular feature of pancreatic carcinogenesis is not sufficient for successful pancreatic cancer treatment. Though inaccessible for therapeutic options, there exists at least one molecular event found in virtually all invasively growing pancreatic tumors and their precursor lesions: The constitutive activation of oncogenic Kras probably demonstrates the most important and best studied event in pancreatic carcinogenesis.

## **5. Impact of Kras activation on pancreatic carcinogenesis**

47]. For instance, Polycomb proteins, which are known for their crucial role in induction of repressive histone modifications, embody oncogenic properties in many human cancers. Polycomb proteins can be divided into two functional biochemical categories, Polycomb repressive complexes (PRC) 1 and 2. While members of the PRC 2 complex initiate gene repression by catalysation of H3K27 trimethylation, proteins belonging to PRC1 maintain the repressive state [48, 49]. Under physiological conditions, the activity of Polycomb proteins is crucial in development as well as in maintenance and proliferation of pluripotent progenitor cells in a variety of tissues. Overexpression of these proteins may promote tumorigenesis by fostering a self-renewing population of cells [50, 51]. Indeed, overexpression of Polycomb proteins is responsible for malignant progression and poor prognosis in breast [52], bladder [53] and prostate [54] cancer and shows strong association with hallmarks of cancer, including induced cellular proliferation [55], angiogenesis [56], survival [57] and migration [58]. Enhancer of Zeste Homolog 2 (EZH2) is the only PRC2 protein member thus far studied in pancreatic cancer. Strong nuclear accumulation of EZH2 was found in 55% of well differenti‐ ated tumors and 98% of poorly differentiated samples in a comprehensive immunohistochem‐ ical analysis of PDACs, indicating a significant correlation between EZH2 expression and dedifferentiation in pancreatic cancer [59]. Additionally, EZH2 overexpression participates in epithelial-to-mesenchymal transition (EMT) and invasion through repression of epithelial

8 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

The third group among the epigenetic players in pancreatic carcinogenesis comprises the MicroRNA (miRNA) family, a class of small non-protein coding RNAs which participate in post-transcriptional control of gene expression in eukaryotic organisms [61]. In the last years, advanced global screening technologies have enabled large scale analyses of miRNA profiles in diverse tissue samples, indicating that miRNAs can function as either onco‐ genes or tumor suppressors in the development of various cancer types, including pancreatic cancer [62, 63]. The analysis of miRNA expression patterns has let to complete‐ ly novel insights into pancreatic cancer biology. Specific miRNAs, such as the miR-200 family, miR-34a and miR-155 are involved in PDAC-biology by regulating genes associat‐

The era of epigenetics in pancreatic cancer has emerged strongly within the last years and deepened our understanding of pancreatic cancer biology. One of the most important charac‐ teristics of epigenetic mechanisms which clearly demarcates them from genetics is their reversibility. This feature provides new targets for novel therapeutic interventions in pancre‐

The manifold genetic and epigenetic events observed in pancreatic carcinogenesis mirror the complexity of this malignancy and lead to the assumption that targeting one molecular feature of pancreatic carcinogenesis is not sufficient for successful pancreatic cancer treatment. Though inaccessible for therapeutic options, there exists at least one molecular event found in virtually all invasively growing pancreatic tumors and their precursor lesions: The constitutive activation of oncogenic Kras probably demonstrates the most important and best studied event

proteins like E-cadherin [60].

ed with metastasis and cell stemness [64, 65].

atic cancer and other epithelial tumors.

in pancreatic carcinogenesis.

The mutation of Kras belongs to the earliest events in pancreatic carcinogenesis. Kras proteins comprise a family of signal-transducing GTPases that mediate a wide variety of cellular functions including proliferation, differentiation and survival and are frequently mutated in human cancers [66]. Although Kras is a GTPase, its intrinsic activity is inefficient and requires GTPase activating proteins to promote GTP hydrolysis and attenuate downstream signaling [1]. Oncogenic mutation of Kras (KrasG12D) is generally accepted to represent the initial key event in pancreatic carcinogenesis and found in virtually all invasively growing pancreatic tumors [7]. Due to its prominent role in pancreatic carcinogenesis Kras is considered to be an attractive therapeutic target of PDAC-treatment, but specific biochemical properties of the protein have made this an elusive goal [67]. Activating Kras point mutations at codon 12 (from GGT to GAT or GTT and more rarely CGT) result in substitution of glycine with aspartate, valine or arginine. Oncogenic Kras mutations lock the protein in its GTP-bound form thus permitting its constitutive interaction with and activation of multiple effectors, independent on growth factor stimulation [67].

The activation of Kras engaged effector pathways, like the RAF-mitogen-activated kinase (MAPK)-cascade, phosphoinositide-3-kinase- (PI3K) signaling and the Ral GDS pathway results in stimulation of proliferation, invasion, metastases and survival thus enabling pancreatic cancer progression [3]. Given the aforementioned limitations in Kras inhibition, these downstream targets may provide alternative effective points of therapeutic intervention and thus are the focus of ongoing studies in pancreatic specific systems.

The impact of constitutive Kras activation is not limited on the epithelial cell but also partici‐ pates in the modulation of the tumor environment. One hallmark of PDAC is an extensive stromal remodeling, the most prominent features of which are the recruitment of inflammatory and mesenchymal cells as well as fibrotic replacement of pancreatic parenchyma [68]. Recent studies revealed that even early stages of PanIN development are associated with a stromal reaction, which is characterized by a robust desmoplastic response and recruitment of immune cells. Subclasses of these immune cells, immature myeloid cells, suppress infiltrating T cells and thus establish an immune privilege in the tumor microenvironment promoting pancreatic carcinogenesis [69, 70]. Mechanistically, constitutive activation of Kras in pancreatic ductal cells triggers the production of the cytokine GM-CSF, which, in turn, promotes the expansion of immunosuppressive myeloid cells, leading to the evasion of CD8+ T-cell-driven-antitumor immunity [69, 70].

Due to its high biological relevance for pancreatic carcinogenesis, a genetically engi‐ neered mouse model (GEMM) with pancreas specific Kras mutation was created, allow‐ ing detailed investigations of morphological as well as molecular features of this disease [71]. This transgenic mouse model bares a mutation of the endogenous murine Kras gene specifically in pancreatic progenitor cells by crossing mice with a conditionally activated Kras allel (LSL-KrasG12D) to transgenic strains that express Cre recombinase in pancreatic lineages (PdxCre or p48Cre). These "KC" mice develop low and high grade PanIN lesions recapitulating pancreatic carcinogenesis in the human situation but only slowly progress to PDAC at an advanced age [71]. This mouse model taught us that in spite of the requirement of Kras-activation for pancreatic cancer development oncogenic Kras muta‐ tion alone fails to transform precursor lesions into invasive cancer due to activation of powerful fail-safe mechanisms (compare Fig. 2).

In agreement with its key role in senescence and tumor suppression, mutational p53 inacti‐ vation is associated with accelerated carcinogenesis in many tumor entities [80]. In the pancreas, p53 inactivation on chromosome 17 has been reported in 50-75% of carcinomas [1]. In the murine pancreas carcinoma model, genetic loss of p53 allows Kras to bypass senescence resulting in 100% penetrance at an early age, thus recapitulating human PDAC including histopathological similarities in neoplastic cells, desmoplasia and occurrence of liver and lung

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**Figure 2. Current model of pancreatic carcinogenesis:** on the background of Kras mutation ADM lesions progress to PanIN-precursor lesions and invasive carcinoma depending on additional signals as loss of tumor suppressor func‐ tion or activation of inflammatory pathways. A: Acinar-ductal metaplasia, B: PanIN-1, C: PanIN-2-3, D: Invasive pancre‐

The p16Ink4a gene, located on the short arm of chromosome 9, is one of the most frequently inactivated tumor suppressor genes in pancreatic cancer [1, 2]. Remarkably, virtually all pancreatic carcinomas bare loss of p16Ink4a function, in 40% of pancreatic cancer through homozygous deletion, in 40% by intragenic mutation coupled with loss of the second allele,

The protein p16Ink4a belongs to the cyclin D-dependent kinase (CDK) inhibitor family and functions to prevent the phosphorylation of Rb-1 by CDK 4 and 6, resulting in a blockage of G1/S-phase transition of the cell cycle [82]. This event is a decisive step in the inhibition of cell

and in 15% by hypermethylation of the p16Ink4a gene promoter [8].

metastases [81].

atic cancer.

Counteracting transformation and growth, cellular senescence, a permanent cell growth arrest, is increasingly recognized as one of the most critical fail-safe programs in pancreatic carcino‐ genesis [72]. A major cause of this permanent growth arrest was found in telomeres, which are non-coding nucleoprotein complexes positioned in the extremes of chromosomes [73]. During successive cellular divisions, telomeres in normal human cells shorten progressively and, when telomeres erode down below a threshold length, the cell ceases to divide itself and becomes senescent. Importantly, senescence can also be observed in the absence of any detectable telomere shortening or dysfunction in numerous conditions such as cellular stress or oncogene activation. Oncogene induced senescence (OIS) has emerged as a powerful tumor suppressor mechanism protecting cells from unrestrained proliferation imposed by oncogenic signaling [74]. It has been proven that normal cells, when forced to express high levels of oncogenic Ras, undergo a permanent and irreversible cell cycle arrest [75]. OIS is frequently found in premalignant lesions but is essentially absent in advanced cancers, suggesting that malignant tumor cells can find ways to bypass or escape senescence [76].

Pancreas specific expression of oncogenic KrasG12D promotes an initial burst of proliferation accompanied by the development of PanIN precursor lesions before cells stop dividing. These precursor lesions then exhibit many features of senescence including positive senescenceassociated ß-galactosidase staining and induction of cell cycle inhibitors [77]. Successful progression of PanIN lesions towards frank adenocarcinoma requires evasion from senes‐ cence. This can result from additional genetic or epigenetic events concerning major tumor suppressor pathways, namely the p19Arf-p53 pathway and the p16Ink4a-Rb cascade [74].

## **6. Role of tumor suppressor inactivation in pancreatic carcinogenesis**

The p53 protein plays a central role in modulating cellular responses to cytotoxic stress by contributing to both, cell cycle arrest and programmed cell death [3]. Signals of mitogenic oncogenes, such as cMyc or Kras lead to activation of p53, which depending on cell type and stimulus induces either apoptosis or senescence and consequently leads to the elimination of cells with oncogenic activation. p53 is integrated in a complex network of upstream sensors and downstream effectors. An important sensor of oncogenic signals for p53 is p19Arf, which is encoded in an alternative reading frame (ARF) by the tumor suppressor locus CDKN2A [78]. Activation of p19Arf antagonizes the effect of the E3 ubiquitin ligase MDM2 that acts upon p53 to initiate its proteasomal degradation, thereby contributing to the stabilization of the tumor suppressor gene [74]. In the nucleus, stabilized p53 binds to promoters of more than 300 target genes with implications for cell growth control. One such important p53 downstream target is p21. p21 binds to and inhibits the activity of Cyclin-CDK2 and Cyclin-CDK1 complexes and thus functions as a negative regulator of cell cycle progression at the G1 phase [79].

In agreement with its key role in senescence and tumor suppression, mutational p53 inacti‐ vation is associated with accelerated carcinogenesis in many tumor entities [80]. In the pancreas, p53 inactivation on chromosome 17 has been reported in 50-75% of carcinomas [1]. In the murine pancreas carcinoma model, genetic loss of p53 allows Kras to bypass senescence resulting in 100% penetrance at an early age, thus recapitulating human PDAC including histopathological similarities in neoplastic cells, desmoplasia and occurrence of liver and lung metastases [81].

to PDAC at an advanced age [71]. This mouse model taught us that in spite of the requirement of Kras-activation for pancreatic cancer development oncogenic Kras muta‐ tion alone fails to transform precursor lesions into invasive cancer due to activation of

10 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Counteracting transformation and growth, cellular senescence, a permanent cell growth arrest, is increasingly recognized as one of the most critical fail-safe programs in pancreatic carcino‐ genesis [72]. A major cause of this permanent growth arrest was found in telomeres, which are non-coding nucleoprotein complexes positioned in the extremes of chromosomes [73]. During successive cellular divisions, telomeres in normal human cells shorten progressively and, when telomeres erode down below a threshold length, the cell ceases to divide itself and becomes senescent. Importantly, senescence can also be observed in the absence of any detectable telomere shortening or dysfunction in numerous conditions such as cellular stress or oncogene activation. Oncogene induced senescence (OIS) has emerged as a powerful tumor suppressor mechanism protecting cells from unrestrained proliferation imposed by oncogenic signaling [74]. It has been proven that normal cells, when forced to express high levels of oncogenic Ras, undergo a permanent and irreversible cell cycle arrest [75]. OIS is frequently found in premalignant lesions but is essentially absent in advanced cancers, suggesting that

Pancreas specific expression of oncogenic KrasG12D promotes an initial burst of proliferation accompanied by the development of PanIN precursor lesions before cells stop dividing. These precursor lesions then exhibit many features of senescence including positive senescenceassociated ß-galactosidase staining and induction of cell cycle inhibitors [77]. Successful progression of PanIN lesions towards frank adenocarcinoma requires evasion from senes‐ cence. This can result from additional genetic or epigenetic events concerning major tumor suppressor pathways, namely the p19Arf-p53 pathway and the p16Ink4a-Rb cascade [74].

**6. Role of tumor suppressor inactivation in pancreatic carcinogenesis**

thus functions as a negative regulator of cell cycle progression at the G1 phase [79].

The p53 protein plays a central role in modulating cellular responses to cytotoxic stress by contributing to both, cell cycle arrest and programmed cell death [3]. Signals of mitogenic oncogenes, such as cMyc or Kras lead to activation of p53, which depending on cell type and stimulus induces either apoptosis or senescence and consequently leads to the elimination of cells with oncogenic activation. p53 is integrated in a complex network of upstream sensors and downstream effectors. An important sensor of oncogenic signals for p53 is p19Arf, which is encoded in an alternative reading frame (ARF) by the tumor suppressor locus CDKN2A [78]. Activation of p19Arf antagonizes the effect of the E3 ubiquitin ligase MDM2 that acts upon p53 to initiate its proteasomal degradation, thereby contributing to the stabilization of the tumor suppressor gene [74]. In the nucleus, stabilized p53 binds to promoters of more than 300 target genes with implications for cell growth control. One such important p53 downstream target is p21. p21 binds to and inhibits the activity of Cyclin-CDK2 and Cyclin-CDK1 complexes and

malignant tumor cells can find ways to bypass or escape senescence [76].

powerful fail-safe mechanisms (compare Fig. 2).

**Figure 2. Current model of pancreatic carcinogenesis:** on the background of Kras mutation ADM lesions progress to PanIN-precursor lesions and invasive carcinoma depending on additional signals as loss of tumor suppressor func‐ tion or activation of inflammatory pathways. A: Acinar-ductal metaplasia, B: PanIN-1, C: PanIN-2-3, D: Invasive pancre‐ atic cancer.

The p16Ink4a gene, located on the short arm of chromosome 9, is one of the most frequently inactivated tumor suppressor genes in pancreatic cancer [1, 2]. Remarkably, virtually all pancreatic carcinomas bare loss of p16Ink4a function, in 40% of pancreatic cancer through homozygous deletion, in 40% by intragenic mutation coupled with loss of the second allele, and in 15% by hypermethylation of the p16Ink4a gene promoter [8].

The protein p16Ink4a belongs to the cyclin D-dependent kinase (CDK) inhibitor family and functions to prevent the phosphorylation of Rb-1 by CDK 4 and 6, resulting in a blockage of G1/S-phase transition of the cell cycle [82]. This event is a decisive step in the inhibition of cell cycle progression and also in senescence initiation. In contrast to that, loss of p16Ink4a results in inappropriate phosphorylation of Rb-1, thereby facilitating progression of the cell cycle through enhanced G1/S transition [1-3, 74].

Ca2+/calcineurin regulated transcription [90]. Thus, NFAT transcription complexes function as signal integrators and detectors. One signal has to be Ca2+/calcineurin, while the second one can have developmental origin or can embody oncogenic qualities as the Ras-MAP kinase pathway [89, 90]. Doing so, the cooperation between NFAT and its partners helps controlling

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**Figure 3. NFAT signaling in pancreatic cancer.** Upon Ca2+ - dependent activation of Calcineurin, NFAT becomes de‐ phosphorylated and shuttles into the nucleus. The calcineurin-inhibitor Cycosporin A (CsA) prevents NFAT activation. In the nucleus GSK3ß-dependent phosphorylation of NFAT either leads to its nuclear export or allows binding to tar‐ get genes in association with partner transcription factors. Ubiquitination of NFAT proteins labels them for proteoso‐

The NFAT family of transcription factors was originally identified as a group of inducible nuclear proteins which regulate transcription during T lymphocyte activation [91]. Following

mal degradation by HDM2.

**7. Oncogenic potential of NFAT signaling**

the specificity of NFAT target gene binding and the resulting mode of action.

Additional to inactivation of tumor suppressor genes, Kras-initiated pancreatic carcinogenesis can be promoted by signals from the inflammatory environment [69, 70]. This type of proin‐ flammatory environment can be provided by chronic pancreatitis, the most relevant risk factor for PDAC development in human [83]. Chronic pancreatitis supports the initiation and progression of this malignancy by direct modification of gene expression networks in pancre‐ atic epithelial cells. For instance, pancreatitis contributes to tumor progression by abrogating the senescence barrier characteristic of low-grade PanIN lesions [84]. Most importantly, chronic pancreatitis induces a wide range of proteins, predominantly inflammatory transcrip‐ tion factors. The majority of these inflammatory transcription factors inhabits oncogenic potential, mediated by inhibition of tumor suppressor genes or synergism with KrasG12D signaling to promote pancreatic carcinogenesis.

By introducing the inflammatory family of Nuclear factor of activated T cells (NFAT) proteins, the following part of the chapter will cite an example how deregulated oncogenes participate in and cooperate with KrasG12D mediated signaling in every single step of pancreatic carcino‐ genesis, beginning from induction of ADM over progression of pancreatic precursor lesions to frank invasive pancreatic ductal adenocarcinoma.

#### **6.1. NFAT proteins and their role in pancreatic carcinogenesis**

#### *6.1.1. The family of NFAT transcription factors and their cellular regulation*

The NFAT family, first described as a regulator of T cell activation and differentiation, comprises four calcium-responsive isoforms named NFATc1, NFATc2, NFATc3 and NFATc4 as well as a more distant relative, NFAT5 [85]. In resting cells, NFAT factors are located in the cytoplasm in a highly phosphorylated, inactive state [85, 86]. Ligand binding to many receptors results in the activation of phospholipase C (PLC), the release of IP3 and in a transient release of Ca2+ from intracellular stores through IP3 receptors. This initial release of Ca2+ demonstrates the prerequisite for increased influx of Ca2+ through specialized Ca2+ released activated channels (termed CRAC) [86]. CRACs provide the persistent Ca2+ signal that is necessary for sufficient activation of the phosphatase calcineurin that targets and dephosphorylates moderately conserved serine rich motifs in the N-terminal homology region of NFAT proteins to unmask its nuclear localization signals [87]. Subsequently, NFAT proteins shuttle into the nucleus where they are either ubiquitinated for HDM2-dependent proteasomal degradation or stabilized by GSK3ß-mediated phosphorylation (compare Fig. 3) [88]. Upon stabilization the transcription factor recognizes its GGAAA consensus sequence within target gene elements and binds DNA either as homodimer or heterodimer [85-88]. In fact, NFAT proteins frequently cooperate with other transcription factors to elicit high-affinity binding on common target genes. GATA Proteins, FoxP3 and members of the MEF family are only few among a wide range of NFAT partner proteins [89]. Additionally, NFAT recruits other signaling regulated transcription factors (e.g. Smad3 and NKkB) to integrate pathway specific signals to Ca2+/calcineurin regulated transcription [90]. Thus, NFAT transcription complexes function as signal integrators and detectors. One signal has to be Ca2+/calcineurin, while the second one can have developmental origin or can embody oncogenic qualities as the Ras-MAP kinase pathway [89, 90]. Doing so, the cooperation between NFAT and its partners helps controlling the specificity of NFAT target gene binding and the resulting mode of action.

cycle progression and also in senescence initiation. In contrast to that, loss of p16Ink4a results in inappropriate phosphorylation of Rb-1, thereby facilitating progression of the cell cycle

12 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Additional to inactivation of tumor suppressor genes, Kras-initiated pancreatic carcinogenesis can be promoted by signals from the inflammatory environment [69, 70]. This type of proin‐ flammatory environment can be provided by chronic pancreatitis, the most relevant risk factor for PDAC development in human [83]. Chronic pancreatitis supports the initiation and progression of this malignancy by direct modification of gene expression networks in pancre‐ atic epithelial cells. For instance, pancreatitis contributes to tumor progression by abrogating the senescence barrier characteristic of low-grade PanIN lesions [84]. Most importantly, chronic pancreatitis induces a wide range of proteins, predominantly inflammatory transcrip‐ tion factors. The majority of these inflammatory transcription factors inhabits oncogenic potential, mediated by inhibition of tumor suppressor genes or synergism with KrasG12D

By introducing the inflammatory family of Nuclear factor of activated T cells (NFAT) proteins, the following part of the chapter will cite an example how deregulated oncogenes participate in and cooperate with KrasG12D mediated signaling in every single step of pancreatic carcino‐ genesis, beginning from induction of ADM over progression of pancreatic precursor lesions

The NFAT family, first described as a regulator of T cell activation and differentiation, comprises four calcium-responsive isoforms named NFATc1, NFATc2, NFATc3 and NFATc4 as well as a more distant relative, NFAT5 [85]. In resting cells, NFAT factors are located in the cytoplasm in a highly phosphorylated, inactive state [85, 86]. Ligand binding to many receptors results in the activation of phospholipase C (PLC), the release of IP3 and in a transient release of Ca2+ from intracellular stores through IP3 receptors. This initial release of Ca2+ demonstrates the prerequisite for increased influx of Ca2+ through specialized Ca2+ released activated channels (termed CRAC) [86]. CRACs provide the persistent Ca2+ signal that is necessary for sufficient activation of the phosphatase calcineurin that targets and dephosphorylates moderately conserved serine rich motifs in the N-terminal homology region of NFAT proteins to unmask its nuclear localization signals [87]. Subsequently, NFAT proteins shuttle into the nucleus where they are either ubiquitinated for HDM2-dependent proteasomal degradation or stabilized by GSK3ß-mediated phosphorylation (compare Fig. 3) [88]. Upon stabilization the transcription factor recognizes its GGAAA consensus sequence within target gene elements and binds DNA either as homodimer or heterodimer [85-88]. In fact, NFAT proteins frequently cooperate with other transcription factors to elicit high-affinity binding on common target genes. GATA Proteins, FoxP3 and members of the MEF family are only few among a wide range of NFAT partner proteins [89]. Additionally, NFAT recruits other signaling regulated transcription factors (e.g. Smad3 and NKkB) to integrate pathway specific signals to

through enhanced G1/S transition [1-3, 74].

signaling to promote pancreatic carcinogenesis.

to frank invasive pancreatic ductal adenocarcinoma.

**6.1. NFAT proteins and their role in pancreatic carcinogenesis**

*6.1.1. The family of NFAT transcription factors and their cellular regulation*

**Figure 3. NFAT signaling in pancreatic cancer.** Upon Ca2+ - dependent activation of Calcineurin, NFAT becomes de‐ phosphorylated and shuttles into the nucleus. The calcineurin-inhibitor Cycosporin A (CsA) prevents NFAT activation. In the nucleus GSK3ß-dependent phosphorylation of NFAT either leads to its nuclear export or allows binding to tar‐ get genes in association with partner transcription factors. Ubiquitination of NFAT proteins labels them for proteoso‐ mal degradation by HDM2.

## **7. Oncogenic potential of NFAT signaling**

The NFAT family of transcription factors was originally identified as a group of inducible nuclear proteins which regulate transcription during T lymphocyte activation [91]. Following their initial discovery, a multitude of studies quickly established that NFAT proteins are also expressed outside the immune system where they participate in the regulation of the expres‐ sion of genes influencing cell growth and differentiation [86]. One of the first studies impli‐ cating NFAT factors in cell proliferation was performed in fibroblasts, in which constitutively active NFATc1 induces cell transformation and colony formation [92]. Similarly, in pancreatic tumor cells proliferation and anchorage-independent growth is - at least in part - dependent on calcineurin activity and nuclear translocation of NFAT proteins [93]. This is consistent with high levels of nuclear NFAT in pancreatic cancer cells and in particular in those cells with accelerated growth. Nowadays, ectopic activation of NFAT members is recognized as an important aspect of oncogenic transformation in several human malignancies, most notably in pancreatic cancer [88, 93]. Proliferation and anchorage-independent growth of cultured pancreatic cancer cells is significantly attenuated by inhibition of Ca2+/Calcineurin signaling with Cyclosporin A or siRNA-technology-mediated depletion of NFATc1 [94]. Besides proliferation and growth, NFAT proteins incorporate additional features of tumor biology. Being downstream mediators of α6ß4 integrin signaling NFATc2 and NFAT5 promote cancer invasion in breast and colon cancer [95]. Stimulation of angiogenesis through upregulation of VEGF and enhancement of tumor cell migration via transcriptional activation of Cox2 are additional oncogenic features of NFAT proteins [86, 96].

recent years, increasing evidence arised that PanIN precursor lesions and invasive PDAC originate from differentiated acinar cells. The development of duct-like PanIN lesions from acinar cells requires massive remodeling of these cells, both morphologically and with respect to gene expression profiles. The transition from acinar to ductal cell properties has been termed acinar-to-ductal metaplasia (ADM) and lineage tracing experiments have confirmed that this process is a result of direct transdifferentiation from adult acinar cells that convert to a ductal phenotype upon expression of constitutive active Kras [97, 98]. In murine and in human samples, ADM development has been shown to precede PanIN formation, suggesting that

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Appreciating the relevance of ADM for pancreatic cancer development, much effort was put into research on the molecular mechanisms facilitating ADM. As a transcription factor that is involved in differentiation processes in many tissues NFAT constitutes a promis‐ ing candidate to mediate ADM. Indeed, NFATc1 is highly operative in pancreatic ADM, while only rare expression of the transcription factor can be found in acinar cells. *In vitro* and *in vivo* studies have revealed that KrasG12D driven ADM requires ligand-dependent activation of the Epidermal growth factor receptor [6, 21]. Careful molecular studies have proven that EGFR signaling – at least in part – is mediated via NFATc1. Most important‐ ly, in spite of active EGFR signaling, pharmacological or genetic inactivation of NFATc1 in acinar cell explants extracted from KrasG12D mice reduces duct formation *in vitro*. Further‐ more, KrasG12D mice harboring a pancreas specific transgenic inactivation of NFATc1 are less susceptible to inflammation induced ADM and show a significant delay of pancreatic carcinogenesis [unpublished data]. These findings clearly indicate a key role of NFAT

ADM represents the first step of pancreatic carcinogenesis.

signaling in the initial steps of pancreatic carcinogenesis.

**9. NFATc1 and STAT3 cooperation in pancreatic carcinogenesis**

in established human pancreatic cancer [Baumgart et al., under review].

Recent investigations established that NFATc1 cooperates with the signal transducer and activator of transcription-3 (STAT3) [Baumgart et al., unpublished data]. Like NFAT proteins, STAT3 is also regulated primarily at the level of its subcellular localization [90]. In resting cells, STAT3 resides in a non-phosphorylated version in the cytoplasm. However, following cytokine or growth factor stimulation, STAT3 proteins are inducibly phosphorylated on critical regulatory tyrosine residues promoting their homodimerization and subsequent translocation into the nucleus where they control gene transcription [99]. Interestingly, genetic depletion of STAT3 attenuates the transformation capacity of NFATc1, suggesting a cooperative function of both transcription factors in pancreatic cancer. From the mechanistic point of view, NFATc1 interacts with STAT3 to form enhancer-promoter communications at jointly regulated genes involved in inflammation and oncogenesis, e.g. EGFR and Wnt-family members. The NFATc1- STAT3 transcription pathway is operative in pancreatitis-mediated carcinogenesis as well as

GEMM with constitutive activation of NFATc1 revealed increased cellular proliferation in pancreata of young mice but mice baring a constitutive activation of NFATc1 failed to develop advanced PanIN lesions within a one-year observations span. In contrast to mice bearing an isolated transgenic induction of NFATc1, mice carrying combined constitutive activation of Kras and NFATc1, a situation found in 70% of human PDACs, surprise with a dramatically shortened survival compared to the KrasG12D animals [Baumgart et al., unpublished data]. Further resembling human PDAC, KrasG12D;NFATc1 mice develop severe cachexia and abdominal distension caused by the accumulation of sanguineous ascites and bile duct obstruction. At necropsy, the pancreata from KrasG12D;NFATc1 mice are enlarged by tumor mass, which contains both solid and cystic regions. Notably, pancreata from KrasG12D;NFATc1 mice express nuclear NFATc1 throughout carcinogenesis and at equivalent levels to those observed in human PDAC.

Beyond doubt, the experience with the described transgenic mouse model which recapitulates human PDAC disease in a very accurate manner clearly shows that activation of NFAT proteins works synergistically with Kras signaling and leads to acceleration of pancreatic carcinogenesis. Further investigations shot light on the NFAT dependent mechanisms facilitating and hastening pancreatic carcinogenesis.

## **8. NFATc1 function in ADM**

The cellular origin of PDAC has been a controversial topic for many decades. PDAC has long been considered to be a disease of pancreatic ducts. However, early efforts to model the disease by forcing Kras expression in pancreatic duct cells did not yield discernable pathology [97]. In recent years, increasing evidence arised that PanIN precursor lesions and invasive PDAC originate from differentiated acinar cells. The development of duct-like PanIN lesions from acinar cells requires massive remodeling of these cells, both morphologically and with respect to gene expression profiles. The transition from acinar to ductal cell properties has been termed acinar-to-ductal metaplasia (ADM) and lineage tracing experiments have confirmed that this process is a result of direct transdifferentiation from adult acinar cells that convert to a ductal phenotype upon expression of constitutive active Kras [97, 98]. In murine and in human samples, ADM development has been shown to precede PanIN formation, suggesting that ADM represents the first step of pancreatic carcinogenesis.

their initial discovery, a multitude of studies quickly established that NFAT proteins are also expressed outside the immune system where they participate in the regulation of the expres‐ sion of genes influencing cell growth and differentiation [86]. One of the first studies impli‐ cating NFAT factors in cell proliferation was performed in fibroblasts, in which constitutively active NFATc1 induces cell transformation and colony formation [92]. Similarly, in pancreatic tumor cells proliferation and anchorage-independent growth is - at least in part - dependent on calcineurin activity and nuclear translocation of NFAT proteins [93]. This is consistent with high levels of nuclear NFAT in pancreatic cancer cells and in particular in those cells with accelerated growth. Nowadays, ectopic activation of NFAT members is recognized as an important aspect of oncogenic transformation in several human malignancies, most notably in pancreatic cancer [88, 93]. Proliferation and anchorage-independent growth of cultured pancreatic cancer cells is significantly attenuated by inhibition of Ca2+/Calcineurin signaling with Cyclosporin A or siRNA-technology-mediated depletion of NFATc1 [94]. Besides proliferation and growth, NFAT proteins incorporate additional features of tumor biology. Being downstream mediators of α6ß4 integrin signaling NFATc2 and NFAT5 promote cancer invasion in breast and colon cancer [95]. Stimulation of angiogenesis through upregulation of VEGF and enhancement of tumor cell migration via transcriptional activation of Cox2 are

14 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

GEMM with constitutive activation of NFATc1 revealed increased cellular proliferation in pancreata of young mice but mice baring a constitutive activation of NFATc1 failed to develop advanced PanIN lesions within a one-year observations span. In contrast to mice bearing an isolated transgenic induction of NFATc1, mice carrying combined constitutive activation of Kras and NFATc1, a situation found in 70% of human PDACs, surprise with a dramatically shortened survival compared to the KrasG12D animals [Baumgart et al., unpublished data]. Further resembling human PDAC, KrasG12D;NFATc1 mice develop severe cachexia and abdominal distension caused by the accumulation of sanguineous ascites and bile duct obstruction. At necropsy, the pancreata from KrasG12D;NFATc1 mice are enlarged by tumor mass, which contains both solid and cystic regions. Notably, pancreata from KrasG12D;NFATc1 mice express nuclear NFATc1 throughout carcinogenesis and at equivalent levels to those

Beyond doubt, the experience with the described transgenic mouse model which recapitulates human PDAC disease in a very accurate manner clearly shows that activation of NFAT proteins works synergistically with Kras signaling and leads to acceleration of pancreatic carcinogenesis. Further investigations shot light on the NFAT dependent mechanisms

The cellular origin of PDAC has been a controversial topic for many decades. PDAC has long been considered to be a disease of pancreatic ducts. However, early efforts to model the disease by forcing Kras expression in pancreatic duct cells did not yield discernable pathology [97]. In

additional oncogenic features of NFAT proteins [86, 96].

facilitating and hastening pancreatic carcinogenesis.

observed in human PDAC.

**8. NFATc1 function in ADM**

Appreciating the relevance of ADM for pancreatic cancer development, much effort was put into research on the molecular mechanisms facilitating ADM. As a transcription factor that is involved in differentiation processes in many tissues NFAT constitutes a promis‐ ing candidate to mediate ADM. Indeed, NFATc1 is highly operative in pancreatic ADM, while only rare expression of the transcription factor can be found in acinar cells. *In vitro* and *in vivo* studies have revealed that KrasG12D driven ADM requires ligand-dependent activation of the Epidermal growth factor receptor [6, 21]. Careful molecular studies have proven that EGFR signaling – at least in part – is mediated via NFATc1. Most important‐ ly, in spite of active EGFR signaling, pharmacological or genetic inactivation of NFATc1 in acinar cell explants extracted from KrasG12D mice reduces duct formation *in vitro*. Further‐ more, KrasG12D mice harboring a pancreas specific transgenic inactivation of NFATc1 are less susceptible to inflammation induced ADM and show a significant delay of pancreatic carcinogenesis [unpublished data]. These findings clearly indicate a key role of NFAT signaling in the initial steps of pancreatic carcinogenesis.

## **9. NFATc1 and STAT3 cooperation in pancreatic carcinogenesis**

Recent investigations established that NFATc1 cooperates with the signal transducer and activator of transcription-3 (STAT3) [Baumgart et al., unpublished data]. Like NFAT proteins, STAT3 is also regulated primarily at the level of its subcellular localization [90]. In resting cells, STAT3 resides in a non-phosphorylated version in the cytoplasm. However, following cytokine or growth factor stimulation, STAT3 proteins are inducibly phosphorylated on critical regulatory tyrosine residues promoting their homodimerization and subsequent translocation into the nucleus where they control gene transcription [99]. Interestingly, genetic depletion of STAT3 attenuates the transformation capacity of NFATc1, suggesting a cooperative function of both transcription factors in pancreatic cancer. From the mechanistic point of view, NFATc1 interacts with STAT3 to form enhancer-promoter communications at jointly regulated genes involved in inflammation and oncogenesis, e.g. EGFR and Wnt-family members. The NFATc1- STAT3 transcription pathway is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer [Baumgart et al., under review].

## **10. Impact of NFAT proteins on the inflammatory tumor environment**

dependent histone acetylation rendering the promoter transcriptionally active. Hyperacety‐ lation of the cMyc promoter is required for recruitment of the Ets-like gene 1 (ELK-1), a protein signaling downstream of Kras, responsible for maximal activation of cMyc [94]. The functional significance of this pathway is emphasized by restoration of TGFß growth suppressor function in cancer cells and impaired cMyc expression indicated by reduced tumor growth and G1 arrest following the pharmacological or genetic inactivation of NFAT proteins [94, 102].

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17

**12. NFAT dependent silencing of tumor suppressor genes by formation of**

Activation of NFAT proteins does not only lead to target gene activation in pancreatic cancer, but also contributes to gene silencing. Being a member of the Ink4 family, p15Ink4b impedes the activation and function of Cyclin dependent kinases (CDK) 4 and 6 which leads to cell cycle inhibition and diminished G1-S phase transition [105]. Therefore, p15Ink4b incorporates important functions as a tumor suppressor in numerous malignancies, most importantly in pancreatic cancer, where p15Ink4b inactivation by genetic or epigenetic events occurs in over 90% of all tumors [9]. NFATc2 targets p15Ink4b for inducible and sequential heterochromatin formation and gene silencing. Sequential Chromatinimmunprecipitation revealed that NFATc2 binding to its putative binding side on the p15Ink4b promoter leads to recruitment of the histone methyltransferase Suv39H1. Local trimethylation of Lysine 9 on histone 3 (H3K9trime) allows docking of heterochromatin protein 1 y (HP1y) which results in stabili‐ zation of the heterochromatin complex on the p15Ink4b promoter. Conflicting with that, inactivation of NFATc2 disrupts the repressor complex and results in restoration of p15Ink4b

**heterochromatin complexes**

expression and function [106].

**Figure 4.** NFAT transcription factors and their impact on hallmarks of cancer

Cancer-associated inflammation plays an important role in restraining anti-tumor immunity, particularly in pancreatic cancer for which a massive infiltration of immunosuppressive leukocytes into the tumor stroma is an early and consistent event in carcinogenesis [84]. In contrast to many other solid tumors, intratumoral T cells are rare in pancreatic cancer, which is associated with an immune escape and bad prognosis [70]. In PDAC, increasing evidence suggests, that oncogenic Kras drives an inflammatory program that establishes immune privilege in the tumor microenvironment [69, 70]. The immune surveillance of pancreatic cancer demonstrates the response to signals from the transformed epithelial pancreatic cell. Cytokines like GM-CSF are secreted by ductal pancreatic cells to modulate the inflammatory tumor environment. Recent work suggests an essential role of NFAT proteins in the transcrip‐ tional induction of a core of cytokines associated with encapsulation of the transformed cell from physiological immune response [100, unpublished data]. Thus, NFAT inactivation might represent a promising possibility to restore pancreatic cancer response to tumor suppressive immune signals.

## **11. NFAT mediated TGFß switch from tumor suppressor to oncogene in pancreatic carcinogenesis**

As mentioned above, an emerging model in cancer biology supports a dual role for TGFß signaling in tumorigenesis, acting as a tumor suppressor in early carcinogenesis and as a strong promoter of cell proliferation, migration and invasion in advanced tumor stages [101, 102]. TGFß blocks cell proliferation in untransformed cells through the induction of a cell cycle arrest at late G1 phase. Two critical molecular events underlie TGFß anti-proliferative response: the transcriptional repression of cMyc and subsequent induction of cell cycle inhibitors like p21 and p15Ink4b [102, 103]. As an immediate early transcription factor proto-oncogenic cMyc functions as a master regulator of G1-S-cell cycle progression and growth promotion in pancreatic cancer [93, 103]. cMyc repression by TGFß requires the activation of a Smad3-4 complex to transduce its stimulus into the nucleus. Here, Smad proteins complex with the transcription factors E2F4/5 and DP1 and corepressor p107 to repress cMyc promoter via binding to its TGFß-inhibitory element (TIE) [104].

During pancreatic carcinogenesis, tumor cells change their transcriptional responsiveness to TGFß and become resistant to the growth inhibitory effects due to functional inactiva‐ tion of the TGFß-Smad pathway [103]. Depending on the cell type and the activation status of a cell, TGFß then signals through Smad-independent pathways (e.g. PI3K and MAPK pathways) to promote the acquisition of a mesenchymal phenotype and stimulate tumor cell migration [102, 103].

TGFß induces expression of NFATc1 and c2, which accumulate in the nucleus and displace pre-existing Smad3 repressor complexes from the cMyc TIE element. Mechanistically, NFATc1 binding to the serum responsive element within the proximal cMyc promoter initiates p300dependent histone acetylation rendering the promoter transcriptionally active. Hyperacety‐ lation of the cMyc promoter is required for recruitment of the Ets-like gene 1 (ELK-1), a protein signaling downstream of Kras, responsible for maximal activation of cMyc [94]. The functional significance of this pathway is emphasized by restoration of TGFß growth suppressor function in cancer cells and impaired cMyc expression indicated by reduced tumor growth and G1 arrest following the pharmacological or genetic inactivation of NFAT proteins [94, 102].

**10. Impact of NFAT proteins on the inflammatory tumor environment**

16 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

immune signals.

**pancreatic carcinogenesis**

cell migration [102, 103].

binding to its TGFß-inhibitory element (TIE) [104].

Cancer-associated inflammation plays an important role in restraining anti-tumor immunity, particularly in pancreatic cancer for which a massive infiltration of immunosuppressive leukocytes into the tumor stroma is an early and consistent event in carcinogenesis [84]. In contrast to many other solid tumors, intratumoral T cells are rare in pancreatic cancer, which is associated with an immune escape and bad prognosis [70]. In PDAC, increasing evidence suggests, that oncogenic Kras drives an inflammatory program that establishes immune privilege in the tumor microenvironment [69, 70]. The immune surveillance of pancreatic cancer demonstrates the response to signals from the transformed epithelial pancreatic cell. Cytokines like GM-CSF are secreted by ductal pancreatic cells to modulate the inflammatory tumor environment. Recent work suggests an essential role of NFAT proteins in the transcrip‐ tional induction of a core of cytokines associated with encapsulation of the transformed cell from physiological immune response [100, unpublished data]. Thus, NFAT inactivation might represent a promising possibility to restore pancreatic cancer response to tumor suppressive

**11. NFAT mediated TGFß switch from tumor suppressor to oncogene in**

As mentioned above, an emerging model in cancer biology supports a dual role for TGFß signaling in tumorigenesis, acting as a tumor suppressor in early carcinogenesis and as a strong promoter of cell proliferation, migration and invasion in advanced tumor stages [101, 102]. TGFß blocks cell proliferation in untransformed cells through the induction of a cell cycle arrest at late G1 phase. Two critical molecular events underlie TGFß anti-proliferative response: the transcriptional repression of cMyc and subsequent induction of cell cycle inhibitors like p21 and p15Ink4b [102, 103]. As an immediate early transcription factor proto-oncogenic cMyc functions as a master regulator of G1-S-cell cycle progression and growth promotion in pancreatic cancer [93, 103]. cMyc repression by TGFß requires the activation of a Smad3-4 complex to transduce its stimulus into the nucleus. Here, Smad proteins complex with the transcription factors E2F4/5 and DP1 and corepressor p107 to repress cMyc promoter via

During pancreatic carcinogenesis, tumor cells change their transcriptional responsiveness to TGFß and become resistant to the growth inhibitory effects due to functional inactiva‐ tion of the TGFß-Smad pathway [103]. Depending on the cell type and the activation status of a cell, TGFß then signals through Smad-independent pathways (e.g. PI3K and MAPK pathways) to promote the acquisition of a mesenchymal phenotype and stimulate tumor

TGFß induces expression of NFATc1 and c2, which accumulate in the nucleus and displace pre-existing Smad3 repressor complexes from the cMyc TIE element. Mechanistically, NFATc1 binding to the serum responsive element within the proximal cMyc promoter initiates p300-

## **12. NFAT dependent silencing of tumor suppressor genes by formation of heterochromatin complexes**

Activation of NFAT proteins does not only lead to target gene activation in pancreatic cancer, but also contributes to gene silencing. Being a member of the Ink4 family, p15Ink4b impedes the activation and function of Cyclin dependent kinases (CDK) 4 and 6 which leads to cell cycle inhibition and diminished G1-S phase transition [105]. Therefore, p15Ink4b incorporates important functions as a tumor suppressor in numerous malignancies, most importantly in pancreatic cancer, where p15Ink4b inactivation by genetic or epigenetic events occurs in over 90% of all tumors [9]. NFATc2 targets p15Ink4b for inducible and sequential heterochromatin formation and gene silencing. Sequential Chromatinimmunprecipitation revealed that NFATc2 binding to its putative binding side on the p15Ink4b promoter leads to recruitment of the histone methyltransferase Suv39H1. Local trimethylation of Lysine 9 on histone 3 (H3K9trime) allows docking of heterochromatin protein 1 y (HP1y) which results in stabili‐ zation of the heterochromatin complex on the p15Ink4b promoter. Conflicting with that, inactivation of NFATc2 disrupts the repressor complex and results in restoration of p15Ink4b expression and function [106].

**Figure 4.** NFAT transcription factors and their impact on hallmarks of cancer

## **13. Perspective**

These examples of NFAT dependent alterations in signaling pathways and transcriptional processes promoting pancreatic carcinogenesis only demonstrate a small insight into how oncogenic transcription factors contribute to pancreatic cancer development. Via transduction of EGFR signaling to downstream targets, by cooperation with other pre inflammatory oncogenes, by modulation of the tumor microenvironment, induction of cell cycle promoting genes as well as via silencing of important tumor suppressor genes, NFAT proteins are highly involved in all phases of pancreatic carcinogenesis reaching from early acinar-to-ductalmetaplasia over establishment of precursor lesions to frank invasive pancreatic adenocarci‐ noma.

Quail MA, Stratton MR, Iacobuzio-Donahue C, Futreal PA. The patterns and dynam‐ ics of genomic instability in metastatic pancreatic cancer. Nature. 2010 Oct

The Molecular Frame of Pancreatic Carcinogenesis

http://dx.doi.org/10.5772/57422

19

[5] Hruban RH, Takaori K, Klimstra DS, Adsay NV, Albores-Saavedra J, Biankin AV, Biankin SA, Compton C, Fukushima N, Furukawa T, Goggins M, Kato Y, Klöppel G, Longnecker DS, Lüttges J, Maitra A, Offerhaus GJ, Shimizu M, Yonezawa S.An illus‐ trated consensus on the classification of pancreatic intraepithelial neoplasia and in‐ traductal papillary mucinous neoplasms. American journal of Surgical Pathology,

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[13] Schutte M, da Costa LT, Hahn SA, Moskaluk C, Hoque AT, Rozenblum E, Weinstein CL, Bittner M, Meltzer PS, Trent JM, et al. Identification by representational differ‐ ence analysis of a homozygous deletion in pancreatic carcinoma that lies within the

creatic adenocarcinoma. J Cell Biochem 2006. 1;97(1): 98-108

BRCA2 region. Proc Natl Acad Sci USA 1995.20;92(13):5950-4

28;467(7319):1109-13.

2004 Aug;28(8):977-87

1998;33(5):432-9

creatology 2001;1(3):254-62

cinoma. Cancer Cell 2012. 22, 318-30

clinical applications. Pancreatology 2007;7(1):9-19

genomic analyses. Science 2008;321(5897):1801-6

As dismal as pancreatic cancer presents itself clinically, as complex and multi-layered are the histopathological and molecular mechanisms responsible for pancreatic carcinogenesis. As the molecular main reason for pancreatic cancer development - the constitutive activation of Kras - evades any pharmacological approach, targeting oncogenic factors like NFAT proteins represents a promising option approaching success in pancreatic cancer treatment.

## **Author details**

Elisabeth Heßmann1 , Sandra Baumgart2 , Nai ming Chen1 , Shiv Singh2 , Garima Singh2 , Alex König1 , Albrecht Neeße1 and Volker Ellenrieder1

1 Department of Gastroenterology II, Georg-August-University, Göttingen, Germany

2 Signaling and Transcription Laboratory, Department of Gastroenterology, Philipps Uni‐ versity, Marburg, Germany

## **References**


Quail MA, Stratton MR, Iacobuzio-Donahue C, Futreal PA. The patterns and dynam‐ ics of genomic instability in metastatic pancreatic cancer. Nature. 2010 Oct 28;467(7319):1109-13.

**13. Perspective**

noma.

**Author details**

Elisabeth Heßmann1

versity, Marburg, Germany

Article ID: 620601:1-16

2010;362:1605-17

Alex König1

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These examples of NFAT dependent alterations in signaling pathways and transcriptional processes promoting pancreatic carcinogenesis only demonstrate a small insight into how oncogenic transcription factors contribute to pancreatic cancer development. Via transduction of EGFR signaling to downstream targets, by cooperation with other pre inflammatory oncogenes, by modulation of the tumor microenvironment, induction of cell cycle promoting genes as well as via silencing of important tumor suppressor genes, NFAT proteins are highly involved in all phases of pancreatic carcinogenesis reaching from early acinar-to-ductalmetaplasia over establishment of precursor lesions to frank invasive pancreatic adenocarci‐

18 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

As dismal as pancreatic cancer presents itself clinically, as complex and multi-layered are the histopathological and molecular mechanisms responsible for pancreatic carcinogenesis. As the molecular main reason for pancreatic cancer development - the constitutive activation of Kras - evades any pharmacological approach, targeting oncogenic factors like NFAT proteins

, Nai ming Chen1

2 Signaling and Transcription Laboratory, Department of Gastroenterology, Philipps Uni‐

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**Chapter 2**

**Stem Cells in Pancreatic Cancer**

Additional information is available at the end of the chapter

CSCs appears as a logical, yet difficult task in anti-cancer strategies.

**2.1. Pancreatic cancer stem stells (CSCs) phenotyping and isolation**

**2. Cancer stem cells: Involvement in the progression, invasion and**

Cancer stem cells from epithelial tissues were identified for the first time in breast cancer in 2003, when Al-Hajj et al. reported that a distinct population of cells, CD44+CD24−/low

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Radu Albulescu

**1. Introduction**

**metastasis**

http://dx.doi.org/10.5772/57530

Cristiana Pistol Tanase, Ana-Maria Enciu, Maria Linda Cruceru, Laura Georgiana Necula, Ana Iulia Neagu, Bogdan Calenic and

Pancreatic cancer is the fourth most frequent cause of cancer-related deaths; it also represents one of the most aggressive cancer types, with a high incidence of distant metastasis and mortality [1]. The detection of pancreatic cancer at early stages, the prediction of the potential resectability, or the response to therapy are the current major challenges in improving the clinical outcome of pancreatic ductal adenocarcinoma (PDAC) [2]. The main issue against successful therapy is represented by the absence of early diagnostic and prognostic markers, as well as the unresponsiveness to radiation and chemotherapies [3]. Among other factors that contribute to the lack of success in the therapy of pancreatic malignancies, cancer stem cells (CSCs) appear to have a major role. Cancer is characterized by cellular heterogeneity; CSCs, which represent a distinct subpopulation of cells, seem to be responsible for tumor initiation and persistency, due to their properties of self-renewal and multilineage differentiation. CSCs are considered as best candidates responsible for tumorigenesis, metastasis, and chemo-and radio-resistance [4]. Understanding and properly addressing the challenge represented by

## **Stem Cells in Pancreatic Cancer**

Cristiana Pistol Tanase, Ana-Maria Enciu, Maria Linda Cruceru, Laura Georgiana Necula, Ana Iulia Neagu, Bogdan Calenic and Radu Albulescu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57530

## **1. Introduction**

Pancreatic cancer is the fourth most frequent cause of cancer-related deaths; it also represents one of the most aggressive cancer types, with a high incidence of distant metastasis and mortality [1]. The detection of pancreatic cancer at early stages, the prediction of the potential resectability, or the response to therapy are the current major challenges in improving the clinical outcome of pancreatic ductal adenocarcinoma (PDAC) [2]. The main issue against successful therapy is represented by the absence of early diagnostic and prognostic markers, as well as the unresponsiveness to radiation and chemotherapies [3]. Among other factors that contribute to the lack of success in the therapy of pancreatic malignancies, cancer stem cells (CSCs) appear to have a major role. Cancer is characterized by cellular heterogeneity; CSCs, which represent a distinct subpopulation of cells, seem to be responsible for tumor initiation and persistency, due to their properties of self-renewal and multilineage differentiation. CSCs are considered as best candidates responsible for tumorigenesis, metastasis, and chemo-and radio-resistance [4]. Understanding and properly addressing the challenge represented by CSCs appears as a logical, yet difficult task in anti-cancer strategies.

## **2. Cancer stem cells: Involvement in the progression, invasion and metastasis**

#### **2.1. Pancreatic cancer stem stells (CSCs) phenotyping and isolation**

Cancer stem cells from epithelial tissues were identified for the first time in breast cancer in 2003, when Al-Hajj et al. reported that a distinct population of cells, CD44+CD24−/low

epithelial-specific antigen (ESA+), develops tumors in immunodeficient mice [5]. In pancreatic cancer, the presence of CSCs was reported in 2007 by Li C *et al,* who showed that CD44+CD24+ESA+cells possess highly tumorigenic potential [6].

expression was increased, neither CD133, nor Notch proteins or ALDH1 reached statistical significance; in turn, Jagged 1 was shown to be a robust marker, along with Nestin [8].

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 31

Mouse models of ductal pancreatic neoplasia seem to harbor a subpopulation of cells express‐ ing high levels of doublecortin-like kinase 1(DCLK1), alpha tubulin acetyltransferase 1(ATAT1), hairy and enhancer of split-1(HES1), hairy/enhancer-of-split related with YRPW motif 1(HEY1), Insulin-like growth factor 1 receptor (IGF1R), and Abelson murine leukemia viral oncogene homolog 1 (ABL1) with cancer-initiating properties. As this subpopulation is identifiable at very early stages during adenocarcinoma development, it provides new targets

All the studies suggest the importance of CSCs in the prognostic and therapeutic responses of pancreatic cancer patients and underline the necessity of stem cell surface marker characteri‐ zation. In this regard, it is useful to better understand the basic genetic and epigenetic processes of cancer stem cell transformation from highly regulated stem cells and also the interaction

Recent studies suggest the involvement of CSCs in the progression, aggressiveness and

*The epithelial-to-mesenchymal transition* concept was first described 40 years ago, in relation to the development of the embryo and germ layer formation [25]. Since then, EMT has been shown to be a key player in several normal biological processes or pathologies, such as: embryogenesis, wound healing or cancer progression. The process is essentially defined by phenotypic changes of epithelial cells towards mesenchymal cells. During embryogenesis, EMT represents the biological process in which cells from the epithelial compartment detach, migrate and acquire a mesenchymal phenotype required for the formation of the mesoderm [26]. EMT also plays a key role upon wounding; the wound healing process is marked by epithelial cell migration to the site following EMT signals from the surrounding tissues and acquisition of the mesenchymal-like phenotype [27]. During this process, changes occur in the expression of specific genes, epithelial cell down-regulation of adherent and tight junction proteins (Claudin1 and 7, Occludin and E-cadherin) and matrix metalloproteinase-increased activity, resulting in increased mobility [28]. The major embryonic signaling pathways Wnt, Notch, Hedgehog and Transforming growth factor beta (TGF-β) are involved in upregulation of EMT-activating transcription factors, including Snail, Twist and Slug families [29].TGF-β signaling, associated with other signaling pathways like Ras/MAPK, is essential for EMT process by repressing junction components like E-cadherin, Claudins, and Occludin via Snail transcription factors. TGF-β is also involved in carcinogenesis, playing dual roles by acting as a tumor suppressor in early tumor development, and paradoxically, by promoting tumor cell

Wnt signaling is also involved in theEMT program, by stabilizing Snail and β-catenin levels and by blocking Glycogen synthase kinase 3 (GSK-3β) activity, processes also related to

epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [23, 24].

for early diagnostic and drug testing [21].

between stem cells and the tumor niche [22].

**2.2. Epithelial-to-mesenchymal transition**

invasion in later stages [30].

Similar to other types of cancer, pancreatic tumor cells apparently grow around a population of CSCs which are capable of promoting tumor growth and progression through many mechanisms, including alteration of adjacent stromal cells and evasion of conventional therapies [7]. Therefore, their identification, isolation and further *in vitro* studies represent the field that provided the most important breakthroughs in pancreatic cancer. The phenotypic characterization of CSCs is an ongoing process, however, there are some biomarkers that are recognized as significant for the stemness phenotype: CD133, Nestin, Notch1-4, Jagged 1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) [8]. Following the model of breast cancer stem cells [5], a pancreatic CSC subpopulation was shown to be epithelial-specific antigen (ESA)+ /CD44+ , but unlike the first, also CD24+ [6]. CD44+ CD24+ ESA+ cells represent 0.5% to 1.0% of all pancreatic cancer cells [4] and show self-renewal capacity *in vitro*, are capable of forming tumor spheres, and can be passaged multiple times without loss of tumor sphere-forming capability [9, 10].

CD133 is a biomarker for putative CSC in several solid tumors [11] and it was used as a marker for flow cytometry to select a subpopulation of tumor cells able to generate tumors in athymic mice [12]; it has been reconfirmed in later studies, by immunohistochemistry, to be present in ductal adenocarcinomas [13]. Furthermore, double positive CD133+ /CXCR4+ seem to be preferentially located in the migration front of pancreatic tumors [12] and demonstrate increased metastatic abilities [14].

Along with CD133, aldehyde dehydrogenase 1 (ALDH1) is also considered a useful marker of stemness, both of which are currently being used for flow cytometry sorting of stem-enriched side populations [15]. Increased activity of ALDH1 was associated with CSCs and has been correlated with invasion, migration and poor overall survival in patients with pancreatic cancer [16]. Therefore, ALDH (+) cells have stem and mesenchymal cell features and are more tumorigenic than CD44+ /CD24+ cells [17]. An intriguing and somewhat discouraging observa‐ tion is that only 0.015% of all tumor cells are concomitantly ALDH+and CD44+ /CD24+ , yet ALDH+cells alone have potent tumorigenic activity, thus, several subsets of tumor-initiating cells might be present within a pancreatic tumor [18].

The majority of CSCs is not positive for cytokeratins (intermediate filament proteins present in differentiated epithelial cells) [12], but for Nestin – an intermediate filament protein and a stem cell marker associated with cell integrity, migration, and differentiation. In pancreatic carcinoma, one third of tumor cells present nestin expression which is correlated with tumor staging and metastasis. Nestin-expressing cells are involved in epithelial-to-mesenchymal transition (EMT) and seem to be the origin of pancreatic intraepithelial neoplasia lesions [19]. Recently, presence of Nestin in various types of malignancy was associated with tumoral angiogenesis and was proposed as an angiogenic marker [20].

Within a recent study, authors comparatively analyzed cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma, yielding surprising results: although expression was increased, neither CD133, nor Notch proteins or ALDH1 reached statistical significance; in turn, Jagged 1 was shown to be a robust marker, along with Nestin [8].

Mouse models of ductal pancreatic neoplasia seem to harbor a subpopulation of cells express‐ ing high levels of doublecortin-like kinase 1(DCLK1), alpha tubulin acetyltransferase 1(ATAT1), hairy and enhancer of split-1(HES1), hairy/enhancer-of-split related with YRPW motif 1(HEY1), Insulin-like growth factor 1 receptor (IGF1R), and Abelson murine leukemia viral oncogene homolog 1 (ABL1) with cancer-initiating properties. As this subpopulation is identifiable at very early stages during adenocarcinoma development, it provides new targets for early diagnostic and drug testing [21].

All the studies suggest the importance of CSCs in the prognostic and therapeutic responses of pancreatic cancer patients and underline the necessity of stem cell surface marker characteri‐ zation. In this regard, it is useful to better understand the basic genetic and epigenetic processes of cancer stem cell transformation from highly regulated stem cells and also the interaction between stem cells and the tumor niche [22].

#### **2.2. Epithelial-to-mesenchymal transition**

epithelial-specific antigen (ESA+), develops tumors in immunodeficient mice [5]. In pancreatic cancer, the presence of CSCs was reported in 2007 by Li C *et al,* who showed that

30 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Similar to other types of cancer, pancreatic tumor cells apparently grow around a population of CSCs which are capable of promoting tumor growth and progression through many mechanisms, including alteration of adjacent stromal cells and evasion of conventional therapies [7]. Therefore, their identification, isolation and further *in vitro* studies represent the field that provided the most important breakthroughs in pancreatic cancer. The phenotypic characterization of CSCs is an ongoing process, however, there are some biomarkers that are recognized as significant for the stemness phenotype: CD133, Nestin, Notch1-4, Jagged 1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) [8]. Following the model of breast cancer stem cells [5], a pancreatic CSC subpopulation was shown to be epithelial-specific antigen

[6]. CD44+

of all pancreatic cancer cells [4] and show self-renewal capacity *in vitro*, are capable of forming tumor spheres, and can be passaged multiple times without loss of tumor sphere-forming

CD133 is a biomarker for putative CSC in several solid tumors [11] and it was used as a marker for flow cytometry to select a subpopulation of tumor cells able to generate tumors in athymic mice [12]; it has been reconfirmed in later studies, by immunohistochemistry, to be present in

preferentially located in the migration front of pancreatic tumors [12] and demonstrate

Along with CD133, aldehyde dehydrogenase 1 (ALDH1) is also considered a useful marker of stemness, both of which are currently being used for flow cytometry sorting of stem-enriched side populations [15]. Increased activity of ALDH1 was associated with CSCs and has been correlated with invasion, migration and poor overall survival in patients with pancreatic cancer [16]. Therefore, ALDH (+) cells have stem and mesenchymal cell features and are more

ALDH+cells alone have potent tumorigenic activity, thus, several subsets of tumor-initiating

The majority of CSCs is not positive for cytokeratins (intermediate filament proteins present in differentiated epithelial cells) [12], but for Nestin – an intermediate filament protein and a stem cell marker associated with cell integrity, migration, and differentiation. In pancreatic carcinoma, one third of tumor cells present nestin expression which is correlated with tumor staging and metastasis. Nestin-expressing cells are involved in epithelial-to-mesenchymal transition (EMT) and seem to be the origin of pancreatic intraepithelial neoplasia lesions [19]. Recently, presence of Nestin in various types of malignancy was associated with tumoral

Within a recent study, authors comparatively analyzed cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma, yielding surprising results: although

tion is that only 0.015% of all tumor cells are concomitantly ALDH+and CD44+

CD24+

ESA+

cells [17]. An intriguing and somewhat discouraging observa‐

cells represent 0.5% to 1.0%

/CXCR4+

seem to be

/CD24+

, yet

CD44+CD24+ESA+cells possess highly tumorigenic potential [6].

, but unlike the first, also CD24+

/CD24+

cells might be present within a pancreatic tumor [18].

angiogenesis and was proposed as an angiogenic marker [20].

ductal adenocarcinomas [13]. Furthermore, double positive CD133+

(ESA)+

/CD44+

capability [9, 10].

increased metastatic abilities [14].

tumorigenic than CD44+

Recent studies suggest the involvement of CSCs in the progression, aggressiveness and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer [23, 24].

*The epithelial-to-mesenchymal transition* concept was first described 40 years ago, in relation to the development of the embryo and germ layer formation [25]. Since then, EMT has been shown to be a key player in several normal biological processes or pathologies, such as: embryogenesis, wound healing or cancer progression. The process is essentially defined by phenotypic changes of epithelial cells towards mesenchymal cells. During embryogenesis, EMT represents the biological process in which cells from the epithelial compartment detach, migrate and acquire a mesenchymal phenotype required for the formation of the mesoderm [26]. EMT also plays a key role upon wounding; the wound healing process is marked by epithelial cell migration to the site following EMT signals from the surrounding tissues and acquisition of the mesenchymal-like phenotype [27]. During this process, changes occur in the expression of specific genes, epithelial cell down-regulation of adherent and tight junction proteins (Claudin1 and 7, Occludin and E-cadherin) and matrix metalloproteinase-increased activity, resulting in increased mobility [28]. The major embryonic signaling pathways Wnt, Notch, Hedgehog and Transforming growth factor beta (TGF-β) are involved in upregulation of EMT-activating transcription factors, including Snail, Twist and Slug families [29].TGF-β signaling, associated with other signaling pathways like Ras/MAPK, is essential for EMT process by repressing junction components like E-cadherin, Claudins, and Occludin via Snail transcription factors. TGF-β is also involved in carcinogenesis, playing dual roles by acting as a tumor suppressor in early tumor development, and paradoxically, by promoting tumor cell invasion in later stages [30].

Wnt signaling is also involved in theEMT program, by stabilizing Snail and β-catenin levels and by blocking Glycogen synthase kinase 3 (GSK-3β) activity, processes also related to

cancer metastasis. On the other hand, Snail can interact with β-catenin and it enhances Wnt signaling [31].

Notch signaling is responsible for cell fate, proliferation, differentiation, apoptosis and the maintenance of stem cells and also for hypoxia, which can activate EMT in cancer [32]. It is also considered that Notch can regulate endothelial and mesenchymal markers to sustain mesenchymal transformation [33]. Notch pathways have been shown to increase cellular migration by activating Nuclear factor kappa β (NF-κB), Matrix metalloproteinase 9 and Vascular endothelial growth factor (VEGF) in pancreatic cancer cells [34]. More studies suggest that Notch inhibition can reverse EMT in the Mesenchymal-to-Epithelial Transition (MET) and can be considered a promising therapeutic strategy in cancer treatment [35].

Hedgehog signaling is also involved in embryonic cell growth and organogenesis as well as in regulating genes associated with cell proliferation, differentiation, and cell motility [36]. Some studies showed that the Hedgehog pathway, normally quiescent in adult organs, is very active in cancer where it can increase stromal hyperplasia, myofibroblast differentiation, and production of extracellular matrix, enabling the EMT process in cancer cells [37].

A solid body of literature shows that the EMT process is actively implicated in tumor metastasis and tumor recurrence and that cancer stem cells that have undergone EMT display resistance to therapy [38, 39]. The accepted theory is that CSCs from solid tumors acquire migratory potential together with mesenchymal transition, migrate from the primary tumor, colonize other tissues and form a new metastatic tumor with similar characteristics as the initial one (Figure 1) [40, 41]. *In vitro* and *in vivo* studies support EMT involvement in early steps of carcinogenesis, by identifying EMT-associated markers such as mesenchymal-specific markers (i.e. Vimentin and Fibronectin), epithelial specific markers (i.e. E-cadherin and Cytokeratin), and transcription factors (i.e. Snail and Slug) in tumor samples [42]. Moreover, the expression of EMT-specific genes has been identified at the level of the invasive front of primary tumors [32] and reversely, the expression of CSCs markers can be induced by overexpressing Snail or Twist, the most important transcrip‐ tion factors involved in the EMT process [43]. From the other point of view, cancer cells from metastasis after the EMT process can show a CSC phenotype and TGF-β signaling is considered to be a crucial factor involved in these processes [44].

**Figure 1.** Epithelial-to-mesenchymal transition process

CSCs [4, 50].

therapy [52].

**2.3. Regulatory pathways in pancreatic cancer stem cells**

integrin signaling, and Ephrin signaling networks [49].

Analysis of expression of CSC-related genes in a purified subpopulation of putative pancreatic CSCs showed that up to 46 canonical pathways are upregulated, including human embryonic stem cell pluripotency, tight junction signaling, NF-kB signaling, Wnt/β-catenin signaling,

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 33

In particular, out of most signaling pathways involved in maintaining self-renewal in normal stem cells, pancreatic CSCs are characterized by overexpression of Sonic Hedgehog (Shh), Wnt, Notch, AKT, NF-kB, and BMI1 Polycomb Ring Finger Oncogene(BMI-1). Further, signaling pathways which are not dysregulated in metastatic tumors are overexpressed in the pancreatic

Hedgehog, Notch, Wnt (Figure 2) are shown to be of particular importance in pancreatic cancer stem cells, due to their role in pancreatic embryonic development and differentiation [51]. These signaling pathways are altered in CSCs and EMT-like cells in pancreatic cancer, being involved in self-renewal of CSCs, tumor growth, invasion, metastasis, and resistance to

Cellular migratory potential is also increased by up-regulation of Mucin-4 (MUC4) and fibroblast growth factor receptor 1 (FGFR-1) stabilization [45]. Other studies show that the process in pancreatic cancer can also be regulated by Forkhead box protein M1 (FoxM1) caveolin [46], GLI-Kruppel family member GLI1 (GLI1) [47], hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF) [48]. Taken into account these observations, EMTtype pancreatic tumor cells represent a highly important research focus for the therapies aiming at reducing or preventing invasion, metastasis and therapeutic resistance in pancreatic cancer.

**Figure 1.** Epithelial-to-mesenchymal transition process

cancer metastasis. On the other hand, Snail can interact with β-catenin and it enhances Wnt

32 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Notch signaling is responsible for cell fate, proliferation, differentiation, apoptosis and the maintenance of stem cells and also for hypoxia, which can activate EMT in cancer [32]. It is also considered that Notch can regulate endothelial and mesenchymal markers to sustain mesenchymal transformation [33]. Notch pathways have been shown to increase cellular migration by activating Nuclear factor kappa β (NF-κB), Matrix metalloproteinase 9 and Vascular endothelial growth factor (VEGF) in pancreatic cancer cells [34]. More studies suggest that Notch inhibition can reverse EMT in the Mesenchymal-to-Epithelial Transition (MET) and

Hedgehog signaling is also involved in embryonic cell growth and organogenesis as well as in regulating genes associated with cell proliferation, differentiation, and cell motility [36]. Some studies showed that the Hedgehog pathway, normally quiescent in adult organs, is very active in cancer where it can increase stromal hyperplasia, myofibroblast differentiation, and

A solid body of literature shows that the EMT process is actively implicated in tumor metastasis and tumor recurrence and that cancer stem cells that have undergone EMT display resistance to therapy [38, 39]. The accepted theory is that CSCs from solid tumors acquire migratory potential together with mesenchymal transition, migrate from the primary tumor, colonize other tissues and form a new metastatic tumor with similar characteristics as the initial one (Figure 1) [40, 41]. *In vitro* and *in vivo* studies support EMT involvement in early steps of carcinogenesis, by identifying EMT-associated markers such as mesenchymal-specific markers (i.e. Vimentin and Fibronectin), epithelial specific markers (i.e. E-cadherin and Cytokeratin), and transcription factors (i.e. Snail and Slug) in tumor samples [42]. Moreover, the expression of EMT-specific genes has been identified at the level of the invasive front of primary tumors [32] and reversely, the expression of CSCs markers can be induced by overexpressing Snail or Twist, the most important transcrip‐ tion factors involved in the EMT process [43]. From the other point of view, cancer cells from metastasis after the EMT process can show a CSC phenotype and TGF-β signaling is

Cellular migratory potential is also increased by up-regulation of Mucin-4 (MUC4) and fibroblast growth factor receptor 1 (FGFR-1) stabilization [45]. Other studies show that the process in pancreatic cancer can also be regulated by Forkhead box protein M1 (FoxM1) caveolin [46], GLI-Kruppel family member GLI1 (GLI1) [47], hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF) [48]. Taken into account these observations, EMTtype pancreatic tumor cells represent a highly important research focus for the therapies aiming at reducing or preventing invasion, metastasis and therapeutic resistance in pancreatic

can be considered a promising therapeutic strategy in cancer treatment [35].

production of extracellular matrix, enabling the EMT process in cancer cells [37].

considered to be a crucial factor involved in these processes [44].

signaling [31].

cancer.

#### **2.3. Regulatory pathways in pancreatic cancer stem cells**

Analysis of expression of CSC-related genes in a purified subpopulation of putative pancreatic CSCs showed that up to 46 canonical pathways are upregulated, including human embryonic stem cell pluripotency, tight junction signaling, NF-kB signaling, Wnt/β-catenin signaling, integrin signaling, and Ephrin signaling networks [49].

In particular, out of most signaling pathways involved in maintaining self-renewal in normal stem cells, pancreatic CSCs are characterized by overexpression of Sonic Hedgehog (Shh), Wnt, Notch, AKT, NF-kB, and BMI1 Polycomb Ring Finger Oncogene(BMI-1). Further, signaling pathways which are not dysregulated in metastatic tumors are overexpressed in the pancreatic CSCs [4, 50].

Hedgehog, Notch, Wnt (Figure 2) are shown to be of particular importance in pancreatic cancer stem cells, due to their role in pancreatic embryonic development and differentiation [51]. These signaling pathways are altered in CSCs and EMT-like cells in pancreatic cancer, being involved in self-renewal of CSCs, tumor growth, invasion, metastasis, and resistance to therapy [52].

Many studies found that pancreatic cancer stem cell resistance to chemotherapy is linked to activated Notch signaling, but the exact mechanism remains unclear [57, 58]. There is more evidence showing that the Notch signaling pathway is essential in supporting KRAS ability to transform normal cells into tumor stem cells. Notch-1 inhibition with specific siRNA or treatment with γ-secretase inhibitors increases apoptosis and decreases proliferative rates, cell migration and invasive properties of pancreatic cancer cells [53]. In this regard, in pancreatic cancer treatment, Notch signaling inhibition can be quite attractive, as long as there is no data arguing that Notch signaling has a critical role in normal adult pancreatic homeostasis [59]. Targeting Notch signaling as a treatment for metastatic pancreatic cancer could prevent the

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 35

*Hedgehog signaling* is another self-renewal pathway, allowing normal stem cells to become independent of control signals; as a result of mutations in this signaling, transformed cells can use Hedgehog for tumor initiation, progression, and metastasis. *In vivo* studies showed that compared to normal pancreatic epithelial cells, CD44+CD24+ESA+pancreatic cancer stem cells present with an up-regulation of Sonic Hedgehog (Shh) transcripts (a ligand of Hedgehog signaling) [61]. Moreover, 70% of pancreatic cancer tissue presents overexpression of Shh, suggesting that Hedgehog signaling may be involved in pancreatic carcinogenesis [51]. Many studies showed that Shh signaling can activate pancreatic stellate cells, promotes fibroblast infiltration, and increases secretion of fibronectin, collagen type I, MMPs, and TGF-β [62]. Studies in the pancreatic cancer cell line PANC-1 showed that inhibition of Hedgehog signaling by Smoothened (Smo) suppression can reverse EMT, induce apoptosis via PI3K/AKT inhibi‐ tion, and inhibit the invasion of pancreatic cancer cells [63]. Moreover, combination of focal irradiation with Hedgehog signaling inhibition reduces lymph node metastasis in an ortho‐

Wnt/β-catenin signaling is involved in cell proliferation, migration, apoptosis, differentiation, and stem cell self-renewal in several types of cancer [65]. Wnt/β-catenin signaling pathway dysregulation is also associated with chemoresistance in pancreatic cancer and recent studies suggest that nuclear β-catenin is essential for the EMT [66, 67]. *In vitro* and *in vivo* studies suggest that activated β-catenin may decrease differentiation of epidermal stem cells, increase self-renewal capacity, and develop epithelial cancers in transgenic mice [68]. Kong D et al. showed that there are some connections between Wnt signaling and Snail, a major regulator of the EMT process. Thus, overexpression of Snail could increase expression of Wnt target

In 2013, Sun L et al. showed that one of the most active signaling pathways in pancreatic cancer stem cells is NF-kB, whose inhibition leads to loss of stem cell properties. This study also showed that aberrant epigenetic processes, like CpG promoter methylation, can be involved in carcinogenesis mediated by cancer stem cells [70]. These results were confirmed by studies conducted on PANC1 and HPAC pancreatic cancer cell lines [51]. Activity of the pro-inflam‐ matory NF-κB induces expression of Shh by pancreatic cancer cells and stromal cells, leading

Another possible marker for pancreatic CSCs is Met Proto-Oncogene (c-Met), whose inhibition has been correlated with a decrease of tumor growth and with preventing the development of

acquisition of the EMT phenotype and resistance to therapy [60].

topic animal model [64].

genes by interaction with β-catenin [69].

to activation of the Hedgehog pathway [71].

**Figure 2.** Factors involved in occurrence of cancer stem cells. The emergence of mutations and aberrant signaling in normal stem cells, progenitors, or differentiated cells triggers the transformation of normal cells into cancer stem cells, losing control of cell division.

*Notch signaling* is involved in the early developmental stages of pancreatic cancer by main‐ taining epithelial cells in a progenitor state. Tumor cells present an overexpression of Notch signaling, high levels of Notch-1 and Notch-2 while normal pancreas shows a weak expression of pathway-related molecules [53, 54]. Notch signaling is involved in cell proliferation, survival, apoptosis and differentiation of pancreatic cells and can promote EMT by controlling some transcription factors and growth factors like Snail, Slug, and TGF-β. Among Notch target genes are found Akt, cyclin D1, c-myc, cyclooxygenase-2 (COX-2), extracellular signalregulated kinase (ERK), matrix metalloproteinase-9 (MMP-9), mammalian target of rapamycin (mTOR), NF-κB, VEGF, p21cip1, p27kip1, and p53, all involved in development and progres‐ sion of human cancer. Gemcitabine-resistant pancreatic cancer cells present overexpression of Notch-2 and Jagged-1, while Notch1, a key downstream mediator of Kirsten rat sarcoma viral oncogene homolog(KRAS), is responsible for pancreatosphere formation [7, 51, 53]. Overex‐ pression of Notch ligand Delta like ligand 4 (Dll-4) in pancreatic cancer cells promotes expression of octamer-binding transcription factor 4(Oct4) and Homeobox Transcription Factor Nanog(Nanog) (transcription factors essential for both early embryonic development and pluripotency maintenance in ES cells) and thus increases the number of CSCs [55, 56]. Many studies found that pancreatic cancer stem cell resistance to chemotherapy is linked to activated Notch signaling, but the exact mechanism remains unclear [57, 58]. There is more evidence showing that the Notch signaling pathway is essential in supporting KRAS ability to transform normal cells into tumor stem cells. Notch-1 inhibition with specific siRNA or treatment with γ-secretase inhibitors increases apoptosis and decreases proliferative rates, cell migration and invasive properties of pancreatic cancer cells [53]. In this regard, in pancreatic cancer treatment, Notch signaling inhibition can be quite attractive, as long as there is no data arguing that Notch signaling has a critical role in normal adult pancreatic homeostasis [59]. Targeting Notch signaling as a treatment for metastatic pancreatic cancer could prevent the acquisition of the EMT phenotype and resistance to therapy [60].

*Hedgehog signaling* is another self-renewal pathway, allowing normal stem cells to become independent of control signals; as a result of mutations in this signaling, transformed cells can use Hedgehog for tumor initiation, progression, and metastasis. *In vivo* studies showed that compared to normal pancreatic epithelial cells, CD44+CD24+ESA+pancreatic cancer stem cells present with an up-regulation of Sonic Hedgehog (Shh) transcripts (a ligand of Hedgehog signaling) [61]. Moreover, 70% of pancreatic cancer tissue presents overexpression of Shh, suggesting that Hedgehog signaling may be involved in pancreatic carcinogenesis [51]. Many studies showed that Shh signaling can activate pancreatic stellate cells, promotes fibroblast infiltration, and increases secretion of fibronectin, collagen type I, MMPs, and TGF-β [62]. Studies in the pancreatic cancer cell line PANC-1 showed that inhibition of Hedgehog signaling by Smoothened (Smo) suppression can reverse EMT, induce apoptosis via PI3K/AKT inhibi‐ tion, and inhibit the invasion of pancreatic cancer cells [63]. Moreover, combination of focal irradiation with Hedgehog signaling inhibition reduces lymph node metastasis in an ortho‐ topic animal model [64].

Wnt/β-catenin signaling is involved in cell proliferation, migration, apoptosis, differentiation, and stem cell self-renewal in several types of cancer [65]. Wnt/β-catenin signaling pathway dysregulation is also associated with chemoresistance in pancreatic cancer and recent studies suggest that nuclear β-catenin is essential for the EMT [66, 67]. *In vitro* and *in vivo* studies suggest that activated β-catenin may decrease differentiation of epidermal stem cells, increase self-renewal capacity, and develop epithelial cancers in transgenic mice [68]. Kong D et al. showed that there are some connections between Wnt signaling and Snail, a major regulator of the EMT process. Thus, overexpression of Snail could increase expression of Wnt target genes by interaction with β-catenin [69].

*Notch signaling* is involved in the early developmental stages of pancreatic cancer by main‐ taining epithelial cells in a progenitor state. Tumor cells present an overexpression of Notch signaling, high levels of Notch-1 and Notch-2 while normal pancreas shows a weak expression of pathway-related molecules [53, 54]. Notch signaling is involved in cell proliferation, survival, apoptosis and differentiation of pancreatic cells and can promote EMT by controlling some transcription factors and growth factors like Snail, Slug, and TGF-β. Among Notch target genes are found Akt, cyclin D1, c-myc, cyclooxygenase-2 (COX-2), extracellular signalregulated kinase (ERK), matrix metalloproteinase-9 (MMP-9), mammalian target of rapamycin (mTOR), NF-κB, VEGF, p21cip1, p27kip1, and p53, all involved in development and progres‐ sion of human cancer. Gemcitabine-resistant pancreatic cancer cells present overexpression of Notch-2 and Jagged-1, while Notch1, a key downstream mediator of Kirsten rat sarcoma viral oncogene homolog(KRAS), is responsible for pancreatosphere formation [7, 51, 53]. Overex‐ pression of Notch ligand Delta like ligand 4 (Dll-4) in pancreatic cancer cells promotes expression of octamer-binding transcription factor 4(Oct4) and Homeobox Transcription Factor Nanog(Nanog) (transcription factors essential for both early embryonic development and pluripotency maintenance in ES cells) and thus increases the number of CSCs [55, 56].

**Figure 2.** Factors involved in occurrence of cancer stem cells. The emergence of mutations and aberrant signaling in normal stem cells, progenitors, or differentiated cells triggers the transformation of normal cells into cancer stem cells,

34 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

losing control of cell division.

In 2013, Sun L et al. showed that one of the most active signaling pathways in pancreatic cancer stem cells is NF-kB, whose inhibition leads to loss of stem cell properties. This study also showed that aberrant epigenetic processes, like CpG promoter methylation, can be involved in carcinogenesis mediated by cancer stem cells [70]. These results were confirmed by studies conducted on PANC1 and HPAC pancreatic cancer cell lines [51]. Activity of the pro-inflam‐ matory NF-κB induces expression of Shh by pancreatic cancer cells and stromal cells, leading to activation of the Hedgehog pathway [71].

Another possible marker for pancreatic CSCs is Met Proto-Oncogene (c-Met), whose inhibition has been correlated with a decrease of tumor growth and with preventing the development of metastases [1, 72]. c-Met is a receptor tyrosine kinases involved in cell survival, growth, angiogenesis and metastasis. c-Met activates many signaling pathways, including Ras-MAPK, PI3K/Akt NF-kB, and Wnt/GSK-3β/β-Catenin and is overexpressed in pancreatic cancer [73].

factors [79]. In AsPC1 tumor xenografts, downregulation of c-MYC and KRAS via let-7a was

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 37

Repression of two tumor-suppressor miRs, miR-143 and miR-145, is reported in pancreatic cancer, as well as in other cancers [80]; moreover, experimental restoration of miR 143/145 levels using nano-vector delivery was demonstrated to inhibit pancreatic cancer cell growth [81]. The miR-143/145 cluster cooperates and inhibits the expression of KRAS2 and ras responsive element binding protein 1 (RREB1), its downstream effector [80]. MiR-145 was demonstrated to inhibit cell proliferation in lung adenocarcinoma, by targeting epidermal growth factor receptor (EGFR). In many cancers, including pancreatic cancer, EGFR is upregulated [82], while inhibition of EGF signaling inhibits cancer initiation and progression [83]. Also a suppressive effect of EGFR on miR-143 and miR-145 was demonstrated on models of colon cancer [84]. These findings are indicators of a negative feedback loop between EGFR

The major role of vascular endothelial growth factor (VEGF) signalling via its receptors, VEGFR1 and VEGFR2, was demonstrated in tumor vascular growth, angiogenesis, and metastasis, while upregulated angiogenic factors in various cancers-colorectal, breast, renal, liver, and ovarian-have been correlated with poor prognosis.Pancreatic ductal adenomacar‐ cinoma (PDAC) exhibits endothelial cell proliferation, a mechanisms that increases angiogenesis. Inhibition of VEGF-A, VEGFR1 and VEGFR2 resulted in inhibition of tumor growth and angiogenesis in mouse models of PDAC. Studies and computational analysis outlined a putative binding site for miR-200 (miR-200a, b and c) in the 3' UTR of VEGFR1

Identification of dysregulated expression of various miRNAs, the existence of regulatory loops between miRNAs and protein regulators of key processes (such as cell growth, angiogenesis, differentiation) suggested the need and potential effectiveness of strategies aiming to restore the "normal phenotype" expression pattern of miRNAs for cancer treatment. Various ap‐ proaches are developed and investigated, such as the delivery of tumor suppressor miRNAs [86], suppression of expression or action of oncomirs [87], targeting the expression of key regulators (such as DCLK1, adenosine monophosphate activated kinase α1(AMPKα1)[88], leading to miRNAs modulation or even to simultaneous modulation of multiple miRNAs, suggesting that using miRNAs as therapeutic agents or addressing miRNAs as targets

Although the presence of stromal tissue is described and accepted as a fact in all types of solid cancers, pancreatic adenocarcinoma displays a particularly dense atmosphere of connective tissue, known as "desmoplastic reaction". Since the new cancer paradigm of "stroma-cancer interaction", more thorough investigations have focused on the pancreatic tumor environ‐ ment, and it is now accepted that the dense connective tissue surrounding malignant cells is at least partially responsible for hindering drug delivery. The pancreatic cancer stroma is now the focus of a new therapeutic approach called "stroma depletion", which can be achieved

observed by a similar mechanism demonstrated in pancreatic cancer cells.

and miR-143/145, which is similar to KRAS/RREB1 − miR-143/145.

represents a potential solution for the therapy of critical cancers.

**2.5. CSCs and tumor environment**

and VEGFR2 [85].

#### **2.4. MicroRNAs in pancreatic adenocarcinoma**

MicroRNAs (miRNAs) are potent regulators of cell function via their role as translational regulators for the synthesis of key proteins. Most often, several miRNAs display different expression profiles in cancer cells, including pancreatic cancers.

MiR-21, miR-155 and miR-17−5p appear upregulated in tumoral cells, and these miRs are often called oncogenic miRNAs [60, 74]. Similarly, a series of miRNAs, referred to as tumor sup‐ pressor miRs (miR-34, miR-15a, miR-16−1 and let-7) are downregulated in cancers [54, 75]. Key cell differentiation programs during development are controlled by the members of lethal-7 (Let-7) and miR-200 families. In cancer, loss of Let-7 leads to disease progression and dedifferentiation. The EMT process is also regulated by miRNA-dependent mechanisms and the same Let-7 family appears as a regulator of EMT and of stem cell maintenance. According to Hasselman et al [75], inhibition of maturation of Let-7 by nuclear receptor for the cytotoxic ligand TNFSF10/TRAIL (TRAILR2) in pancreatic cancer cell lines, increases their proliferation. This is consistent with high levels of nuclear TRAIL2 in tissue samples from poor outcome patients.

Pancreatic neoplasms seem also to exhibit their own pattern of miR overexpression, when compared to normal pancreatic tissue: upregulation of miR-93, miR-95, miR-135b, miR-181c, miR-181d, miR-182, miR-183, miR-190, miR-196b and miR-203, miR-767 and miR-1269 and downregulation of miR-20a and miR-29c [76]. In human pancreatic cancer, DCLK1 regulates EMT by a mechanism dependent on miR-200a [77].

MiRNAs were recently considered to have a role in regulation of CSCs [51]. The population of BxPC-3-LN cells (lymph node metastatic pancreatic cells) contains a 5-fold increased population of CD133+/CXCR4+cells (stem-like cells) compared with the parental (nonmetastatic) BxPC-3 cells. Remarkably, a different miRNA pattern is displayed in CSC-like compared with the regular cells: up-regulated miR-572, miR-206, miR-449a, miR-489 and miR-184 were found, as well as downregulated let-7g-3p, let-7i-3p, let-7a-3p, miR-107, miR-128 and miR-141−5p[14].

The miR-200 family members are identified as key regulators of cell maintenance and EMT. It is considered possible that tumor progression is a process resulting in progressive dedifferentiation towards a cell type having a stem cell-like phenotype. This process appears to be regulated by miRNA-dependent mechanisms. DCLK1 (a putative marker for pancreatic and intestinal cancer stem cells) regulates EMT in human pancreatic cancer cells via a miR-200a-dependent mechanism [77]; it also acts as a regulator of Let-7a in pancreatic and colorectal cancer cells, supporting the concept that these miRNAs may be novel and relevant targets in solid tumor cancers [78]. Sureban et al demonstrated that DCLK1 inhibition results in up-regulation of miRNAs that negatively regulate some key angiogenic and pluripotency factors [79]. In AsPC1 tumor xenografts, downregulation of c-MYC and KRAS via let-7a was observed by a similar mechanism demonstrated in pancreatic cancer cells.

Repression of two tumor-suppressor miRs, miR-143 and miR-145, is reported in pancreatic cancer, as well as in other cancers [80]; moreover, experimental restoration of miR 143/145 levels using nano-vector delivery was demonstrated to inhibit pancreatic cancer cell growth [81]. The miR-143/145 cluster cooperates and inhibits the expression of KRAS2 and ras responsive element binding protein 1 (RREB1), its downstream effector [80]. MiR-145 was demonstrated to inhibit cell proliferation in lung adenocarcinoma, by targeting epidermal growth factor receptor (EGFR). In many cancers, including pancreatic cancer, EGFR is upregulated [82], while inhibition of EGF signaling inhibits cancer initiation and progression [83]. Also a suppressive effect of EGFR on miR-143 and miR-145 was demonstrated on models of colon cancer [84]. These findings are indicators of a negative feedback loop between EGFR and miR-143/145, which is similar to KRAS/RREB1 − miR-143/145.

The major role of vascular endothelial growth factor (VEGF) signalling via its receptors, VEGFR1 and VEGFR2, was demonstrated in tumor vascular growth, angiogenesis, and metastasis, while upregulated angiogenic factors in various cancers-colorectal, breast, renal, liver, and ovarian-have been correlated with poor prognosis.Pancreatic ductal adenomacar‐ cinoma (PDAC) exhibits endothelial cell proliferation, a mechanisms that increases angiogenesis. Inhibition of VEGF-A, VEGFR1 and VEGFR2 resulted in inhibition of tumor growth and angiogenesis in mouse models of PDAC. Studies and computational analysis outlined a putative binding site for miR-200 (miR-200a, b and c) in the 3' UTR of VEGFR1 and VEGFR2 [85].

Identification of dysregulated expression of various miRNAs, the existence of regulatory loops between miRNAs and protein regulators of key processes (such as cell growth, angiogenesis, differentiation) suggested the need and potential effectiveness of strategies aiming to restore the "normal phenotype" expression pattern of miRNAs for cancer treatment. Various ap‐ proaches are developed and investigated, such as the delivery of tumor suppressor miRNAs [86], suppression of expression or action of oncomirs [87], targeting the expression of key regulators (such as DCLK1, adenosine monophosphate activated kinase α1(AMPKα1)[88], leading to miRNAs modulation or even to simultaneous modulation of multiple miRNAs, suggesting that using miRNAs as therapeutic agents or addressing miRNAs as targets represents a potential solution for the therapy of critical cancers.

#### **2.5. CSCs and tumor environment**

metastases [1, 72]. c-Met is a receptor tyrosine kinases involved in cell survival, growth, angiogenesis and metastasis. c-Met activates many signaling pathways, including Ras-MAPK, PI3K/Akt NF-kB, and Wnt/GSK-3β/β-Catenin and is overexpressed in pancreatic cancer [73].

36 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

MicroRNAs (miRNAs) are potent regulators of cell function via their role as translational regulators for the synthesis of key proteins. Most often, several miRNAs display different

MiR-21, miR-155 and miR-17−5p appear upregulated in tumoral cells, and these miRs are often called oncogenic miRNAs [60, 74]. Similarly, a series of miRNAs, referred to as tumor sup‐ pressor miRs (miR-34, miR-15a, miR-16−1 and let-7) are downregulated in cancers [54, 75]. Key cell differentiation programs during development are controlled by the members of lethal-7 (Let-7) and miR-200 families. In cancer, loss of Let-7 leads to disease progression and dedifferentiation. The EMT process is also regulated by miRNA-dependent mechanisms and the same Let-7 family appears as a regulator of EMT and of stem cell maintenance. According to Hasselman et al [75], inhibition of maturation of Let-7 by nuclear receptor for the cytotoxic ligand TNFSF10/TRAIL (TRAILR2) in pancreatic cancer cell lines, increases their proliferation. This is consistent with high levels of nuclear TRAIL2 in tissue samples from poor outcome

Pancreatic neoplasms seem also to exhibit their own pattern of miR overexpression, when compared to normal pancreatic tissue: upregulation of miR-93, miR-95, miR-135b, miR-181c, miR-181d, miR-182, miR-183, miR-190, miR-196b and miR-203, miR-767 and miR-1269 and downregulation of miR-20a and miR-29c [76]. In human pancreatic cancer, DCLK1 regulates

MiRNAs were recently considered to have a role in regulation of CSCs [51]. The population of BxPC-3-LN cells (lymph node metastatic pancreatic cells) contains a 5-fold increased population of CD133+/CXCR4+cells (stem-like cells) compared with the parental (nonmetastatic) BxPC-3 cells. Remarkably, a different miRNA pattern is displayed in CSC-like compared with the regular cells: up-regulated miR-572, miR-206, miR-449a, miR-489 and miR-184 were found, as well as downregulated let-7g-3p, let-7i-3p, let-7a-3p, miR-107, miR-128

The miR-200 family members are identified as key regulators of cell maintenance and EMT. It is considered possible that tumor progression is a process resulting in progressive dedifferentiation towards a cell type having a stem cell-like phenotype. This process appears to be regulated by miRNA-dependent mechanisms. DCLK1 (a putative marker for pancreatic and intestinal cancer stem cells) regulates EMT in human pancreatic cancer cells via a miR-200a-dependent mechanism [77]; it also acts as a regulator of Let-7a in pancreatic and colorectal cancer cells, supporting the concept that these miRNAs may be novel and relevant targets in solid tumor cancers [78]. Sureban et al demonstrated that DCLK1 inhibition results in up-regulation of miRNAs that negatively regulate some key angiogenic and pluripotency

**2.4. MicroRNAs in pancreatic adenocarcinoma**

EMT by a mechanism dependent on miR-200a [77].

patients.

and miR-141−5p[14].

expression profiles in cancer cells, including pancreatic cancers.

Although the presence of stromal tissue is described and accepted as a fact in all types of solid cancers, pancreatic adenocarcinoma displays a particularly dense atmosphere of connective tissue, known as "desmoplastic reaction". Since the new cancer paradigm of "stroma-cancer interaction", more thorough investigations have focused on the pancreatic tumor environ‐ ment, and it is now accepted that the dense connective tissue surrounding malignant cells is at least partially responsible for hindering drug delivery. The pancreatic cancer stroma is now the focus of a new therapeutic approach called "stroma depletion", which can be achieved through Hedgehog inhibitors [89]. What stromal cells are responsible for Hedgehog signaling responsiveness is currently under investigation, as it would designate them as new anti-cancer targets. Stromal cells are also of importance when considering the concept of stem cell nichea unique microenvironment involved in generating hierarchies to maintainself-renewal and to control cell fate. The relationship between CSCs and a putative malignant niche is less well stated than for normal stem cells. CSCs are capable of migrating from the original tumor to distance, behavior that is not common for adult, normal stem cells, but is well documented for the hematopoietic stem cell. Stroma of hematopoietic tissue is a particular one, based on reticular connective tissue, unlike most malignant stromas, rich in dense irregular connective tissue. This would possibly indicate the partial independence of CSC from stem-cell niche [90]. From tumor-stroma interactions new lessons were learned in diagnostics and therapeutics of pancreatic cancer. Secreted Protein, Acidic, Cysteine-Rich (SPARC) (a member of the family of matricellular glycoproteins that is highly expressed in PSCs and the tumour/stroma interface) is now proposed as marker for accurate diagnostic, as 80% of pancreatic ductal adenocarcinomas seem to express it [105]. Due to its ability to bind to basement membrane collagen IV and fibrillar collagens I, III, V and also to bind albumin [106], it has been used to increase distribution of the chemotherapeutic agent paclitaxel within the tumoral mass [107].

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 39

Changes within the stem niche, such as hypoxia, are "tuning" the behavior of stem cells,

Mesenchymal stem cells (MSCs) are pluripotent cells with homing abilities that are involved in tissue repair, including outside their native niche, that reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues [108]. They also exhibit a natural tendency of homing into tumors – ability that is starting to be exploited in anticancer treatment, using these versatile cells as cargo delivery for cytotoxic drugs or gene therapy [109]. This behavior has been also reported in pancreatic cancer, by the use of genetically engineered labeled MSCs that efficiently accumulatewithin

Very recent reports have demonstrated that mesenchymal stem cells (MSCs) can function as precursors for CAFs [111, 112]. Interestingly, not all types of MSCs have this particular ability, a recent report from Subramanian et al. arguing that this is not a feature of umbilical-cord derived pluripotent cells[113]. In pancreatic cancer, like in any other type of cancer, these myofibroblast-like cells contribute to inducing EMT in side population cells, maintain tumorinitiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere-forming activity, promoting tumor formation in

Bone marrow derived progenitor cells were found to participate to neovascularization of tumors [115], a process that was shown to be dependent on Hedgehog signaling [116]. The recruitment of these progenitors is accomplished by CAFs through stroma-cell derived factor

An increasing number of reports show that MSCs have the ability of negatively influencing tumor behaviour, in terms of proliferation and invasiveness. Cell cultures co-cultivated or treated with MSCs conditioned media showed inhibited growth [118-120] and co-injection of tumor cells and MSCs in nude animals showed that tumor growth was significantly inhibited [120]. Some authors explain this activity by MSCs to inhibit the expression of Wnt signaling pathway-related factors in tumor cells, consequently unbalancing cellular proliferation and

inducing the activation of survival, proliferation, differentiation and angiogenesis.

**b.** Mesenchymal stem cells – dual facets in cancer

*Pro-tumor effect of MSCs*

1(SDF-1) signaling [117].

*Anti-tumor activity*

apoptosis [121].

the pancreatic tumor, when injected into tumor-bearing mice [110].

a mouse xenograft model, and showing resistance to anticancer drugs [114].

#### **a.** Pancreatic stellate cells

There is a proven interaction between the CSCs and the tumor stroma, at least in part respon‐ sible for increased metastatic abilities of cancer cells. Tumor-stroma interaction is the new cancer paradigm and in the particular case of pancreatic cancer is supported by the presence of pancreatic stellate cells (PSCs) – a subpopulation of desmin-positive periacinar cells, found as well, but in inactive state, in the normal pancreas [91]. Studied at first in relationship with pancreatic fibrosis [92], they were more recently increasingly investigated in the progression of pancreatic cancer [93-95]. In the activated form, stellate cells secrete an array of proinflammatory cytokines and promote an immunosuppresive microenvironment [96], secrete various growth factors (e.g. platelet-derived growth factor, stromal-derived factor 1, epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor) [97], as well as matrix adhesion molecules (collagen type I, secreted protein acidic and rich in cysteine (SPARC), small leucine-rich proteoglycans, periostin) and matrix metalloproteinases (MMP-2 and MMP-9), that have been associated with the invasive phenotype of pancreatic cancer cell lines [41]. This particular pattern of pancreatic cell secretome mediates effects on tumor growth, invasion, metastasis and resistance to chemotherapy and is modulated by CSCs, through release of mitogenic and fibrogenic stimulants, such as Transforming Growth Factor β1 platelet-derived growth factor, sonic hedgehog, galectin 3, endothelin 1 and serine protease inhibitor nexin 2 [97]. Recognition of their importance in tumoral behaviour led efforts to isolate, cultivate and immortalize them for further manipulation with therapeutic purposes [98-100]. Upon activa‐ tion, pancreatic stellate cells suffer a shift of phenotype towards myofibroblast morphology and a subsequent switch of protein expression [101]. Indirect co-culture of pancreatic cancer cells with PSCs seem to favor the stem phenotype of cancer cells, as evaluated by Hamada et al. by the spheroid-forming ability of cancer cells and expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells *in vivo* [90]. The presence of α smooth muscle actin (αSMA) in activated pancreatic stellate cells leads to association with cancer-associated fibroblasts (CAFs) – a cancer modified subpopulation of fibroblasts, identified by the very same marker, that was shown to sustain tumor cells metabolism and favor tumor progression [102]. CAFs also mediate EMT of tumor cells, possibly through a pro-inflammatory signature [103] – secretome that has also been reported in pancreatic stellate cells, not only in cancer but also in chronic pancreatitis [104].

From tumor-stroma interactions new lessons were learned in diagnostics and therapeutics of pancreatic cancer. Secreted Protein, Acidic, Cysteine-Rich (SPARC) (a member of the family of matricellular glycoproteins that is highly expressed in PSCs and the tumour/stroma interface) is now proposed as marker for accurate diagnostic, as 80% of pancreatic ductal adenocarcinomas seem to express it [105]. Due to its ability to bind to basement membrane collagen IV and fibrillar collagens I, III, V and also to bind albumin [106], it has been used to increase distribution of the chemotherapeutic agent paclitaxel within the tumoral mass [107].

Changes within the stem niche, such as hypoxia, are "tuning" the behavior of stem cells, inducing the activation of survival, proliferation, differentiation and angiogenesis.

**b.** Mesenchymal stem cells – dual facets in cancer

Mesenchymal stem cells (MSCs) are pluripotent cells with homing abilities that are involved in tissue repair, including outside their native niche, that reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues [108]. They also exhibit a natural tendency of homing into tumors – ability that is starting to be exploited in anticancer treatment, using these versatile cells as cargo delivery for cytotoxic drugs or gene therapy [109]. This behavior has been also reported in pancreatic cancer, by the use of genetically engineered labeled MSCs that efficiently accumulatewithin the pancreatic tumor, when injected into tumor-bearing mice [110].

#### *Pro-tumor effect of MSCs*

through Hedgehog inhibitors [89]. What stromal cells are responsible for Hedgehog signaling responsiveness is currently under investigation, as it would designate them as new anti-cancer targets. Stromal cells are also of importance when considering the concept of stem cell nichea unique microenvironment involved in generating hierarchies to maintainself-renewal and to control cell fate. The relationship between CSCs and a putative malignant niche is less well stated than for normal stem cells. CSCs are capable of migrating from the original tumor to distance, behavior that is not common for adult, normal stem cells, but is well documented for the hematopoietic stem cell. Stroma of hematopoietic tissue is a particular one, based on reticular connective tissue, unlike most malignant stromas, rich in dense irregular connective tissue. This would possibly indicate the partial independence of CSC from stem-cell niche [90].

38 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

There is a proven interaction between the CSCs and the tumor stroma, at least in part respon‐ sible for increased metastatic abilities of cancer cells. Tumor-stroma interaction is the new cancer paradigm and in the particular case of pancreatic cancer is supported by the presence of pancreatic stellate cells (PSCs) – a subpopulation of desmin-positive periacinar cells, found as well, but in inactive state, in the normal pancreas [91]. Studied at first in relationship with pancreatic fibrosis [92], they were more recently increasingly investigated in the progression of pancreatic cancer [93-95]. In the activated form, stellate cells secrete an array of proinflammatory cytokines and promote an immunosuppresive microenvironment [96], secrete various growth factors (e.g. platelet-derived growth factor, stromal-derived factor 1, epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor) [97], as well as matrix adhesion molecules (collagen type I, secreted protein acidic and rich in cysteine (SPARC), small leucine-rich proteoglycans, periostin) and matrix metalloproteinases (MMP-2 and MMP-9), that have been associated with the invasive phenotype of pancreatic cancer cell lines [41]. This particular pattern of pancreatic cell secretome mediates effects on tumor growth, invasion, metastasis and resistance to chemotherapy and is modulated by CSCs, through release of mitogenic and fibrogenic stimulants, such as Transforming Growth Factor β1 platelet-derived growth factor, sonic hedgehog, galectin 3, endothelin 1 and serine protease inhibitor nexin 2 [97]. Recognition of their importance in tumoral behaviour led efforts to isolate, cultivate and immortalize them for further manipulation with therapeutic purposes [98-100]. Upon activa‐ tion, pancreatic stellate cells suffer a shift of phenotype towards myofibroblast morphology and a subsequent switch of protein expression [101]. Indirect co-culture of pancreatic cancer cells with PSCs seem to favor the stem phenotype of cancer cells, as evaluated by Hamada et al. by the spheroid-forming ability of cancer cells and expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells *in vivo* [90]. The presence of α smooth muscle actin (αSMA) in activated pancreatic stellate cells leads to association with cancer-associated fibroblasts (CAFs) – a cancer modified subpopulation of fibroblasts, identified by the very same marker, that was shown to sustain tumor cells metabolism and favor tumor progression [102]. CAFs also mediate EMT of tumor cells, possibly through a pro-inflammatory signature [103] – secretome that has also been reported in pancreatic stellate cells, not only in cancer but also in chronic

**a.** Pancreatic stellate cells

pancreatitis [104].

Very recent reports have demonstrated that mesenchymal stem cells (MSCs) can function as precursors for CAFs [111, 112]. Interestingly, not all types of MSCs have this particular ability, a recent report from Subramanian et al. arguing that this is not a feature of umbilical-cord derived pluripotent cells[113]. In pancreatic cancer, like in any other type of cancer, these myofibroblast-like cells contribute to inducing EMT in side population cells, maintain tumorinitiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere-forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs [114].

Bone marrow derived progenitor cells were found to participate to neovascularization of tumors [115], a process that was shown to be dependent on Hedgehog signaling [116]. The recruitment of these progenitors is accomplished by CAFs through stroma-cell derived factor 1(SDF-1) signaling [117].

#### *Anti-tumor activity*

An increasing number of reports show that MSCs have the ability of negatively influencing tumor behaviour, in terms of proliferation and invasiveness. Cell cultures co-cultivated or treated with MSCs conditioned media showed inhibited growth [118-120] and co-injection of tumor cells and MSCs in nude animals showed that tumor growth was significantly inhibited [120]. Some authors explain this activity by MSCs to inhibit the expression of Wnt signaling pathway-related factors in tumor cells, consequently unbalancing cellular proliferation and apoptosis [121].

To conclude, the presence of MSC within the tumor site is a fact, but its role is still to be determined.

Some *in vitro* studies showed that blocking *cis*-acting elements, that are common for pluripo‐ tency maintaining Transcription Factor SOX-2 (Sox2), Oct4, and proto-Oncogene C-Myc (c-Myc), dramatically decreased CSCs proliferation and their ability to generate tumors in nude mice [15]. Equally, simultaneous knockdown of OCT4 and its target Nanog led to decreased proliferation, migration, invasiveness and tumorigenesis of putative pancreatic cancer stem cells [129]. Inhibition of the Nodal/Activin receptor Alk4/7 in CSCs decreased almost to zero their self-renewal capacity and tumorigenicity, and reversed the resistance of CSCs to gemci‐ tabine. Concordant with previous reports on stroma-tumor interaction, Lonardo *et al.* also found the response to gemcitabine was dependent on the amount of stroma which hindered drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor (HHI) en‐ hanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 41

The *Hedgehog* signaling pathway is usually targeted in experimental designs as adjuvant to classic chemotherapy. The combined blockade of Shh and mTOR signaling together with gemcitabine is capable of eliminating pancreatic CSCs [131]. Inhibition of Smoothen (Smo), combined with gemcitabine and mTOR inhibitor rapamycin, led to abrogation of cancer stem cells and the authors reported a long-term disease stabilization or regression and subsequent

*Notch* pathway inhibition by selective γ-secretase inhibitors, such as PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine, inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1 and induced tumor regression in xenograft tumor models. The authors argue that the observed effects are due to PF-03084014 targeting of putative aggressive cancer stem cells [59]. Another potent and selective γ-secretase inhibitor, MRK-003, also led to downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumorinitiating cells capable of extensive self-renewal. Pretreatment of a pancreatic adenocarcinoma cell line with MRK-003 significantly inhibited the subsequent engraftment in immunocom‐ promised mice and mixed regimen MRK-003 and gemcitabine of engrafted mice reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis [133].However, some of such pathways are common to normal and CSCs, raising the problem of increasing

Most clinical studies addressing molecular therapies in pancreatic cancer report usage of monoclonal antibodies,for several simple rationales: i)they are already tested as drugs in other types of pathologies, tumoral or not; ii) they block proliferative oversignaling – a characteris‐ tic feature of malignancy; iii) some of them address phenotypic anomalies given by genetic dysregulations, such as EFGR overexpression/ oversignaling. However, these antibodies do not address specifically stem cells, but the larger category of cancer cells. There are some constructs that are, however, effective on the side population of CSCs. A combination of tigatuzumab, a fully humanized death receptor5 (DR5) agonist monoclonal antibody, with gemcitabine proved to be more efficacious in killing both CSCs and adenocarcinoma bulk cells.

survival [130].

long-term survival [132].

the selectivity towards cancer stem cells.

**3.2. Clinical studies**

## **3. CSCs and therapy outcomes**

In pancreatic cancer, surgery is usually accompanied by other complementary treatments such as multi-chemotherapy regimens and radiotherapy. Despite clear progress in detection and treatment of cancer, current strategies fail to completely remove the tumor and prevent recurrence and metastasis. Existing therapies are toxic and non-specific, being directed towards both normal cells and tumor cells. Most chemotherapeutic regimens are based on gemcitabine, but provided a modest improvement in median survival. The response rate was increased by using more than two chemotherapeutic agents [122]. Human pancreatic cancer tissue contains CSCs defined by CD133 and CXCR4 expression and these cells are highly resistant to standard chemotherapy and are involved in metastasis [12]. Features of CSCs have also been confirmed in brain and colon cancers [9].Therapy failure for other highly malignant tumors has been explained, at least partially, by the chemo-[10, 123] and radio-resistant [124] nature of CSCs. Cancer stem cells therapy resistance is considered to be the result of inappro‐ priate activation of several proliferative signaling pathways, including EGFR, PDGFR(plateletderived growth factor receptor), stem cell factor (SCF) receptor KIT [125], and activation of Hedgehog and Wnt/*β*-catenin signaling [50]. Another well sustained argument for chemo‐ therapy resistance is the expression of multidrug resistance-linked genes, out of which most are ATP-binding cassette (ABC) drug transporters [126]. High levels ofABC transporters were documented in pancreatic CSCs and chemotherapeutic agents such as etoposide, doxorubicin, vincristine and paclitaxel are direct substrates of ABC transporters [127]. Gemcitabine uptake, the golden standard for pancreatic adenocarcinoma chemiotherapy, seems to be negatively influenced by expression of ABCG2, though there is no clear evidence that ABC transporters directly efflux gemcitabine or its metabolites in pancreatic cancer cells [90]. Several reports indicate that conventional chemotherapy itself could propagate the CSC population in pancreatic cancer, through exerting a positive selection pressure of CD24/CD44/ESA triple positive CSC fraction [12, 128].

Differential expression of some CSCs biomarkers can be indicative of particular characteristics, such as responsiveness to different therapies or outcomes.

#### **3.1. CSCs as therapeutic targets**

Different strategies are developed to target specifically CSCs, thus eliminating this particular set of cells. Several key regulatory pathways operating in the stem cells have been proposed and demonstrated to considerably improve the therapy outcomes; relevant examples are Sonic Hedgehog, Notch/Jagged, CD133, TGF beta signaling; specifically addressing such pathways, by small molecule inhibitors, monoclonal antibodies or siRNAs results in increasing the efficacy of therapies, as suggested by *in vitro* studies, as well as by clinical outcomes.

Some *in vitro* studies showed that blocking *cis*-acting elements, that are common for pluripo‐ tency maintaining Transcription Factor SOX-2 (Sox2), Oct4, and proto-Oncogene C-Myc (c-Myc), dramatically decreased CSCs proliferation and their ability to generate tumors in nude mice [15]. Equally, simultaneous knockdown of OCT4 and its target Nanog led to decreased proliferation, migration, invasiveness and tumorigenesis of putative pancreatic cancer stem cells [129]. Inhibition of the Nodal/Activin receptor Alk4/7 in CSCs decreased almost to zero their self-renewal capacity and tumorigenicity, and reversed the resistance of CSCs to gemci‐ tabine. Concordant with previous reports on stroma-tumor interaction, Lonardo *et al.* also found the response to gemcitabine was dependent on the amount of stroma which hindered drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor (HHI) en‐ hanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival [130].

The *Hedgehog* signaling pathway is usually targeted in experimental designs as adjuvant to classic chemotherapy. The combined blockade of Shh and mTOR signaling together with gemcitabine is capable of eliminating pancreatic CSCs [131]. Inhibition of Smoothen (Smo), combined with gemcitabine and mTOR inhibitor rapamycin, led to abrogation of cancer stem cells and the authors reported a long-term disease stabilization or regression and subsequent long-term survival [132].

*Notch* pathway inhibition by selective γ-secretase inhibitors, such as PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine, inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1 and induced tumor regression in xenograft tumor models. The authors argue that the observed effects are due to PF-03084014 targeting of putative aggressive cancer stem cells [59]. Another potent and selective γ-secretase inhibitor, MRK-003, also led to downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumorinitiating cells capable of extensive self-renewal. Pretreatment of a pancreatic adenocarcinoma cell line with MRK-003 significantly inhibited the subsequent engraftment in immunocom‐ promised mice and mixed regimen MRK-003 and gemcitabine of engrafted mice reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis [133].However, some of such pathways are common to normal and CSCs, raising the problem of increasing the selectivity towards cancer stem cells.

#### **3.2. Clinical studies**

To conclude, the presence of MSC within the tumor site is a fact, but its role is still to be

40 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

In pancreatic cancer, surgery is usually accompanied by other complementary treatments such as multi-chemotherapy regimens and radiotherapy. Despite clear progress in detection and treatment of cancer, current strategies fail to completely remove the tumor and prevent recurrence and metastasis. Existing therapies are toxic and non-specific, being directed towards both normal cells and tumor cells. Most chemotherapeutic regimens are based on gemcitabine, but provided a modest improvement in median survival. The response rate was increased by using more than two chemotherapeutic agents [122]. Human pancreatic cancer tissue contains CSCs defined by CD133 and CXCR4 expression and these cells are highly resistant to standard chemotherapy and are involved in metastasis [12]. Features of CSCs have also been confirmed in brain and colon cancers [9].Therapy failure for other highly malignant tumors has been explained, at least partially, by the chemo-[10, 123] and radio-resistant [124] nature of CSCs. Cancer stem cells therapy resistance is considered to be the result of inappro‐ priate activation of several proliferative signaling pathways, including EGFR, PDGFR(plateletderived growth factor receptor), stem cell factor (SCF) receptor KIT [125], and activation of Hedgehog and Wnt/*β*-catenin signaling [50]. Another well sustained argument for chemo‐ therapy resistance is the expression of multidrug resistance-linked genes, out of which most are ATP-binding cassette (ABC) drug transporters [126]. High levels ofABC transporters were documented in pancreatic CSCs and chemotherapeutic agents such as etoposide, doxorubicin, vincristine and paclitaxel are direct substrates of ABC transporters [127]. Gemcitabine uptake, the golden standard for pancreatic adenocarcinoma chemiotherapy, seems to be negatively influenced by expression of ABCG2, though there is no clear evidence that ABC transporters directly efflux gemcitabine or its metabolites in pancreatic cancer cells [90]. Several reports indicate that conventional chemotherapy itself could propagate the CSC population in pancreatic cancer, through exerting a positive selection pressure of CD24/CD44/ESA triple

Differential expression of some CSCs biomarkers can be indicative of particular characteristics,

Different strategies are developed to target specifically CSCs, thus eliminating this particular set of cells. Several key regulatory pathways operating in the stem cells have been proposed and demonstrated to considerably improve the therapy outcomes; relevant examples are Sonic Hedgehog, Notch/Jagged, CD133, TGF beta signaling; specifically addressing such pathways, by small molecule inhibitors, monoclonal antibodies or siRNAs results in increasing the

efficacy of therapies, as suggested by *in vitro* studies, as well as by clinical outcomes.

determined.

**3. CSCs and therapy outcomes**

positive CSC fraction [12, 128].

**3.1. CSCs as therapeutic targets**

such as responsiveness to different therapies or outcomes.

Most clinical studies addressing molecular therapies in pancreatic cancer report usage of monoclonal antibodies,for several simple rationales: i)they are already tested as drugs in other types of pathologies, tumoral or not; ii) they block proliferative oversignaling – a characteris‐ tic feature of malignancy; iii) some of them address phenotypic anomalies given by genetic dysregulations, such as EFGR overexpression/ oversignaling. However, these antibodies do not address specifically stem cells, but the larger category of cancer cells. There are some constructs that are, however, effective on the side population of CSCs. A combination of tigatuzumab, a fully humanized death receptor5 (DR5) agonist monoclonal antibody, with gemcitabine proved to be more efficacious in killing both CSCs and adenocarcinoma bulk cells.

The combination therapy produced remarkable reduction in pancreatic CSCs, tumor remis‐ sions, and significant improvements in time to tumor progression [134]. Signaling pathways can also be inhibited by small molecule kinase inhibitors that act downstream of the extracellu‐ lar domain of the receptor. Sunitinib targets multiple receptor tyrosine kinases, including stem cell factor receptor (c-KIT) and it has been shown to have antitumor efficacy in *in vivo*. The combination of gemcitabine with sunitinib could not surpass the effects of the single agent sunitinib [135]. Cabozantinib – a small kinase inhibitor that targets c-Met and VEGFR2 inhibited viability and spheroid formation and induced apoptosis in pancreatic malignant cells with minor effects in non-malignant cells. In primary, CSC-enriched spheroidal cultures cabozantinibdownregulatedCSCmarkers SOX2, c-Met andCD133 andinducedapoptosis [73]. Most clinical studies, so far, do not seem to report any significant improvement with various regimens employed [136]. Early clinical data for the Shh inhibitor, GDC-0449 (vismodegib), in combination with either gemcitabine or erlotinib, indicate that these regimens are feasible and well tolerated [137]. However, a phase II trial of gemcitabine plus saridegib versus gemcita‐ bineplusplacebo inpreviouslyuntreatedpatients withmetastaticpancreatic cancer was halted early based on a shorter overall survival rate in the gemcitabine plus saridegib arm [106].

apoptosis along with inhibition of self-renewing potential, ALDH1 activity, clonogenicity, xenograft growth and relapse of gemcitabinetreated tumor cells in nude mice [146]. The flavonoid Quercetin enhances TRAIL-mediated apoptosis, acts as a chemosensitizer for the ABC pump-proteins, and can enhance the effects of sulforaphane in inhibiting the pancreatic

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 43

Targeted therapeutic delivery is a way to ensure that drugs reach the designated target at the highest concentration within safety margins, limiting in the same time undesired side effects resulting from unspecific diffusion in well vascularized tissues. This aim is now being resolved with the use of nanomedicine –a multidisciplinary field that aims to utilize nanoscale (up to 100 nm) particles to improve delivery of chemotherapeutics [148]. These constructs fall into several categories – micelles, microemulsions, liposomes, polymers [149] silica and carbonbased nanoparticles [150] and dendrimers [151]. This coating of a nanoparticle can be improved with stabilizing agents (such as polyethylene glycol – PEG) or ligands to direct them to a specific target (such as an antibody towards a cancer cell type). Liposome delivery of active agents has been recently paired with ultrasound technology, by development of ultrasoundresponsive stable liposomes. Ultrasound-induced heating triggers phase transition in the phospholipid membrane, leading to drug release in the targeted region [152]. To date, there are at least twelve FDA (Food and Drug Administration) approved liposome-based drugs, most of them being chemotherapeutics for breast, ovarian and pancreatic cancer [153].

Generation of magnetic/metallic nanoparticles was considered a step-forward in magnetic resonance imaging and diagnostics [154], adding a new utility to biomedical nanoscience. Another type of imaging strategy using nanoparticles is optical, through use of carbon nanomaterials that display natural fluorescence emission [155], or use of other infrared light emission agents [156], forming upconversion nanoparticles [157], or incorporated in a wide variety of coating surfaces, such as gold [158] and polymer-based [159]. Photoacoustic imaging is another nanomedical promising technology that combines the benefits of optical imaging methods with the clinically available and cost-effective ultrasound imaging modality [160]. Originally used for investigation of vascularization pattern, based on high endogenous contrast of blood *versus* surrounding tissues [161] and or/vascular wall/lumen alterations [162], it has been increasingly used in tumor assessment, providing further molecular information on cancer, given by the chemical composition of tissues and by targeted nanoparticles that can

By incorporating active drugs into imaging nanoparticles, a dual therapeutic and diagnostic agent was generated, thus the emerging field of "theragnostic", is widely used especially in cancer research. Most nanoparticles accumulate in tumors due to their intense and leaky neovascularization, but some can be retained there with the use of cancer-specific antigens [164] and stimulated into releasing their chemotherapeutic cargo. Cancer diagnostic and concomitant treatment through nanoparticles benefits from real-time assessment of drug

CSC characteristics [147].

**4. Nanotheragnostics in pancreatic cancer**

interact with extravascular tissues at the receptor level [163].

bioavailability and more accurate monitoring of tumor evolution.

A very interesting new trend in advanced, chemotherapy-resistant cancers, aiming for a different approach, tests personalized peptide vaccination (PPV) – a method to generate an immune response against tumor-associated antigens and so far employed for aggressive cancers such as lung cancer [138] and biliary tract cancer [139]. For advanced pancreatic cancer a phase II clinical trial was also conducted in which vaccine antigens were selected and administered based on the pre-existing IgG responses to 31 different pooled peptides [140]. Other vaccines are aimed at increasing the patient's immune response against tumor cells – targeting cancer markers with the aid of specialized antigen-presenting cells such as dendritic cells. Currently, there are several vaccines for human pancreatic cancer in clinical trials including: i) whole-cell vaccines, ii) combined dendritic cells with antigen to present to patient leukocytes iii) peptide and DNA vaccines, iv) Ras peptide vaccine; v) vaccine against common cancer mutations, targetable by CD4/8 T cells; vi) Telomerase peptide vaccine; vii) carcinoem‐ brionar antigen (CEA) and Mucin 1; viii) Survivin-targeted vaccine [141]. Also, it was shown that boosting the immune response by additional treatment with dendritic cells (LANEX-DC®) is highly effective and extends the median survival times up to 8.9 months [142].

Lack of response to all of the above mentioned types of therapies led to an investigation of *non-conventional therapies*. Salinomycin, an anti-protozoa agent that was recently shown to preferentially kill breast CSCs [143], and later investigated in other types of malignancies, was shown to inhibit growth of pancreatic adenocarcinoma CSCs *in vitro. In vivo* xenografting studies showed that salinomycin combined with gemcitabine could eliminate the engraftment of human pancreatic cancer more effectively than the individual agents [144]. Adamantylsubstituted retinoid-related molecules (ARRs) inhibit growth and induce apoptosis in the pancreatic stem-like cell population, possibly through decreased IGF-1R and *β*-catenin expression [145]. Isothiocyanate sulforaphane (SF) was used as sensitizer of pancreatic CSCs to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)-induced apoptosis, by quercetin and sorafenib. The combination of SF with a cytotoxic drug efficiently induced apoptosis along with inhibition of self-renewing potential, ALDH1 activity, clonogenicity, xenograft growth and relapse of gemcitabinetreated tumor cells in nude mice [146]. The flavonoid Quercetin enhances TRAIL-mediated apoptosis, acts as a chemosensitizer for the ABC pump-proteins, and can enhance the effects of sulforaphane in inhibiting the pancreatic CSC characteristics [147].

#### **4. Nanotheragnostics in pancreatic cancer**

The combination therapy produced remarkable reduction in pancreatic CSCs, tumor remis‐ sions, and significant improvements in time to tumor progression [134]. Signaling pathways can also be inhibited by small molecule kinase inhibitors that act downstream of the extracellu‐ lar domain of the receptor. Sunitinib targets multiple receptor tyrosine kinases, including stem cell factor receptor (c-KIT) and it has been shown to have antitumor efficacy in *in vivo*. The combination of gemcitabine with sunitinib could not surpass the effects of the single agent sunitinib [135]. Cabozantinib – a small kinase inhibitor that targets c-Met and VEGFR2 inhibited viability and spheroid formation and induced apoptosis in pancreatic malignant cells with minor effects in non-malignant cells. In primary, CSC-enriched spheroidal cultures cabozantinibdownregulatedCSCmarkers SOX2, c-Met andCD133 andinducedapoptosis [73]. Most clinical studies, so far, do not seem to report any significant improvement with various regimens employed [136]. Early clinical data for the Shh inhibitor, GDC-0449 (vismodegib), in combination with either gemcitabine or erlotinib, indicate that these regimens are feasible and well tolerated [137]. However, a phase II trial of gemcitabine plus saridegib versus gemcita‐ bineplusplacebo inpreviouslyuntreatedpatients withmetastaticpancreatic cancer was halted early based on a shorter overall survival rate in the gemcitabine plus saridegib arm [106].

42 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

A very interesting new trend in advanced, chemotherapy-resistant cancers, aiming for a different approach, tests personalized peptide vaccination (PPV) – a method to generate an immune response against tumor-associated antigens and so far employed for aggressive cancers such as lung cancer [138] and biliary tract cancer [139]. For advanced pancreatic cancer a phase II clinical trial was also conducted in which vaccine antigens were selected and administered based on the pre-existing IgG responses to 31 different pooled peptides [140]. Other vaccines are aimed at increasing the patient's immune response against tumor cells – targeting cancer markers with the aid of specialized antigen-presenting cells such as dendritic cells. Currently, there are several vaccines for human pancreatic cancer in clinical trials including: i) whole-cell vaccines, ii) combined dendritic cells with antigen to present to patient leukocytes iii) peptide and DNA vaccines, iv) Ras peptide vaccine; v) vaccine against common cancer mutations, targetable by CD4/8 T cells; vi) Telomerase peptide vaccine; vii) carcinoem‐ brionar antigen (CEA) and Mucin 1; viii) Survivin-targeted vaccine [141]. Also, it was shown that boosting the immune response by additional treatment with dendritic cells (LANEX-DC®)

is highly effective and extends the median survival times up to 8.9 months [142].

Lack of response to all of the above mentioned types of therapies led to an investigation of *non-conventional therapies*. Salinomycin, an anti-protozoa agent that was recently shown to preferentially kill breast CSCs [143], and later investigated in other types of malignancies, was shown to inhibit growth of pancreatic adenocarcinoma CSCs *in vitro. In vivo* xenografting studies showed that salinomycin combined with gemcitabine could eliminate the engraftment of human pancreatic cancer more effectively than the individual agents [144]. Adamantylsubstituted retinoid-related molecules (ARRs) inhibit growth and induce apoptosis in the pancreatic stem-like cell population, possibly through decreased IGF-1R and *β*-catenin expression [145]. Isothiocyanate sulforaphane (SF) was used as sensitizer of pancreatic CSCs to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)-induced apoptosis, by quercetin and sorafenib. The combination of SF with a cytotoxic drug efficiently induced Targeted therapeutic delivery is a way to ensure that drugs reach the designated target at the highest concentration within safety margins, limiting in the same time undesired side effects resulting from unspecific diffusion in well vascularized tissues. This aim is now being resolved with the use of nanomedicine –a multidisciplinary field that aims to utilize nanoscale (up to 100 nm) particles to improve delivery of chemotherapeutics [148]. These constructs fall into several categories – micelles, microemulsions, liposomes, polymers [149] silica and carbonbased nanoparticles [150] and dendrimers [151]. This coating of a nanoparticle can be improved with stabilizing agents (such as polyethylene glycol – PEG) or ligands to direct them to a specific target (such as an antibody towards a cancer cell type). Liposome delivery of active agents has been recently paired with ultrasound technology, by development of ultrasoundresponsive stable liposomes. Ultrasound-induced heating triggers phase transition in the phospholipid membrane, leading to drug release in the targeted region [152]. To date, there are at least twelve FDA (Food and Drug Administration) approved liposome-based drugs, most of them being chemotherapeutics for breast, ovarian and pancreatic cancer [153].

Generation of magnetic/metallic nanoparticles was considered a step-forward in magnetic resonance imaging and diagnostics [154], adding a new utility to biomedical nanoscience. Another type of imaging strategy using nanoparticles is optical, through use of carbon nanomaterials that display natural fluorescence emission [155], or use of other infrared light emission agents [156], forming upconversion nanoparticles [157], or incorporated in a wide variety of coating surfaces, such as gold [158] and polymer-based [159]. Photoacoustic imaging is another nanomedical promising technology that combines the benefits of optical imaging methods with the clinically available and cost-effective ultrasound imaging modality [160]. Originally used for investigation of vascularization pattern, based on high endogenous contrast of blood *versus* surrounding tissues [161] and or/vascular wall/lumen alterations [162], it has been increasingly used in tumor assessment, providing further molecular information on cancer, given by the chemical composition of tissues and by targeted nanoparticles that can interact with extravascular tissues at the receptor level [163].

By incorporating active drugs into imaging nanoparticles, a dual therapeutic and diagnostic agent was generated, thus the emerging field of "theragnostic", is widely used especially in cancer research. Most nanoparticles accumulate in tumors due to their intense and leaky neovascularization, but some can be retained there with the use of cancer-specific antigens [164] and stimulated into releasing their chemotherapeutic cargo. Cancer diagnostic and concomitant treatment through nanoparticles benefits from real-time assessment of drug bioavailability and more accurate monitoring of tumor evolution.

Pancreatic cancer treatement benefits from development of biomedical nanotechnology, in both clinical practice and fundamental research. A PEGylated polymeric nanoparticle containing a potent antagonist of the Hedgehog transcription factor Gli1 combined with gemcitabine significantly impeded the growth of orthotopic pancreatic cancer xenografts [165]. In *in vivo* studies, squalene-conjugated gemcitabine nanoparticles decreased tumor growth significantly, prevented tumor cell invasion, and prolonged the survival time of mice bearing orthotopic pancreatic tumors [166]. Liposomal delivery of tissue transglutami‐ nase 2 siRNA effectively blocked the growth of pancreatic adenocarcinoma in nude mice [167]. EGFR monoclonal antibody or peptidylglycine alpha-amidating monooxygenase (PAM4)-conjugated gold nanoparticles induced significant tumor destruction in a murine model of pancreatic carcinoma after radiofrequency radiation [168]. Paclitaxel, one of firstline chemotherapeutic agents before the gemcitabine era, is now available as a positively charged lipid-based complex (known as EndoTAG-1) [169] that in combination with gemcitabine was able to inhibit the incidence of metastasis in pancreatic cancer animal models [170]. A controlled phase II clinical trial for pancreatic cancer showed significant‐ ly increased survival rates of patients treated with EndoTAG®-1 and gemcitabine combina‐ tion therapy [171]. An ongoing phase I study (NCT00968604) of advanced pancreatic cancer is currently investigating the effects of intravenous injection of the liposome nanoparticle BikDD, which contains a pro-apoptotic agent [172].

**Acknowledgements**

**Author details**

Romania

**References**

This work was partly supported by Grants POS CCE 685-152/2010.

Cristiana Pistol Tanase1\*, Ana-Maria Enciu1,2, Maria Linda Cruceru2

3 Stefan S. Nicolau Institute of Virology, Bucharest, Romania

\*Address all correspondence to: bioch@vbabes.ro

Independentei, Bucharest, Romania

2012;18(38):5321-3.

ers-in-pancreatic-cancer.

Laura Georgiana Necula1,3, Ana Iulia Neagu1,3, Bogdan Calenic1,2 and Radu Albulescu1,4

1 Victor Babes National Institute of Pathology, Dept. of Biochemistry-Proteomics, Splaiul

2 Carol Davila University of Medicine and Pharmacy, Eroilor Sanitari, Bucharest, Romania

4 National Institute for Chemical Pharmaceutical Research and Development, Bucharest,

[1] Herreros-Villanueva M, Zubia-Olascoaga A, Bujanda L. c-Met in pancreatic cancer stem cells: therapeutic implications. World journal of gastroenterology : WJG.

[2] Dima SO TC, Albulescu R, Botezatu A and Popescu I (2012). Novel Biomarkers in Pancreatic Cancer, Pancreatic Cancer-Clinical Management, Prof. Sanjay Srivastava (Ed.), ISBN: 978-953-51-0394-3, InTech, DOI: 10.5772/29001. Available from: http:// www.intechopen.com/books/pancreatic-cancer-clinical-management/novel-biomark‐

[3] Kaur S, Baine MJ, Jain M, Sasson AR, Batra SK. Early diagnosis of pancreatic cancer: challenges and new developments. Biomarkers in medicine. 2012;6(5):597-612.

[4] Lee CJ, Dosch J, Simeone DM. Pancreatic cancer stem cells. Journal of clinical oncolo‐ gy : official journal of the American Society of Clinical Oncology. 2008;26(17):2806-12.

[5] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Acade‐

my of Sciences of the United States of America. 2003;100(7):3983-8.

,

Stem Cells in Pancreatic Cancer http://dx.doi.org/10.5772/57530 45

#### **4.1. Nanoparticles for cancer stem cell targeted therapy**

In the same manner that nanoparticles are targeted for the bulk tumor, they can be targeted for CSCs, through the use of antigens against specific CSCs markers (e.g CD-133). Such targeted therapy has already been tested *in vitro*, against targeting CD133-expressing cancer cells of colon and pancreatic origin, with encouraging results [56]. Breast CSCs-targeted nanoparticle delivery of doxorubicin reduced their mammosphere formation capacity and cancer initiation activity, eliciting tumor growth inhibition in animal models[173].

Apart from cytotoxic drug delivery, nanoparticles can be used to target and modify certain characteristics of CSCs, such as activation of signaling pathways that confer renewal proper‐ ties, targeting metabolism and inhibiting drug efflux transporters in an attempt to sensitize them to therapy [174]. Multi-lamellar vesicle liposomes targeted against CSCs, containing a steroid nucleus, were formulated to disrupt mitochondrial integrity and to facilitate release of cytochrome c to attain programmed cell death [175].

## **5. Conclusions**

CSCs represent key components in the heterogeneous cellular system represented by pancre‐ atic tumors. Their biological features configure them as one of the major players and major targets for investigation; they offer sets of additional and reliable biomarkers for prognosis and stratification. Discovery of target mechanisms and molecules within cancer stem cells is plausible to provide the needed boost for therapy improvement.

## **Acknowledgements**

Pancreatic cancer treatement benefits from development of biomedical nanotechnology, in both clinical practice and fundamental research. A PEGylated polymeric nanoparticle containing a potent antagonist of the Hedgehog transcription factor Gli1 combined with gemcitabine significantly impeded the growth of orthotopic pancreatic cancer xenografts [165]. In *in vivo* studies, squalene-conjugated gemcitabine nanoparticles decreased tumor growth significantly, prevented tumor cell invasion, and prolonged the survival time of mice bearing orthotopic pancreatic tumors [166]. Liposomal delivery of tissue transglutami‐ nase 2 siRNA effectively blocked the growth of pancreatic adenocarcinoma in nude mice [167]. EGFR monoclonal antibody or peptidylglycine alpha-amidating monooxygenase (PAM4)-conjugated gold nanoparticles induced significant tumor destruction in a murine model of pancreatic carcinoma after radiofrequency radiation [168]. Paclitaxel, one of firstline chemotherapeutic agents before the gemcitabine era, is now available as a positively charged lipid-based complex (known as EndoTAG-1) [169] that in combination with gemcitabine was able to inhibit the incidence of metastasis in pancreatic cancer animal models [170]. A controlled phase II clinical trial for pancreatic cancer showed significant‐ ly increased survival rates of patients treated with EndoTAG®-1 and gemcitabine combina‐ tion therapy [171]. An ongoing phase I study (NCT00968604) of advanced pancreatic cancer is currently investigating the effects of intravenous injection of the liposome nanoparticle

44 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

In the same manner that nanoparticles are targeted for the bulk tumor, they can be targeted for CSCs, through the use of antigens against specific CSCs markers (e.g CD-133). Such targeted therapy has already been tested *in vitro*, against targeting CD133-expressing cancer cells of colon and pancreatic origin, with encouraging results [56]. Breast CSCs-targeted nanoparticle delivery of doxorubicin reduced their mammosphere formation capacity and cancer initiation

Apart from cytotoxic drug delivery, nanoparticles can be used to target and modify certain characteristics of CSCs, such as activation of signaling pathways that confer renewal proper‐ ties, targeting metabolism and inhibiting drug efflux transporters in an attempt to sensitize them to therapy [174]. Multi-lamellar vesicle liposomes targeted against CSCs, containing a steroid nucleus, were formulated to disrupt mitochondrial integrity and to facilitate release of

CSCs represent key components in the heterogeneous cellular system represented by pancre‐ atic tumors. Their biological features configure them as one of the major players and major targets for investigation; they offer sets of additional and reliable biomarkers for prognosis and stratification. Discovery of target mechanisms and molecules within cancer stem cells is

BikDD, which contains a pro-apoptotic agent [172].

cytochrome c to attain programmed cell death [175].

**5. Conclusions**

**4.1. Nanoparticles for cancer stem cell targeted therapy**

activity, eliciting tumor growth inhibition in animal models[173].

plausible to provide the needed boost for therapy improvement.

This work was partly supported by Grants POS CCE 685-152/2010.

## **Author details**

Cristiana Pistol Tanase1\*, Ana-Maria Enciu1,2, Maria Linda Cruceru2 , Laura Georgiana Necula1,3, Ana Iulia Neagu1,3, Bogdan Calenic1,2 and Radu Albulescu1,4

\*Address all correspondence to: bioch@vbabes.ro

1 Victor Babes National Institute of Pathology, Dept. of Biochemistry-Proteomics, Splaiul Independentei, Bucharest, Romania

2 Carol Davila University of Medicine and Pharmacy, Eroilor Sanitari, Bucharest, Romania

3 Stefan S. Nicolau Institute of Virology, Bucharest, Romania

4 National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania

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**Chapter 3**

**miRNAs in Pancreatic Cancer**

Daniela Ionela Popescu, Simona Mihai and

Additional information is available at the end of the chapter

Cristiana Tanase

**1. Introduction**

outcomes.

http://dx.doi.org/10.5772/58397

Radu Albulescu, Adrian Claudiu Popa, Elena Codrici,

mRNAs as a miRNA-mRNA dimer to a degradative complex.

miRNAs are seen inhibiting or even reversing the process.

"candidate" therapeutic targets or "candidate" therapeutic tools.

Pancreatic cancer and especially PDAC (Pancreatic Ductal AdenoCarcinoma) is among the most difficult to treat cancer, characterized by invasiveness, metastatic potential and bad

miRNAs emerged in recent years as potent regulators of cellular activities, playing a central role in controlling the protein expression at the post-transcriptional level. They have significant implication in pathology in general and most relevantly in cancers. Their main role is the control of the process of proteosynthesis at the translational level, by leading their target

Deregulation in expression levels of miRNAs and some genetic alterations were demonstrated in various cancers, including PDAC. Investigations on tissue samples provided a considerable amount of knowledge, leading to the identification of miRNAs with altered expression associated with tumorigenesis and tumor progression. Tumor-inducing and tumor-promoting miRNAs were significantly up-regulated, while sets of tumor-suppressor miRNAs are downregulated or suppressed. By targeting major protein players in cell regulatory networks, some miRNAs appear to have the ability to shift the balance towards tumorigenesis, while other

Tissular and soluble miRNAs were demonstrated as potential biomarkers, serving as diag‐ nostic, stratification or prognostic tools, while other representatives were identified as

MicroRNAs (miRs, miRNAs) form a class of small-sized but powerful cell regulators. Pres‐ ently, the family of human miRNAs comprises 1872 precursors and 2578 mature forms, but

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **Chapter 3**

## **miRNAs in Pancreatic Cancer**

Radu Albulescu, Adrian Claudiu Popa, Elena Codrici, Daniela Ionela Popescu, Simona Mihai and Cristiana Tanase

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58397

## **1. Introduction**

Pancreatic cancer and especially PDAC (Pancreatic Ductal AdenoCarcinoma) is among the most difficult to treat cancer, characterized by invasiveness, metastatic potential and bad outcomes.

miRNAs emerged in recent years as potent regulators of cellular activities, playing a central role in controlling the protein expression at the post-transcriptional level. They have significant implication in pathology in general and most relevantly in cancers. Their main role is the control of the process of proteosynthesis at the translational level, by leading their target mRNAs as a miRNA-mRNA dimer to a degradative complex.

Deregulation in expression levels of miRNAs and some genetic alterations were demonstrated in various cancers, including PDAC. Investigations on tissue samples provided a considerable amount of knowledge, leading to the identification of miRNAs with altered expression associated with tumorigenesis and tumor progression. Tumor-inducing and tumor-promoting miRNAs were significantly up-regulated, while sets of tumor-suppressor miRNAs are downregulated or suppressed. By targeting major protein players in cell regulatory networks, some miRNAs appear to have the ability to shift the balance towards tumorigenesis, while other miRNAs are seen inhibiting or even reversing the process.

Tissular and soluble miRNAs were demonstrated as potential biomarkers, serving as diag‐ nostic, stratification or prognostic tools, while other representatives were identified as "candidate" therapeutic targets or "candidate" therapeutic tools.

MicroRNAs (miRs, miRNAs) form a class of small-sized but powerful cell regulators. Pres‐ ently, the family of human miRNAs comprises 1872 precursors and 2578 mature forms, but

the discovery process is adding further members at a rapid rate [1, 2]. The conventional nomenclature of miRNAs establishes some rules: a mature miRNA is designated in the form hsa-miR-121, where the first three characters encode the species. The letters that may occur at the end of the name refer to the different locations where the coding gene is located (the same miRNA can be encoded on multiple chromosomes and on either + or – strands). At the same time, the precursors are designated in the form hsa-mir-121. The rate of discovery is quite fast, so usually the numbers are assigned in the sequential order of discovery. However, if a new miRNA has a similar sequence to an existing one, it will acquire identical names, the differ‐ entiation being made by the letter. For historical reasons, the first miRNAs discovered, let-7 and lin-4, are exempt from the rule.

have been described and several reviews thoroughly describe the processes involving

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 63

**•** miRNAs are encoded in various locations (both in protein-coding and non-coding gene sequences); often, the location of these coding sequences is in fragile chromosomal regions;

**•** miRNA sequences are transcribed by RNA polymerase II as larger primary-microRNA molecules, which are further processed by Rnase III endonucleases Drosha and DGCR8 to form precursor miRNAs (pre-microRNAs, stem-loop structures containing about 70

**•** Processing in the cytoplasm is performed by Dicer (an RNAse III endonuclease), which removes the loop of the pre-miRNA and generates an imperfect duplex, formed by the mature miRNA sequence and a fragment of similar size derived from the opposite side of

**•** The counter strand is separated and most often degraded; however, in many cases this

**•** Perfect or near-perfect complementarity targets mRNA for degradation by RISC (RNA-

The majority (if not all) of miRNAs are multivalent. That is, almost every miRNA has the ability to interfere with multiple genes. Often a "cross talk" between miRNAs and other cellregulatory or effector proteins is encountered, generating a mutual modification of expression,

A novel pathway, translation activation, was demonstrated by Vasudevan et al. (2007) for miR-369-3. Cell cycle arrest by serum starvation transforms the TNFα AU-rich element (ARE) into a translation activator signal. AGO2 (Argonaute2) and FXR1 (fragile X mental retardation– related protein 1 (FXR1) are associated with ARE on translation activation; both proteins are required to increase translation efficiency. The seed sequence (the nucleotides 2-8 at the 5' end of the miRNA [13] of miR-369-3 was demonstrated to be able to form base-pairs with two target sites on the minimal TNFα ARE required for translation activation. The formation of basepairs between mir-369-3 and the target sites was demonstrated to be required for translation activation by knock-down experiments and by experiments using mutant ARE, as well as modified sequences of miR-369-3 (in order to restore complementarity to modified targets on

Gene expression is controlled by regulation of mRNA translation and degradation:

A brief description of the basic mechanisms of biogenesis may be given as follows:

therefore, they are highly susceptible to molecular modification.

**•** Exportin 5 transfers pre-microRNAs to the cytoplasm.

counter strand can also function as a regulator.

**•** Imperfect complementarity blocks translation by the ribosome.

miRNAs [11, 12].

nucleotides).

the loop, (miRNA\*).

Induced Silencing Complex).

resulting in negative regulatory loops.

mutant ARE) [14].

miRNAs act in the post-transcriptional regulation of protein expression and their involvement was demonstrated in normal processes as well as in pathology. Most of them are "multivalent", so that one single miRNA is able to "target" multiple genes, thus regulating the expression of several proteins. miRNAs are non-coding RNA molecules, 18-28 nucleotides lengths in the mature form, that regulate a variety of cellular processes including cell differentiation, cell cycle progression and apoptosis. miRNAs can function either as oncogenes or tumor suppres‐ sors [3]; oncogenic miRNAs (oncomiRs) are up-regulated in cancer cells [1].

In cancer, several miRNAs are situated "upstream" of the carcinogenesis process – acting as triggers for carcinogenesis or for progression; other miRNAs are situated "downstream" of the carcinogenic process, their modified expression appearing as the outcome of carcinogenetic transformation or progression. miRNAs play major roles in the multistep processes of carcinogenesis, either by oncogenic or tumor-suppressor functions. The study of miRNAs has been extended into many kinds of tumors, including those of the pancreas [4, 5]. Those studies have revealed that miRNAs may be potential diagnostic or prognostic tools for cancer [6, 7]. miRNAs are important tools due to their suitability for detection in both tissues [either fresh or Formalin-Fixed-Paraffin-Embedded (FFPE)] and in other biological samples (blood, serum, plasma, saliva, feces).

The discovery of miRNAs opened new opportunities for non-invasive tests for the early diagnosis of cancer [8, 9]. It has been recently revealed that, once detected, the miRNAs (being differentially expressed in blood) can be used as diagnostic and prognostic circulating biomarkers [10].

In the present chapter we summarize some of the existing knowledge regarding miRNAs involved in tumorigenesis and progression of pancreatic cancer. We have focused on the possible diagnostic role of miRNAs and their tissue-related expression in correlation with their soluble forms. We have summarized recent evidence regarding the assessment of their diagnostic value in pancreatic cancer patients.

## **2. miRNAs: Biogenesis and mechanisms of action**

miRNAs act as post-transcriptional regulators of gene expression in eukaryotic cells. Their biological roles in development, normal cell function and in pathology, including cancer, have been described and several reviews thoroughly describe the processes involving miRNAs [11, 12].

A brief description of the basic mechanisms of biogenesis may be given as follows:


the discovery process is adding further members at a rapid rate [1, 2]. The conventional nomenclature of miRNAs establishes some rules: a mature miRNA is designated in the form hsa-miR-121, where the first three characters encode the species. The letters that may occur at the end of the name refer to the different locations where the coding gene is located (the same miRNA can be encoded on multiple chromosomes and on either + or – strands). At the same time, the precursors are designated in the form hsa-mir-121. The rate of discovery is quite fast, so usually the numbers are assigned in the sequential order of discovery. However, if a new miRNA has a similar sequence to an existing one, it will acquire identical names, the differ‐ entiation being made by the letter. For historical reasons, the first miRNAs discovered, let-7

62 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

miRNAs act in the post-transcriptional regulation of protein expression and their involvement was demonstrated in normal processes as well as in pathology. Most of them are "multivalent", so that one single miRNA is able to "target" multiple genes, thus regulating the expression of several proteins. miRNAs are non-coding RNA molecules, 18-28 nucleotides lengths in the mature form, that regulate a variety of cellular processes including cell differentiation, cell cycle progression and apoptosis. miRNAs can function either as oncogenes or tumor suppres‐

In cancer, several miRNAs are situated "upstream" of the carcinogenesis process – acting as triggers for carcinogenesis or for progression; other miRNAs are situated "downstream" of the carcinogenic process, their modified expression appearing as the outcome of carcinogenetic transformation or progression. miRNAs play major roles in the multistep processes of carcinogenesis, either by oncogenic or tumor-suppressor functions. The study of miRNAs has been extended into many kinds of tumors, including those of the pancreas [4, 5]. Those studies have revealed that miRNAs may be potential diagnostic or prognostic tools for cancer [6, 7]. miRNAs are important tools due to their suitability for detection in both tissues [either fresh or Formalin-Fixed-Paraffin-Embedded (FFPE)] and in other biological samples (blood, serum,

The discovery of miRNAs opened new opportunities for non-invasive tests for the early diagnosis of cancer [8, 9]. It has been recently revealed that, once detected, the miRNAs (being differentially expressed in blood) can be used as diagnostic and prognostic circulating

In the present chapter we summarize some of the existing knowledge regarding miRNAs involved in tumorigenesis and progression of pancreatic cancer. We have focused on the possible diagnostic role of miRNAs and their tissue-related expression in correlation with their soluble forms. We have summarized recent evidence regarding the assessment of their

miRNAs act as post-transcriptional regulators of gene expression in eukaryotic cells. Their biological roles in development, normal cell function and in pathology, including cancer,

sors [3]; oncogenic miRNAs (oncomiRs) are up-regulated in cancer cells [1].

and lin-4, are exempt from the rule.

plasma, saliva, feces).

biomarkers [10].

diagnostic value in pancreatic cancer patients.

**2. miRNAs: Biogenesis and mechanisms of action**


Gene expression is controlled by regulation of mRNA translation and degradation:


The majority (if not all) of miRNAs are multivalent. That is, almost every miRNA has the ability to interfere with multiple genes. Often a "cross talk" between miRNAs and other cellregulatory or effector proteins is encountered, generating a mutual modification of expression, resulting in negative regulatory loops.

A novel pathway, translation activation, was demonstrated by Vasudevan et al. (2007) for miR-369-3. Cell cycle arrest by serum starvation transforms the TNFα AU-rich element (ARE) into a translation activator signal. AGO2 (Argonaute2) and FXR1 (fragile X mental retardation– related protein 1 (FXR1) are associated with ARE on translation activation; both proteins are required to increase translation efficiency. The seed sequence (the nucleotides 2-8 at the 5' end of the miRNA [13] of miR-369-3 was demonstrated to be able to form base-pairs with two target sites on the minimal TNFα ARE required for translation activation. The formation of basepairs between mir-369-3 and the target sites was demonstrated to be required for translation activation by knock-down experiments and by experiments using mutant ARE, as well as modified sequences of miR-369-3 (in order to restore complementarity to modified targets on mutant ARE) [14].

## **3. miRNAs in tumor progression**

#### **3.1. Cell growth and proliferation**

Low levels of expression of miR-34a, 34b and 34c were found in cultivated pancreatic cancer cells (MiaPaCa2 and BxpC3), while the levels of the target genes Bcl2 (Apoptosis regulator Bcl-2) and Notch1 (Neurogenic locus notch homolog protein 1) were elevated. Restoration of miR-34 levels by transfection with miR-34 mimics down-regulation of the target genes, inhibits clonogenic growth and activates apoptosis via the caspase-3 pathway [15].

in microRNA expression in tumor stages were investigated [20]. A synthesis of these data is

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 65

**miRNA Modification Significance Ref.** let-7 Down-regulated [21, 22] let-7d Up-regulated [23, 24] let-7f-1 Up-regulated [21] miR-10a Up-regulated Metastasis [22] [20, 25] miR-10b Up-regulated [25] miR-15b Up-regulated Metastasis, Hyperplasia [20, 25] miR-16-1 Up-regulated [24] miR-17-5p Up-regulated Angiogenesis, Hyperplasia [20] miR-18a Up-regulated [26] miR-19b Up-regulated Angiogenesis [20] miR-20a Up-regulated Hyperplasia, angiogenesis [20] miR-21 Up-regulated Angiogenesis [24, 26] miR-23a Up-regulated [25] miR-23b Up-regulated Metastasis [20, 25] miR-24 Up-regulated Metastasis [20] miR-24-1,2 Up-regulated [24] miR-25 Up-regulated Hyperplasia, dysplasia [20] miR-27b Up-regulated Metastasis [20] miR-29c Down-regulated [26] miR-31 Up-regulated [26] Mir-92 Up-regulated Hyperplasia, dysplasia, metastasis [20] miR-92-1 Up-regulated [24] miR-93 Up-regulated [26] miR-95 Up-regulated [26] miR-96 Down-regulated [24] miR-99 Up-regulated [25] miR-100 Up-regulated [24, 25] miR-100-1/2 Up-regulated [25] miR-103-2 Up-regulated [25] miR-106a Up-regulated Hyperplasia, dysplasia [20] miR-107 Up-regulated [24, 25] miR-124a Up-regulated Tumor signature, metastasis [20] miR-125a Up-regulated [25] miR-125b-1 Up-regulated [24, 25] miR-126 Up-regulated Metastasis [20]

presented in table 1.

miR-21 over-expression is demonstrated in PDAC. Its presence and over-expression is associated with poor survival, invasiveness and resistance to gemcitabine. The findings relating to miR-21's role and mechanism in tumor tissue were confirmed *in vitro*, on primary cultures and cancer cell lines, fibroblasts and normal pancreatic ductal cell lines [16]. Enhance‐ ment of miR-21 levels (by pre-miR-21 transfection) decreased the anti-proliferative and antiapoptotic effects of gemcitabine and up-regulated the expression of MMP2 (Matrix-MetalloProteinase 2) and MMP9 (Matrix-MetalloProteinase 9) [16].

#### **3.2. Tumorigenesis**

In the case of pancreatic cancer, as is the case in other cancers, distinct patterns of expression of miRNAs occur, depending on disease stage. The expression changes during progression. From these miRNAs, some are common to many cancers, while a few are tissue-specific and can help to track more precisely the tissue in which a carcinogenic process takes place [17]. There is a clear distinction between pre-malignant lesions, primary tumors and metastasis in the pattern of expression of miRNAs. Moreover, some of these distinctions can also be made in exosomal miRNAs.

Deregulated expression of miRNAs may represent an early modification in pancreatic tumorigenesis, generating progression of PanIN (Pancreatic Intraepithelial Neoplasia) lesions to more invasive forms. Ryu et al. investigated three candidates (selected on the basis of previous reports as over-expressed in pancreatic cancer). mir-155 was significantly overexpressed in PanIN-2 and 3 (2.6-fold, p=0.02 and 7.4-fold p=0.049, respectively); miR-21 was over-expressed only in PanIN-3 (2.5-fold, p=0.02), while no modification was found for miR-221 in PanIN lesions compared to normal duct epithelium [18]. Another set of miRNAs were investigated by du Rieu et al. in laser-dissected tissue samples from PanIN lesions (from a mouse model and from human patients). miR-21, 205 and 200 paralleled PanIN progression in mouse models. mir-21 and miR-205 preceded phenotypic changes of the duct. In precursor lesions, miR-21 achieved the highest relative concentrations. In human samples, miR-21,-221, 222 and let-7a increased with lesion grade, with maximal expression in two thirds of lesions. Up-regulation of miR-21 was controlled by KRAS and EGFR in PDAC-(Pancreatic Ductal AdenoCarcinoma) derived cell lines [19].

Another complex investigation of miRNA signatures during tumorigenesis and progression in pancreatic cancer was reported by Olson et al. The study stressed the down-regulation of the miR-200 family in metastases and metastasis-like primary tumors. Also, multiple changes in microRNA expression in tumor stages were investigated [20]. A synthesis of these data is presented in table 1.

**3. miRNAs in tumor progression**

Low levels of expression of miR-34a, 34b and 34c were found in cultivated pancreatic cancer cells (MiaPaCa2 and BxpC3), while the levels of the target genes Bcl2 (Apoptosis regulator Bcl-2) and Notch1 (Neurogenic locus notch homolog protein 1) were elevated. Restoration of miR-34 levels by transfection with miR-34 mimics down-regulation of the target genes, inhibits

64 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

miR-21 over-expression is demonstrated in PDAC. Its presence and over-expression is associated with poor survival, invasiveness and resistance to gemcitabine. The findings relating to miR-21's role and mechanism in tumor tissue were confirmed *in vitro*, on primary cultures and cancer cell lines, fibroblasts and normal pancreatic ductal cell lines [16]. Enhance‐ ment of miR-21 levels (by pre-miR-21 transfection) decreased the anti-proliferative and antiapoptotic effects of gemcitabine and up-regulated the expression of MMP2 (Matrix-

In the case of pancreatic cancer, as is the case in other cancers, distinct patterns of expression of miRNAs occur, depending on disease stage. The expression changes during progression. From these miRNAs, some are common to many cancers, while a few are tissue-specific and can help to track more precisely the tissue in which a carcinogenic process takes place [17]. There is a clear distinction between pre-malignant lesions, primary tumors and metastasis in the pattern of expression of miRNAs. Moreover, some of these distinctions can also be made

Deregulated expression of miRNAs may represent an early modification in pancreatic tumorigenesis, generating progression of PanIN (Pancreatic Intraepithelial Neoplasia) lesions to more invasive forms. Ryu et al. investigated three candidates (selected on the basis of previous reports as over-expressed in pancreatic cancer). mir-155 was significantly overexpressed in PanIN-2 and 3 (2.6-fold, p=0.02 and 7.4-fold p=0.049, respectively); miR-21 was over-expressed only in PanIN-3 (2.5-fold, p=0.02), while no modification was found for miR-221 in PanIN lesions compared to normal duct epithelium [18]. Another set of miRNAs were investigated by du Rieu et al. in laser-dissected tissue samples from PanIN lesions (from a mouse model and from human patients). miR-21, 205 and 200 paralleled PanIN progression in mouse models. mir-21 and miR-205 preceded phenotypic changes of the duct. In precursor lesions, miR-21 achieved the highest relative concentrations. In human samples, miR-21,-221, 222 and let-7a increased with lesion grade, with maximal expression in two thirds of lesions. Up-regulation of miR-21 was controlled by KRAS and EGFR in PDAC-(Pancreatic Ductal

Another complex investigation of miRNA signatures during tumorigenesis and progression in pancreatic cancer was reported by Olson et al. The study stressed the down-regulation of the miR-200 family in metastases and metastasis-like primary tumors. Also, multiple changes

clonogenic growth and activates apoptosis via the caspase-3 pathway [15].

MetalloProteinase 2) and MMP9 (Matrix-MetalloProteinase 9) [16].

**3.1. Cell growth and proliferation**

**3.2. Tumorigenesis**

in exosomal miRNAs.

AdenoCarcinoma) derived cell lines [19].


**miRNA Modification Significance Ref.** miR-200b Down-regulated Metastasis [20, 27] miR-200c Up-regulated Metastasis [20] miR-203 Up-regulated [26] miR-205 Up-regulated [25, 26] miR-210 Up-regulated [25] miR-212 Up-regulated [24] miR-213 Up-regulated [25] miR-216 Down-regulated [26] miR-217 Down-regulated [26] miR-220 Up-regulated [25] miR-221 Up-regulated [24-27] miR-222 Up-regulated [25-27] miR-223 Up-regulated [25] miR-224 Up-regulated [26] miR-301 Up-regulated [24] miR-329 Up-regulated Metastasis [20] miR-335 Down-regulated Tumor signature [20] miR-344 Up-regulated Metastasis [20] miR-345 Down-regulated [24] miR-365 Down-regulated Metastasis [24] miR-375 Down-regulated [25, 26] miR-376a Up-regulated [24] miR-376 Up-regulated Tumor signature [24] 409-3p Up-regulated Tumor signature [20]

miR-410 Down-regulated

**Table 1.** miRNAs involved in tumorigenesis of the pancreas

**3.3. Apoptosis, cell viability**

Up-regulated

miR-424 Up-regulated Angiogenesis [20, 24] miR-429 Down-regulated Metastasis [20] miR-431 Up-regulated Metastasis [20] miR-434-3p Up-regulated Tumor signature [20] miR-449 Up-regulated Metastasis [20]

miR-21, miR-155 and miR-221 over-expression was reported by Lee et al. (2007) for pancreatic tumors compared to paired normal samples. Since the same miRNAs were also over-expressed in other cancers, the authors hypothesized that deregulation of these miRNAs represents a common feature in cancer. For other miRNAs, the pattern of differential expression appeared

Hyperplasia Metastasis

[20]

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 67



**Table 1.** miRNAs involved in tumorigenesis of the pancreas

#### **3.3. Apoptosis, cell viability**

**miRNA Modification Significance Ref.** miR-126\* Up-regulated Angiogenesis [20] miR-129 Up-regulated Metastasis [20] miR-129-3p Up-regulated Angiogenesis [20] miR-130b Down-regulated [24] miR-132 Up-regulated Metastasis, tumor signature [20] miR-137 Up-regulated Metastasis [20] miR-139 Down-regulated [24] miR-141 Down-regulated Metastasis [20] miR-142-p Down-regulated [24] miR-142-3p Down-regulated Hyperplasia, angiogenesis, tumor

66 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

miR-142-5p Up-regulated

Down-regulated

miR-146a Down-regulated Angiogenesis

signature

Metastasis

miR-143 Down-regulated Metastasis [25],18,[26] miR-145 Down-regulated Metastasis [20, 24, 26] miR-146 Up-regulated [25]

miR-148a Down-regulated Metastasis [25, 26] miR-148b Down-regulated Metastasis [25, 26] miR-150 Down-regulated Tumor signature [20, 26] miR-152 Down-regulated Metastasis [20] miR-155 Up-regulated Hyperplasia/dysplasia [20, 25, 26] miR-181a Up-regulated Metastasis [20, 24, 25] miR-181b Up-regulated Metastasis [20, 25] miR-181b-1 Up-regulated [25] miR-181b-2 Up-regulated [25] miR-181c Up-regulated [25] miR-181d Up-regulated [25] MiR-182 Down-regulated Metastasis [20] miR-184 Down-regulated Tumor signature [20] miR-186 Up-regulated [27] miR-189 Up-regulated Metastasis [20] miR-190 Up-regulated [27] miR-196a Up-regulated [26] miR-196b Up-regulated [26] miR-199a-1 Up-regulated [25] miR-199a-2 Up-regulated [25] miR-200a Down-regulated Metastasis [20]

Angiogenesis Tumor signature [20]

[20]

[20, 26]

miR-21, miR-155 and miR-221 over-expression was reported by Lee et al. (2007) for pancreatic tumors compared to paired normal samples. Since the same miRNAs were also over-expressed in other cancers, the authors hypothesized that deregulation of these miRNAs represents a common feature in cancer. For other miRNAs, the pattern of differential expression appeared different in pancreatic cancer compared to other cancers; modification of miR-376a and miR-301 expression was reported as a distinctive feature of pancreatic cancer [24].

**4. miRNAs in tumor stem cells**

in tissue samples from poor outcome patients [36].

miR-107, miR-128 and miR-141-5p [37].

In gemcitabine-resistant cells with fibroblast morphology, high levels of vimentin and ZEB1 and low levels of E-cadherin, miR-200b, miR-200c, let-7b, 7c, 7d and 7e were found to be downregulated, according to Li et al. (2009). As in the case of miR-146, DIM (3, 3' DiIndolylMethane) and isoflavone were demonstrated to restore a less invasive phenotype [33]. ZEB1 was demonstrated to repress expression of stemness-inhibiting miR-203; candidate targets of miR-200 family members are also stem cell factors, such as Sox2 (Transcription factor SOX-2) and Klf4 (Krueppel-like factor 4). miR-200c, miR-203 and miR-183 cooperate to suppress expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as dem‐

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 69

onstrated for the polycomb repressor Bmi1 (Polycomb complex protein BMI-1) [32].

Key cell differentiation programs during development are controlled by the members of let-7 and miR-200 families. In cancer, loss of let-7 leads to disease progression and de-differentiation [33]. The same let-7 family appears as a regulator of EMT and of stem cell maintenance. The EMT process is regulated by miRNA-dependent mechanisms. In human pancreatic cancer, DCLK1 (Serine/threonine-protein kinase DCLK1) regulates EMT by a mechanism dependent on miR-200a [33-35]. According to Hasselman et al. [36], inhibition of maturation of let-7 by nuclear TRAIL-R2 (TNF-Related Apoptosis-Inducing Ligand Receptor 2) in pancreatic cancer cell lines increases their proliferation. This is consistent with high levels of nuclear TRAIL-R2

The population of BxPC-3-LN cells (lymph node metastatic pancreatic cells) contains a fivefold increased population of CD133+/CXCR4+cells (stem cell-like cells) compared with the parental (non-metastatic) BxPC-3 cells. Remarkably, a different miRNA pattern is displayed in CSC-like cells compared with the regular cells: up-regulated miR-572, miR-206, miR-449a, miR-489 and miR184 were found, as well as down-regulated let-7g-3p, let-7i-3p, let-7a-3p,

The miR-200 family members are identified as key regulators of cell maintenance and EMT. It is considered possible that tumor progression is a process resulting in progressive dedifferentiation towards a cell type which has a stem cell-like phenotype. This process appears to be regulated by miRNA-dependent mechanisms. DCLK1 (a putative marker for pancreatic and intestinal cancer stem cells) regulates EMT in human pancreatic cancer cells via a miR-200a-dependent mechanism [38]; it also acts as a regulator of let-7a in pancreatic and colorectal cancer cells, supporting the idea that these miRNAs may be novel and relevant targets in solid tumor cancers [33-35]. Sureban et al. [39] demonstrated that DCLK1 inhibition results in up-regulation of miRNAs that negatively regulate some key angiogenic and pluripotency factors. In AsPC1 (metastatic adenocarcinoma cell line) tumor xenografts, the down-regulation of c-MYC (Myc Proto-oncogene Protein) and KRAS (GTP-ase Kras) via let-7a

was observed, by a similar mechanism demonstrated in pancreatic cancer cells.

Inhibiting miR-21 and miR-221 with antisense nucleotides resulted in reduced proliferation and increased apoptosis [28].

Hanoun et al. reported the identification of 29 miRNA encoding genes that are susceptible to inactivation by hypermethylation. "In-depth" investigations on miR-148a showed that its production was repressed due to hypermethylation. Hypermethylation analysis was demon‐ strated as a potential tool for differential diagnostics for PDAC and pancreatitis [29].

Down-regulation of miR-146 was demonstrated by Li et al. (2010) in pancreatic cancer cells compared with normal duct epithelium. Re-expression of miR-146 inhibits the invasive capacity of cancer cells, concomitant with down-regulation of EGFR (Epidermal Growth Factor Receptor) and IRAK-1 (Interleukin 1 Receptor-Associated Kinase). Treatment with two natural compounds, diindolylmethane and isoflavone, were demonstrated to activate miR-146a and inhibit invasion [30].

#### **3.4. Tumor suppressors**

Mees et al. (2009) applied microarray, TLDA (Taq-Man Low Density Array) and RT-PCR (Real Time Polymerase Chain Reaction) methods to investigate microRNA profiles in pancreatic cancer cell lines. Fifty-six miRNAs with modified expression were identified: 27 (by microar‐ ray) and 19 (by TLDA) miRNAs were over-expressed in highly metastatic cell lines compared to less metastatic ones. Down-regulation (investigated by TLDA) revealed 35 down-regulated microRNAs. Eight of these were tumor-suppressor gene-related miRNAs: miR-21 (PTEN-Phosphatase and TENsin Homolog), miR-26b (EP300-E1A binding protein p300, PTEN), miR-194 (EP300), miR-200b (EP300), miR-200c (EP300), miR-320 (PTEN), miR-374 (EP300) and miR-429 (EP300) [31].

The influence of miR-10a on the behavior of pancreatic tumors was investigated by Weiss et al. in a zebrafish animal model (zebrafish with transplanted tumors) [22]. miR-10a promotes metastatic potential and miR-10 repression is sufficient to inhibit invasions and metastasis. mir-10a is a retinoic acid (RA) target and RA receptor antagonists are effective repressors of miR-10a expression. The anti-metastatic effect is blocked by the knockdown of HOXB1 (Homeodomain containing DNA-binding Box protein 1) and HOXB3 (HOXB3=Homeodomain containing DNA-binding Box protein 3) genes. The epithelial to mesenchymal cell transition (EMT) program triggers cellular mobility and promotes invasion and metastasis. ZEB1 (zinc finger E box binding homeobox1), an EMT activator, promotes cell mobility by disrupting stemness maintenance and promoting mobile, migrating stem cells. ZEB1 was demonstrated to inhibit miR-200 family members and miR-203 [32].

A specific miRNA signature differentiates between pancreatic adenocarcinoma, normal pancreas and chronic pancreatitis [25]. In total, 21 over-expressed and four down-regulated miRNAs allow a differential diagnosis among these three pathologic conditions. In addition, Szafranska et al. reported that miR-196a and-196b levels are high in pancreatic ductal adeno‐ carcinoma but not in normal or inflamed pancreatic tissues [26].

## **4. miRNAs in tumor stem cells**

different in pancreatic cancer compared to other cancers; modification of miR-376a and

Inhibiting miR-21 and miR-221 with antisense nucleotides resulted in reduced proliferation

Hanoun et al. reported the identification of 29 miRNA encoding genes that are susceptible to inactivation by hypermethylation. "In-depth" investigations on miR-148a showed that its production was repressed due to hypermethylation. Hypermethylation analysis was demon‐

Down-regulation of miR-146 was demonstrated by Li et al. (2010) in pancreatic cancer cells compared with normal duct epithelium. Re-expression of miR-146 inhibits the invasive capacity of cancer cells, concomitant with down-regulation of EGFR (Epidermal Growth Factor Receptor) and IRAK-1 (Interleukin 1 Receptor-Associated Kinase). Treatment with two natural compounds, diindolylmethane and isoflavone, were demonstrated to activate miR-146a and

Mees et al. (2009) applied microarray, TLDA (Taq-Man Low Density Array) and RT-PCR (Real Time Polymerase Chain Reaction) methods to investigate microRNA profiles in pancreatic cancer cell lines. Fifty-six miRNAs with modified expression were identified: 27 (by microar‐ ray) and 19 (by TLDA) miRNAs were over-expressed in highly metastatic cell lines compared to less metastatic ones. Down-regulation (investigated by TLDA) revealed 35 down-regulated microRNAs. Eight of these were tumor-suppressor gene-related miRNAs: miR-21 (PTEN-Phosphatase and TENsin Homolog), miR-26b (EP300-E1A binding protein p300, PTEN), miR-194 (EP300), miR-200b (EP300), miR-200c (EP300), miR-320 (PTEN), miR-374 (EP300) and

The influence of miR-10a on the behavior of pancreatic tumors was investigated by Weiss et al. in a zebrafish animal model (zebrafish with transplanted tumors) [22]. miR-10a promotes metastatic potential and miR-10 repression is sufficient to inhibit invasions and metastasis. mir-10a is a retinoic acid (RA) target and RA receptor antagonists are effective repressors of miR-10a expression. The anti-metastatic effect is blocked by the knockdown of HOXB1 (Homeodomain containing DNA-binding Box protein 1) and HOXB3 (HOXB3=Homeodomain containing DNA-binding Box protein 3) genes. The epithelial to mesenchymal cell transition (EMT) program triggers cellular mobility and promotes invasion and metastasis. ZEB1 (zinc finger E box binding homeobox1), an EMT activator, promotes cell mobility by disrupting stemness maintenance and promoting mobile, migrating stem cells. ZEB1 was demonstrated

A specific miRNA signature differentiates between pancreatic adenocarcinoma, normal pancreas and chronic pancreatitis [25]. In total, 21 over-expressed and four down-regulated miRNAs allow a differential diagnosis among these three pathologic conditions. In addition, Szafranska et al. reported that miR-196a and-196b levels are high in pancreatic ductal adeno‐

miR-301 expression was reported as a distinctive feature of pancreatic cancer [24].

68 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

strated as a potential tool for differential diagnostics for PDAC and pancreatitis [29].

and increased apoptosis [28].

inhibit invasion [30].

**3.4. Tumor suppressors**

miR-429 (EP300) [31].

to inhibit miR-200 family members and miR-203 [32].

carcinoma but not in normal or inflamed pancreatic tissues [26].

In gemcitabine-resistant cells with fibroblast morphology, high levels of vimentin and ZEB1 and low levels of E-cadherin, miR-200b, miR-200c, let-7b, 7c, 7d and 7e were found to be downregulated, according to Li et al. (2009). As in the case of miR-146, DIM (3, 3' DiIndolylMethane) and isoflavone were demonstrated to restore a less invasive phenotype [33]. ZEB1 was demonstrated to repress expression of stemness-inhibiting miR-203; candidate targets of miR-200 family members are also stem cell factors, such as Sox2 (Transcription factor SOX-2) and Klf4 (Krueppel-like factor 4). miR-200c, miR-203 and miR-183 cooperate to suppress expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as dem‐ onstrated for the polycomb repressor Bmi1 (Polycomb complex protein BMI-1) [32].

Key cell differentiation programs during development are controlled by the members of let-7 and miR-200 families. In cancer, loss of let-7 leads to disease progression and de-differentiation [33]. The same let-7 family appears as a regulator of EMT and of stem cell maintenance. The EMT process is regulated by miRNA-dependent mechanisms. In human pancreatic cancer, DCLK1 (Serine/threonine-protein kinase DCLK1) regulates EMT by a mechanism dependent on miR-200a [33-35]. According to Hasselman et al. [36], inhibition of maturation of let-7 by nuclear TRAIL-R2 (TNF-Related Apoptosis-Inducing Ligand Receptor 2) in pancreatic cancer cell lines increases their proliferation. This is consistent with high levels of nuclear TRAIL-R2 in tissue samples from poor outcome patients [36].

The population of BxPC-3-LN cells (lymph node metastatic pancreatic cells) contains a fivefold increased population of CD133+/CXCR4+cells (stem cell-like cells) compared with the parental (non-metastatic) BxPC-3 cells. Remarkably, a different miRNA pattern is displayed in CSC-like cells compared with the regular cells: up-regulated miR-572, miR-206, miR-449a, miR-489 and miR184 were found, as well as down-regulated let-7g-3p, let-7i-3p, let-7a-3p, miR-107, miR-128 and miR-141-5p [37].

The miR-200 family members are identified as key regulators of cell maintenance and EMT. It is considered possible that tumor progression is a process resulting in progressive dedifferentiation towards a cell type which has a stem cell-like phenotype. This process appears to be regulated by miRNA-dependent mechanisms. DCLK1 (a putative marker for pancreatic and intestinal cancer stem cells) regulates EMT in human pancreatic cancer cells via a miR-200a-dependent mechanism [38]; it also acts as a regulator of let-7a in pancreatic and colorectal cancer cells, supporting the idea that these miRNAs may be novel and relevant targets in solid tumor cancers [33-35]. Sureban et al. [39] demonstrated that DCLK1 inhibition results in up-regulation of miRNAs that negatively regulate some key angiogenic and pluripotency factors. In AsPC1 (metastatic adenocarcinoma cell line) tumor xenografts, the down-regulation of c-MYC (Myc Proto-oncogene Protein) and KRAS (GTP-ase Kras) via let-7a was observed, by a similar mechanism demonstrated in pancreatic cancer cells.

## **5. miRNA Polymorphisms**

Single nucleotide polymorphisms (SNPs) were demonstrated to affect the functional capacity of miRNAs, influencing MIR processing and miR-mRNA interactions. SNPs in miR-196a2 and miR-146a were differentially expressed between patients with T1/T2 stage pancreatic tumors compared with T3/T4 stages [40].

appear in all body fluids and interest in studying them has increased since they contain functional proteins, mRNA and miRNAs. Thus, exosomal populations of different origins may be identified by their protein and miRNA signatures. Moreover, they appear to be actively involved in cell communication. In the case of cancer, they will be, for instance, involved in tumorigenesis, differentiation of stem cells, metastasis and angiogenesis [49]. Most of the reports on exosomes so far concern other cancers, like ovarian [50], prostate [51] and glioblas‐ toma [52]; however, there is also a study concerning exosomes in pancreatic cancer [53].

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 71

miRNAs, already described as potent regulators of genes, can be viewed as both therapeutic targets and therapy agents [54]. Recently, the potential to target miRNAs was demonstrated

In pancreatic cancer (and other epithelial tumors as well), a loss of epithelial differentiation and acquisition of the mesenchymal phenotype occurs, leading to enhanced invasion and migration [55]. Another feature of pancreatic cancer is its drug resistance characteristics, often

miR-21 overexpression is associated with resistance to gemcitabine and is generally associated with poor survival [57]. Inhibition of miR-21 decreases cell proliferation and promotes apoptosis [58] but also correlates with 5-FU (5-Fluoro-Uracyl) sensitivity [59]. Another study points out that miR-200 down-regulation and over-expression of miR-21 associates with gemcitabine resistance and their restoration renders the pancreatic cell lines responsive to

miR-34 is a tumor-suppressor miRNA, which can restore chemo-and radio-sensitivity in tumor cell lines; overexpression of miR-34 reduces the tumor-initiating cells and tumor-sphere

Another candidate is miR-155, which also appears down-regulated in pancreatic cancers; its overexpression appears to suppress tumor growth [18, 62]. Similar findings are also published for miR-20 and miR-146, with regard to their impact on invasiveness and chemo-sensitivity

The published literature dealing with miRNAs as therapeutic targets in digestive tract cancers does not abound and relies mostly on results obtained on cell lines. miRNAs as therapeutic targets are foreseen in chemotherapy resistance [65-67], silencing oncogenic miRNAs and

Another important set of studies focuses on miRNAs' oncogenic function and on the modalities of intervening using miRNA silencing, antisense blocking and miRNA modifications [54].

The miRNAs with tumor-suppression functions can represent new strategies for inhibiting tumor growth in pancreatic cancer, liver cancer and colorectal cancer [68], while miRNAs as

oncogenes can be targeted leading to controlling multiple genes [69].

**7. miRNAs: Therapeutic targets and drugs**

associated with epithelial-to-mesenchymal transition (EMT) [56].

by a series of *in vitro* studies.

gemcitabine [60].

[63, 64].

formation significantly [61].

intervention on tumor-suppressive miRNAs.

## **6. Circulating miRNAs**

Some serum tumor markers, such as carcinoembryonic antigen and carbohydrate antigen 19– 9, are used as convenient diagnostic markers. Other factors involved in cancer progression, among which are angiogenic factors such as VEGF (Vascular Endothelial Growths Factor) and bFGF (Basic Fibroblast Growth Factor), have drawn attention for the detection of pancreatic cancers [41, 42]. However, these conventional serum markers lack the sensitivity and specif‐ icity to facilitate the early detection of cancer. Several studies have identified tumor-specific alterations in plasma/serum nucleic acids in cancer patients and have shown the potential of plasma-circulating nucleic acids to act as new non-invasive biomarkers in patients with various cancers [43]. Recently, several studies have demonstrated that miRNAs are stably detectable in plasma/serum and have discussed key aspects regarding experimental design, such as extraction from biological material, different techniques for miRNA evaluation (TLDA, arrays, etc.) [44-46]. Mitchell et al. clearly showed that circulating miRNAs originate from cancer tissues and are protected from endogenous RNase activity. They also demonstrated that the circulating plasma miRNAs are not associated with circulating tumor cells [45].

Li et al. investigated a set of 735 miRNAs by RT-qPCR (Reverse-Transcriptase quantitative Polymersase Chain Reaction) using microarrays. Eighteen candidates were further validated. The best classifier was miR-1290, with ROC-AUC (Receiver Operator Characteristics Area Under the Curve) of 0.96, while other miRNAs (miR-24, miR-134, miR-146a, miR-378, miR-484, miR-628-3p and miR-1825) also displayed considerable accuracy (ROC-AUC > 0.7). miR-1290 could differentiate between normal pancreas, chronic pancreatitis, pancreatic adenocarcinoma and pancreatic neuroendocrine tumors. Remarkably, miR-1290 is a better classifier than the classical biomarker CA 19-9, distinguishing with greater accuracy low-grade pancreatic cancer from normal subjects [47].

Morimura et al. demonstrated the value of miR-18a as a biomarker for pancreatic cancer; they demonstrated higher levels of expression of this miRNA in cancer tissue and cancer cell lines (compared to normal tissue) and also reported higher plasma levels in patients with PC, with ROC-AUC of 0.9369 [46].

A signature of seven miRNAs was established as a good biomarker for early detection by Liu R et al. [48]. The panel comprised miR-20a, miR-21, miR-24, miR-25, miR-99a, miR-185 and miR-191; the levels of overexpression in plasma ranged between 2.1 and 5.08.

Exosomal miRNAs represents a more recent field of investigation. Exosomes are 40-100 nm vesicles derived from the fusion of multivesicular bodies with the plasma membrane. They appear in all body fluids and interest in studying them has increased since they contain functional proteins, mRNA and miRNAs. Thus, exosomal populations of different origins may be identified by their protein and miRNA signatures. Moreover, they appear to be actively involved in cell communication. In the case of cancer, they will be, for instance, involved in tumorigenesis, differentiation of stem cells, metastasis and angiogenesis [49]. Most of the reports on exosomes so far concern other cancers, like ovarian [50], prostate [51] and glioblas‐ toma [52]; however, there is also a study concerning exosomes in pancreatic cancer [53].

## **7. miRNAs: Therapeutic targets and drugs**

**5. miRNA Polymorphisms**

compared with T3/T4 stages [40].

**6. Circulating miRNAs**

from normal subjects [47].

ROC-AUC of 0.9369 [46].

Single nucleotide polymorphisms (SNPs) were demonstrated to affect the functional capacity of miRNAs, influencing MIR processing and miR-mRNA interactions. SNPs in miR-196a2 and miR-146a were differentially expressed between patients with T1/T2 stage pancreatic tumors

70 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Some serum tumor markers, such as carcinoembryonic antigen and carbohydrate antigen 19– 9, are used as convenient diagnostic markers. Other factors involved in cancer progression, among which are angiogenic factors such as VEGF (Vascular Endothelial Growths Factor) and bFGF (Basic Fibroblast Growth Factor), have drawn attention for the detection of pancreatic cancers [41, 42]. However, these conventional serum markers lack the sensitivity and specif‐ icity to facilitate the early detection of cancer. Several studies have identified tumor-specific alterations in plasma/serum nucleic acids in cancer patients and have shown the potential of plasma-circulating nucleic acids to act as new non-invasive biomarkers in patients with various cancers [43]. Recently, several studies have demonstrated that miRNAs are stably detectable in plasma/serum and have discussed key aspects regarding experimental design, such as extraction from biological material, different techniques for miRNA evaluation (TLDA, arrays, etc.) [44-46]. Mitchell et al. clearly showed that circulating miRNAs originate from cancer tissues and are protected from endogenous RNase activity. They also demonstrated that the

circulating plasma miRNAs are not associated with circulating tumor cells [45].

Li et al. investigated a set of 735 miRNAs by RT-qPCR (Reverse-Transcriptase quantitative Polymersase Chain Reaction) using microarrays. Eighteen candidates were further validated. The best classifier was miR-1290, with ROC-AUC (Receiver Operator Characteristics Area Under the Curve) of 0.96, while other miRNAs (miR-24, miR-134, miR-146a, miR-378, miR-484, miR-628-3p and miR-1825) also displayed considerable accuracy (ROC-AUC > 0.7). miR-1290 could differentiate between normal pancreas, chronic pancreatitis, pancreatic adenocarcinoma and pancreatic neuroendocrine tumors. Remarkably, miR-1290 is a better classifier than the classical biomarker CA 19-9, distinguishing with greater accuracy low-grade pancreatic cancer

Morimura et al. demonstrated the value of miR-18a as a biomarker for pancreatic cancer; they demonstrated higher levels of expression of this miRNA in cancer tissue and cancer cell lines (compared to normal tissue) and also reported higher plasma levels in patients with PC, with

A signature of seven miRNAs was established as a good biomarker for early detection by Liu R et al. [48]. The panel comprised miR-20a, miR-21, miR-24, miR-25, miR-99a, miR-185 and

Exosomal miRNAs represents a more recent field of investigation. Exosomes are 40-100 nm vesicles derived from the fusion of multivesicular bodies with the plasma membrane. They

miR-191; the levels of overexpression in plasma ranged between 2.1 and 5.08.

miRNAs, already described as potent regulators of genes, can be viewed as both therapeutic targets and therapy agents [54]. Recently, the potential to target miRNAs was demonstrated by a series of *in vitro* studies.

In pancreatic cancer (and other epithelial tumors as well), a loss of epithelial differentiation and acquisition of the mesenchymal phenotype occurs, leading to enhanced invasion and migration [55]. Another feature of pancreatic cancer is its drug resistance characteristics, often associated with epithelial-to-mesenchymal transition (EMT) [56].

miR-21 overexpression is associated with resistance to gemcitabine and is generally associated with poor survival [57]. Inhibition of miR-21 decreases cell proliferation and promotes apoptosis [58] but also correlates with 5-FU (5-Fluoro-Uracyl) sensitivity [59]. Another study points out that miR-200 down-regulation and over-expression of miR-21 associates with gemcitabine resistance and their restoration renders the pancreatic cell lines responsive to gemcitabine [60].

miR-34 is a tumor-suppressor miRNA, which can restore chemo-and radio-sensitivity in tumor cell lines; overexpression of miR-34 reduces the tumor-initiating cells and tumor-sphere formation significantly [61].

Another candidate is miR-155, which also appears down-regulated in pancreatic cancers; its overexpression appears to suppress tumor growth [18, 62]. Similar findings are also published for miR-20 and miR-146, with regard to their impact on invasiveness and chemo-sensitivity [63, 64].

The published literature dealing with miRNAs as therapeutic targets in digestive tract cancers does not abound and relies mostly on results obtained on cell lines. miRNAs as therapeutic targets are foreseen in chemotherapy resistance [65-67], silencing oncogenic miRNAs and intervention on tumor-suppressive miRNAs.

Another important set of studies focuses on miRNAs' oncogenic function and on the modalities of intervening using miRNA silencing, antisense blocking and miRNA modifications [54].

The miRNAs with tumor-suppression functions can represent new strategies for inhibiting tumor growth in pancreatic cancer, liver cancer and colorectal cancer [68], while miRNAs as oncogenes can be targeted leading to controlling multiple genes [69].

Recently, the inhibition of miR-21 and miR-17-92 activity was reported as being associated with reduced tumor growth, invasion, angiogenesis and metastasis in PDAC [70, 71].

so far of these possible drugs, therefore only preclinical studies were reported, showing that upon the insertion of a functional miR-34, inhibition of cell growth, chemo-sensitization and apoptosis are triggered along with the abolishment of cancer stem cell characteristics [79].

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 73

Modification in the expression of miRNAs is consistently associated with the process of tumorigenesis. Such deregulation of miRNAs encompasses early-stage or tumor-initiating events, triggers invasion and metastasis, or alternatively it may also represent the outcome of complex alterations specific to tumor cells. Deregulated miRNAs were demonstrated to have potential and several have been already validated as biomarkers for cancer diagnostics or prognosis, including several for pancreatic cancer, especially in tissue-based investigations.

A great number of miRNAs have similar expression patterns in cancers, but several have been

A considerable effort is directed towards the development of miRNA-based instruments for diagnostics, prognostics or monitoring the disease and great hope is placed in the exosomal miRNAs. Assays based on exosomal or plasma miRNAs have potential clinical uses in

They also prove useful in evaluating the completeness of tumor resection and the evaluation of adjuvant therapy. As biomarkers, they show important advantages over other nucleic acids. Compared to the mRNA, in the case of miRNAs a considerably smaller number of molecules can establish an effective screen to differentiate normal from tumoral disease. At the same time, circulating miRNAs are more stable for detection, by comparison with other classes of markers,

Meanwhile, the presence of specific miRNAs in pathological tissue opens a new perspective in therapy. As has already been proved, targeting deregulated miRNAs with specific instru‐ ments, like miR-mimics, antago-miRs or miRNAs, restores the phenotype from tumoral to normal and the results so far suggest that controlling the expression of miRNA modifies clinical features of tumor cells, such as growth rate, apoptotic susceptibility, drug resistance, mobility

screening patients at risk of cancer or monitoring recurrence post-resection.

**8. Conclusions**

demonstrated to be tumor-specific.

like mRNAs or proteins.

**9. Abbreviations**

AGO2=Protein Argonaute2

ARE=TNFα AU-rich element

Bcl2=Apoptosis regulator Bcl-2

**Proteins:**

and invasiveness of metastatic potential.

As therapeutic targets, miRNAs can be manipulated with silencing methodology or recovery of altered microRNAs. Using miRNAs as therapeutic targets may result in several clinical goals: prevent recurrence of the disease, control the growth of advanced metastatic tumors and sensitize tumor cells to chemo-and radiotherapy. There are few studies on *in vivo* experimental models and no clinical trial have commenced using these small molecules as targets [54]. Thus, miRNAs may be possible drugs and/or drug targets and they could potentially be used as molecular therapeutic agents that could inhibit oncogenes or restore the expression of silenced tumor-suppressor genes [1, 72].

Taking into account the updated findings, miRNA-based cancer gene therapy is to be used as follows: RNA or DNA drugs against messenger RNA-encoding genes involved in the patho‐ genesis of cancers or by directly targeting ncRNAs (non-coding RNAs) that participate in cancer pathogenesis, as reported in colorectal cancers [73].

The reported stages of miRNAs as drugs are generally at the preclinical phase. Groups are using cell lines or even primary cells in a workflow comprising *in vitro* treatment and after‐ wards detecting the alteration of proliferation, increase in apoptosis and/or abolishment of cancer stem cell characteristics. In animal models treating tumor-bearing mice with specific siRNA, the overall effect on tumor development and survival was tested along with the excretion route of the drug. Very few clinical studies are reported and mostly only in phase I [74, 75]

Up to now, the reported inhibitory RNAs drugs have been: antisense oligonucleotides (ASOs), ribozymes or DNAzymes, siRNAs, microRNAs mimetics, LNAs (Locked Nucleic Acids), antimiRNAs or antagomiRs (small synthetic oligonucleotides blocking the binding of miRNAs to their targets [28, 76].

siRNAs represent a double strand RNA homologous to the mRNA of a target gene. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC). The antisense strand guides this complex to its homologous mRNA target for endo-nucleasic cleavage of messenger RNA. ERBB2 (ErbB2 protein encoding gene) amplification was demonstrated in gastrointestinal adenocarcinomas, while in cellular and animal models, siRNA was used to knock down ERBB2 in cell lines, demonstrating that this treatment decreased ERBB2 protein levels and apoptotic pathways were triggered [77].

siRNA specific for bcl-2 (Apoptosis regulator Bcl-2) was also used as a possible therapeutic tool in pancreatic cancer in cells lines and xenografts and the bcl-2 gene was inhibited [78].

MicroRNAs mimics represent small single-strand 19–24 nucleotide RNA produced from the cleavage of a hairpin structure by RNAse III enzymes. These possible therapeutic agents act by the inhibition of protein production by either mRNA degradation or translational block after the formation of miRNA<mRNA duplexes.

miR-34 can target p53, Notch, HMGA2 (High Mobility Group Protein HMGI-C) and Bcl-2, genes mainly involved in cancer stem cells characteristics. There are no clinical trials published so far of these possible drugs, therefore only preclinical studies were reported, showing that upon the insertion of a functional miR-34, inhibition of cell growth, chemo-sensitization and apoptosis are triggered along with the abolishment of cancer stem cell characteristics [79].

## **8. Conclusions**

Recently, the inhibition of miR-21 and miR-17-92 activity was reported as being associated

As therapeutic targets, miRNAs can be manipulated with silencing methodology or recovery of altered microRNAs. Using miRNAs as therapeutic targets may result in several clinical goals: prevent recurrence of the disease, control the growth of advanced metastatic tumors and sensitize tumor cells to chemo-and radiotherapy. There are few studies on *in vivo* experimental models and no clinical trial have commenced using these small molecules as targets [54]. Thus, miRNAs may be possible drugs and/or drug targets and they could potentially be used as molecular therapeutic agents that could inhibit oncogenes or restore the

Taking into account the updated findings, miRNA-based cancer gene therapy is to be used as follows: RNA or DNA drugs against messenger RNA-encoding genes involved in the patho‐ genesis of cancers or by directly targeting ncRNAs (non-coding RNAs) that participate in

The reported stages of miRNAs as drugs are generally at the preclinical phase. Groups are using cell lines or even primary cells in a workflow comprising *in vitro* treatment and after‐ wards detecting the alteration of proliferation, increase in apoptosis and/or abolishment of cancer stem cell characteristics. In animal models treating tumor-bearing mice with specific siRNA, the overall effect on tumor development and survival was tested along with the excretion route of the drug. Very few clinical studies are reported and mostly only in phase I

Up to now, the reported inhibitory RNAs drugs have been: antisense oligonucleotides (ASOs), ribozymes or DNAzymes, siRNAs, microRNAs mimetics, LNAs (Locked Nucleic Acids), antimiRNAs or antagomiRs (small synthetic oligonucleotides blocking the binding of miRNAs to

siRNAs represent a double strand RNA homologous to the mRNA of a target gene. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC). The antisense strand guides this complex to its homologous mRNA target for endo-nucleasic cleavage of messenger RNA. ERBB2 (ErbB2 protein encoding gene) amplification was demonstrated in gastrointestinal adenocarcinomas, while in cellular and animal models, siRNA was used to knock down ERBB2 in cell lines, demonstrating that this treatment

siRNA specific for bcl-2 (Apoptosis regulator Bcl-2) was also used as a possible therapeutic tool in pancreatic cancer in cells lines and xenografts and the bcl-2 gene was inhibited [78]. MicroRNAs mimics represent small single-strand 19–24 nucleotide RNA produced from the cleavage of a hairpin structure by RNAse III enzymes. These possible therapeutic agents act by the inhibition of protein production by either mRNA degradation or translational block

miR-34 can target p53, Notch, HMGA2 (High Mobility Group Protein HMGI-C) and Bcl-2, genes mainly involved in cancer stem cells characteristics. There are no clinical trials published

decreased ERBB2 protein levels and apoptotic pathways were triggered [77].

after the formation of miRNA<mRNA duplexes.

with reduced tumor growth, invasion, angiogenesis and metastasis in PDAC [70, 71].

72 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

expression of silenced tumor-suppressor genes [1, 72].

cancer pathogenesis, as reported in colorectal cancers [73].

[74, 75]

their targets [28, 76].

Modification in the expression of miRNAs is consistently associated with the process of tumorigenesis. Such deregulation of miRNAs encompasses early-stage or tumor-initiating events, triggers invasion and metastasis, or alternatively it may also represent the outcome of complex alterations specific to tumor cells. Deregulated miRNAs were demonstrated to have potential and several have been already validated as biomarkers for cancer diagnostics or prognosis, including several for pancreatic cancer, especially in tissue-based investigations.

A great number of miRNAs have similar expression patterns in cancers, but several have been demonstrated to be tumor-specific.

A considerable effort is directed towards the development of miRNA-based instruments for diagnostics, prognostics or monitoring the disease and great hope is placed in the exosomal miRNAs. Assays based on exosomal or plasma miRNAs have potential clinical uses in screening patients at risk of cancer or monitoring recurrence post-resection.

They also prove useful in evaluating the completeness of tumor resection and the evaluation of adjuvant therapy. As biomarkers, they show important advantages over other nucleic acids. Compared to the mRNA, in the case of miRNAs a considerably smaller number of molecules can establish an effective screen to differentiate normal from tumoral disease. At the same time, circulating miRNAs are more stable for detection, by comparison with other classes of markers, like mRNAs or proteins.

Meanwhile, the presence of specific miRNAs in pathological tissue opens a new perspective in therapy. As has already been proved, targeting deregulated miRNAs with specific instru‐ ments, like miR-mimics, antago-miRs or miRNAs, restores the phenotype from tumoral to normal and the results so far suggest that controlling the expression of miRNA modifies clinical features of tumor cells, such as growth rate, apoptotic susceptibility, drug resistance, mobility and invasiveness of metastatic potential.

## **9. Abbreviations**

**Proteins:**

AGO2=Protein Argonaute2 ARE=TNFα AU-rich element Bcl2=Apoptosis regulator Bcl-2

FFPE=Formalin-Fixed, Paraffin Embedded

PanIN=Pancreatic Intraepithelial Neoplasia PDAC=Pancreatic Duct AdenoCarcinoma

RISC=RNA-Induced Silencing Complex

SNPs=Single Nucleotide Polymorphisms

Radu Albulescu1,2, Adrian Claudiu Popa3

726-31. Epub 2009/01/01.

and Cristiana Tanase1

3 Army Center for Medical Research, Bucharest, Romania

[2] 2013; Available from: http://www.mirbase.org/.

2007;43(10):1529-44. Epub 2007/05/29.

2008;299(4):425-36. Epub 2008/01/31.

TLDA=Taq-Man Low Density Array

ROC-AUC=Receiver Operator Characteristics Area Under the Curve

1 National Institute of Pathology "Victor Babes", Bucharest, Romania

2 National Institute for Chemical Pharmaceutical R&D, Bucharest, Romania

RA=Retinoic Acid

**Author details**

Simona Mihai1

**References**

LNA=Locked Nucleic Acids: conformationally-restricted nucleic acid analogue in which the ribose ring is 'locked' with a methylene bridge connecting the 2´-O atom with the 4´-C atom.

, Elena Codrici1

[1] Saito Y, Friedman JM, Chihara Y, Egger G, Chuang JC, Liang G. Epigenetic therapy upregulates the tumor suppressor microRNA-126 and its host gene EGFL7 in human cancer cells. Biochemical and biophysical research communications. 2009;379(3):

[3] Wiemer EA. The role of microRNAs in cancer: no small matter. Eur J Cancer.

[4] Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N, et al. Micro‐ RNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA : the journal of the American Medical Association.

, Daniela Ionela Popescu1

,

miRNAs in Pancreatic Cancer http://dx.doi.org/10.5772/58397 75

bFGF=Basic Fibroblast Growth Factor Bmi1=Polycomb complex protein BMI-1 c-MYC=Myc Proto-oncogene Protein DCLK1=Serine/threonine-protein kinase DCLK1 DGCR8=Integral membrane protein DGCR2/IDD EGFR=Epidermal Growth Factor Receptor EP300=E1A binding protein p300 ERBB2=ErbB2 protein encoding gene EMT=Epithelial-Mesenchymal Transition FXR1=Fragile X Mental Retardation–related Protein 1 HMGA2=High Mobility Group Protein HMGI-C HOXB1=Homeodomain containing DNA-binding Box protein 1 HOXB3=Homeodomain containing DNA-binding Box protein 3 IRAK-1=Interleukin 1 Receptor-Associated Kinase Klf4=Krueppel-like factor 4 KRAS=GTP-ase Kras MMP2=Matrix-MetalloProteinase 2 MMP9=Matrix-MetalloProteinase 9 Notch1=Neurogenic locus notch homolog protein 1 PTEN=Phosphatase and TENsin homolog Sox2=Transcription factor SOX-2 TRAIL2=TNF-Related Apoptosis-Inducing Ligand 2 TRAILR2=TNF-Related Apoptosis-Inducing Ligand Receptor 2 VEGF=Vascular Endothelial Growths Factor ZEB1=Zinc finger E box Binding homeobox1 **Other Abbreviations** 5-FU=5 Fluoro-Uracil CSC=Cancer Stem Cells DIM=3, 3' DiIndolylMethane

FFPE=Formalin-Fixed, Paraffin Embedded

LNA=Locked Nucleic Acids: conformationally-restricted nucleic acid analogue in which the ribose ring is 'locked' with a methylene bridge connecting the 2´-O atom with the 4´-C atom.

PanIN=Pancreatic Intraepithelial Neoplasia

PDAC=Pancreatic Duct AdenoCarcinoma

RA=Retinoic Acid

bFGF=Basic Fibroblast Growth Factor

c-MYC=Myc Proto-oncogene Protein

Bmi1=Polycomb complex protein BMI-1

EGFR=Epidermal Growth Factor Receptor

EP300=E1A binding protein p300

Klf4=Krueppel-like factor 4

MMP2=Matrix-MetalloProteinase 2 MMP9=Matrix-MetalloProteinase 9

Sox2=Transcription factor SOX-2

KRAS=GTP-ase Kras

**Other Abbreviations** 5-FU=5 Fluoro-Uracil

CSC=Cancer Stem Cells

DIM=3, 3' DiIndolylMethane

ERBB2=ErbB2 protein encoding gene

EMT=Epithelial-Mesenchymal Transition

FXR1=Fragile X Mental Retardation–related Protein 1

HOXB1=Homeodomain containing DNA-binding Box protein 1 HOXB3=Homeodomain containing DNA-binding Box protein 3

HMGA2=High Mobility Group Protein HMGI-C

IRAK-1=Interleukin 1 Receptor-Associated Kinase

Notch1=Neurogenic locus notch homolog protein 1

TRAIL2=TNF-Related Apoptosis-Inducing Ligand 2

TRAILR2=TNF-Related Apoptosis-Inducing Ligand Receptor 2

PTEN=Phosphatase and TENsin homolog

VEGF=Vascular Endothelial Growths Factor ZEB1=Zinc finger E box Binding homeobox1

DCLK1=Serine/threonine-protein kinase DCLK1 DGCR8=Integral membrane protein DGCR2/IDD

74 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

RISC=RNA-Induced Silencing Complex

ROC-AUC=Receiver Operator Characteristics Area Under the Curve

SNPs=Single Nucleotide Polymorphisms

TLDA=Taq-Man Low Density Array

#### **Author details**

Radu Albulescu1,2, Adrian Claudiu Popa3 , Elena Codrici1 , Daniela Ionela Popescu1 , Simona Mihai1 and Cristiana Tanase1

1 National Institute of Pathology "Victor Babes", Bucharest, Romania

2 National Institute for Chemical Pharmaceutical R&D, Bucharest, Romania

3 Army Center for Medical Research, Bucharest, Romania

#### **References**


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**Chapter 4**

**Linking Obesity and Pancreatic Cancer**

Cancer of the pancreas is the tenth most common form of cancer in the United States and the fourth leading cause of cancer-related death with a stunningly low 5-year survival rate of less that 6% [1-4]. Although there are genetic links with pancreatic cancer (10-15% of patients diagnosed will have a family history) [5], chronic pancreatitis [6], cigarette smoking and smokeless tobacco [7, 8], obesity [9-11], and type 2 diabetes mellitus (T2DM) [12-14] are the strongest environmental risk factors linked to this malignancy. Recently high fructose corn syrup (HFCS) consumption which also contributes to obesity, T2DM, and non-alcoholic fatty liver disease (NAFLD), has also been directly linked to pancreatic cancer [15]. The development of the industrial age and subsequent loss of the "hunter-gatherer" life-style has resulted in a world-wide epidemic of obesity and its associated chronic diseases including: atherosclerotic heart disease, stroke, diabetes, and multiple obesity-associated malignancies including cancer of the pancreas. Epidemiologic studies have demonstrated that as underdeveloped countries progress into industrialized economies and life-styles change (especially consumption of high density fat/carbohydrate diets coupled with decreased physical activity), the prevalence of obesity and obesity-related chronic diseases increases. The direct link between obesity, chronic inflammation, and oncogenesis is becoming increasingly more appreciated and the underlying cellular mechanisms involved this process are currently intensively being investigated and reviewed [16, 17]. In addition to the direct role of obesity in oncogenesis, obese individuals also demonstrate worse outcomes and shorter cancer survival compared to persons with normal body mass indexes (BMIs) [16]. These observations suggest that the abnormal hormo‐ nal and inflammatory milieu of obesity is directly involved in oncogenesis, promotes tumor growth, spread, and metastasis while possibly also increasing resistance to therapeutic intervention [16]. This chapter is meant to review the links between obesity, abnormal adipose tissue function, induction of abnormal hormonal and chronic inflammatory signaling path‐

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Kelly McCall, Anthony L Schwartz and

Additional information is available at the end of the chapter

Frank L Schwartz

**1. Introduction**

http://dx.doi.org/10.5772/58546


## **Linking Obesity and Pancreatic Cancer**

Kelly McCall, Anthony L Schwartz and Frank L Schwartz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58546

#### **1. Introduction**

[76] Frampton AE, Castellano L, Colombo T, Giovannetti E, Krell J, Jacob J, et al. Micro‐ RNAs Cooperatively Inhibit a Network of Tumor Suppressor Genes to Promote Pan‐ creatic Tumor Growth and Progression. Gastroenterology. 2014;146(1):268-77 e18.

82 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

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2008;8:266. Epub 2008/09/23.

Cancer of the pancreas is the tenth most common form of cancer in the United States and the fourth leading cause of cancer-related death with a stunningly low 5-year survival rate of less that 6% [1-4]. Although there are genetic links with pancreatic cancer (10-15% of patients diagnosed will have a family history) [5], chronic pancreatitis [6], cigarette smoking and smokeless tobacco [7, 8], obesity [9-11], and type 2 diabetes mellitus (T2DM) [12-14] are the strongest environmental risk factors linked to this malignancy. Recently high fructose corn syrup (HFCS) consumption which also contributes to obesity, T2DM, and non-alcoholic fatty liver disease (NAFLD), has also been directly linked to pancreatic cancer [15]. The development of the industrial age and subsequent loss of the "hunter-gatherer" life-style has resulted in a world-wide epidemic of obesity and its associated chronic diseases including: atherosclerotic heart disease, stroke, diabetes, and multiple obesity-associated malignancies including cancer of the pancreas. Epidemiologic studies have demonstrated that as underdeveloped countries progress into industrialized economies and life-styles change (especially consumption of high density fat/carbohydrate diets coupled with decreased physical activity), the prevalence of obesity and obesity-related chronic diseases increases. The direct link between obesity, chronic inflammation, and oncogenesis is becoming increasingly more appreciated and the underlying cellular mechanisms involved this process are currently intensively being investigated and reviewed [16, 17]. In addition to the direct role of obesity in oncogenesis, obese individuals also demonstrate worse outcomes and shorter cancer survival compared to persons with normal body mass indexes (BMIs) [16]. These observations suggest that the abnormal hormo‐ nal and inflammatory milieu of obesity is directly involved in oncogenesis, promotes tumor growth, spread, and metastasis while possibly also increasing resistance to therapeutic intervention [16]. This chapter is meant to review the links between obesity, abnormal adipose tissue function, induction of abnormal hormonal and chronic inflammatory signaling path‐

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ways involved pancreatic cancer origin, growth, spread, and resistance to treatment. Our research efforts have been focused on the role of pathologic expression of toll like receptors (TLRs) in this process which links increasing visceral obesity to these processes.

stasis, and resistance to chemotherapeutics. Cigarette smoking also increases the risk for T2DM by inducing insulin resistance as well. Finally, as will be reviewed below, increasing BMI and obesity are also clearly risk factors for the development of hyperlipidemia, T2DM, chronic

Linking Obesity and Pancreatic Cancer http://dx.doi.org/10.5772/58546 85

There are multiple epidemiologic studies in the US and world-wide linking the epidemic of obesity and higher BMI to increa`sed risk for multiple malignancies including carcinoma of the pancreas [10, 27]. The American Cancer Society calculates that of the 1.5 million new cancer cases diagnosed each year, at least 20% are due to obesity [2]. The risk of pancreatic cancer in both men and women is increased in those who have a BMI > 25 but is most pronounced in those with a BMI of 35 or greater [11, 28]. The risk has been shown to increase by 10 per cent for every five-point increase in BMI. The strongest environmental risk factors related to pancreatic cancer as stated previously are cigarette smoking [8] and obesity [9]; both of which are also linked to inducing chronic inflammation, insulin resistance and T2DM [14]. As stated previously, individuals with T2DM are also twice as likely to develop acute pancreatitis and pancreatic cancer compared to non-diabetics [9, 10]. Studies looking at the components of diet and pancreatic cancer link increased risk with consumption of high fat diets, processed and/or organ meats, the glycemic index of food, and recently high-fructose corn syrup (HFCS) as important factors contributing to obesity, T2DM and risk for carcinoma of the pancreas [15]. As many as 40-50 % of patients with chronic pancreatitis will develop diabetes mellitus (DM) from the chronic destruction of beta cell function as well (insulin deficiency rather than the hyperinsulinemia discussed later). Furthermore, 40% of patients with carcinoma of the pancreas develop insulin deficiency from tumor replacement of beta cells and the DM often

In contrast, there is a reciprocal relationship between the amount of exercise and risk for obesity, T2DM, and pancreatic cancer. Exercise alone burns calories and reduces the risk and/ or severity of obesity, reduces insulin resistance, and promotes the production of antiinflammatory cytokines which counter all of the proinflammatory and oncogenic processes

**5. Molecular pathways linking obesity, inflammation, diabetes, and**

When caloric intake exceeds normal metabolic demand there is a need to store this excess energy and that is the principle function of the adipocyte. Adipose tissue however, is more than just a storage depot. Adipose tissue (especially **visceral fat**) is composed of multiple cell types (adipocytes, pre-adipocytes, macrophages, fibroblasts, and blood vessels), and is now recognized as a significant endocrine organ that expresses and secretes multiple hormones

pancreatitis, and a 2-fold higher prevalence of pancreatic cancer.

precedes the diagnosis of the cancer.

which are discussed below [10].

**pancreatic cancer**

**4. Epidemiology of obesity, T2DM, and pancreatic cancer**

#### **2. Genetic linkage to pancreatic cancer**

Family aggregation of pancreatic cancers suggests a genetic linkage and several important pancreatic cancer susceptibility genes have been identified including high-penetrance genes: **BRCA2, PALB2, PRSS1, SPINK1, STK11** have recently been reviewed [5], and DNA missmatch repair genes. Genome-wide association studies (GWAS) are also finding single-gene polymorphisms (snps) that are also associated with increased risk for pancreatic cancer including: **ABO, 1q32.1, 13q22.1, CLPTM1/TERT, CFTR** [18, 19].

**Chronic pancreatitis** is the strongest independent risk factor for cancer of the pancreas and there are environmentally induced forms as well as rare inherited forms. Autosomal dominant mutations of the cationic trypsinogen gene **PRSS1** causes a hereditary form of chronic pancreatitis [20] while an autosomal recessive defect in the serine protease inhibitor gene **SPINK1** also causes hereditary pancreatitis [21]. These familial forms of chronic pancreatitis exhibit the greatest risk for pancreatic cancer (50-fold increase compared to the general population) and these individuals also experience the longest duration of chronic pancreatitis as well. As life expectancy from cystic fibrosis (CF) has increased from childhood into adult‐ hood, individuals with the cystic fibrosis transmembrane conductance regulator (**CFTR**) gene now exhibit a 5-fold increased risk for pancreatic cancer from their early onset exocrine pancreatic disease and chronic pancreatitis [22, 23]. These are the major genes associated with risk for pancreatic cancer to date and most investigators anticipate that gene-gene and geneenvironmental interactions coupled with the chronic inflammation are cooperatively involved in the pathogenesis of such complex cancers.

#### **3. Environmental causes of chronic pancreatitis**

Patients with chronic pancreatitis from any cause are at increased risk for pancreatic cancer with severity and duration of chronic pancreatitis (>20 years), age of the patient, and concom‐ itant tobacco use being the major associated co-factors. Although alcohol abuse is causally linked to the development of chronic pancreatitis, interestingly it does not appear to be an independent risk factor for pancreatic cancer which has been confirmed by multiple recent epidemiologic met-analysis studies [24, 25]. **Cigarette smoke contains numerous carcinogenic compounds** including nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone **(NNK) [26]**. One of the most well-known features of NNK is the ability of its metabolites to bind to DNA and induce activating point mutations in the RAS gene [26]. Nicotine itself has also been shown to stimulate Src kinase activity which facilitates the induction of the inhibitor of differentiation-1 (Id1) transcription factor which promotes pancreatic tumor growth, meta‐ stasis, and resistance to chemotherapeutics. Cigarette smoking also increases the risk for T2DM by inducing insulin resistance as well. Finally, as will be reviewed below, increasing BMI and obesity are also clearly risk factors for the development of hyperlipidemia, T2DM, chronic pancreatitis, and a 2-fold higher prevalence of pancreatic cancer.

## **4. Epidemiology of obesity, T2DM, and pancreatic cancer**

ways involved pancreatic cancer origin, growth, spread, and resistance to treatment. Our research efforts have been focused on the role of pathologic expression of toll like receptors

Family aggregation of pancreatic cancers suggests a genetic linkage and several important pancreatic cancer susceptibility genes have been identified including high-penetrance genes: **BRCA2, PALB2, PRSS1, SPINK1, STK11** have recently been reviewed [5], and DNA missmatch repair genes. Genome-wide association studies (GWAS) are also finding single-gene polymorphisms (snps) that are also associated with increased risk for pancreatic cancer

**Chronic pancreatitis** is the strongest independent risk factor for cancer of the pancreas and there are environmentally induced forms as well as rare inherited forms. Autosomal dominant mutations of the cationic trypsinogen gene **PRSS1** causes a hereditary form of chronic pancreatitis [20] while an autosomal recessive defect in the serine protease inhibitor gene **SPINK1** also causes hereditary pancreatitis [21]. These familial forms of chronic pancreatitis exhibit the greatest risk for pancreatic cancer (50-fold increase compared to the general population) and these individuals also experience the longest duration of chronic pancreatitis as well. As life expectancy from cystic fibrosis (CF) has increased from childhood into adult‐ hood, individuals with the cystic fibrosis transmembrane conductance regulator (**CFTR**) gene now exhibit a 5-fold increased risk for pancreatic cancer from their early onset exocrine pancreatic disease and chronic pancreatitis [22, 23]. These are the major genes associated with risk for pancreatic cancer to date and most investigators anticipate that gene-gene and geneenvironmental interactions coupled with the chronic inflammation are cooperatively involved

Patients with chronic pancreatitis from any cause are at increased risk for pancreatic cancer with severity and duration of chronic pancreatitis (>20 years), age of the patient, and concom‐ itant tobacco use being the major associated co-factors. Although alcohol abuse is causally linked to the development of chronic pancreatitis, interestingly it does not appear to be an independent risk factor for pancreatic cancer which has been confirmed by multiple recent epidemiologic met-analysis studies [24, 25]. **Cigarette smoke contains numerous carcinogenic compounds** including nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone **(NNK) [26]**. One of the most well-known features of NNK is the ability of its metabolites to bind to DNA and induce activating point mutations in the RAS gene [26]. Nicotine itself has also been shown to stimulate Src kinase activity which facilitates the induction of the inhibitor of differentiation-1 (Id1) transcription factor which promotes pancreatic tumor growth, meta‐

(TLRs) in this process which links increasing visceral obesity to these processes.

84 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

**2. Genetic linkage to pancreatic cancer**

in the pathogenesis of such complex cancers.

**3. Environmental causes of chronic pancreatitis**

including: **ABO, 1q32.1, 13q22.1, CLPTM1/TERT, CFTR** [18, 19].

There are multiple epidemiologic studies in the US and world-wide linking the epidemic of obesity and higher BMI to increa`sed risk for multiple malignancies including carcinoma of the pancreas [10, 27]. The American Cancer Society calculates that of the 1.5 million new cancer cases diagnosed each year, at least 20% are due to obesity [2]. The risk of pancreatic cancer in both men and women is increased in those who have a BMI > 25 but is most pronounced in those with a BMI of 35 or greater [11, 28]. The risk has been shown to increase by 10 per cent for every five-point increase in BMI. The strongest environmental risk factors related to pancreatic cancer as stated previously are cigarette smoking [8] and obesity [9]; both of which are also linked to inducing chronic inflammation, insulin resistance and T2DM [14]. As stated previously, individuals with T2DM are also twice as likely to develop acute pancreatitis and pancreatic cancer compared to non-diabetics [9, 10]. Studies looking at the components of diet and pancreatic cancer link increased risk with consumption of high fat diets, processed and/or organ meats, the glycemic index of food, and recently high-fructose corn syrup (HFCS) as important factors contributing to obesity, T2DM and risk for carcinoma of the pancreas [15]. As many as 40-50 % of patients with chronic pancreatitis will develop diabetes mellitus (DM) from the chronic destruction of beta cell function as well (insulin deficiency rather than the hyperinsulinemia discussed later). Furthermore, 40% of patients with carcinoma of the pancreas develop insulin deficiency from tumor replacement of beta cells and the DM often precedes the diagnosis of the cancer.

In contrast, there is a reciprocal relationship between the amount of exercise and risk for obesity, T2DM, and pancreatic cancer. Exercise alone burns calories and reduces the risk and/ or severity of obesity, reduces insulin resistance, and promotes the production of antiinflammatory cytokines which counter all of the proinflammatory and oncogenic processes which are discussed below [10].

## **5. Molecular pathways linking obesity, inflammation, diabetes, and pancreatic cancer**

When caloric intake exceeds normal metabolic demand there is a need to store this excess energy and that is the principle function of the adipocyte. Adipose tissue however, is more than just a storage depot. Adipose tissue (especially **visceral fat**) is composed of multiple cell types (adipocytes, pre-adipocytes, macrophages, fibroblasts, and blood vessels), and is now recognized as a significant endocrine organ that expresses and secretes multiple hormones (leptin, adiponectin, resistin), inflammatory cytokines (TNF-α, IL-6, and IFN-β), components of complement, plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF) and other proteins such as monocyte chemoattractant protein (MCP-1). These adipose tissue-derived factors (Figure 1) are now thought to contribute dramatically to the induction of chronic inflammation which is expressed as insulin resistance [29], hyperinsulinemia, T2DM, hyperlipidemia, hypertension, and atherosclerosis [30], and also contributing to the oncogenesis of many solid tumors [11, 16]. Visceral obesity is the fat depot most closely associated with the production of these substances and the subsequent development of insulin resistance, T2DM, and pancreatic cancer oncogenesis.

now the leading cause of cirrhosis of the liver and primary hepatocellular cancer. Diets high in HFCS have also been linked directly to increased risk for pancreatic cancer [39]. Mechanisms by which HFCS induces insulin resistance are thought to be due to its unique metabolism in the liver via pathways identical to alcohol. Fructose binds to only one of the glucose trans‐ porters (GLUT 5) which is present only in enterocytes of the intestine and in the liver. Thus, although it is selectively concentrated in the liver, fructose cannot be utilized as a carbohydrate for energy in any other cell or organ of the body. Acutely, fructose ingestion results in the shunting of fructose-1-phosphate into dihydroxyacetone-phosphate and glyceraldehyde which enters the TCA cycle from pyruvate and citrate to excessively increase *de novo* hepatic lipogenesis and the over-production of TGs and FFAs [40]. Fructose-1-phosphate also directly induces janus kinase-1 (**JNK-1**) signaling, increasing serine phosphorylation of insulin receptor substrate-1 (**IRS-1**) in the liver and preventing normal insulin-stimulated tyrosine phosphor‐ ylation of IRS-1 [41]. TG and FFAs derived from HFCS intake also induce insulin resistance in the liver as the FFAs precipitate in hepatocytes (lipid droplet accumulation), also stimulating excessive TLR4 signaling and further amplification of multiple inflammatory cytokine pathways. Dihydroxyacetone-phosphate and glyceraldehyde are also both directly hepato‐ toxic while the excessive accumulation of lipid droplets in the liver induces steatosis further amplifying inflammatory cytokine release. All of these processes are thought to contribute to progressive development of hepatic fibrosis, cirrhosis, and primary hepatic cancer. Elevated TGs and FFAs produced by the liver or which cannot be cleared from the portal vein by the liver accumulate in the peripheral circulation, exerting similar effects on the insulin receptor signaling in other target tissues such as adipose tissue, skeletal muscle, and the exocrine

Linking Obesity and Pancreatic Cancer http://dx.doi.org/10.5772/58546 87

With regard to pancreatic cancer, there is increasing evidence of a specific dose-dependent linkage between HFCS intake and its occurrence and this risk is independent of obesity or BMI [15]. Furthermore, fructose directly stimulates increased nucleic acid synthesis through the pentose phosphate pathway (catalyzed by transketolase) which is necessary for proliferation of malignant cells and consumption of HFCS is now linked both to oncogenesis as well as

High intake of processed meats containing heterocyclic amines and benzo (a) pyrines or have been prepared at high temperatures (fried or grilled) have been linked to pancreatic cancer [42] as have other foods containing aflatoxins [43] and other mutagens, however their link to

Inflammatory cytokines (adipokines) such as **TNF-α, IL-6, IL8, VEGF, and IFN-β** have been shown to be elevated in states of visceral obesity [16], as well as acute and chronic pancreatitis, and pancreatic cancer [11]. Visceral adipocytes/macrophages are major sources of the obesityassociated cytokines which are thought to promote insulin resistance [29] (see below) as well

pancreas [40].

tumor spread and metastasis [15].

pancreatic cancer are fairly weak at this time.

**a. Adipocyte-Derived Inflammatory Proteins:**

**5.2. Molecular pathways triggered by dietary constituents**

**c. Carcinogens in Foods:**

#### **5.1. Dietary contributions**

#### **a. High Fat Diets (HFDs) and Excess Free Fatty Acids (FFAs):**

Dietary fats (triglycerides, glycerol, and FFAs) are directly absorbed from the small intestine as chylomicrons into the thoracic duct into the subclavian vein and then into the general circulation. Chylomicrons are taken up by adipocytes and hepatocytes [31]. However, once the adipocyte storage capacity is exceeded, excess TG's and FFA's stimulate adipogenesis and are deposited ectopically into the liver where these excess fats accumulate in small vacuoles within hepatocytes which is the first stage of fatty liver disease (steatosis) [32, 33]. There is also increased *de novo* hepatic lipogenesis with consequent endogenous over-production of triglycerides (TGs) and free fatty acids (FFAs). Excess fats are also deposited in skeletal muscle and other insulin target tissues (even beta cells of pancreas) where they initiate acute inflam‐ matory processes (lipotoxicity) with the activation of multiple inflammatory cytokines [16]. Inflammatory cytokines in turn, directly contribute to the induction of insulin resistance through down regulation of the insulin receptor (IR) and post-receptor signaling pathways in insulin target tissues [33]. In the liver, the ectopic dietary fat also initiates an inflammatory response (steatohepatitis) which contributes to the development of non-alcoholic fatty liver disease (NAFLD) [33].

Within visceral fat cells themselves, FFAs (palmitate, etc.) directly induce the release of inflammatory cytokines [16] and also trigger the pathologic signaling of toll-like receptors (TLRs); activation of TLR4, in particular, increases additional inflammatory cytokine produc‐ tion, contributing to the initiation of insulin resistance [34] and adipogenesis, further increasing adipocyte mass, and the chronic inflammatory state now associated with obesity, T2DM, and oncogenesis.

#### **b. High Fructose Corn Syrup (HFCS):**

Fructose is a dietary carbohydrate normally derived from plant sources (tree and vine fruits, flowers, berries, and most root vegetables) which is much sweeter than glucose or sucrose. It is commonly used commercially in prepared foods due to its sweetness, effects on prepared food texture, and browning of baked foods. Commercially it is derived from sugar cane, sugar beets, and corn. HFCS is a mixture of glucose and fructose as monosaccharaides and as a food supplement it is now being vilified for its role in the obesity epidemic as well as induction of insulin resistance, T2DM and non-alcoholic fatty liver disease (NAFLD) [35-38]. NAFLD is now the leading cause of cirrhosis of the liver and primary hepatocellular cancer. Diets high in HFCS have also been linked directly to increased risk for pancreatic cancer [39]. Mechanisms by which HFCS induces insulin resistance are thought to be due to its unique metabolism in the liver via pathways identical to alcohol. Fructose binds to only one of the glucose trans‐ porters (GLUT 5) which is present only in enterocytes of the intestine and in the liver. Thus, although it is selectively concentrated in the liver, fructose cannot be utilized as a carbohydrate for energy in any other cell or organ of the body. Acutely, fructose ingestion results in the shunting of fructose-1-phosphate into dihydroxyacetone-phosphate and glyceraldehyde which enters the TCA cycle from pyruvate and citrate to excessively increase *de novo* hepatic lipogenesis and the over-production of TGs and FFAs [40]. Fructose-1-phosphate also directly induces janus kinase-1 (**JNK-1**) signaling, increasing serine phosphorylation of insulin receptor substrate-1 (**IRS-1**) in the liver and preventing normal insulin-stimulated tyrosine phosphor‐ ylation of IRS-1 [41]. TG and FFAs derived from HFCS intake also induce insulin resistance in the liver as the FFAs precipitate in hepatocytes (lipid droplet accumulation), also stimulating excessive TLR4 signaling and further amplification of multiple inflammatory cytokine pathways. Dihydroxyacetone-phosphate and glyceraldehyde are also both directly hepato‐ toxic while the excessive accumulation of lipid droplets in the liver induces steatosis further amplifying inflammatory cytokine release. All of these processes are thought to contribute to progressive development of hepatic fibrosis, cirrhosis, and primary hepatic cancer. Elevated TGs and FFAs produced by the liver or which cannot be cleared from the portal vein by the liver accumulate in the peripheral circulation, exerting similar effects on the insulin receptor signaling in other target tissues such as adipose tissue, skeletal muscle, and the exocrine pancreas [40].

With regard to pancreatic cancer, there is increasing evidence of a specific dose-dependent linkage between HFCS intake and its occurrence and this risk is independent of obesity or BMI [15]. Furthermore, fructose directly stimulates increased nucleic acid synthesis through the pentose phosphate pathway (catalyzed by transketolase) which is necessary for proliferation of malignant cells and consumption of HFCS is now linked both to oncogenesis as well as tumor spread and metastasis [15].

#### **c. Carcinogens in Foods:**

(leptin, adiponectin, resistin), inflammatory cytokines (TNF-α, IL-6, and IFN-β), components of complement, plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF) and other proteins such as monocyte chemoattractant protein (MCP-1). These adipose tissue-derived factors (Figure 1) are now thought to contribute dramatically to the induction of chronic inflammation which is expressed as insulin resistance [29], hyperinsulinemia, T2DM, hyperlipidemia, hypertension, and atherosclerosis [30], and also contributing to the oncogenesis of many solid tumors [11, 16]. Visceral obesity is the fat depot most closely associated with the production of these substances and the subsequent development of insulin

86 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Dietary fats (triglycerides, glycerol, and FFAs) are directly absorbed from the small intestine as chylomicrons into the thoracic duct into the subclavian vein and then into the general circulation. Chylomicrons are taken up by adipocytes and hepatocytes [31]. However, once the adipocyte storage capacity is exceeded, excess TG's and FFA's stimulate adipogenesis and are deposited ectopically into the liver where these excess fats accumulate in small vacuoles within hepatocytes which is the first stage of fatty liver disease (steatosis) [32, 33]. There is also increased *de novo* hepatic lipogenesis with consequent endogenous over-production of triglycerides (TGs) and free fatty acids (FFAs). Excess fats are also deposited in skeletal muscle and other insulin target tissues (even beta cells of pancreas) where they initiate acute inflam‐ matory processes (lipotoxicity) with the activation of multiple inflammatory cytokines [16]. Inflammatory cytokines in turn, directly contribute to the induction of insulin resistance through down regulation of the insulin receptor (IR) and post-receptor signaling pathways in insulin target tissues [33]. In the liver, the ectopic dietary fat also initiates an inflammatory response (steatohepatitis) which contributes to the development of non-alcoholic fatty liver

Within visceral fat cells themselves, FFAs (palmitate, etc.) directly induce the release of inflammatory cytokines [16] and also trigger the pathologic signaling of toll-like receptors (TLRs); activation of TLR4, in particular, increases additional inflammatory cytokine produc‐ tion, contributing to the initiation of insulin resistance [34] and adipogenesis, further increasing adipocyte mass, and the chronic inflammatory state now associated with obesity, T2DM, and

Fructose is a dietary carbohydrate normally derived from plant sources (tree and vine fruits, flowers, berries, and most root vegetables) which is much sweeter than glucose or sucrose. It is commonly used commercially in prepared foods due to its sweetness, effects on prepared food texture, and browning of baked foods. Commercially it is derived from sugar cane, sugar beets, and corn. HFCS is a mixture of glucose and fructose as monosaccharaides and as a food supplement it is now being vilified for its role in the obesity epidemic as well as induction of insulin resistance, T2DM and non-alcoholic fatty liver disease (NAFLD) [35-38]. NAFLD is

resistance, T2DM, and pancreatic cancer oncogenesis.

**a. High Fat Diets (HFDs) and Excess Free Fatty Acids (FFAs):**

**5.1. Dietary contributions**

disease (NAFLD) [33].

**b. High Fructose Corn Syrup (HFCS):**

oncogenesis.

High intake of processed meats containing heterocyclic amines and benzo (a) pyrines or have been prepared at high temperatures (fried or grilled) have been linked to pancreatic cancer [42] as have other foods containing aflatoxins [43] and other mutagens, however their link to pancreatic cancer are fairly weak at this time.

#### **5.2. Molecular pathways triggered by dietary constituents**

#### **a. Adipocyte-Derived Inflammatory Proteins:**

Inflammatory cytokines (adipokines) such as **TNF-α, IL-6, IL8, VEGF, and IFN-β** have been shown to be elevated in states of visceral obesity [16], as well as acute and chronic pancreatitis, and pancreatic cancer [11]. Visceral adipocytes/macrophages are major sources of the obesityassociated cytokines which are thought to promote insulin resistance [29] (see below) as well

cellular repair mechanisms), (3) inhibition of apoptosis (*Bcl-xL, Mcl-1*), (4) decreased cellular

Linking Obesity and Pancreatic Cancer http://dx.doi.org/10.5772/58546 89

**Leptin** is also secreted by adipocytes and plays a key role in regulating metabolism and appetite. Leptin is known as the satiety hormone however serum leptin levels are elevated in obesity due to central leptin receptor resistance (by mechanisms similar to insulin discussed below). Leptin has mitogenic actions in many cancer cell lines which appear to be via mitogenactivated-protein-kinase (**MAPK**) mediated pathways; however in certain pancreatic cancer

**Adiponectin** is exclusively secreted by adipocytes and has both anti-inflammatory and insulin-sensitizing effects. Known as the "good adipokine" serum levels of leptin are inversely related to BMI and levels are reduced obese patients and in many cancers. High levels of

**PAI-1** is a serine protease inhibitor produced by adipocytes and stromal cells in visceral fat, is associated with tumor cell invasion, metastasis, and angiogenesis of many malignancies, and over-expression of PAI-1 has been demonstrated in many obesity-related tumors suggesting it contributes to the spread of malignancies [50]. Interestingly, high expression of the plasmi‐ nogen activator inhibitor-2 (PAI-2) was a predictor of improved survival in patients with

**VEGF** is another adipocyte-derived polypeptide that has been implicated in cancer growth, shown to be over-expressed in many pancreatic cancers, and its expression in these tumors is

**b. Insulin Resistance, Hyperinsulinemia, and Increased Insulin/IGF-1 Receptor Signaling**

The FFA's and inflammatory cytokines produced by visceral obesity discussed earlier directly induce insulin resistance at the insulin receptor (IR) level [34, 54] resulting in compensatory beta cell insulin secretion (hyperinsulinemia) in an attempt to maintain euglycemia. The hyperinsulinemia becomes a self-perpetuating vicious cycle, in turn, as it directly contributes to insulin resistance by down-regulating its own receptor. Insulin resistance can originate anywhere in the insulin-action cascade; from a direct reduction in IR number or affinity, to reduced phosphorylation/activation of the insulin receptor itself, to down-regulation of the intracellular protein-kinase cascade normally triggered by insulin action following interaction with the IR (post-receptor signaling) [55]. Over-stimulation of the IR by hyperinsulinemia itself results in high levels of **STAT3** activation, which then up-regulates suppressors of cytokine signaling-3 (*socs-3*); which in turn, inhibits post-receptor insulin signaling as a negative "feedback" inhibitory mechanism, thereby down-regulating its own receptor system [56]. We have shown that excessive TLR4 signaling and inflammatory cytokine release up-regulates *socs-3* which contributes to insulin resistance [34]. Overall decreased insulin signaling then leads to decreased activation of GLUT4 transporters and decreased insulin-stimulated suppression of hepatic gluconeogenesis and glucose uptake into peripheral target tissues such as adipocytes and skeletal muscle which leads to the development of T2DM. Although IRmediated pathways associated with carbohydrate and fat metabolism are down-regulated,

cell lines it inhibits growth [47] so its role in this cancer is unclear at present [48, 49].

adiponectin are inversely related to the incidence of pancreatic cancer [49].

pancreatic adenocarcinoma [51].

linked to poorer survival [52, 53].

**Pathways**

adhesion, and/or (5) stimulation of angiogenesis (VEGF) [46].

**Figure 1.** The role of dysfunctional adipose tissue in obesity. Dysfunctional adipose tissue is a critical source of mole‐ cules that mediate inflammation, cancer, insulin resistance and angiogenesis. PAI-1 (plasminogen activator inhibi‐ tor-1); FFAs (free fatty acids); IGF-1 (insulin-like growth factor 1); VEGF (vascular endothelial growth factor); IL-6 (interleukin 6); TNF-α (tumor necrosis factor alpha); TLR4 (toll-like receptor 4).

as directly contribute to oncogenesis via several pathways [16] including other growth factor receptors, cytokine receptors, or non-receptor tyrosine kinases. Each of these pathways can increase Janus kinase (JAK)/signal transduction and activator of transcription Signal Trans‐ ducer and Activator of Transcription (STATs) of which **STAT3** [44, 45] is directly linked to cancer of the pancreas. Both of these pathways can stimulate cellular proliferation—transfor‐ mation through (1) up-regulation of genes encoding cell cycle regulators (cyclins D1/D2, c-Myc), (2) increasing the probability of mutation, (e.g., cellular proto-oncogenes, DNA, and cellular repair mechanisms), (3) inhibition of apoptosis (*Bcl-xL, Mcl-1*), (4) decreased cellular adhesion, and/or (5) stimulation of angiogenesis (VEGF) [46].

**Leptin** is also secreted by adipocytes and plays a key role in regulating metabolism and appetite. Leptin is known as the satiety hormone however serum leptin levels are elevated in obesity due to central leptin receptor resistance (by mechanisms similar to insulin discussed below). Leptin has mitogenic actions in many cancer cell lines which appear to be via mitogenactivated-protein-kinase (**MAPK**) mediated pathways; however in certain pancreatic cancer cell lines it inhibits growth [47] so its role in this cancer is unclear at present [48, 49].

**Adiponectin** is exclusively secreted by adipocytes and has both anti-inflammatory and insulin-sensitizing effects. Known as the "good adipokine" serum levels of leptin are inversely related to BMI and levels are reduced obese patients and in many cancers. High levels of adiponectin are inversely related to the incidence of pancreatic cancer [49].

**PAI-1** is a serine protease inhibitor produced by adipocytes and stromal cells in visceral fat, is associated with tumor cell invasion, metastasis, and angiogenesis of many malignancies, and over-expression of PAI-1 has been demonstrated in many obesity-related tumors suggesting it contributes to the spread of malignancies [50]. Interestingly, high expression of the plasmi‐ nogen activator inhibitor-2 (PAI-2) was a predictor of improved survival in patients with pancreatic adenocarcinoma [51].

**VEGF** is another adipocyte-derived polypeptide that has been implicated in cancer growth, shown to be over-expressed in many pancreatic cancers, and its expression in these tumors is linked to poorer survival [52, 53].

#### **b. Insulin Resistance, Hyperinsulinemia, and Increased Insulin/IGF-1 Receptor Signaling Pathways**

The FFA's and inflammatory cytokines produced by visceral obesity discussed earlier directly induce insulin resistance at the insulin receptor (IR) level [34, 54] resulting in compensatory beta cell insulin secretion (hyperinsulinemia) in an attempt to maintain euglycemia. The hyperinsulinemia becomes a self-perpetuating vicious cycle, in turn, as it directly contributes to insulin resistance by down-regulating its own receptor. Insulin resistance can originate anywhere in the insulin-action cascade; from a direct reduction in IR number or affinity, to reduced phosphorylation/activation of the insulin receptor itself, to down-regulation of the intracellular protein-kinase cascade normally triggered by insulin action following interaction with the IR (post-receptor signaling) [55]. Over-stimulation of the IR by hyperinsulinemia itself results in high levels of **STAT3** activation, which then up-regulates suppressors of cytokine signaling-3 (*socs-3*); which in turn, inhibits post-receptor insulin signaling as a negative "feedback" inhibitory mechanism, thereby down-regulating its own receptor system [56]. We have shown that excessive TLR4 signaling and inflammatory cytokine release up-regulates *socs-3* which contributes to insulin resistance [34]. Overall decreased insulin signaling then leads to decreased activation of GLUT4 transporters and decreased insulin-stimulated suppression of hepatic gluconeogenesis and glucose uptake into peripheral target tissues such as adipocytes and skeletal muscle which leads to the development of T2DM. Although IRmediated pathways associated with carbohydrate and fat metabolism are down-regulated,

as directly contribute to oncogenesis via several pathways [16] including other growth factor receptors, cytokine receptors, or non-receptor tyrosine kinases. Each of these pathways can increase Janus kinase (JAK)/signal transduction and activator of transcription Signal Trans‐ ducer and Activator of Transcription (STATs) of which **STAT3** [44, 45] is directly linked to cancer of the pancreas. Both of these pathways can stimulate cellular proliferation—transfor‐ mation through (1) up-regulation of genes encoding cell cycle regulators (cyclins D1/D2, c-Myc), (2) increasing the probability of mutation, (e.g., cellular proto-oncogenes, DNA, and

(interleukin 6); TNF-α (tumor necrosis factor alpha); TLR4 (toll-like receptor 4).

**Figure 1.** The role of dysfunctional adipose tissue in obesity. Dysfunctional adipose tissue is a critical source of mole‐ cules that mediate inflammation, cancer, insulin resistance and angiogenesis. PAI-1 (plasminogen activator inhibi‐ tor-1); FFAs (free fatty acids); IGF-1 (insulin-like growth factor 1); VEGF (vascular endothelial growth factor); IL-6

88 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

other signaling pathways are not suppressed but rather continuously stimulated by insulin resulting in activation of the Ras/Raf/mitogen-activated-protein-kinase (**MAPK**) system and **mTOR** pathways which are known to promote abnormal cell growth and proliferation [57, 58]. Thus, in states of obesity and FFA/TLR4/cytokine-mediated insulin resistance, the principle functions of insulin action via the IR (glucose transport and suppression of gluco‐ neogenesis) are impaired while insulin-stimulated abnormal cell growth and proliferation in target tissues continues [58]. Secondly, hyperinsulinemia induces the synthesis of insulin-like growth factor-1 (**IGF-1**) in liver and the high serum levels of free IGF-1 also results in overstimulation of its own receptor (IGF-1R). Excess IGF-1R signaling also stimulates abnormal cell proliferation through the same downstream signaling networks which are being chroni‐ cally stimulated by insulin; including the phosphatidylinositol 3-kinase **(PI3-K)-AKT** system [58]. Thus obesity induced insulin resistance results in excess insulin and IGF-1 promotion of abnormal cell growth and proliferation in multiple organ systems. Expression of IGF-1 receptors has also been demonstrated in multiple malignant tumors including pancreatic cancer, and IGF-1 contributes to cell migration and invasion in some human pancreatic carcinomas.

adipocytes which stimulates adipocyte differentiation, high fat diet (HFD)-mediated induction of visceral obesity, TLR4-mediated cytokine signaling, insulin resistance, and glucose intoler‐ ance [34, 65]. This in turn stimulates insulin/IGF-1 signaling pathways which also promote tumor growth. Fructose also stimulates abnormal TLR4 signaling [36] and as mentioned earlier, HFCS diets are associated with induction of visceral obesity, T2DM, chronic pancrea‐ titis, and cancer of the pancreas as well. Since both FFA's and fructose are potent ligands for TLR4 and both are present in high concentrations in the diets of developed countries it is logical that they could promote pancreatic oncogenesis via TLR mediated pathways to be described. Finally, as just mentioned hyperglycemia in the form of glucose intolerance and overt T2DM also stimulates abnormal TLR4 signaling [66] as well as EGFR transactivation in pancreatic tissue in a glucose dependent manner thus also serving as a ligand to promote tumor growth

Linking Obesity and Pancreatic Cancer http://dx.doi.org/10.5772/58546 91

Chronic inflammation has been shown to be an important risk factor for the onset and progression of multiple cancers, including pancreatic cancer [67-72] [72-75]. Chronic inflam‐ mation is thought to induce malignant transformation via activation of oncogenes, induction of immunosuppression, and inhibition of tumor suppressor genes and lymphocytes. Patho‐ logic activation of TLRs play a critical role in the inflammatory response induced by high fat diets and HFCS by inducing the production of multiple pro-inflammatory cytokines and they have been shown to be important for the induction, proliferation, survival, metastasis, and escape from immune surveillance of many of these cancers as well [70, 76]. Some of the most important TLR-induced cytokines implicated in cancer include **TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-23.** Proinflammatory cytokine production then leads to the activation of many tumor promoting transcription factors and anti-apoptotic genes. Nuclear factor kappa beta **(NF-κB**) and **signal** transducer and activator of transcription 3 (**STAT3**) are two of the most well studied

**7. Pathologic toll-like receptor signaling, pancreatic cancer growth, and**

We have previously described the relationship between obesity and pancreatic cancer risk as well as the direct correlation between increasing BMI and hyperglycemia to lower responses to treatment and over-all worse outcomes in this all too common disease. Obesity-induced TLR activation of NF-κB and STAT3 signaling pathways are major mediators of this process in multiple cancers including pancreatic cancer. NF-κB and STAT3 are activated by a variety of similar stimuli (stressors, cytokines, etc.) and both control expression of proliferationenhancing, anti-apoptotic, angiogenic, and immune-modulating genes; however they are regulated by entirely different signaling mechanisms. NF-κB's pro-inflammatory cytokine receptors such as; **TNF-α** and **IL-1** [77-80] promote not only tumor transformation, but also proliferation, angiogenesis, invasion, metastasis, and chemo/radio resistance [81-89]. STAT3 activation by TLR-mediated cytokines also activates the **IL-6 family** (IL-6, IL-11, IL-27, etc.), **IL-10 family** (IL-10, IL-22, IL-19, IL-20), and the epidermal growth factor (**EGF**) family (VEGF, IL-21, IL-23, HGF) of growth factors which also stimulate tumor transformation, growth and

and spread.

oncogenic transcription factors.

**resistance to therapy**

#### **c. Hyperglycemia Induces Pancreatic Cancer Epidermal Growth Factor Expression**

As we have previously discussed in this chapter, diabetes is associated with an increased risk of pancreatic cancer by a variety of cytokine and hormone receptor signaling pathways and that large numbers of patients with pancreatic cancer develop diabetes and elevated glucoses. The direct effect of hyperglycemia on oncogenesis, pancreatic cancer growth and spread is of interest as well. Epidemiologic studies have demonstrated that glucose control in patients with pancreatic cancer results in improved survival, suggesting that high glucose levels might directly promote tumor growth and progression [59]. Recent *in vitro* cell culture studies have demonstrated that glucose in a dose-dependent manner promotes different pancreatic cancer cell line growth and perineural invasion through the regulation of expression of glial cell linederived neurotropic factor (GDNF) and epidermal growth factor (EGF) via increased epider‐ mal growth factor receptor (EGFR) transactivation [60]. These observations support intensive glucose control as a potential target for improving patient survival in pancreatic cancer.

#### **6. Obesity, toll-like receptors, and pancreatic oncogenesis**

Toll-Like Receptors (**TLRs**) are pathogen recognition receptors (PRRs) critical for the activation of the innate and adaptive immune responses to foreign pathogens. Functional TLRs are not only expressed in immune cells but also in many non-immune cells [61]. Their activation, signaling, and proinflammatory responses have been shown to be mediators of multiple inflammatory and autoimmune diseases, as well as, contribute to oncogenesis, tumor growth and metastasis. Pathologic signaling of multiple TLRs have been implicated in many cancers including; melanoma, breast, prostate cancer, colorectal, lung, cervical, liver, and pancreatic cancer [62-64]. Obesity and T2DM are associated with an increased risk for many of these same malignancies; especially pancreatic cancer. FFA's are capable of activating TLR4 signaling in adipocytes which stimulates adipocyte differentiation, high fat diet (HFD)-mediated induction of visceral obesity, TLR4-mediated cytokine signaling, insulin resistance, and glucose intoler‐ ance [34, 65]. This in turn stimulates insulin/IGF-1 signaling pathways which also promote tumor growth. Fructose also stimulates abnormal TLR4 signaling [36] and as mentioned earlier, HFCS diets are associated with induction of visceral obesity, T2DM, chronic pancrea‐ titis, and cancer of the pancreas as well. Since both FFA's and fructose are potent ligands for TLR4 and both are present in high concentrations in the diets of developed countries it is logical that they could promote pancreatic oncogenesis via TLR mediated pathways to be described. Finally, as just mentioned hyperglycemia in the form of glucose intolerance and overt T2DM also stimulates abnormal TLR4 signaling [66] as well as EGFR transactivation in pancreatic tissue in a glucose dependent manner thus also serving as a ligand to promote tumor growth and spread.

other signaling pathways are not suppressed but rather continuously stimulated by insulin resulting in activation of the Ras/Raf/mitogen-activated-protein-kinase (**MAPK**) system and **mTOR** pathways which are known to promote abnormal cell growth and proliferation [57, 58]. Thus, in states of obesity and FFA/TLR4/cytokine-mediated insulin resistance, the principle functions of insulin action via the IR (glucose transport and suppression of gluco‐ neogenesis) are impaired while insulin-stimulated abnormal cell growth and proliferation in target tissues continues [58]. Secondly, hyperinsulinemia induces the synthesis of insulin-like growth factor-1 (**IGF-1**) in liver and the high serum levels of free IGF-1 also results in overstimulation of its own receptor (IGF-1R). Excess IGF-1R signaling also stimulates abnormal cell proliferation through the same downstream signaling networks which are being chroni‐ cally stimulated by insulin; including the phosphatidylinositol 3-kinase **(PI3-K)-AKT** system [58]. Thus obesity induced insulin resistance results in excess insulin and IGF-1 promotion of abnormal cell growth and proliferation in multiple organ systems. Expression of IGF-1 receptors has also been demonstrated in multiple malignant tumors including pancreatic cancer, and IGF-1 contributes to cell migration and invasion in some human pancreatic

90 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

**c. Hyperglycemia Induces Pancreatic Cancer Epidermal Growth Factor Expression**

**6. Obesity, toll-like receptors, and pancreatic oncogenesis**

As we have previously discussed in this chapter, diabetes is associated with an increased risk of pancreatic cancer by a variety of cytokine and hormone receptor signaling pathways and that large numbers of patients with pancreatic cancer develop diabetes and elevated glucoses. The direct effect of hyperglycemia on oncogenesis, pancreatic cancer growth and spread is of interest as well. Epidemiologic studies have demonstrated that glucose control in patients with pancreatic cancer results in improved survival, suggesting that high glucose levels might directly promote tumor growth and progression [59]. Recent *in vitro* cell culture studies have demonstrated that glucose in a dose-dependent manner promotes different pancreatic cancer cell line growth and perineural invasion through the regulation of expression of glial cell linederived neurotropic factor (GDNF) and epidermal growth factor (EGF) via increased epider‐ mal growth factor receptor (EGFR) transactivation [60]. These observations support intensive glucose control as a potential target for improving patient survival in pancreatic cancer.

Toll-Like Receptors (**TLRs**) are pathogen recognition receptors (PRRs) critical for the activation of the innate and adaptive immune responses to foreign pathogens. Functional TLRs are not only expressed in immune cells but also in many non-immune cells [61]. Their activation, signaling, and proinflammatory responses have been shown to be mediators of multiple inflammatory and autoimmune diseases, as well as, contribute to oncogenesis, tumor growth and metastasis. Pathologic signaling of multiple TLRs have been implicated in many cancers including; melanoma, breast, prostate cancer, colorectal, lung, cervical, liver, and pancreatic cancer [62-64]. Obesity and T2DM are associated with an increased risk for many of these same malignancies; especially pancreatic cancer. FFA's are capable of activating TLR4 signaling in

carcinomas.

Chronic inflammation has been shown to be an important risk factor for the onset and progression of multiple cancers, including pancreatic cancer [67-72] [72-75]. Chronic inflam‐ mation is thought to induce malignant transformation via activation of oncogenes, induction of immunosuppression, and inhibition of tumor suppressor genes and lymphocytes. Patho‐ logic activation of TLRs play a critical role in the inflammatory response induced by high fat diets and HFCS by inducing the production of multiple pro-inflammatory cytokines and they have been shown to be important for the induction, proliferation, survival, metastasis, and escape from immune surveillance of many of these cancers as well [70, 76]. Some of the most important TLR-induced cytokines implicated in cancer include **TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-23.** Proinflammatory cytokine production then leads to the activation of many tumor promoting transcription factors and anti-apoptotic genes. Nuclear factor kappa beta **(NF-κB**) and **signal** transducer and activator of transcription 3 (**STAT3**) are two of the most well studied oncogenic transcription factors.

## **7. Pathologic toll-like receptor signaling, pancreatic cancer growth, and resistance to therapy**

We have previously described the relationship between obesity and pancreatic cancer risk as well as the direct correlation between increasing BMI and hyperglycemia to lower responses to treatment and over-all worse outcomes in this all too common disease. Obesity-induced TLR activation of NF-κB and STAT3 signaling pathways are major mediators of this process in multiple cancers including pancreatic cancer. NF-κB and STAT3 are activated by a variety of similar stimuli (stressors, cytokines, etc.) and both control expression of proliferationenhancing, anti-apoptotic, angiogenic, and immune-modulating genes; however they are regulated by entirely different signaling mechanisms. NF-κB's pro-inflammatory cytokine receptors such as; **TNF-α** and **IL-1** [77-80] promote not only tumor transformation, but also proliferation, angiogenesis, invasion, metastasis, and chemo/radio resistance [81-89]. STAT3 activation by TLR-mediated cytokines also activates the **IL-6 family** (IL-6, IL-11, IL-27, etc.), **IL-10 family** (IL-10, IL-22, IL-19, IL-20), and the epidermal growth factor (**EGF**) family (VEGF, IL-21, IL-23, HGF) of growth factors which also stimulate tumor transformation, growth and resistance to therapy. NF-κB and STAT3 activate anti-apoptotic genes such as Bcl-xL, Bcl-2, and c-IAP2 [90-92] and also interact and mediate crosstalk between tumor cells and inflam‐ matory cells within the tumor microenvironment to promote the development and progression of multiple types of human cancers including but not limited to pancreatic, colon, gastric, skin, head and neck, and liver cancers [44, 90, 93-96]. Finally, Wnt5a a member of the Wnt family has also been implicated in carcinogenesis and inflammation. Non-canonical Wnt5a activates β-catenin-independent pathways important for cell migration and polarity. Wnt5a has been found in tissue samples of pancreatic adenocarcinomas [97] and is highly expressed in advanced pancreatic cancer [98]. Recently, a TLR / IL-6 / STAT3 / Wnt5a signaling loop was described [62, 99].

metastasis via TLR signaling. TLR antagonists might also decrease the level of activation of stromal cells such as tumor-associated macrophages. Macrophages express an array of TLRs and are able to produce several growth factors via TLR signaling [107]. Moreover, abrogation of TLR-4 signaling in tumor-associated macrophages decrease tumor growth *in vivo* [108]. Our group demonstrated that in papillary thyroid carcinoma cells, IL-6, a TLR3 signaling product, activates STAT3, results in overexpression of **Wnt5a** which mediates tumor growth and spread [62]. Further, we demonstrated that phenylmethimazole (C10), a small molecule derivative of methimazole, blocked TLR3 signaling, and subsequent IL-6 production, STAT3 activation, Wnt5a overexpression, and subsequent growth and migration of papillary thyroid carcinoma cells [62]. Toll-like receptors were first implicated in the pathogenesis of pancreatic cancer in 2009. Our laboratory demonstrated that TLR3 and Wnt5a were coordinately consti‐ tutively expressed in a human pancreatic cell line (PANC-1), activation of signaling also played a key role in the regulation of pancreatic cancer growth and migration and that C10, inhibited its growth and migration both in vitro and *in vivo* [63]. Another study reported that activation of the TLR4 signaling pathway-increased invasiveness of pancreatic cancer cells while blockade of TLR4 signaling decreased invasive ability [109]. These studies were the first to implicate both TLR3 or TLR4 expression and signaling as playing a role in pancreatic tumor growth and migration and demonstrated that inhibition of TLR signaling pathways were potential therapeutic targets. Gemcitabine is currently the standard of care chemotherapeutic for pancreatic cancer; however, its efficacy is diminished due to toxicity and the chemoresist‐ ance of the tumors. Recently, another group combined TLR4/NF-κB antagonist with gemcita‐ bine in an orthotopic model of pancreatic cancer and the combination therapy significantly delayed tumor growth and decreased tumor size compared to gemcitabine alone or the control groups. Thus, TLR antagonists, when combined with other chemotherapeutic agents may prove to be effective adjunctive therapies to suppress the inflammatory cytokine/growth factor microenvironment which contributes to the induction and/or support of tumor growth and

Linking Obesity and Pancreatic Cancer http://dx.doi.org/10.5772/58546 93

progression and reduce the dose/toxicity of established agents.

**9. Prevention of obesity associated pancreatic cancer**

growth and response to treatment.

There is now compelling evidence that obesity, chronic inflammation, and the associated secretion of numerous inflammatory cytokines, hormones and growth factors described herein contribute both directly and indirectly to the increased risk for pancreatic cancer, more aggressive tumor growth, as well as poor response to therapeutic intervention. Thus, in addition to smoking cessation and moderation in alcohol consumption, life-style modification with exercise, maintenance of normal BMI's, consumption of higher amounts of fresh fruits and vegetables, less animal fat and processed foods; especially those fortified with HFCS are obvious recommendations. In addition, there is increasing evidence that other anti-inflamma‐ tory agents such as the non-steroidal anti-inflammatory drugs (NSAIDS) [110], the Statin lipidlowering medications, and T2DM medications such as the thiazolidinediones (TZD's) [111] and metformin [112, 113] have specific protective effects against oncogenesis as well as tumor

## **8. TLRs as a potential therapeutic target**

Several recent studies have evaluated the potential therapeutic use of TLR activators and inhibitors in multiple cancer models. The theory for activation of TLR signaling pathways in a tumor environment is that it would possibly induce tumor cell apoptosis or inhibit the production of various factors described in this review that control tumor growth. In addition, it induction of TLR signaling could elicit an antitumor immune response that could lead to tumor cell destruction by the host's immune system. Treatment with TLR agonists have shown to induce an antitumor response by enhancing dendritic cell (DC) vaccination or T cell adoptive therapies. A recent study reported that the use of TLR agonists such as poly(I:C) or CpG combined with adoptive transfer immunotherapy directly to a B16F10 melanoma model inhibited tumor growth [100]. Also, in a mouse breast xenograft model, the antitumor effect of the TLR3 activator was shown to be dependent on the expression of TLR3 expression in tumor cells. This was further validated in humans where treatment with dsRNA improved outcomes in patients harboring TLR3-positive breast tumors [101]. Similarly, CpG treatment via TLR9 activation induced tumor cell death in human neuroblastoma cells, and tumortargeted delivery of this TLR9 agonist increased survival in a xenograft model of neuroblas‐ toma [102].

In contrast, it has also been shown that TLR agonists can promote cancer cell survival and migration, and tumor progression. For example, TLR agonists have been shown to increase tumor viability and metastasis of human lung cancer (TLR7/8) [103] ; proliferation of human myeloma (TLR3) [104] ; adhesion and metastasis of human colorectal cancer (TLR4) [105] ; and migration of human glioblastoma (TLR4) or human breast cancer (TLR2) [106]. In regards to pancreatic cancer, TLR7 was recently reported not only be highly expressed in mouse and human pancreatic cancers, but ligation of TLR7 led to accelerated tumor progression through the STAT3 growth pathways previously discussed. Thus, there appears to be a double edged sword between reducing or promoting tumor growth using agonists based therapies for different TLRs.

On the other hand, the use of TLR antagonists has shown to be beneficial at inhibiting tumor growth in animal models in which the tumor microenvironment promotes survival and metastasis via TLR signaling. TLR antagonists might also decrease the level of activation of stromal cells such as tumor-associated macrophages. Macrophages express an array of TLRs and are able to produce several growth factors via TLR signaling [107]. Moreover, abrogation of TLR-4 signaling in tumor-associated macrophages decrease tumor growth *in vivo* [108].

resistance to therapy. NF-κB and STAT3 activate anti-apoptotic genes such as Bcl-xL, Bcl-2, and c-IAP2 [90-92] and also interact and mediate crosstalk between tumor cells and inflam‐ matory cells within the tumor microenvironment to promote the development and progression of multiple types of human cancers including but not limited to pancreatic, colon, gastric, skin, head and neck, and liver cancers [44, 90, 93-96]. Finally, Wnt5a a member of the Wnt family has also been implicated in carcinogenesis and inflammation. Non-canonical Wnt5a activates β-catenin-independent pathways important for cell migration and polarity. Wnt5a has been found in tissue samples of pancreatic adenocarcinomas [97] and is highly expressed in advanced pancreatic cancer [98]. Recently, a TLR / IL-6 / STAT3 / Wnt5a signaling loop was

92 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment

Several recent studies have evaluated the potential therapeutic use of TLR activators and inhibitors in multiple cancer models. The theory for activation of TLR signaling pathways in a tumor environment is that it would possibly induce tumor cell apoptosis or inhibit the production of various factors described in this review that control tumor growth. In addition, it induction of TLR signaling could elicit an antitumor immune response that could lead to tumor cell destruction by the host's immune system. Treatment with TLR agonists have shown to induce an antitumor response by enhancing dendritic cell (DC) vaccination or T cell adoptive therapies. A recent study reported that the use of TLR agonists such as poly(I:C) or CpG combined with adoptive transfer immunotherapy directly to a B16F10 melanoma model inhibited tumor growth [100]. Also, in a mouse breast xenograft model, the antitumor effect of the TLR3 activator was shown to be dependent on the expression of TLR3 expression in tumor cells. This was further validated in humans where treatment with dsRNA improved outcomes in patients harboring TLR3-positive breast tumors [101]. Similarly, CpG treatment via TLR9 activation induced tumor cell death in human neuroblastoma cells, and tumortargeted delivery of this TLR9 agonist increased survival in a xenograft model of neuroblas‐

In contrast, it has also been shown that TLR agonists can promote cancer cell survival and migration, and tumor progression. For example, TLR agonists have been shown to increase tumor viability and metastasis of human lung cancer (TLR7/8) [103] ; proliferation of human myeloma (TLR3) [104] ; adhesion and metastasis of human colorectal cancer (TLR4) [105] ; and migration of human glioblastoma (TLR4) or human breast cancer (TLR2) [106]. In regards to pancreatic cancer, TLR7 was recently reported not only be highly expressed in mouse and human pancreatic cancers, but ligation of TLR7 led to accelerated tumor progression through the STAT3 growth pathways previously discussed. Thus, there appears to be a double edged sword between reducing or promoting tumor growth using agonists based therapies for

On the other hand, the use of TLR antagonists has shown to be beneficial at inhibiting tumor growth in animal models in which the tumor microenvironment promotes survival and

described [62, 99].

toma [102].

different TLRs.

**8. TLRs as a potential therapeutic target**

Our group demonstrated that in papillary thyroid carcinoma cells, IL-6, a TLR3 signaling product, activates STAT3, results in overexpression of **Wnt5a** which mediates tumor growth and spread [62]. Further, we demonstrated that phenylmethimazole (C10), a small molecule derivative of methimazole, blocked TLR3 signaling, and subsequent IL-6 production, STAT3 activation, Wnt5a overexpression, and subsequent growth and migration of papillary thyroid carcinoma cells [62]. Toll-like receptors were first implicated in the pathogenesis of pancreatic cancer in 2009. Our laboratory demonstrated that TLR3 and Wnt5a were coordinately consti‐ tutively expressed in a human pancreatic cell line (PANC-1), activation of signaling also played a key role in the regulation of pancreatic cancer growth and migration and that C10, inhibited its growth and migration both in vitro and *in vivo* [63]. Another study reported that activation of the TLR4 signaling pathway-increased invasiveness of pancreatic cancer cells while blockade of TLR4 signaling decreased invasive ability [109]. These studies were the first to implicate both TLR3 or TLR4 expression and signaling as playing a role in pancreatic tumor growth and migration and demonstrated that inhibition of TLR signaling pathways were potential therapeutic targets. Gemcitabine is currently the standard of care chemotherapeutic for pancreatic cancer; however, its efficacy is diminished due to toxicity and the chemoresist‐ ance of the tumors. Recently, another group combined TLR4/NF-κB antagonist with gemcita‐ bine in an orthotopic model of pancreatic cancer and the combination therapy significantly delayed tumor growth and decreased tumor size compared to gemcitabine alone or the control groups. Thus, TLR antagonists, when combined with other chemotherapeutic agents may prove to be effective adjunctive therapies to suppress the inflammatory cytokine/growth factor microenvironment which contributes to the induction and/or support of tumor growth and progression and reduce the dose/toxicity of established agents.

## **9. Prevention of obesity associated pancreatic cancer**

There is now compelling evidence that obesity, chronic inflammation, and the associated secretion of numerous inflammatory cytokines, hormones and growth factors described herein contribute both directly and indirectly to the increased risk for pancreatic cancer, more aggressive tumor growth, as well as poor response to therapeutic intervention. Thus, in addition to smoking cessation and moderation in alcohol consumption, life-style modification with exercise, maintenance of normal BMI's, consumption of higher amounts of fresh fruits and vegetables, less animal fat and processed foods; especially those fortified with HFCS are obvious recommendations. In addition, there is increasing evidence that other anti-inflamma‐ tory agents such as the non-steroidal anti-inflammatory drugs (NSAIDS) [110], the Statin lipidlowering medications, and T2DM medications such as the thiazolidinediones (TZD's) [111] and metformin [112, 113] have specific protective effects against oncogenesis as well as tumor growth and response to treatment.

#### **10. Conclusion**

Obesity contributes to increased risk for multiple solid cancers including pancreatic cancer. For pancreatic cancer in particular, obesity promotes a proinflammatory environment which promotes oncogenesis, tumor growth, metastatic spread as well as resistance to therapy through a variety of molecular pathways. The principle obesity-linked pathways include increases in TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-23 as well as activation of NF-κB and STAT3. The current diets of industrialized nations which contain too much low glycemic-index carbohydrates, saturated fats, and HFCS are major environmental triggers of pathologic TLR3 and TLR4 signaling pathways in adipocytes which then contribute to the development of insulin resistance, ectopic fat deposition in multiple tissues including the pancreas which in turn amplify the growth and signaling pathways described herein which lead to oncogenesis and tumor spread.

[6] Lowenfels AB, Maisunneuve, P., DiMagno, E.P., Elitser, Y., Gates, L. K., Perralt, J., Whticomb, D. C. Heriditary Pancreatitis and risk for Pancreatic Cancer. J Nat Cancer

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#### **Author details**

Kelly McCall, Anthony L Schwartz and Frank L Schwartz\*

\*Address all correspondence to: schwartf@ohio.edu

Ohio University Heritage College of Osteopathic Medicine, Ohio, USA

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**10. Conclusion**

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\*Address all correspondence to: schwartf@ohio.edu

Obesity contributes to increased risk for multiple solid cancers including pancreatic cancer. For pancreatic cancer in particular, obesity promotes a proinflammatory environment which promotes oncogenesis, tumor growth, metastatic spread as well as resistance to therapy through a variety of molecular pathways. The principle obesity-linked pathways include increases in TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-23 as well as activation of NF-κB and STAT3. The current diets of industrialized nations which contain too much low glycemic-index carbohydrates, saturated fats, and HFCS are major environmental triggers of pathologic TLR3 and TLR4 signaling pathways in adipocytes which then contribute to the development of insulin resistance, ectopic fat deposition in multiple tissues including the pancreas which in turn amplify the growth and signaling pathways described herein which lead to oncogenesis

94 Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment


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**Section 2**

**Novel Approaches to Earlier Detection**


**Novel Approaches to Earlier Detection**

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**Chapter 5**

**Endoscopic Ultrasound in**

Andrada Seicean

**1. Introduction**

influence on survival [2].

with a mean of 66 days until diagnosis [4].

**2. Detection**

EUS vs CT

http://dx.doi.org/10.5772/57182

**Pancreatic Cancer: The New Perspective**

Pancreatic cancer is one of the most deadly forms of cancer worldwide, with median survival of less than 6 months and a 5-year survival rate of 35%. Endoscopic ultrasound (EUS) was first introduced for assessment of pancreatic pathology more than 30 years ago, as transabdominal imaging yields limited information. EUS has a role in the detection, staging and sampling of pancreatic tumor. Curative-intent surgery, chemotherapy, and radiation therapy of pancreatic cancer are all performed more frequently in patients with EUS evaluation [1]. Palliative EUSguided treatments are also possible. However, a recent large observational study reported no

The detection rate for pancreatic tumors by EUS is 90-100%, with good detection for tumors less than 2 cm in diameter, but EUS does not definitively rule out the presence of malignancy. In certain situations EUS may give false-negative results, especially when there is concomitant chronic pancreatitis, if the examination is performed too soon after an acute episode of acute pancreatitis, or in the presence of diffusely infiltrating carcinoma or a prominent ventral/dorsal split [3]. For patients with false-negative endoscopic ultrasound fine-needle aspiration (EUS-FNA), the risk for malignancy is higher when vascular involvement or lymph nodes are seen,

Two studies showed that the detection of small pancreatic tumors (diameter less than 3 cm) by EUS is better than by CT or MRI (accuracy 93% vs 53% vs 67%) [5] or than by CT or US

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

**Chapter 5**
