Role of Estrogen Receptor-b Methylation in Breast Cancer

1. Abstract Estrogen and its receptors play an important role in breast carcinogenesis. In humans, there are two subtypes of estrogen receptors (ER), ERa and ERb, both of which are expressed in normal breast and breast cancer. Breast tumorigenesis has been reported to be associated with an increase in ERa and a decrease in ERb (1). Thus, it is hypothesized that ERb plays a role as a tumor suppressor in breast cancer. The mechanism involved in ERb downregulation is poorly understood, however epigenetic changes such as DNA methylation may be involved in gene regulation (2). We (3) demonstrated that loss of ERb expression in prostate cancer was associated with hypermethylation of the ERbON promoter. The goal of this study was to determine if increased CpG island (CGI) methylation in the ERbON promoter correlated with loss of ERb expression in breast cancers. Sections of breast cancer and adjacent normal tissue were stained for ERb expression. Representative breast cancer and adjacent normal foci were microdissected using laser capture microscopy. Bisulfite genomic sequencing was then used to examine the methylation status of all 41 CGIs in the ERbON promoter. Immunocytochemistry data revealed 1) normal epithelial cells expressed high levels of nuclear and cytosolic ERb, 2) well differentiated tumors expressed weak nuclear but significant cytosolic staining, and 3) poorly differentiated tumors had either no or very weak nuclear staining and some cytosolic staining. Bisulfite genomic analyses revealed that well differentiated breast tumors had a lower degree of ERbON promoter CGI methylation in both cancerous and normal cells, whereas, poorly differentiated tumors had a higher degree of cytosine methylation in both cancerous and normal cells. However, an apparent mono-allelic methylation pattern was observed in the normal epithelial cells, whereas bi-allelic methylation seemed to occur in the cancerous cells. These results provide support that hypermethylation of the ERbON promoter may contribute to loss of ERb expression in breast tumorigenesis. Also, it suggests that the ERbON promoter in the normal epithelial cells in poorly differentiated tumors may have already undergone aberrant methylation when compared to well differentiated tumors. The difference in the methylation status of the ER-bON promoter in normal epithelial cells found between the poorly differentiated and well differentiated breast tumors suggest that each may evolve through a separate pathway. References: • Balfe et al Eur J Surg Oncol. 30:1043-1050 (2004).   • Zhao et al Oncogene. 22:7600-7606 (2003).   • Zhu X et al, Am J Pathology 164: 2003-2012, 2004

2. Introduction • Breast cancer is the second leading cause of cancer death in women in the USA • Estrogen and its receptors play important roles in the genesis and malignant progression of breast cancer • Estrogen receptors (ER): • Are members of the nuclear hormone receptor superfamily and act as ligand-activated nuclear transcription factors • The first ER was cloned in 1986 and named ERa • The second ER, named ERb, was identified in 1996 • Estrogen Receptor-b (ERb): • Various isoforms of ERb have been described which are alternatively spliced variants (ERb1-5) • Another level of complexity provided by diversity in the 5’ untranslated region-2 alternatively spliced exons-ON and OK • Breast Cancer: • There appears to be a relative increase in ERa and a decrease in ERb expression in breast cancer compared with normal breast • It is hypothesized that ERb might play a role as a tumor suppressor in breast carcinogenesis • Epigenetic modifications such as DNA methylation could be involved in ERb downregulation • DNA methylation: • Mammalian cells can epigenetically modify their genome via covalent addition of a methyl group to the 5’-position of the cytosine ring • About 3-5% of cytosines in the human genome are methylated • 70-80% of the 5-methylcytosines are located in CpG islands found in the 5’ promoter region and first exon • Methylation of CpG islands is frequently associated with gene silencing

3. CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CG TG CG UG TG TG TG CG UG CG CG CG UG CG UG CG CG CG CG CG CG CG CG CG Protocol For Determining Methylation Status of ERb Promoter/Exon ON Immunohistochemistry to identify ERb status in normal breast and breast cancer Laser Capture Microscopy of representative areas Bisulfite Sequencing DNA Extraction Protocol For Bisulfite Sequencing Methylated CpG Island Unmethylated CpG Island Step 1: Bisulfite treatment Step 2: PCR Step 3: Sequencing

4. Methylation Status of ERb Promoter/Exon ON in Prostate Cancer (Zhu et al, Am J of Path, 2004)

5. Immunohistochemistry of ERb in Breast Tissue Well differentiated Breast Ca-40X Adjacent normal-10X Adjacent normal-40X Poorly differentiated Breast Ca-100X Normal Breast (breast reduction)-40X Normal Breast (breast reduction)-10X

6. Methylation Status of ERb Promoter/Exon ON in Normal Breast Tissue promoter exon ON IHC 10 30 20 40 Case #1 • ERb +++ Case #2 • ERb +++ Case #3 • ERb +++ Breast Tissue from Reduction Mammoplasty Case #4 • ERb +++ Case #5 • ERb +++

7. Methylation Status of ERb Promoter/Exon ON in Breast Cancer and Adjacent Normal promoter exon ON IHC 10 30 20 40 • ERb +++ to ++++ N Case #1 • ERb 95% - • ERa- , PR - C N • ERb +++ Case #2 C • ERb ++ to +++ • ERa- , PR - Poorly Differentiated N • ERb +++ to ++++ Case #3 • ERb 75% +++, 25% - • ERa- , PR - C N • ERb +++ Case #4 • ERb +++ • ERa- , PR - C N • ERb +++ Well Differentiated Case #5 • ERb +++ • ERa +++, PR - C

8. Summary • Normal breast tissue has a different cytosine methylation pattern of promoter/exon ON compared with normal prostate tissue • The methylation pattern suggests that there may be mono-allelic methylation in normal breast tissue • Well differentiated tumors have a lower degree of cytosine methylation of the promoter/exon ON in, which correlates with a higher ERb expression • Poorly differentiated tumors have a higher degree of cytosine methylation of the promoter/exon ON, which correlates with lower ERb expression • The methylation pattern suggests that there may be mono-allelic methylation in adjacent normal breast tissue, whereas bi-allelic methylation seems to occur in the cancerous cells Normal Breast Tissue Luminal cells Myoepithelial cells Stromal cells Poorly Differentiated Breast Cancer Well Differentiated Breast Cancer Adjacent Normal Breast Adjacent Normal Breast Breast Cancer Breast Cancer