1. Estrogen and its receptors play an important role in breast carcinogenesis. In humans, there are two subtypes of estrogen receptors (ER), ER  and ER , both of which are expressed in normal breast and breast cancer. Breast tumorigenesis has been reported to be associated with an increase in ER  and a decrease in ER  (1). Thus, it is hypothesized that ER  plays a role as a tumor suppressor in breast cancer. The mechanism involved in ER  downregulation is poorly understood, however epigenetic changes such as DNA methylation may be involved in gene regulation (2). We (3) demonstrated that loss of ER  expression in prostate cancer was associated with hypermethylation of the ER  ON promoter. The goal of this study was to determine if increased CpG island (CGI) methylation in the ER  ON promoter correlated with loss of ER  expression in breast cancers. Sections of breast cancer and adjacent normal tissue were stained for ER  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 ER  ON promoter. Immunocytochemistry data revealed 1) normal epithelial cells expressed high levels of nuclear and cytosolic ER , 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 ER  ON 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 ER  ON promoter may contribute to loss of ER  expression in breast tumorigenesis. Also, it suggests that the ER  ON 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-  ON 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: 1.Balfe et al Eur J Surg Oncol. 30:1043-1050 (2004). 2.Zhao et al Oncogene. 22:7600-7606 (2003). 3.Zhu X et al, Am J Pathology 164: 2003-2012, 2004 Abstract

2. 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 ER  –The second ER, named ER , was identified in 1996 Estrogen Receptor-  (ER  ): –Various isoforms of ER  have been described which are alternatively spliced variants (ER  1-5) –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 ER  and a decrease in ER  expression in breast cancer compared with normal breast – It is hypothesized that ER  might play a role as a tumor suppressor in breast carcinogenesis – Epigenetic modifications such as DNA methylation could be involved in ER  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 Introduction

3. Immunohistochemistry to identify ER  status in normal breast and breast cancer Laser Capture Microscopy of representative areas DNA Extraction Bisulfite Sequencing Protocol For Determining Methylation Status of ER  Promoter/Exon ON Methylated CpG Island Unmethylated CpG Island CH 3 CG CH 3 CG CH 3 CG CH 3 CG CGCGCGCGCGCG CGCG CH 3 CG CH 3 CG CH 3 CG CH 3 CG TGTGTGTGTGTG TGTG UGUGUGUGUGUG UGUG Step 1: Bisulfite treatment treatment Step 2: PCR Step 3: Sequencing Protocol For Bisulfite Sequencing

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

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

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

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

8. 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 ER  expression Poorly differentiated tumors have a higher degree of cytosine methylation of the promoter/exon ON, which correlates with lower ER  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 Summary Normal Breast Tissue Luminal cells Myoepithelial cells Stromal cells Breast Cancer Well Differentiated Breast CancerPoorly Differentiated Breast Cancer Adjacent Normal Breast Breast Cancer