A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2‐positive breast cancer

Abstract

Purpose Adhesion G protein coupled receptors (aGPCRs) remains least explored GPCRs family for their role in health and disease. The aGPCR, ADGRF1 (GPR110), is overexpressed and predicts poor survival in various cancer types. However, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We have reported that ADGRF1 is overexpressed in tumorigenic population and in anti-HER2 therapy-resistant derivatives of various HER2+ breast cancer (BC) cell line models. Further, we have reported that ADGRF1 knockdown reduces tumorigenesis and metastasis in HER2+ BC cells. In this study, we evaluated the effects of ADGRF1 overexpression (OE) on tumorigenesis, signaling pathways, and outcomes in HER2+ BC. Methods Stable clones of 2 HER2+ BC cell lines, BT474 and SKBR3, were generated to overexpress ADGRF1 construct in doxycycline-inducible manner. The effect of ADGRF1 on tumorigenesis was evaluated in vitro using soft agar assays, mammosphere assay, and Aldefluor assay. The effect on the HER pathway was evaluated by assessing expression of phospho- and total-HER1 and -HER2 and potency of anti-HER2 drugs. G protein coupling of ADGRF1 was determined by co-immunoprecipitation of Gαs and Gαq and detecting second messengers, cAMP and IP1, in presence/absence of synaptamide, an ADGRF1 agonist. Downstream pathways of ADGRF1 were explored by RNAseq and Reverse Phase Protein Arrays (RPPA) and functional assays. ADGRF1 expression was assessed in BC patients from The Cancer Genome Atlas. ADGRF1 OE effects on survival was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium dataset. Results ADGRF1 OE enhanced the number of colonies and mammospheres and increased Aldefluor+ tumorigenic cell population, suggesting a role in tumorigenesis. ADGRF1 OE did not alter the expression total- or phospho-HER1/-HER2 proteins or efficacy of anti-HER2 drugs. Overexpressed ADGRF1 co-immunoprecipitated with Gαs and Gαq proteins and increased cAMP and IP1. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 OE, suggesting pro-tumorigenic effects of the ADGRF1-Gαs pathway. Synaptamide, did not alter cAMP or IP1 with/without ADGRF1 OE or the number of mammospheres. Integration of RNA-Seq and RPPA data showed that ADGRF1 OE inhibited cell cycle. We detected G0/1 arrest by cell cycle analysis with ADGRF1 OE. The protein level of the proliferation marker Ki67 was reduced upon ADGRF1 OE. Also, ADGRF1 OE led to a 10-fold reduction in potency of docetaxel, a chemotherapy drug used in HER2+ BC. ADGRF1 was overexpressed and amplified in basal and HER2 compared to luminal A and B BCs. High ADGRF1 expression predicted poor survival in HER2 subtype, but not in basal BC. Conclusions: ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.