Abstract A72: Exploring the role of miR-223 loss in mammary epithelial cell transformation
Authors: Citron Francesca , Segatto Ilenia , Vinciguerra Gian Luca Rampioni , Baldassarre Gustavo , Belletti Barbara 
Published: August 2018

Abstract
Despite novel treatment strategies and prevention, one of the major clinical challenges in breast cancer (BC) is the development of local and distant recurrences. These are linked not only to genetic and epigenetic alteration in cancer cells, but also to tumor-microenvironment and tumor-host crosstalk. In this context, surgical removal of primary tumor, inducing an inflammatory response and a wound healing process, may stimulate residual cancer cell survival, growth, and invasion. Radiotherapy, representing a standard care after surgery in BC, exerts its function by killing residual tumor cells and altering the local microenvironment. We have recently demonstrated that intraoperative radiotherapy strongly induced miR-223 expression that, by targeting EGF released in the peritumoral context, dampened the activation of EGFR/HER2 pathway, eventually resulting as a potent restrainer of residual cancer cell survival and growth.
We also observed that miR-223 is highly expressed in normal mammary tissue, while all BC specimens analyzed expressed remarkably low levels. In line with these results, normal mammary epithelial cells of basal (HMEC) or luminal (BPEC) subtype expressed high levels of miR-223 compared to all tested BC cell lines. These data supported the possibility that loss of miR-223 expression could represent a common and possibly early event during mammary epithelial cell transformation. To explore this possibility, we transduced HMEC and BPEC cells using several oncogenes and tested the expression of miR-223. Transient expression of HER2, vSrc, and KRasG12D resulted in immediate drop of miR-223. Furthermore, to mimic oncogene-driven miR-223 down-modulation, we silenced its expression in HMEC and BPEC cells. Intriguingly, the loss of miR-223 per se induced the acquisition of transformed-like phenotypes, such as an increased proliferation in 2D-culture and a loss of polarized acinar structure with the formation of disorganized colonies in 3D-context.
Interrogation of the TCGA BC dataset, comprising a large panel of normal and BC samples, confirmed our data and showed that, among all BC subtypes, miR-223 was particularly decreased in HER2+ and luminal BC. Notably, clustering HER2+ BC for miR-223HIGH/pY1068-EGFRLOW stratified patients with a significantly better overall survival.
We thus explored the contribution of miR-223 in HER2-driven cell transformation. We overexpressed miR-223 in normal mammary epithelial cells and transformed them with HER2WT, ∆16HER2 (a particularly aggressive alternative splicing form of HER2), or HER2V659E (constitutively active form of HER2). Overexpression of miR-223 was able to efficiently counteract HER2-driven transformation, both in in vitro and in vivo models, not only in the presence of the WT oncogene, but also in the presence of the two aggressive mutants, the ∆16HER2 and HER2V659E.
Altogether, our results indicate that loss or decreased expression of miR-223 may represent an early and common event during mammary epithelial cell transformation, contributing to loss of polarity and altering the maintenance of the differentiate phenotype, features profoundly linked to the tumorigenic process. Discovering the molecular mechanism by which miR-223 (and/or its targets) exerts their functions in cellular transformation and their possible interference in the tumor/stroma crosstalk may lead to identification of novel prognostic markers for early lesions and new promising therapeutic targets.
Citation Format: Francesca Citron, Ilenia Segatto, Gian Luca Rampioni Vinciguerra, Gustavo Baldassarre, Barbara Belletti. Exploring the role of miR-223 loss in mammary epithelial cell transformation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A72.