The alignment was performed using STAR aligner v.2.7.5b (https://github.com/alexdobin/STAR/releases(last accessed on 13 October 2021) [39]. Gene level read counts were obtained by using Salmon v.1.2.1 (https://github.com/COMBINE-lab/Salmon(last accessed on 13 October 2021) for all libraries [40]. the attenuated growth of tumors with TMEM176B gene silencing compared with controls. We found that the AKT/mTOR signaling pathway was activated or repressed in cells overexpressing or silenced for TMEM176B, respectively. Overall, our results suggest that TMEM176B expression in breast cancer cells regulates key signaling pathways and genes that contribute to cancer cell growth and progression, and is a potential target for therapeutic antibodies. Keywords:TMEM176B, calcium channel, triple negative breast cancer, AKT/mTOR signaling, RNA-seq, therapeutic antibodies == 1. Introduction == TMEM176B is a tetraspanin membrane protein that belongs to the membrane-spanning 4A (MS4A) protein family [1,2]. Although TMEM176B was described in human lung fibroblasts more than 20 years ago [3,4], the understanding of its role in cancer biology remains limited. Previous studies have described TMEM176B as an acid-sensitive cation channel, and research has primarily focused on its role in immune cell regulation [4,5,6]. Myeloid lineage immune cells express TMEM176B, in which it regulates dendritic cell maturation and antigen presentation MMP3 inhibitor 1 [6,7]. Higher TMEM176B expression has been found in immature dendritic cells in allograft tolerance models and in patients with spinal cord injuries [8,9], while lower expression has been reported in mature dendritic cells. In dendritic cells, TMEM176B was reported to contribute to their suppressive function by permitting the sodium counterflux required for the acidification of endophagosomes [4]. It has additionally been reported to traffic through the Golgi apparatus in TMEM176B transfected HeLa cells, although there are conflicting results regarding its subcellular localization in different cell types [4,5,6,10]. The whole-body deletion or pharmacological inhibition of MMP3 inhibitor 1 TMEM176B in murine cancer models decreased tumor growth through enhanced anti-tumor T cell immunity and improved the response to immune checkpoint inhibitors [4,10]. Although the importance of TMEM176B in immune regulation is emerging [4,10], much remains to be understood about its role in intracellular processes. Phylogenetic analysis has proposed that TMEM176 genes first appeared in cartilaginous fish, and were expressed in nonimmune cells, prior to expression expanding to immune cells in mammalian species [11]. A small number of human and rodent studies have examined TMEM176B in nonimmune cells [12,13]. Importantly, an altered expression of TMEM176B has been found in a number of cancer types, while abnormal methylation of the CpG islands associated with TMEM176B was reported in breast cancers [12,14]. The chromosome 7q36.1-3 region within which lies the gene for TMEM176B exhibits frequent gain/amplification in a number of human cancers [15]. Decreased overall survival in gastric cancer was found to correlate with higher levels ofTMEM176BmRNA [16]. In contrast, the overexpression of TMEM176B led to decreased proliferation of the androgen-sensitive LNCaP prostate cancer cell line and reduced the growth of NIH3T3 cells transfected with constitutively active H-Ras [17,18]. The regulation of endosomal pH by cation channels in cancer cells, and their importance in cancer cell signaling, is an emerging field [19]. Overall, much remains to be understood regarding the role of TMEM176B in cancer biology. Our interest in TMEM176B began when we identified it as the most upregulated gene in a c-Myc/VEGFA-expressing murine breast cancer cell line (Mvt1) sorted by flow cytometry based on the positive expression of the sialoglycoprotein CD24 [20]. We found that orthotopic tumors derived from the CD24-positive (CD24+) subset grew more rapidly than the CD24-negative cells [20]. CD24 has recently been identified as a putative oncogene, a marker of resistance to chemotherapy and a dont eat me signal in ovarian cancer and triple-negative breast cancer (TNBC) [21,22,23]. In this study, we aimed to develop a greater understanding of the role of TMEM176B in TNBC cell processes. == 2. Methods == == 2.1. Expression and Survival Studies in Publicly Available Datasets == We used cBioportal for Cancer Genomics to examineTMEM176Bcopy number amplification in breast cancer subtypes in the METABRIC dataset [24,25,26,27]. The analysis of gene expression subtype in the TCGA dataset was performed using the UALCAN cancer database [28]. Gene MMP3 inhibitor 1 expression by breast cancer grade was examined using the Gene Expression database of the Normal and Tumor Tissues 2 (GENT2) dataset [29]. We used KaplanMeier plotter to examine the relapse-free survival according to theTMEM176Blow and high mRNA expression from individual breast cancer studies within the dataset [30]. Detailed information of these studies can be found in the Gene Expression Omnibus Rabbit Polyclonal to VEGFB (GEO), National Center for Biotechnology Information (NCBI). == 2.2. Cell Lines == Murine and human TNBC cell lines were used in these experiments. The Mvt1, Met-1, and M-wnt cell lines were.