Genes with Dual Tumor Suppressor and Oncogenic Activities

*Genes and Cancer*

nature15818

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**45**

**Chapter 3**

**Abstract**

targeted therapy

**1. Introduction**

Tumour Suppressor Genes with

Oncogenic Roles in Lung Cancer

*Leigha D. Rock, Daiana D. Becker-Santos, Adam P. Sage,* 

**Keywords:** tumour-suppressor genes, oncogenes, dual roles, lung cancer,

signalling networks and often oncogene addiction [3].

Cancer cells arise in non-malignant tissue due to the sequential acquisition of molecular alterations that drive proliferation, permit the evasion of growth suppression and apoptosis signals and promote angiogenesis, invasion and metastasis [1]. This process is stochastic, and over time the tumour continues to evolve in a dynamic manner, generating a group of cells harbouring different genetic and epigenetic features [2]. The resulting heterogeneity is the basis of tumour evolution and leads to the selection of tumour cells. These cells often present with rewired

The uncontrolled growth of cancer cells can in part be explained by their aberrant gene expression patterns. While most cancer genes are characterized as either oncogenes or tumour suppressors based on their typical behaviour in tumours, some genes display dual oncogenic and tumour suppressive functions [4, 5]. The majority of these genes encode multiple isoforms, which are further post-translationally modified and form a variety of protein complexes, generating a context-dependent cellular network [6]. In diploid organisms, gain-of-function (GOF) mutations in oncogenes are typically dominant (single events are sufficient to promote tumourigenesis), while loss-of-function alterations are recessive in TSGs

Lung cancer is one of the most common cancers and the leading cause of cancerrelated deaths worldwide. High-throughput sequencing efforts have uncovered the molecular heterogeneity of this disease, unveiling several genetic and epigenetic disruptions driving its development. Unlike oncogenes, tumour suppressor genes negatively regulate cell cycle control and exhibit loss-of-function alterations in cancer. Although tumour suppressor genes are more frequently disrupted, oncogenes are more likely to be drug-targeted. Many genes are described as presenting both tumour suppressive and oncogenic functions in different tumour types or even within the natural history of the disease in a single tumour. In this chapter, we describe current knowledge of tumour suppressor genes in lung tissues, focusing on

*Mateus Camargo Barros-Filho, Florian Guisier,* 

*Erin A. Marshall and Wan L. Lam*

tumour suppressor/oncogene duality.

#### **Chapter 3**

## Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer

*Mateus Camargo Barros-Filho, Florian Guisier, Leigha D. Rock, Daiana D. Becker-Santos, Adam P. Sage, Erin A. Marshall and Wan L. Lam*

#### **Abstract**

Lung cancer is one of the most common cancers and the leading cause of cancerrelated deaths worldwide. High-throughput sequencing efforts have uncovered the molecular heterogeneity of this disease, unveiling several genetic and epigenetic disruptions driving its development. Unlike oncogenes, tumour suppressor genes negatively regulate cell cycle control and exhibit loss-of-function alterations in cancer. Although tumour suppressor genes are more frequently disrupted, oncogenes are more likely to be drug-targeted. Many genes are described as presenting both tumour suppressive and oncogenic functions in different tumour types or even within the natural history of the disease in a single tumour. In this chapter, we describe current knowledge of tumour suppressor genes in lung tissues, focusing on tumour suppressor/oncogene duality.

**Keywords:** tumour-suppressor genes, oncogenes, dual roles, lung cancer, targeted therapy

#### **1. Introduction**

Cancer cells arise in non-malignant tissue due to the sequential acquisition of molecular alterations that drive proliferation, permit the evasion of growth suppression and apoptosis signals and promote angiogenesis, invasion and metastasis [1]. This process is stochastic, and over time the tumour continues to evolve in a dynamic manner, generating a group of cells harbouring different genetic and epigenetic features [2]. The resulting heterogeneity is the basis of tumour evolution and leads to the selection of tumour cells. These cells often present with rewired signalling networks and often oncogene addiction [3].

The uncontrolled growth of cancer cells can in part be explained by their aberrant gene expression patterns. While most cancer genes are characterized as either oncogenes or tumour suppressors based on their typical behaviour in tumours, some genes display dual oncogenic and tumour suppressive functions [4, 5]. The majority of these genes encode multiple isoforms, which are further post-translationally modified and form a variety of protein complexes, generating a context-dependent cellular network [6]. In diploid organisms, gain-of-function (GOF) mutations in oncogenes are typically dominant (single events are sufficient to promote tumourigenesis), while loss-of-function alterations are recessive in TSGs (requires two inactivation events) [7]. For example, for a TSG with dual oncogenic roles, one gain-of-function mutation can potentially cease its tumour suppressive function and turn on oncogenic signalling [5].

Recently, genes with both oncogenic and tumour-suppressive functions were described across 12 main cancer types using The Cancer Genome Atlas (TCGA) database [5]. Using a text mining approach, the authors identified genes mainly represented by kinases (e.g. *BCR*, *CHEK2*, *MAP2K4*, *NTRK3* and *SYK*) or transcription factors (e.g. *BRCA1, EZH2, NOTCH1, NOTCH2, STAT3* and *TP53*) and evaluated them at the genomic and gene expression levels. Using an *in silico* analysis, it was shown that genes with dual functions interact with more partners and are more important hub-genes in protein**-**protein interaction networks.

In this chapter, we discuss TSGs with both tumour suppressive and oncogenic functions in lung cancer.

#### **1.1 Lung cancer classification**

Lung cancer is one of the most common cancers and the leading cause of cancerrelated deaths worldwide [8]. In the United States, lung cancer accounts for 13.5% of all new cancer cases and 25.3% of all cancer deaths. The five-year survival rate is dismal, with only 18.6% of patients surviving 5 years [9]. The majority of lung cancer cases (approximately 80%) are attributed to cigarette smoking [10]. About 10–25% of cases occur in people who have never smoked [11]. The aetiology behind these cases is most likely a combination of genetic factors, as well as the effects of exposure to environmental carcinogens such as asbestos, radon gas or other forms of pollution [12].

Lung cancer is classified according to histological type. There are two major types: small cell lung cancer (SCLC), which accounts for 15–20% of lung cancer patients, and non-small cell lung cancer (NSCLC), comprising the remaining 80–85% (**Figure 1**) [13]. SCLC, primarily originating from the central airways, is thought to be derived from neuroendocrine cells [14]. NSCLC is composed of three major histological

#### **Figure 1.**

*Histological classification of lung cancer. (A) Lung cancer histological types. (B) Location of the tumours and cell origins. SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; LCC, large cell carcinoma.*

**47**

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer*

subtypes: adenocarcinoma (LUAD), squamous cell carcinoma (LUSC) and large cell carcinoma (LCC). LUAD is the most common, accounting for approximately 40% of all lung cases [15]. LUAD typically arises from glandular epithelium, from bronchioalveolar stem cells, club (Clara) cells or type II pneumocytes in the lung periphery [13]. LUAD is also the predominant subtype that arises in patients who have never smoked [15]. LUSC develops primarily in the central airways and segmental bronchi, strongly associates with a history of smoking and accounts for approximately 20% of all lung cancer cases. LCC may arise anywhere in the lung and are classified as tumours

*Mutational frequency of TSGs in small cell lung cancer (SCLC; n = 110) [16], lung adenocarcinoma (LUAD; n = 660) [23] and lung squamous cell carcinoma (LUSC; n = 484) [23]. TSGs were defined according to COSMIC Cancer Gene Census (https://cancer.sanger.ac.uk/census) and mutation frequency of the most commonly disrupted TSGs in these subtypes of lung cancer were retrieved using cBioPortal (http://www.*

Beyond the histological heterogeneity of lung cancer, genomic studies of large cohorts have uncovered the complex molecular landscape of lung tumours. Indeed, it has been observed that a wide variety of oncogenes and TSGs can be altered in lung cancer, and these molecular events are vastly different between histological

Clinical studies have shown that molecularly defined lung cancer subgroups can correlate with characteristics such as ethnicity [18], smoking history [19], treatment sensitivity [20] or prognosis [21]. Many of the commonly identified gain-of-function alterations in proto-oncogenes have been actively investigated for therapeutic purposes. For example, *EGFR, ALK, ROS1, BRAF, MET, RET* and *HER2* are routinely assessed in the clinic to offer targeted therapy for eligible LUAD patients [22]. Three TSGs are frequently mutated in all three major lung cancer subtypes: *TP53, LRP1B* and *CSMD3*. Other TSGs of particular interest in lung cancer are as follows *RB1* and *CREBBP* in SCLC, *KEAP1* and *STK11* in LUAD, *CDKN2A* in LUSC, *NOTCH1* and *PTEN* in both SCLC and LUSC and NF1 in both LUAD and LUSC (**Figure 2**). Mutations in these TSGs are usually mutually exclusive, indicating that

without general features associated with SCLC, LUAD or LUSC [13].

individual genes are capable of driving lung cancer progression.

Several TSGs in lung cancer have also been shown to behave as oncogenes, depending on the molecular context and/or the mechanism by which they are

**2. TSGs with oncogenic roles in lung cancer**

**1.2 TSG mutation spectrum in lung cancer**

subtypes [16, 17].

**Figure 2.**

*cbioportal.org/).*

*DOI: http://dx.doi.org/10.5772/intechopen.85017*

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.85017*

#### **Figure 2.**

*Genes and Cancer*

functions in lung cancer.

**1.1 Lung cancer classification**

function and turn on oncogenic signalling [5].

**46**

**Figure 1.**

*Histological classification of lung cancer. (A) Lung cancer histological types. (B) Location of the tumours and cell origins. SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; LUAD, lung adenocarcinoma;* 

(requires two inactivation events) [7]. For example, for a TSG with dual oncogenic roles, one gain-of-function mutation can potentially cease its tumour suppressive

Recently, genes with both oncogenic and tumour-suppressive functions were described across 12 main cancer types using The Cancer Genome Atlas (TCGA) database [5]. Using a text mining approach, the authors identified genes mainly represented by kinases (e.g. *BCR*, *CHEK2*, *MAP2K4*, *NTRK3* and *SYK*) or transcription factors (e.g. *BRCA1, EZH2, NOTCH1, NOTCH2, STAT3* and *TP53*) and evaluated them at the genomic and gene expression levels. Using an *in silico* analysis, it was shown that genes with dual functions interact with more partners and are

In this chapter, we discuss TSGs with both tumour suppressive and oncogenic

Lung cancer is one of the most common cancers and the leading cause of cancerrelated deaths worldwide [8]. In the United States, lung cancer accounts for 13.5% of all new cancer cases and 25.3% of all cancer deaths. The five-year survival rate is dismal, with only 18.6% of patients surviving 5 years [9]. The majority of lung cancer cases (approximately 80%) are attributed to cigarette smoking [10]. About 10–25% of cases occur in people who have never smoked [11]. The aetiology behind these cases is most likely a combination of genetic factors, as well as the effects of exposure to environmental carcinogens such as asbestos, radon gas or other forms of pollution [12]. Lung cancer is classified according to histological type. There are two major types: small cell lung cancer (SCLC), which accounts for 15–20% of lung cancer patients, and non-small cell lung cancer (NSCLC), comprising the remaining 80–85% (**Figure 1**) [13]. SCLC, primarily originating from the central airways, is thought to be derived from neuroendocrine cells [14]. NSCLC is composed of three major histological

more important hub-genes in protein**-**protein interaction networks.

*LUSC, lung squamous cell carcinoma; LCC, large cell carcinoma.*

*Mutational frequency of TSGs in small cell lung cancer (SCLC; n = 110) [16], lung adenocarcinoma (LUAD; n = 660) [23] and lung squamous cell carcinoma (LUSC; n = 484) [23]. TSGs were defined according to COSMIC Cancer Gene Census (https://cancer.sanger.ac.uk/census) and mutation frequency of the most commonly disrupted TSGs in these subtypes of lung cancer were retrieved using cBioPortal (http://www. cbioportal.org/).*

subtypes: adenocarcinoma (LUAD), squamous cell carcinoma (LUSC) and large cell carcinoma (LCC). LUAD is the most common, accounting for approximately 40% of all lung cases [15]. LUAD typically arises from glandular epithelium, from bronchioalveolar stem cells, club (Clara) cells or type II pneumocytes in the lung periphery [13]. LUAD is also the predominant subtype that arises in patients who have never smoked [15]. LUSC develops primarily in the central airways and segmental bronchi, strongly associates with a history of smoking and accounts for approximately 20% of all lung cancer cases. LCC may arise anywhere in the lung and are classified as tumours without general features associated with SCLC, LUAD or LUSC [13].

#### **1.2 TSG mutation spectrum in lung cancer**

Beyond the histological heterogeneity of lung cancer, genomic studies of large cohorts have uncovered the complex molecular landscape of lung tumours. Indeed, it has been observed that a wide variety of oncogenes and TSGs can be altered in lung cancer, and these molecular events are vastly different between histological subtypes [16, 17].

Clinical studies have shown that molecularly defined lung cancer subgroups can correlate with characteristics such as ethnicity [18], smoking history [19], treatment sensitivity [20] or prognosis [21]. Many of the commonly identified gain-of-function alterations in proto-oncogenes have been actively investigated for therapeutic purposes. For example, *EGFR, ALK, ROS1, BRAF, MET, RET* and *HER2* are routinely assessed in the clinic to offer targeted therapy for eligible LUAD patients [22].

Three TSGs are frequently mutated in all three major lung cancer subtypes: *TP53, LRP1B* and *CSMD3*. Other TSGs of particular interest in lung cancer are as follows *RB1* and *CREBBP* in SCLC, *KEAP1* and *STK11* in LUAD, *CDKN2A* in LUSC, *NOTCH1* and *PTEN* in both SCLC and LUSC and NF1 in both LUAD and LUSC (**Figure 2**). Mutations in these TSGs are usually mutually exclusive, indicating that individual genes are capable of driving lung cancer progression.

#### **2. TSGs with oncogenic roles in lung cancer**

Several TSGs in lung cancer have also been shown to behave as oncogenes, depending on the molecular context and/or the mechanism by which they are altered (**Table 1**). Among them are *TP53*, *NFIB*, members of the NOTCH family, *NKX2-1*, *NFE2L2*, as well as some non-coding RNAs (*MALAT1*, *mir*-125, and *mir*-378), which will be discussed in detail below.

#### **2.1** *TP53*

*TP53* is a well-known TSG, representing the most common somatically mutated gene in human cancer, especially in lung tumours [24]. The classic functions of the encoded p53 protein are cell cycle regulation, DNA repair, senescence mediated by stress, apoptosis and angiogenesis. These functions mainly occur through


*TF, transcription factor; OE, overexpression; EMT, epithelial-mesenchymal transition. Numbers in brackets refer to the list of reference.*

**49**

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer*

the binding of a p53 tetramer to the promoter of target genes [25]. In many cancer types, *TP53* mutation is associated with poor prognosis, including local and distant

that activating *TP53* alterations can act to promote cancer development and progression [25, 28]. Depending on the location of the mutation within the *TP53* gene, protein structure and subsequent DNA binding activity can be lost or altered, resulting in either loss or gain of function [25]. In contrast to the majority of TSGs, *TP53* is not commonly inactivated by deletions or truncating mutations. Indeed, 74% of mutations within the *TP53* locus are missense point mutations, which can be found in proteins in human tumours [25]. In fact, altered *TP53* was initially considered as a cancer antigen with putative oncogenic properties [25]. Together, this highlights the dichotomous role of *TP53* disruptions, in that both the loss of wild-type p53 and gain-of-function mutations can provide a growth advantage to tumours [28]. Lung cancer is commonly associated with tobacco use, where the prolonged exposure to carcinogens damages the DNA of the exposed cells. These alterations are especially enriched in missense mutations in *TP53*, leading to GOF-p53 [29]. The oncogenic GOF mutation in p53 was previously shown to be related with the inactivation of AMP-activated protein kinase (AMPK) signalling in head and neck cancer and another tobacco-related cancer [30]. AMPK is a master regulator of metabolic homeostasis and GOF-mutated p53 is able to physically interact and inhibit AMPK, stimulating aerobic glycolysis under energetic stress conditions and

In lung cancer mouse models, prevention of tumour formation by inhibiting GOF p53 mutants has been demonstrated [53]. Although the highly aberrant genomes in p53-mutated tumours should lead to unfeasible mitosis, these mutations facilitate the survival and proliferation of these cells through stabilizing replication

GOF p53 mutants are most likely involved in multiple mechanisms that coordinate tumour progression. For example, GOF-p53 (R175H, R273H and D281G) was demonstrated to upregulate *CXCL5, CXCL8* and *CXCL12* through its transcription factor activity, promoting migration of lung cancer cell lines [54]. *CXCL5* expression was shown to be elevated in human lung tumour samples harbouring GOF-p53, and its inhibition could reverse cell motility in lung cancer and melanoma cell lines [54]. In NSCLC, it was recently reported that GOF-p53 can physically interact with HIF-1 and binds to the SWI/SNF chromatin remodelling complex, inducing the expression of hypoxia-responsive genes [55]. Importantly, specific extracellular matrix components are upregulated by this process and mediate pro-tumourigenic

Nuclear factor I (NFI) is a transcription factor family, comprising NFIA, NFIB, NFIC and NFIX, that plays important roles in normal development and in numerous diseases [56]. These proteins bind to specific DNA sequences leading to repression or activation of gene expression in a context-dependent manner, regulating cell differentiation and proliferation through their target genes [57]. *NFIB*, in particular, has been implicated in a wide range of malignancies, being described as both an

Using an *in vivo* model, it was demonstrated that NFIB is a metastatic driver in SCLC, inducing global chromatin reprogramming during metastasis [33]. The authors isolated tumour cells from primary and metastatic sites of genetically engineered mice, and using genome-wide analysis, they showed a pronounced increase

Despite having a reputation as a 'guardian of the genome', recent work has shown

metastases events, resistance to treatment and decreased survival [26, 27].

*DOI: http://dx.doi.org/10.5772/intechopen.85017*

leading to invasive growth.

features in NSCLC [55].

oncogene and a potential TSG [58].

**2.2** *NFIB*

forks and promoting micronuclei arrangement [31].

#### **Table 1.**

*Main TSGs with dual functions reported in lung cancer.*

#### *Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.85017*

*Genes and Cancer*

**2.1** *TP53*

*TP53* TF: regulates cell cycle,

*NFIB* TF: crucial in lung

*NOTCH1/NOTCH2* Transmembrane

*NFE2L2* TF: cellular defense

*NKX2-1* TF: essential for lung

*TGFB* Cytokine: regulates

*TUSC3* Endoplasmic reticulum

*WT1* TF: role in urogenital

*STK11* Serine-threonine kinase:

polarity

development, differentiation and homeostasis

DNA repair, senescence and apoptosis

receptors: proliferation, differentiation and

mechanism against oxidative stress

regulation of energetic metabolism and cell

protein in magnesium uptake, glycosylation and embryonic development

system development

*Main TSGs with dual functions reported in lung cancer.*

*MALAT1* Long non-coding RNA OE reduces invasiveness in

miR-378 microRNA OE reverses chemoresistance

development

development

378), which will be discussed in detail below.

survival

**Gene Main function Role as TSG Role as oncogene**

altered (**Table 1**). Among them are *TP53*, *NFIB*, members of the NOTCH family, *NKX2-1*, *NFE2L2*, as well as some non-coding RNAs (*MALAT1*, *mir*-125, and *mir*-

[34]

[36]

[40]

[47]

*miR-125b* microRNA OE induces apoptosis [49] OE promotes metastasis [50]

*TF, transcription factor; OE, overexpression; EMT, epithelial-mesenchymal transition. Numbers in brackets refer to* 

TSG in several tissues: frequently lost through mutations [24]

*TP53* is a well-known TSG, representing the most common somatically mutated gene in human cancer, especially in lung tumours [24]. The classic functions of the encoded p53 protein are cell cycle regulation, DNA repair, senescence mediated by stress, apoptosis and angiogenesis. These functions mainly occur through

> Underexpressed in NSCLC and associated with poor survival in LUAD [32]

Inactivated by inhibitor ligands and through mutations, especially in SCLC

Protects lung tissue against exposure to oxidative stress

Acts as a TSG in *KRAS-*driven p53-mutant LUAD [38]

Mutational inactivation promotes cancer development

Expression loss leads to growth arrest in early-stage lung and other cancers [41]

Hypermethylation; expression loss in NSCLC; inhibits cell proliferation and promotes apoptosis [43]

Loss of function enhances cell viability and proliferation in Wilms' tumour [45]

PTEN expressing tumours

to cisplatin in LUAD [51]

Missense mutations confer gain-of-function oncogenic

Maintains stem cell features; promotes proliferation in LUAD

Mutational activation: aids cells to escape from endogenous tumour suppression [37]

Enhanced oncogenic signals in *EGFR*-driven LUAD [39]

OE promotes tumour growth in advanced cancer stages [42]

OE in NSCLC accelerates cancer growth; induces EMT [44]

OE promotes survival in *KRAS*mutated NSCLC [46]

OE is associated with invasion and brain metastasis [52]

OE associated with chemotherapy resistance in

NSCLC [48]

OE maintains metabolic homeostasis and attenuates oxidative stress [40]

properties [31]

[35]

Amplified and OE in SCLC: inducing chromatin reprogramming during metastasis [33]

**48**

**Table 1.**

*the list of reference.*

the binding of a p53 tetramer to the promoter of target genes [25]. In many cancer types, *TP53* mutation is associated with poor prognosis, including local and distant metastases events, resistance to treatment and decreased survival [26, 27].

Despite having a reputation as a 'guardian of the genome', recent work has shown that activating *TP53* alterations can act to promote cancer development and progression [25, 28]. Depending on the location of the mutation within the *TP53* gene, protein structure and subsequent DNA binding activity can be lost or altered, resulting in either loss or gain of function [25]. In contrast to the majority of TSGs, *TP53* is not commonly inactivated by deletions or truncating mutations. Indeed, 74% of mutations within the *TP53* locus are missense point mutations, which can be found in proteins in human tumours [25]. In fact, altered *TP53* was initially considered as a cancer antigen with putative oncogenic properties [25]. Together, this highlights the dichotomous role of *TP53* disruptions, in that both the loss of wild-type p53 and gain-of-function mutations can provide a growth advantage to tumours [28].

Lung cancer is commonly associated with tobacco use, where the prolonged exposure to carcinogens damages the DNA of the exposed cells. These alterations are especially enriched in missense mutations in *TP53*, leading to GOF-p53 [29]. The oncogenic GOF mutation in p53 was previously shown to be related with the inactivation of AMP-activated protein kinase (AMPK) signalling in head and neck cancer and another tobacco-related cancer [30]. AMPK is a master regulator of metabolic homeostasis and GOF-mutated p53 is able to physically interact and inhibit AMPK, stimulating aerobic glycolysis under energetic stress conditions and leading to invasive growth.

In lung cancer mouse models, prevention of tumour formation by inhibiting GOF p53 mutants has been demonstrated [53]. Although the highly aberrant genomes in p53-mutated tumours should lead to unfeasible mitosis, these mutations facilitate the survival and proliferation of these cells through stabilizing replication forks and promoting micronuclei arrangement [31].

GOF p53 mutants are most likely involved in multiple mechanisms that coordinate tumour progression. For example, GOF-p53 (R175H, R273H and D281G) was demonstrated to upregulate *CXCL5, CXCL8* and *CXCL12* through its transcription factor activity, promoting migration of lung cancer cell lines [54]. *CXCL5* expression was shown to be elevated in human lung tumour samples harbouring GOF-p53, and its inhibition could reverse cell motility in lung cancer and melanoma cell lines [54]. In NSCLC, it was recently reported that GOF-p53 can physically interact with HIF-1 and binds to the SWI/SNF chromatin remodelling complex, inducing the expression of hypoxia-responsive genes [55]. Importantly, specific extracellular matrix components are upregulated by this process and mediate pro-tumourigenic features in NSCLC [55].

#### **2.2** *NFIB*

Nuclear factor I (NFI) is a transcription factor family, comprising NFIA, NFIB, NFIC and NFIX, that plays important roles in normal development and in numerous diseases [56]. These proteins bind to specific DNA sequences leading to repression or activation of gene expression in a context-dependent manner, regulating cell differentiation and proliferation through their target genes [57]. *NFIB*, in particular, has been implicated in a wide range of malignancies, being described as both an oncogene and a potential TSG [58].

Using an *in vivo* model, it was demonstrated that NFIB is a metastatic driver in SCLC, inducing global chromatin reprogramming during metastasis [33]. The authors isolated tumour cells from primary and metastatic sites of genetically engineered mice, and using genome-wide analysis, they showed a pronounced increase in chromatin accessibility during tumour progression, resulting from *NFIB* copy number amplifications. Interestingly, the distal regions that became accessible upon *NFIB* upregulation were similar to open regions found in neural tissue. Recently, the same group described two metastatic models in SCLC, one dependent and other independent of *NFIB* amplification [59]. *NFIB* was likewise reported as amplified and/or overexpressed in melanoma [60], breast [61], oesophagus [62] and salivary gland malignancies [63].

A gene fusion involving *NFIB* (*MYB-NFIB*) is frequently found in adenoid cystic carcinomas from salivary glands [64] and in adenoid cystic carcinoma from other topologies [65]. Despite the putative oncogenic function of *NFIB*, studies have focused on its fusion partner *MYB* as the main oncogenic driver in these cancers [66]. Given the fact that other fusion partners of *NFIB* have been reported in adenoid cystic carcinomas [67] and that *MYB-NFIB* fusions lead to *NFIB* truncation [68], *NFIB* may have a possible independent role as a TSG in these malignancies.

While the *MYB-NFIB* fusion is not observed in lung cancers, *NFIB* is frequently underexpressed in NSCLC tissues [32] and during epithelial-to-mesenchymal transition in NSCLC cell lines [69]. NFIB is an essential transcriptional factor in lung development [70] and was demonstrated to be targeted by many microRNAs that recapitulate their foetal lung expression patterns in NSCLC [32]. Lower expression of this gene was associated with shorter overall survival, less-differentiated tumour features and repressed expression of cell differentiation markers in LUAD patients [32]. Therefore, contrary to the established oncogenic role of NFIB in SCLC, these observations suggest a tumour suppressive role in NSCLC.

#### **2.3** *NOTCH* **gene family**

The Notch signalling pathway is important in the regulation of cell fate during embryogenesis and maintenance of homeostasis in adult tissues [71]. It includes Notch receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) and ligands from the DSL family, which suppress or induce tumour-related mechanisms under specific cellular contexts [71].

In SCLC, Notch signalling is frequently inactivated by either a mutation in Notch receptors or the overexpression of ligands that inhibit downstream signalling [34]. Despite this potential role as a TSG, Notch signalling in lung tumours is complex, as it has also been shown to be related to chemoresistance in SCLC [72]. In addition, the overactivation of this pathway through several mechanisms acts like an oncogene in LUAD by preserving stem cell features and promoting proliferation [35, 73]. Notch1 expression is required in Kras-driven LUAD carcinogenesis, suppressing apoptosis via the p53 pathway [35]. The inhibition of the Notch pathway is able to restrain lung cancer stem cell maintenance, which is characterized by subpopulations of cells expressing aldehyde dehydrogenase [74].

Conversely, loss-of-function mutations of Notch receptors generating truncated receptors imply a TSG role in LUSC [75]. Although functional studies to further corroborate this hypothesis are still needed, reports in other squamous cell carcinomas substantiate the idea that the inactivation of this signalling pathway promotes tumourigenesis [76].

#### **2.4** *NKX2-1* **(also known as** *TTF-1***)**

Nkx2-1 is a homeobox-containing transcription factor that is essential for lung development and is expressed in type II pneumocytes and bronchiolar cells in adults [77]. It is expressed in 40–50% of lung cancers and is amplified and overexpressed in 6–11% of LUAD [78].

**51**

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer*

Nkx2-1 acts as a lineage-specific oncogene in some LUAD cases [79], enhancing cell viability and proliferation in lung cancer cell lines [78]. This function relies on the activation of (i) the pro-survival PI3K-AKT pathway, through ROR1 kinasedependent c-Src activation as well as maintaining the EGFR-ERBB3 association [80], and (ii) LMO3, a member of the LMO family of oncogenes that is translocated

On the other hand, *Nkx2-1* expression has been associated with good patient outcome [82] and the loss of *Nkx2-1* expression was associated with the aggressive behaviour of NSCLCs [83]. Mechanistically, tumour suppressive functions of Nkx2-1 in lung adenocarcinoma rely on the restriction of cell motility, invasion and metastatic ability, through the inhibition of the TGF-β [41] and IKK-B/NFk-B [39] pathways. The dual role of Nkx2-1 is dependent on *EGFR, KRAS* and *TP53* status in LUAD: *NKX2-1* acts as a TSG in *KRAS*-driven and *TP53*-mutant tumours, whereas it

*NFE2L2* encodes a transcription factor that regulates proteins involved in cellular defense mechanisms against metabolic, xenobiotic and oxidative stress [86]. *NFE2L2* has been often considered a TSG due to its protective role against genomedamaging agents, the higher propensity to cancer development in *NFE2L2*-deficient

Due to the constant exposure to oxidative stress in the lung, the *NFE2L2* pathway is important to guarantee the genomic stability of these cells [88]. However, once transformation of normal to cancer cells occurs, *NFE2L2* favours tumour development by acting to protect against oxidative stress resulting from the tumour microenvironment and exposure to genotoxic agents during patient treatment [86]. In fact, mutations in *NFE2L2* and *KEAP1,* an important member of the *NFE2L2* signalling, are very common and mutually exclusive in NSCLC [89]. Curiously, a recent study demonstrated that lung cancer patients presenting *NFE2L2* or *KEAP1* mutations are highly resistant to chemotherapy [89]. However, the relation between the *NFE2L2* pathway and treatment response prediction needs further investigation.

While large-scale genomic sequencing efforts have uncovered an invaluable number of genetic alterations related to cancer biology, in the past, they were commonly focused on the 2% of the genome that encodes protein [90]. In the last decade, noncoding RNA transcripts have been shown to have important regulatory functions in normal and disease biology [91]. Indeed, many non-coding genes have been shown to

play tumour-suppressive or oncogenic roles in numerous cancer types [92].

Metastasis-associated lung adenocarcinoma transcript 1 (*MALAT1*) was one of the first cancer-related long non-coding RNAs to be described [93]. *MALAT1* is broadly expressed in normal cells, where it has been shown to regulate the alternative splicing of pre-mRNAs by changing the distribution of splicing regulators in nuclear speckles [94]. *MALAT1* was primarily identified as an oncogenic transcript in lung cancer and has since been widely considered a marker of metastasis, poor patient survival [93] and chemotherapy resistance in NSCLC [48]. Mechanistically, *MALAT1* has been shown to promote carcinogenesis through P53 deacetylation [95] and enhance cell migration through Akt/mTOR signalling [96] and TGF-β-induced endothelial-to-mesenchymal transition [97]. Conversely, *MALAT1* has also been shown to reduce invasiveness by modulating the expression of EpCAM and ITGB4 in PTEN-expressing tumours [47] and by downregulation of MMP2 and inactivation

*DOI: http://dx.doi.org/10.5772/intechopen.85017*

enhances *EGFR*-driven tumourigenesis [84, 85].

**2.6** *MALAT1* **and other non-coding RNAs**

mice and its protective effects in cancer chemoprevention [87].

in T-ALL [81].

**2.5** *NFE2L2*

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.85017*

Nkx2-1 acts as a lineage-specific oncogene in some LUAD cases [79], enhancing cell viability and proliferation in lung cancer cell lines [78]. This function relies on the activation of (i) the pro-survival PI3K-AKT pathway, through ROR1 kinasedependent c-Src activation as well as maintaining the EGFR-ERBB3 association [80], and (ii) LMO3, a member of the LMO family of oncogenes that is translocated in T-ALL [81].

On the other hand, *Nkx2-1* expression has been associated with good patient outcome [82] and the loss of *Nkx2-1* expression was associated with the aggressive behaviour of NSCLCs [83]. Mechanistically, tumour suppressive functions of Nkx2-1 in lung adenocarcinoma rely on the restriction of cell motility, invasion and metastatic ability, through the inhibition of the TGF-β [41] and IKK-B/NFk-B [39] pathways. The dual role of Nkx2-1 is dependent on *EGFR, KRAS* and *TP53* status in LUAD: *NKX2-1* acts as a TSG in *KRAS*-driven and *TP53*-mutant tumours, whereas it enhances *EGFR*-driven tumourigenesis [84, 85].

#### **2.5** *NFE2L2*

*Genes and Cancer*

gland malignancies [63].

**2.3** *NOTCH* **gene family**

specific cellular contexts [71].

tumourigenesis [76].

in 6–11% of LUAD [78].

**2.4** *NKX2-1* **(also known as** *TTF-1***)**

in chromatin accessibility during tumour progression, resulting from *NFIB* copy number amplifications. Interestingly, the distal regions that became accessible upon *NFIB* upregulation were similar to open regions found in neural tissue. Recently, the same group described two metastatic models in SCLC, one dependent and other independent of *NFIB* amplification [59]. *NFIB* was likewise reported as amplified and/or overexpressed in melanoma [60], breast [61], oesophagus [62] and salivary

A gene fusion involving *NFIB* (*MYB-NFIB*) is frequently found in adenoid cystic carcinomas from salivary glands [64] and in adenoid cystic carcinoma from other topologies [65]. Despite the putative oncogenic function of *NFIB*, studies have focused on its fusion partner *MYB* as the main oncogenic driver in these cancers [66]. Given the fact that other fusion partners of *NFIB* have been reported in adenoid cystic carcinomas [67] and that *MYB-NFIB* fusions lead to *NFIB* truncation [68], *NFIB* may have a possible independent role as a TSG in these malignancies. While the *MYB-NFIB* fusion is not observed in lung cancers, *NFIB* is frequently underexpressed in NSCLC tissues [32] and during epithelial-to-mesenchymal transition in NSCLC cell lines [69]. NFIB is an essential transcriptional factor in lung development [70] and was demonstrated to be targeted by many microRNAs that recapitulate their foetal lung expression patterns in NSCLC [32]. Lower expression of this gene was associated with shorter overall survival, less-differentiated tumour features and repressed expression of cell differentiation markers in LUAD patients [32]. Therefore, contrary to the established oncogenic role of NFIB in SCLC, these

The Notch signalling pathway is important in the regulation of cell fate during embryogenesis and maintenance of homeostasis in adult tissues [71]. It includes Notch receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) and ligands from the DSL family, which suppress or induce tumour-related mechanisms under

In SCLC, Notch signalling is frequently inactivated by either a mutation in Notch receptors or the overexpression of ligands that inhibit downstream signalling [34]. Despite this potential role as a TSG, Notch signalling in lung tumours is complex, as it has also been shown to be related to chemoresistance in SCLC [72]. In addition, the overactivation of this pathway through several mechanisms acts like an oncogene in LUAD by preserving stem cell features and promoting proliferation [35, 73]. Notch1 expression is required in Kras-driven LUAD carcinogenesis, suppressing apoptosis via the p53 pathway [35]. The inhibition of the Notch pathway is able to restrain lung cancer stem cell maintenance, which is characterized by subpopula-

Conversely, loss-of-function mutations of Notch receptors generating truncated receptors imply a TSG role in LUSC [75]. Although functional studies to further corroborate this hypothesis are still needed, reports in other squamous cell carcinomas substantiate the idea that the inactivation of this signalling pathway promotes

Nkx2-1 is a homeobox-containing transcription factor that is essential for lung development and is expressed in type II pneumocytes and bronchiolar cells in adults [77]. It is expressed in 40–50% of lung cancers and is amplified and overexpressed

observations suggest a tumour suppressive role in NSCLC.

tions of cells expressing aldehyde dehydrogenase [74].

**50**

*NFE2L2* encodes a transcription factor that regulates proteins involved in cellular defense mechanisms against metabolic, xenobiotic and oxidative stress [86]. *NFE2L2* has been often considered a TSG due to its protective role against genomedamaging agents, the higher propensity to cancer development in *NFE2L2*-deficient mice and its protective effects in cancer chemoprevention [87].

Due to the constant exposure to oxidative stress in the lung, the *NFE2L2* pathway is important to guarantee the genomic stability of these cells [88]. However, once transformation of normal to cancer cells occurs, *NFE2L2* favours tumour development by acting to protect against oxidative stress resulting from the tumour microenvironment and exposure to genotoxic agents during patient treatment [86]. In fact, mutations in *NFE2L2* and *KEAP1,* an important member of the *NFE2L2* signalling, are very common and mutually exclusive in NSCLC [89]. Curiously, a recent study demonstrated that lung cancer patients presenting *NFE2L2* or *KEAP1* mutations are highly resistant to chemotherapy [89]. However, the relation between the *NFE2L2* pathway and treatment response prediction needs further investigation.

#### **2.6** *MALAT1* **and other non-coding RNAs**

While large-scale genomic sequencing efforts have uncovered an invaluable number of genetic alterations related to cancer biology, in the past, they were commonly focused on the 2% of the genome that encodes protein [90]. In the last decade, noncoding RNA transcripts have been shown to have important regulatory functions in normal and disease biology [91]. Indeed, many non-coding genes have been shown to play tumour-suppressive or oncogenic roles in numerous cancer types [92].

Metastasis-associated lung adenocarcinoma transcript 1 (*MALAT1*) was one of the first cancer-related long non-coding RNAs to be described [93]. *MALAT1* is broadly expressed in normal cells, where it has been shown to regulate the alternative splicing of pre-mRNAs by changing the distribution of splicing regulators in nuclear speckles [94]. *MALAT1* was primarily identified as an oncogenic transcript in lung cancer and has since been widely considered a marker of metastasis, poor patient survival [93] and chemotherapy resistance in NSCLC [48]. Mechanistically, *MALAT1* has been shown to promote carcinogenesis through P53 deacetylation [95] and enhance cell migration through Akt/mTOR signalling [96] and TGF-β-induced endothelial-to-mesenchymal transition [97]. Conversely, *MALAT1* has also been shown to reduce invasiveness by modulating the expression of EpCAM and ITGB4 in PTEN-expressing tumours [47] and by downregulation of MMP2 and inactivation

of ERK/MAPK signalling [98]. *MALAT1* also binds the nuclear p65/p50 heterodimer and thus inhibits NF-κB-dependent pathways [99] and is thought to be involved in the response to DNA damage [100]. Furthermore, *MALAT1* reduces the invasiveness of cerebral metastases by sustaining the blood-brain barrier [101]. *MALAT1* expression and subcellular location is finely tuned through various regulatory mechanisms [102], which may drive its pro- or anti-tumour effects [103]. Analysis of the dual role of *MALAT1* highlights not only the complexity of non-coding RNA function but also their relevance to broad areas of cancer biology and management.

MicroRNAs (miRNAs) are short transcripts that typically regulate coding genes post-transcriptionally through direct interaction with mRNA transcripts. Many are deregulated in lung cancer [104], where they have documented tumour-suppressive and oncogenic roles [105]. For example, miRNA-125b has been shown to have a multifaceted function as a tumour suppressor and oncogene, being underexpressed in bladder [106] and ovarian cancer [107] and overexpressed in glioma [108] and prostate cancer [109]. It was shown that miRNA-125b induces apoptosis in cancer cell lines exposed to nutrient starvation and chemotherapy, including in lung cancer [49]. On the other hand, miRNA-125b may also function as an oncogene in NSCLC, as it is able to promote metastasis by targeting TP53INP1 [50]. In addition, inhibition of miR-125b can also decrease the invasive potential and leads to cell cycle arrest and apoptosis in NSCLC [110]. Similarly, miR-378 was reported to be overexpressed in lung cancer and other tumour types, inducing cell migration, invasion and tumour angiogenesis [111]. However, it was previously demonstrated that upregulation of this miRNA sensitizes lung cancer cell lines to cisplatin [51].

#### **3. Conclusions and future directions**

Here, we summarize the commonly disrupted genes in lung cancer with dual roles as both tumour suppressors and oncogenes. These conflicting roles are a result from the complexity of biological pathways and the heterogeneity of cancer cells.

Most of the current molecular therapies are based on hyperactivated oncogene inhibitors. In lung cancer, only a fraction of the cases exhibit alterations in targetable genes, such as *EGFR, BRAF* and *MET* mutations and *ALK, RET* and *ROS1* fusions [112]. Therefore, there is an urgent need for the development of novel therapeutic strategies exploiting non-oncogene alterations of lung tumour cells.

Considering that TSGs are found altered more frequently than oncogenes in human tumours [113], the existence of TSGs with dual oncogenic roles opens a new window of opportunities for the development of new targeted therapies. However, therapeutic action against TSGs remains challenging, as many are not amenable to current pharmacologic inactivation strategies. Most of the TSGs are not a kinase that can be pharmacologically blocked and are not located at the cell surface to be targeted by an antibody.

In summary, there is an unmet need to clarify the ambiguity found within genes, both coding and non-coding, with both pro- and anti-tumour functions. Broadening our understanding of these features may enable the development of novel and specific therapeutic strategies that consider both molecular and tissue contexts.

#### **Acknowledgements**

This work was supported by grants from the Canadian Institutes for Health Research (CIHR FDN-143345) and scholarships from CIHR, the BC Cancer Foundation, the Ligue nationale contre le cancer, the Fonds de Recherche en Santé

**53**

**Author details**

Mateus Camargo Barros-Filho1,2\*

Centre, Vancouver, BC, Canada

Daiana D. Becker-Santos1

Vancouver, BC, Canada

†

, Adam P. Sage1

2 International Research Center, A.C. Camargo Cancer Center, SP, Brazil

3 Pneumology Department, Rouen University Hospital, Rouen, France

University of British Columbia, Vancouver, BC, Canada

\*Address all correspondence to: mbarros@bccrc.ca

† These authors contributed equally to this work

4 Department of Cancer Control Research, British Columbia Cancer Research

5 Department of Oral and Biological Medical Sciences, Faculty of Dentistry,

, Florian Guisier1,3†

1 Department of Integrative Oncology, British Columbia Cancer Research Centre,

, Erin A. Marshall1

, Leigha D. Rock1,4,5†

and Wan L. Lam1

,

provided the original work is properly cited.

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer*

Respiratoire (appel d'offres 2018 emis en commun avec la Fondation du Souffle), the Fondation Charles Nicolle and the São Paulo Research Foundation (FAPESP 2015/17707-5 and 2018/06138-8). D.D.B.S. and E.A.M. are Vanier Canada Scholars.

*DOI: http://dx.doi.org/10.5772/intechopen.85017*

The authors have no conflicts to declare.

**Conflict of interest**

© 2019 The Author(s). Licensee IntechOpen. 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,

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.85017*

Respiratoire (appel d'offres 2018 emis en commun avec la Fondation du Souffle), the Fondation Charles Nicolle and the São Paulo Research Foundation (FAPESP 2015/17707-5 and 2018/06138-8). D.D.B.S. and E.A.M. are Vanier Canada Scholars.

### **Conflict of interest**

*Genes and Cancer*

of ERK/MAPK signalling [98]. *MALAT1* also binds the nuclear p65/p50 heterodimer and thus inhibits NF-κB-dependent pathways [99] and is thought to be involved in the response to DNA damage [100]. Furthermore, *MALAT1* reduces the invasiveness of cerebral metastases by sustaining the blood-brain barrier [101]. *MALAT1* expression and subcellular location is finely tuned through various regulatory mechanisms [102], which may drive its pro- or anti-tumour effects [103]. Analysis of the dual role of *MALAT1* highlights not only the complexity of non-coding RNA function but also

MicroRNAs (miRNAs) are short transcripts that typically regulate coding genes post-transcriptionally through direct interaction with mRNA transcripts. Many are deregulated in lung cancer [104], where they have documented tumour-suppressive and oncogenic roles [105]. For example, miRNA-125b has been shown to have a multifaceted function as a tumour suppressor and oncogene, being underexpressed in bladder [106] and ovarian cancer [107] and overexpressed in glioma [108] and prostate cancer [109]. It was shown that miRNA-125b induces apoptosis in cancer cell lines exposed to nutrient starvation and chemotherapy, including in lung cancer [49]. On the other hand, miRNA-125b may also function as an oncogene in NSCLC, as it is able to promote metastasis by targeting TP53INP1 [50]. In addition, inhibition of miR-125b can also decrease the invasive potential and leads to cell cycle arrest and apoptosis in NSCLC [110]. Similarly, miR-378 was reported to be overexpressed in lung cancer and other tumour types, inducing cell migration, invasion and tumour angiogenesis [111]. However, it was previously demonstrated that upregulation of this miRNA sensitizes lung cancer cell lines to cisplatin [51].

Here, we summarize the commonly disrupted genes in lung cancer with dual roles as both tumour suppressors and oncogenes. These conflicting roles are a result from the complexity of biological pathways and the heterogeneity of cancer cells. Most of the current molecular therapies are based on hyperactivated oncogene inhibitors. In lung cancer, only a fraction of the cases exhibit alterations in targetable genes, such as *EGFR, BRAF* and *MET* mutations and *ALK, RET* and *ROS1* fusions [112]. Therefore, there is an urgent need for the development of novel therapeutic strategies exploiting non-oncogene alterations of lung tumour cells. Considering that TSGs are found altered more frequently than oncogenes in human tumours [113], the existence of TSGs with dual oncogenic roles opens a new window of opportunities for the development of new targeted therapies. However, therapeutic action against TSGs remains challenging, as many are not amenable to current pharmacologic inactivation strategies. Most of the TSGs are not a kinase that can be pharmacologically blocked and are not located at the cell surface to be

In summary, there is an unmet need to clarify the ambiguity found within genes, both coding and non-coding, with both pro- and anti-tumour functions. Broadening our understanding of these features may enable the development of novel and specific therapeutic strategies that consider both molecular and tissue contexts.

This work was supported by grants from the Canadian Institutes for Health Research (CIHR FDN-143345) and scholarships from CIHR, the BC Cancer Foundation, the Ligue nationale contre le cancer, the Fonds de Recherche en Santé

their relevance to broad areas of cancer biology and management.

**3. Conclusions and future directions**

targeted by an antibody.

**Acknowledgements**

**52**

The authors have no conflicts to declare.

#### **Author details**

Mateus Camargo Barros-Filho1,2\* † , Florian Guisier1,3† , Leigha D. Rock1,4,5† , Daiana D. Becker-Santos1 , Adam P. Sage1 , Erin A. Marshall1 and Wan L. Lam1

1 Department of Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada

2 International Research Center, A.C. Camargo Cancer Center, SP, Brazil

3 Pneumology Department, Rouen University Hospital, Rouen, France

4 Department of Cancer Control Research, British Columbia Cancer Research Centre, Vancouver, BC, Canada

5 Department of Oral and Biological Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada

\*Address all correspondence to: mbarros@bccrc.ca

† These authors contributed equally to this work

© 2019 The Author(s). Licensee IntechOpen. 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.

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[89] Frank R, Scheffler M, Merkelbach-Bruse S, Ihle MA, Kron A, Rauer M, et al. Clinical and pathological characteristics of KEAP1- and NFE2L2 mutated non-small cell lung carcinoma (NSCLC). Clinical Cancer Research. 2018;**24**(13):3087-3096

[90] Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al. The transcriptional landscape of the mammalian genome. Science. 2005;**309**(5740):1559-1563

[91] Esteller M. Non-coding RNAs in human disease. Nature Reviews Genetics. 2011;**12**(12):861-874

[92] Zhang W, Bojorquez-Gomez A, Velez DO, Xu G, Sanchez KS, Shen JP, et al. A global transcriptional network connecting noncoding mutations to changes in tumor gene expression. Nature Genetics. 2018;**50**(4):613-620

[93] Ji P, Diederichs S, Wang W, Boing S, Metzger R, Schneider PM, et al. MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene. 2003;**22**(39):8031-8041

[94] Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, et al. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Molecular Cell. 2010;**39**(6):925-938

[95] Chen R, Liu Y, Zhuang H, Yang B, Hei K, Xiao M, et al. Quantitative proteomics reveals that long non-coding RNA MALAT1 interacts with DBC1 to regulate p53 acetylation. Nucleic Acids Research. 2017;**45**(17):9947-9959

[96] Tang Y, Xiao G, Chen Y, Deng Y. LncRNA MALAT1 promotes migration and invasion of non-small-cell lung cancer by targeting miR-206 and activating Akt/mTOR signaling. Anti-Cancer Drugs. 2018;**29**(8):725-735

[97] Xiang Y, Zhang Y, Tang Y, Li Q. MALAT1 modulates TGF-beta1 induced endothelial-to-mesenchymal transition through downregulation of miR-145. Cellular Physiology and Biochemistry. 2017;**42**(1):357-372

[98] Han Y, Wu Z, Wu T, Huang Y, Cheng Z, Li X, et al. Tumorsuppressive function of long noncoding RNA MALAT1 in glioma cells by downregulation of MMP2 and inactivation of ERK/MAPK signaling. Cell Death & Disease. 2016;**7**:e2123

[99] Zhao G, Su Z, Song D, Mao Y, Mao X. The long noncoding RNA MALAT1 regulates the lipopolysaccharideinduced inflammatory response through its interaction with NF-kappaB. FEBS Letters. 2016;**590**(17):2884-2895

[100] Gao C, He Z, Li J, Li X, Bai Q, Zhang Z, et al. Specific long noncoding RNAs response to occupational PAHs exposure in coke oven workers. Toxicology Reports. 2016;**3**:160-166

[101] Ma J, Wang P, Yao Y, Liu Y, Li Z, Liu X, et al. Knockdown of long noncoding RNA MALAT1 increases the blood-tumor barrier permeability by up-regulating miR-140. Biochimica et Biophysica Acta. 2016;**1859**(2):324-338

[102] Gutschner T, Hammerle M, Diederichs S. MALAT1—A paradigm for long noncoding RNA function in

cancer. Journal of Molecular Medicine. 2013;**91**(7):791-801

[103] Guo F, Guo L, Li Y, Zhou Q, Li Z. MALAT1 is an oncogenic long non-coding RNA associated with tumor invasion in non-small cell lung cancer regulated by DNA methylation. International Journal of Clinical and Experimental Pathology. 2015;**8**(12):15903-15910

[104] Cinegaglia NC, Andrade SC, Tokar T, Pinheiro M, Severino FE, Oliveira RA, et al. Integrative transcriptome analysis identifies deregulated microRNA-transcription factor networks in lung adenocarcinoma. Oncotarget. 2016;**7**(20):28920-28934

[105] Svoronos AA, Engelman DM, Slack FJ. OncomiR or tumor suppressor? The duplicity of micrornas in cancer. Cancer Research. 2016;**76**(13):3666-3670

[106] Ichimi T, Enokida H, Okuno Y, Kunimoto R, Chiyomaru T, Kawamoto K, et al. Identification of novel microRNA targets based on microRNA signatures in bladder cancer. International Journal of Cancer. 2009;**125**(2):345-352

[107] Guan Y, Yao H, Zheng Z, Qiu G, Sun K. MiR-125b targets BCL3 and suppresses ovarian cancer proliferation. International Journal of Cancer. 2011;**128**(10):2274-2283

[108] Pogue AI, Cui JG, Li YY, Zhao Y, Culicchia F, Lukiw WJ. Micro RNA-125b (miRNA-125b) function in astrogliosis and glial cell proliferation. Neuroscience Letters. 2010;**476**(1):18-22

[109] Ozen M, Creighton CJ, Ozdemir M, Ittmann M. Widespread deregulation of microRNA expression in human prostate cancer. Oncogene. 2008;**27**(12):1788-1793

[110] Wang X, Zhang Y, Fu Y, Zhang J, Yin L, Pu Y, et al. MicroRNA-125b may

**61**

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.85017*

function as an oncogene in lung cancer cells. Molecular Medicine Reports.

[111] Ho CS, Yap SH, Phuah NH, In LL, Hasima N. MicroRNAs associated with tumour migration, invasion and angiogenic properties in A549 and SK-Lu1 human lung adenocarcinoma cells. Lung Cancer. 2014;**83**(2):154-162

[112] Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;**511**(7511):543-550

[113] Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science. 2013;**339**(6127):1546-1558

2015;**11**(5):3880-3887

*Tumour Suppressor Genes with Oncogenic Roles in Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.85017*

function as an oncogene in lung cancer cells. Molecular Medicine Reports. 2015;**11**(5):3880-3887

*Genes and Cancer*

[95] Chen R, Liu Y, Zhuang H, Yang B, Hei K, Xiao M, et al. Quantitative proteomics reveals that long non-coding RNA MALAT1 interacts with DBC1 to regulate p53 acetylation. Nucleic Acids Research. 2017;**45**(17):9947-9959

cancer. Journal of Molecular Medicine.

[104] Cinegaglia NC, Andrade SC, Tokar T, Pinheiro M, Severino FE, Oliveira RA, et al. Integrative transcriptome analysis identifies deregulated microRNA-transcription factor networks in lung adenocarcinoma. Oncotarget. 2016;**7**(20):28920-28934

[105] Svoronos AA, Engelman DM, Slack FJ. OncomiR or tumor suppressor? The duplicity of micrornas in cancer. Cancer Research. 2016;**76**(13):3666-3670

[106] Ichimi T, Enokida H, Okuno Y, Kunimoto R, Chiyomaru T, Kawamoto K, et al. Identification of novel microRNA targets based on microRNA signatures in bladder cancer. International Journal of Cancer.

[107] Guan Y, Yao H, Zheng Z, Qiu G, Sun K. MiR-125b targets BCL3 and suppresses ovarian cancer proliferation.

[108] Pogue AI, Cui JG, Li YY, Zhao Y, Culicchia F, Lukiw WJ. Micro RNA-125b (miRNA-125b) function in astrogliosis and glial cell proliferation. Neuroscience

Ozdemir M, Ittmann M. Widespread deregulation of microRNA expression in human prostate cancer. Oncogene.

[110] Wang X, Zhang Y, Fu Y, Zhang J, Yin L, Pu Y, et al. MicroRNA-125b may

International Journal of Cancer.

2011;**128**(10):2274-2283

Letters. 2010;**476**(1):18-22

[109] Ozen M, Creighton CJ,

2008;**27**(12):1788-1793

2009;**125**(2):345-352

[103] Guo F, Guo L, Li Y, Zhou Q, Li Z. MALAT1 is an oncogenic long non-coding RNA associated with tumor invasion in non-small cell lung cancer regulated by DNA methylation. International Journal of Clinical and Experimental Pathology.

2013;**91**(7):791-801

2015;**8**(12):15903-15910

[96] Tang Y, Xiao G, Chen Y, Deng Y. LncRNA MALAT1 promotes migration and invasion of non-small-cell lung cancer by targeting miR-206 and activating Akt/mTOR signaling. Anti-Cancer Drugs. 2018;**29**(8):725-735

[97] Xiang Y, Zhang Y, Tang Y, Li Q. MALAT1 modulates TGF-beta1 induced endothelial-to-mesenchymal transition through downregulation of miR-145. Cellular Physiology and Biochemistry. 2017;**42**(1):357-372

[98] Han Y, Wu Z, Wu T, Huang Y, Cheng Z, Li X, et al. Tumorsuppressive function of long noncoding RNA MALAT1 in glioma cells by downregulation of MMP2 and inactivation of ERK/MAPK signaling. Cell Death & Disease. 2016;**7**:e2123

[99] Zhao G, Su Z, Song D, Mao Y, Mao X. The long noncoding RNA MALAT1 regulates the lipopolysaccharide-

induced inflammatory response through its interaction with NF-kappaB. FEBS Letters. 2016;**590**(17):2884-2895

[100] Gao C, He Z, Li J, Li X, Bai Q, Zhang Z, et al. Specific long noncoding RNAs response to occupational PAHs exposure in coke oven workers. Toxicology Reports. 2016;**3**:160-166

[101] Ma J, Wang P, Yao Y, Liu Y, Li Z, Liu X, et al. Knockdown of long noncoding RNA MALAT1 increases the blood-tumor barrier permeability by up-regulating miR-140. Biochimica et Biophysica Acta. 2016;**1859**(2):324-338

[102] Gutschner T, Hammerle M, Diederichs S. MALAT1—A paradigm for long noncoding RNA function in

**60**

[111] Ho CS, Yap SH, Phuah NH, In LL, Hasima N. MicroRNAs associated with tumour migration, invasion and angiogenic properties in A549 and SK-Lu1 human lung adenocarcinoma cells. Lung Cancer. 2014;**83**(2):154-162

[112] Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;**511**(7511):543-550

[113] Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science. 2013;**339**(6127):1546-1558

**63**

**Chapter 4**

Cancer

*Amal Qattan*

**1. Introduction**

**Abstract**

Duplicitous Dispositions of

Micro-RNAs (miRs) in Breast

In 1993, a gene silencer known as lin-4 was first discovered in *Caenorhabditis elegans* and demonstrated to be critical for larval development. Lin-4 belongs to a family of signaling molecules known as non-protein coding microRNAs (miRNAs) which are not only highly conserved in humans, but also involved in the fundamental processes of oncogenesis. While miRNAs are not translated to proteins themselves, they are capable of regulating the expression and translation of other genes thus affecting a multitude of biological and pathological pathways as well as those essential to the malignant landscape. The aim of this chapter is to explore the diverse roles of miRNAs in the context of breast cancer. Following a brief overview of miRNA biogenesis, this chapter covers the production of miRNAs by tumor cells and stromal cells, onco-suppressor miRNAs, use as therapeutics, contribution to

**Keywords:** microRNAs (miRs), breast cancer epigenetic alteration, microRNA-based therapy, miRNA pharmacogenomics, miRSNPs, miR-polymorphisms, clinical trials

A gene silencer known as *lin-4* was first discovered in *Caenorhabditis elegans* and demonstrated to be critical for larval development [1]. *Lin-4* belongs to a family of signaling molecules known as non-protein coding microRNA (miRNAs) which are not only highly conserved in humans, but also involved in the fundamental processes of oncogenesis [2]. Approximately 2000 miRNAs are present in the human genome [3]. While miRNAs are not translated into proteins themselves, they are implicated in the regulation of 30% of all genes and are thereby capable of regulating the expression and translation of other genes influencing a multitude of biological and pathological pathways [4]. This chapter explores the diverse roles of miRNAs in the most frequent cancer among women in the world: breast cancer (BC). BC impacts 2.1 million women yearly [5] and it also causes the greatest number of cancer-related deaths among women. Early detection and diagnosis are critical to survival. In the context of BC, miRNAs are dynamically regulated implicating their use in diagnosis, prognosis and tracking of drug efficacy during treatment. Following a brief overview of miRNA biogenesis, this chapter covers the production of miRNAs by tumor cells, onco-suppressor and tumor-suppressor miRNAs, their contribution to therapeutic resistance, therapeutic miRNAs (as well as therapeutics targeting of miRNAs), and finally their emerging role as biomarkers for BC prognosis, treatment responsiveness and efficacy.

therapeutic resistance, and finally their emerging role as biomarkers.

#### **Chapter 4**

## Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer

*Amal Qattan*

#### **Abstract**

In 1993, a gene silencer known as lin-4 was first discovered in *Caenorhabditis elegans* and demonstrated to be critical for larval development. Lin-4 belongs to a family of signaling molecules known as non-protein coding microRNAs (miRNAs) which are not only highly conserved in humans, but also involved in the fundamental processes of oncogenesis. While miRNAs are not translated to proteins themselves, they are capable of regulating the expression and translation of other genes thus affecting a multitude of biological and pathological pathways as well as those essential to the malignant landscape. The aim of this chapter is to explore the diverse roles of miRNAs in the context of breast cancer. Following a brief overview of miRNA biogenesis, this chapter covers the production of miRNAs by tumor cells and stromal cells, onco-suppressor miRNAs, use as therapeutics, contribution to therapeutic resistance, and finally their emerging role as biomarkers.

**Keywords:** microRNAs (miRs), breast cancer epigenetic alteration, microRNA-based therapy, miRNA pharmacogenomics, miRSNPs, miR-polymorphisms, clinical trials

#### **1. Introduction**

A gene silencer known as *lin-4* was first discovered in *Caenorhabditis elegans* and demonstrated to be critical for larval development [1]. *Lin-4* belongs to a family of signaling molecules known as non-protein coding microRNA (miRNAs) which are not only highly conserved in humans, but also involved in the fundamental processes of oncogenesis [2]. Approximately 2000 miRNAs are present in the human genome [3]. While miRNAs are not translated into proteins themselves, they are implicated in the regulation of 30% of all genes and are thereby capable of regulating the expression and translation of other genes influencing a multitude of biological and pathological pathways [4]. This chapter explores the diverse roles of miRNAs in the most frequent cancer among women in the world: breast cancer (BC). BC impacts 2.1 million women yearly [5] and it also causes the greatest number of cancer-related deaths among women. Early detection and diagnosis are critical to survival. In the context of BC, miRNAs are dynamically regulated implicating their use in diagnosis, prognosis and tracking of drug efficacy during treatment. Following a brief overview of miRNA biogenesis, this chapter covers the production of miRNAs by tumor cells, onco-suppressor and tumor-suppressor miRNAs, their contribution to therapeutic resistance, therapeutic miRNAs (as well as therapeutics targeting of miRNAs), and finally their emerging role as biomarkers for BC prognosis, treatment responsiveness and efficacy.

#### **2. MiRNA biogenesis and mechanisms of action**

Since 1993, researchers have proceeded to learn that miRNAs were of ancient evolutionary origins. Single stranded, non-protein coding miRNAs with genetic suppression activities were found in algae, plants, invertebrates, vertebrates and even viruses [6]. Further characterization has revealed that miRNAs are not only critical for normal human development, but their aberrant expression is associated with diseases such as cancer [7, 8].

The miRNAs are encoded by genetic sequences which may be located within the introns of protein coding genes as well as in the exons and introns of long noncoding RNAs, and even intergenic regions [9]. According to the miRIAD database, 1157 (61.5%) miRNAs are intragenic (169 exonic and 988 intronic) and 724 (38.5%) are intergenic [10]. MiRNA's are single-stranded RNA transcripts that are transcribed from DNA sequences and are usually around 22 nucleotides in length. They often form distinct secondary folding conformational motifs. Most miRNAs are first transcribed into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and mature miRNAs. Usually they bind to the 3′-untranslated region (UTR) of target mRNAs to suppress their target's expression by inhibiting its translation. However, they can also interact with coding sequences, the 5′UTR and gene promoter regions. Though less common, some are involved in the translation activation and stabilization of target transcripts. Furthermore, the shuttling of miRNAs between different cellular compartments can also control rates of transcription and translation of their targets.

In the canonical pathway of miRNA biogenesis, RNA polymerase II transcribes miRNAs into primary miRNAs (pri-miRNAs) greater than 200 nucleotides long. Pri-mRNAs are then cleaved into pre-mRNAs by the RNAse III enzyme, Drosha with the help of double stranded RNA binding proteins Pasha and DiGeorge Syndrome Critical Region 8 (DGCR8). The 60–70 nucleotides long pre-mRNAs are then exported out of the nucleus and into the cell cytoplasm by exportin-5 and Ran GTPase. Once in the cytoplasm, pre-mRNAs are cleaved by the RNAse III enzyme Dicer which removes hairpin loops resulting in miRNA duplexes composed of a guide strand and a passenger strand. The passenger strand is discarded and the guide strand associates with Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) which brings the miRNA to its target mRNA. A 6–8 nucleotide sequence on the miRNA, referred to as the "seed sequence" locates the corresponding sequence of the target mRNA. A double stranded complex is formed which impedes the ribosome from translating the target [11]. Imperfect complementarity between the seed sequence and the target mRNA can also cause target degradation indirectly via deadenylation at the 3′-UTR. Non-canonical miRNA biogenesis is less common and can generally be grouped into Drosha/DGCR8-independent and Dicer-independent pathways which are outside the scope of this chapter. In addition to the inhibition of target miRNAs, there is evidence indicating that some miRNAs directly increase target translation via recruitment of protein synthesis complexes to the translation initiation region. Alternatively, target mRNA expression can also be increased due to inhibition of modulating repressors that block translation. Moreover, some miRNAs enhance ribosome biogenesis resulting in increased protein synthesis [12].

In summary, miRNA biogenesis is a multi-step process that requires various enzymes and shuttling proteins to reach a final product. Mature miRNAs are either stable molecules with half-lives of greater than 24 h or they display shorter half-lives of less than 12 h, depending on the functionality of the product [13]. More on the regulation of miRNA expression is discussed in the next section.

**65**

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

to dysregulated expression of miRNAs by cancer cells.

in organs such as the liver and heart [22].

**4. miRNAs (miRs) production by breast cancer cells**

In general, just as protein-coding genes are regulated by transcription factors (TF), TFs are one of the central ways by which miRNA expression is regulated. Tissue and developmental stage specific TFs can control the transcription of miRNA genes. Many miRNAs and TFs form autoregulatory loops, in which they mutually regulate each other [14]. In addition, various physiological and pathological stimuli, such as steroid hormones, retinoids, hypoxia, interferons, stress, as well as estrogen, can affect miRNA expression [15]. Finally, while transcription regulates the magnitude of miRNA expression, decay rates influence miRNA dynamic regulation. Slow decay leads to a high level of accumulation while fast decay leads quick changes in miRNA expression levels implying that fast turnover may be involved in

Epigenetic mechanisms are heritable changes in gene expression that occur without any modifications in the DNA sequence itself and include DNA methylation and histone modifications as well as miRNAs themselves [16]. The covalent binding of methyl groups to cytosine bases located among CpG dinucleotide sequences is the major modification of eukaryotic genomes which results in down regulation of gene expression. DNA methylation controls embryonic cell fate lineages and prevents reversion to an undifferentiated state [17]. Frequency of methylation is nearly one order of magnitude higher in human miRNA genes compared to the methylation of other protein-coding genes [18, 19]. This indicates strict epigenetic control of miRNA expression and also reveals how epigenetic changes in cancer cells can lead

Genome variations include genetic mutations and polymorphisms; defined as a DNA variation in which a possible sequence is present in at least 1% of people. Single nucleotide polymorphisms (SNPs) constitute approximately 1% of the human genome. SNPs contribute to phenotypic diversity within a species as well as disease susceptibility. MiRSNPs/miR-polymorphisms are a new mechanism and novel class of functional SNPs. As miRNA molecular interactions with their targets are affected via base pairing as well as genetic variation, such as changes in genome sequence; which influences binding energy and annealing strength, SNPs can result in no change, off target or absence of miRNA binding to the predicted target [20]. Carcinogens such as those from cigarettes, dietary elements and other foreign chemical toxicities referred to as "xenobiotics," can also affect miRNA expression. Importantly, many more changes in miRNA expression were observed in cancertarget tissues than in the non-target tissues following acute or chronic exposure to carcinogens thus implicating their use as potential biomarkers for exposure to xenobiotics [21]. Finally, circadian rhythm control of miRNA expression has significant consequences for circadian timing as some miRNAs have promoter sequences inducible by circadian clock proteins. Moreover, some miRNAs can even be regulated by light and dark cycles which confer important rhythmic expressions

In summary, miRNA regulation is similar to other protein coding gene regulation as changes in expression can occur based on the presences or exposure to TFs, genetic polymorphisms, epigenetic factors, xenobiotics and carcinogens. How miRNA expression is regulated in the context of BC is discussed in the next section.

As summarized above, TF, SNPs, epigenetics, hormones and xenobiotics all affect the regulation of miRNAs; therefore, it is not surprising that breast cancer

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

**3. Regulation of miRNA expression**

transient biological processes.

#### **3. Regulation of miRNA expression**

*Genes and Cancer*

targets.

**2. MiRNA biogenesis and mechanisms of action**

with diseases such as cancer [7, 8].

Since 1993, researchers have proceeded to learn that miRNAs were of ancient evolutionary origins. Single stranded, non-protein coding miRNAs with genetic suppression activities were found in algae, plants, invertebrates, vertebrates and even viruses [6]. Further characterization has revealed that miRNAs are not only critical for normal human development, but their aberrant expression is associated

The miRNAs are encoded by genetic sequences which may be located within the introns of protein coding genes as well as in the exons and introns of long noncoding RNAs, and even intergenic regions [9]. According to the miRIAD database, 1157 (61.5%) miRNAs are intragenic (169 exonic and 988 intronic) and 724 (38.5%) are intergenic [10]. MiRNA's are single-stranded RNA transcripts that are transcribed from DNA sequences and are usually around 22 nucleotides in length. They often form distinct secondary folding conformational motifs. Most miRNAs are first transcribed into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and mature miRNAs. Usually they bind to the 3′-untranslated region (UTR) of target mRNAs to suppress their target's expression by inhibiting its translation. However, they can also interact with coding sequences, the 5′UTR and gene promoter regions. Though less common, some are involved in the translation activation and stabilization of target transcripts. Furthermore, the shuttling of miRNAs between different cellular compartments can also control rates of transcription and translation of their

In the canonical pathway of miRNA biogenesis, RNA polymerase II transcribes miRNAs into primary miRNAs (pri-miRNAs) greater than 200 nucleotides long. Pri-mRNAs are then cleaved into pre-mRNAs by the RNAse III enzyme, Drosha with the help of double stranded RNA binding proteins Pasha and DiGeorge Syndrome Critical Region 8 (DGCR8). The 60–70 nucleotides long pre-mRNAs are then exported out of the nucleus and into the cell cytoplasm by exportin-5 and Ran GTPase. Once in the cytoplasm, pre-mRNAs are cleaved by the RNAse III enzyme Dicer which removes hairpin loops resulting in miRNA duplexes composed of a guide strand and a passenger strand. The passenger strand is discarded and the guide strand associates with Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) which brings the miRNA to its target mRNA. A 6–8 nucleotide sequence on the miRNA, referred to as the "seed sequence" locates the corresponding sequence of the target mRNA. A double stranded complex is formed which impedes the ribosome from translating the target [11]. Imperfect complementarity between the seed sequence and the target mRNA can also cause target degradation indirectly via deadenylation at the 3′-UTR. Non-canonical miRNA biogenesis is less common and can generally be grouped into Drosha/DGCR8-independent and Dicer-independent pathways which are outside the scope of this chapter. In addition to the inhibition of target miRNAs, there is evidence indicating that some miRNAs directly increase target translation via recruitment of protein synthesis complexes to the translation initiation region. Alternatively, target mRNA expression can also be increased due to inhibition of modulating repressors that block translation. Moreover, some miRNAs enhance ribosome biogenesis resulting in increased

In summary, miRNA biogenesis is a multi-step process that requires various enzymes and shuttling proteins to reach a final product. Mature miRNAs are either stable molecules with half-lives of greater than 24 h or they display shorter half-lives of less than 12 h, depending on the functionality of the product [13]. More on the

regulation of miRNA expression is discussed in the next section.

**64**

protein synthesis [12].

In general, just as protein-coding genes are regulated by transcription factors (TF), TFs are one of the central ways by which miRNA expression is regulated. Tissue and developmental stage specific TFs can control the transcription of miRNA genes. Many miRNAs and TFs form autoregulatory loops, in which they mutually regulate each other [14]. In addition, various physiological and pathological stimuli, such as steroid hormones, retinoids, hypoxia, interferons, stress, as well as estrogen, can affect miRNA expression [15]. Finally, while transcription regulates the magnitude of miRNA expression, decay rates influence miRNA dynamic regulation. Slow decay leads to a high level of accumulation while fast decay leads quick changes in miRNA expression levels implying that fast turnover may be involved in transient biological processes.

Epigenetic mechanisms are heritable changes in gene expression that occur without any modifications in the DNA sequence itself and include DNA methylation and histone modifications as well as miRNAs themselves [16]. The covalent binding of methyl groups to cytosine bases located among CpG dinucleotide sequences is the major modification of eukaryotic genomes which results in down regulation of gene expression. DNA methylation controls embryonic cell fate lineages and prevents reversion to an undifferentiated state [17]. Frequency of methylation is nearly one order of magnitude higher in human miRNA genes compared to the methylation of other protein-coding genes [18, 19]. This indicates strict epigenetic control of miRNA expression and also reveals how epigenetic changes in cancer cells can lead to dysregulated expression of miRNAs by cancer cells.

Genome variations include genetic mutations and polymorphisms; defined as a DNA variation in which a possible sequence is present in at least 1% of people. Single nucleotide polymorphisms (SNPs) constitute approximately 1% of the human genome. SNPs contribute to phenotypic diversity within a species as well as disease susceptibility. MiRSNPs/miR-polymorphisms are a new mechanism and novel class of functional SNPs. As miRNA molecular interactions with their targets are affected via base pairing as well as genetic variation, such as changes in genome sequence; which influences binding energy and annealing strength, SNPs can result in no change, off target or absence of miRNA binding to the predicted target [20]. Carcinogens such as those from cigarettes, dietary elements and other foreign chemical toxicities referred to as "xenobiotics," can also affect miRNA expression. Importantly, many more changes in miRNA expression were observed in cancertarget tissues than in the non-target tissues following acute or chronic exposure to carcinogens thus implicating their use as potential biomarkers for exposure to xenobiotics [21]. Finally, circadian rhythm control of miRNA expression has significant consequences for circadian timing as some miRNAs have promoter sequences inducible by circadian clock proteins. Moreover, some miRNAs can even be regulated by light and dark cycles which confer important rhythmic expressions in organs such as the liver and heart [22].

In summary, miRNA regulation is similar to other protein coding gene regulation as changes in expression can occur based on the presences or exposure to TFs, genetic polymorphisms, epigenetic factors, xenobiotics and carcinogens. How miRNA expression is regulated in the context of BC is discussed in the next section.

#### **4. miRNAs (miRs) production by breast cancer cells**

As summarized above, TF, SNPs, epigenetics, hormones and xenobiotics all affect the regulation of miRNAs; therefore, it is not surprising that breast cancer (BC) leads to significant, dynamic changes in miRNA expression both by tumor cells and by surrounding stromal cells. This section describes BC tumor cell production of miRNAs as well as the surrounding non-cancerous stromal cells. In general, miRNAs either support or suppress tumorigenesis and are often dysregulated due to tumor-specific epigenetic changes. Likewise, tumor secreted factors such as exosomes and cytokines can also lead to aberrant signaling in the surrounding stromal cells. Furthermore, while all BCs begin in the breast, there are many subtypes which are named to reflect their particular molecular pathogenesis. Subtype diagnosis can help select appropriate therapies. Likewise, aberrant regulation of miRNAs can be subtype specific. Therefore, this section begins with a brief overview of cancer subtypes.

Breast carcinoma can begin either in the ducts or the lobules and as such, termed either ductal carcinoma in situ (DCIS), or lobular carcinoma in situ (LCIS). Both can either stay contained to the area or travel to surrounding tissue and lymph nodes in which case the clinical diagnosis is either invasive ductal carcinoma (IDC) or invasive lobular carcinoma (ILC). IDC is the most common type of BC (50–75%) followed by ILC (5–15%) [23]. Rare BC is characterized by tumor origination in the mucinous, papillary, medullary or cribriform compartments of the breast [24]. Metastasis of breast cancer to other organs is the main cause of mortality and up to 5% of patients will already have experienced metastasis at the time of diagnosis [25].

MiRNA microarray performed on 1542 breast tissue samples procured via the Molecular Taxonomy of Breast Cancer International Consortium and the Akershus University Hospital (AHUS) revealed that no miRNAs were differentially expressed in DCIS patients relative to IDC, supporting the idea that miRNA dysregulation occurs at an early stage of BC development [26]. Among the invasive subtypes, however, expression of seven miRNAs was consistently downregulated, including tumor suppressors let-7c-5p, miR-125b-5p, miR140-3p, miR-145-3p, miR-145-5p, miR-193a-5p, and miR378a-3p while expression of four oncogenic miRNAs was consistently upregulated including miR-106b-5p, miR-142, miR-342-3p, and miR425-5p. Taken together these miRNAs may significantly contribute to the transition to an invasive BC subtype [26].

While Bloom and Richardson's histologic grading system which was modified by Elston and Ellis in 1991 is the most commonly used system to gain prognostic insight, hormone receptors status, tumor size, nodal status and whether tumorous cells have invaded the lymph or blood vessels is also considered during initial diagnosis. Hormone receptor statuses including estrogen receptor (ER) and progesterone receptor (PR) as well as the tyrosine kinase receptor, human epidermal growth receptor type two (HER2) are always measured on newly diagnosed invasive BCs. Subtypes are identified via immunohistochemical staining for hormone receptors, HER2 expression status, and Ki-67 proliferation index as: luminal A (ER-positive and/or PR-positive, HER2-negative, low proliferation), luminal B (ER-positive and/or PR-positive, HER2-negative, high proliferation; or hormone receptor (HR) positive and HER2-positive), HER2-positive (HR-negative and HER2-positive) and finally TNBC type (HR-negative and HER2-negative) [27]. ER+ breast cancer subtype is particularly prevalent in postmenopausal women taking hormone replacement therapy (HRT) which activates the transcription factor estrogen receptor alpha (ERα) which promotes the expression of numerous oncogenic genes. While ERα-signaling is targeted by miRNAs for degradation, aberrant activation of this receptor leads to aberrant expression of miRNAs controlled by ERα-signaling [28].

Several miRNAs are both tissue and cancer specific. As the primary role of miRNA is to decrease target mRNA expression, miRNAs that are upregulated by

**67**

observed [35].

resistant is listed in **Table 1**.

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

cancerous cells are often those that support cancer growth and are referred to as oncomiRs. miR-10b, miR-21 and miR-155 are well characterized oncomiRs in BC [29]. Their main role is to downregulate tumor suppressor genes which results in the promotion of cancer cell proliferation, de-differentiation and invasion [30]. BC cells also produce less tumor-suppressor miRNAs (miR-31, miR-125b, miR-200 and miR-205) which downregulate oncogenic proteins. Cancer-initiating cells (CSCs) were first isolated from breast cancer tumors and are considered the seed-cells of tumor development [31]. While CSCs are similar to normal somatic stem cells in that they are capable of asymmetric cell division and the efflux of small molecules, they have more phenotypic plasticity. The family of miRNAs known as let-7 was demonstrated to be a master regulator of self-renewal and tumor-seeding ability [32]. Likewise, the process of epithelial to mesenchymal transition (EMT) which enables tumorigenicity and invasion, was facilitated via transforming growth factor β2 (TGF-β2) and Zeb1 transcription factor mediated repression of the miR-200 and miR-141; two miRNAs which are responsible for epithelial differentiation [33]. In summary, reflecting the cancer cells aim of aberrant, dysregulated gene expression needed for tumor cell survival and proliferation, a global downregulation of all miRNAs is observed in cancer. In tumor cells, the main mechanism by which global miRNA production is suppressed is via the upregulation of miRNAs that target the crucial miRNA biogenesis enzyme Dicer, miR-103 and miR-107 [34]. Likewise, chromatin remodeling that results in an increase in miRNAs that support EMT and self-renewal rather than continuation of a differentiated cell type is

**5. miRNAs affecting breast cancer chemotherapy efficacy and resistance**

Chemoresistance is the primary cause of treatment failure in breast cancer. Dysregulation of some miRNAs can result in increases in drug efflux, alter drug targets and energy metabolism, stimulate DNA repair pathways and evasion of apoptosis and result in loss of cell cycle control. The first BC drug was a DNAreplication blocker called doxorubicin. Resistance to doxorubicin correlated with downregulation of miR-505, miR-128, and miR-145 tumor suppressors [36–41]. In contrast, miR-663, miR-181a, and miR-106b are oncogenic miRNAs whose downregulation resulted in enhancement of doxorubicin sensitivity in formerly resistant cells [41–43]. Like doxorubicin, cisplatin inhibits DNA replication and was also one of the first established therapies for BC. Upregulation of miR-345 and miR-7 contribute to cisplatin-resistance, while miR-302b can sensitize resistant cells to cisplatin therapy [44, 45]. A list of miRNA expression levels and targets of BC drug

In addition to doxorubicin and cisplatin, efficacy of the chemotherapeutic agents docetaxel and paclitaxel which inhibit microtubule formation during cell division, can also be compromised by miRNAs. Downregulation of miR-34a, miR-100, and miR-30c were observed in paclitaxel-resistant BC cell while the upregula-

In ER+ breast cancer, *de novo* and acquired resistance to conventional endocrine therapies such as aromatase inhibitors, fulvestrant and tamoxifen, can occur in more than 30% of patients [63]. Evidence suggests that resistance to these drugs is in part mediated by miRNAs. As most BC patients have high estrogen receptor-α (ER-α) expression, targeting ER-α signaling is a critical therapy. Resistance to tamoxifen, an agent which blocks interaction between estrogen and estrogen receptor is associated with the downregulation of the following tumor suppressor miRNAs: miR-15a, miR-16, miR-214, miR-320, miR-342, miR-451, miR-873,

tion of miR-129-3p was found to contribute to resistance [57–61].

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

#### *Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.88466*

*Genes and Cancer*

subtypes.

time of diagnosis [25].

transition to an invasive BC subtype [26].

(BC) leads to significant, dynamic changes in miRNA expression both by tumor cells and by surrounding stromal cells. This section describes BC tumor cell production of miRNAs as well as the surrounding non-cancerous stromal cells. In general, miRNAs either support or suppress tumorigenesis and are often dysregulated due to tumor-specific epigenetic changes. Likewise, tumor secreted factors such as exosomes and cytokines can also lead to aberrant signaling in the surrounding stromal cells. Furthermore, while all BCs begin in the breast, there are many subtypes which are named to reflect their particular molecular pathogenesis. Subtype diagnosis can help select appropriate therapies. Likewise, aberrant regulation of miRNAs can be subtype specific. Therefore, this section begins with a brief overview of cancer

Breast carcinoma can begin either in the ducts or the lobules and as such, termed either ductal carcinoma in situ (DCIS), or lobular carcinoma in situ (LCIS). Both can either stay contained to the area or travel to surrounding tissue and lymph nodes in which case the clinical diagnosis is either invasive ductal carcinoma (IDC) or invasive lobular carcinoma (ILC). IDC is the most common type of BC (50–75%) followed by ILC (5–15%) [23]. Rare BC is characterized by tumor origination in the mucinous, papillary, medullary or cribriform compartments of the breast [24]. Metastasis of breast cancer to other organs is the main cause of mortality and up to 5% of patients will already have experienced metastasis at the

MiRNA microarray performed on 1542 breast tissue samples procured via the Molecular Taxonomy of Breast Cancer International Consortium and the Akershus University Hospital (AHUS) revealed that no miRNAs were differentially expressed in DCIS patients relative to IDC, supporting the idea that miRNA dysregulation occurs at an early stage of BC development [26]. Among the invasive subtypes, however, expression of seven miRNAs was consistently downregulated, including tumor suppressors let-7c-5p, miR-125b-5p, miR140-3p, miR-145-3p, miR-145-5p, miR-193a-5p, and miR378a-3p while expression of four oncogenic miRNAs was consistently upregulated including miR-106b-5p, miR-142, miR-342-3p, and miR425-5p. Taken together these miRNAs may significantly contribute to the

While Bloom and Richardson's histologic grading system which was modified by Elston and Ellis in 1991 is the most commonly used system to gain prognostic insight, hormone receptors status, tumor size, nodal status and whether tumorous cells have invaded the lymph or blood vessels is also considered during initial diagnosis. Hormone receptor statuses including estrogen receptor (ER) and progesterone receptor (PR) as well as the tyrosine kinase receptor, human epidermal growth receptor type two (HER2) are always measured on newly diagnosed invasive BCs. Subtypes are identified via immunohistochemical staining for hormone receptors, HER2 expression status, and Ki-67 proliferation index as: luminal A (ER-positive and/or PR-positive, HER2-negative, low proliferation), luminal B (ER-positive and/or PR-positive, HER2-negative, high proliferation; or hormone receptor (HR) positive and HER2-positive), HER2-positive (HR-negative and HER2-positive) and finally TNBC type (HR-negative and HER2-negative) [27]. ER+ breast cancer subtype is particularly prevalent in postmenopausal women taking hormone replacement therapy (HRT) which activates the transcription factor estrogen receptor alpha (ERα) which promotes the expression of numerous oncogenic genes. While ERα-signaling is targeted by miRNAs for degradation, aberrant activation of this receptor leads to aberrant expression of miRNAs controlled by

Several miRNAs are both tissue and cancer specific. As the primary role of miRNA is to decrease target mRNA expression, miRNAs that are upregulated by

**66**

ERα-signaling [28].

cancerous cells are often those that support cancer growth and are referred to as oncomiRs. miR-10b, miR-21 and miR-155 are well characterized oncomiRs in BC [29]. Their main role is to downregulate tumor suppressor genes which results in the promotion of cancer cell proliferation, de-differentiation and invasion [30]. BC cells also produce less tumor-suppressor miRNAs (miR-31, miR-125b, miR-200 and miR-205) which downregulate oncogenic proteins. Cancer-initiating cells (CSCs) were first isolated from breast cancer tumors and are considered the seed-cells of tumor development [31]. While CSCs are similar to normal somatic stem cells in that they are capable of asymmetric cell division and the efflux of small molecules, they have more phenotypic plasticity. The family of miRNAs known as let-7 was demonstrated to be a master regulator of self-renewal and tumor-seeding ability [32]. Likewise, the process of epithelial to mesenchymal transition (EMT) which enables tumorigenicity and invasion, was facilitated via transforming growth factor β2 (TGF-β2) and Zeb1 transcription factor mediated repression of the miR-200 and miR-141; two miRNAs which are responsible for epithelial differentiation [33].

In summary, reflecting the cancer cells aim of aberrant, dysregulated gene expression needed for tumor cell survival and proliferation, a global downregulation of all miRNAs is observed in cancer. In tumor cells, the main mechanism by which global miRNA production is suppressed is via the upregulation of miRNAs that target the crucial miRNA biogenesis enzyme Dicer, miR-103 and miR-107 [34]. Likewise, chromatin remodeling that results in an increase in miRNAs that support EMT and self-renewal rather than continuation of a differentiated cell type is observed [35].

#### **5. miRNAs affecting breast cancer chemotherapy efficacy and resistance**

Chemoresistance is the primary cause of treatment failure in breast cancer. Dysregulation of some miRNAs can result in increases in drug efflux, alter drug targets and energy metabolism, stimulate DNA repair pathways and evasion of apoptosis and result in loss of cell cycle control. The first BC drug was a DNAreplication blocker called doxorubicin. Resistance to doxorubicin correlated with downregulation of miR-505, miR-128, and miR-145 tumor suppressors [36–41]. In contrast, miR-663, miR-181a, and miR-106b are oncogenic miRNAs whose downregulation resulted in enhancement of doxorubicin sensitivity in formerly resistant cells [41–43]. Like doxorubicin, cisplatin inhibits DNA replication and was also one of the first established therapies for BC. Upregulation of miR-345 and miR-7 contribute to cisplatin-resistance, while miR-302b can sensitize resistant cells to cisplatin therapy [44, 45]. A list of miRNA expression levels and targets of BC drug resistant is listed in **Table 1**.

In addition to doxorubicin and cisplatin, efficacy of the chemotherapeutic agents docetaxel and paclitaxel which inhibit microtubule formation during cell division, can also be compromised by miRNAs. Downregulation of miR-34a, miR-100, and miR-30c were observed in paclitaxel-resistant BC cell while the upregulation of miR-129-3p was found to contribute to resistance [57–61].

In ER+ breast cancer, *de novo* and acquired resistance to conventional endocrine therapies such as aromatase inhibitors, fulvestrant and tamoxifen, can occur in more than 30% of patients [63]. Evidence suggests that resistance to these drugs is in part mediated by miRNAs. As most BC patients have high estrogen receptor-α (ER-α) expression, targeting ER-α signaling is a critical therapy. Resistance to tamoxifen, an agent which blocks interaction between estrogen and estrogen receptor is associated with the downregulation of the following tumor suppressor miRNAs: miR-15a, miR-16, miR-214, miR-320, miR-342, miR-451, miR-873,


*Abbreviations: Expression level of miRs: upregulation (↑) or downregulation (↓) of miRNAs in breast cancer therapy. The reference of each miR is included in the table. Table adapted from Hu et al. [62].*

#### **Table 1.**

*miRNAs involved in the regulation of common breast cancer drugs.*

miRNA-375, miR-378a-3p, and miR-574-3p [64–71] .In contrast, oncogenic miRs: miR-101, miR-221/222, miR-301, and miRNAs-C19MC were highly expressed in tamoxifen resistant cells [72–75]. In addition, both the humanized monoclonal antibody targeting HER2 named trastuzumab, as well as lapatinib, which is a smallmolecule tyrosine kinase inhibitor targeting both HER2 and epithelial growth factor receptor (EGFR), improve therapeutic outcome but result in resistance after 1 year.

**69**

their targets [88–91].

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

Resistance to these two drugs is correlated with an upregulation of miR-21, miR-221

The role of miRNA in chemotherapeutic resistance is associated with the modification of drug transporters which has a net effect of drug efflux out of the cell via exosomes as well as modifications of autophagy and apoptosis pathways which lead to enhanced survival, the promotion of growth factors and activation EMT [81]. The tumor microenvironment which consists of the surrounding stromal cells serve as the normal foundation upon which the deviant tumor "house" is constructed supplying it with blood vessels, signaling molecules and ECM. Exosomes transport bioactive molecules and mediate cellular communication in the tumor microenvironment, facilitating a more cancerous and recalcitrant milieu [82]. For example, exosome-derived miRNAs such as miR-222 transfer doxorubicinresistance by inhibiting PTEN in recipient cells, 22 miRNAs were concentrated in exosomes and correlated to chemotherapy resistance [83]. While the major function of exosomes in the context of BC and drug resistance is the shuttling of drugs out of the tumor, exosomes can also be bio-hacked for use as a prime chemo-

In summary, in the context of breast cancer, tumor cells regulate miRNAs in a way that promotes tumor survival, growth and invasion. Aside from a global downregulation of most miRNAs and especially tumor suppressor miRNAs, oncogenic miRNAs are increased and often exported via exosomes where they are taken up by non-cancerous cells, transforming the local environment to a pro-cancer milieu. Knowing how BC cells regulate miRNAs opens the door for potential therapies that target oncogenic miRNAs (antagomirs) or add back tumor suppresser miRNAs (mimic miRNAs). The targeting of miRs in breast cancer is discussed in the follow-

As reviewed in this chapter, miRNAs are dynamically regulated in BC and can also contribute to drug resistance. Therefore, interventions that disrupt activities of dysregulated miRNAs offer promising targets for novel therapeutics in the form of mimics or antagomirs. In addition, mature miRNAs and their precursors can also be targeted by small molecules. In general, there are two strategies for targeting miRNA in BC. In the first strategy, tumor suppressor miRNAs which are down regulated by tumor cells can be added back to the tumor microenvironment using chemically synthesized miRNA mimics which imitate endogenous mature double-stranded miRNA [87]. MiRNA mimics could be delivered in viral vectors which would allow extended expression. The second strategy is to target oncogenic miRNAs which are highly expressed and exported by tumor cells. In this strategy, oligonucleotides, locked-nucleic-acids antisense oligonucleotides (LNAs), miRNA sponges, multiple-target anti-miRNA antisense oligo-deoxyribonucleotides (MTg-AMOs), miRNA-masking and nanoparticles are used to target for degradation or impede aberrantly expressed oncogenic miRNAs from reaching

As previously mentioned, the majority of highly expressed, dysregulated miRNAs

in tumor cells are oncomirs, or those that support tumorigenesis, while tumor suppressor miRNAs are suppressed [92]. For example, miR-155 is an oncogenic miRNA upregulated in BC tumor tissue. Targeting of miR-155 with an antisense oligonucleotide (miR-155) in a BC cell line blocked proliferation and augmented apoptosis [93]. MiR-892b is an example of a tumor suppressor miRNA that is significantly downregulated in BC tissue specimens. By supplementing miR-892b

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

therapy delivery system [84–86].

**6. miRNAs as breast cancer therapy**

ing section.

and miR-375 [76–80].

*Genes and Cancer*

miR-7

miR-106b~25 cluster

**68**

**Table 1.**

miRNA-375, miR-378a-3p, and miR-574-3p [64–71] .In contrast, oncogenic miRs: miR-101, miR-221/222, miR-301, and miRNAs-C19MC were highly expressed in tamoxifen resistant cells [72–75]. In addition, both the humanized monoclonal antibody targeting HER2 named trastuzumab, as well as lapatinib, which is a smallmolecule tyrosine kinase inhibitor targeting both HER2 and epithelial growth factor receptor (EGFR), improve therapeutic outcome but result in resistance after 1 year.

**miRNA BC therapy Targets Level Mechanism/Refs.** miR-200 Carboplatin Zeb ↓ Reverses EMT [46] miR-345 Cisplatin MRP1 ↓ Not yet characterized [45]

miR-302b Cisplatin E2F1 (direct) ↓ Inhibit cell cycle progression

miR-24 Cisplatin BimL F1H1 ↑ Promotes EMT and cancer stem

miR-128 Doxorubicin Bmi-1 ABCC5 ↓ Increases apoptosis [48] miR-145 Doxorubicin MRP1 ↓ Induces intracellular

miR-181a Doxorubicin Bcl-2 ↓ Increases apoptosis [41] miR-181a Doxorubicin Bax ↑ Inhibits apoptosis [49] miR-25 Doxorubicin ULK1 ↑ Inhibits autophagy [50] miR-326 Doxorubicin MDR-1 ↓ Downregulates MRP-1 [51] miR-505 Doxorubicin Akt3 (indirect) ↓ Not yet investigated [37] miR-644a Doxorubicin CTBP1 ↓ Inhibits EMT [52] miR-663 Doxorubicin HSPG2 ↑ Inhibits apoptosis [42] miR-129-3p Docetaxel CP100 ↑ Reduces cell cycle arrest and

miR-34a Docetaxel BCL-2 CCND1 ↑ Inhibit apoptosis [54]

miR-218 MDR Survivin ↓ Enhance apoptosis [56] miR-100 Paclitaxel mTOR ↓ Enhance cell cycle arrest and

miR-125b Paclitaxel Sema4C ↓ Reverses EMT [58] miR-125b Taxol Bak1 ↑ Inhibits apoptosis [59]

IL-11

*The reference of each miR is included in the table. Table adapted from Hu et al. [62].*

miR-30c Doxorubicin TWF1 (PTK9) VIM

*miRNAs involved in the regulation of common breast cancer drugs.*

Paclitaxel

miR-484 Gemcitabine CDA ↓ Promote proliferation and cell-

miR-34a Doxorubicin HDAC1HDAC7 ↑ Inhibits autophagic cell death [61] Cisplatin *Abbreviations: Expression level of miRs: upregulation (↑) or downregulation (↓) of miRNAs in breast cancer therapy.* 

Doxorubicin EP300 ↑ Activates EMT [43]

[44] ATM (indirect)

cells [47]

apoptosis [53]

apoptosis [57]

↓ Reverses EMT [60]

cycle redistribution [55]

doxorubicin accumulation [36]

Resistance to these two drugs is correlated with an upregulation of miR-21, miR-221 and miR-375 [76–80].

The role of miRNA in chemotherapeutic resistance is associated with the modification of drug transporters which has a net effect of drug efflux out of the cell via exosomes as well as modifications of autophagy and apoptosis pathways which lead to enhanced survival, the promotion of growth factors and activation EMT [81]. The tumor microenvironment which consists of the surrounding stromal cells serve as the normal foundation upon which the deviant tumor "house" is constructed supplying it with blood vessels, signaling molecules and ECM. Exosomes transport bioactive molecules and mediate cellular communication in the tumor microenvironment, facilitating a more cancerous and recalcitrant milieu [82]. For example, exosome-derived miRNAs such as miR-222 transfer doxorubicinresistance by inhibiting PTEN in recipient cells, 22 miRNAs were concentrated in exosomes and correlated to chemotherapy resistance [83]. While the major function of exosomes in the context of BC and drug resistance is the shuttling of drugs out of the tumor, exosomes can also be bio-hacked for use as a prime chemotherapy delivery system [84–86].

In summary, in the context of breast cancer, tumor cells regulate miRNAs in a way that promotes tumor survival, growth and invasion. Aside from a global downregulation of most miRNAs and especially tumor suppressor miRNAs, oncogenic miRNAs are increased and often exported via exosomes where they are taken up by non-cancerous cells, transforming the local environment to a pro-cancer milieu. Knowing how BC cells regulate miRNAs opens the door for potential therapies that target oncogenic miRNAs (antagomirs) or add back tumor suppresser miRNAs (mimic miRNAs). The targeting of miRs in breast cancer is discussed in the following section.

#### **6. miRNAs as breast cancer therapy**

As reviewed in this chapter, miRNAs are dynamically regulated in BC and can also contribute to drug resistance. Therefore, interventions that disrupt activities of dysregulated miRNAs offer promising targets for novel therapeutics in the form of mimics or antagomirs. In addition, mature miRNAs and their precursors can also be targeted by small molecules. In general, there are two strategies for targeting miRNA in BC. In the first strategy, tumor suppressor miRNAs which are down regulated by tumor cells can be added back to the tumor microenvironment using chemically synthesized miRNA mimics which imitate endogenous mature double-stranded miRNA [87]. MiRNA mimics could be delivered in viral vectors which would allow extended expression. The second strategy is to target oncogenic miRNAs which are highly expressed and exported by tumor cells. In this strategy, oligonucleotides, locked-nucleic-acids antisense oligonucleotides (LNAs), miRNA sponges, multiple-target anti-miRNA antisense oligo-deoxyribonucleotides (MTg-AMOs), miRNA-masking and nanoparticles are used to target for degradation or impede aberrantly expressed oncogenic miRNAs from reaching their targets [88–91].

As previously mentioned, the majority of highly expressed, dysregulated miRNAs in tumor cells are oncomirs, or those that support tumorigenesis, while tumor suppressor miRNAs are suppressed [92]. For example, miR-155 is an oncogenic miRNA upregulated in BC tumor tissue. Targeting of miR-155 with an antisense oligonucleotide (miR-155) in a BC cell line blocked proliferation and augmented apoptosis [93]. MiR-892b is an example of a tumor suppressor miRNA that is significantly downregulated in BC tissue specimens. By supplementing miR-892b

"mimics" in BC cells, a decrease in tumor growth, metastases rate, and angiogenesis was observed. MiR-892b mimic blocked impeded tumorigenesis by attenuating nuclear transcription factor kappa B (NF-kB) signaling [94]. Artificial miRNAs can also be constructed to inhibit targets that are not normally targeted by endogenous miRNAs. For example, a novel artificial miRNA (amiRNA) called miR-p-27-5p, which targets the 3′-UTR of cyclin-dependent kinase 4 (CDK4) mRNA, inhibited cell cycle progression via downregulation of CDK4 expression and suppression of retinoblastoma protein (RB1) phosphorylation [95]. Likewise, an a miRNA against a C-X-C motif chemokine receptor 4 (CXCR4) inserted into an expression vector reduced CXCR4 expression and suppressed migration and invasion of BC cells [96]. While in vitro experiments provide proof of concept for further development of miRNA targeting in oncogenic diseases, only clinical trial results can determine whether miRNA therapy is truly efficacious. Patents, clinical trials and biopharmaceutical companies invested in the development of miRNA therapies are summarized by Chakraborty *et al*, [97]. A seminal trial for miRNA replacement therapy took place employing the tumor suppressor miR-34 mimic (MRX34). MRX34 was formulated for intravenous injection using a liposome delivery system for patients with metastatic liver cancer. MRX34 along with dexamethasone was associated with safety and showed evidence of antitumor activity in a subset of patients with refractory advanced solid tumors [98]. However, there were adverse events in the trial which indicate the need for alternative approaches in formulation design and delivery.

In summary, there is much research to be done in the emerging field of miRNA therapeutics. Drug developers, pharmacists, physicians and molecular biologists must work together to develop novel strategies for miRNA delivery that is more targeted and controlled in order to mitigate off-target effects by affecting only cell signaling of targeted tumor cells.

#### **7. miRNAs as breast cancer biomarkers**

MiRNAs that maintain a stable presence in the serum are referred to as "circulating" miRNAs. Thus, in addition to therapeutic targeting, many studies have reported utility of miRNAs in the context of BC as biomarkers for diagnostic, prognostic, or predictive of drug efficacy. In this final section, miRNAs currently being used as biomarkers in the context of BC are discussed.

In the context of diagnostics, the current gold standard for BC is mammography. However, many women avoid mammograms for fear of pain or inconvenience in scheduling thus rendering assays performed on less invasive, routine blood draws amenable to early screening for BC. Global profiling of circulating miRNAs in early-stage ER+ BC (*n* = 48) and age-matched healthy controls (*n* = 24) revealed a panel of nine miRNAs (miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365 and miR-425) that discriminated between patients with early-stage ER+ BC and healthy controls [99]. A study in Japan performed on serum (*n* = 1280 BC, *n* = 2836 non-cancer controls) found a combination of five miRNAs: miR-1246, miR-1307-3p, miR-4634, miR-6861-5p and miR-6875-5p, could predict breast cancer with a sensitivity of 97.3% overall, 98% sensitivity for early stage BC and a specificity of 82.9% and accuracy of 89.7% [100]. A study based in Prague (*n* = 63 early stage BC, *n* = 21 non cancer controls) found that several oncogenic miRNAs were significantly elevated in early stage BC; including: miR-155, miR-19a, miR-181b, and miR-24 and unsurprisingly, their expression dropped following surgical resection of the tumor [101]. A study in Singapore performed global profiling

**71**

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

Serum miR-214 Indicates malignant

miR-4634 and -6875-5p ↓ in BC [100]

Serum miR-155 Correlates w/PR

miR-4270, -1225-5p, -188-5p, -1202, -4281, -1207-5p, -642b-3p, -1290, and -3141

Serum miR-202 and let-7b ↑ expression in BC

Serum miR-18b, -103, -107, and -652 Associated w/tumor

Plasma miR-10b and -373 ↑ in breast cancer w/

Serum miR-10b, 34a, and -155 Correlates w/

miR-10b-5p ↑ levels correlate w/

**Source miRNA Expression/Refs DX PX PR VA** Blood miR-195, let-7 and -155 ↑ in BC [108] Y N N N

Plasma miR-148b, -133a, and -409-3p ↑ in BC [111] Y N N Y Serum miR-15a ↑ in BC [99] Y N N Y

↓ in BC [99]

Serum miR-484 ↑ in BC [112] Y N N Y

Serum miR-155, -19a, -181b, and -24 ↑ in BC [101] Y N N N Serum miR-1, -92a, -133a, and -133b ↑ in BC [102] Y N N Y

Serum let-7c ↓ in BC [103] Y N N N Serum miR-182 ↑ in BC [114] Y N N N Blood miR-138 ↑ in BC [115] Y N N N

status [116]

[118]

Serum miR-148b-3p and -652-3p ↓ in the BC [120] Y Y N Y

[105]

Associated w/ histological tumor grade and sex hormone receptor expression [117]

↑ in BC and correlates w/stage and molecular subtype

and correlates w/ tumor aggressive and overall survival [119]

poor prognosis [120]

relapse and overall survival in TNBC

LN metastasis [121]

tumor stage and/or metastasis [122]

from benign and healthy [109]

Y N N N

Y N N N

Y N N N

Y N N Y

Y Y N N

Y Y N Y

Y Y N Y

Y Y N N

↑ in BC [110] Y N N Y

↑ in BC [100] Y N N Y

↑ in BC [113] Y N N Y

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

Plasma miR-127-3p, -376a, -148b, -409-3p, -652 and -801

Serum miR-1246, -1307-3p, and -6861-5p

Plasma miR-505-5p, -125b-5p, -21- 5p, and -96-5p

Serum miR-21, -126, -155, -199a, and -335

Serum; Plasma

miR-18a, -107, -425, -133a, -139-5p, -143, -145, and -365


*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.88466*

*Genes and Cancer*

delivery.

signaling of targeted tumor cells.

**7. miRNAs as breast cancer biomarkers**

being used as biomarkers in the context of BC are discussed.

"mimics" in BC cells, a decrease in tumor growth, metastases rate, and angiogenesis was observed. MiR-892b mimic blocked impeded tumorigenesis by attenuating nuclear transcription factor kappa B (NF-kB) signaling [94]. Artificial miRNAs can also be constructed to inhibit targets that are not normally targeted by endogenous miRNAs. For example, a novel artificial miRNA (amiRNA) called miR-p-27-5p, which targets the 3′-UTR of cyclin-dependent kinase 4 (CDK4) mRNA, inhibited cell cycle progression via downregulation of CDK4 expression and suppression of retinoblastoma protein (RB1) phosphorylation [95]. Likewise, an a miRNA against a C-X-C motif chemokine receptor 4 (CXCR4) inserted into an expression vector reduced CXCR4 expression and suppressed migration and invasion of BC cells [96]. While in vitro experiments provide proof of concept for further development of miRNA targeting in oncogenic diseases, only clinical trial results can determine whether miRNA therapy is truly efficacious. Patents, clinical trials and biopharmaceutical companies invested in the development of miRNA therapies are summarized by Chakraborty *et al*, [97]. A seminal trial for miRNA replacement therapy took place employing the tumor suppressor miR-34 mimic (MRX34). MRX34 was formulated for intravenous injection using a liposome delivery system for patients with metastatic liver cancer. MRX34 along with dexamethasone was associated with safety and showed evidence of antitumor activity in a subset of patients with refractory advanced solid tumors [98]. However, there were adverse events in the trial which indicate the need for alternative approaches in formulation design and

In summary, there is much research to be done in the emerging field of miRNA therapeutics. Drug developers, pharmacists, physicians and molecular biologists must work together to develop novel strategies for miRNA delivery that is more targeted and controlled in order to mitigate off-target effects by affecting only cell

MiRNAs that maintain a stable presence in the serum are referred to as "circulating" miRNAs. Thus, in addition to therapeutic targeting, many studies have reported utility of miRNAs in the context of BC as biomarkers for diagnostic, prognostic, or predictive of drug efficacy. In this final section, miRNAs currently

In the context of diagnostics, the current gold standard for BC is mammography. However, many women avoid mammograms for fear of pain or inconvenience in scheduling thus rendering assays performed on less invasive, routine blood draws amenable to early screening for BC. Global profiling of circulating miRNAs in early-stage ER+ BC (*n* = 48) and age-matched healthy controls (*n* = 24) revealed a panel of nine miRNAs (miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365 and miR-425) that discriminated between patients with early-stage ER+ BC and healthy controls [99]. A study in Japan performed on serum (*n* = 1280 BC, *n* = 2836 non-cancer controls) found a combination of five miRNAs: miR-1246, miR-1307-3p, miR-4634, miR-6861-5p and miR-6875-5p, could predict breast cancer with a sensitivity of 97.3% overall, 98% sensitivity for early stage BC and a specificity of 82.9% and accuracy of 89.7% [100]. A study based in Prague (*n* = 63 early stage BC, *n* = 21 non cancer controls) found that several oncogenic miRNAs were significantly elevated in early stage BC; including: miR-155, miR-19a, miR-181b, and miR-24 and unsurprisingly, their expression dropped following surgical resection of the tumor [101]. A study in Singapore performed global profiling

**70**


*Abbreviations: DX, diagnostic; PX, prognostic; PR, predictive; VA, validated; BC, breast cancer; ddPCR, droplet digital PCR; DS, deep sequencing; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LN, lymph node; miRNA (miR), microRNA; PR, progesterone receptor; qRT-PCR, quantitative reverse transcriptase realtime PCR; TNBC, triple-negative breast cancer; NR, non-relapse; pCR, Pathologic complete response.*

#### **Table 2.**

*Circulating miRNAs; diagnostic, prognostic, predictive and validated biomarkers in breast cancer.*

of miRNA expression in BC tumor tissue, non-tumor tissue and serum samples obtained from BC patients (*n* = 132) and from healthy controls (*n* = 123) revealed miR-1, miR-92a, miR-133a and miR-133b as significantly upregulated diagnostic markers in BC sera [102]. In addition to upregulation of oncogenic miRNAs, tumor suppressor Let-7c was decreased in BC tissue and sera according to a study performed in China (*n* = 90 BC, *n* = 64 controls) [103]. Although some studies have suggested that let-7 and miR-195 restoration may be therapeutic, results of Qattan et al. in 2017 [104] supported literature indicating that tumor cells export hsamiR-195 and let-7 miRNAs. While the data of this study did not generally support the use of these miRNAs as therapies, it suggested that these markers may be the most robust markers to use in a blood-based screen for the early detection of TNBC and luminal breast cancer [104].

The definition of a prognostic biomarker is one that indicates recurrence or progression; such as chance of survival, independent of the course of therapy. In a study based in Germany, pre-operative serum (*n* = 102) and post-operative serum (*n* = 34) of BC patients was compared to healthy women (*n* = 37) or those with benign breast disease (*n* = 26). The mean follow-up time of for BC patients was 6.2 years. In this study, high expression of miR-202 positively correlated with reduced overall survival (poor prognosis). In a European study, genome-wide miRNA expression profiling using serum from TNBC patients (*n* = 130) and healthy controls (*n* = 30), revealed a four-miRNA signature (miR-18b, miR-103, miR-107

**73**

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

and miR-652) that predicted tumor recurrence and overall survival [105]. While few studies have investigated the use of miRNA serum expression levels as a predictive metric for treatment response, clinically relevant outcomes were revealed in the studies performed indicating the need for incentivizing investigations into miRNA biomarkers. For example, elevated miR-125b expression predicts poor prognosis, is associated with tumor size and TNM stage in HER2+ BC as well as poor responsiveness to paclitaxel-based neoadjuvant chemotherapy [106]. Therefore, miR-125b may be a potential predictor of clinical outcome, particularly in HER2+ BC patients receiving paclitaxel-based neoadjuvant chemotherapy. In another example, miR-155 was significantly increased in BC patients (*n* = 103) compared with healthy normal (*n* = 55). Post-surgical resection and four cycles of chemotherapy, a subset of BC patient sera (*n* = 29) were collected to evaluate the effects of clinical treatment on serum levels of candidate miRNAs. Decreased levels of circulating miR-155 posttreatment was associated with response to therapy and stable disease [107].

In summary, the data from these studies and others suggest that BC patients with novel miRNA signatures correlating with poor prognosis are not receiving adequate treatment and should be selected for inclusion in novel randomized clinical trials for the chance to receive alternative life-saving therapies. **Table 2** summarizes studies revealing statistically significant regulation of circulating miRNAs with diagnostic (DX), prognostic (PX), predictive biomarkers (PR) potential for

In conclusion, this chapter provided an overview of the most recent studies describing the dynamic roles of miRNAs in the context of BC. This overview demonstrates that just as miRNAs are integral to maintaining normal homeostasis, they are simultaneously sensitive to changes in overall physiology and local micro-environments thus studying them will likely lead to insight into the unique manifestation of BC in an individual. Given that they are actively released by tumor cells into the circulatory system, both monitoring and targeting miRNAs enables the diagnosis and monitoring of BC as well as the opportunity for the development of novel therapeutics. Future studies should employ well standardized methods for sample collection and multi-center global miRNA profiling to reveal novel nuances and robust results regarding miRNA signaling in the context of BC. Taken together, the emerging field of precision oncology may rely on understanding miRNA

BC. Some studies were validated (VA) with alternative cohorts.

**8. Conclusions**

profiles.

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.88466*

and miR-652) that predicted tumor recurrence and overall survival [105]. While few studies have investigated the use of miRNA serum expression levels as a predictive metric for treatment response, clinically relevant outcomes were revealed in the studies performed indicating the need for incentivizing investigations into miRNA biomarkers. For example, elevated miR-125b expression predicts poor prognosis, is associated with tumor size and TNM stage in HER2+ BC as well as poor responsiveness to paclitaxel-based neoadjuvant chemotherapy [106]. Therefore, miR-125b may be a potential predictor of clinical outcome, particularly in HER2+ BC patients receiving paclitaxel-based neoadjuvant chemotherapy. In another example, miR-155 was significantly increased in BC patients (*n* = 103) compared with healthy normal (*n* = 55). Post-surgical resection and four cycles of chemotherapy, a subset of BC patient sera (*n* = 29) were collected to evaluate the effects of clinical treatment on serum levels of candidate miRNAs. Decreased levels of circulating miR-155 posttreatment was associated with response to therapy and stable disease [107].

In summary, the data from these studies and others suggest that BC patients with novel miRNA signatures correlating with poor prognosis are not receiving adequate treatment and should be selected for inclusion in novel randomized clinical trials for the chance to receive alternative life-saving therapies. **Table 2** summarizes studies revealing statistically significant regulation of circulating miRNAs with diagnostic (DX), prognostic (PX), predictive biomarkers (PR) potential for BC. Some studies were validated (VA) with alternative cohorts.

#### **8. Conclusions**

*Genes and Cancer*

Serum miR-29b-2, miR-155, miR -197 and miR -205

Serum miR-21-5p, -375, -205-5p, and -194-5p


Serum miR-34a, -93, -373, -17, and -155

miR-382-5p, -376c-3p, and

Serum miR-92a ↓ in BC, LN

miR-21 ↑ in BC, LN

Serum miR-125b ↑ expression in non-

Serum miR-122 ↓ in NR and pCR

Serum miR-155 ↑ in BC; ↓ post chemo

miR-375 ↑ in NR and pCR

**72**

**Table 2.**

and luminal breast cancer [104].

of miRNA expression in BC tumor tissue, non-tumor tissue and serum samples obtained from BC patients (*n* = 132) and from healthy controls (*n* = 123) revealed miR-1, miR-92a, miR-133a and miR-133b as significantly upregulated diagnostic markers in BC sera [102]. In addition to upregulation of oncogenic miRNAs, tumor suppressor Let-7c was decreased in BC tissue and sera according to a study performed in China (*n* = 90 BC, *n* = 64 controls) [103]. Although some studies have suggested that let-7 and miR-195 restoration may be therapeutic, results of Qattan et al. in 2017 [104] supported literature indicating that tumor cells export hsamiR-195 and let-7 miRNAs. While the data of this study did not generally support the use of these miRNAs as therapies, it suggested that these markers may be the most robust markers to use in a blood-based screen for the early detection of TNBC

*time PCR; TNBC, triple-negative breast cancer; NR, non-relapse; pCR, Pathologic complete response.*

*Circulating miRNAs; diagnostic, prognostic, predictive and validated biomarkers in breast cancer.*

**Source miRNA Expression/Refs DX PX PR VA**

[123]

[125]

[125]

[128]

[128]

[107]

*Abbreviations: DX, diagnostic; PX, prognostic; PR, predictive; VA, validated; BC, breast cancer; ddPCR, droplet digital PCR; DS, deep sequencing; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LN, lymph node; miRNA (miR), microRNA; PR, progesterone receptor; qRT-PCR, quantitative reverse transcriptase real-*

Correlates w/tumor grade and metastasis

metastasis [124]

metastasis [124]

↑ in recurrent BC

↓ in recurrent BC

responsive [127]

Expression correlated w/metastasis and HER2, PR, and ER status [126]

Y Y N N

Y Y N N

Y Y N Y

Y N N N

Y N Y N

N N Y Y

Y N Y N

The definition of a prognostic biomarker is one that indicates recurrence or progression; such as chance of survival, independent of the course of therapy. In a study based in Germany, pre-operative serum (*n* = 102) and post-operative serum (*n* = 34) of BC patients was compared to healthy women (*n* = 37) or those with benign breast disease (*n* = 26). The mean follow-up time of for BC patients was 6.2 years. In this study, high expression of miR-202 positively correlated with reduced overall survival (poor prognosis). In a European study, genome-wide miRNA expression profiling using serum from TNBC patients (*n* = 130) and healthy controls (*n* = 30), revealed a four-miRNA signature (miR-18b, miR-103, miR-107

In conclusion, this chapter provided an overview of the most recent studies describing the dynamic roles of miRNAs in the context of BC. This overview demonstrates that just as miRNAs are integral to maintaining normal homeostasis, they are simultaneously sensitive to changes in overall physiology and local micro-environments thus studying them will likely lead to insight into the unique manifestation of BC in an individual. Given that they are actively released by tumor cells into the circulatory system, both monitoring and targeting miRNAs enables the diagnosis and monitoring of BC as well as the opportunity for the development of novel therapeutics. Future studies should employ well standardized methods for sample collection and multi-center global miRNA profiling to reveal novel nuances and robust results regarding miRNA signaling in the context of BC. Taken together, the emerging field of precision oncology may rely on understanding miRNA profiles.

*Genes and Cancer*

#### **Author details**

Amal Qattan1,2

1 Department of Molecular Oncology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

2 Department of Biochemistry and Molecular Medicine, School of Medicine and Health Science (SMHS), GW University, Washington, DC, USA

\*Address all correspondence to: aqattan5@gwu.edu; akattan@kfshrc.edu.sa

© 2019 The Author(s). Licensee IntechOpen. 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.

**75**

*Duplicitous Dispositions of Micro-RNAs (miRs) in Breast Cancer*

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[11] Ha M, Kim VN. Regulation of microRNA biogenesis. Nature Reviews. Molecular Cell Biology. 2014;**15**:509-524.

[12] Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: MicroRNAs can

up-regulate translation. Science (80-). 2007;**318**:1931-1934. DOI: 10.1126/

[13] Rüegger S, Großhans H. MicroRNA turnover: When, how, and why. Trends in Biochemical Sciences. 2012;**37**:436- 446. DOI: 10.1016/j.tibs.2012.07.002

[14] Tsang J, Zhu J, van Oudenaarden A. MicroRNA-mediated feedback and feedforward loops are recurrent

network motifs in mammals. Molecular Cell. 2007;**26**:753-767. DOI: 10.1016/j.

[15] Gulyaeva LF, Kushlinskiy NE. Regulatory mechanisms of microRNA expression. Journal of Translational Medicine. 2016;**14**:143. DOI: 10.1186/

[16] Goldberg AD, Allis CD, Bernstein E. Epigenetics: A landscape takes shape. Cell. 2007;**128**:635-638. DOI: 10.1016/j.

[17] Messerschmidt DM, Knowles BB, Solter D. DNA methylation dynamics

DOI: 10.1038/nrm3838

science.1149460

molcel.2007.05.018

s12967-016-0893-x

cell.2007.02.006

gr.2722704

bau099

*DOI: http://dx.doi.org/10.5772/intechopen.88466*

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#### **References**

*Genes and Cancer*

**74**

**Author details**

Amal Qattan1,2

Centre, Riyadh, Saudi Arabia

provided the original work is properly cited.

1 Department of Molecular Oncology, King Faisal Specialist Hospital and Research

© 2019 The Author(s). Licensee IntechOpen. 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,

2 Department of Biochemistry and Molecular Medicine, School of Medicine

\*Address all correspondence to: aqattan5@gwu.edu; akattan@kfshrc.edu.sa

and Health Science (SMHS), GW University, Washington, DC, USA

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Section 3

Tumor Suppressor

Proteins in Cell Signalling

Pathways

Section 3
