Abstract P2-03-02: Modulatory action of melatonin and miR-17 on ROCK-1 in breast cancer metastasis model

Abstract

Abstract Breast cancer is the most common cancer in women is often associated with high morbidity and mortality rates, with great financial impact on health system. Besides, the disease is characterized by the high rates of metastasis, which worsen significantly the prognosis. This process is associated with two regulatory molecules, microRNAs (miR), specially miR-17, and ROCK-1, which overexpression has been associated to tumor growth and metastasis. In contrast, melatonin has shown oncostatic and anti-metastatic properties by reducing the cell ability to migrate and invade the tissue, besides the inhibition of cell proliferation. The aim of this study was to investigate the effect of melatonin to modulate miR-17 and ROCK-1, a possible candidate gene to miR-17 target in metastatic breast cancer cell line, MDA-MB-231. To determine the effect of melatonin to modulate miR-17 and ROCK-1, MDA-MB-231 cells were treated with melatonin and anti-miR-17-5p. ROCK-1 and miR-17 gene expression were accessed by real time PCR and ROCK-1 protein expression verified by immunocytochemistry and western blotting. Migration and invasion assay was performed to verify the action of melatonin and anti-miR-17-5p to inhibit these processes. In the in vivo study, was developed pulmonary metastasis model followed for six weeks, the tumor induction was continued for 4 weeks, and treatment with anti-miR-17-5p inhibitor for two more weeks. At the end of treatment, animals were euthanized, the lungs removed and used for analysis of miR-17-5p and Let-7c (positive control) and ROCK-1 gene expression. ROCK-1 protein was analyzed by immunohistochemistry. MiR17-5p inhibition managed directly modulate gene and protein expression of ROCK-1 in MDA-MB-231 cells, as well, the gene expression of MYC. In additional, the migration and invasion were decreased after melatonin and anti-miR-17-5p treatment. To validate the findings of the study in vitro with miR-17, was used for lung metastasis model in athymic nude mice. According to our findings, normal animals without metastasis (negative control) had lower levels of miR-17 compared to animals with metastasis and without treatment (positive control). In contrast, animals with metastasis who received anti-miR17-5p treatment, interestingly also had low miR-17 levels compared to positive control animals. Furthermore, it was observed fewer metastases and a reduction in ROCK-1 protein expression in these treated animals compared to positive control animals. Our results demonstrated that miR-17-5p inhibition can modulate ROCK-1 gene and protein in MDA-MB-231 cells and decreasing the number of lung metastases in treated animals. Furthermore, melatonin can act as a synergic mechanism to decrease migration and invasion on metastasis processes mediated by ROCK-1. Citation Format: Borin TF, Pongeluppi RI, Gelaleti GB, Leonel C, Moschetta MG, Ferreira LC, Zuccari DAPdC. Modulatory action of melatonin and miR-17 on ROCK-1 in breast cancer metastasis model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-03-02.