**8. PcG and cancer epigenetics**

Other epigenetic modifiers have been identified, including the Polycomb group (PcG) proteins and small non-coding RNAs. PcG repressors serve as a docking platform for DNA methyltransferases and target a gene for permanent silencing by methylation of hisone H3 on lysine 27 (H3K27). Reversal of permanent silencing is only overcome by dedifferentiation processes in the germline. Small non-coding RNA molecules, such as microRNAs, regulate gene expression by targeting RNA degradation (Luczak & Jagodzinski, 2006). These RNAs have also been found to also target gene promoters and result in transcriptional gene silencing (Balch et al., 2007; Han et al., 2007).

Increasing evidence from cancer epigenomic studies suggests a critical role for PcG factors in abnormal epigenetic silencing of tumor suppressor genes in cancer cells (Baylin & Ohm, 2006;Jones & Baylin, 2007;Lund & van Lohuizen, 2004;Valk-Lingbeek et al., 2004;Ting et al., 2006). There are at least four different PcG complexes identified in mammalian, including the maintenance complex, PRC1, composed of RING, HPC, HPH, and BMI1, and three different initiation complexes, PRC2 through PRC4, which are formed by enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and different isoforms of embryonic ectoderm development (EED) (Baylin & Ohm, 2006;Ting et al., 2006;Kuzmichev et al., 2004;Kuzmichev et al., 2005). In particular, PRC4 exists in embryonic, stem, progenitor and cancer cells and associates with a class III HDAC called SIRT1 ((Baylin & Ohm, 2006;Ting et al., 2006). The crucial function of PRC complexes in H3K27 methylation is mediated by EZH2, a histone lysine methyltransferase, that catalyzes this lysine methylation (Cao et al., 2002;Cao & Zhang, 2004;Martin & Zhang, 2005). Methylation of H3K27 possibly stabilizes the binding of PcG complexes to this histone mark to facilitate long-term gene silencing (Fischle et al., 2003;Martin & Zhang, 2005). Importantly, H3K27me is often present at the promoters of the DNA hypermethylated and silenced cancer genes investigated thus far (McGarvey et al., 2006), indicating that PcG proteins play an essential role in aberrant gene silencing in cancer cells. A recent study also showed that PcG-targeted genes in normal cells are closely associated with de novo DNA methylation in cancer cells, suggesting that PcG may preprogram its targeted genes as targets of subsequent DNA methylation in cancer cells (Keshet et al., 2006;Schlesinger et al., 2007).

In addition, several studies have shown that expression of PcG proteins such as EZH2, SUZ12 and BMI1 is aberrantly elevated in breast cancer and other cancers (Dimri et al., 2002;Kleer et al., 2003), suggesting deregulation of components of nucleosomal remodeling complexes can also be a mechanism resulting in gene silencing in cancer cells. In the case of

expression via interaction with specific transcription factors (Kim et al., 2007;Yang et al., 2003). Differential DNA methylation of the parental ICRs is one of the most common features associated with imprinted genes (Kim et al., 2003;Liang et al., 2000;Mancini-Dinardo et al., 2003). Typical disorders associated with imprinted genes include Prader-Willi and Angelman syndromes, Beckwith-Wiedemann syndrome and multiple forms of neoplasia (Weksberg et al., 2003;Zeschnigk et al., 1997). In addition to that, X inactivation is a mechanism that functionally equalizes the difference of X-linked genes between XX females and XY males by silencing one of the two X chromosomes in females. Dosage compensation is a widely known method of silencing the X chromosome in females. This is achieved epigenetically through a cascade of CpG methylation superimposed by global histone deacetylation (Avner & Heard, 2001;Lyon, 1999;Monk, 2002;Pfeifer et al., 1990).

Other epigenetic modifiers have been identified, including the Polycomb group (PcG) proteins and small non-coding RNAs. PcG repressors serve as a docking platform for DNA methyltransferases and target a gene for permanent silencing by methylation of hisone H3 on lysine 27 (H3K27). Reversal of permanent silencing is only overcome by dedifferentiation processes in the germline. Small non-coding RNA molecules, such as microRNAs, regulate gene expression by targeting RNA degradation (Luczak & Jagodzinski, 2006). These RNAs have also been found to also target gene promoters and

Increasing evidence from cancer epigenomic studies suggests a critical role for PcG factors in abnormal epigenetic silencing of tumor suppressor genes in cancer cells (Baylin & Ohm, 2006;Jones & Baylin, 2007;Lund & van Lohuizen, 2004;Valk-Lingbeek et al., 2004;Ting et al., 2006). There are at least four different PcG complexes identified in mammalian, including the maintenance complex, PRC1, composed of RING, HPC, HPH, and BMI1, and three different initiation complexes, PRC2 through PRC4, which are formed by enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and different isoforms of embryonic ectoderm development (EED) (Baylin & Ohm, 2006;Ting et al., 2006;Kuzmichev et al., 2004;Kuzmichev et al., 2005). In particular, PRC4 exists in embryonic, stem, progenitor and cancer cells and associates with a class III HDAC called SIRT1 ((Baylin & Ohm, 2006;Ting et al., 2006). The crucial function of PRC complexes in H3K27 methylation is mediated by EZH2, a histone lysine methyltransferase, that catalyzes this lysine methylation (Cao et al., 2002;Cao & Zhang, 2004;Martin & Zhang, 2005). Methylation of H3K27 possibly stabilizes the binding of PcG complexes to this histone mark to facilitate long-term gene silencing (Fischle et al., 2003;Martin & Zhang, 2005). Importantly, H3K27me is often present at the promoters of the DNA hypermethylated and silenced cancer genes investigated thus far (McGarvey et al., 2006), indicating that PcG proteins play an essential role in aberrant gene silencing in cancer cells. A recent study also showed that PcG-targeted genes in normal cells are closely associated with de novo DNA methylation in cancer cells, suggesting that PcG may preprogram its targeted genes as targets of subsequent DNA methylation in cancer

In addition, several studies have shown that expression of PcG proteins such as EZH2, SUZ12 and BMI1 is aberrantly elevated in breast cancer and other cancers (Dimri et al., 2002;Kleer et al., 2003), suggesting deregulation of components of nucleosomal remodeling complexes can also be a mechanism resulting in gene silencing in cancer cells. In the case of

result in transcriptional gene silencing (Balch et al., 2007; Han et al., 2007).

**8. PcG and cancer epigenetics** 

cells (Keshet et al., 2006;Schlesinger et al., 2007).

another repressive histone mark, H3K9me2 (me3), this lysine methylation is catalyzed by several histone lysine methyltransferases, including SUV39H, SETDB1, G9a and GLP among others (Schultz et al., 2002;Lehnertz et al., 2003;Tachibana et al., 2005). Although the defined role of H3K9 methylation in epigenetic gene silencing remains elusive, one possible mechanism is that this mark can serve as a binding site for heterochromatin protein HP1, which has an intrinsic ability to recruit DNA methyltransferases to the silenced genes (Fuks et al., 2003;Lachner et al., 2001).

To establish DNA methylation in a subset of genes, polycomb protein EZH2 must associate with DNMTs (Esteller, 2007). It is thought that polycomb proteins could collaborate with DNMTs by recruiting them to silenced promoters to establish long-term silencing (Matarazzo et al., 2007). Leu et al (2004) investigated whether the removal of ERα signaling could cause changes in DNA methylation and chromatin structure of ERα target promoters. They used RNAi to transiently disable ERα in breast cancer cells and found that polycomb repressors and histone deacetylases assemble in the promoter of an ERα target gene. Accumulation of DNA methylation in these silenced targets like the PR promoter region then occurs and can be stably transmitted to cell progeny for long-term silencing. Both ERα expression and DNA demethylation appear to be required to restore PR expression. They also observed a trend that more ERα negative tumors had more methylated loci than ERα positive tumors (Leu et al., 2004). This indicates that dysregulation of normal signaling in cancer cells may result in stable silencing of downstream targets maintained by epigenetic machinery (Dworkin et al., 2009).

The epigenetic mechanisms for gene silencing involve the interplay between DNA methylation, histone modifications and nucleosomal remodeling. The families of methyl-CpG binding proteins (MBD and Kaiso families) have been identified to play a key role in this interplay. The molecular functions of methyl-CpG binding proteins are dependent on their ability to recognize and bind methylated DNA (Clouaire & Stancheva, 2008;Meehan et al., 1989; ing et al., 2006). Accumulating evidence suggests that methyl-CpG binding proteins can associate directly or indirectly with DNMTs, HDACs and HMTs and cooperate with them to modify chromatin structure and suppress initiation of gene transcription (Fuks et al., 2003;Jones et al., 1998;Kimura & Shiota, 2003;Sarraf & Stancheva, 2004). The associated partners of methyl-CpG binding proteins have also been found to include many nucleosomal remodeling complexes such as NuRD, CoREST, NCoR/SMRT, Sin3A, SUV39H and SWI/SNF (Fujita et al., 2003;Harikrishnan et al., 2005;Le Guezennec et al., 2006;Yoon et al., 2003;Wade et al., 1999;Zhang et al., 1999). The significant role of methyl-CpG binding proteins in cancer epigenetics is supported by the findings that they are localized to DNA hypermethylated and aberrantly silenced cancer genes (Bakker et al., 2002; Lopez-Serra et al., 2006; Nguyen et al., 2001).

Thus, it has been postulated that methyl-CpG binding proteins initially recognize and bind to methylated DNA, and then bring down nucleosomal remodeling complexes to modify chromatin to the repressive compact heterochromatin structure, which causes gene silencing. Inversely, the results from some other studies show that chromatin remodeling activities can further facilitate binding of methyl-CpG binding proteins to methylated DNA sites (Feng & Zhang, 2001;Harikrishnan, et al., 2005), suggesting interaction between methyl-CpG binding proteins and nucleosomal remodeling complexes results in mutual stimulation of each others' activity. Taken together, methyl-CpG binding proteins represent an important class of chromosomal proteins that associate with multiple protein partners to modify surrounding chromatin and silence transcription, providing a functional link between DNA methylation and chromatin modification and remodeling (Lo & Sukumar, 2008).

Epigenetics and Breast Cancer 301

RASSF1A is transcribed from a CpG island promoter region, and is one of the most frequently hypermethylated genes thus far described in human cancer. The CpG island of RASSF1A is hypermethylated in 60–77% of breast cancers (Lewis et al., 2005;Vincent-Salomon et al., 2007) resulting in gene silencing in cancer cell lines and primary tissues. Its diverse functions include regulation of apoptosis, growth regulation, and microtubule dynamics during mitotic progression. Specifically, RASSF1A is a Ras effector and induces apoptosis through its interactions with pro-apoptotic kinase MST1. When cells lacking RASSF1A expression are treated with a DNA methyltransferase, such as 5-aza-2′ deoxycytidine, expression can be reactivated (Pfeifer & Dammann, 2005). Mouse knockout studies show that RASSF1A−/− mice are prone to spontaneous development of lung adenomas, lymphomas and breast adenocarcinomas. These mice are prone to early spontaneous tumorigenesis and show a severe tumor susceptibility phenotype compared to

Furthermore, it has been reported that the DNA methylation assay might be used for risk assessment and prognosis of breast cancer. Lewis et al. studied five frequently methylated genes, including RASSF1A, APC, H-cadherin, RARβ, and cyclin D2, and found a higher methylation frequency of both RASSF1A and APC genes in unaffected women at high risk for breast cancer compared with those at low or intermediate risk based on the Gail model analysis. This suggests that promoter hypermethylation of these genes is associated with epidemiologic markers of increased breast cancer risk (Lewis et al., 2005). This finding needs confirmation that such alterations do indeed occur earlier than abnormal histological findings, and by follow-up studies to examine whether these changes are associated with subsequent development of breast cancer (Lo & Sukumar, 2008). The prognostic significance of aberrant DNA methylation has been investigated by Muller et al. (2003) after screening 39 genes in DNA from serum of normal control patients and patients with primary or metastatic breast cancer, they identified two genes, RASSF1A and APC, whose methylation has a statistically significant association with poor outcome. Other methylated genes, such as GSTP1, SFRP1, have also been identified to be associated with poor prognosis (Arai et al.,

In breast cancer, multiple genes are hypermethylated compared to non-cancerous tissue (Agrawal & Murphy, 2007). 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) (Han et al., 2007; Yan et al., 2001; Widschwendter & Jones, 2002). These genes are not only hypermethylated in tumor cells, but show increased epigenetic silencing in normal epithelium surrounding the tumor site. The first observations of this phenomenon were in oral cancer. Slaughter et al (1953) was the first group to use the term "field cancerization" which refers to the presence of cancer causing changes in apparently normal tissue surrounding a neoplasm. They theorized the existence of (pre-) neoplastic processes at multiple sites, with the unproven assumption that these have developed independently (Slaughter et al., 1953). In subsequent years, the presence of field cancerization has been described in head and neck squamous cell carcinoma, lung, esophagus, vulva, cervix, colon, bladder, skin, and breast cancers (Yan et al., 2006). Studies have demonstrated that normal adjacent cells to tumors frequently harbor loss of heterozygosity, microsatellite and chromosome instability, and gene mutations (Braakhuis et al., 2003). Recently DNA methylation has been added to list as hypermethylated normal tissue

immediately adjacent to tumor sites has been found (Ushijima, 2007).

that of littermate wild-type mice (Pfeifer & Dammann, 2005).

2006;Veeck et al., 2006).

Again, cancer generally has been viewed as a disease that is driven by progressive genetic abnormalities, involving chromosomal abnormalities, mutations in oncogenes and tumor suppressor genes (Hanahan & Weinberg, 2000;Vogelstein & Kinzler 2004). Nevertheless, it has been shown that breast cancer, similar to other types of cancer, is also a disease that is driven by epigenetic alterations, which do not affect the primary DNA sequence (Widschwendter & Jones, 2002;Polyak, 2007). The result of these alterations is aberrant transcriptional regulation that leads to a modify in expression patterns of genes implicated in survival, differentiation and cellular proliferation (Baylin & Ohm, 2006;Esteller, 2007;Widschwendter & Jones, 2002). In transformed cells, epigenetic alterations occur at the chromosomal level. These involve changes in DNA methylation, histone modifications, altered expression and function of factors implicated in regulating assembly and remodeling of nucleosomes (Baylin & Ohm, 2006;Esteller, 2007;Jones & Baylin, 2002;Jones & Baylin, 2007;Ting et al., 2006). Alterations in DNA methylation include global hypomethyation and focal hypermethylation.

Global hypomethylation has been found to increase with age and is linked to genomic instability and activation of oncogene expression (Eden et al., 2003;Feinberg & Tycko, 2004;Richardson, 2002). Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. DNA Methylation silencing of tumor suppressor genes, aberrant expression of DNMT1 or demethylation of oncogenes can lead to the conversion of a normal cell to a malignant cell. In addition chromosomal instability and inactivation of the DNA repair system has both the genetic and epigenetic backgrounds (Esteller & Herman, 2002;Szyf, 2008). Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. Hypermethylation of the mismatch repair gene MLH1 is associated with tumors exhibiting microsatellite instability, and hypermethylation of the breast cancer gene BRCA1 is found in 10%- 15% of women with non-familial breast cancer (Jones & Baylin, 2002).

#### **9. Epigenetic modifications and breast cancer**

Epigenetic modifications are believed to be early events in cancer development (Leu et al., 2004) and breast cancer is a disease characterized by both genetic and epigenetic alterations. It is thought that once epigenetic alterations are established in premalignant tissues, the extent of modifications will accumulate as the disease progresses (Dworkin et al., 2009). Varying theories have been proposed on how this field defect arises. One theory is based on the self-metastasis model and the idea that the primary tumor is composed of multiple selfmetastases that form around a seed from the tumor to itself (Norton, 2005). A second theory has been seen in gastric cancers and is based on cell methylation profiles influencing H. pylori infection which leads to additional methylation of promoters in gastric mucosal cells and accompanying increases in risk for gastric cancer (Maekita et al., 2006). Another theory has supportive evidence in breast cancer and is based on the idea that early epigenetic changes are associated with a large area of pre-malignant changes, and the "epicenter" appears to accumulate additional epigenetic changes (Yan et al., 2006).

Allelic losses of 3p, including a critical region at 3p21.3, are frequently detected in many cancers including breast cancer. The Ras-associated domain family member 1 gene (RASSF1) maps to the region of frequent loss. It is comprised of eight exons and through different promoter usage and alternative splicing generates seven unique transcripts, RASSF1A-G.

Again, cancer generally has been viewed as a disease that is driven by progressive genetic abnormalities, involving chromosomal abnormalities, mutations in oncogenes and tumor suppressor genes (Hanahan & Weinberg, 2000;Vogelstein & Kinzler 2004). Nevertheless, it has been shown that breast cancer, similar to other types of cancer, is also a disease that is driven by epigenetic alterations, which do not affect the primary DNA sequence (Widschwendter & Jones, 2002;Polyak, 2007). The result of these alterations is aberrant transcriptional regulation that leads to a modify in expression patterns of genes implicated in survival, differentiation and cellular proliferation (Baylin & Ohm, 2006;Esteller, 2007;Widschwendter & Jones, 2002). In transformed cells, epigenetic alterations occur at the chromosomal level. These involve changes in DNA methylation, histone modifications, altered expression and function of factors implicated in regulating assembly and remodeling of nucleosomes (Baylin & Ohm, 2006;Esteller, 2007;Jones & Baylin, 2002;Jones & Baylin, 2007;Ting et al., 2006). Alterations in DNA methylation include global hypomethyation and

Global hypomethylation has been found to increase with age and is linked to genomic instability and activation of oncogene expression (Eden et al., 2003;Feinberg & Tycko, 2004;Richardson, 2002). Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. DNA Methylation silencing of tumor suppressor genes, aberrant expression of DNMT1 or demethylation of oncogenes can lead to the conversion of a normal cell to a malignant cell. In addition chromosomal instability and inactivation of the DNA repair system has both the genetic and epigenetic backgrounds (Esteller & Herman, 2002;Szyf, 2008). Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. Hypermethylation of the mismatch repair gene MLH1 is associated with tumors exhibiting microsatellite instability, and hypermethylation of the breast cancer gene BRCA1 is found in

Epigenetic modifications are believed to be early events in cancer development (Leu et al., 2004) and breast cancer is a disease characterized by both genetic and epigenetic alterations. It is thought that once epigenetic alterations are established in premalignant tissues, the extent of modifications will accumulate as the disease progresses (Dworkin et al., 2009). Varying theories have been proposed on how this field defect arises. One theory is based on the self-metastasis model and the idea that the primary tumor is composed of multiple selfmetastases that form around a seed from the tumor to itself (Norton, 2005). A second theory has been seen in gastric cancers and is based on cell methylation profiles influencing H. pylori infection which leads to additional methylation of promoters in gastric mucosal cells and accompanying increases in risk for gastric cancer (Maekita et al., 2006). Another theory has supportive evidence in breast cancer and is based on the idea that early epigenetic changes are associated with a large area of pre-malignant changes, and the "epicenter"

Allelic losses of 3p, including a critical region at 3p21.3, are frequently detected in many cancers including breast cancer. The Ras-associated domain family member 1 gene (RASSF1) maps to the region of frequent loss. It is comprised of eight exons and through different promoter usage and alternative splicing generates seven unique transcripts, RASSF1A-G.

10%- 15% of women with non-familial breast cancer (Jones & Baylin, 2002).

appears to accumulate additional epigenetic changes (Yan et al., 2006).

**9. Epigenetic modifications and breast cancer** 

focal hypermethylation.

RASSF1A is transcribed from a CpG island promoter region, and is one of the most frequently hypermethylated genes thus far described in human cancer. The CpG island of RASSF1A is hypermethylated in 60–77% of breast cancers (Lewis et al., 2005;Vincent-Salomon et al., 2007) resulting in gene silencing in cancer cell lines and primary tissues. Its diverse functions include regulation of apoptosis, growth regulation, and microtubule dynamics during mitotic progression. Specifically, RASSF1A is a Ras effector and induces apoptosis through its interactions with pro-apoptotic kinase MST1. When cells lacking RASSF1A expression are treated with a DNA methyltransferase, such as 5-aza-2′ deoxycytidine, expression can be reactivated (Pfeifer & Dammann, 2005). Mouse knockout studies show that RASSF1A−/− mice are prone to spontaneous development of lung adenomas, lymphomas and breast adenocarcinomas. These mice are prone to early spontaneous tumorigenesis and show a severe tumor susceptibility phenotype compared to that of littermate wild-type mice (Pfeifer & Dammann, 2005).

Furthermore, it has been reported that the DNA methylation assay might be used for risk assessment and prognosis of breast cancer. Lewis et al. studied five frequently methylated genes, including RASSF1A, APC, H-cadherin, RARβ, and cyclin D2, and found a higher methylation frequency of both RASSF1A and APC genes in unaffected women at high risk for breast cancer compared with those at low or intermediate risk based on the Gail model analysis. This suggests that promoter hypermethylation of these genes is associated with epidemiologic markers of increased breast cancer risk (Lewis et al., 2005). This finding needs confirmation that such alterations do indeed occur earlier than abnormal histological findings, and by follow-up studies to examine whether these changes are associated with subsequent development of breast cancer (Lo & Sukumar, 2008). The prognostic significance of aberrant DNA methylation has been investigated by Muller et al. (2003) after screening 39 genes in DNA from serum of normal control patients and patients with primary or metastatic breast cancer, they identified two genes, RASSF1A and APC, whose methylation has a statistically significant association with poor outcome. Other methylated genes, such as GSTP1, SFRP1, have also been identified to be associated with poor prognosis (Arai et al., 2006;Veeck et al., 2006).

In breast cancer, multiple genes are hypermethylated compared to non-cancerous tissue (Agrawal & Murphy, 2007). 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) (Han et al., 2007; Yan et al., 2001; Widschwendter & Jones, 2002). These genes are not only hypermethylated in tumor cells, but show increased epigenetic silencing in normal epithelium surrounding the tumor site. The first observations of this phenomenon were in oral cancer. Slaughter et al (1953) was the first group to use the term "field cancerization" which refers to the presence of cancer causing changes in apparently normal tissue surrounding a neoplasm. They theorized the existence of (pre-) neoplastic processes at multiple sites, with the unproven assumption that these have developed independently (Slaughter et al., 1953). In subsequent years, the presence of field cancerization has been described in head and neck squamous cell carcinoma, lung, esophagus, vulva, cervix, colon, bladder, skin, and breast cancers (Yan et al., 2006). Studies have demonstrated that normal adjacent cells to tumors frequently harbor loss of heterozygosity, microsatellite and chromosome instability, and gene mutations (Braakhuis et al., 2003). Recently DNA methylation has been added to list as hypermethylated normal tissue immediately adjacent to tumor sites has been found (Ushijima, 2007).

Epigenetics and Breast Cancer 303

(Esteller et al., 2000). More recently, 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 tumors (Ai et al., 2006; Lo et al., 2006).). Thus, in addition to the genetic mutation-mediated mechanism, epigenetic gene silencing is another mechanism that fosters malignant transformation of the mammary gland by aberrantly activating oncogenic

There are two main reasons RASSF1A methylation is a good biomarker for breast cancer. First, RASSF1A methylation is rare in normal tissue providing a marker with high specificity. Second, the frequency of methylation is observed in 60 to 77% of cells from a tumor which provides a high frequency of diagnostic coverage (Campan et al., 2006; Muller et al., 2003). In addition to breast tumors, hypermethylation of RASSF1A can be detected in non-malignant breast cells and patient sera. In one study, hypermethylation of sera in breast cancer patients was detected in six out of 26 cases (Pfeifer & Dammann, 2005). Promoter methylation of RASSF1A was observed in 70% of samples from women at high-risk of developing breast cancer versus only 29% of samples from women at low-risk. Women with a previous history of benign breast growths are statistically more likely to have RASSF1A methylation (Lewis et al., 2005). Thus, hypermethylation of RASSFIA could be used as a form of breast cancer screening to detect breast cancer at its earliest stages (Dworkin et al.,

However, it is well reported that prolonged exposure of undifferentiated (immature) breast cells to estrogen or estrogen-mimetic compounds during early development increases breast cancer risk in adult life. This phenomenon is called estrogen imprinting (Fenton, 2006). These studies can explain why, in addition to genetic factors, the risk of breast cancer is affected by pregnancy, lifestyle in terms of intake of food and drink, and environment. Although the tumorigenic mechanism underlying this phenomenon and its connection with epigenetic regulation are still largely unknown, recently published findings provide insight into this mechanism. One line of evidence is from the study of DNA methylation patterns in several subtypes of breast cells. Bloushtain-Qimron et al. found that several transcription factor genes involved in stem cell function were hypomethylated and highly expressed in breast progenitor/stem (undifferentiated) cells compared with differentiated breast epithelial cells (Bloushtain-Qimron et al., 2008), suggesting the epigenetic programs define mammary epithelial cell phenotypes. Since breast progenitor/stem cells possess self-renewal and proliferating ability and more sensitively respond to estrogenic action, this subtype of cells has been thought to be potent targets of malignant transformation (Shipitsin et al., 2007). The second line of evidence is from the study of the effects of estrogen exposure on breast progenitor/stem cells, using a primary culture system to decipher the phenomenon of estrogen imprinting. Recent study compared the DNA methylation profiles of epithelial progeny of estrogen-exposed breast progenitor cells with those of epithelial progeny of nonestrogen-exposed progenitor cells. They found that estrogen exposure caused epithelial progeny to exhibit a cancer-like methylome, leading to silencing of some tumor suppressor genes (Cheng et al., 2008). Even though the dose of estradiol (E2) used in their study was higher than normal physiological levels, their findings suggest abnormal exposure to estrogen or estrogenic chemicals induces epigenetic alterations in breast progenitor cells, which have

been previously implicated in breast cancer (Lo & Sukumar, 2008).

signaling pathways (Lo & Sukumar, 2008).

**10. Breast cancer epigenetic markers** 

2009).

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 (Baylin & Ohm, 2006; Esteller, 2007; Jones & Baylin, 2002; Jones & Baylin, 2007). These genes are selectively hypermethylated in tumorigenesis for inactivation owing to their functional involvement in various cellular pathways that prevent cancer formation. Some of the methylated genes identified in human cancers are classic tumor suppressor genes in which one mutationally inactivated allele is inherited. According to Knudson's two-hit model, complete inactivation of a tumor suppressor gene requires loss-of-function of both gene copies (Knudson, 2000). Epigenetic silencing of the remaining wild-type allele of the tumor suppressor gene, thus, can be considered as the second hit in this model. For example, some well-known tumor suppressor genes, such as p16INK4a, APC and BRCA1, are mutationally inactivated in the germline occasionally lose function of the remaining functional allele in breast epithelial cells through DNA hypermethylation (Birgisdottir et al., 2006; Jin et al., 2001; Knudson, 2000). Since the consequence of aberrant DNA methylation is transcriptional silencing, novel tumor suppressor genes can be identified using methylated CpG islands as a marker.

As a result of that, hypermethylated genes identified from breast neoplasms now form a long list. Their biological functions encompass cell cycle regulation (p16INK4a, p14ARF, 14−3−3σ, cyclin D2, p57KIP2), apoptosis (APC, DAPK1, HIC1, HOXA5, TWIST, TMS1), DNA repair (GSTP1, MGMT, BRCA1), hormone regulation (ERα, PR), cell adhesion and invasion (CDH1, APC, TIMP3), angiogenesis (maspin, THBS1), cellular growth-inhibitory signaling (RARβ, RASSF1A, SYK, TGFβRII, HIN1, NES1, SOCS1, SFRP1 and WIF1). In addition to protein-coding genes, recent studies showed that microRNAs with tumorsuppressor function could be silenced in breast cancer cells through DNA methylation (Lehmann et al., 2008). These breast-genome methylation patterns have been developed as biomarkers for early detection and the classification of subtype of breast tumors, as predictors for risk assessment and for monitoring prognosis, and as indicators of susceptibility or response to therapy (Widschwendter & Jones, 2002;Lo & Sukumar, 2008).

These advances in the knowledge of the breast methylome strongly indicate that DNA hypermethylation plays a crucial role in initiation, promotion and maintenance of breast carcinogenesis, which cooperatively and synergistically interact with other genetic alterations to promote the development of breast cancer. For example, human mammary epithelial cells (HMECs) that gained the ability to emerge from the first transient growth plateau lost p16INK4A expression concurrently with hypermethylation of p16INK4A promoter, indicating that loss of tumor-suppressor function of p16INK4A is required for HMECs to gain growth competency by successfully bypassing the stage of cell senescence (Widschwendter & Jones, 2002; Tlsty et al., 2004). This finding is consistent with other studies where the life span of stem cells could be extended by germline loss of this gene (Janzen et al., 2006). Deregulation of cell cycle control by inhibiting the function of the cyclin-dependent kinase inhibitor, p16INK4A, could create a context for facilitating early abnormal clonal expansion of cells at risk for cancer. It is believed that loss of p16INK4A gene is permissive for enabling such expanding cells to develop genomic instability (Kiyono et al, 1998).

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 tumor suppressor genes or activation of oncogenes, which further drive breast tumorigenesis

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 (Baylin & Ohm, 2006; Esteller, 2007; Jones & Baylin, 2002; Jones & Baylin, 2007). These genes are selectively hypermethylated in tumorigenesis for inactivation owing to their functional involvement in various cellular pathways that prevent cancer formation. Some of the methylated genes identified in human cancers are classic tumor suppressor genes in which one mutationally inactivated allele is inherited. According to Knudson's two-hit model, complete inactivation of a tumor suppressor gene requires loss-of-function of both gene copies (Knudson, 2000). Epigenetic silencing of the remaining wild-type allele of the tumor suppressor gene, thus, can be considered as the second hit in this model. For example, some well-known tumor suppressor genes, such as p16INK4a, APC and BRCA1, are mutationally inactivated in the germline occasionally lose function of the remaining functional allele in breast epithelial cells through DNA hypermethylation (Birgisdottir et al., 2006; Jin et al., 2001; Knudson, 2000). Since the consequence of aberrant DNA methylation is transcriptional silencing, novel

tumor suppressor genes can be identified using methylated CpG islands as a marker.

develop genomic instability (Kiyono et al, 1998).

As a result of that, hypermethylated genes identified from breast neoplasms now form a long list. Their biological functions encompass cell cycle regulation (p16INK4a, p14ARF, 14−3−3σ, cyclin D2, p57KIP2), apoptosis (APC, DAPK1, HIC1, HOXA5, TWIST, TMS1), DNA repair (GSTP1, MGMT, BRCA1), hormone regulation (ERα, PR), cell adhesion and invasion (CDH1, APC, TIMP3), angiogenesis (maspin, THBS1), cellular growth-inhibitory signaling (RARβ, RASSF1A, SYK, TGFβRII, HIN1, NES1, SOCS1, SFRP1 and WIF1). In addition to protein-coding genes, recent studies showed that microRNAs with tumorsuppressor function could be silenced in breast cancer cells through DNA methylation (Lehmann et al., 2008). These breast-genome methylation patterns have been developed as biomarkers for early detection and the classification of subtype of breast tumors, as predictors for risk assessment and for monitoring prognosis, and as indicators of susceptibility or response to therapy (Widschwendter & Jones, 2002;Lo & Sukumar, 2008). These advances in the knowledge of the breast methylome strongly indicate that DNA hypermethylation plays a crucial role in initiation, promotion and maintenance of breast carcinogenesis, which cooperatively and synergistically interact with other genetic alterations to promote the development of breast cancer. For example, human mammary epithelial cells (HMECs) that gained the ability to emerge from the first transient growth plateau lost p16INK4A expression concurrently with hypermethylation of p16INK4A promoter, indicating that loss of tumor-suppressor function of p16INK4A is required for HMECs to gain growth competency by successfully bypassing the stage of cell senescence (Widschwendter & Jones, 2002; Tlsty et al., 2004). This finding is consistent with other studies where the life span of stem cells could be extended by germline loss of this gene (Janzen et al., 2006). Deregulation of cell cycle control by inhibiting the function of the cyclin-dependent kinase inhibitor, p16INK4A, could create a context for facilitating early abnormal clonal expansion of cells at risk for cancer. It is believed that loss of p16INK4A gene is permissive for enabling such expanding cells to

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 tumor suppressor genes or activation of oncogenes, which further drive breast tumorigenesis (Esteller et al., 2000). More recently, 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 tumors (Ai et al., 2006; Lo et al., 2006).). Thus, in addition to the genetic mutation-mediated mechanism, epigenetic gene silencing is another mechanism that fosters malignant transformation of the mammary gland by aberrantly activating oncogenic signaling pathways (Lo & Sukumar, 2008).
