Abstract P5-03-07: CD24 epigenetic regulation in breast cancer tissues and tumor initiating cells: Promoter specific analysis using next generation sequencing

Abstract

Abstract CD24 negativity together with a positive expression of CD44 is considered as a marker for breast tumor initiating cells (breast cancer stem cells). We have previously reported that estrogen dynamically down regulate CD24 expression through an estrogen response element (ERE) present in the CD24 distal promoter. Further analysis of clinical breast cancer tissues suggested a frequent loss of CD24 expression in invasive breast cancers compared to the adjacent carcinoma in situ. Also, treatment with 5-AzaC could reverse the CD24 expression in many CD24 negative breast cancer cell lines. In order to understand the epigenetic regulation of CD24 expression and its correlation to hormonal status, the proximal promoter hypermethylation of CD24 gene was analyzed by Next Generation Sequencing (NGS) in DNA from clinical samples and established cell lines from breast cancer patients. DNA from blood, non-cancerous, and cancerous tissues from 73 breast cancer patients were used. There were approximately equal number of estrogen receptor alpha (ERa) positive and negative patients. DNA was also obtained from CD44+/CD24- and CD44+/CD24+ subpopulation FACS sorted from MCF7, SUM159 and MDA-MB231 breast cancer cell lines. CD24 proximal promoter region was PCR amplified from bisulfite converted DNA and prevalence of individual CpG methylation was quantified by NGS. The methylation pattern of individual CpGs suggested that CpGs close to the CD24 transcription start site was more methylated compared to the distant CpGs. Promoter methylation was significantly higher in breast cancer tissues compared to blood DNA. Analysis of clinical parameters showed a significantly higher methylation in ERα positive tumors compared to the negative tumors suggesting a hormonal regulation in CD24 epigenetic silencing. Analysis of ONCOMINE data confirmed this lower CD24 expression in ERα positive tumors. Importantly, analysis of sorted subpopulation from established breast cancer cell lines suggested that the CD24 epigenetic silencing is the major regulatory mechanism in maintaining low CD44+/CD24- subpopulation in cancer cell lines. Interestingly, though MCF7 cell lines have high CD24 expression with mostly unmethylated CD24 promoter, it contain less than 1% CD44+/CD24- subpopulation with stem cell like phenotype. NGS analysis discovered hypermethylation (>90%) of CD24 proximal promoter in that stem cell like subpopulation. Functional studies like mammosphere formation assay suggested that stem cell properties are higher in subpopulation with CD24 proximal promoter hypermethylation. Altogether, our results suggest that CD24 proximal promoter methylation maybe a hormonally regulated mechanism and epigenetic regulation of CD24 is important in maintaining the phenotype of the low prevalent breast tumor initiating population. Current studies are in progress to further understand the clinical and therapeutic significance of this regulation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-07.