title: The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer.
abstract: Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive disease with poor survival and is the sixth most common cancer worldwide. Gastroesophageal reflux is a common event in SCCHN patients. GPR4 is a proton-sensing G-protein coupled receptor, which can be activated by acidosis. The objective of this study was to explore the role of GPR4 in acid exposure and tumor angiogenesis in SCCHN. In this study, we confirmed that overexpressing GPR4 in SCCHN cells could increase the expression and secretion of IL6, IL8 and VEGFA at pH 5.9. This effect could be inhibited by SB203580 (a p38 inhibitor). Western blot analysis indicated that phosphorylation of p38 increased in GPR4 infected cells at pH 5.9, which could be inhibited by SB203580. In tube formation assay, HMEC-1 cells were incubated with conditioned medium (CM, pH 5.9, 6.5, 7.4) derived from control and GPR4 infected SCCHN cells. Tube length was significantly increased in HMEC-1 cells incubated with CM from GPR4 infected cells compared with control cells at pH5.9, which indicated the pro-angiogenic effect of GPR4 in acidic pH. The neutralizing antibodies of IL6, IL8 and VEGFA could inhibit tube formation of HMEC-1 cells. In vivo, the effect of GPR4 on angiogenesis was investigated with the chick chorioallantoic membrane (CAM) model. Control and GPR4 infected SCCHN cells were seeded onto the upper CAM surface (n = 5 in each group) and 5 μL DMEM/F12 (pH 5.9, 6.5, 7.4) was added to the surface of the cell every 24 h. Four days later, the upper CAM were harvested and the ratio of the vascular area to the CAM area was quantified using Image-Pro Plus 6.0 software. GPR4 infected cells could recruit more vascular than control cells at pH5.9. In conclusion, we suggested that GPR4 induces angiogenesis via GPR4-induced p38-mediated IL6, IL8 and VEGFA secretion at acidic extracellular pH in SCCHN.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Jing
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhong
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Junwei
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Ding
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Zhigang
authors.identifier: http://orcid.org/0000-0002-4438-7417
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000098:blood supply / Q000473:pathology*; D045744:Cell Line, Tumor; D005110:Extracellular Space / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000098:blood supply / Q000473:pathology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D000077195:Squamous Cell Carcinoma of Head and Neck; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C091670:GPR4 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D043562:Receptors, G-Protein-Coupled; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A