**10. Breast cancer epigenetic markers**

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., 2009).

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).

Epigenetics and Breast Cancer 305

showed neither LOH nor promoter methylation (Press et al., 2008). Another study of 47 breast tumors from hereditary breast cancer families identified three BRCA1 carriers of which two showed BRCA1 promoter methylation in their tumors (Birgisdottir et al., 2006). All these investigated studies suggest that methylation of BRCA1 may be serve as a second

Furthermore, 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 (Xu et al., 2008). However, they also found a higher prevalence of BRCA1 promoter methylation in cases with at least one node involved and with tumor size greater than 2cm. Based on their findings higher methylation levels may correlate with more advanced tumor stage at diagnosis. They also observed a 45% increase in mortality of individuals with BRCA1 methylation positive tumors compared those who had unmethylated BRCA1 promoters (Xu et al., 2008). Another recent 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 tumors from BRCA1 mutation negative families. However, seven individuals had both promoter hypermethylation and LOH; the majority of these tumors had a basal-like phenotype and

Moreover, much of the research effort to date has concentrated on the identification of silenced genes implicated in breast tumorigenesis. Evron et al. successfully used a three-gene panel (Cyclin D2, RARβ and TWIST) to detect malignant breast cancer cells in ductal fluid from routine operative breast endoscopy (ROBE) and ductal lavage (Evron et al., 2001). Fackler et al. improved this method and tested a four-gene panel (RASSF1A, TWIST, HIN1 and Cyclin D2) using the QM-MSP assay to examine clinical tissue samples (Fackler et al., 2004). The cumulative methylation of these four genes is commonly observed to be higher in primary invasive breast cancers compared with reduction mammoplasty specimens from healthy women (Fackler et al., 2004). Fackler et al. further used the same technique but adopted a ninegene panel (RASSF1A, TWIST, HIN1, Cyclin D2, RARβ, APC, BRCA1, BRCA2 and p16) to examine ductal lavage samples from women with or without breast cancer. This trial demonstrated that methylation-marker detection was twice as sensitive as cytological diagnosis of ductal lavage cells (Fackler et al., 2006). In addition to biopsied tissue sections and ductal fluid, methylated DNA is also detected in blood since the blood of patients with manifest breast cancer contains detectable amounts of circulating methylated DNA (Widschwendte & Menon, 2006). The blood detection of tumor-specific methylated DNA has been pursued for its potential for prognostic prediction and monitoring relapse of breast cancer after therapy (Widschwendte & Menon, 2006; Muller et al., 2003; Silva et al., 2002). The analysis of methylation profiles in human cancer indicates that hypermethylation of some of the CpG islands is shared by multiple tumour types, whereas others are methylated in a tumour type-specific manner (Bae et al., 2004; Costello et al., 2000; Esteller et al., 2001; Nass et al., 2000; Parrella et al., 2004; Parrella, 2010). Promoter-aberrant methylation seems to be an early event in tumorigenesis, and an increase in the number of methylated genes during progression has been observed in several tumour types including breast cancer (Lehmann et al., 2002; Subramaniam et al., 2009). Hoque et al (2009) have shown there were differences in the patterns of methylation in pre-invasive breast lesions (atypical ductal hyperplesia and

hit in tumors from a subset of BRCA1 mutation carriers (Dworkin et al., 2009).

were triple negative (Honrado et al., 2007).

**11. Analysis of DNA methylation in breast cancer** 

Even though the aberrant activation of estrogen signaling can lead to tumor-associated alterations in the epigenome of breast progenitor cells, approximately 30% of diagnosed breast cancer cases lack estrogen signaling due to loss or downregulation of estrogen receptor (ER)-α, also subject to epigenetic silencing (Lapidus et al., 1998; Ottaviano et al., 1994). ER-negative breast cancers exhibit more aggressive characteristics than ER-positive breast cancers and are resistant to anti-estrogen therapy. How ER-negative breast cancer cells acquire more aggressive properties after loss of estrogen signaling is a very important issue in the field of breast cancer research. Another study provides evidence to link loss of ER signaling to epigenetic silencing of ERα downstream target genes (Leu et al., 2004). Their study showed that abrogation of ERα signaling by small interfering RNA-mediated knockdown of ERα expression resulted in epigenetic inactivation of ERα targets, which began from recruiting PcG repressors and HDACs to their promoters and was then progressively followed by DNA methylation of their promoters (Leu et al., 2004). Their results suggest that epigenetic regulation on ERα target genes is required for establishing ERα-independent growth and other characteristics of ER-negative breast cancer cells (Lo & Sukumar, 2008).

Other post-translational modifications of ERα such as phosphorylation, ubiquitination, glycosylation, and acetylation are believed to play a role in breast cancer promotion. ERα is modified by p300 on two lysine residues (302 and 303) located in the hinge region (between DNA- and ligand binding domains). When these lysine residues are mutated, ERα had increased hormone sensitivity. Thirty-four percent of atypical breast hyperplasia samples have mutations of the lysine at 303 (K303R) of the ERα (Margueron et al., 2004; Popov et al., 2007; Wang et al., 2001) explaining a functional role of these mutations in breast cancer promotion.

Furthermore, BRCA1 is a tumor suppressor gene for both breast and ovarian cancer (Campan et al., 2006). It encodes a multifunctional protein with roles in DNA repair, cell cycle check point control, protein ubiquitization, and chromatin remodeling (Mirza et al., 2007). In vitro experiments showed that decreased BRCA1 expression in cells led to increased levels of tumor growth, while increased expression of BRCA1 led to growth arrest and apoptosis. Recent studies indicate that BRCA1 methylation is an important marker for prognosis. The magnitude of the decrease of functional BRCA1 protein correlates with disease prognosis (Mirza et al., 2007; Vincent-Salomon et al., 2007). Tumors with BRCA1 mutations are usually more likely to be higher-grade, poorly differentiated, highly proliferative, estrogen receptor (ER) negative, and progesterone receptor (PR) negative, and harbor p53 mutations. BRCA1 mutated breast cancers are also associated with poor survival in some studies (Chappuis et al., 2000; Robson et al., 2004; Stoppa-Lyonnet et al., 2000). Phenotypically, BRCA1-methylated tumors are similar to tumors from carriers of germline BRCA1 mutations.

BRCA1 is thought to be a classical tumor suppressor gene for which Knudson's two-hit hypothesis holds true. About 20% of individuals with a strong personal and family history of breast and ovarian cancer carry germline mutations in the BRCA1 gene (Birgisdottir et al., 2006; Tapia et al., 2008). A second hit is thought to be required in the wild-type BRCA1 allele for the development of BRCA-associated cancer (Chenevix-Trench et al., 2006; Osorio et al., 2002; Osorio et al., 2007). However, about 20% of all tumors from BRCA mutation carriers do not show LOH of the wildtype BRCA1 (Chenevix-Trench et al., 2006; Meric-Bernstam, 2007; Osorio et al., 2002; Osorio et al., 2007).). Other studies have looked at the rate of BRCA1 methylation in germline carriers. BRCA1 promoter hypermethylation was observed in one of two tumors from BRCA1 carriers lacking LOH (Esteller et al., 2001). In other study of population-based ovarian tumors, two of eight tumors with germline BRCA1 mutations

Even though the aberrant activation of estrogen signaling can lead to tumor-associated alterations in the epigenome of breast progenitor cells, approximately 30% of diagnosed breast cancer cases lack estrogen signaling due to loss or downregulation of estrogen receptor (ER)-α, also subject to epigenetic silencing (Lapidus et al., 1998; Ottaviano et al., 1994). ER-negative breast cancers exhibit more aggressive characteristics than ER-positive breast cancers and are resistant to anti-estrogen therapy. How ER-negative breast cancer cells acquire more aggressive properties after loss of estrogen signaling is a very important issue in the field of breast cancer research. Another study provides evidence to link loss of ER signaling to epigenetic silencing of ERα downstream target genes (Leu et al., 2004). Their study showed that abrogation of ERα signaling by small interfering RNA-mediated knockdown of ERα expression resulted in epigenetic inactivation of ERα targets, which began from recruiting PcG repressors and HDACs to their promoters and was then progressively followed by DNA methylation of their promoters (Leu et al., 2004). Their results suggest that epigenetic regulation on ERα target genes is required for establishing ERα-independent growth and other characteristics of ER-negative breast cancer cells (Lo &

Other post-translational modifications of ERα such as phosphorylation, ubiquitination, glycosylation, and acetylation are believed to play a role in breast cancer promotion. ERα is modified by p300 on two lysine residues (302 and 303) located in the hinge region (between DNA- and ligand binding domains). When these lysine residues are mutated, ERα had increased hormone sensitivity. Thirty-four percent of atypical breast hyperplasia samples have mutations of the lysine at 303 (K303R) of the ERα (Margueron et al., 2004; Popov et al., 2007; Wang et al., 2001) explaining a functional role of these mutations in breast cancer promotion. Furthermore, BRCA1 is a tumor suppressor gene for both breast and ovarian cancer (Campan et al., 2006). It encodes a multifunctional protein with roles in DNA repair, cell cycle check point control, protein ubiquitization, and chromatin remodeling (Mirza et al., 2007). In vitro experiments showed that decreased BRCA1 expression in cells led to increased levels of tumor growth, while increased expression of BRCA1 led to growth arrest and apoptosis. Recent studies indicate that BRCA1 methylation is an important marker for prognosis. The magnitude of the decrease of functional BRCA1 protein correlates with disease prognosis (Mirza et al., 2007; Vincent-Salomon et al., 2007). Tumors with BRCA1 mutations are usually more likely to be higher-grade, poorly differentiated, highly proliferative, estrogen receptor (ER) negative, and progesterone receptor (PR) negative, and harbor p53 mutations. BRCA1 mutated breast cancers are also associated with poor survival in some studies (Chappuis et al., 2000; Robson et al., 2004; Stoppa-Lyonnet et al., 2000). Phenotypically, BRCA1-methylated tumors are

BRCA1 is thought to be a classical tumor suppressor gene for which Knudson's two-hit hypothesis holds true. About 20% of individuals with a strong personal and family history of breast and ovarian cancer carry germline mutations in the BRCA1 gene (Birgisdottir et al., 2006; Tapia et al., 2008). A second hit is thought to be required in the wild-type BRCA1 allele for the development of BRCA-associated cancer (Chenevix-Trench et al., 2006; Osorio et al., 2002; Osorio et al., 2007). However, about 20% of all tumors from BRCA mutation carriers do not show LOH of the wildtype BRCA1 (Chenevix-Trench et al., 2006; Meric-Bernstam, 2007; Osorio et al., 2002; Osorio et al., 2007).). Other studies have looked at the rate of BRCA1 methylation in germline carriers. BRCA1 promoter hypermethylation was observed in one of two tumors from BRCA1 carriers lacking LOH (Esteller et al., 2001). In other study of population-based ovarian tumors, two of eight tumors with germline BRCA1 mutations

similar to tumors from carriers of germline BRCA1 mutations.

Sukumar, 2008).

showed neither LOH nor promoter methylation (Press et al., 2008). Another study of 47 breast tumors from hereditary breast cancer families identified three BRCA1 carriers of which two showed BRCA1 promoter methylation in their tumors (Birgisdottir et al., 2006). All these investigated studies suggest that methylation of BRCA1 may be serve as a second hit in tumors from a subset of BRCA1 mutation carriers (Dworkin et al., 2009).

Furthermore, 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 (Xu et al., 2008). However, they also found a higher prevalence of BRCA1 promoter methylation in cases with at least one node involved and with tumor size greater than 2cm. Based on their findings higher methylation levels may correlate with more advanced tumor stage at diagnosis. They also observed a 45% increase in mortality of individuals with BRCA1 methylation positive tumors compared those who had unmethylated BRCA1 promoters (Xu et al., 2008). Another recent 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 tumors from BRCA1 mutation negative families. However, seven individuals had both promoter hypermethylation and LOH; the majority of these tumors had a basal-like phenotype and were triple negative (Honrado et al., 2007).
