The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer

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.头颈部鳞状细胞癌（Squamous cell carcinoma of the head and neck, SCCHN）是一种侵袭性疾病，预后不良，为全球第六大常见癌症。胃食管反流在SCCHN患者中较为常见。GPR4是一种质子敏感型G蛋白偶联受体，可被酸中毒激活。本研究旨在探讨GPR4在SCCHN中酸性暴露及肿瘤血管生成中的作用。本研究证实，在pH 5.9条件下，于SCCHN细胞中过表达GPR4可提升IL6（白细胞介素6）、IL8（白细胞介素8）及VEGFA（血管内皮生长因子A）的表达与分泌水平，该效应可被SB203580（p38抑制剂）抑制。蛋白质免疫印迹实验结果显示，在pH 5.9条件下，转染GPR4的细胞中p38的磷酸化水平升高，且该现象可被SB203580阻断。在管形成实验中，将人微血管内皮细胞-1（HMEC-1）与取自对照组及转染GPR4的SCCHN细胞的条件培养基（CM，pH 5.9、6.5、7.4）共培养。在pH 5.9条件下，与对照组相比，用转染GPR4的SCCHN细胞的条件培养基培养的HMEC-1细胞的管长度显著增加，这表明GPR4在酸性pH环境下具有促血管生成作用。IL6、IL8及VEGFA的中和抗体可抑制HMEC-1细胞的管形成能力。体内实验中，本研究采用鸡胚绒毛尿囊膜（CAM）模型探究GPR4对血管生成的影响。将对照组及转染GPR4的SCCHN细胞接种于CAM上层表面（每组n=5），并每24小时向细胞表面添加5 μL DMEM/F12培养基（pH 5.9、6.5、7.4）。培养四天后，收集上层CAM组织，采用Image-Pro Plus 6.0软件定量分析血管面积与CAM面积的比值。在pH 5.9条件下，转染GPR4的细胞较对照组可募集更多新生血管。综上，本研究表明，在SCCHN中，GPR4可通过酸性细胞外pH环境下GPR4介导的p38信号通路促进IL6、IL8及VEGFA的分泌，进而诱导血管生成。