Abstract P6-05-05: Epigenetic regulation of ER through miR-18a shows ECM activation and identifies poor prognostic subtype within ER+ve HER2-ve breast cancer

Abstract

Introduction Although endocrine therapy has improved the survival of patients with hormone receptor (HR) positive breast cancer considerably, patients with weaker HR expression tend to have poorer outcomes. There exists significant intra tumoral heterogeneity of ER expression and a recent study suggested that patients with high intra tumoral heterogeneity of ER had an increased long term risk of recurrence (Lindstrom et al JNCI 2018). Multiple reports have supported a role for alterations in microRNA levels having a role in promoting tumor invasion and metastasis. In this study we have explored the epigenetic regulation of ER by microRNA hsa-miR-18a-5p and its role as a prognostic marker in ER positive tumors. Methods 123 surgically excised specimens of ER positive primary breast cancers were analyzed. Samples were segregated into high and low ER positive groups using 50% positivity as the cut-off. Relative abundance of hsa-miR-18a-5p in these samples was assessed using a TaqMan qRT-PCR. hsa-miR-18a-5p was over-expressed in MCF7 cells using a synthetic mimic and downstream changes in gene and protein expression were assessed using q-RT PCR, immunofluorescence and western blot. Migratory and proliferative ability was assessed using wound healing and MTT assay respectively. Microarray based global gene expression analysis of hsa-miR-18a-5p over-expressing cells was performed. Disease free survival analysis was done using Kaplan-Meier survival analysis. Result We estimated the relative abundance of hsa-miR-18a-5p in 123 ER positive primary breast cancers and found the distribution of this miRNA to be highest in the lower ERgroup (p<0.05). hsa-miR-18a-5p also correlated negatively with ESR1 and PGR (p<0.05).To further probe the role of miR-18a-5p in invasion and metastasis, we over-expressed this microRNA using a synthetic mimic in a luminal cell line MCF7. Microarray analysis revealed an increase in the expression of ECM associated genes, and the Cadherin signalling pathway. We observed a decrease in the expression of luminal genes (PGR, TFF1, GREB1; p<0.05), loss of Tff1 and increase in the levels of basal cytokeratin 14 at the protein level in addition to an increase in proliferative rate of upto 35% (p<0.05). Further, we observed a 15 % (p<0.05) increase in the invasive ability by wound healing assay with a significant loss in the levels of E-cadherin protein. In order to study the prognostic importance of hsa-miR-18a-5p, we performed Kaplan-Meier survival analysis and found that stratification of the ER+ve tumor samples by hsa-miR-18a-5p levels produced significant separation of the groups based on disease-free survival (log rank p <0.05). The prognostic value was also validated with multivariate Cox-proportional hazard analysis (Hazard Ratio (HR)of 3.18 (1.07-9.42); p=0.03). Conclusion The results from the over-expression of miR-18a-5p in MCF7 cells support the existence of an epigenetic pathway of repression of the luminal phenotype and increased acquisition of traits associated with basal-like and mesenchymal characteristics. It is possible that the over-expression of miR-18a-5p plays a role in patients with poorer outcomes. Citation Format: Nair MG, Prabhu JS, Korlimarla A, Hari P, Rajarajan S, Kaul R, Alexander A, Raghavan RS, Rakesh S, Correa M, Prasad M, Srinath B, Tirumalai SS. Epigenetic regulation of ER through miR-18a shows ECM activation and identifies poor prognostic subtype within ER+ve HER2-ve breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-05.