Abstract 5240: Epigenetic dysregulation of miR-196b in breast cancer
Authors: Rebecca T. Marquez, Amber R. Smith, Bryan Tsao, Liang Xu
Journal: Cancer Research (2014)

Abstract Dysregulation of microRNA (miRNA) expression levels is a major cause of cancer development. miRNA expression profiles can determine tumor type, patient prognosis, reoccurrence and treatment outcome, with greater accuracy as compared to mRNA expression profiles. The expression of miRNAs can be epigenetically dysregulated by aberrant DNA methylation and histone deacetylation in breast cancer cells. Furthermore, one third of miRNA promoters are targets for aberrant methylation in breast cancer. Identifying miRNAs that are epigenetically silenced in breast cancer will provide new insight into breast cancer development as well as define novel signatures for predicting therapeutic response to epigenetic modifying agents. New studies have shown that using transient low doses of demethylating agents, such as decitabine (DAC), result in sustained changes in major cancer cell signaling pathways, therefore reducing tumorigenicity and cancer stem cell self-renewal. The goal of this project is to identify epigenetically-silenced miRNAs that can be reactivated upon low dose treatment of DAC. miR-196b has been shown to inhibit metastasis by targeting HOXC8. Using online datasets, we determined miR-196b is hypermethylated in breast cancer patient samples compared to normal breast samples. We used Taqman miRNA assays to measure the expression of miR-196b in breast cancer patient samples and found it was down-regulated in tumor samples compared to paired normal breast tissue. miR-196b is also down-regulated in MDA-MB-231 cells. We treated MDA-MB-231 cells with low dose (100 nM) DAC for 3 days and found miR-196b was upregulated 5-fold compared to DMSO control. The methylated CpG content of miR-196b-associated CpG island was measured using bisulfite sequencing DNA purified from DMSO and DAC treated cells. The miR-196b CpG loci in the DAC treatment was demethylated by 33% compared to DMSO control. Therefore, reactivation of miR-196b is associated with demethylation of miR-196b CpG loci. Interestingly, fourteen days after ending DAC treatment, miR-196b re-expression continued to increase up to 13-fold above DMSO control. In addition, we found that exogenously re-introduced mature miR-196b into MDA-MB-231 breast cancer cells resulted in upregulation of endogenous primary miR-196b. This suggests a novel positive feedback loop that allows miR-196b to regulate its own expression, possibly through epigenetic mechanisms. Future studies will involve identifying the mechanism associated with this re-expression, determining whether miR-196b regulates additional gene promoters, and the affect this has on breast cancer metastatic phenotype. Citation Format: Rebecca T. Marquez, Amber R. Smith, Bryan Tsao, Liang Xu. Epigenetic dysregulation of miR-196b in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2014-5240