Tumor Suppressor miR-16 Is Modulated by ErbB-2 in Breast Cancer and May Serve as an Alternative Therapy for Trastuzumab-Resistant Breast Carcinoma

Progesterone receptor (PR), estrogen receptor (ER) and tyrosine kinase receptor, ErbB-2, are all major players in the breast cancer (BC) scenario. MicroRNAs (miRNAs) are a recently discovered class of short noncoding endogenous RNAs with regulatory functions, whose role in cancer and metastasis has been identified only in the past few years. In this context, we recently revealed the first progestin-regulated miRNA expression profile and identified a novel role for microRNA-16 (miR-16) as a tumor suppressor in progestin- and growth factor-induced growth in BC. Moreover, we identified cyclins E1 (CCNE1) and D1 (CCND1) as relevant miR-16 targets in BC. On the other hand, it has been demonstrated that estrogen induces miR-16 down-regulation, leading to an augmentation in BC cell proliferation. In spite of ErbB-2’s acknowledged relevance in BC, the relationship between ErbB-2 and miR-16 has not yet been addressed. The potential role of this relationship in the resistance to anti-ErbB-2 directed therapy has not been explored either. Trastuzumab (TZ) is an effective targeted therapy in ErbB-2 overexpressing BC. However, many ErbB-2-positive patients initially are or eventually become resistant to TZ. Thus, elucidating mechanisms of TZ resistance in BC cells is clinically important. Here, we found that inhibition of cell proliferation induced by ErbB-2 abrogation (using an RNA interference strategy) was associated to miR-16 up-regulation, both in TZ- sensitive (BT-474 and SKBR-3) and -resistant (HCC-1569 and JIMT-1) cells. CCNE1 expression, whose role in TZ resistance has been reported previously, was reduced by miR-16 in all cell lines at mRNA and protein levels. Interestingly, in TZ-sensitive cell lines, TZ treatment also up-regulated miR-16, reduced CCND1 and CCNE1 expression levels and, consequently, inhibited cell growth. In contrast, in TZ-resistant cell lines, we did not observe miR-16 up-regulation upon TZ treatment. Taking this into account, we explored the effects of miR-16 precursor in vitro transfection and found that it successfully inhibited cell proliferation in various TZ-resistant cell lines (HCC-1569, JIMT-1, HCC-1419 and MDA-MB-453). Finally, we developed a preclinical trial using JIMT-1 cells, and our results indicate that intratumoral miR-16 administration significantly inhibited in vivo growth of these TZ-resistant cells. Our study demonstrates for the first time that ErbB-2 is implicated in miR-16 expression in BC and provides evidence that miR-16 administration may function as a novel and alternative therapy for TZ-resistant BC.