**4. Mechanisms of chemoresistance in PC**

Cancer chemoresistance is a current PC challenge. Intrinsic chemoresistance occurs when chemotherapy is ineffective from the start of treatment, whereas acquired chemoresistance develops only after exposure to anticancer drugs. Although PC cells are more susceptible to Gemcitabine when compared with other anticancer agents, most patients develop resistance within weeks of treatment initiation, leading to poor survival [2]. Mechanisms of cancer chemoresistance include drug modification, reduction or inhibition of drug-induced apoptosis, overexpression of drug efflux proteins, increased expression of survival factors and deregulation of pathways, such as Notch, and expansion of cancer stem cells (CSCs), among others [43].

#### **4.1. Pancreatic cancer stem cells (PCSCs)**

The hierarchical model of cancer states that tumors arise from CSC or cancer-initiating cells that can reproduce all tumor cell types. CSCs have common characteristics associated to normal stem cells. CSCs are tumorigenic, show self-renewal capabilities, and can be differentiated into multiple cancer cell types. CSCs hide in the tumor niche causing relapse and metastasis. The tumor niche is composed of stromal and inflammatory cells, cytokines, ECM, and vasculature. It provides signals helping CSCs to maintain their undifferentiated state. The accumulation of ECM destroys the normal PC architecture and enhances the expression of PCSC markers [44].

PCSCs express various markers, including CD24+CD44+, CD133+, CD24+CD44+ESA+, ALDH+, or c-Met+. Metastatic PCSCs express CXCR4+CD133+. PCSC markers CD133 and CD44 correlated to CXCR1 expression. PCSC could be identified using Hoechst 33342 dye by flow cytometry. Hoechst-negative cells were called "side population" and were linked to chemoresistance [45]. ALDHs are a class of enzymes that oxidize aldehydes. ALDH + PCSC show clonogenic and metastatic potential that affects survival in PC. Positive PC cells for PCSC markers form tumors in mice, in contrast to negative PC cells. ALDH1 mediates resistance to Cyclophosphamide and Gemcitabine in PC. TGF-β negatively regulates ALDH1 in PC in a SMAD-dependent manner. That can be disrupted by SMAD4 mutations and deletions. Therefore, targeting PCSC could induce sensitization of PC to chemotherapeutic treatment [46].

Chemotherapeutic agents target the bulk of the tumor but unfortunately allow the proliferation of CSC that exhibits chemoresistance. Gemcitabine kills tumor cells but increases PCSC (CD24+ and CD133+) that expresses stemness-associated genes, such as Bmi1, Sox2, and Nanog. PCSC expansion increased cell migration, chemoresistance, and tumorigenesis [47]. Drug resistant cells showed activated c-Met and increased expression of CD24, CD44, and ESA. The use of a c-Met+ cell inhibitor (Cabozantinib) abrogated Gemcitabine resistance in PC patients [48]. Administration of anti-CD44 monoclonal antibody to a human PC xenograft mouse model increased Gemcitabine sensitivity [49]. Similarly, Metformin enhanced the antiproliferation effects of Gemcitabine by inhibiting the proliferation of CD133+ cells in PC [50].

Another PCSC marker, Dclk1, was found in PanIN lesions, and PC at invasive stages [51], suggesting that PCSC may be used as diagnosis biomarkers. PCSCs show transcription factors found on embryonic stem cells (Oct-4, Sox-2, and Nanog). Increased levels of Oct-4 and Nanog correlate with early stages of carcinogenesis and worse prognosis. Oct-4 contributes to metastasis and cancer multidrug resistance. Sox-2 expression alone in PC could induce selfrenewal and differentiation [24].

PCSC marker expression correlates with lymph node metastasis and poor survival. There are several factors that could affect PCSC maintenance and proliferation. For example, PCSC maintenance and survival are affected by miRNA34. In addition, stem cell factor (SCF) binding to its receptor, c-Kit, induces an increase in HIF-1α synthesis, which is involved in PC progression and chemoresistance [26].

Our data suggest that 5-FU (a common chemotherapeutic used in PC treatment) decreased PC tumorsphere formation. PC cells that expressed CD24 + CD44+, CD24 + CD44 + ESA+, and pluripotency (Oct-4, Sox-2, Nanog) markers were spared by the 5-FU treatment [30]. Therefore, the development of specific treatments against PCSC remains a challenge.

#### **4.2. ATP-binding cassette proteins**

long isoform, Ob-Rb, is found more often in cancer cells and has full signaling capabilities, in contrast to the short isoform. Leptin and Ob-R have absolute affinity for binding. Leptin binding to Ob-R activates canonical (JAK2/STAT3, MAPK, PI-3 K/AKT1) and noncanonical signaling pathways (p38MAK, JNK, AMPK). The first leptin signaling event is the activation of JAK2, which phosphorylates Ob-R intracytoplasmic tail, leading to the phosphorylation of a tyrosine residue of STAT3 (pSTAT3). pSTAT3 forms a dimer that is translocated to the nucleus, inducing the transcription of specific genes, such as SOCS3, which acts as a potent negative feedback regulator of the JAK/STAT pathway [26]. Recently, it was reported that the central or peripheral administration of an Ob-R antagonist induced comparable changes in food intake, body weight, and hypothalamic SOCS3 expression in lean and diet-induced obesity (DIO) mice. These results suggest that endogenous Ob-R signaling may not be reduced in the context of DIO, thus challenging the established concept of leptin resistance under

Cancer chemoresistance is a current PC challenge. Intrinsic chemoresistance occurs when chemotherapy is ineffective from the start of treatment, whereas acquired chemoresistance develops only after exposure to anticancer drugs. Although PC cells are more susceptible to Gemcitabine when compared with other anticancer agents, most patients develop resistance within weeks of treatment initiation, leading to poor survival [2]. Mechanisms of cancer chemoresistance include drug modification, reduction or inhibition of drug-induced apoptosis, overexpression of drug efflux proteins, increased expression of survival factors and deregulation of pathways, such as

The hierarchical model of cancer states that tumors arise from CSC or cancer-initiating cells that can reproduce all tumor cell types. CSCs have common characteristics associated to normal stem cells. CSCs are tumorigenic, show self-renewal capabilities, and can be differentiated into multiple cancer cell types. CSCs hide in the tumor niche causing relapse and metastasis. The tumor niche is composed of stromal and inflammatory cells, cytokines, ECM, and vasculature. It provides signals helping CSCs to maintain their undifferentiated state. The accumulation of ECM destroys the normal PC architecture and enhances the expression

PCSCs express various markers, including CD24+CD44+, CD133+, CD24+CD44+ESA+, ALDH+, or c-Met+. Metastatic PCSCs express CXCR4+CD133+. PCSC markers CD133 and CD44 correlated to CXCR1 expression. PCSC could be identified using Hoechst 33342 dye by flow cytometry. Hoechst-negative cells were called "side population" and were linked to chemoresistance [45]. ALDHs are a class of enzymes that oxidize aldehydes. ALDH + PCSC show clonogenic and metastatic potential that affects survival in PC. Positive PC cells for PCSC markers form tumors in mice, in contrast to negative PC cells. ALDH1 mediates resistance to Cyclophosphamide and

dietary-induced conditions [42].

36 Advances in Pancreatic Cancer

**4. Mechanisms of chemoresistance in PC**

**4.1. Pancreatic cancer stem cells (PCSCs)**

of PCSC markers [44].

Notch, and expansion of cancer stem cells (CSCs), among others [43].

Overexpression of drug efflux proteins (ATP-binding cassette proteins and ABC family of proteins) increases the elimination of anticancer drugs and decreases their accumulation inside the cancer cells. ABC proteins (ABCB1, ABCC1, and ABCG2) are found in PCSC and contribute to their resistance to Gemcitabine [52]. Indeed, ABCB1 was significantly increased in CD44+ PC cells during the acquisition of resistance to Gemcitabine [53]. PC chemoresistance correlated with increased expression of CXCR4, CD133, and ABCB1 by PCSC [54]. Interestingly, ABCG2 localization and activity were not confined only to the plasma membrane, as intracellular vesicles containing ABCG2 were detected within CSC in PC, colorectal, and hepatocellular cancers. Moreover, a direct relationship between the presence of these vesicles in CSCs and the maintenance of their stem-like properties, including chemoresistance, was found. Furthermore, the vesicles accumulated ABCG2-dependent substrates, such as the fluorescent vitamin riboflavin (vitamin B2). In addition, the vesicles could accumulate ABCG2-depedent therapeutics, such as Mitoxantrone, to avoid apoptotic cell death [55]. Our data showed that PC tumorspheres treated with 5-FU were enriched in cells that overexpressed ABCC5 and ABCC11 efflux proteins [30].

β-catenin into the cytoplasm. Increased β-catenin levels and activity have been found in PC but not in the normal pancreas [58]. Wnt pathway induces PC formation by actions not only on the tumor cells but also on the stromal compartment through increases in ECM formation [59].

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There are other dysregulated pathways in PC. The nuclear factor-κB (NF-κB) proteins constitute a family of transcription factors associated with mediating inflammatory responses. However, these transcription factors also control diverse genes involved in development, apoptosis, and cell proliferation. NF-κB has an important role in PC. Additionally, Notch and IL-1 induce NF-κB in PC [60]. NF-κB signaling crosstalks with other signaling pathways, oncogenic or cancer-related proteins, such as STAT3, p53, ALDH1, PI-3 K, and MAPK. A recent study that evaluated a large number of human PC samples along with a few PanIN lesions found amplification of c-Myc in 30% of the tumors [61]. c-Myc deregulation, in cooperation with other oncogenic pathways, such as Kras, is sufficient to promote tumorigenesis [62]. The complexity of the PC altered signaling pathways affects pathogenesis and could explain why there is no successful PC treatment. Relationships among tumor cells, stroma, and signaling pathway crosstalks demonstrate the importance of developing combined therapies targeting

Apoptosis or programmed cell death regulates the tissue homeostasis. Chemoresistance is in part due to impairment of apoptosis in cancer cells. Antiapoptotic protein Bcl-2 is not frequently overexpressed in PC, which differs from other cancer types. In contrast, an imbalance between antiapoptotic Bcl-XL and proapoptotic Bax was found in the TGF-α murine model of PC [63]. Moreover, inhibitors of apoptosis, such as survivin, are overexpressed in PC when compared with normal pancreatic tissue. Resistant PC cells can be sensitized to death receptor–mediated apoptosis by inhibiting the NF-κB prosurvival pathway or by decreasing the expression of antiapoptotic proteins. The p53 pathway plays an important role in cancer cells avoiding the apoptosis, with mutations in p53 gene leading to increased drug resistance in PC cell lines and poor survival in PC patients [63]. Our data showed that 5-FU treatment of PC tumorspheres reduced RIP and Bcl-XL levels and increased Bax. Moreover, 5-FU increased caspase-3 activation and decreased uncleaved PARP in PC [30]. These data indicate that 5-FU actions on PC induce apoptosis through several components of the pathway. Numerous chemotherapeutic drugs target DNA synthesis in cancer cells, leading to increased apoptosis.

Leptin induces a wide range of prooncogenic effects. We have shown, for the first time, that leptin could be secreted by PC cells and derived tumorspheres. Moreover, leptin induced PCSC in tumorspheres [28]. In line with these data, a study of a pool analysis from PC patients showed that leptin levels and elevated Ob-R expression correlated to Oct-4 [64]. Our data demonstrated that leptin increased PC cell proliferation, tumorsphere formation, and xenograft growth in an immunocompromised mouse model. Moreover, leptin induced cell cycle progression, PCSC markers (CD24 + CD44 + ESA+, ALDH+), and ATP-binding cassette protein expression (ABCB1) in PC cells [28]. Leptin has been shown to increase the expression of miR21, while the tumor suppressors (miR200a, miR200b, and miR200c) decrease the expression of Ob-R. Furthermore, these tumor suppressors could also interact with some of the

both compartments and altered signaling in PC.

**4.7. Leptin and chemoresistance mechanisms in pancreatic cancer**

**4.6. Inhibition of apoptosis**

#### **4.3. Epithelial to mesenchymal transition (EMT) and PC metastasis**

To gain invasive and migratory capacity, and resistance to apoptosis, cancer epithelial cells undergo EMT. The expression of transcription factors, including Snail, Slug, zinc finger E-boxbinding homeobox 1 (ZEB1), and Twist, among others, induces EMT. ZEB1 deletion had a negative effect on tumor progression, invasiveness, and metastasis, reaffirming EMT's role in PC metastasis [55]. Gemcitabine-resistant PC cells had increased Vimentin and decreased E-cadherin expression. These alterations are hallmarks of EMT.

Our data showed that the use of 5-FU rendered different outcomes on EMT markers in tumorspheres derived from different PC cell lines. In BxPC-3 tumorspheres, 5-FU did not change the levels of expression of EMT markers (Vimentin and N-cadherin), while in MiaPaCa-2 tumorspheres, it slightly increased the expression of N-cadherin. Moreover, 5-FU spared PC cells that were N-cadherin+ [30]. Recently, the EMT concept was challenged by studies demonstrating the existence of a hybrid epithelial/mesenchymal phenotype in cells transitioning from EMT to mesenchymal to epithelial transition (MET). Because MET has been considered crucial for metastasis seeding in distant organs, this hybrid phenotype seems to be linked to drug resistance and tumor-initiating potential. Moreover, MET could allow tumor cells to collectively migrate in clusters to form metastases in a more effective way than pure EMT single cells [55].

#### **4.4. Tumor microenvironment**

PC desmoplasia results from proliferation of cancer-associated fibroblasts and increased deposit of ECM. This process reduces elasticity of tumor tissue and increases interstitial pressure, leading to decreased perfusion of chemotherapeutic agents [56]. The proliferative pancreatic stellate cells are the primary source of many of the ECM components in PC. These cells show increased proliferation and sensitivity to mitogenic factors. Fibrous proteins (e.g., collagen) and polysaccharide chain glycosaminoglycans (e.g., hyaluronan) are ECM factors that constitute the noncellular components of PC desmoplastic tissue. A significant overproduction of ECM components can be described as the failed resolution of a healing wound, which leads to fibrosis in PC. Immune cells (macrophages, neutrophils, and regulatory T cells [Treg]) contribute to PC desmoplasia. Therapeutics reducing the contribution of the desmoplastic reaction to chemoresistance are being actively pursued as a potential therapeutic approach [57].

#### **4.5. Changes in signaling pathways**

From the early lesions, PC cells harbor alterations in signaling pathways that remain throughout carcinogenesis. These changes not only impact tumor cells but also the surrounding stromal cells. Components of the Hedgehog (Hh) signaling pathway have essential roles in PC pathogenesis. In a global genomic analysis of PC, all tumors tested had alterations in at least one of the Hedgehog signaling genes. Hh signaling induced desmoplasia, playing a key role in chemoresistance [56]. Wnt signaling pathway is mainly involved in PC cell growth. The Wnt pathway is activated when ligands bind to the cell membrane Wnt receptor, resulting in the release of β-catenin into the cytoplasm. Increased β-catenin levels and activity have been found in PC but not in the normal pancreas [58]. Wnt pathway induces PC formation by actions not only on the tumor cells but also on the stromal compartment through increases in ECM formation [59].

There are other dysregulated pathways in PC. The nuclear factor-κB (NF-κB) proteins constitute a family of transcription factors associated with mediating inflammatory responses. However, these transcription factors also control diverse genes involved in development, apoptosis, and cell proliferation. NF-κB has an important role in PC. Additionally, Notch and IL-1 induce NF-κB in PC [60]. NF-κB signaling crosstalks with other signaling pathways, oncogenic or cancer-related proteins, such as STAT3, p53, ALDH1, PI-3 K, and MAPK. A recent study that evaluated a large number of human PC samples along with a few PanIN lesions found amplification of c-Myc in 30% of the tumors [61]. c-Myc deregulation, in cooperation with other oncogenic pathways, such as Kras, is sufficient to promote tumorigenesis [62]. The complexity of the PC altered signaling pathways affects pathogenesis and could explain why there is no successful PC treatment. Relationships among tumor cells, stroma, and signaling pathway crosstalks demonstrate the importance of developing combined therapies targeting both compartments and altered signaling in PC.

#### **4.6. Inhibition of apoptosis**

ABCG2-depedent therapeutics, such as Mitoxantrone, to avoid apoptotic cell death [55]. Our data showed that PC tumorspheres treated with 5-FU were enriched in cells that overex-

To gain invasive and migratory capacity, and resistance to apoptosis, cancer epithelial cells undergo EMT. The expression of transcription factors, including Snail, Slug, zinc finger E-boxbinding homeobox 1 (ZEB1), and Twist, among others, induces EMT. ZEB1 deletion had a negative effect on tumor progression, invasiveness, and metastasis, reaffirming EMT's role in PC metastasis [55]. Gemcitabine-resistant PC cells had increased Vimentin and decreased

Our data showed that the use of 5-FU rendered different outcomes on EMT markers in tumorspheres derived from different PC cell lines. In BxPC-3 tumorspheres, 5-FU did not change the levels of expression of EMT markers (Vimentin and N-cadherin), while in MiaPaCa-2 tumorspheres, it slightly increased the expression of N-cadherin. Moreover, 5-FU spared PC cells that were N-cadherin+ [30]. Recently, the EMT concept was challenged by studies demonstrating the existence of a hybrid epithelial/mesenchymal phenotype in cells transitioning from EMT to mesenchymal to epithelial transition (MET). Because MET has been considered crucial for metastasis seeding in distant organs, this hybrid phenotype seems to be linked to drug resistance and tumor-initiating potential. Moreover, MET could allow tumor cells to collectively migrate in clusters to form metastases in a more effective way than pure EMT single cells [55].

PC desmoplasia results from proliferation of cancer-associated fibroblasts and increased deposit of ECM. This process reduces elasticity of tumor tissue and increases interstitial pressure, leading to decreased perfusion of chemotherapeutic agents [56]. The proliferative pancreatic stellate cells are the primary source of many of the ECM components in PC. These cells show increased proliferation and sensitivity to mitogenic factors. Fibrous proteins (e.g., collagen) and polysaccharide chain glycosaminoglycans (e.g., hyaluronan) are ECM factors that constitute the noncellular components of PC desmoplastic tissue. A significant overproduction of ECM components can be described as the failed resolution of a healing wound, which leads to fibrosis in PC. Immune cells (macrophages, neutrophils, and regulatory T cells [Treg]) contribute to PC desmoplasia. Therapeutics reducing the contribution of the desmoplastic reaction

to chemoresistance are being actively pursued as a potential therapeutic approach [57].

From the early lesions, PC cells harbor alterations in signaling pathways that remain throughout carcinogenesis. These changes not only impact tumor cells but also the surrounding stromal cells. Components of the Hedgehog (Hh) signaling pathway have essential roles in PC pathogenesis. In a global genomic analysis of PC, all tumors tested had alterations in at least one of the Hedgehog signaling genes. Hh signaling induced desmoplasia, playing a key role in chemoresistance [56]. Wnt signaling pathway is mainly involved in PC cell growth. The Wnt pathway is activated when ligands bind to the cell membrane Wnt receptor, resulting in the release of

pressed ABCC5 and ABCC11 efflux proteins [30].

38 Advances in Pancreatic Cancer

**4.4. Tumor microenvironment**

**4.5. Changes in signaling pathways**

**4.3. Epithelial to mesenchymal transition (EMT) and PC metastasis**

E-cadherin expression. These alterations are hallmarks of EMT.

Apoptosis or programmed cell death regulates the tissue homeostasis. Chemoresistance is in part due to impairment of apoptosis in cancer cells. Antiapoptotic protein Bcl-2 is not frequently overexpressed in PC, which differs from other cancer types. In contrast, an imbalance between antiapoptotic Bcl-XL and proapoptotic Bax was found in the TGF-α murine model of PC [63]. Moreover, inhibitors of apoptosis, such as survivin, are overexpressed in PC when compared with normal pancreatic tissue. Resistant PC cells can be sensitized to death receptor–mediated apoptosis by inhibiting the NF-κB prosurvival pathway or by decreasing the expression of antiapoptotic proteins. The p53 pathway plays an important role in cancer cells avoiding the apoptosis, with mutations in p53 gene leading to increased drug resistance in PC cell lines and poor survival in PC patients [63]. Our data showed that 5-FU treatment of PC tumorspheres reduced RIP and Bcl-XL levels and increased Bax. Moreover, 5-FU increased caspase-3 activation and decreased uncleaved PARP in PC [30]. These data indicate that 5-FU actions on PC induce apoptosis through several components of the pathway. Numerous chemotherapeutic drugs target DNA synthesis in cancer cells, leading to increased apoptosis.

#### **4.7. Leptin and chemoresistance mechanisms in pancreatic cancer**

Leptin induces a wide range of prooncogenic effects. We have shown, for the first time, that leptin could be secreted by PC cells and derived tumorspheres. Moreover, leptin induced PCSC in tumorspheres [28]. In line with these data, a study of a pool analysis from PC patients showed that leptin levels and elevated Ob-R expression correlated to Oct-4 [64]. Our data demonstrated that leptin increased PC cell proliferation, tumorsphere formation, and xenograft growth in an immunocompromised mouse model. Moreover, leptin induced cell cycle progression, PCSC markers (CD24 + CD44 + ESA+, ALDH+), and ATP-binding cassette protein expression (ABCB1) in PC cells [28]. Leptin has been shown to increase the expression of miR21, while the tumor suppressors (miR200a, miR200b, and miR200c) decrease the expression of Ob-R. Furthermore, these tumor suppressors could also interact with some of the PCSC markers (c-Met, ABCB1, and CD44), which decrease their expression. Oncogenic miR21 increases the expression of ABCB1, ALDH, and CD44.

and Oct-4), ABCB1 (MDR1), tumorsphere formation, cell cycle progression, proliferation, and tumorigenesis. These effects were reduced by GSI [28]. Moreover, mouse and human PC and cell lines treated with adiponectin, or an adiponectin receptor agonist, AdipoRon, suppressed leptin-induced STAT3 signaling in vitro and reduced PC growth in vivo [74]. The addition of leptin to 5-FU treated tumorspheres decreased 5-FU-induced cytotoxicity and increased colony forming ability, number of cells expressing pluripotency and EMT markers, drug efflux proteins (ABCC5 and ABCC11), and Notch. Leptin also reduced the 5-FU effects on apoptosis by decreasing proapoptotic (Bax, caspase-3 activation, and PARP degradation) and increasing antiapoptotic factors (RIP and Bcl-XL). Leptin's effects on PC tumorspheres were mainly Notch signaling dependent [30]. Therefore, the leptin-Notch axis could be a target to develop

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To decrease the risk of local and distant metastasis, adjuvant therapy is usually started 1–2months after PC surgery. Although no regimen has been proven significantly more effective than others, a regimen based on 5-FU or Gemcitabine for 6 months is usually the option used to reduce PC patients' mortality [75]. The activity of 5-FU/Leucovorin has been compared to Gemcitabine as an adjuvant therapy in the European Study Group for PC (ESPAC)-3 trial [76]. However, the study showed that median overall survival for patients treated with 5-FU/ Leucovorin was 23 months when compared with 23.6 months for patients treated with Gemcitabine. The ESPAC-4 study measured the efficacy of a combination treatment with Gemcitabine plus Capecitabine when compared with monotherapy with Gemcitabine alone. The results showed a survival of 28 months in the combined therapy when compared with 25.5 months in the monotherapy group. Because the dual therapy was well tolerated, the combination of Gemcitabine and Capecitabine has been used as a standard in the clinical setting [77]. Currently, regimens with Gemcitabine plus nanoparticle albumin-bound Paclitaxel (nab-Paclitaxel) and a combination of 5-FU, Irinotecan, and Oxaliplatin (FOLFIRINOX) are evaluated in the clinical setting [78]. Gemcitabine has usually some efficacy as an adjuvant therapy, but often patients develop chemoresistance. Nab-Paclitaxel, a water-soluble compound, has enhanced distribution properties within the tumor microenvironment when compared with Paclitaxel. However, studies have shown that nab-Paclitaxel treatment neither decreased tumor stroma nor increased tumor vascular perfusion in a mouse patient-derived xenograft (PDX) tumor model [79]. The infiltration of neoplastic lesions by CD8+ T lymphocytes is associated with improved prognosis. However, a CD40 monoclonal antibody that activated CD8+ T cells in Phase I clinical trial had only a partial response [80]. FOLFIRINOX and nab-Paclitaxel plus Gemcitabine have the potential to downstage local advanced disease and to improve tumor resection rates. The use of chemoradiation therapy as an adjuvant is controversial and with minimal effects on survival in clinical trials so far [81]. New studies that incorporate modern radiation techniques and current chemotherapy regimens are still needed to deter-

novel strategies for PC treatment.

**5.1. Chemotherapy**

**5. Pancreatic cancer treatment**

mine if radiation is beneficial in PC treatment.

Leptin can directly regulate the expression of HDAC4 and HDAC5 and indirectly affect the expression of other HDAC via microRNA or PCSC markers. We have suggested that leptin can increase the expression of miR21, which in turn can increase the expression of HDAC3. Analysis of data from PC biopsies (TCGA databank) suggested that HDAC, miRNA21/200, and leptin could have complex signaling crosstalk that could be a novel therapeutic target for obese PC patients. We further determined the effects of leptin on HDAC expression in PC tumorspheres. HDAC3 and HDAC8 expression was increased by leptin. Furthermore, the Gemcitabine-induced decreased expression of HDAC2, HDAC3, and HDAC8 was reversed by leptin. Thus, we have shown that leptin through its effects on PCSC, ABCB1, and HDAC could be involved in PC chemoresistance [65]. Moreover, using another chemotherapeutic agent commonly used in PC treatment, 5-FU, we demonstrated that leptin impaired 5-FU cytotoxicity by increasing the expression and number of PCSC+, pluripotency+, and EMT+ PC cells. ABCC5 and ABCC11 expression as well as the number of positive cells for these ATPbinding cassette proteins were increased by leptin in PC tumorspheres. These leptin's effects protected the survival of PC tumorspheres treated with 5-FU and reduced its cytotoxicity. The survival of PC tumorspheres treated with 5-FU and leptin was linked to reduced apoptosis. Leptin increased the levels of PARP, Bcl-XL, and RIP and decreased Bax. 5-FU increased caspase-3 activation, which was reduced by leptin. These data could help to unravel the multiple mechanisms through which leptin signaling contributes to drug resistance in PC [30].

#### *4.7.1. Leptin-Notch crosstalk in pancreatic cancer*

Notch signaling controls the cell proliferation, PCSC maintenance and differentiation, apoptosis, invasion, and metastasis in cancer. Overexpression of Notch receptors (Notch1 and Notch2) was found in PCSC when compared with nonmalignant pancreatic stem cells [66]. DLL4 increase in PC cells stimulated the expression of Oct-4, Nanog, and stem cells [67]. PCSCs that express Oct-4, Sox-2, and Nanog show an increased aggressivity and chemoresistance. Notch4 overexpression was linked to PC chemoresistance to Docetaxel [68]. Expression of Notch3 and Hey1 was associated with reduced survival in PC [69]. Resistance to Gemcitabine correlated with Notch2, Notch4, and JAG1 overexpression [70]. The inhibition of Notch1 by siRNA suppressed proliferation, induced apoptosis, and reduced migration and invasion of PC cells [71].

Notch signaling induced EMT phenotype in Gemcitabine-resistant PC cells overexpressing Notch2, Notch4, and JAG1. Furthermore, the inhibition of Notch signaling decreased EMT markers, including Vimentin, Snail, Slug, and ZEB1, in human PC cell lines [72]. MiR200 members increased Notch activation by ZEB1 that regulates the expression of JAG1 and the mastermind-like coactivators (Maml2 and Maml3). In PC cells, miR200 expression showed an inverse correlation with JAG1 and ZEB1 levels [73]. Therefore, miR200 inhibits EMT by interacting with ZEB1/2 and the Notch pathway and represses self-renewal and differentiation in CSC. MiR200 is also involved in apoptosis [72].

Our data showed that leptin induced the expression of Notch family components in PC (Notch1–4, DLL4, JAG1, survivin, and Hey2), PCSC markers (CD24CD44ESA, ALDH, CD133, and Oct-4), ABCB1 (MDR1), tumorsphere formation, cell cycle progression, proliferation, and tumorigenesis. These effects were reduced by GSI [28]. Moreover, mouse and human PC and cell lines treated with adiponectin, or an adiponectin receptor agonist, AdipoRon, suppressed leptin-induced STAT3 signaling in vitro and reduced PC growth in vivo [74]. The addition of leptin to 5-FU treated tumorspheres decreased 5-FU-induced cytotoxicity and increased colony forming ability, number of cells expressing pluripotency and EMT markers, drug efflux proteins (ABCC5 and ABCC11), and Notch. Leptin also reduced the 5-FU effects on apoptosis by decreasing proapoptotic (Bax, caspase-3 activation, and PARP degradation) and increasing antiapoptotic factors (RIP and Bcl-XL). Leptin's effects on PC tumorspheres were mainly Notch signaling dependent [30]. Therefore, the leptin-Notch axis could be a target to develop novel strategies for PC treatment.
