Study On The Epigenetic Regulation Of Progesterone Receptor In Endometrial Cancer Cells-chromatin Composition Alterations Associated With DNA Methylation And Expression Silencing

Background:Endometrial carcinoma is the most common gynecologic malignancy threatening many women's health and lives. It is noteworthy that results from some studies indicated while the incidence rate appears to be stable, the death rate is rising. The challenge we are facing is how to increase the survival rate and extend the life of patients. The therapeutic value of progestational treatment for endometrial hyperplasia and early stage cancers has been well recognized. However, in advanced and recurrent cancers the progesterone receptor is often lost, leading to a decreased sensitivity to progestational therapy. Therefore how to improve the efficacy of progestins for treating advanced and recurrent cancers has become an important topic. While aberrant DNA methylation of a CpG island in PR-B promoter is considered the major cause for PR negativity, the epigenetic mechanism has not been not well understood. It is not clear if DNA methylation play a decisive role, and what occurs in the chromatin during the epigenetic silencing of progesterone receptor gene. Better understanding of the pathway will not only help us to improve the treatment of endometrial cancer, but also help us to understand the epigenetic mechanism controlling gene expression.Objective:In this study, we determine the specific changes in chromatin composition that are associated with the PR-B transcriptional silencing in endometrial cancer cells. We performed for parts of studies:1) to confirm the PR-B expression status in several endometrial cancer cell lines and observe the reactivation of PR-B transcription by DAN methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors.2) To determine the relationship between PR-B expression and epigenetic silencing. Using chromatin immunoprecipitation assay, we compared the methyl CpG-binding proteins (MBD) binding and histone modification alterations among cells with different PR-B methylation and expression status, as well as cells before and after treatment with DNA methyltransferase or histone deacetylase inhibitors.3) To observe the relationship between MBD binding/histone modification PR-B expression.4) To further investigate the role of MeCP2 in the expression control of PR-B. Methods:We first determined the PR expression status in 18 different endometrial cancer cell lines, and chose PR-B negative and positive cell lines for further analysis. We applied methylation specific PCR to determine the methylation status of PR-B in these cells. Chromatin Immumoprecipitation (ChIP) assay was used to examine the MBD binding and histone posttranslational modification in the PR-B chromatin from different cells with different PR-B expression patterns, or cells before and after drug treatment.Results:We have found methyl CpG-binding proteins, including MeCP2, MBD1, MBD2, MBD3, and MBD4 exhibit diverged binding profiles for methylated/silenced and unmethylated/active progesterone receptor gene. In addition, treatment with DNA methyltransferase and histone deacetylase inhibitors causes dynamic changes in both MBD binding and the histone modifications within chromatins associated with progesterone receptor gene.1) In KLE and HEC-1B cells with methylated PR-B gene, MeCP2 was found to bind to PR-B promoter. In contrast, no MeCP2 binding was observed in the Ishikawa cells in which PR-B gene is unmethylated.2) MBD1 and MBD2 bind to both methylted and unmethylated PR-B gene. ADC treatment induced the MBD1 binding of methylated, but not unmethylated PR-B promoters. The same treatment induced MBD2 binding in KLE cells.3) No change was found in the binding of MBD3 and MBD4.4) TSA treatment did not lead to alteration in MBD binding. Therefore, different MBD family members display varied binding pattern and dynamics upon their binding to PR-B gene.We also used the ChIP assay to examine the H3 and H4 acetylation and methylation at K4 and K9A positions. We found that 1) H3 and H4 acetylation is at their low levels in methylated PR-B promoters when compared to unmethylated promoter.2) ADC and TSA significantly affect the H3 and H4 acetylation levels.3) H3-K9 methylation level in the methylated PR-B is higher than in unmethylated PR-B. The H3 K4 methylation showed an opposite pattern.4) ADC and TSA treatment of cells led to decreased methylation of K9 but increased methylation in K4 position.5) In contrast to the findings in the methylated PR-B gene, ADC and TSA treatment had no significant effect on the histone acetylation and methylation in the unmethylated PR-B gene. Conclusions:We found that the low levels of histone H3 K4 methylation and high levels of K9 methylation is the characteristics of the methylated and silenced PR-B promoter. Treatment of PR-B negative cells with DNA methyltransferase and histone deacetylase inhibitors resulted in reversal of the MeCP2 occupancy and histone covalent modifications, and reactivation of the silenced PR-B gene. Importantly, Western blot analysis showed that MBD expression levels remain unchanged following treatment with DNA methyltransferase and histone deacetylase inhibitors. Thus, the observed changes in the chromatin composition are not caused by global drug effects, rather, they are most likely mediated by specific action associated with local changes on PR-B gene. To further confirm the important role of MeCP2 in epigenetic regulation of PR-B, we performed siRNA-mediated MeCP2 knockdown and measured the MeCP2 binding to PR-B promoter and PR-B mRNA levels. These experiments demonstrated that MeCP2 binding to methylated PR-B gene is critical for PR-B silencing.These findings have for the first time, provided new insights into the epigenetic mechanism leading to PR-B silencing in endometrial cancer. The information will be useful for the design epigenetic interference for reaction of PR-B, and enhancement of the sensitivity of endometrial cancer cells to hormonal therapy. For example, we may be able to more efficiently kill the cancer cells by pretreatment of cancer patients with DNMT and HDAC inhibitors. Since the advanced and recurrent cancer cause most of deaths of patient and many of these cancers are R-B negative, this strategy appears to be highly attractive. Using the current data, we will be able to design animal model to test the efficacy of this new treatment modality.