ADGRF1 signaling pathways in Breast Cancer

Background Adhesion G‐protein‐coupled receptors (GPCRs) form the second largest GPCR subfamily. They control many cellular processes involved in cancer, such as cell adhesion, invasion/migration, and stem cell function. Most of adhesion GPCRs are still orphans without any known endogenous ligands. In breast cancer, ADGRF1, previously called GPR110, is overexpressed in 2 aggressive subtypes of breast cancers, HER2+ and triple‐negative. We have found that HER2+ cells with ADGRF1 overexpression in doxycycline‐inducible manner formed more secondary mammospheres and more colonies in the soft agar assay and had higher % of Aldefluor+ cell population. However, ADGRF1‐overexpressing BT474 cells formed fewer tumors in mice and had reduced tumor growth compared to control. Aim To understand the downstream signaling of ADGRF1 that may contribute to the opposing findings in vitro and in vivo . Methods The experiments were conducted using two HER2+ breast cancer cell lines: BT474 and SKBR3 stable cells overexpressing ADGRF1 in doxycycline‐inducible manner and MDA‐MB‐231, a triple‐negative breast cancer cell line with endogenous ADGRF1 overexpression. The coupling of ADGRF1 to Gαs and Gαq pathway was assessed by measuring cAMP (AlphaScreen assay) and IP1 (Homogeneous Time‐Resolved Fluorescence assay), respectively and by co‐immunoprecipitation and immunoblotting. The effect of synaptamide, an ADGRF1 agonist, was also assessed on cAMP and IP1. The effects of Gαs or Gαq pathway activators were assessed on the potential of mammosphere formation (using Mammosphere assay). Results The basal levels of cAMP and IP1 was significantly higher in doxycycline‐treated (+Dox) BT474 cells overexpressing ADGRF1 compared to cells without doxycycline (−Dox). Synaptamide (1–100 nM) did not further increase cAMP or IP1 in these cells. On the other hand, synaptamide increased cAMP but not IP1 in a concentration‐dependent manner in MDA‐MB‐231 cells. Immunoprecipitation with both anti‐Gαs and anti‐Gαq antibodies pulled down ADGRF1 in +Dox but not −Dox treated BT474 and SKBR3 cells. Forskolin, an activator of adenylyl cyclase that increases cAMP, reduced mammosphere formation in MDA‐MB‐231 cells. The effect of Gαs or Gαq pathway activators on mammosphere formation in BT474 and SKBR3 clones is being assessed in ongoing experiments. Conclusion Our results show that ADGRF1 couples to both Gαs and Gαq pathways. However, our data do not indicate that differential coupling leads to opposing in vitro versus in vivo effects. Additional studies with pathway activators as well as acute versus chronic activation of ADGRF1 may elucidate ADGRF1 pathways relevant in breast cancer biology. Support or Funding Information Department of Defense Grants W81XWH‐14‐1‐0340 and W81XWH‐14‐1‐0341 to Drs. Trivedi and Schiff, respectively.