Abstract
TMEM176B is a member of the membrane spanning 4-domains (MS4) family of transmembrane proteins, and a putative ion channel that is expressed in immune cells and certain cancers. We aimed to understand the role of TMEM176B in cancer cell signaling, gene expression, cell proliferation, and migration in vitro, as well as tumor growth in vivo. We generated breast cancer cell lines with overexpressed and silenced TMEM176B, and a therapeutic antibody targeting TMEM176B. Proliferation and migration assays were performed in vitro, and tumor growth was evaluated in vivo. We performed gene expression and Western blot analyses to identify the most differentially regulated genes and signaling pathways in cells with TMEM176B overexpression and silencing. Silencing TMEM176B or inhibiting it with a therapeutic antibody impaired cell proliferation, while overexpression increased proliferation in vitro. Syngeneic and xenograft tumor studies revealed 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.
Highlights
TMEM176B is a tetraspanin membrane protein that belongs to the membrane-spanningTMEM176B was reported to contribute to their suppressive function by permitting the sodium counterflux required for the acidification of endophagosomes [4]
176Bsh1 and 176Bsh2 cells had 48% and 47% fewer stained cells than controls (Figure 1G,H). We further repeated these experiments in three murine breast cancer cell lines: Mvt1, Met-1, and M-wnt cells, using two Tmem176b short hairpin RNAs (shRNA) constructs (176bsh1 and 176bsh2)
These results suggest that the cation channel TMEM176B regulates the AKT/mTOR signaling in the TMEM176B-silenced cells, we found reductions in AKT (Thr308 ) phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), p70S6K, and ribosomal protein S6 (RPS6) phosphorylation pathway
Summary
TMEM176B is a tetraspanin membrane protein that belongs to the membrane-spanningTMEM176B was reported to contribute to their suppressive function by permitting the sodium counterflux required for the acidification of endophagosomes [4]. The importance of TMEM176B in immune regulation is emerging [4,10], much remains to be understood about its role in intracellular processes. A small number of human and rodent studies have examined TMEM176B in nonimmune cells [12,13]. The chromosome 7q36.1-3 region within which lies the gene for TMEM176B exhibits frequent gain/amplification in a number of human cancers [15]. 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]. Much remains to be understood regarding the role of TMEM176B in cancer biology
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