Abstract 4302: Human endometrial cancer cell CD133+ cell fractions are regulated by methylation

Like other solid tumors, endometrial tumors have been shown to contain a subset of tumor initiating cells although little is known about how these rare sub-fractions are regulated. Our primary objective was to analyze potential epigenetic regulation of such a tumor initiating cell population in human endometrial cancer cells. To accomplish this, we demonstrated by flow cytometry that primary endometrial tumors contain CD133+ cells. To assess their tumor initiating capacity, serially transplanted endometrial human tumor explants generated in NOD/SCID mice were harvested, enzymatically dissociated, depleted of H-2Kd+ mouse cells and sorted via flow cytometry to generate relatively pure (> 98.8 %) CD133+ and CD133− fractions. These positive and negative fractions were serially diluted and subcutaneously injected into immunocompromised mice. The CD133+ fractions had a significantly increased capacity for tumor formation relative to the CD133− fractions and this difference was more pronounced as the number of injected cells decreased. Interestingly, the level of CD133+ tumor cells appeared to be enriched following serial transplantation as evidenced by flow cytometric and immunohistochemical analyses. It has been proposed that methylation may play a role in regulation of tumor initiating cells. To investigate this possibility in endometrial cancer, we isolated DNA from serially transplanted tumors and analyzed the methylation status of CpG islands located upstream of the CD133 transcription start site. This region was shown to be a target of methylation, which led us to determine whether changing the methylation status would alter CD133 expression in endometrial cancer cells. We treated 4 individual human endometrial cancer cell lines with either vehicle or 5 μM 5-aza-2′-deoxyctidine (5-aza-DC), for 72 hours, and evaluated post-treatment levels of CD133 expression by RT-PCR and flow cytometry. In the tested cell lines, CD133 mRNA levels, as measured by RT-PCR, were increased following treatment with 5-aza-DC suggesting that methylation of the CD133 promoter was suppressing its expression. To extend this finding, we analyzed the percentage of CD133 expressing cells in either vehicle treated or 5-aza-DC by flow cytometry. Consistent with the RT-PCR results, the frequency of CD133-expressing cells was increased in 3 of the 4 cell lines following treatment with 5-aza-DC. It is not clear however, if the 5-aza-DC mediated demethylation and subsequent shift in the percentage of CD133+ cells correlates with a shift in the frequency of cells that have increased tumor initiating capacity. Nevertheless, CD133 expression in human endometrial cancer cells is regulated at least in part by methylation and altering this may cause these tumor cells to become more sensitive to standard chemotherapy regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4302.