Abstract C22: Next-generation sequencing reveals CpG island shore methylation regulates caveolin-1 expression in breast cancer
Authors: Rao Xi , Evans Jared , Chae Heejoon , Rhee Je-Keun , Kim Sun , Huang Tim H-M , Nephew Kenneth P. 
Published: June 2012

Caveolin-1 (Cav1), a multifunctional membrane protein associated with cell surface caveolae, regulates caveolae-dependent lipid trafficking, vesicular transport and signal transduction. While reported to function as a tumor suppressor at early stages of cancer progression, a growing body of evidence indicates that Cav1 also performs a tumor-promoting role in various cancer types. Cav1 expression level is decreased in breast carcinomas compared to normal tissue, and loss of Cav1 expression promotes mammary tumorigenesis and is associated with the development of tamoxifen resistance. However, gene expression profiling of breast cancer cell lines revealed an inverse relationship between expression of Cav1 and estrogen receptor-alpha (ERα), and Cav1 overexpression was observed in a subset of basal-like breast carcinomas. To investigate the mechanism regulating Cav1 expression, we performed genome-wide profiling of DNA methylation using affinity purification and subsequent next-generation sequencing of eluted DNA (MethylCap-seq) on 30 breast cancer cell lines. In breast cancer cell lines displaying low Cav1 expression (“Cav1-low;” n=15 of primarily luminal subtype), Cav1 promoter CpG island (CGI) hypermethylation was observed in only four cell lines. In the remaining Cav1-low lines, dense methylation at “CGI shores” (methylation at regions outside of CGI) was seen, and Cav1 promoter CGI methylation was strikingly low or absent. In “Cav1-high” cell lines (n=15 of basal-like subtype), hypomethylation of both promoter CGI and CGI shores was observed. By integrating the methylome data with Cav1 mRNA expression profiles, we found a significant negative correlation between Cav1 CGI shore methylation and Cav1 gene expression. Treatment of Cav1-low cells with the DNA methyltransferase inhibitor 5-aza-deoxycytidine resulted in demethylation of CGI shores and Cav1 re-expression, further confirming the regulation of Cav1 expression by CGI shore methylation. MethylCap-seq results were validated by pyrosequencing, and we observed a similar phenomenon in a panel of antiestrogen-sensitive and -resistant cell lines previously generated by our lab, with hypermethylation at CGI shores for a Cav1-low, tamoxifen-resistant line, and CGI shore hypomethylation in Cav1-high, fulvestrant-resistant lines (basal-like). SiRNA-mediated Cav1 knockdown resulted in a significant cell growth inhibition, indicating an important role of Cav1 in supporting breast cancer cell proliferation. Furthermore, by using an on-line survival analysis tool (Kaplan Meier Plotter), we determined a significant positive correlation between Cav1 levels and poor overall survival for patients with ERα-negative breast tumors. The results of this study demonstrate that DNA methylation at Cav1 CGI shores is associated with tumor progression, displaying hypomethylation in normal breast epithelial cells, hypermethylation in luminal breast cancer cells, and hypomethylation in basal-like breast cancer cells. In addition, Cav1 CGI shore methylation may represent a novel prognostic factor for ERα-negative, fulvestrant-resistant breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C22.