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Women with breast cancer has the tumor progression and angiogenesis-induced metastasis as the main cause of death. MicroRNAs (miRNAs) are small noncoding mRNA molecules that play an important role in gene regulation and once deregulated, these molecules may be involved with the progression of different human tumor types, including breast cancer. These miRNAs play an oncostatic role on tumor suppressor genes and regulate the process of angiogenesis, tumor growth and metastasis. So as new possible adjuvant treatment against breast cancer our group have shown melatonin, that is a hormone secreted by the pineal gland, by exhibiting several anti-tumor and antiangiogenic effects. Therefore, the aim of this study was to evaluate the potential therapeutic of melatonin on miRNAs regulation to verify breast cancer progression and potent tumor suppressor miR-148a-3p. In silico analysis was performed for selection of miRNAs involved in breast cancer. The MDA-MB-231 breast cancer cell line (metastatic negative estrogen receptor) was grown and separated in two different experimental conditions maintained for 24 hours: control group and melatonin-treated group (concentration of 1 mM). After this period the extraction of total RNA was performed (Qiagen®) and the total concentration of miRNAs of each sample was determined (NanoDrop Spectrophotometer 2000C - Thermo Scientific®). Differential expressions of these miRNAs was evaluate using miScript miRNA PCR-Array (Qiagen®) containing 84 miRNAs associated with breast cancer. The overexpression of miR-148a-3p in MDA-MB-231 cells was performed by bacterial cloning vector and the relative quantification of gene expression of their target IGF-1R and VEGF by real-time PCR. Moreover, the quantification of protein expression was performed by immunocytochemistry of IGF-1R and VEGF. Lastly, the migration and invasion cell assays were carried out to assess the potential capacity of these cells to undergo metastasis. The analysis of miRNAs MDA-MB-231 cell line by PCR-array showed 24 upregulated miRNAs and 8 miRNAs downregulated after the treatment with melatonin. Relative quantification shows that the melatonin treatment increases the gene expression of miR-148a-3p and decreases the gene and protein levels of IGF-1R and VEGF. The capability of migration and invasion of the breast cancer cells decreased after treatment with melatonin and overexpression of miR-148a-3p. Our results confirm the action of melatonin on the miR-148a-3p regulation known being involved in the progression of breast cancer. Thus the establishment of this therapeutic protocols can control these cellular events essential for the prognosis of patients with breast cancer. Citation Format: Debora Zuccari, Jéssica Zani Lacerda, Lívia Carvalho Ferreira, Beatriz Camargo Lopes, Andrés Felipe Aristizábal-Pachón, Marcio Chaim Bajgelman. Melatonin regulates the tumor suppressor miR-148a-3p involved in angiogenesis and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2017-1477

Regulatory T cells (Treg) play an important role controlling the balance between tolerance and surveillance. Regarding its potential to inhibit T cell proliferation, Treg can antagonize antitumor immunity. Therefore, inhibition of Treg can potentiate antitumor immunity. There are several strategies to inhibit Treg function or traffic to tumor site; however these approaches aren't specific for Treg and can impair antitumor response. In this way, the exploration of molecular targets could assure more selectivity for Treg inhibition. The FoxP3 transcription factor is considered an important Treg marker and a master key which is associated with the determination of immunosuppressive phenotype. Silencing FoxP3 expression can lead inactivation of immunosuppressive phenotype of Tregs. Here in we developed an assay to validate RNAi candidates targeting FoxP3. The assay is based in two steps. In the first step we generate shRNA candidates that are cloned in expression vectors. These vectors are co-transfected with a luciferase reporter vector encoding synthetic targets, in cell culture. A decrease in luciferase activity indicates efficiency of the shRNA sequence. Using this screening tool we could find shRNA candidates that exhibited more than 95% of knockdown efficiency. In the second step we evaluated the activity of the best shRNA candidates to knockdown endogenous FoxP3 in primary cells. We generated lentiviral vectors encoding both the shRNA and an expression cassette of CD90.1 reporter gene to transduce primary CD4 T cells. These transduced cells were incubated with a TGF beta cocktail which induces FoxP3 expression and adaptive Treg generation. FoxP3 expression was checked by flow cytometry, gating CD90.1 positive cells. We found up to 75% of inhibition, for the RNAi candidate #183. On the other hand, we also observed that a highly efficient shRNA sequence indicated by the luciferase reporter assay could be inefficient to silence the endogenous FoxP3 in primary cells. We conclude this assay is a reliable tool which can be used to generate RNAi to knockdown FoxP3 expression in primary cells, overcoming issues associated with transfection or transduction efficiency. Transduced cells can be labeled on cell surface with an ati-CD90.1 marker and stained for membrane, intracellular markers or even other kind of analysis as cell cycle. The validated RNAi candidates are active against the endogenous target and can be used to explore strategies to inhibit the immunosuppressive phenotype of regulatory T cells and to potentiate antitumor immunity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A149. Citation Format: Marcio C. Bajgelman, Randall J. Brenneman, Dawn Seales, Alexey Berezhnoy, Ana Carolina P. Carvalho, Eli Gilboa. Assay development to validate RNAi candidates targeting FoxP3 transcription factor in adaptive regulatory T cell. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A149.