miR-16 is a tumor suppressor in progestin-induced breast cancer

LEANDRO VENTURUTTI; MARTIN A. RIVAS; ROXANA SCHILLACI; TIM HUANG; PATRICIA V. ELIZALDE

Chicago

Congreso; 101st Annual Meeting of the American Association for Cancer Research; 2012

AACR

Breast cancer is the most common type of cancer among women in the US and the second leading cause of death associated to cancer. microRNA (miRNA) are short ribonucleic acids with important regulatory functions and with an increasingly acknowledged role in cancer. We recently found that progestins modulate miRNA expression in a progestin-dependent murine breast cancer model. In particular, miR-16 was found to be downregulated by progestins. We here demonstrate that the treatment of T47D and BT-474 human breast cancer cells with the synthetic progestin medroxyprogesterone acetate (MPA) results in a significant miR-16 decrease and a concomitant cell proliferation augment. Interestingly, MPA treatment of T47D-Y cells, a variant of T47D lacking progesterone receptor (PR) expression, did not modify miR-16 levels. Furthermore, transfection of T47D-Y with PR-mPRO, a mutant PR which lacks the ability to activate nongenomic signaling pathways (but retains the transcriptional activity of PR), or with PR-C587A, a variant with the capacity to activate nongenomic pathways but uncapable of binding to DNA, did not recover the ability of progestins to downregulate miR-16, suggesting that both PR actions are necessary for miR-16 dowregulation by progestins. In addition, MPA induced a potent increase in the levels of the oncogenic transcription factor c-Myc, a previously reported suppressor of miR-16. Silencing of the signal transducer and activator of transcription 3 (Stat3) by siRNA, abrogated MPA capacity to induce c-Myc expression. Cyclin E, a cell cycle promoter with a major role in breast cancer progression, has been shown to be a miR-16 target. In this study, we not only demonstrate that MPA induces Cyclin E upregulation (both at the mRNA and protein level), but also that this can be prevented by the inhibition of Stat3 and/or c-Myc. We then wondered whether this regulation would occur in vivo. Thus, we injected 2 x 107 BT-474 cells in nude mice carrying a 17β-Estradiol (E2) pellet, in the absence of matrigel. As previously reported, the tumors first grew but started to regress soon afterwards. At that time point (7 days post-injection), half of the animals were administered MPA. The tumors in the group treated with MPA started to grow at a higher rate than the group supplied with E2 alone. Seven days after MPA supply, we excised some of the tumors and measured miR-16 levels, which showed that the treatment with MPA in vivo, produced a profound downregulation of miR-16. Furthermore, these tumors exhibited a higher level of cyclin E by western blot, validating cyclin E as a target of miR-16 in vivo. Our results are the first ones to identify miR-16 as a tumor suppressor modulated by progestins in human breast cancer cells, demonstrating as well that this regulation is relevant for the proliferative biological effect of progestins.