Melatonin is a hormone produced by the pineal gland and has been shown different antitumor effects, as immunomodulatory, antioxidant, pro-apoptotic, anti-proliferative, antimetastatic and antiangiogenic, however, the pathways by which melatonin exerts its action need to be identified. Thus, the aims of this study were to perform the transcriptome analysis to evaluate the pathways of melatonin action in triple-negative breast cancer. Triple-negative breast cancer cells (MDA-MB-231) were injected into the mammary gland of the athymic nude mice (n=10), which were treated with melatonin (40 mg/kg) or vehicle during 21 days. RNA-Seq libraries were created using Truseq RNA-Seq Library Prep Kit v2. The experiment was paired-end with 100nt read length, performed on the Illumina HiSeq2500 sequencer, producing about 30 million reads per library. To differentiate human and mouse expression, the alignment was performed to filter out mouse-like reads before mapping to the human reference and vice versa, and data were mapped against human (GRCh37/hg19) and mouse (NCBI37/mm9) genomes separately, using the TopHat software. The HTSeq was employed for analyses of read counts and DESeq2 was used to identify genes differentially expressed between melatonin treated and control tumors. Differentially expressed genes (DEGs) were identified based on a false discovery rate (FDR) q-value threshold of less than 0.05. Also, we applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). Results showed that animals treated with melatonin had smaller tumors volume than controls (p<0.05). RNA-Seq data showed that 57.24% of reads mapped uniquely to human, 29.66% reads in mouse and 11% reads mapping to both human and mouse genomes. In human tumor cells, there was no DEGs between melatonin treated and control group (adjP>0.05). In mouse cells, which represent the tumor microenvironment, there were 34 DEGs between animals treated with melatonin and controls (adjP<0.05). In tumors cells, we detected 714 differentially co-expressed genes (IKdiffI>0.6), which were functionally enriched for GO terms like lipid metabolic process, response to drug, oxidoreductase activity and PPAR signaling (adjP<0.1). Also, we identified 3 gene modules strongly associated with melatonin treatment, which were related with metabolic pathways (adjP<0.1). In mouse cells, were detected 1345 differentially co-expressed genes, which were enriched for signaling pathways like Wnt receptor, Hedgehog and TGF-beta (adjP<0.1). There were 3 gene modules strongly associated with melatonin treatment, which are enriched for regulation of translation and cell cycle, immune system process and T cell differentiation, regulation of action cytoskeleton and ErbB signaling pathway (adjP<0.1). Also, potential regulator genes for melatonin treatment were detected by generating clusters of co-expressed genes and individual analysis confirms these results. Transcriptomic network analysis coupled with other results showed that melatonin treatment controls the tumor growth, acting especially by metabolic pathways in tumor cells and modulating the tumor microenvironment.

Citation Format: Jardim-Perassi BV, Sonehara NM, de Paula-Junior R, Chammas R, Coutinho LL, Reis Júnior O, Alexandre PA, Fukumasu H, Zuccari DAPC. Melatonin treatment: A transcriptomic networks in a xenograft model of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-28.