Contents

#### **Preface XI**


## Chapter 9 **Enantiomerically Pure Substituted Benzo-Fused Heterocycles — A New Class of Anti-Breast Cancer Agents 203**

Joaquín M. Campos, M. Eugenia García-Rubiño, Nawal Mahfoudh and César Lozano-López

## Preface

Chapter 9 **Enantiomerically Pure Substituted Benzo-Fused Heterocycles — A New Class of Anti-Breast Cancer Agents 203**

and César Lozano-López

**VI** Contents

Joaquín M. Campos, M. Eugenia García-Rubiño, Nawal Mahfoudh

Cancer is the leading cause of death in most countries and continues to increase mainly be‐ cause of the aging and growth of the world population as well as habitation of cancer-caus‐ ing behaviors such as smoking and alcohol. Based on statistics of the GLOBOCAN 2012, about 14.1 million cancer cases and 8.2 million cancer deaths are estimated to have occurred in 2012 (Torre LA et al. Ca Cancer J Clin 65:87-108, 2015). Breast cancer is one of the most frequently diagnosed cancer and the leading cause of cancer death, accounting for 25% of the total cancer cases and 15% of the cancer deaths among females. Thus researches on can‐ cer especially for breast cancer are important to overcome both economical and physiologi‐ cal burden. The current book for breast cancer aims at providing information of recent molecular researches in the field. The current book covers topics such as gene regulation and abnormalities in DNA in breast cancer cells, role of miRNA and its potential use, impor‐ tance of bioinformatics and co-association other cancer types with this cancer. We hope that the book will provide concise recent developments for breast cancer and lead the scientists, researchers and educators in the field.

> **Prof. Dr. Mehmet Gunduz** Turgut Ozal University Medical School, Turkey

## **Chapter 1**

## **Breast Cancer- It's All in the DNA**

Somaira Nowsheen, Khaled Aziz, Asef Aziz and Alexandros G Georgakilas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60033

## **1. Introduction**

Breast cancer is the leading cause of cancer death in women, the second most common cancer worldwide, and the fifth most common cause of cancer-related deaths [1-3]. Not only are the incidence rates of breast cancer increasing, partly due to improved screening and detection techniques, but also the global burden of breast cancer exceeds all other cancers. So it is imperative to improve the quality of life of these patients.

Our knowledge of the process of tumorigenesis has increased significantly over the last decade thanks to continued funding from federal and private organizations, improved technologies enabling affordable sequencing of the entire genome, analysis of large data sets as well as gene expression profiles of human tumor samples, and improved animal models that attempt to resemble tumor formation in humans. The predisposing risk factors, precancerous lesions, and disease progression vary significantly across the tissues of origin. However, common themes have been described that drive a normal cell to undergo transformation and generate a tumor. We plan to lay the groundwork for our discussion utilizing the widely recognized models of colorectal cancer by Bert Vogelstein, the two hit hypothesis by Alfred Knudson, and the common characteristics of cancer cells described by Doug Hanahan and Robert Weinberg.

Furthermore, in this chapter we aim to discuss the early events that cause a normal breast epithelial cell to initiate the process of tumor formation and delineate them from later stage insults to the cell that cause it to progress to advanced metastatic disease. We particularly plan to focus on the role of oxidative stress and one major environmental agent i.e. ionizing radiation inducing DNA damage and chromosomal instability. At the same time we will discuss the cell cycle changes that ensue and the implications of loss of a tumor suppressor gene. Concurrently, there are morphological changes that can be witnessed in experiments performed with cancer cells in vitro which we will tie in with the underlying molecular mechanisms. We will trace

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the damaged cell along its course to metastasis by focusing on the molecular mechanisms that cause loss of cell-cell adhesion, loss of cellular polarity, ability to migrate through the stroma and gain access to the vascular or lymphatic system, resistance to anoikis and ability to seed a tumor in a new environment. A myriad of hypotheses exists in literature that attempts to explain the process of cancer formation and progression.

Next, we will classify breast tumors as malignant or non-malignant while describing the subtypes of each in a concise manner. Since the therapeutic options available in the clinic are targeted to particular genetic subtypes such as BRCA1 positive, estrogen receptor (ER) positive or triple negative (Her2-/-, ER -/-, PR -/-) etc., we will also discuss these molecular signatures. The clinical diagnosis criteria and imaging modalities will be mentioned concisely. A limited number of clinical trials that have a promising premise behind the study and considered to be ground breaking will be described.

Therapeutic options for breast cancer have expanded in the past 10 years to improve the survival outcomes for the disease. Existing FDA approved pharmacologic agents, small molecule inhibitors in clinical trials and drugs shown to have efficacy in preclinical studies will be methodically described in the final section. In the process, we hope to summarize where we are now with respect to this potent disease that affects millions.

## **2. How does cancer arise?**

As a cell achieves a neoplastic phenotype, its genetic sequence is usually vastly altered and multiple genes are mutated, amplified, or lost. Several models have been proposed regarding what leads to tumorigenesis. One of the models proposed by Dr. Bert Vogelstein proposes the loss of function of tumor suppressors [4-7]. According to his model, loss of function of tumor suppressors such as p53 leads to genomic instability which eventually leads to tumorigenesis via alterations in metabolism, loss of sensitivity to apoptotic signals, and increased invasive‐ ness [8, 9]. Loss of function of the tumor suppressor, p53, is associated with the development of most, if not all, tumor types [10-12]. An inactivating mutation in a tumor suppressor not only leads to hyper-proliferation of epithelial cells, it may also inactivate DNA repair genes. Mutations in proto-oncogene can either create an oncogene or lead to a cascade of inactivation of several more tumor suppressor genes before resulting in cancer. Figure 1 shows this model for colon carcinogenesis.

An alternate theory that accounts for both hereditary and non-hereditary cancer is the two-hit theory of cancer causation proposed by Dr. Alfred Knudson [13, 14]. Normal cells have two undamaged chromosomes, one inherited from each parent. People with a hereditary suscept‐ ibility to cancer inherit a damaged gene on one of the chromosomes at conception which is their 'first hit' or mutation. Others receive the 'first hit' in their lifetime. Damage to the same gene on the second chromosome in their lifetime may lead to cancer. An overview of this model is given in Figure 2 and is seen in cancer such as retinoblastoma.

Weinberg and Hanahan have proposed the hallmarks of cancer which helps explain oncogen‐ esis. These are biological capabilities acquired during the complex multistep development of

**Figure 1.** The cascade of events that lead to oncogenesis.

the damaged cell along its course to metastasis by focusing on the molecular mechanisms that cause loss of cell-cell adhesion, loss of cellular polarity, ability to migrate through the stroma and gain access to the vascular or lymphatic system, resistance to anoikis and ability to seed a tumor in a new environment. A myriad of hypotheses exists in literature that attempts to

Next, we will classify breast tumors as malignant or non-malignant while describing the subtypes of each in a concise manner. Since the therapeutic options available in the clinic are targeted to particular genetic subtypes such as BRCA1 positive, estrogen receptor (ER) positive or triple negative (Her2-/-, ER -/-, PR -/-) etc., we will also discuss these molecular signatures. The clinical diagnosis criteria and imaging modalities will be mentioned concisely. A limited number of clinical trials that have a promising premise behind the study and considered to be

Therapeutic options for breast cancer have expanded in the past 10 years to improve the survival outcomes for the disease. Existing FDA approved pharmacologic agents, small molecule inhibitors in clinical trials and drugs shown to have efficacy in preclinical studies will be methodically described in the final section. In the process, we hope to summarize where

As a cell achieves a neoplastic phenotype, its genetic sequence is usually vastly altered and multiple genes are mutated, amplified, or lost. Several models have been proposed regarding what leads to tumorigenesis. One of the models proposed by Dr. Bert Vogelstein proposes the loss of function of tumor suppressors [4-7]. According to his model, loss of function of tumor suppressors such as p53 leads to genomic instability which eventually leads to tumorigenesis via alterations in metabolism, loss of sensitivity to apoptotic signals, and increased invasive‐ ness [8, 9]. Loss of function of the tumor suppressor, p53, is associated with the development of most, if not all, tumor types [10-12]. An inactivating mutation in a tumor suppressor not only leads to hyper-proliferation of epithelial cells, it may also inactivate DNA repair genes. Mutations in proto-oncogene can either create an oncogene or lead to a cascade of inactivation of several more tumor suppressor genes before resulting in cancer. Figure 1 shows this model

An alternate theory that accounts for both hereditary and non-hereditary cancer is the two-hit theory of cancer causation proposed by Dr. Alfred Knudson [13, 14]. Normal cells have two undamaged chromosomes, one inherited from each parent. People with a hereditary suscept‐ ibility to cancer inherit a damaged gene on one of the chromosomes at conception which is their 'first hit' or mutation. Others receive the 'first hit' in their lifetime. Damage to the same gene on the second chromosome in their lifetime may lead to cancer. An overview of this model

Weinberg and Hanahan have proposed the hallmarks of cancer which helps explain oncogen‐ esis. These are biological capabilities acquired during the complex multistep development of

explain the process of cancer formation and progression.

2 A Concise Review of Molecular Pathology of Breast Cancer

we are now with respect to this potent disease that affects millions.

is given in Figure 2 and is seen in cancer such as retinoblastoma.

ground breaking will be described.

**2. How does cancer arise?**

for colon carcinogenesis.

**Figure 2.** The two-hit model of carcinogenesis.

cancer. Figure 3 summarizes the 8 hallmarks of cancer. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming of energy metab‐ olism, and evading immune destruction [15]. All these hallmarks lead to genomic instability and persistent inflammation, possibly fueling further genetic diversity, as well as propagation, acquisition and fostering of multiple hallmark functions.

A possible contributing factor that hasn't gained much attention is the role of fragile sites. Common fragile sites (CFSs) are regions of the genome with a predisposition to DNA doublestrand breaks in response to intrinsic (oncogenic) or extrinsic replication stress. CFS breakage

**Figure 3.** The 8 possible hallmarks of cancer.

is a common feature in carcinogenesis from its earliest stages and through its evolutions. In a recent article the association of several fragile sites stability with key DNA damage response (DDR) and DNA repair proteins like breast cancer type 1 susceptibility protein (BRCA1), Ataxia telangiectasia and Rad3 related (ATR), and Ataxia telangiectasia mutated (ATM) opens another possibility for the induction and/or acceleration of instability in breast tissue [16]. For example *FRA3B*, one of the most frequently expressed fragile sites in the human genome, is located within the tumor suppressor gene *FHIT* region. Deletions within *FHIT* have been associated with various human cancers including breast [17].

## **3. Events that cause a normal breast epithelial cell to start the process of tumor formation and eventually progress to advanced metastatic disease**

A proto-oncogene is a normal gene that can convert to an oncogene due to mutations (generally dominant mutations) or increased expression [18-20]. Proto-oncogenes function in promoting cell division and inhibiting cell differentiation. Oncogenes, however, promote all the markers of a cancer cell such as increased cell division and replication stress, decreased cell differen‐ tiation, and inhibition of cell death (usually apoptosis). A proto-oncogene can convert into an oncogene due to various reasons including chromosomal translocation (such as BCR-ABL that is seen in leukemia), gene amplification, point mutations, deletions, alterations in promoter region leading to increased transcription, and insertions that lead to a hyperactive gene product. Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene that is amplified in about 30% of breast cancer [18]. This is discussed in detail in a subsequent section. To balance the effect of oncogenes, tumor suppressors are present as well to regulate cell growth and cell death but mutations in them can lead to tumor formation. The guardian of the genome, p53, is the most commonly mutated tumor suppressor gene in human cancer [21, 22]. It is involved in multiple pathways including maintenance of genomic stability by causing cell cycle arrest as the cell attempts to repair the damaged DNA, apoptosis, tumor progression, and metastasis [23]. Not surprisingly, a lot of breast cancers harbor mutations in this tran‐ scription factor as well. Since p53 has been linked to how BRCA1 dictates DNA repair and cell death, it may have a role in tumor response to treatment as well [24].

Checkpoints are present throughout the cell cycle that halt further progression of DNA replication and cell division, either permanently (senescence) or transiently, when damaged DNA is detected. This activates specific DNA repair pathways (discussed below). ATM and ATR are key proteins in the DNA damage response pathway. ATM is recruited to and activated by DNA double strand breaks while ATR is recruited to and activated by replication protein A-coated double stranded DNA. Two of the best studied ATM/ATR targets are the protein kinases checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2). Together with ATM and ATR, these proteins reduce cyclin dependent kinase (CDK) activity which slows down or arrests cell-cycle progression at the G1–S, intra-S and G2–M cell cycle checkpoints allowing more time for DNA repair before progression of replication or mitosis. Moreover, ATM/ATR can promote DNA repair by a variety of methods including induction of DNA repair proteins transcriptionally or post-transcriptionally, by recruiting repair factors to the damage-site, and by activating DNA-repair proteins by modulating their post-transcriptional modifications such as phosphorylation, acetylation, ubiquitylation or SUMOylation.

is a common feature in carcinogenesis from its earliest stages and through its evolutions. In a recent article the association of several fragile sites stability with key DNA damage response (DDR) and DNA repair proteins like breast cancer type 1 susceptibility protein (BRCA1), Ataxia telangiectasia and Rad3 related (ATR), and Ataxia telangiectasia mutated (ATM) opens another possibility for the induction and/or acceleration of instability in breast tissue [16]. For example *FRA3B*, one of the most frequently expressed fragile sites in the human genome, is located within the tumor suppressor gene *FHIT* region. Deletions within *FHIT* have been

**3. Events that cause a normal breast epithelial cell to start the process of tumor formation and eventually progress to advanced metastatic disease**

A proto-oncogene is a normal gene that can convert to an oncogene due to mutations (generally dominant mutations) or increased expression [18-20]. Proto-oncogenes function in promoting cell division and inhibiting cell differentiation. Oncogenes, however, promote all the markers of a cancer cell such as increased cell division and replication stress, decreased cell differen‐ tiation, and inhibition of cell death (usually apoptosis). A proto-oncogene can convert into an oncogene due to various reasons including chromosomal translocation (such as BCR-ABL that is seen in leukemia), gene amplification, point mutations, deletions, alterations in promoter region leading to increased transcription, and insertions that lead to a hyperactive gene product. Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene that is amplified in about 30% of breast cancer [18]. This is discussed in detail in a subsequent section.

associated with various human cancers including breast [17].

**Figure 3.** The 8 possible hallmarks of cancer.

4 A Concise Review of Molecular Pathology of Breast Cancer

Continuous DNA damage checkpoint activation may lead to selective suppression of the DNA-damage response-induced antitumor barriers. This may be due to inactivating muta‐ tions. This process promotes genomic instability and tumor progression [25-28]. Prolonged overexpression of licensing factors such as hCdt1 and hCdc6 prevent cell death and lead to a more aggressive phenotype. Overexpression of the replication licensing factor Cdc6 led to phenotypic changes with mesenchymal features and loss of E-cadherin. Analysis in various types of human cancer revealed a strong correlation between increased Cdc6 expression and reduced E-cadherin levels [29]. Cells possessing re-replicated DNA above a critical threshold are typically neutralized by cell death mechanisms but cells with re-replicated elements below a critical threshold are prone to recombination processes leading to genomic instability. As a result these cells are much more resistant to therapy [30].

DNA can be damaged spontaneously during replication stress and cell division as well as due to exogenous/environmental agents. This leads to thousands of DNA lesions/cell per day. In some cases of high oxidative or environmental stresses, repair resistant complex DNA damage can be induced as analytically discussed in a recent review by Kryston et al. 2011 [31]. As little as one unrepaired DNA double strand break can be lethal to the cell. Thus, the DDR and DNA repair pathways are in place to maintain the genomic integrity. This response pathway detects the DNA damage, signals their presence to recruit repair factors and halt cell cycle progression, and promote DNA repair. DNA lesions can block genomic replication and transcription and lead to mutations. Most of the time, cells undergo death in the form of apoptosis or necrosis when there is unrepaired DNA. Cells defective in DNA repair are hypersensitive towards DNA damaging agents. For example, breast cancer cells with defective BRCA proteins are sensitive to poly ADP ribose polymerase (PARP) inhibitors. This is an active area of research with promising results thus far. This is discussed further in a later section. DNA repair pathways include base excision repair (BER), nucleotide excision repair (NER), double strand break repair via homologous recombination (HR) or non-homologous end joining (NHEJ), and mismatch repair (MMR) [32-34]. Frequently, multiple proteins are involved in the repair of the damaged DNA. The repair pathways are briefly described below.

In MMR-mediated repair, nuclease, polymerase and ligase enzymes fix a single-strand cut that is induced upon detection of mismatches and insertion/deletion loops. DNA glycosylase detects a damaged base in BER-mediated repair. This is subsequently removed before nuclease, polymerase and ligase proteins complete the repair. NER-mediated repair recognizes helix-distorting base lesions. The damage is excised as a 22–30-base oligonucleotide, producing single-stranded DNA that is a substrate for DNA polymerases and associated factors. The process ends with ligation. There are 2 major DNA double strand break repair pathways. NHEJ is predominantly used in the repair of radiation induced DNA damage. It is highly efficient but error-prone. The Ku proteins recognize and bind to the damaged site and activate the protein kinase DNA-PKcs, leading to recruitment and activation of end-processing enzymes, polymerases and DNA ligase IV. In contrast, HR uses sister-chromatid sequences as the template to mediate faithful repair. It is used in repair of replicative stress-induced lesions, stalled replication forks, and inter-strand DNA crosslinks. HR starts with single strand DNA generation, which is promoted by various proteins including the MRE11–RAD50–NBS1 (MRN) complex. In events catalyzed by RAD51 and the breast-cancer susceptibility proteins BRCA1 and BRCA2, the single strand DNA then invades the undamaged template and, following the actions of proteins mentioned above such as polymerases, nucleases, helicases, etc., the DNA is repaired.

One of the most famous mutations in cancer is the BRCA family of genes which are critical for HR-mediated repair of DNA double strand breaks [35, 36]. Mutations in the BRCA genes lead to an increased risk for breast cancer as part of the hereditary breast-ovarian cancer syndrome. Women with mutated BRCA1 or BRCA2 gene have up to a 60% risk of developing breast cancer [37, 38]. Hypermethylation of the BRCA1 promoter may be an inactivating mechanism for BRCA1 expression [39, 40]. Many of the mutations in BRCA1 or BRCA2 that predispose to breast cancer cause premature termination of the amino acid coding sequences, resulting in a truncated, dysfunctional protein.

Mutations in ATM, a critical DNA repair protein, lead to Ataxia Telangiectasia (AT). As mentioned above, ATM is a serine/threonine protein kinase that is recruited and activated by DNA double strand breaks and phosphorylates proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2 and H2AX are tumor suppressors which explains why AT sufferers are predisposed to breast cancer and are hypersensitive to radiation [41, 42]. Another example is the Werner syndrome which is marked by mutations in Werner syndrome ATPdependent helicase (WRN) and Rad51 genes leading to deficiency in HR- and NHEJ mediated DNA double strand break repair which, as expected, leads to increased incidence of breast cancer.

when there is unrepaired DNA. Cells defective in DNA repair are hypersensitive towards DNA damaging agents. For example, breast cancer cells with defective BRCA proteins are sensitive to poly ADP ribose polymerase (PARP) inhibitors. This is an active area of research with promising results thus far. This is discussed further in a later section. DNA repair pathways include base excision repair (BER), nucleotide excision repair (NER), double strand break repair via homologous recombination (HR) or non-homologous end joining (NHEJ), and mismatch repair (MMR) [32-34]. Frequently, multiple proteins are involved in the repair of the

In MMR-mediated repair, nuclease, polymerase and ligase enzymes fix a single-strand cut that is induced upon detection of mismatches and insertion/deletion loops. DNA glycosylase detects a damaged base in BER-mediated repair. This is subsequently removed before nuclease, polymerase and ligase proteins complete the repair. NER-mediated repair recognizes helix-distorting base lesions. The damage is excised as a 22–30-base oligonucleotide, producing single-stranded DNA that is a substrate for DNA polymerases and associated factors. The process ends with ligation. There are 2 major DNA double strand break repair pathways. NHEJ is predominantly used in the repair of radiation induced DNA damage. It is highly efficient but error-prone. The Ku proteins recognize and bind to the damaged site and activate the protein kinase DNA-PKcs, leading to recruitment and activation of end-processing enzymes, polymerases and DNA ligase IV. In contrast, HR uses sister-chromatid sequences as the template to mediate faithful repair. It is used in repair of replicative stress-induced lesions, stalled replication forks, and inter-strand DNA crosslinks. HR starts with single strand DNA generation, which is promoted by various proteins including the MRE11–RAD50–NBS1 (MRN) complex. In events catalyzed by RAD51 and the breast-cancer susceptibility proteins BRCA1 and BRCA2, the single strand DNA then invades the undamaged template and, following the actions of proteins mentioned above such as polymerases, nucleases, helicases,

One of the most famous mutations in cancer is the BRCA family of genes which are critical for HR-mediated repair of DNA double strand breaks [35, 36]. Mutations in the BRCA genes lead to an increased risk for breast cancer as part of the hereditary breast-ovarian cancer syndrome. Women with mutated BRCA1 or BRCA2 gene have up to a 60% risk of developing breast cancer [37, 38]. Hypermethylation of the BRCA1 promoter may be an inactivating mechanism for BRCA1 expression [39, 40]. Many of the mutations in BRCA1 or BRCA2 that predispose to breast cancer cause premature termination of the amino acid coding sequences, resulting in a

Mutations in ATM, a critical DNA repair protein, lead to Ataxia Telangiectasia (AT). As mentioned above, ATM is a serine/threonine protein kinase that is recruited and activated by DNA double strand breaks and phosphorylates proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2 and H2AX are tumor suppressors which explains why AT sufferers are predisposed to breast cancer and are hypersensitive to radiation [41, 42]. Another example is the Werner syndrome which is marked by mutations in Werner syndrome ATPdependent helicase (WRN) and Rad51 genes leading to deficiency in HR- and NHEJ mediated

damaged DNA. The repair pathways are briefly described below.

6 A Concise Review of Molecular Pathology of Breast Cancer

etc., the DNA is repaired.

truncated, dysfunctional protein.

Breast cancer often metastasizes to bones, lungs, liver and brain [43-47]. The metastatic cascade is a series of biological steps that tumor cells must complete to exit the primary tumor and develop a new tumor at a distant site. One of the most critical steps involves invasion of the basement membrane and surrounding tissue and enter the bloodstream or lymphatic system. Cells that survive, eventually move into the tissue and establish a new colony that may form a tumor down the line. The host defense system is able to fend off millions of cancer cells that enter the blood stream but a few may escape nonetheless. Invasion involves the loss of cellcell adhesion which may be mediated by matrix metalloproteinases and urokinases which break down integrins which attach tumor cells to their microenvironment and plasminogen respectively [48-54]. Cadherins are an intricate part of cell-cell adhesion and so downregulation of e-cadherin and upregulation of n-cadherin, involved in epithelial and mesenchymal phenotypes respectively, can promote metastasis [55-60].

Circulating tumor cells (CTCs) which like breast cancer is a heterogeneous population on cells, have a crucial role in the metastatic cascade, tumor dissemination and progression. Epithelialto-mesenchymal transition (EMT) has an important role in the generation of CTCs and the acquisition of resistance to therapy [61-63]. Fibroblasts and myofibroblasts represent the majority of stromal cells within breast cancer. These cells promote the growth of cells by creating the perfect environment for cell survival and proliferation including enhanced angiogenesis. Tumor cells can express chemokine receptors that not only help direct migrating tumor cells to specific sites, they also determine if the cells will thrive and colonize at those sites. The bloodstream is highly unfavorable to tumor cells owing not only to the presence of immune cells, but also physical forces and anoikis, which combats metastasis. Interestingly, binding of tumor cells to coagulation factors, including tissue factor, fibrinogen, fibrin and thrombin, creates an embolus and facilitates arrest in capillary beds followed by the estab‐ lishment of metastasis [64].

EMT is an important process in metastasis. Here, epithelial cells lose cell-to-cell contacts and cell polarity, downregulate epithelial-associated genes, upregulate mesenchymal-genes, and undergo major changes in their cytoskeleton. This confers greater motility and invasiveness. Expression of stem-cell markers and acquisition of stem-cell characteristics are important processes in this pathway as well. Once the tumor cells seed at the secondary site, they undergo redifferentiation to an epithelial phenotype [65]. One of the factors involved in EMT is epithelial derived growth factor (EGFR) which induces tissue factor which in turn promotes tumor seeding via the process described above. The transcription factor Twist-related protein 1 (TWIST1), the receptor ligand tumor derived growth factor β (TGFβ), Hypoxia-inducible factor 1 (HIF1), HER2, and Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ Protein kinase B (AKT) signaling pathways have also been implicated in metastases. In preclinical models, expression of TWIST reduces metastasis and number of CTCs. CTCs often express NOTCH1 which confers self-renewal abilities. Some cells also express Aldehyde dehydrogen‐ ase 1 (ALDH1), another gene associated with stem cell like properties. Interleukin 6 (IL6) and Interleukin 8 (IL8) attract CTCs while Matrix metalloproteinase-1 (MMP1)–collagenase 1 and the actin cytoskeleton component fascin 1 help CTCs infiltrate into tumors. Overexpression of the chemokine receptor C-X-C chemokine receptor type 1 (CXCR1) in CTCs is associated with decreased metastases and may be a therapeutic target.

## **4. Risk factors for breast cancer**

Risk factors for malignant breast tumors include increased estrogen exposure which can be due to a number of reasons. For example, a woman can be exposed to increased estrogen due to increased total number of menstrual cycles, older age at 1st live birth, and obesity (increased estrogen exposure as adipose tissue converts androstenedione to estrone). BRCA1 and BRCA2 gene mutations also increase the risk of breast cancer and much research has been done in this avenue. Interestingly, increased incidence of triple negative breast cancer is seen in the African American population. Breast cancer risk is also increased with increased alcohol intake. Research suggests alcohol stimulates tumor growth by fuelling the production of growth factors that promote angiogenesis and by suppressing the immune system [66].

## **5. Classification of breast tumors as malignant or non-malignant**

The breast is an organized organ and diseases may arise at any of its structural subunits. The stroma provides a supporting environment and this is where fibroadenoma and phyllodes tumor can arise. The smallest subunit is the lobule where we can see lobular carcinoma. Lobules give rise to terminal ducts where we can see tubular carcinoma. Next are major ducts where fibrocystic changes, DCIS, and invasive ductal carcinoma are often seen. These join to form the lactiferous sinus where intraductal papilloma may arise. Finally, Paget disease can be seen at the nipple. Figure 4 summarizes the different breast pathologies.

**Figure 4.** Pathologies that can affect the different breast tissues.

Not all breast tumors are malignant. Fibroadenoma are small, mobile, firm mass with sharp edges. They are most common in those <35 years old and increase in size and tenderness in response to estrogen as is seen in pregnancy and prior to menstruation. As mentioned, it does not lead to breast cancer. Similarly, intraductal papillomas are small benign tumors that grow in lactiferous ducts, typically beneath the areola. They can cause serous (faintly yellow and thin) or bloody nipple discharge. Of note, they do increase the risk for carcinoma be approxi‐ mately 2-fold [67]. Phyllodes tumor are large bulky mass of connective tissue and cysts with leaf-like projections. They are most common in the 6th decade of life and similar to intraductal papilloma, can become malignant.

Malignant breast tumors are more common in postmenopausal women. They usually arise from terminal duct lobular unit. Overexpression of different proteins such as HER2 and EGFR are often seen. As discussed in a later section, receptor status can affect the therapy and prognosis. Since approximately 70% of the breast is drained by the axillary lymph node, involvement of this node indicating metastasis is the single most important prognostic factor. Since there is more tissue in the upper outer quadrant of the breast, tumors often arise here.

Malignant breast tumors can be subdivided into noninvasive and invasive tumors. Noninva‐ sive tumors include ductal carcinoma in situ (DCIS), Paget disease, and comedocarcinoma. Comedocarcinoma is a subtype of DCIS where ductal caseous necrosis is seen. DCIS fills the ductal lumen and arises from ductal atypia. They are often seen as microcalcification on mammography due to necrosis. Paget disease results from underlying DCIS and results in eczematous patches on the nipple. Invasive breast tumors include invasive ductal and lobular cancer. A firm, fibrous mass with sharp margins and small, glandular, duct-like cells are seen in invasive ductal tumors. They are the worst and most invasive of the tumors as well as the most common, comprising of over 70% of all breast cancer. Invasive lobular cancer often presents bilaterally with multiple lesions in the same location. Pathologically, they present as an orderly row of cells. Fleshy, cellular lymphocytic infiltrate is seen with medullary breast carcinoma and it has a good prognosis. Finally, inflammatory breast tumor presents with dermal lymphatic invasion and has approximately 50% survival at 5 years. Due to blockage of the lymphatic drainage, Peau d'orange is often seen with this condition.

The classification is important because treatment varies based on the type of cancer. When a tumor is diagnosed as benign, it is often left alone. With malignant tumors, biopsy is performed to determine the severity and aggressiveness of the tumor.

#### **5.1. Subtypes of breast cancer**

Molecular subtypes of breast cancer may be useful in planning treatment and developing new therapies and so a lot of research is being conducted in this field. Figure 5 depicts some of the more common subtypes. Most studies divide breast cancer into six major molecular subtypes:

**i.** Luminal A

the actin cytoskeleton component fascin 1 help CTCs infiltrate into tumors. Overexpression of the chemokine receptor C-X-C chemokine receptor type 1 (CXCR1) in CTCs is associated with

Risk factors for malignant breast tumors include increased estrogen exposure which can be due to a number of reasons. For example, a woman can be exposed to increased estrogen due to increased total number of menstrual cycles, older age at 1st live birth, and obesity (increased estrogen exposure as adipose tissue converts androstenedione to estrone). BRCA1 and BRCA2 gene mutations also increase the risk of breast cancer and much research has been done in this avenue. Interestingly, increased incidence of triple negative breast cancer is seen in the African American population. Breast cancer risk is also increased with increased alcohol intake. Research suggests alcohol stimulates tumor growth by fuelling the production of growth

factors that promote angiogenesis and by suppressing the immune system [66].

**5. Classification of breast tumors as malignant or non-malignant**

the nipple. Figure 4 summarizes the different breast pathologies.

**Figure 4.** Pathologies that can affect the different breast tissues.

The breast is an organized organ and diseases may arise at any of its structural subunits. The stroma provides a supporting environment and this is where fibroadenoma and phyllodes tumor can arise. The smallest subunit is the lobule where we can see lobular carcinoma. Lobules give rise to terminal ducts where we can see tubular carcinoma. Next are major ducts where fibrocystic changes, DCIS, and invasive ductal carcinoma are often seen. These join to form the lactiferous sinus where intraductal papilloma may arise. Finally, Paget disease can be seen at

decreased metastases and may be a therapeutic target.

**4. Risk factors for breast cancer**

8 A Concise Review of Molecular Pathology of Breast Cancer


**Figure 5.** Subtypes of breast cancer.

Some of the less common subtypes include apocrine molecular type. Molecular apocrine breast cancers are aggressive estrogen receptor negative tumors overexpressing either HER2 or gross cystic disease fluid protein-15 (GCDFP15) [68]. Breast cancers that do not fall into any of these subtypes are often listed as unclassified.

#### **i. Luminal A**

Most breast cancers are luminal tumors. Luminal tumor cells look the most like the cells of breast cancers that start in the inner (luminal) cells lining the mammary ducts. Luminal A tumors tend to be ER+ and/or PR+, HER2-, and tumor grade 1 or 2. Less than 15% of luminal A tumors have p53 mutations. Hence, luminal A tumors tend to have the best prognosis, with fairly high survival rates and fairly low recurrence rates. Since luminal A tumors tend to be ER+, treatment often includes hormonal therapy which is discussed in a subsequent section.

#### **ii. Luminal B**

As mentioned above, luminal tumors have cells that look like those of breast cancers that start in the inner (luminal) cells lining the mammary ducts. Luminal B tumors tend to be ER+ and/ or PR+. Since they have highly mitotically active cells, they are positive for Ki67. They are often HER2+ as well. Interestingly, women with luminal B tumors are often diagnosed at a younger age than those with luminal A tumors and have a poorer prognosis due to poorer tumor grade, larger tumor size and lymph node involvement. About 30% of the tumors also have mutations in p53.

## **iii. Triple negative/basal-like**

Triple negative breast cancers are: ER-, PR-, and HER2-; hence the name triple negative. There are several subsets of triple negative breast cancer. One subset is referred to as basal-like because the tumors have cells with features similar to those of the outer (basal) cells surround‐ ing the mammary ducts. Most basal-like tumors have mutations in p53. About 15 to 20% of breast cancers are triple negative or basal-like. These tumors tend to occur more often in younger and African American women. Of note, most BRCA1 breast cancers are both triple negative and basal-like. Triple negative/basal-like tumors are often aggressive and have a poorer prognosis. These tumors are usually treated with some combination of surgery, radiation therapy and chemotherapy.

#### **iv. HER2 type**

**iv.** HER2 positive **v.** Claudin low **vi.** Normal-like

10 A Concise Review of Molecular Pathology of Breast Cancer

**Figure 5.** Subtypes of breast cancer.

**i. Luminal A**

**ii. Luminal B**

subtypes are often listed as unclassified.

Some of the less common subtypes include apocrine molecular type. Molecular apocrine breast cancers are aggressive estrogen receptor negative tumors overexpressing either HER2 or gross cystic disease fluid protein-15 (GCDFP15) [68]. Breast cancers that do not fall into any of these

Most breast cancers are luminal tumors. Luminal tumor cells look the most like the cells of breast cancers that start in the inner (luminal) cells lining the mammary ducts. Luminal A tumors tend to be ER+ and/or PR+, HER2-, and tumor grade 1 or 2. Less than 15% of luminal A tumors have p53 mutations. Hence, luminal A tumors tend to have the best prognosis, with fairly high survival rates and fairly low recurrence rates. Since luminal A tumors tend to be ER+, treatment often includes hormonal therapy which is discussed in a subsequent section.

As mentioned above, luminal tumors have cells that look like those of breast cancers that start in the inner (luminal) cells lining the mammary ducts. Luminal B tumors tend to be ER+ and/ or PR+. Since they have highly mitotically active cells, they are positive for Ki67. They are often HER2+ as well. Interestingly, women with luminal B tumors are often diagnosed at a younger age than those with luminal A tumors and have a poorer prognosis due to poorer tumor grade,

The molecular subtype HER2 type is not the same as HER2+ and is not used to guide treatment. Although most HER2 type tumors are HER2+ (and named for this reason), about 30 percent are HER2-. HER2 type tumors tend to be ER-, PR-, with lymph node involvement and poor tumor grade. About 10% to 15% of breast cancers fall under this category and about 75% of HER2 type tumors contain p53 mutations. HER2 type tumors have a fairly poor prognosis and are prone to early and frequent recurrence and metastases. Women with HER2 type tumors appear to be diagnosed at a younger age than those with luminal A and luminal B tumors. HER2/neu-positive tumors can be treated with the drug trastuzumab (Herceptin) and this is discussed in further detail in a subsequent section.

#### **v. Claudin-low**

Claudin low is often triple-negative, but distinct in that there is low expression of cell-cell junction proteins including E-cadherin and frequently there is infiltration of lymphocytes. It is also enriched in mesenchymal and stem cell features [69].

#### **vi. Normal-like**

About 6 to 10% of all breast cancers are classified as normal-like. These tumors are usually small and tend to have a good prognosis.

## **6. Clinical diagnosis criteria and imaging modalities for breast cancer**

Breast cancer is divided into different stages. Table 1 summarizes these stages.

The extent of cancer can be used to stratify patients. Patients with clinical stage I, IIA, or a subset of stage IIB disease (T2N1 where T= tumor, N= node) are classified as having early-stage breast cancer. Patients with a T3 tumor without nodal involvement or stage IIIA to IIIC disease are classified as having locally advanced breast cancer. Stage IV is when there are distant metastases present and is seen in about 5% of newly diagnosed patients.


**Table 1.** Stages of breast cancer

#### **i. Early-stage breast cancer**

The surgical approach to the primary tumor depends on the size of the tumor, whether or not multifocal disease is present, and the size of the breast. Options include breast-conserving therapy or mastectomy and both have similar outcomes.

The risk for metastatic disease in the regional nodes is related to tumor size, histologic grade, and the presence of lymphatic invasion within the primary tumor. As mentioned above, the axillary nodes drain most of the breast tissue. Tumor characteristics are used to select adjuvant treatment for patients with breast cancer. Patients with hormone receptor-positive breast cancer should receive adjuvant endocrine therapy. For patients with triple-negative breast cancer, treatment option includes adjuvant chemotherapy if the tumor size is >0.5 cm. Patients with HER2-positive breast cancer >1 cm in size typically receive a combination of chemother‐ apy plus HER2-directed therapy. Following chemotherapy, patients with ER-positive disease generally receive adjuvant endocrine therapy.

#### **ii. Locally advanced breast cancer**

Most patients with locally advanced, inoperable breast cancer should receive neoadjuvant systemic therapy rather than proceeding with primary surgery in an attempt to shrink the tumor. Typically, these patients are usually not candidates for breast conservation. Neoadju‐ vant treatment improves the rate of breast conservation without compromising survival outcomes and so most patients get chemotherapy in the neoadjuvant setting rather than endocrine therapy. Due to its greater toxicity to cancer cells, chemotherapy is associated with higher response rates in a faster time frame. As mentioned earlier, HER2-directed agent (ie, trastuzumab) should be added to the chemotherapy regimen for tumors that are HER2 positive. Following surgery, all patients who undergo breast-conserving surgery generally undergo adjuvant radiation therapy (RT) to maximize locoregional control. Some patients treated by a mastectomy should receive postmastectomy RT in order to kill any cancer cells that may have escaped during the procedure.

Patients with hormone receptor-positive breast cancer should receive adjuvant endocrine therapy. The selection of endocrine therapy is made according to menopausal status. In patients with ER-positive breast cancer, in whom surgery is not an option or life expectancy is limited, primary hormonal treatment with either tamoxifen or an aromatase inhibitor without surgery is generally used.

**Figure 6.** Different treatments available for breast cancer.

## **7. Therapeutic options for breast cancer**

The heterogeneity of breast cancers makes it a challenge to diagnose and treat this solid tumor.

The main types of treatment for breast cancer are:

**i.** Surgery

**Stage Description**

**Table 1.** Stages of breast cancer

**0** Restricted to membrane of the milk duct (DCIS, LCIS)

**2** 2-5 cm tumor +/- metastasis to draining lymph node

therapy or mastectomy and both have similar outcomes.

generally receive adjuvant endocrine therapy.

that may have escaped during the procedure.

without surgery is generally used.

**ii. Locally advanced breast cancer**

**3** Metastasis to the lymph nodes +/- superficial skin and surrounding muscles

The surgical approach to the primary tumor depends on the size of the tumor, whether or not multifocal disease is present, and the size of the breast. Options include breast-conserving

The risk for metastatic disease in the regional nodes is related to tumor size, histologic grade, and the presence of lymphatic invasion within the primary tumor. As mentioned above, the axillary nodes drain most of the breast tissue. Tumor characteristics are used to select adjuvant treatment for patients with breast cancer. Patients with hormone receptor-positive breast cancer should receive adjuvant endocrine therapy. For patients with triple-negative breast cancer, treatment option includes adjuvant chemotherapy if the tumor size is >0.5 cm. Patients with HER2-positive breast cancer >1 cm in size typically receive a combination of chemother‐ apy plus HER2-directed therapy. Following chemotherapy, patients with ER-positive disease

Most patients with locally advanced, inoperable breast cancer should receive neoadjuvant systemic therapy rather than proceeding with primary surgery in an attempt to shrink the tumor. Typically, these patients are usually not candidates for breast conservation. Neoadju‐ vant treatment improves the rate of breast conservation without compromising survival outcomes and so most patients get chemotherapy in the neoadjuvant setting rather than endocrine therapy. Due to its greater toxicity to cancer cells, chemotherapy is associated with higher response rates in a faster time frame. As mentioned earlier, HER2-directed agent (ie, trastuzumab) should be added to the chemotherapy regimen for tumors that are HER2 positive. Following surgery, all patients who undergo breast-conserving surgery generally undergo adjuvant radiation therapy (RT) to maximize locoregional control. Some patients treated by a mastectomy should receive postmastectomy RT in order to kill any cancer cells

Patients with hormone receptor-positive breast cancer should receive adjuvant endocrine therapy. The selection of endocrine therapy is made according to menopausal status. In patients with ER-positive breast cancer, in whom surgery is not an option or life expectancy is limited, primary hormonal treatment with either tamoxifen or an aromatase inhibitor

**1** <2cm tumor restricted to the breast

12 A Concise Review of Molecular Pathology of Breast Cancer

**4** Metastasis to other parts of the body

**i. Early-stage breast cancer**


Treatments can be classified into broad groups (Figure 6), based on how they work and when they are used.

#### **a. Local and systemic therapy**

As the name implies, local therapy is intended to treat a tumor at the site without affecting the rest of the body. Examples include surgery and radiation therapy. Systemic therapy refers to drugs which can be given by mouth or directly into the bloodstream to reach cancer cells anywhere in the body. Chemotherapy, hormone therapy, and targeted therapy are systemic therapies that are widely used.

#### **b. Adjuvant and neoadjuvant therapy**

Since even in the early stages of breast cancer, cancer cells may break away from the primary breast tumor and begin to spread, adjuvant therapy is often given to patients with no detectable cancer after surgery. A small number of cells can't be 'felt' on a physical exam or seen on Xrays or other imaging tests, and they cause no symptoms until they reach a certain number but, menacingly, they can go on to become new tumors in nearby tissues, other organs, and bones. Hence, adjuvant therapy is a mainstay following surgery. Both systemic therapy like chemotherapy, hormone therapy, and targeted therapy, and radiation can be used as adjuvant therapy.

In neoadjuvant therapy, patients are treated with chemotherapy or hormonal therapy prior to surgery. The goal of this treatment is to shrink the tumor in the hope it will allow a less extensive operation to be done. This also lowers the chance of the cancer coming back later.

#### **i. Surgery**

For both DCIS and early-stage invasive breast cancer, doctors generally recommend surgery to remove the tumor. To make sure that the entire tumor is removed, the surgeon will also remove a small area of normal tissue around the tumor until a negative margin is achieved. A lumpectomy is the removal of the tumor and a small cancer-free margin while a mastectomy is the removal of the entire breast. It is important to lower the risk of recurrence and to get rid of any remaining cancer cells that can lead to both local and distant recurrence of cancer. Adjuvant therapies include radiation therapy, chemotherapy, targeted therapy, and/or hormonal therapy which are described below. Surgical treatment for breast cancer involves removal of the lymph nodes and can also include resection of the surrounding axillary nodes.

#### **ii. Radiation therapy**

This involves killing the cancer cells by inducing clustered DNA damage using ionizing radiation. By overwhelming the cell with DNA damage, the cell undergoes apoptosis. As little as one DNA double strand break can be lethal to the cell. By giving multiple doses of radiation broken up into fractions, the hope is to prolong survival. Some of the side effects include dermatologic issues, fibrosis, nausea etc. due to the radiation. Although most side effects usually go away after radiation therapy has been concluded, some long-term side effects may occur months or even years after treatment ends. These late effects which usually associate with persistent inflammation and oxidative stress may include develop‐ ing a secondary primary cancer. However, we must mention that the risk of developing a second cancer because of radiation therapy is relatively low, and this risk is generally outweighed by the benefit of treating the primary, existing cancer and offering survival to the patient.

#### **iii. Chemotherapy**

This involves using drugs and small molecules to selectively kill the cancer cells. Examples include: carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, fluorouracil (5-FU), gemcitabine, methotrexate, paclitaxel, etc. A patient may receive one drug at a time or combinations of different drugs at the same time. Research has shown that combinations of certain drugs are sometimes more effective than single drugs for adjuvant treatment and so combinations are often used. Carboplatin and cisplatin are alkylating agents and belong to the group of platinum-based antineoplastic agents. They interact with DNA to interfere with DNA repair. These drugs cross-link with the DNA strands, mostly to guanine groups. This causes intra- and inter-strand DNA cross-links, resulting in inhibition of DNA, RNA and protein synthesis. Antimetabolites, such as methotrexate, are more active against S-phase cells where they block DNA synthesis whereas vinca alkaloids are more active in the M-phase where they inhibit spindle formation and alignment of chromosomes. Antimetabolites are compounds that bear a structural similarity to naturally occurring substances such as vitamins, nucleosides or amino acids. They compete with the natural substrate for the active site on an essential enzyme or receptor. Methotrexate competitively inhibits dihydrofolate reductase, which is responsible for the formation of tetrahydrofolate from dihydrofolate. This plays an important role in the synthesis of, among others, purines and methionine. Anthracyclines such as doxorubicin intercalate with DNA and affect the topoisomerase II enzyme. This DNA gyrase splits the DNA helix and reconnects it to overcome the torsional forces that would interfere with replication. The anthracyclines stabilize the DNA topoisomerase II complex and thus prevent reconnection of the strands. Paclitaxel promotes assembly of microtubules and inhibits their disassembly which interferes with cell division.

**b. Adjuvant and neoadjuvant therapy**

14 A Concise Review of Molecular Pathology of Breast Cancer

therapy.

**i. Surgery**

**ii. Radiation therapy**

the patient.

**iii. Chemotherapy**

Since even in the early stages of breast cancer, cancer cells may break away from the primary breast tumor and begin to spread, adjuvant therapy is often given to patients with no detectable cancer after surgery. A small number of cells can't be 'felt' on a physical exam or seen on Xrays or other imaging tests, and they cause no symptoms until they reach a certain number but, menacingly, they can go on to become new tumors in nearby tissues, other organs, and bones. Hence, adjuvant therapy is a mainstay following surgery. Both systemic therapy like chemotherapy, hormone therapy, and targeted therapy, and radiation can be used as adjuvant

In neoadjuvant therapy, patients are treated with chemotherapy or hormonal therapy prior to surgery. The goal of this treatment is to shrink the tumor in the hope it will allow a less extensive

For both DCIS and early-stage invasive breast cancer, doctors generally recommend surgery to remove the tumor. To make sure that the entire tumor is removed, the surgeon will also remove a small area of normal tissue around the tumor until a negative margin is achieved. A lumpectomy is the removal of the tumor and a small cancer-free margin while a mastectomy is the removal of the entire breast. It is important to lower the risk of recurrence and to get rid of any remaining cancer cells that can lead to both local and distant recurrence of cancer. Adjuvant therapies include radiation therapy, chemotherapy, targeted therapy, and/or hormonal therapy which are described below. Surgical treatment for breast cancer involves removal of the lymph nodes and can also include resection of the surrounding axillary nodes.

This involves killing the cancer cells by inducing clustered DNA damage using ionizing radiation. By overwhelming the cell with DNA damage, the cell undergoes apoptosis. As little as one DNA double strand break can be lethal to the cell. By giving multiple doses of radiation broken up into fractions, the hope is to prolong survival. Some of the side effects include dermatologic issues, fibrosis, nausea etc. due to the radiation. Although most side effects usually go away after radiation therapy has been concluded, some long-term side effects may occur months or even years after treatment ends. These late effects which usually associate with persistent inflammation and oxidative stress may include develop‐ ing a secondary primary cancer. However, we must mention that the risk of developing a second cancer because of radiation therapy is relatively low, and this risk is generally outweighed by the benefit of treating the primary, existing cancer and offering survival to

This involves using drugs and small molecules to selectively kill the cancer cells. Examples include: carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, fluorouracil (5-FU), gemcitabine, methotrexate, paclitaxel, etc. A patient may receive one drug at a time or

operation to be done. This also lowers the chance of the cancer coming back later.

One of the more recent treatment options for breast are PARP inhibitors which showed initial promise in patients with tumors that have BRCA1 or BRCA2 mutations and therefore deficient double strand break repair. PARP inhibitors achieve an enhanced or synthetic lethality for tumor cells by blocking DNA repair pathways. PARP, which has multiple family members, detects single strand DNA breaks and participates in BER. It forms poly (ADP-ribose) polymers on itself and a number of substrates which can alter a number of pathways including DNA repair. Inhibition of PARP leads to persistent single strand break which converts to a double strand break as the cell attempts to replicate the DNA. Normal cells have an intact HRmediated repair pathway and so are able to repair the DNA double strand break. However, in the absence of intact HR-mediated repair pathway which can happen with loss of or mutation in BRCA proteins, the cell is unable to repair the double strand break. As a result, typically, the cell undergoes apoptosis. A phase II study of the PARP inhibitor olaparib in patients with advanced breast cancer with BRCA1 or BRCA2 mutations has shown promising results with a response rate of 11/27, a progression-free survival of 5.7 months, and a median objective response duration of 144 days [70]. Phase III trials are currently in progress to evaluate olaparib in breast cancer [71]. TNBC also demonstrates BRCAness and so PARP inhibitors may be useful in this setting as well. Data from clinical trials have not been conclusive in this regard thus far.

Phosphatase and tensin homolog (PTEN) regulates RAD51 mediated DNA repair to maintain genomic stability. PTEN mutations, which occur in 30–50% of breast cancers, cause genomic instability similar to that seen in BRCA-deficient cells and so may be targets of PARP inhibitors as well [72].

#### **iv. Hormonal therapy**

Hormonal therapy is widely used in breast cancer treatment. These are used in the setting of ER+ and PR+ tumors. Since these tumors use hormones to fuel their growth, blocking the hormones can help prevent or at least slow down the growth of the tumor.

Selective estrogen receptor modulators (SERMs) are a class of compounds that act on the estrogen receptor. Tamoxifen blocks estrogen from binding to breast cancer cells. It is effective for not only lowering the risk of recurrence in the breast that had cancer, it also reduces the risk of developing cancer in the other breast, and the risk of distant recurrence. It is also approved to reduce the risk of breast cancer in women at high risk for developing breast cancer and for lowering the risk of a local recurrence for women with DCIS who have had a lumpec‐ tomy. Tamoxifen is also an effective treatment for metastatic hormone receptor-positive breast cancer. However, chronic Tamoxifen use has been linked with some toxicity and adverse effects like persistent oxidative stress and others as reviewed in [73].

Aromatase inhibitors (AIs) decrease the amount of estrogen made by tissues other than the ovaries in postmenopausal women by blocking the aromatase enzyme, which converts androgens into estrogen. These drugs include anastrozole and exemestane. Similar to Tamox‐ ifen, AIs are also an effective treatment for metastatic hormone receptor positive breast cancer.

Fulvestrant, a SERM, is an additional hormonal therapy approved for patients with metastatic breast cancer. Fulvestrant is an estrogen-receptor targeting therapy that is used for the treatment of advanced-stage breast cancer in postmenopausal women with endocrinesensitive cancer [74-77].

#### **v. Targeted therapy**

Targeted therapy is a treatment that targets specific genes or proteins. One of the advantages of this is that it limits damage to healthy cells. Trastuzumab, a monoclonal antibody, is approved for both the treatment of advanced breast cancer and as an adjuvant therapy for early-stage HER2+ breast cancer. Trastuzumab does have cardio toxic effects. Pertuzumab is a monoclonal antibody marketed by Genentech for the treatment of HER2+ breast cancer, in combination with trastuzumab and docetaxel. It inhibits the dimerization of HER2 with other HER receptors, which reduces tumor growth. Lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2/neu and epidermal growth factor receptor (EGFR) pathways, is com‐ monly used for women with HER2-positive metastatic breast cancer when trastuzumab and pertuzumab in combination with docetaxel are no longer effective at controlling the cancer's growth. Lapatinib decreases tumor-causing breast cancer stem cells and inhibits receptor signal processes by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain, preventing auto-phosphorylation and subsequent activation of the signal mechanism.

Table 2 lists some of the current trials evaluating different therapies for breast cancer.


**iv. Hormonal therapy**

16 A Concise Review of Molecular Pathology of Breast Cancer

sensitive cancer [74-77].

**v. Targeted therapy**

Hormonal therapy is widely used in breast cancer treatment. These are used in the setting of ER+ and PR+ tumors. Since these tumors use hormones to fuel their growth, blocking the

Selective estrogen receptor modulators (SERMs) are a class of compounds that act on the estrogen receptor. Tamoxifen blocks estrogen from binding to breast cancer cells. It is effective for not only lowering the risk of recurrence in the breast that had cancer, it also reduces the risk of developing cancer in the other breast, and the risk of distant recurrence. It is also approved to reduce the risk of breast cancer in women at high risk for developing breast cancer and for lowering the risk of a local recurrence for women with DCIS who have had a lumpec‐ tomy. Tamoxifen is also an effective treatment for metastatic hormone receptor-positive breast cancer. However, chronic Tamoxifen use has been linked with some toxicity and adverse

Aromatase inhibitors (AIs) decrease the amount of estrogen made by tissues other than the ovaries in postmenopausal women by blocking the aromatase enzyme, which converts androgens into estrogen. These drugs include anastrozole and exemestane. Similar to Tamox‐ ifen, AIs are also an effective treatment for metastatic hormone receptor positive breast cancer.

Fulvestrant, a SERM, is an additional hormonal therapy approved for patients with metastatic breast cancer. Fulvestrant is an estrogen-receptor targeting therapy that is used for the treatment of advanced-stage breast cancer in postmenopausal women with endocrine-

Targeted therapy is a treatment that targets specific genes or proteins. One of the advantages of this is that it limits damage to healthy cells. Trastuzumab, a monoclonal antibody, is approved for both the treatment of advanced breast cancer and as an adjuvant therapy for early-stage HER2+ breast cancer. Trastuzumab does have cardio toxic effects. Pertuzumab is a monoclonal antibody marketed by Genentech for the treatment of HER2+ breast cancer, in combination with trastuzumab and docetaxel. It inhibits the dimerization of HER2 with other HER receptors, which reduces tumor growth. Lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2/neu and epidermal growth factor receptor (EGFR) pathways, is com‐ monly used for women with HER2-positive metastatic breast cancer when trastuzumab and pertuzumab in combination with docetaxel are no longer effective at controlling the cancer's growth. Lapatinib decreases tumor-causing breast cancer stem cells and inhibits receptor signal processes by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain, preventing auto-phosphorylation and subsequent activation of the signal mechanism.

Table 2 lists some of the current trials evaluating different therapies for breast cancer.

hormones can help prevent or at least slow down the growth of the tumor.

effects like persistent oxidative stress and others as reviewed in [73].


**Table 2.** Current clinical trials evaluating therapies for breast cancer

#### **8. Conclusion**

Breast cancer continues to be a threat and a challenge to treat. While a lot has been accom‐ plished in the past decade, there is more that can be done. Further understanding of tumor evolution will lead to the eradication and effective prevention of this disease. At the same time delineating the breast oncogenic mechanisms like DNA damage response, conversion of DNA lesions to mutations, etc. will help us target initiating events and further optimize personalized therapies and possibly develop new ones. Therefore we believe that it is the 'DNA' which plays the dominant role and holds the key for effective treatment of the whole phenomenon of breast carcinogenesis.

## **Abbreviations**

**ClinicalTrials.gov Identifier Description**

18 A Concise Review of Molecular Pathology of Breast Cancer

**NCT01509625**

**NCT01534455**

**NCT01880385**

**NCT01881230**

**NCT02000622**

**NCT02202746**

**8. Conclusion**

carcinogenesis.

**NCT01351597** Evaluate the efficacy and safety of combination chemotherapy with DoceTaxel

the adjuvant and/or metastatic setting.

patients with inflammatory breast cancer.

metastatic triple negative breast cancer.

cancer.

**Table 2.** Current clinical trials evaluating therapies for breast cancer

breast cancer patients with germline BRCA 1/2 mutations.

Breast cancer continues to be a threat and a challenge to treat. While a lot has been accom‐ plished in the past decade, there is more that can be done. Further understanding of tumor evolution will lead to the eradication and effective prevention of this disease. At the same time delineating the breast oncogenic mechanisms like DNA damage response, conversion of DNA lesions to mutations, etc. will help us target initiating events and further optimize personalized therapies and possibly develop new ones. Therefore we believe that it is the 'DNA' which plays the dominant role and holds the key for effective treatment of the whole phenomenon of breast

(Detaxel) and Oxaliplatin (Oxalitin) in recurrent or metastatic breast cancer

dose after having progressed with a previous anti-estrogen therapy.

Assess the response to treatment with fulvestrant at a dose of 500 mg/month with a loading dose of 500 mg, in terms of progression free survival, overall survival, and clinical benefit rate, in post-menopausal women with advanced breast cancer and estrogen receptor positive, who were treated with this medicinal product and at said

Compare the efficacy and tolerability of two dose-schedules of eribulin (a ketone analog) plus lapatinib in HER2-positive breast cancer, pre-treated with trastuzumab in

Evaluating the treatment of bevacizumab in association with pre-operative chemotherapy, followed by surgery, adjuvant chemotherapy and radiotherapy in

Compare the safety and efficacy of nab-paclitaxel in combination with either gemcitabine or carboplatin to the combination of gemcitabine and carboplatin as first line treatment in female subjects with triple negative metastatic breast cancer or

Assess the efficacy and safety of single agent olaparib, a PARP inhibitor, vs standard of care based on physician's choice of capecitabine (that is converted to 5-FU during metabolism), vinorelbine (anti-mitotic drug) or eribulin (a ketone analog) in metastatic

Determine whether lucitanib, a potent tyrosine kinase inhibitor, is safe and effective in the treatment of patients with fibroblast growth factor aberrant metastatic breast


HIF1: Hypoxia-inducible factor 1 HR: Homologous recombination IL6: Interleukin 6 IL8: Interleukin 8 MMP1: Matrix metalloproteinase-1 MMR: Mismatch repair MRN: MRE11–RAD50–NBS1 NER: Nucleotide excision repair NHEJ: Non-homologous end joining PARP: Poly ADP ribose polymerase PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase PTEN: Phosphatase and tensin homolog RT: Radiation therapy SERM: Selective estrogen receptor modulator TGFβ: Tumor derived growth factor β TWIST: Twist-related protein WRN: Werner syndrome ATP-dependent helicase

## **Acknowledgements**

Somaira Nowsheen and Khaled Aziz thank the Mayo Clinic Medical Scientist Training Program for fostering an outstanding environment for physician-scientist training. Somaira Nowsheen was supported by the Laura J. Siegel Breast Cancer Fellowship Award from the Foundation for Women's Wellness. Dr. Georgakilas was supported by an EU Marie Curie Reintegration Grant MC-CIG-303514, Greek National funds through the Operational Program Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)- Research Funding Program: THALES (Grant number MIS 379346) and COST Action CM1201 Biomimetic Radical Chemistry.

## **Author details**

HIF1: Hypoxia-inducible factor 1

20 A Concise Review of Molecular Pathology of Breast Cancer

HR: Homologous recombination

MMP1: Matrix metalloproteinase-1

IL6: Interleukin 6

IL8: Interleukin 8

MMR: Mismatch repair

RT: Radiation therapy

MRN: MRE11–RAD50–NBS1

NER: Nucleotide excision repair

NHEJ: Non-homologous end joining

PARP: Poly ADP ribose polymerase

PTEN: Phosphatase and tensin homolog

TGFβ: Tumor derived growth factor β

TWIST: Twist-related protein

**Acknowledgements**

Biomimetic Radical Chemistry.

SERM: Selective estrogen receptor modulator

WRN: Werner syndrome ATP-dependent helicase

Somaira Nowsheen and Khaled Aziz thank the Mayo Clinic Medical Scientist Training Program for fostering an outstanding environment for physician-scientist training. Somaira Nowsheen was supported by the Laura J. Siegel Breast Cancer Fellowship Award from the Foundation for Women's Wellness. Dr. Georgakilas was supported by an EU Marie Curie Reintegration Grant MC-CIG-303514, Greek National funds through the Operational Program Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)- Research Funding Program: THALES (Grant number MIS 379346) and COST Action CM1201

PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase

Somaira Nowsheen1 , Khaled Aziz1 , Asef Aziz2 and Alexandros G Georgakilas3\*

\*Address all correspondence to: alexg@mail.ntua.gr

1 Medical Scientist Training Program, Mayo Graduate School, Mayo Medical School, Mayo Clinic, Rochester, MN, USA

2 Baylor University, Waco, TX, USA

3 Department of Physics, School of Applied Mathematical and Physical Sciences, National Technical University of Athens, Zografou Campus, GR, Athens, Greece

### **References**


[26] Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, et al. Oncogeneinduced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature. 2006;444(7119):633-7.

[11] Whibley C, Pharoah PD, Hollstein M. p53 polymorphisms: cancer implications. Na‐

[12] Muller PA, Vousden KH. Mutant p53 in Cancer: New Functions and Therapeutic Op‐

[13] Knudson AG. Two genetic hits (more or less) to cancer. Nature Reviews Cancer.

[14] Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nature Reviews

[15] Hanahan D, Weinberg Robert A. Hallmarks of Cancer: The Next Generation. Cell.

[16] Georgakilas AG, Tsantoulis P, Kotsinas A, Michalopoulos I, Townsend P, Gorgoulis VG. Are common fragile sites merely structural domains or highly organized "func‐ tional" units susceptible to oncogenic stress? Cellular and Molecular Life Sciences.

[17] Dillon LW, Burrow AA, Wang Y-H. DNA instability at chromosomal fragile sites in

[18] Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER2/neu proto-oncogene in human breast and ovarian cancer. Science.

[19] Finlay CA, Hinds PW, Levine AJ. The p53 proto-oncogene can act as a suppressor of

[20] Franke TF, Yang S-I, Chan TO, Datta K, Kazlauskas A, Morrison DK, et al. The pro‐ tein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated

[21] Malkin D, Li FP, Strong LC, Fraumeni J, Nelson CE, Kim DH, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms.

[22] Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers.

[23] Brosh R, Rotter V. When mutants gain new powers: news from the mutant p53 field.

[24] Yang ES, Nowsheen S, Rahman MA, Cook RS, Xia F. Targeting BRCA1 localization to augment breast tumor sensitivity to poly(ADP-ribose) polymerase inhibition. Can‐

[25] Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage re‐ sponse as a candidate anti-cancer barrier in early human tumorigenesis. Nature.

ture Reviews Cancer. 2009;9(2):95-107.

2001;1(2):157-62.

2011;144(5):646-74.

1989;244(4905):707-12.

2014:1-26.

Cancer. 2004;4(3):197-205.

22 A Concise Review of Molecular Pathology of Breast Cancer

portunities. Cancer Cell. 2014;25(3):304-17.

cancer. Current genomics. 2010;11(5):326.

transformation. Cell. 1989;57(7):1083-93.

Science. 1990;250(4985):1233-8.

Science. 1991;253(5015):49-53.

cer Res. 2012; 72(21): 5547-5555.

2005;434(7035):864-70.

Nature Reviews Cancer. 2009;9(10):701-13.

phosphatidylinositol 3-kinase. Cell. 1995;81(5):727-36.


[55] Oka H, Shiozaki H, Kobayashi K, Inoue M, Tahara H, Kobayashi T, et al. Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relation‐ ship to metastasis. Cancer Research. 1993;53(7):1696-701.

[40] Marsit CJ, Liu M, Nelson HH, Posner M, Suzuki M, Kelsey KT. Inactivation of the Fanconi anemia//BRCA pathway in lung and oral cancers: implications for treatment

[41] Alderton G. Radiation sensitivity: Tolerance is not a virtue. Nat Rev Cancer.

[42] Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev

[43] Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate

[44] Coleman R, Rubens R. The clinical course of bone metastases from breast cancer.

[45] Weigelt B, Peterse JL, Van't Veer LJ. Breast cancer metastasis: markers and models.

[46] Mehrotra J, Vali M, McVeigh M, Kominsky SL, Fackler MJ, Lahti-Domenici J, et al. Very high frequency of hypermethylated genes in breast cancer metastasis to the

[47] Bos PD, Zhang XH-F, Nadal C, Shu W, Gomis RR, Nguyen DX, et al. Genes that me‐ diate breast cancer metastasis to the brain. Nature. 2009;459(7249):1005-9.

[48] Gomez D, Alonso D, Yoshiji H, Thorgeirsson U. Tissue inhibitors of metalloprotei‐ nases: structure, regulation and biological functions. European journal of cell biolo‐

[49] Nagase H, Woessner JF. Matrix metalloproteinases. Journal of Biological Chemistry.

[50] Gialeli C, Theocharis AD, Karamanos NK. Roles of matrix metalloproteinases in can‐ cer progression and their pharmacological targeting. FeBS Journal. 2011;278(1):16-27.

[51] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor

[52] Bourboulia D, Stetler-Stevenson WG, editors. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in

[53] Leblanc R, Lee S, David M, Bordet J, Norman D, Patil R, et al. Interaction of plateletderived autotaxin with tumor integrin αVβ3 controls metastasis of breast cancer cells

[54] Lorger M, Felding-Habermann B. Integrin Signaling in Angiogenesis and Metastatic Cancer Progression in the Brain. Signaling Pathways and Molecular Mediators in

tumor cell adhesion. Seminars in cancer biology; 2010: Elsevier.

breast cancer metastasis to lung. Nature. 2005;436(7050):518-24.

bone, brain, and lung. Clinical Cancer Research. 2004;10(9):3104-9.

and survival. Oncogene. 2003;23(4):1000-4.

British journal of cancer. 1987;55(1):61.

Nature Reviews Cancer. 2005;5(8):591-602.

microenvironment. Cell. 2010;141(1):52-67.

Metastasis: Springer; 2012. p. 311-29.

2007;7(4):230-1.

Cancer. 2003;3(3):155-68.

24 A Concise Review of Molecular Pathology of Breast Cancer

gy. 1997;74(2):111-22.

1999;274(31):21491-4.

to bone. Blood. 2014.


mors overexpressing either HER2 or GCDFP15. Breast Cancer Research. 2013;15(3):R37.


## **Chapter 2**

## **DNA Methylation**

mors overexpressing either HER2 or GCDFP15. Breast Cancer Research.

[69] Prat A, Parker J, Karginova O, Fan C, Livasy C, Herschkowitz J, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer.

[70] Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN, et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. The Lancet.

[71] Tutt A, Balmana J, Robson M, Garber J, Kaufman B, Geyer C, et al. 331TiPOlympia, Neo-Olympia and Olympiad: Randomized phase III trials of Olaparib in patients with breast cancer and a germline BRCA1/2 mutation. Annals of Oncology.

[72] Zhang HY, Liang F, Jia ZL, Song ST, Jiang ZF. PTEN mutation, methylation and ex‐

[73] Yang G, Nowsheen S, Aziz K, Georgakilas AG. Toxicity and adverse effects of Ta‐ moxifen and other anti-estrogen drugs. Pharmacology & therapeutics. 2013;139(3):

[74] Clemons MJ, Cochrane B, Pond GR, Califaretti N, Chia SK, Dent RA, et al. Rando‐ mised, phase II, placebo-controlled, trial of fulvestrant plus vandetanib in postmeno‐ pausal women with bone only or bone predominant, hormone-receptor-positive metastatic breast cancer (MBC): the OCOG ZAMBONEY study. Breast Cancer Re‐

[75] Massarweh S, Romond E, Black EP, Van Meter E, Shelton B, Kadamyan-Melkumian V, et al. A phase II study of combined fulvestrant and everolimus in patients with metastatic estrogen receptor (ER)-positive breast cancer after aromatase inhibitor

[76] Schwartzberg LS, Wang G, Somer BG, Blakely LJ, Wheeler BM, Walker MS, et al. Phase II Trial of Fulvestrant With Metronomic Capecitabine for Postmenopausal Women With Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer.

[77] Bachelot T, McCool R, Duffy S, Glanville J, Varley D, Fleetwood K, et al. Compara‐ tive efficacy of everolimus plus exemestane versus fulvestrant for hormone-receptorpositive advanced breast cancer following progression/recurrence after endocrine therapy: a network meta-analysis. Breast Cancer Research and Treatment.

(AI) failure. Breast Cancer Research and Treatment. 2014;143(2):325-32.

pression in breast cancer patients. Oncology letters. 2013;6(1):161-8.

2013;15(3):R37.

26 A Concise Review of Molecular Pathology of Breast Cancer

2010;376(9737):235-44.

2014;25(suppl 4):iv109.

392-404.

Breast Cancer Research. 2010;12(5):R68.

search and Treatment. 2014;146(1):153-62.

Clinical breast cancer. 2014;14(1):13-9.

2014;143(1):125-33.

Majed S. Alokail and Amal M. Alenad

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59467

## **1. Introduction**

DNA methylation is a major epigenetic modification that is strongly involved in the physio‐ logical control of genome expression. Developmental processes and proper biological func‐ tions are tightly dependent on hierarchical and regulated gene expression patterns. Numerous molecular processes control gene expression. DNA methylation is a physiological epigenetic process that leads to long term-repression of gene expression. DNA methylation is a common epigenetic modification involving the methylation of 5'-cytosine residues and is often detected in the dinucleotides of CpG sequences. Methylation is often localized in promoter regions and occasionally in transcriptional regulatory regions in mammals, plants and even prokaryotes. DNA methylation may be classified as hyper-and hypomethylation, according to increased and decreased levels of genomic modification, respectively. Hypermethylation is an epigenetic alteration often leading to gene-inactivating deletions and translocations. Hypermethylated cells may exhibit a phenotype of drug-resistance or malignant proliferation. Aberrant meth‐ ylation in eukaryotic cells may lead to silencing of important genes, such as tumour suppressor genes, affecting their related transcriptional pathways and ultimately leading to the develop‐ ment of disease such as cancer. Therefore, it is considered to be a hallmark of cancer, it is detected in several types of cancer cells, including colon, breast, ovarian and cervical cancer cells and is associated with alterations in specific gene expression.

Hypermethylation of tumour suppressor gene promoters and global disruption of many histone modifications are characteristic features of cancer. Deregulation of the epigenetic profile alters the transcription profile of many genes. In the case of tumour suppressors DNA methylation reduces gene expression and subsequently removes regulatory proteins required for normal cell growth and development. Therefore, DNA methylation in cancer would be predicted to influence multiple gene networks rather than single genes. Because of heteroge‐ neity of breast cancer at both histological and molecular levels staging breast cancer fails to predict prognosis or therapeutic response of the disease, therefore, DNA methylation targeted

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

therapies, in recent years, play an increased role in the treatment of breast cancer. DNA methylation targeted therapies, in recent years, play an increased role in the treatment of breast cancer. Two groups of agents targeting epigenetic modifications have been studied previously, namely histone deacetylase inhibitors and DNA methyl transferase inhibitors. The associations between DNA methylation mechanism and breast cancer classification and prognosis will be reviewed in this chapter in detail by describing the DNA methylation mechanism and gene expression in breast cancer, as well as functional genomics and genome wide DNA methyla‐ tion in breast cancer.

#### **2. What is epigenetics?**

The term epigenetic was introduced by Conard Waddington in 1942 as a concept of environ‐ mental influence in inducing phenotype modification. His work on developmental plasticity states that the environmental influences during development could induce alternative phenotypes from one genotype, one of the clearest examples is polyphenisms in insects. He showed that exposing the pupae of wild type Drosophila melanogaster to heat shock treat‐ ment, results in altered wing vein patterns [1,2]. Breeding individuals who have been exposed to these environmentally induced changes led to a stable population exhibiting the phenotype without the environmental stimulus. The concept of epigenetics was not clarified until the late 1990s when Wolffe and Matzkeset the modern definition, which was 'the study of heritable changes in gene expression that occur without a change in DNA sequence'[4]. Bird came with a wider definition of epigenetic which is 'the structural adaptation of chromosomal regions so as to register, signal or perpetuate activity states' [5]. The term epigenome has emerged to describe the epigenetic modifications all over the epigenome, thus, the epigenome controls the genome in both normal and abnormal cellular processes and events [6]. Epigenetic mecha‐ nisms include; DNA methylation, histone modification and non-coding RNAs, which work cooperatively to control gene expression.

#### **3. DNA methylation**

DNA methylation is a well conserved process that occurs in eukaryotes and prokaryotes [7]. DNA methylation refers to the covalent addition of a methyl group to carbon number five in the nitrogenous base cytosine at the DNA strand. Only cytosine residues where adjacent to guanine are targets for the methylation by the methyltransferases enzymes and the distribution of methylated and unmethylated CpGs is tissue-specific which leads to cell-specific pattern of DNA methylation [8]. The CpG may occur in multiple repeats which are known as CpG islands [9]. These regions are often associated with the promoter regions of genes. Almost half of the genes in our genome have CpG rich promoter regions. In the whole genome, about 80% of the CpG dinucleotides not associated with CpG islands are heavily methylated [10]. In contrast, the CpG islands associated with gene promoters are usually unmethylated [11].

There are a number of factors that may maintain the undermethylated state of CpG islands, such as sequence feature, SP1 binding sites, specific acting enhancer elements, as well as specific histone methylation mark H3K4me3, which prevents the binding of de novo methyl‐ ation complexes [12]. Methylation of the CpG islands in the promoter region silences gene expression, and the absence of methylation is associated with active transcription. Thus unmethylated CpG islands are associated with the promoters of transcriptionally active genes, such as housekeeping genes and many regulated genes, such as genes showing tissue specific expression [13]. DNA methylation information at every cytosine can be determined, but it was targeted at few candidate genes using methylation-sensitive restriction enzymes or genespecific DNA methylation mapping by sequencing bisulfite-converted DNA. In contrast, development of advance technology in DNA methylation mapping, including high-density oligonucleotide arrays, illumina bead arrays and next-generation high-throughput sequenc‐ ing, together with advances in bioinformatics, have enable examination of broad regions of the genome and provide high-content profiles of DNA methylation.

#### **3.1. DNA Methyltransferases (DNMTs)**

therapies, in recent years, play an increased role in the treatment of breast cancer. DNA methylation targeted therapies, in recent years, play an increased role in the treatment of breast cancer. Two groups of agents targeting epigenetic modifications have been studied previously, namely histone deacetylase inhibitors and DNA methyl transferase inhibitors. The associations between DNA methylation mechanism and breast cancer classification and prognosis will be reviewed in this chapter in detail by describing the DNA methylation mechanism and gene expression in breast cancer, as well as functional genomics and genome wide DNA methyla‐

The term epigenetic was introduced by Conard Waddington in 1942 as a concept of environ‐ mental influence in inducing phenotype modification. His work on developmental plasticity states that the environmental influences during development could induce alternative phenotypes from one genotype, one of the clearest examples is polyphenisms in insects. He showed that exposing the pupae of wild type Drosophila melanogaster to heat shock treat‐ ment, results in altered wing vein patterns [1,2]. Breeding individuals who have been exposed to these environmentally induced changes led to a stable population exhibiting the phenotype without the environmental stimulus. The concept of epigenetics was not clarified until the late 1990s when Wolffe and Matzkeset the modern definition, which was 'the study of heritable changes in gene expression that occur without a change in DNA sequence'[4]. Bird came with a wider definition of epigenetic which is 'the structural adaptation of chromosomal regions so as to register, signal or perpetuate activity states' [5]. The term epigenome has emerged to describe the epigenetic modifications all over the epigenome, thus, the epigenome controls the genome in both normal and abnormal cellular processes and events [6]. Epigenetic mecha‐ nisms include; DNA methylation, histone modification and non-coding RNAs, which work

DNA methylation is a well conserved process that occurs in eukaryotes and prokaryotes [7]. DNA methylation refers to the covalent addition of a methyl group to carbon number five in the nitrogenous base cytosine at the DNA strand. Only cytosine residues where adjacent to guanine are targets for the methylation by the methyltransferases enzymes and the distribution of methylated and unmethylated CpGs is tissue-specific which leads to cell-specific pattern of DNA methylation [8]. The CpG may occur in multiple repeats which are known as CpG islands [9]. These regions are often associated with the promoter regions of genes. Almost half of the genes in our genome have CpG rich promoter regions. In the whole genome, about 80% of the CpG dinucleotides not associated with CpG islands are heavily methylated [10]. In contrast,

the CpG islands associated with gene promoters are usually unmethylated [11].

tion in breast cancer.

**2. What is epigenetics?**

28 A Concise Review of Molecular Pathology of Breast Cancer

cooperatively to control gene expression.

**3. DNA methylation**

The methylation process is catalysed by the DNA methyltransferases enzymes (DNMTs) which are known as DNMTs; DNMT1, DNMT3A, DNMT3B, and DNMT3L [14]. DNMT3A and DNMT3B are the de novo methyltransferases while DNMT1 maintains the methylation patterns during DNA replication (mitosis) [15]. However, the actual function of DNMT2 is not clear, bur several forms of DNMT1 have been detected which differ in their translation start sites and prefer hemimethylated DNA. Overexpression of DNMT1 has been reported in human tumours and may contribute to the global methylation abnormalities seen in cancer cells although increased expression of the DNMTs is likely to be only partially responsible for the observed methylation abnormalities since not all tumours overexpress these enzymes [10]. Cytosine (C5 )-DNA methyltransferases catalyze the transfer of a methyl group from Sadenosyl-methionine onto cytosine residues in specific sequences of duplex DNA, with production of 5-methyl cytosine and S-adenosyl-homocystein (SAMe) (Figure1). For most proteins, cytosine (C5 )-DNA methyltransferases have up to 10 conservative regions arranged in a strictly defined sequence [16]. Comparison of the primary structures of cytosine (C5 )-DNA methyltransferases reveals the association of their major functions with their conservative motifs, whereas the site-specific recognition belongs to a variable region of the target-recog‐ nizing domain (TRD) [17]. Among ten conservative blocks of amino acids in cytosine (C5 )-DNA methyltransferases, the N-terminal domain of DNMT1 contains varied specific functional sequences, such as the nuclear localization signal (NLS), the cysteine–enriched zinc-binding motif, and a special sequence directing the methylase into the area of DNA replication. In addition, DNMT1 interact with the proliferating cell nuclear antigen (PCNA) which is required for DNA replication, and the DNMT1-PCNA interaction allow rapid remethylation of the newly synthesised daughter strands before packed into chromatin [18]. A null mutation of the mouse methylase DNMT1 gene resulted in a significant (up to 70%) decrease in the genome methylation and death of developing embryos [19]. The remaining 30% level of DNA meth‐ ylation and the ability of embryonic stem cells deprived of the DNMT1 methylase for de novo methylation of DNA suggest that these functions were performed by other DNA methylases [19]. Such methylases were searched for in animals, and new enzymes of the DNMT2 and DNMT3 families were found [20]. Cell-cycle regulators p21 and retinoblastoma gene product Rb can bind to DNMT1 and inhibit its methyltransferase activity during DNA replication in the cell cycle [18]. This observations show complex interaction between DNMT1 and cellular proteins involved in gene regulation and epigenetic signalling during cell replication [21].

The DNMT3 family consists of two genes, DNMT3a and DNMT3b, which are highly expressed in undifferentiated ES cells but downregulated after differentiation and expressed at low levels in adult somatic tissues and are overexpressed in tumour cells [22]. Both DNMT3a and DNMT3b are required for genome-wide de novo methylation and are essential for mammalian development [22]. Both DNMT3a and DNMT3b had been mapped by the unigene consortium via polymorphisms in 3' –untranslated region sequences. DNMT3b mapped to the region of chromosome 20q that contains the trait for ICFNS (immunodeficiency centromeric instability, facial ubnormalities) syndrome. This syndrome presents with variable combined immunode‐ ficiency, mild facial anomalies and extravagant cytogenetic abnormalities which largely affect the pericentric region of chromosomes 1, 9 and 16. These pericentric regions contain a type of satellite DNA termed classical satellite, or satellites 2 and 3. It is normally heavily methylated, but is nearly completely unmethylated in the DNA of ICF patients. It was found that immu‐ nodeficiency centromeric instability (ICF) patients had mutations in the C-terminal DNA methyltransferase domain of DNMT3b. DNMT3b remains the only DNA methyltransferase shown to be mutated in a human disease [15]. DNMT3b has been shown to play a crucial role in hypermethylation of promoter CpG-rich regions of tumour suppressor genes and thus its inactivation within human cancer cells [22].

#### **3.2. How does demethylation occur?**

The key question is how the enzymes know where to methylate? Two theories have been suggested. Firstly, it has been suggested that all genes are methylated by default except for active genes [23]. Actively transcribed genes have a preponderance of attached transcriptional factors, giving no physical access to the methyltransferses to reach their targets. On the other hand, inactive DNA is susceptible to the methyltransferases and subsequently become methylated. This model was confirmed by the study of the transcription factor SP1. It has been shown that as long as SP1 is attached to its site, no methylation could occur in the adjacent CpG sites, and removal of the SP1 leads to de novo methylation at this site [24]. The second theory is that methylation is directed by sequence specific binding proteins so the methyl‐ transferases bind with certain proteins such as a histone deacetylases (HDACs) and other transcription repressors, and form a complex would bind to specific sequence on the DNA [23].

Methylated genes may need to be activated in response to environmental signals and thus demethylation is an important dynamic epigenetic mechanism and it was originally thought that demethylation only occured through passive demethylation (Figure 2). However, the rapid demethylation of the paternal genomes upon fertilization and examples of rapid demethylation of genes in post-mitotic neurons suggest that an active demethylase must exist [23,25]. A number of enzymes have been suggested to have demethylase activity these include MBD2b, MBD4, the DNA repair endonucleases XPG (Gadd45a) and a G/T mismatch repair DNA glycosylase which is glycosidase dependent. In this mechanism, the methylated cytosine is recognized by glycosidase which cleaves the bond between the DNA back bone and base. The base is subsequently removed and replaced with unmethylated cytosine by the DNA repair system.

## **4. Histone Deacetylases (HDACs)**

[19]. Such methylases were searched for in animals, and new enzymes of the DNMT2 and DNMT3 families were found [20]. Cell-cycle regulators p21 and retinoblastoma gene product Rb can bind to DNMT1 and inhibit its methyltransferase activity during DNA replication in the cell cycle [18]. This observations show complex interaction between DNMT1 and cellular proteins involved in gene regulation and epigenetic signalling during cell replication [21].

The DNMT3 family consists of two genes, DNMT3a and DNMT3b, which are highly expressed in undifferentiated ES cells but downregulated after differentiation and expressed at low levels in adult somatic tissues and are overexpressed in tumour cells [22]. Both DNMT3a and DNMT3b are required for genome-wide de novo methylation and are essential for mammalian development [22]. Both DNMT3a and DNMT3b had been mapped by the unigene consortium via polymorphisms in 3' –untranslated region sequences. DNMT3b mapped to the region of chromosome 20q that contains the trait for ICFNS (immunodeficiency centromeric instability, facial ubnormalities) syndrome. This syndrome presents with variable combined immunode‐ ficiency, mild facial anomalies and extravagant cytogenetic abnormalities which largely affect the pericentric region of chromosomes 1, 9 and 16. These pericentric regions contain a type of satellite DNA termed classical satellite, or satellites 2 and 3. It is normally heavily methylated, but is nearly completely unmethylated in the DNA of ICF patients. It was found that immu‐ nodeficiency centromeric instability (ICF) patients had mutations in the C-terminal DNA methyltransferase domain of DNMT3b. DNMT3b remains the only DNA methyltransferase shown to be mutated in a human disease [15]. DNMT3b has been shown to play a crucial role in hypermethylation of promoter CpG-rich regions of tumour suppressor genes and thus its

The key question is how the enzymes know where to methylate? Two theories have been suggested. Firstly, it has been suggested that all genes are methylated by default except for active genes [23]. Actively transcribed genes have a preponderance of attached transcriptional factors, giving no physical access to the methyltransferses to reach their targets. On the other hand, inactive DNA is susceptible to the methyltransferases and subsequently become methylated. This model was confirmed by the study of the transcription factor SP1. It has been shown that as long as SP1 is attached to its site, no methylation could occur in the adjacent CpG sites, and removal of the SP1 leads to de novo methylation at this site [24]. The second theory is that methylation is directed by sequence specific binding proteins so the methyl‐ transferases bind with certain proteins such as a histone deacetylases (HDACs) and other transcription repressors, and form a complex would bind to specific sequence on the DNA [23].

Methylated genes may need to be activated in response to environmental signals and thus demethylation is an important dynamic epigenetic mechanism and it was originally thought that demethylation only occured through passive demethylation (Figure 2). However, the rapid demethylation of the paternal genomes upon fertilization and examples of rapid demethylation of genes in post-mitotic neurons suggest that an active demethylase must exist [23,25]. A number of enzymes have been suggested to have demethylase activity these include MBD2b, MBD4, the DNA repair endonucleases XPG (Gadd45a) and a G/T mismatch repair

inactivation within human cancer cells [22].

30 A Concise Review of Molecular Pathology of Breast Cancer

**3.2. How does demethylation occur?**

Histones are five basic nuclear proteins that form the core of the nucleosome and the histone octamer contains two molecules each of histones H2A, H2B, H3 and H4. Histone H1 the linker histone is located outside the core and involve in the packing of DNA [26]. Histone modifica‐ tions play a major role in regulating gene expression and extend the information potential of the DNA which explains the growing interest of the 'Histone Code' [27]. Modifications to amino acids on the N-terminal tails of histones protruding from the nucleosome core can induce both an open or closed chromatin structure and these affect the ability of transcription factors to access promoter regions to activate transcription. The covalent modification can be acetylation, methylation, phosphorylation and ubiquitination. Methylation of some residues is associated with both transcriptional repression, such as methylation of histone 3 lysine 9 (H3 K9) and others with transcriptional activation, such as methylation of histone 3 lysine 4 (H3 K4) [28,29].

Histone methylation is performed by histone methltransferase (HMTs) which can transfer up to three methyl groups to lysine residues within the tails of the histones with different effects on gene activity. Acetylation which occurs at lysine residue is associated with transcriptional activation [30]. This modification is performed by histone acetylases (HATs) and removed by the HDACs [31]. The HDACs are critical in the regulation of expression of genes important for cell survival, proliferation, differentiation, and apoptosis [32]. HDACs also act as members of a protein complex responsible for recruitment of transcription factors to the promoter region of genes, including those of tumour suppressors, and regulation of acetylation status of specific cell cycle regulatory proteins [33]. High HDAC expression and histone hypoacetylation have been observed in cancer with associated transcriptional repression of genes, providing a rationale for the investigation of HDAC inhibitors in cancer therapeutics [34].

Additionally, acetylation of histones has been extensively studied as one of the key regulatory mechanisms of gene expression [35]. Histone acetylation was found to affect RNA transcription as early as the 1960s [36]. The highly conserved lysine residue at the N-terminal of H3 at position 9, 14, 18 and 23, and H4 lysine 5,8,12 and 16, are frequently targeted for modification [37]. Acetylations of the lysine residues neutralize the positive charge of the histone tails. Therefore, decrease their affinity for DNA which results in open chromatin conformation allowing the transcriptional machinery to reach its target [38]. The acetyltransferases added the acetyl groups from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues [39]. There are eighteen HDAC enzymes in mammalian cells which are divided into two families: a) zinc metalloenzymes that catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs, and b) NAD-dependent Sir2 deactylases which are considered as class III HDACs [40,41].

Figure 1. Methylation of DNA by DNA methyltransferases enzymes (DNMTs) DNMT1, DNMT3A, DNMT3B. A methyl group transfer from S-adenosyl-methionine onto cytosine residues leading to production of 5-methyl cytosine and S-adenosyl-homocystein (SAMe). **Figure 1.** Methylation of DNA by DNA methyltransferases enzymes (DNMTs) DNMT1, DNMT3A, DNMT3B. A meth‐ yl group transfer from S-adenosyl-methionine onto cytosine residues leading to production of 5-methyl cytosine and Sadenosyl-homocystein (SAMe).

Figure 2. DNA demethylation appears to be a shared attribute of reprogramming events, and understanding DNA methylation dynamics is thus of considerable interest. Some enzymes such as MBD2b and MBD4 convert 5-methylcytosine (5mC) to 5 hydroxymethylcytosine (5hmC). **Figure 2.** DNA demethylation appears to be a shared attribute of reprogramming events, and understanding DNA methylation dynamics is thus of considerable interest. Some enzymes such as MBD2b and MBD4 convert 5-methylcy‐ tosine (5mC) to 5-hydroxymethylcytosine (5hmC).

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus [40]. Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues [40]. Class III includes the NAD-dependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones besides other proteins [42]. Class IV contains one member which is HDAC11 which is closely related to class I thus some reviewers consider it as a member of that class. The function of HDAC11 has not been characterized yet [43], however, there is increasing evidence 3

Additionally, acetylation of histones has been extensively studied as one of the key regulatory mechanisms of gene expression [35]. Histone acetylation was found to affect RNA transcription as early as the 1960s [36]. The highly conserved lysine residue at the Nterminal of H3 at position 9, 14, 18 and 23, and H4 lysine 5,8,12 and 16, are frequently targeted for modification [37]. Acetylations of the lysine residues neutralize the positive charge of the histone tails. Therefore, decrease their affinity for DNA which results in open chromatin conformation allowing the transcriptional machinery to reach its target [38]. The acetyltransferases added the acetyl groups from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues [39]. There are eighteen HDAC enzymes in mammalian cells which are divided into two families: a) zinc metalloenzymes that catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs, and b) NAD-dependent Sir2 deactylases which are

considered as class III HDACs [40,41].

4

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus [40]. Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues [40]. Class III includes the NAD-dependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones besides other proteins [42]. Class IV contains one member which is HDAC11 which is closely related to class I thus some reviewers

showing that changes in chromatin structure would alter DNA methylation patterns. The targeting of DNA methylation enzymes to gene promoters is guided by chromatin modifying enzymes. The fact is that chromatin configuration is dynamic and that chromatin modifying enzymes are activated by cellular signalling pathways. This provides a link between the extracellular environment and the state of DNA methylation [44]. Evidence of the link between chromatin modelling and DNA methylation in humans and mice arises from mutations of the SWI-SNF proteins which are involved in chromatin remodelling. These mutations result in defects in DNA methylation [44]. A number of histone methyltransferases, such as G9a, SUV39H1 and EZH2, a member of the multi-protein polycomb complex PRC2 can regulate DNA methylation by either recruiting or regulating the stability of DNMTs. DNMTs in turn can recruit HDACs and MBPs to achieve chromatin condensation and gene silencing [45]. This relationship between the epigenetic machinery makes the epigenetic mechanisms of genome expression a tightly regulated process.

## **5. DNA methylation and breast cancer**

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus [40]. Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues [40]. Class III includes the NAD-dependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones besides other proteins [42]. Class IV contains one member which is HDAC11 which is closely related to class I thus some reviewers consider it as a member of that class. The function of HDAC11 has not been characterized yet [43], however, there is increasing evidence

**Figure 2.** DNA demethylation appears to be a shared attribute of reprogramming events, and understanding DNA methylation dynamics is thus of considerable interest. Some enzymes such as MBD2b and MBD4 convert 5-methylcy‐

**O2 O**

Figure 2. DNA demethylation appears to be a shared attribute of reprogramming events, and understanding DNA methylation dynamics is thus of considerable interest. Some enzymes such as MBD2b and MBD4 convert 5-methylcytosine (5mC) to 5-

**Demethylation**

**MBD2b MBD4**

4

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus [40]. Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues [40]. Class III includes the NAD-dependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones besides other proteins [42]. Class IV contains one member which is HDAC11 which is closely related to class I thus some reviewers

Additionally, acetylation of histones has been extensively studied as one of the key regulatory mechanisms of gene expression [35]. Histone acetylation was found to affect RNA transcription as early as the 1960s [36]. The highly conserved lysine residue at the Nterminal of H3 at position 9, 14, 18 and 23, and H4 lysine 5,8,12 and 16, are frequently targeted for modification [37]. Acetylations of the lysine residues neutralize the positive charge of the histone tails. Therefore, decrease their affinity for DNA which results in open chromatin conformation allowing the transcriptional machinery to reach its target [38]. The acetyltransferases added the acetyl groups from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues [39]. There are eighteen HDAC enzymes in mammalian cells which are divided into two families: a) zinc metalloenzymes that catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs, and b) NAD-dependent Sir2 deactylases which are

3

Figure 1. Methylation of DNA by DNA methyltransferases enzymes (DNMTs) DNMT1, DNMT3A, DNMT3B. A methyl group transfer from S-adenosyl-methionine onto cytosine residues leading to production of 5-methyl cytosine and S-adenosyl-homocystein

**Figure 1.** Methylation of DNA by DNA methyltransferases enzymes (DNMTs) DNMT1, DNMT3A, DNMT3B. A meth‐ yl group transfer from S-adenosyl-methionine onto cytosine residues leading to production of 5-methyl cytosine and S-

**Methylation**

**O**

**N**

2

**N**

2

3

3

**NH2**

4

**5**

**CH3**

6

1

**NH2**

4

**5**

**OH**

6

1

**Hydroxymethylcytosine (5hmC)** 

**5-methylcytosine (5mC)**

**S-Adenosyl Methionine (SAMe) O**

**CH3**

**H**

**DNMT1 DNMT3A DNMT3B**

(SAMe).

**O**

**N**

2

3

**N**

2

3

**NH2**

4

**5**

32 A Concise Review of Molecular Pathology of Breast Cancer

6

1

**Cytosine (C)**

adenosyl-homocystein (SAMe).

**NH2**

4

**5**

6

tosine (5mC) to 5-hydroxymethylcytosine (5hmC).

hydroxymethylcytosine (5hmC).

1

**5-methylcytosine (5mC)** 

considered as class III HDACs [40,41].

During the last decade, the study of epigenetic mechanisms in cancer, such as DNA methyla‐ tion, histone modification, nucleosome positioning, and micro RNA expression, has provided extensive information about the mechanisms that contribute to the neoplastic phenotype through the regulation of expression of genes critical to transformation pathways. Regarding DNA methylation, the low level of CpG methylation in tumours compared with that in their normal-tissue counterparts was one of the first epigenetic alterations to be found in human cancer this let us to think that the cancer cells have a specific epigenome [46]. Hypomethylation in cancer cells is associated with a number of adverse products, including chromosome instability, activation of transposable elements, and loss of genomic imprinting [47].

Breast cancer has traditionally been staged by histopathological standards that are based on size, level of invasiveness and lymph node infiltration, and by immunochemical characteri‐ zation of cell surface receptors, including oestrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). However, in many instances staging breast cancer fails to predict prognosis or therapeutic response because of the hetero‐ geneity of the disease. Changes in gene expression that reset a cell program from a normal to a diseased state involve multiple genetic circuitries, creating a characteristic signature of gene expression that defines the cell's unique identity and to classify subtypes of breast cancers [48]. Detailed knowledge of the DNA methylation status of all cytosines (the methylome) is paramount for understanding the mechanisms and functions underlying DNA methylation and led to extend our ability to classify breast cancer and the outcome prediction. DNA methylation is a forceful biomarker, greatly more stable than proteins or RNA, and is therefore a promising target for the development of new approaches for diagnosis and prognosis of breast cancer and other diseases. Because DNA methylation is critical in gene expression programming, a change in methylation from a normal to diseased state should be similarly reflected in a signature of DNA methylation that involves multiple gene pathways. Wholegenome approaches have been used with different levels of success to distinguish breastcancer-specific DNA methylation signatures, and to test whether they can classify breast cancer and whether they could be associated with specific clinical outcomes [48].

Application of DNA methylation profiling becomes important for breast cancer diagnosis and prognosis only if it provides additional classification value to other currently used methods like immunohistochemistry and mRNA expression analysis. A recent whole-genome DNA methylation analysis by using the Illumina 27 K arrays suggests that DNA methylation profiling might expand current classifications of breast cancer subtypes [49,50]. The analysis of 248 breast cancer tumour samples, comprising a 'main set' of 123 samples (4 normal and 119 infiltrating ductal carcinomas (IDCs)), and a 'validation set' of 125 samples (8 normal and 117 IDCs), revealed an immune 'signature' in a mixed tumour stromal population, as also reported [51]. Methylome analysis performed on frozen primary tumour samples, led to the identifica‐ tion of six different methylation clusters [52]. It was shown for the first time that DNA methylation profiles can reflect the cell-type composition of the tumour microenvironment, with a T lymphocyte infiltration of these tumours in particular in HER2-enriched and basallike tumours. High expression of certain immune-related genes were found to be associated with improved relapse-free survival providing further insight into the importance of the immune system and tumour microenvironment in certain breast cancer subtypes [53].

Furthermore, aberrations in DNA methylation patterns of the CpG islands in the promoter regions of tumour-suppressor genes are accepted as being a common feature of human cancer [54]. CpG island promoter hypermethylation affects genes from a wide range of cellular pathways, such as cell cycle, DNA repair, toxic catabolism, cell adherence, apoptosis, and angiogenesis, among others [54], and may occur at various stages in the development of cancer [55]. The CpG-island-containing gene promoters are usually unmethylated in normal cells to maintain euchromatic structure, which is the transcriptionally active conformation allowing gene expression. Yet, during cancer development, many of these genes are hypermethylated at their CpG-island-containing promoters to inactivate their expression by changing open euchromatic structure to compact heterochromatic structure [56,57]. These genes are selec‐ tively hypermethylated in tumourigenesis for inactivation owing to their functional involve‐ ment in various cellular pathways that prevent cancer formation. Some of the methylated genes identified in human cancers are classic tumour suppressor genes in which one mutationally inactivated allele is inherited. According to Knudson's (2000) two-hit model, complete inactivation of a tumour suppressor gene requires loss-of-function of both gene copies [58]. Epigenetic silencing of the remaining wild-type allele of the tumour suppressor gene, thus, can be considered as the second hit in this model. For example, some well-known tumour suppressor genes, such as the cyclin-dependent kinase inhibitorp16INK4a, APC and BRCA1, are mutationally inactivated in the germline occasionally lose function of the remaining functional allele in breast epithelial cells through DNA hypermethylation [59].These advances in the knowledge of the breast methylome strongly indicate that DNA hypermethylation mechanism plays a crucial role in initiation, promotion and maintenance of breast carcino‐ genesis, which cooperatively and synergistically interact with other genetic alterations to promote the development of breast cancer. In addition to cell-cycle regulatory genes, DNA methylation-mediated silencing of DNA repair genes, such as BRCA1 and MGMT, could result in further inactivation of tumour suppressor genes or activation of oncogenes, which further drive breast tumourigenesis [60]. The genes that function as inhibitors of WNT oncogenic pathway such as SFRP1 and WIF1 have been found to be frequently hypermethylated in primary breast tumours [61]. Accordingly, epigenetic gene silencing is another mechanism that fosters malignant transformation of the mammary gland by aberrantly activating onco‐ genic signalling pathways in addition to the genetic mutation-mediated mechanism [62].

cancer-specific DNA methylation signatures, and to test whether they can classify breast cancer

Application of DNA methylation profiling becomes important for breast cancer diagnosis and prognosis only if it provides additional classification value to other currently used methods like immunohistochemistry and mRNA expression analysis. A recent whole-genome DNA methylation analysis by using the Illumina 27 K arrays suggests that DNA methylation profiling might expand current classifications of breast cancer subtypes [49,50]. The analysis of 248 breast cancer tumour samples, comprising a 'main set' of 123 samples (4 normal and 119 infiltrating ductal carcinomas (IDCs)), and a 'validation set' of 125 samples (8 normal and 117 IDCs), revealed an immune 'signature' in a mixed tumour stromal population, as also reported [51]. Methylome analysis performed on frozen primary tumour samples, led to the identifica‐ tion of six different methylation clusters [52]. It was shown for the first time that DNA methylation profiles can reflect the cell-type composition of the tumour microenvironment, with a T lymphocyte infiltration of these tumours in particular in HER2-enriched and basallike tumours. High expression of certain immune-related genes were found to be associated with improved relapse-free survival providing further insight into the importance of the immune system and tumour microenvironment in certain breast cancer subtypes [53].

Furthermore, aberrations in DNA methylation patterns of the CpG islands in the promoter regions of tumour-suppressor genes are accepted as being a common feature of human cancer [54]. CpG island promoter hypermethylation affects genes from a wide range of cellular pathways, such as cell cycle, DNA repair, toxic catabolism, cell adherence, apoptosis, and angiogenesis, among others [54], and may occur at various stages in the development of cancer [55]. The CpG-island-containing gene promoters are usually unmethylated in normal cells to maintain euchromatic structure, which is the transcriptionally active conformation allowing gene expression. Yet, during cancer development, many of these genes are hypermethylated at their CpG-island-containing promoters to inactivate their expression by changing open euchromatic structure to compact heterochromatic structure [56,57]. These genes are selec‐ tively hypermethylated in tumourigenesis for inactivation owing to their functional involve‐ ment in various cellular pathways that prevent cancer formation. Some of the methylated genes identified in human cancers are classic tumour suppressor genes in which one mutationally inactivated allele is inherited. According to Knudson's (2000) two-hit model, complete inactivation of a tumour suppressor gene requires loss-of-function of both gene copies [58]. Epigenetic silencing of the remaining wild-type allele of the tumour suppressor gene, thus, can be considered as the second hit in this model. For example, some well-known tumour suppressor genes, such as the cyclin-dependent kinase inhibitorp16INK4a, APC and BRCA1, are mutationally inactivated in the germline occasionally lose function of the remaining functional allele in breast epithelial cells through DNA hypermethylation [59].These advances in the knowledge of the breast methylome strongly indicate that DNA hypermethylation mechanism plays a crucial role in initiation, promotion and maintenance of breast carcino‐ genesis, which cooperatively and synergistically interact with other genetic alterations to promote the development of breast cancer. In addition to cell-cycle regulatory genes, DNA methylation-mediated silencing of DNA repair genes, such as BRCA1 and MGMT, could result

and whether they could be associated with specific clinical outcomes [48].

34 A Concise Review of Molecular Pathology of Breast Cancer

In vitro experiments showed that decreased BRCA1expression in cells led to increased levels of tumour growth, while increased expression of BRCA1 led to growth arrest and apopto‐ sis. The magnitude of the decrease of functional BRCA1 protein correlates with disease prognosis [63]. Phenotypically, BRCA1-methylated tumours are similar to tumours from carriers of germline BRCA1 mutations. BRCA1 promoter hypermethylation was observed in one of two tumours from BRCA1 carriers lacking LOH [64]. In other study of populationbased ovarian tumours, two of eight tumours with germline BRCA1mutations showed neither LOH nor promoter methylation [65]. Another study of 47 breast tumours from hereditary breast cancer families identified three BRCA1 carriers of which two showed BRCA1 promoter methylation in their tumours [66]. All these investigated studies sug‐ gest that methylation of BRCA1 may be serve as a second hit in tumours from a subset of BRCA1 mutation carriers [67].Tumours with BRCA1 mutations are usually more likely to be higher-grade, poorly differentiated, highly proliferative, ER negative, and PR negative, and p53 mutations. BRCA1 mutated breast cancers are also associated with poor survival in some studies [68]. BRCA1 promoter methylation was more frequent in invasive than in situ carcinoma and there were no correlation between BRCA1 promoter methylation and ER/PR status in a subset population [69]. However, they also found a higher prevalence of BRCA1 promoter methylation in cases with at least one node involved and with tumour size greater than 2cm. Based on their findings higher methylation levels may correlate with more advanced tumour stage at diagnosis. They also observed a 45% increase in mortali‐ ty of individuals with BRCA1 methylation positive tumours compared those who had unmethylated BRCA1 promoters [69]. Another study conducted a familial breast cancer based study and found contradicting results. They found no overall correlation of ER, PR, or grade with hypermethylation of BRCA1 in the tumours from BRCA1 mutation nega‐ tive families. However, seven individuals had both promoter hypermethylation and LOH; the majority of these tumours had a basal-like phenotype and were triple negative [70].

In addition, discriminate between tumour and normal or histologically non-malignant breast tissue has been applied widely by genome wide DNA methylation. One of the first genome wide DNA methylation studies in breast cancer developed methylation-specific digital karyotyping (MSDK) to assess epithelial, myoepithelial, and stromal fibroblasts from normal abreast and cancer tissues [71]. Furthermore, genome wide DNA methylation studies in breast cancer identified gene families that were commonly identified as differentially methylated between non-malignant and tumour included transcription factors (FOX, KLF, PRDM, ZBTB, and ZNF) and gene families involved in cell transport of proteins or vesicles(RAB and SLC) or involvement in cell adhesion (CDH and PCDH) [71-74]. The pathways and gene families do not appear to have a strong link to hormone metabolism or signalling, it is likely that these genes are not drivers of cancer but rather are secondary events that occur as part of the tumourigenic process [75,76].

Genome wide DNA methylation studies have supported correlation between DNA methyla‐ tion and gene expression, particularly the association between CpG islands DNA hyperme‐ thylation and gene repression [49,74,77,78]. Using familial breast cancers and BRCA1/2 mutated tumours combined DNA methylation profiles that alone predicted BRCA status, with gene expression and copy number variation (CNV) and found that genes with reduced expression were more likely to be in genomic regions with loss of heterozygosity and/or high levels of DNA methylation. It has also been shown that the combination of gene dosage in breast cancer cell lines, allelic status, and DNA methylation explains more gene expression changes than either genomic element alone [79]. Combining DNA methylation profiling with CNV and gene expression can be promising tool to facilitate the identification of critical genes involved in tumourigenesis. In genome wide methylation analysis, several platforms have been recently developed to allow genome wide methylation analysis. The Golden Gate methylation array was the first platform which allowed methylation of 1536 CpG loci to be investigated. The Infinium Human Methylation 27 increased CpG investigation with the use of 27,578 probes. Most recently was the Infinium Human Methylation 450K array, designed by Illumina. This array utilises florescence microarray hybridisation technique, often associ‐ ated with expression studies, to provide a methylation profile of 485,764 CpG loci including CpG associated in CpG islands, shores, shelves and the isolated loci in the open sea regions of the genome and promoter regionshave used Illumina Infinium Human Methylation 27 Bead Chip to analyse normal breast tissues from ten healthy individuals and compared this to 62 breast tumour samples (19 were inflammatory breast cancer) [73].

Further studies have also compared tumour to non-malignant tissue and the number of genes identified that discriminates the two depends on the filtering or analyses utilized. For instance, Kim et al. (2012) used several filtering processes to identify six genes [80], whereas, Faryna et al, (2012) identified 214 CpG islands but only one CpG island (TAC1) was methylated in all ten cancer samples [81]. The DNA methylation profiles divided the samples into three groups based on high, intermediate, and low DNA methylation levels, with the normal samples having low DNA methylation levels. When comparing DNA methylation between normal and tumour samples, 1352 CpG loci (1134 genes) were differentially methylated [73]. There was significantly greater methylation in tumours compared with normal and 77% of these are CpG loci. Another study using the same technology found 6309 CpGs differentially methylated between 119 tumours and four normal breast tissue samples identified several hundred differentially methylated loci between 11 adjacent non-malignant breast tissues and 108 tumours [49;74]. Kim et al, (2011) pooled DNA from ten cancers and ten non-malignant matched adjacent tissues and identified 1181 differentially methylated CpGs (corresponding to 1043 genes) with the vast majority (972) hypermethylated [82]. Another study found 291 probes (264 genes) hypermethylated in breast cancer (n=39) compared with non-malignant breast tissue (n=4) after removal of imprinted genes and X chromosome genes [83].

In addition, numbers of studies have investigated whether genome wide DNA methylation profiling can cluster breast cancers into hormone receptor status (ER/PR positive or negative) or subtype (luminal A or B, basal or HER2). These investigations differentiate hormone receptor-positive breast cancers from hormone receptor-negative cases using DNA methyla‐ tion profiles [49,77,83-85]. The majority of genome wide DNA methylation studies have found that ER+PR+tumours have higher levels of DNA methylation compared with ER−PR− tumours [77,82,85,86]. Li et al, (2010) found 148 altered CpG sites (93 hypermethylated and 55 hypo‐ methylated) in ER+PR+breast cancers relative to ER−PR− tumours [85]. Other study have identified 40 CpG probes that had an overall specificity of 89% and sensitivity of 90% for classifying ER+from ER− tumours [86].

Moreover, Hill et al, (2011) have used cluster analysis to show that ER+PR+tumours had high methylation, whereas triple-negative breast cancers had low methylation status [83]. Breast cancer cell lines have also shown clustering according to hormone receptor status based on DNA methylation levels [78]. Thus, all these genome wide DNA methylation studies demon‐ strate that an adequately results of appropriate clinical samples should identify methylation differences based on hormone receptor status. These studies may serve with additional future studies as a basis for the development of an improved clinical test to identify the hormone status of breast cancers.

In addition, in DNA methylation cluster analysis found that one cluster was predominantly luminal A (22/30 samples), the second cluster was highly correlated with basal-like (7/8 samples), and the third cluster contained a mixture of subtypes [74]. Recently, the Cancer Genome Atlas (TCGA) [87] and genome-wide profiling of DNA methylation has been also performed in primary breast tumours and revealed genes whose hypermethylation was significantly correlated with relapse-free survival, including RECK, SFRP2 and ACADL. Tumour specificity of methylation was confirmed for these genes by sequencing of an independent set of normal/breast tumour samples. Other investigation observed that the reduction of RECK methylation has been associated with worst prognosis in other tumours [88]. Genome-wide analysis has also been employed to characterize the DNA methylation profile of primary breast cancer with different metastatic potential. A global breast CpG island methylation phenotype (B-CIMP) was identified as an epigenetic profile associated with low risk of metastasis. Parallel gene expression analyses identified genes with both significant hypermethylation and down-regulation in B-CIMP tumours, including those involved in epithelial-mesenchymal transition (EMT), such as LYN, MMP7, KLK10 and WNT6 and the genes in the B-CIMP repression signature showed genes whose differential expression correlated with prognosis across several BC cohorts [89].

### **6. HDAC inhibitors and breast cancer**

genes are not drivers of cancer but rather are secondary events that occur as part of the

Genome wide DNA methylation studies have supported correlation between DNA methyla‐ tion and gene expression, particularly the association between CpG islands DNA hyperme‐ thylation and gene repression [49,74,77,78]. Using familial breast cancers and BRCA1/2 mutated tumours combined DNA methylation profiles that alone predicted BRCA status, with gene expression and copy number variation (CNV) and found that genes with reduced expression were more likely to be in genomic regions with loss of heterozygosity and/or high levels of DNA methylation. It has also been shown that the combination of gene dosage in breast cancer cell lines, allelic status, and DNA methylation explains more gene expression changes than either genomic element alone [79]. Combining DNA methylation profiling with CNV and gene expression can be promising tool to facilitate the identification of critical genes involved in tumourigenesis. In genome wide methylation analysis, several platforms have been recently developed to allow genome wide methylation analysis. The Golden Gate methylation array was the first platform which allowed methylation of 1536 CpG loci to be investigated. The Infinium Human Methylation 27 increased CpG investigation with the use of 27,578 probes. Most recently was the Infinium Human Methylation 450K array, designed by Illumina. This array utilises florescence microarray hybridisation technique, often associ‐ ated with expression studies, to provide a methylation profile of 485,764 CpG loci including CpG associated in CpG islands, shores, shelves and the isolated loci in the open sea regions of the genome and promoter regionshave used Illumina Infinium Human Methylation 27 Bead Chip to analyse normal breast tissues from ten healthy individuals and compared this to 62

Further studies have also compared tumour to non-malignant tissue and the number of genes identified that discriminates the two depends on the filtering or analyses utilized. For instance, Kim et al. (2012) used several filtering processes to identify six genes [80], whereas, Faryna et al, (2012) identified 214 CpG islands but only one CpG island (TAC1) was methylated in all ten cancer samples [81]. The DNA methylation profiles divided the samples into three groups based on high, intermediate, and low DNA methylation levels, with the normal samples having low DNA methylation levels. When comparing DNA methylation between normal and tumour samples, 1352 CpG loci (1134 genes) were differentially methylated [73]. There was significantly greater methylation in tumours compared with normal and 77% of these are CpG loci. Another study using the same technology found 6309 CpGs differentially methylated between 119 tumours and four normal breast tissue samples identified several hundred differentially methylated loci between 11 adjacent non-malignant breast tissues and 108 tumours [49;74]. Kim et al, (2011) pooled DNA from ten cancers and ten non-malignant matched adjacent tissues and identified 1181 differentially methylated CpGs (corresponding to 1043 genes) with the vast majority (972) hypermethylated [82]. Another study found 291 probes (264 genes) hypermethylated in breast cancer (n=39) compared with non-malignant

breast tissue (n=4) after removal of imprinted genes and X chromosome genes [83].

In addition, numbers of studies have investigated whether genome wide DNA methylation profiling can cluster breast cancers into hormone receptor status (ER/PR positive or negative)

breast tumour samples (19 were inflammatory breast cancer) [73].

tumourigenic process [75,76].

36 A Concise Review of Molecular Pathology of Breast Cancer

As we mentioned previously, abnormal HDAC activity has been documented in a variety of tumour types and led to the development of HDAC inhibitors as anticancer therapeutics. Currently available HDAC inhibitors target a variety of HDAC isoenzymes with class 1 (HDAC 1, 2, 3 and 8), class 2 (HDAC 4–7 and 9–10), and class 4 (HDAC 11) activity. Modest clinical benefits were previously reported with relatively weak HDAC inhibitors such as valproic acid and phenylbutyrate in advanced solid tumours or hematologic malignancies [89]. Laboratory research conducted to date supports the investigation of HDAC inhibitors for the treatment of breast cancer. Recently, vorinostat as HDAC inhibitor induces differentiation or arrests growth of a wide variety of human carcinoma cells including breast cancer cells [90].Vorinostat also reduced tumour incidence in NMU-induced rat mammary tumourigene‐ sis by 40 % [91]. In vitro studies demonstrated that vorinostat inhibits clonogenic growth of both ER-positive and ER-negative breast cancer cell lines by inducing G1 and G2/M cell cycle arrest and subsequent apoptosis [92].

The ability of the HDAC inhibitors to relieve transcriptional repression in preclinical breast cancer models has also been investigated. The accumulation of acetylated H3 and H4 histone tails in conjunction with re-expression of a functional ER in ER-negative breast cancer cell lines has been observed with a novel HDAC inhibitor known as scriptaid [93].Treatment of ERnegative breast cancer cell lines with vorinostat is associated with reactivation of silenced ER, as well as down regulation of DNMT1 and EGFR protein expression [94]. The significance of an epigenetically reactivated ER was demonstrated when tamoxifen sensitivity was restored in the ER-negative MDA-MB-231 breast cancer cells following treatment with both HDAC (trichostatin A) and DNMT inhibitors (DAC) [95]. Entinostat has been shown to induce not only re-expression of ERα, but also the androgen receptor and the aromatase enzyme (CYP19) both in vitro and in triple-negative breast cancer xenografts [96]. In addition, the combination ofletrozole and entinostat resulted in a significant and durable reduction in the xenograft tumour volume when compared to treatment with either agent alone. These experiments have provided the strong rationale for combining epigenetic modifiers with hormonal therapy in breast cancer clinical trials [96]. Interestingly, many of these studies also indicate that a strategy which combines HDAC and DNMT inhibitors is more efficacious than either agent alone with respect to both re-expression of silenced genes and restoration of response to tamoxifen and aromatase inhibitors [93.97].

Moreover, pretreatment of various tumour cell lines with HDAC inhibitors increases the cytotoxicity of chemotherapy. Administering the HDAC inhibitor after chemotherapy did not achieve the same results, suggesting that pretreatment with these agents may open the chromatin structure and thus facilitate an enhanced anti-cancer effect of chemotherapy drugs that target DNA [98]. In breast cancer cell lines with amplification and overexpression of HER2, HDAC inhibitor use depleted HER2 by attenuation of its mRNA levels and promotion of proteosomal degradation. HDAC inhibition also had been reported to enhance apoptosis induction by trastuzumab, docetaxel, epothilone B, and gemcitabine [99]. HDAC inhibitors also significantly enhance trastuzumab-induced growth inhibition in trastuzumab-sensitive, HER2-overexpressing breast cancer cells, providing a strong rationale for clinical studies with this combination in patients with HER2-positive disease [100].

Additionally, HDAC inhibitors such as entinostat or valproic acid, have been tested in breast cancer cells and efficiently restored both ERα expression and letrozole sensibility in ER-BC in vitro and in vivo [101,102].The association of HDAC inhibitors or 5-azadeoxycytidine with a treatment inducing overexpression of TFAP2C might improve ESR1 expression in ERpatients. A combined HDAC inhibitors and 5-azadeoxycytidine treatment induces the most significant increase in ERα content. Surprisingly however, addition of tamoxifen does not produce a tumourigenic response in ER-BC cells demonstrated that a better response to tamoxifen in BC cells, correlated with a lower level of the RNA-stabilizing HuR protein [103]. Tamoxifen treatment increased HuR content, and contributed to its own resistance while HDAC inhibi‐ tors /5-azadeoxycytidine decreased HuR. Preliminary treatment with HDAC inhibitors /5 azadeoxycytidine was given before delivering tamoxifen to attempt to obtain the best tamoxifen sensitivity. The precise roles of tamoxifen are complex: although it competes with 17β-estradiol to bind to ERα, ERα bound to tamoxifen is still able to target the TFF1 (also called pS2) promoter without constitutive activation of gene transcription. The loss of transcriptional activity of the tamoxifen-ERα complex is mediated by changes in the balance of co-activators/ co-repressors and ERα-interacting partners [104].

## **7. DNMTs inhibitors and breast cancer**

valproic acid and phenylbutyrate in advanced solid tumours or hematologic malignancies [89]. Laboratory research conducted to date supports the investigation of HDAC inhibitors for the treatment of breast cancer. Recently, vorinostat as HDAC inhibitor induces differentiation or arrests growth of a wide variety of human carcinoma cells including breast cancer cells [90].Vorinostat also reduced tumour incidence in NMU-induced rat mammary tumourigene‐ sis by 40 % [91]. In vitro studies demonstrated that vorinostat inhibits clonogenic growth of both ER-positive and ER-negative breast cancer cell lines by inducing G1 and G2/M cell cycle

The ability of the HDAC inhibitors to relieve transcriptional repression in preclinical breast cancer models has also been investigated. The accumulation of acetylated H3 and H4 histone tails in conjunction with re-expression of a functional ER in ER-negative breast cancer cell lines has been observed with a novel HDAC inhibitor known as scriptaid [93].Treatment of ERnegative breast cancer cell lines with vorinostat is associated with reactivation of silenced ER, as well as down regulation of DNMT1 and EGFR protein expression [94]. The significance of an epigenetically reactivated ER was demonstrated when tamoxifen sensitivity was restored in the ER-negative MDA-MB-231 breast cancer cells following treatment with both HDAC (trichostatin A) and DNMT inhibitors (DAC) [95]. Entinostat has been shown to induce not only re-expression of ERα, but also the androgen receptor and the aromatase enzyme (CYP19) both in vitro and in triple-negative breast cancer xenografts [96]. In addition, the combination ofletrozole and entinostat resulted in a significant and durable reduction in the xenograft tumour volume when compared to treatment with either agent alone. These experiments have provided the strong rationale for combining epigenetic modifiers with hormonal therapy in breast cancer clinical trials [96]. Interestingly, many of these studies also indicate that a strategy which combines HDAC and DNMT inhibitors is more efficacious than either agent alone with respect to both re-expression of silenced genes and restoration of response to tamoxifen and

Moreover, pretreatment of various tumour cell lines with HDAC inhibitors increases the cytotoxicity of chemotherapy. Administering the HDAC inhibitor after chemotherapy did not achieve the same results, suggesting that pretreatment with these agents may open the chromatin structure and thus facilitate an enhanced anti-cancer effect of chemotherapy drugs that target DNA [98]. In breast cancer cell lines with amplification and overexpression of HER2, HDAC inhibitor use depleted HER2 by attenuation of its mRNA levels and promotion of proteosomal degradation. HDAC inhibition also had been reported to enhance apoptosis induction by trastuzumab, docetaxel, epothilone B, and gemcitabine [99]. HDAC inhibitors also significantly enhance trastuzumab-induced growth inhibition in trastuzumab-sensitive, HER2-overexpressing breast cancer cells, providing a strong rationale for clinical studies with

Additionally, HDAC inhibitors such as entinostat or valproic acid, have been tested in breast cancer cells and efficiently restored both ERα expression and letrozole sensibility in ER-

vitro and in vivo [101,102].The association of HDAC inhibitors or 5-azadeoxycytidine with a

A combined HDAC inhibitors and 5-azadeoxycytidine treatment induces the most significant

treatment inducing overexpression of TFAP2C might improve ESR1 expression in ER-

BC in

patients.

this combination in patients with HER2-positive disease [100].

arrest and subsequent apoptosis [92].

38 A Concise Review of Molecular Pathology of Breast Cancer

aromatase inhibitors [93.97].

The human DNMTs 1, 3A, and 3B coordinate mRNA expression in normal tissues and overexpression in tumours and the expression levels of these DNMTs are reportedly elevated in breast cancer [105,106]. The mean levels of DNMT1, DNMT3a, and DNMT3b overexpression have turned out to be quite similar among different tumour types. The DNMT3b gene has shown the highest range of expression (81.8 for DNMT3a compared with 16.6 and 14 for DNMT1 and DNMT3a, respectively). About 30% of patients revealed overexpression of DNMT3b in the tumour tissue as compared to normal breast tissue. Taking only these overexpressing tumours into account, the DNMT3b expression change was 82-fold, thus being significantly higher [106]. Interestingly, DNMT1 and DNMT3a were overexpressed in only 5 and 3% of breast carcinomas [107]. As a result of these studies, DNMT3b plays the predominant role over DNMT3a and DNMT1 in breast tumourigene‐ sis. This is consistent with a recent study in breast cancer cell lines, which demonstrated a strong correlation between total DNMT activity and overexpression of DNMT3b, but not with the expression of DNMT3a or DNMT1 [107,108].

Cancer was the first group of diseases to be associated with DNA methylation and to be considered for DNA-methylation-targeted therapeutics, and it serves as a prototype for determining the role of DNA methylation and DNA-methylation-targeted therapeutics in other diseases [109]. As we mentioned previously, several types of aberration in DNA methylation and in the proteins involved in DNA methylation occur in cancer: hypermethy‐ lation of tumour suppressor genes, aberrant expression of DNMT1 and other DNMTs, and hypomethylation of unique genes and repetitive sequences [110,111]. Silencing of tumour suppressor genes by DNA methylation provides a powerful molecular mechanism by which DNA methylation can trigger cancer, and also provides a rationale for therapeutics aimed at inhibition of DNA methylation and re-expression of silenced tumour suppressor genes. Multiple genes are hypermethylatedin breast cancer compared to non-cancerous tissue [112]. These include genes involved in evasion of apoptosis (RASSF1A, HOXA5, TWIST1), limitless replication potential (CCND2, p16, BRCA1, RARβ), growth (ERα, PGR), and tissue invasion and metastasis (CDH1) [113]. These genes are not only hypermethylated in tumour cells, but show increased epigenetic silencing in normal epithelium surrounding the tumour site.

Unlike genetic alterations which are almost impossible to revert, DNA methylation is a reversible event. Reactivation of hypermethylated tumour-suppressor genes can be consid‐ ered as a possible therapeutic target which will lead to develop pharmacological inhibitors of DNA methylation. Moreover, the use of DNMT inhibitors is good tools for cancer treatment because the restoration of expression of tumour-suppressor genes could restore the protective effect of these genes on tumour divisions [114]. The nucleoside analogues, 5-azacytidine (vidaza or AZA,) and 5-aza-2'-deoxycytidine (decitabine or DAC) are two DNMT inhibitors that are effective hypomethylating agent that inhibit cell proliferation [115]. These two drugs represent the two most prominent DNMT inhibitors being under preclinical and clinical investigation for over 30 years [116]. Moreover, these agents are pro-drugs that need to be incorporated into DNA to act as inhibitors of DNMTs [116]. The nucleoside analogues are first phosphorylated to the triphosphate nucleotide and incorporated into DNA during DNA synthesis. DNMT1 forms a covalent bond with the carbon at position 6 of the cytosine as well as 5-aza-cytosine ring. Under normal conditions, as mentioned previously, the enzyme transfers the methyl group from SAMe to the fifth carbon position of the cytosine ring. This enables the release of the enzyme from its covalent bond with cytosine. When a 5′-aza-cytosine ring replaces cytosine in the DNA, the methyl transfer does not take place and the DNMT is trapped on the DNA (Figure 3). The replication fork progresses in the absence of DNMT1 resulting in passive loss of DNA methylation in the nascent strand but not the template [116].

Because they are cytidine analogues, both agents are incorporated into DNA after activation to a triphosphate moiety. After formation of an irreversible complex with DNMT1, degrada‐ tion of the enzyme occurs [117]. This prevents methylation of daughter DNA in CpG islands during DNA replication. In addition, AZA (but not DAC) is converted into a ribonucleoside moiety and is incorporated into RNA, interfering with protein translation. At low concentra‐ tions (e.g. 30nM DAC, 300nM AZA), these inhibitors exhibit potent DNA hypomethylation properties, whereas high concentrations (≈3–10 μM) are cytotoxic [119]. The doses of AZA and DAC that were employed in many of the early clinical trials in solid tumours were cytotoxic, reflecting maximum tolerated doses, which likely accounts for the excessive toxicity, and possibly also to lack of overall efficacy, observed in these studies [120]. Previous study indicated that the DNMT inhibitors were associated with response rates as high as 18% in breast cancer [120]. The doses of AZA that were employed in these studies, however, were far higher than doses used in clinical trials today and likely exerted cytotoxic activity as opposed to relief of transcriptional repression as an anti-cancer strategy [120].

Current clinical studies with administration of DNMT inhibitors at the presumed optimal epigenetic dose aim to elucidate the biological effects of these agents, and to assess clinical efficacy, alone or in combination with other anti-cancer agents. The ability of single agent AZA to induce expression of the ER and PR genes in patients with triple-negative breast cancer who are awaiting definitive breast cancer surgery is under investigation using a 75 mg/m2 /day dosing schedule [121]. Based on the preclinical evidence previously described which suggests that a combination of epigenetic modifiers may be more successful in re-expression of silenced genes and restoration of hormonal therapy responsiveness, patients with advanced triplenegative and hormone-resistant breast cancer are being enrolled in an ongoing multi-center phase 2 clinical trial and receive the combination of low dose AZA (40 mg/m2 ) on days 1–5 and 8–10, and entinostat 7 mg on days 3 and 10 of a 28 day cycle. Tumour biopsies prior to and after therapy are collected to assess modulation of candidate gene methylation and expression, such as the ER gene. Patients may transition to an optional continuation phase at the time of disease progression in which the same epigenetic therapy is administered with the addition of hormonal therapy [122].

and metastasis (CDH1) [113]. These genes are not only hypermethylated in tumour cells, but show increased epigenetic silencing in normal epithelium surrounding the tumour site.

40 A Concise Review of Molecular Pathology of Breast Cancer

Unlike genetic alterations which are almost impossible to revert, DNA methylation is a reversible event. Reactivation of hypermethylated tumour-suppressor genes can be consid‐ ered as a possible therapeutic target which will lead to develop pharmacological inhibitors of DNA methylation. Moreover, the use of DNMT inhibitors is good tools for cancer treatment because the restoration of expression of tumour-suppressor genes could restore the protective effect of these genes on tumour divisions [114]. The nucleoside analogues, 5-azacytidine (vidaza or AZA,) and 5-aza-2'-deoxycytidine (decitabine or DAC) are two DNMT inhibitors that are effective hypomethylating agent that inhibit cell proliferation [115]. These two drugs represent the two most prominent DNMT inhibitors being under preclinical and clinical investigation for over 30 years [116]. Moreover, these agents are pro-drugs that need to be incorporated into DNA to act as inhibitors of DNMTs [116]. The nucleoside analogues are first phosphorylated to the triphosphate nucleotide and incorporated into DNA during DNA synthesis. DNMT1 forms a covalent bond with the carbon at position 6 of the cytosine as well as 5-aza-cytosine ring. Under normal conditions, as mentioned previously, the enzyme transfers the methyl group from SAMe to the fifth carbon position of the cytosine ring. This enables the release of the enzyme from its covalent bond with cytosine. When a 5′-aza-cytosine ring replaces cytosine in the DNA, the methyl transfer does not take place and the DNMT is trapped on the DNA (Figure 3). The replication fork progresses in the absence of DNMT1 resulting in passive loss of DNA methylation in the nascent strand but not the template [116].

Because they are cytidine analogues, both agents are incorporated into DNA after activation to a triphosphate moiety. After formation of an irreversible complex with DNMT1, degrada‐ tion of the enzyme occurs [117]. This prevents methylation of daughter DNA in CpG islands during DNA replication. In addition, AZA (but not DAC) is converted into a ribonucleoside moiety and is incorporated into RNA, interfering with protein translation. At low concentra‐ tions (e.g. 30nM DAC, 300nM AZA), these inhibitors exhibit potent DNA hypomethylation properties, whereas high concentrations (≈3–10 μM) are cytotoxic [119]. The doses of AZA and DAC that were employed in many of the early clinical trials in solid tumours were cytotoxic, reflecting maximum tolerated doses, which likely accounts for the excessive toxicity, and possibly also to lack of overall efficacy, observed in these studies [120]. Previous study indicated that the DNMT inhibitors were associated with response rates as high as 18% in breast cancer [120]. The doses of AZA that were employed in these studies, however, were far higher than doses used in clinical trials today and likely exerted cytotoxic activity as opposed

Current clinical studies with administration of DNMT inhibitors at the presumed optimal epigenetic dose aim to elucidate the biological effects of these agents, and to assess clinical efficacy, alone or in combination with other anti-cancer agents. The ability of single agent AZA to induce expression of the ER and PR genes in patients with triple-negative breast cancer who are awaiting definitive breast cancer surgery is under investigation using a 75 mg/m2

dosing schedule [121]. Based on the preclinical evidence previously described which suggests that a combination of epigenetic modifiers may be more successful in re-expression of silenced

/day

to relief of transcriptional repression as an anti-cancer strategy [120].

The DNMT inhibitors combination with standard chemotherapy has not been extensively evaluated in the breast cancer setting and preclinical evidence have shown the AZA could overcome platinum resistance through DNA hypomethylation, patients with both platinum resistant and refractory ovarian cancer received the combination of AZA and carboplatin after being enrolled [122,123]. Since DNMT inhibitors like AZA and DAC are known to be effective in the clinic for diseases like myelodys plastic syndromes that may result in part from tran‐ scriptional dysregulation due to epigenetic changes, there is interest in developing novel DNMT inhibitors that would be more effective and less toxic. One such putative agent is zebularine, a cytidine which has been reported to prevent early tumour development and also to inhibit growth of mammary gland tumours and breast cancer cells lines [124,125]. Zebular‐ ine is a novel DNMT inhibitor, which was developed as a more stable and less toxic drug [126]. Zebularine, similar to AZA-CR and 5-AZA-CdR, incorporates into DNA and forms a covalent irreversible complex with DNMT preventing the enzyme from methylating position 5 of cytosines clustered in regulatory CpG islands [127]. Recent studies showed the ability of zebularine to sustain the demethylation state of the 5′ region of the tumour suppressor gene CDKN2A/p16 and other methylated genes in T24, HCT15, CFPAC-1, SW48, and HT-29 cells [127]. It was also reported that zebularine inhibits growth of cancer cell lines but not normal cells [128].

Zebularine acts as a cytidine analogue containing a 2-(1H)-pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor to prevent deamination of nucleoside analogues [129,130]. Zebularine is also a versatile starting material for the synthesis of complex nucleosides and is a mechanism based DNA cytosine methyltransferase inhibitor [131]. It acts primarily as a trap for DNMT protein by forming tight covalent complexes between DNMT protein and zebularine-substituted DNA [132]. In contrast, to other DNMT inhibitors, it has low toxicity in most tested cell lines and is quite stable with a half-life of 510 h at pH 7.4 [131, 133,134]. Because of its low toxicity, continuous administration of effective doses of zebularine alone or in combination with other DNMT inhibitors is feasible and this can result in the enhanced re-expression of epigenetically silenced genes in cancer cells [128].

Zebularine treatment led to increased p21 protein expression coupled with decreased cyclin B and D protein expression in MCF-7 cells and an increased percentage of cells in S-phase that indicates a zebularine induced S-phase arrest [135].This finding suggests errors in chromatin assembly that contribute to genome instability [136]. S-phase arrest can also be triggered by repression of histone synthesis in human cells [137]. The genomic instability induced by DNMT1 down regulation and repression of histone synthesis triggers the activation of S-phase

**Figure 3.** Activation of gene expression by nucleoside analogues, 5-azacytidine (vidaza or AZA,) and 5-aza-2'-deoxycy‐ tidine (decitabine or DAC), both are DNMTs inhibitors. (A) In active transcription is characterized by the presence of methylated cytosines within CpG dinucleotides (CH3) which is sustained by DNMTs. (B) When a 5′-aza-cytosine ring replaces cytosine in the DNA, the methyl transfer does not take place and the DNMT is trapped on the DNA and the gene expression could restored again.

check point proteins like p21 (in MCF-7 cells) and/or down regulates cyclin-D to permit DNA repair before entering G2 phase.

The zebularine-mediated decrease in expression of global acetylated histones observed in our studies further supports our hypothesis. Several preclinical studies have evaluated zebularine as a possible therapeutic in cancer cell lines. Zebularine preferentially incorporates into DNA, leading to cell growth inhibition and increased expression of cell cycle regulatory genes in cancer cell lines compared with normal fibroblasts [135]. Additionally, to determine the ability of zebularine to prevent or treat breast cancer, Min et al, 2012 tested if daily oral treatment with zebularine affects mammary tumour growth in these MMTV-PyMT mice [124]. They observed a significant delay in tumour growth and a reduction of total tumour burden in the zebularinetreated mice. They have reported that the depletion of DNMTs in tumours excised from zebularine-treated mice and identified upregulation of 12 genes previously characterized as silenced by DNA hypermethylation. Zebularine treatment was shown to be associated with a dose-dependent depletion of DNMT1, DNMT3a, and DNMT3b proteins in the breast cancer cell lines MCF-7 and MDA-MB-231 [124]. Zebularine also depletes DNMT1 in T24 bladder carcinoma cells after 24 hours of treatment and partially depletes DNMT3b after 3 days of drug exposure [128]. Recently, Chen et al, (2012) have proofed in in vivo study that DNMT1 was depleted, and DNMT3b was significantly lowered (50% depletion) in the mammary tumours derived from zebularine-treated mice as compared with untreated mice [138]. Regardless of the mechanism of tumour growth inhibition, tumour cells eventually develop resistance to zebularine treatment. Because it has been shown that zebularine and the HDAC inhibitor depsipeptide have a synergistic effect on the inhibition of breast cancer growth a combinatorial treatment with DNMT inhibitors and a combinatorial treatment with DNMT inhibitors and HDAC inhibitors may be warranted to overcome resistance to single-drug therapy.

Moreover, zebularine have been reported to depleted expression of all three DNMT proteins post-transcriptionally in both breast cancer cell lines at most doses tested. It has been reported that human cancer cells lacking DNMT1 or DNMT3b retain significant global methylation and gene silencing, but those lacking both DNMT1 and DNMT3b had >95% reduction in genomic DNA methylation and virtually absent DNMT activity [135]. The zebularine treatment specifically targets DNMT1, and reduced DNMT 3a and 3b protein expression, implying that treated cells may still retain substantial methylation [139]. Another study observed similar results in T24 bladder cancer cells continuously treated with zebularine for 40 days. In these cells zebularine had no effect on the expression of DNMT1, 3a or 3b mRNA but complete loss of DNMT1 and partial depletion of DNMT 3a and 3b protein were observed [128].

Previous findings observed that ER can be epigenetically silenced in some human breast cancer cell lines and HDAC or DNMT inhibitors could reexpress functional ER in ER negative breast cancer cells [140,141]. Further investigation demonstrated that treatment of ER negative MDA-MB-231 breast cancer cells with zebularine results in functional ER reactivation as manifested by expression of ER mRNA and its target gene, PR. This has been reported with a dose as low as 50μM, far lower than doses that induced apoptosis. Chromatin immunoprecipitation analysis of the ER promoter in zebularine-treated cells showed characteristics of an active chromatin as manifested by accumulation of acetylated H3 and H4 and release of DNMT1, 3a and 3b from the ER promoter region. Although reexpression of ER with zebularine was not as robust as with 5-azaDc, the low toxicity could enable continuous administration for sustained re-expression of ER cells [141].

However, several studies have shown that zebularine has some potential limitations such as less potent than the two FDA-approved DNMT inhibitors, azaC and 5-azaDc [133]. It is hypothesized that the reduced inhibitor potency is due to sequestration of the drug by cytidine deaminase, competitive inhibition of zebularine incorporation into DNA by increased cytidine and deoxycytidine that accumulate as a consequence of its cytidine deaminase properties, and preferential incorporation of zebularine into RNA over DNA [142]. For these reasons, the drug is effective only at very high doses, making administration more problematic. Its efficacy combined with a low toxicity profile makes it an attractive agent for combination or sequential therapy with other DNMT or HDAC inhibitors [143].

## **8. Combination of DNMT inhibitors**

check point proteins like p21 (in MCF-7 cells) and/or down regulates cyclin-D to permit DNA

**Figure 3.** Activation of gene expression by nucleoside analogues, 5-azacytidine (vidaza or AZA,) and 5-aza-2'-deoxycy‐ tidine (decitabine or DAC), both are DNMTs inhibitors. (A) In active transcription is characterized by the presence of methylated cytosines within CpG dinucleotides (CH3) which is sustained by DNMTs. (B) When a 5′-aza-cytosine ring replaces cytosine in the DNA, the methyl transfer does not take place and the DNMT is trapped on the DNA and the

The zebularine-mediated decrease in expression of global acetylated histones observed in our studies further supports our hypothesis. Several preclinical studies have evaluated zebularine as a possible therapeutic in cancer cell lines. Zebularine preferentially incorporates into DNA, leading to cell growth inhibition and increased expression of cell cycle regulatory genes in cancer cell lines compared with normal fibroblasts [135]. Additionally, to determine the ability of zebularine to prevent or treat breast cancer, Min et al, 2012 tested if daily oral treatment with zebularine affects mammary tumour growth in these MMTV-PyMT mice [124]. They observed a significant delay in tumour growth and a reduction of total tumour burden in the zebularinetreated mice. They have reported that the depletion of DNMTs in tumours excised from zebularine-treated mice and identified upregulation of 12 genes previously characterized as silenced by DNA hypermethylation. Zebularine treatment was shown to be associated with a dose-dependent depletion of DNMT1, DNMT3a, and DNMT3b proteins in the breast cancer cell lines MCF-7 and MDA-MB-231 [124]. Zebularine also depletes DNMT1 in T24 bladder carcinoma cells after 24 hours of treatment and partially depletes DNMT3b after 3 days of drug exposure [128]. Recently, Chen et al, (2012) have proofed in in vivo study that DNMT1 was depleted, and DNMT3b was significantly lowered (50% depletion) in the mammary tumours derived from zebularine-treated mice as compared with untreated mice [138]. Regardless of the mechanism of tumour growth inhibition, tumour cells eventually develop resistance to

repair before entering G2 phase.

gene expression could restored again.

42 A Concise Review of Molecular Pathology of Breast Cancer

Based on the preclinical evidence previously described which suggests that a combination of epigenetic modifiers may be more successful in re-expression of silenced genes and restoration of hormonal therapy responsiveness, we have mentioned previously that the patients with advanced triple-negative and hormone-resistant breast cancer are being enrolled in an ongoing multi-center phase 2 clinical trial and receive the combination of low dose of AZA [122]. Tumour biopsies prior to and after therapy are collected to assess modulation of candidate gene methylation and expression, such as the ER. Patients may transition to an optional continuation phase at the time of disease progression in which the same epigenetic therapy is administered with the addition of hormonal therapy [123]. Indeed, in a recently published trial exploring the combination of AZA and entinostat in advanced non-small cell lung cancer patients, investigators observed that the regimen was well tolerated and associated with a number of objective responses [144]. These included a complete response as well as a partial response in a patient without progression of disease for 2 years after completing the clinical trial. Interestingly, a number of patients were found to have unexpected major objective responses to subsequent anti-cancer strategies, raising the question as to whether these agents may prime tumour cells to respond to subsequent therapies. A phase 1/2 Canadian trial investigating the combination of decitabine and vorinostat in patients with advanced solid tumours or hematologic malignancies has also indicated clinical activity. Stabilization of disease for 4 or more cycles was observed in 29 % evaluable patients; two of these patients had metastatic breast cancer [145].

Moreover, cytidine deaminase destabilizes DNMT inhibitors like 5-azaDc, resulting in complete loss of their antineoplastic ability [146]. Hence administration of cytidine deaminase inhibitors like zebularine should theoretically potentiate therapeutic effects of 5-azaDc by slowing its degradation and stabilizing activity. Indeed, the combination of 5-aza-Dc and zebularine produced greater inhibition in cell proliferation and clonogenicity than either drug alone in leukemic L1210 and HL-60 cell lines [147]. Similarly, treatment of the AML-193 acute myeloid leukemic cell line, which has a densely methylated p15INK4B CpG island, with zebularine followed by the HDAC inhibitor, trichostatin-A, synergistically enhanced p15INK4B expression [134]. Consistent with these results, the combination of 50μMzebularine and 1μM 5-azaDc in breast cancer cells significantly inhibited cell proliferation compared with either drug alone. Similarly, zebularine significantly inhibited cell proliferation and colony formation in combination with low doses of vorinostat. Cheishvili et al, (2014) have investi‐ gated the combination of methylated DNA binding protein 2 (MBD2) depletion and DNMT inhibitor 5-azaCdR in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumour growth arrest on one hand while inhibiting invasiveness triggered by 5 azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by combination of MBD2 and DNMT inhibitors [148].

The combination of DNMT inhibitors with standard chemotherapy has not been extensively evaluated in the breast cancer setting. Based on strong preclinical evidence that the addition of AZA could overcome platinum resistance through DNA hypomethylation, patients with both platinum resistant and refractory ovarian cancer received the combination of AZA and carboplatin after being enrolled into a phase 1b/2 study. The overall response rate of 22 % was observed in the platinum-resistant patients (disease progression within 6 months of platinum, n=18) suggesting that further evaluation of the combination was warranted [149]. Whether combining DNMT inhibitors with standard therapies or novel agents will result in clinical benefit for patients with breast cancer remains to be seen. In the meantime, robust preclinical data should support the development of new concepts in order to maximize the chance of success with these agents in the solid tumour arena.

## **9. Conclusion**

multi-center phase 2 clinical trial and receive the combination of low dose of AZA [122]. Tumour biopsies prior to and after therapy are collected to assess modulation of candidate gene methylation and expression, such as the ER. Patients may transition to an optional continuation phase at the time of disease progression in which the same epigenetic therapy is administered with the addition of hormonal therapy [123]. Indeed, in a recently published trial exploring the combination of AZA and entinostat in advanced non-small cell lung cancer patients, investigators observed that the regimen was well tolerated and associated with a number of objective responses [144]. These included a complete response as well as a partial response in a patient without progression of disease for 2 years after completing the clinical trial. Interestingly, a number of patients were found to have unexpected major objective responses to subsequent anti-cancer strategies, raising the question as to whether these agents may prime tumour cells to respond to subsequent therapies. A phase 1/2 Canadian trial investigating the combination of decitabine and vorinostat in patients with advanced solid tumours or hematologic malignancies has also indicated clinical activity. Stabilization of disease for 4 or more cycles was observed in 29 % evaluable patients; two of these patients had

Moreover, cytidine deaminase destabilizes DNMT inhibitors like 5-azaDc, resulting in complete loss of their antineoplastic ability [146]. Hence administration of cytidine deaminase inhibitors like zebularine should theoretically potentiate therapeutic effects of 5-azaDc by slowing its degradation and stabilizing activity. Indeed, the combination of 5-aza-Dc and zebularine produced greater inhibition in cell proliferation and clonogenicity than either drug alone in leukemic L1210 and HL-60 cell lines [147]. Similarly, treatment of the AML-193 acute myeloid leukemic cell line, which has a densely methylated p15INK4B CpG island, with zebularine followed by the HDAC inhibitor, trichostatin-A, synergistically enhanced p15INK4B expression [134]. Consistent with these results, the combination of 50μMzebularine and 1μM 5-azaDc in breast cancer cells significantly inhibited cell proliferation compared with either drug alone. Similarly, zebularine significantly inhibited cell proliferation and colony formation in combination with low doses of vorinostat. Cheishvili et al, (2014) have investi‐ gated the combination of methylated DNA binding protein 2 (MBD2) depletion and DNMT inhibitor 5-azaCdR in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumour growth arrest on one hand while inhibiting invasiveness triggered by 5 azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by

The combination of DNMT inhibitors with standard chemotherapy has not been extensively evaluated in the breast cancer setting. Based on strong preclinical evidence that the addition of AZA could overcome platinum resistance through DNA hypomethylation, patients with both platinum resistant and refractory ovarian cancer received the combination of AZA and carboplatin after being enrolled into a phase 1b/2 study. The overall response rate of 22 % was observed in the platinum-resistant patients (disease progression within 6 months of platinum, n=18) suggesting that further evaluation of the combination was warranted [149]. Whether combining DNMT inhibitors with standard therapies or novel agents will result in clinical

metastatic breast cancer [145].

44 A Concise Review of Molecular Pathology of Breast Cancer

combination of MBD2 and DNMT inhibitors [148].

Future studies need to include a more detailed investigation of the methylation differences between breast cancer subtypes to determine whether there is a methylation signature that can identify breast cancer subtypes. It is also possible that DNA methylation subtypes are different to the subtypes identified by gene expression and may provide additional information that assists in the clinical setting. Further research is required to delineate these options and determine how subtypes identified by DNA methylation profiling differ to subtypes identified by gene expression. Laboratory studies have shown that AZA and DAC optimally inhibit DNA methylation when used at lower than cytotoxic doses with prolonged exposures. The exact impact of using epigenetic modifiers at an optimally epigenetic dose instead of a cytotoxic dose is yet unknown in solid tumours, despite the supposition that anti-cancer activity will be enhanced. Ongoing clinical trials in breast cancer patients aim to elucidate this question. Optimizing the use of the clinically available epigenetic modifiers is clearly important. An oral form of AZA is currently in development which may be far more convenient for patients than the intravenous and subcutaneous routes employed at this time. A number of new agents are also in development which may circumvent some of the limitations of the currently available drugs such as their in vivo deamination by cytidine deaminase and tendency to be subject to drug resistance.

## **Author details**

Majed S. Alokail1\* and Amal M. Alenad2

\*Address all correspondence to: malokail@ksu.edu.sa

1 Biomarker Research Group, Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia

2 School of Biological Sciences, Life Science Building, University of Southampton, Southampton, UK

## **References**

[1] Waddington CH. Selection of the genetic basis for an acquired character. Nature 1952;169:625-626.


[19] Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R, Li E. De novo DNA cy‐ tosine methyltransferase activities in mouse embryonic stem cells. Development 1996;122:3195-3205.

[2] Waddington CH. Evolutionary adaptation. Perspectives in Biology and Medi‐

[3] Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science

[4] Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science

[6] Vaissiere T, Sawan C, Herceg Z. Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutation Research 2008;659:40-48.

[7] Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends in

[8] RazinA, Szyf M. DNA methylation patterns. Formation and function. Biochimical et

[9] Fuks F. DNA methylation and histone modifications: teaming up to silence genes.

[10] Robertson KD, Jones PA. DNA methylation: past, present and future directions. Car‐

[12] Straussman R, Nejman D, Roberts D, Steinfeld I, Blum B, et al,. Developmental pro‐ gramming of CpG island methylation profiles in the human genome. Nature Struc‐

[13] Bird AP. CpG-rich islands and the function of DNA methylation. Nature

[14] Jin B, Li Y, Robertson KD. DNA methylation: superior or subordinate in the epige‐

[15] Bestor TH. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. The European Molecular Biology Organization Journal

[16] Buryanov YI, Shevchuk TV. DNA methyltransferases and structural-functional spe‐ cificity of eukaryotic DNA modification. Biochemistry (Mosc) 2005;70: 730-742.

[17] Lauster R, Trautner TA, and Noyer-Weidner M. Cytosine-specific type II DNA meth‐ yltransferases. A conserved enzyme core with variable target-recognizing domains.

[18] Subramaniam D, Thombre R, Dhar A, Anant S. DNA methyltransferases: a novel tar‐

get for prevention and therapy.Frontiers in Oncology2014;1(4):80.

[5] Bird A. Perceptions of epigenetics. Nature2007;447:396-398.

Current Opinion in Genetics & Development 2005;15:490-495.

[11] Singal R, Ginder GD. DNA methylation. Blood 1999;93:4059-4070.

cine1959;2:379-401.

46 A Concise Review of Molecular Pathology of Breast Cancer

1999;286:481-486.

1999;286:481-486.

Biochemical Scinces 2006;31:89-97.

Biophysical Acta 1984;782(4):331-342.

tural & Molecular Biology2009;16:564-571.

netic hierarchy? Genes & Cancer 2011;2:607–17.

Journal Molecular Biology 1989;206: 305-312.

cinogenesis 2000;21:461-467.

1986;321:209-213.

1992;11:2611-2617.


[48] Szyf M. DNA methylation signatures for breast cancer classification and prognosis. Genome Medicine 2012;4(3): 26.

[33] Arts J, de Schepper S, Van Emelen K. Histone deacetylase inhibitors: from chromatin remodeling to experimental cancer therapeutics. Current Medicinal Chemis‐

[34] Prince HM, Bishton MJ, Harrison SJ. Clinical studies of histone deacetylase inhibi‐

[35] Grant PA. A tale of histone modifications. Genome Biology 2001;2(4): reviews0003-

[36] Allfrey VG, Faulknerr R, Mirsky AE. Acetylation and methylation the histones and their possible role in the regulation of RNA synthesis. Proceedings of the National

[37] Roth SY, Denu JM, Allis CD. Histone acetyltransferases. Annual Review in Biochem‐

[38] Hong L, Schroth GP, Matthews HR, Yau P, Bradbury EM. Studies of the DNA bind‐ ing properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. Jour‐

[39] Kim Y, Tanner KG, Denu JM. A continuous, nonradioactive assay for histone acetyl‐

[40] Glaser KB. HDAC inhibitors: clinical update and mechanism-based potential. Bio‐

[41] Vigushin DM, Ali S, Pace PE, Mirsaidi N, Ito K.Trichostatin A is a histone deacety‐ lase inhibitor with potent antitumor activity against breast cancer in vivo. Clinical

[42] Kyrylenko S, Kyrylenko O, Suuronen T, Salminen A. Differential regulation of the Sir2 histone deacetylase gene family by inhibitors of class I and II histone deacetylas‐

[43] Crabb SJ, Howell M, Rogers H, Ishfaq M, Yurek-George A, et al. C,aracterisation of the in vitro activity of the depsipeptide histone deacetylase inhibitor spiruchostatin

[44] Szyf M. The dynamic epigenome and its implications in toxicology. Toxicology Sci‐

[45] Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis 2010;31:27-36.

cancers from their normal counterparts. Nature 1983;301:89–92.

[46] Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human

[47] BerdascoM,Esteller M. Aberrant epigenetic landscape in cancer: how cellular identity

Academy of Sciences of the United States of America1964;51: 786-794.

try2003;10:2343–2350.

48 A Concise Review of Molecular Pathology of Breast Cancer

istry 2001; 70: 81-120.

reviews0003.6

tors. Clinical Cancer Research 2009;15:3958–3969.

nal of Biological Chemistry1993;268: 305-314.

chemical Pharmacology 2007;74:659-671.

Cancer Research 2001;7: 971-976.

ence 2007;100: 7-23.

transferases.Analytical Biochemistry 2000;280(2):308-314.

es. Cellular and molecular life sciences2003;60: 1990-1997.

A. Biochemical Pharmacology 2008;76: 463-475.

goes awry. Developmental cell 2010;19:698-711.


[75] DayTK, Bianco-MiottoT. Common gene pathways and families altered by DNA methylation in breast and prostate cancers. Endocrine Related Cancer 2013; 20(5):R215-32.

[63] Mirza S, Sharma G, Prasad CP, Parshad R, Srivastava, A et al. Promoter hypermethy‐ lation of TMS1, BRCA1, ERalpha and PRB in serum and tumor DNA of invasive duc‐

[64] Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of hu‐

[65] Press JZ, De Luca A, Boyd N, Young S, Troussard A. et al. Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities. BMC Can‐

[66] Birgisdottir V, Stefansson OA, Bodvarsdottir SK, Hilmarsdottir H, Jonasson JG. et al. Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer. Breast

[67] Dworkin AM, Huang TH-M, Tolandbcd AE. Epigenetic alterations in the breast: Im‐ plications for breast cancer detection, prognosis and treatment. Seminar in Cancer Bi‐

[68] ChappuisPO, Kapusta L, Begin LR, Wong N, Brunet JS, et al. Germline BRCA1/2 mu‐ tations and p27(Kip1) protein levels independently predict outcome after breast can‐

[69] Xu X, Gammon MD, Zhang Y, Bestor TH, Zeisel SH. Et al. BRCA1 promoter methyla‐ tion is associated with increased mortality among women with breast cancer. Breast

[70] Honrado, E., Osorio, A., Milne, RL., Paz MF, Melchor L, et al. Immunohistochemical classification of non-BRCA1/2 tumors identifies different groups that demonstrate

[71] Hu M, Yao J, Cai L, Bachman KE, van den Brule F, et al. Distinct epigenetic changes

[72] Tommasi S, Karm DL, Wu X, Yen Y, Pfeifer GP. Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer. Breast Cancer Research 2009;11:

[73] Van der Auwera I, Yu W, Suo L, Van Neste L, van Dam P, et al., Array-based DNA methylation profiling for breast cancer subtype discrimination. PLOS ONE 2010;5:

[74] Kamalakaran S, Varadan V, GierckskyRussnes HE, Levy D, Kendall J. DNA methyla‐ tion patterns in luminal breast cancers differ from non-luminal subtypes and can identify relapse risk independent of other clinical variables. Molecular Oncology

the heterogeneity of BRCAX families. Modern Pathology 2007;20:1298–1306.

in the stromal cells of breast cancers. Nature Genetics 2005;37:899–905.

tal breast carcinoma patients. Life Science 2007; 81:280–287.

man cancer. Cancer Research 2001; 61:3225–3229.

cer. Journal of Clinical Oncology 2000;18:4045–4052.

cancer research and treatment 2009;115:397-404.

cer 2008;8:17.

Cancer Research 2006;8: R38.

50 A Concise Review of Molecular Pathology of Breast Cancer

ology 2009;3:165-171.

R14.

e12616.

2011;5: 77–92.


[100] Bali P, Pranpat M, Swaby R, Fiskus W, Yamaguchi H, et al. Activity of suberoylanili‐ dehydroxamic Acid against human breast cancer cells with amplification of her-2. Clinical Cancer Research 2005;11:6382–6389.

[87] Trape AP, Gonzalez-AnguloAM. Breast Cancer and Metastasis: On the Way Toward

[88] Van't Veer LJ, Dai H, van de Vijver MJ, He YD, et al Gene expression profiling pre‐

[89] Smid M, Wang Y, Klijn JG, Sieuwerts AM, Zhang Y, et al. Genes associated with breast cancer metastatic to bone. Journal Clinical Oncology 2006;24:2261–2267.

[91] Olsen EA, Kim YH, Kuzel TM, Pacheco TR, Foss FM, et al. Phase IIbmulticenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutane‐

[92] Munster PN, Troso-Sandoval T, Rosen N, Rifkind R, Marks PA, et al. The histone de‐ acetylase inhibitor suberoylanilidehydroxamic acid induces differentiation of human

[93] Cohen LA, Amin S, Marks PA, Rifkind RA, Desai D, et al. Chemoprevention of carci‐ nogen-induced mammary tumorigenesis by the hybrid polar cytodifferentiation agent, suberanilohydroxamic acid (SAHA) Anticancer Research 1999;19:4999–5005.

[94] Huang L, Pardee AB. Suberoylanilidehydroxamic acid as a potential therapeutic agent for human breast cancer treatment. Molecular Medicine 2000;6:849–866.

[95] Keen JC, Yan L, Mack KM, Pettit C, Smith D, et al. A novel histone deacetylase inhib‐ itor, scriptaid, enhances expression of functional estrogen receptor alpha (ER) in ER negative human breast cancer cells in combination with 5-aza 2′-deoxycytidine.

[96] Zhou Q, Shaw PG, Davidson NE. Inhibition of histone deacetylase suppresses EGF signaling pathways by destabilizing EGFR mRNA in ER-negative human breast can‐

[97] Sharma D, Saxena NK, Davidson NE, Vertino PM. Restoration of tamoxifen sensitivi‐ ty in estrogen receptor-negative breast cancer cells: tamoxifen-bound reactivated ER recruits distinctive corepressor complexes. Cancer Research 2006;66:6370–6378. [98] Kim MS, Blake M, Baek JH, Kohlhagen G, Pommier Y, et al. Inhibition of histone de‐ acetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Research

[99] Fuino L, Bali P, Wittmann S, Donapaty S, Guo F, et al. Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzu‐ mab, taxotere, gemcitabine, and epothilone B. Molecular Cancer Therapeutics

cer cells. Breast Cancer Research and Treatment 2009;117(2):443–451.

[90] Jenuwein T, Allis CD. Translating the histone code.Science. 2001;293:1074–1080.

ous T-cell lymphoma. Journal of Clinical Oncology 2007;25:3109–3115.

breast cancer cells. Cancer Research 2001;61:8492–8497.

Breast Cancer Research and Treatment 2003;81:177–186.

2003;63:7291–7300.

2003;2:971–984.

Individualized Therapy. Cancer Genomics Proteomics 2012;9(5):297-310.

dicts clinical outcome of breast cancer. Nature 2002;415: 530–536.

52 A Concise Review of Molecular Pathology of Breast Cancer


[127] Cheng JC, Yoo CB, Weisenberger DJ, Chuang J, Wozniak C, et al. Preferential re‐ sponse of cancer cells to zebularine. Cancer Cell 2004;6:151–158. Cheng JC, Weisen‐ berger DJ, Gonzales FA, Liang G, Xu GL. et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Molecular and Cel‐ lular Biology 2004;24:1270–1278.

[113] Widschwendter M, Jones PA. DNA methylation and breast carcinogenesis.Onco‐

[115] Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methyla‐

[116] Wu JC, SantiDV.On the mechanism and inhibition of DNA cytosine methyltransfer‐

[117] el-Deiry WS, Nelkin BD, Celano P, Yen RW, Falco JP, et al. High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. Proceedings of the National Academy of Sciences USA

[118] Patra SK, Patra A, Zhao H, Dahiya R. DNA methyltransferase and demethylase in

[119] Girault I, Tozlu S, Lidereau R, Bieche I. Expression analysis of DNA methyltransfer‐ ases 1, 3A, and 3B in sporadic breast carcinomas. Clinical Cancer Research 2003;9(12):

[120] Broske AM, Vockentanz L, Kharazi S, Huska MR, Mancini E, et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Na‐

[121] Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA. DNMT1 maintains progenitor function in self-renewing somatic tissue. Nature 2010;463(7280):563–710.

[122] Fu S, Hu W, Iyer R, Kavanagh JJ, Coleman RL,et al. Phase 1b-2a study to reverse plat‐ inum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer. Cancer

[123] Connolly R, Stearns V. Epigenetics as a therapeutic target in breast cancer. Journal of

[124] Chen M, Shabashvili D, Nawab A, Yang SX, Dyer LM, et al. DNA methyltransferase inhibitor, zebularine, delays tumor growth and induces apoptosis in a genetically en‐ gineered mouse model of breast cancer. Molecular Cancer Therapeutics2012;11(2):

[125] Billam M, Sobolewski MD, Davidson NE. Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Research Treatment

[126] Yoo CB, Cheng JC, Jones PA.Zebularine: a new drug for epigenetic therapy. Bio‐

Mammary Gland Biology and Neoplasia 2012; 17(3-4):191–204.

chemical Society Transactions 2004;32:910–912.

[114] Esteller M. Epigenetics in Biology and Medicine. Boca Raton: In CRC: 2009.

ases. Progress Clinical and Biological Research 1985;198:119–129.

human prostate cancer. Molecular Carcinogenesis 2002;33(3):163.

gene. 2002;21(35):5462-5482.

54 A Concise Review of Molecular Pathology of Breast Cancer

tion.Cell. 1980;20(1):85-93.

1991;88(8):3470–3410.

ture Genetics 2009; 41(11):1207–1510.

4415–4422.

2011;117:1661–1669.

370-382.

2010;120:581–592.


2011; doi: 10.1158/1535-7163.MCT-11-0458 Molecular Cancer Therapeutics 2012;11; 370.


**Chapter 3**
