Abstract P4-09-10: Epigenetically altered microRNA mediated pathway dysregulation in ER negative breast cancer
Authors: Worsham MJ , Chen KM , Datta I , Stephen JK , Chitale D , Divine G 
Published: May 2016

Background: Micro RNAs (miRNA) are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Compared to mRNA signatures, miRNAs have better and stronger biomarker properties with 20 times more power in biomarker studies as compared to mRNAs (when comparing 20,000 mRNAs to ∼1,000 miRNAs). Emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer, representing a novel class of potential biomarkers for diagnosis, prediction of treatment, or prognosis. ER-negative breast cancer (BC) is an aggressive histological subtype with limited treatment options and very poor prognosis. Our long term objective is to derive a diagnostic, prognostic, and predictive ER-negative specific miRNA panel for detection of early cancer, recurrence/metastasis, and as potential therapeutic targets for better management of ER-negative BC.

Methods: The initial discovery step profiled 39 primary ER negative and 40 ER positive BC cases using the Illumina Infinium HumanMethylation450 BeadChip followed by a subanalysis focusing on 2249 miRNA CpGs assigned to 615 unique miRNAs. T-tests were used to compare the means of the M-values for the ER-positive and ER-negative groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER positive and ER negative of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). Because miRNAs perform their important functions via their targets, the targets of miRNAs were assessed for functional enrichment analysis in IPA for biologic involvement.

Results: Over half of the miRNA CpGs (1224/2249, 54%) were differentially methylated between ER negative and ER positive BC with significant aFDR levels. The 1224 CpGs at Tier 1 were associated with 379 miRNAs; the 24 and 2 CpGs for Tiers 2 and 3 with 22 and 2 miRNAs, respectively. The 22 miRNA genes were assigned to 4621 targets using online databases that predict miRNA targets. The degree of confidence that a target gene is associated with a miRNA is characterized in these databases as either "experimentally observed", or just as "high" (predicted). Of these 4621 targets, 87 were designated as experimentally observed and were examined in IPA. Top pathways and networks designated by miRNA targets included the cell cycle G1/S checkpoint regulation canonical pathway, and the cell-to-cell interaction/cancer networks among others. MiRNA targets in top pathways and networks were circled back to their respective miRNAs revealing cooperatively mediated pathway dysregulation of ER negative BC.

Conclusion: Aberrantly methylated miRNAs showed perturbation of biologically significant pathways and networks, suggesting that miRNAs mediate pathway dysregulation in a coordinated manner, strengthening the case for utility of miRNAs as viable biomarkers in ER negative BC. Support: Komen Foundation: KG110218.

Citation Format: Worsham MJ, Chen KM, Datta I, Stephen JK, Chitale D, Divine G. Epigenetically altered microRNA mediated pathway dysregulation in ER negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-10.