Abstract 2013: microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis

Abstract

Metastases derived from primary tumors are responsible for the high cancer-associated death rates. Lymph nodes near to the primary breast tumor have a high chance to develop a secondary tumor, representing one of the first signs of metastasis. For this reason, inhibition of metastasis should be a major goal for breast cancer treatment. Metastasis is a non-random process that initiates with a phenotypic change of the tumor cells, named Epithelial-Mesenchymal Transition (EMT), and is leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. Because these changes in tumor cells must be plastic and reversible, epigenetic alterations are necessary for EMT. In this sense, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets associated to metastasis. The aim of this study is to identify microRNAs that regulate the expression of EMT-transcription factors in breast cancer tumors, and that are involved in lymph node metastasis. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors, 28 from patients with lymph node metastasis. Transcription factor expression was assayed by immunohistochemistry. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAs down-regulated in breast tumors expressing EMT-transcription factors (p<0.05). Using in silico analysis, we predicted 28 microRNAs to be regulators of SNAIL, SLUG and/or TWIST. Four not previously validated microRNAs were selected: miR-202, miR-210, miR-331 and miR-34b. We used luciferase reporter assays to assess the regulation of the 3´UTR of selected transcription factors by each microRNA, at two different concentrations: 10nM and 50nM. We found that miR-210 caused a 50% decrease in luciferase activity through SNAIL and SLUG 3'UTR when tested at 50nM. miR-331, predicted as regulator of SLUG 3'UTR, decreased luciferase activity at both evaluated concentrations in a dose-dependent manner, in a 20% and a 37%. In the case of TWIST, miR-34b at 50nM, decreased luciferase activity in a 65%. No inhibition of luciferase activity was found with mir202. In addition to transcription factors, two of these microRNAs have been predicted as regulators of chemokine receptors CXCR4 and CCR7. Both receptors direct lymphocyte migration to lymphoid organs, and their role has been also implicated in cancer. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of primary breast tumor cells, promoting invasion and colonization of lymph nodes. In this sense, changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients. Citation Format: Elisa Pérez-Moreno, Valentina Zavala, Gabriela Valarezo, Wanda Fernández, Pilar Carvallo. microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2013.