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ADGRF1 promotes tumorigenesis via the activation of STAT3 pathway in HER2+ and triple‐negative breast cancers

Adhesion G protein‐coupled receptors (aGPCR) regulate many cellular processes involved in cancer. ADGRF1, a class VI aGPCR, is overexpressed and/or predicts poor survival in many cancer types. We previously reported that in breast cancer (BC), ADGRF1 is highly expressed in human epidermal growth factor receptor‐2 positive (HER2+) and basal‐like subtype, 85% of which are triple‐negative. Also, higher ADGRF1 expression predicts worse BC‐specific survival in both HER2+ and triple‐negative BCs patients. We previously showed that inducible ADGRF1 overexpression promoted and knockdown inhibited tumorigenesis in HER2+ BC cells. Consistently, ADGRF1 overexpression promoted tumor growth in vivo. We also reported that ADGRF1 coupling to Gαs was responsible for its pro‐tumorigenic effects. However, the downstream mediators of tumorigenesis promoted by ADGRF1 signaling remain largely unknown in cancer. Since STAT3 is a well‐known mediator of tumorigenesis in many cancers, and previous studies have shown that ADGRF1 modulates STAT3 activation in some cancers, we aimed to evaluate the effects of genetic and pharmacologic modulation of ADGRF1 on STAT3 phosphorylation (Y705) in HER2+ and triple‐negative breast cancers models. In a HER2+ BC cell line model (BT474), we found that ADGRF1 overexpression increased STAT3 phosphorylation (Y705) in vitro and in vivo. ADGRF1 knockdown also decreased STAT3 phosphorylation in BT474 cells and MDA‐MB‐231 cells, a triple‐negative BC cell line with high endogenous ADGRF1 gene expression and amplification. ADGRF1 knockdown inhibited cell growth as well as mammosphere and colony formation (a measure of tumorigenesis) in triple‐negative BC cell lines. Synaptamide increased secondary mammosphere formation, STAT3 phosphorylation (Y705), and cAMP but not IP1 in MDA‐MB‐231 cells while it had no effect on BT474 cells with inducible ADGRF1 overexpression. Synaptamide‐induced STAT3 phosphorylation was reversed by SQ22536, an adenylyl cyclase inhibitor. Our data suggest that ADGRF1 promotes tumorigenesis by coupling to Gas, which in turn activates STAT3 in HER2+ and triple‐negative BCs.