Head and neck cancer is the sixth most common malignancy, and there is an urgent need to identify physiological processes contributing to tumorigenesis. Extracellular acidification caused by aerobic glycolysis within tumor microenvironments can stimulate proton-sensing receptors. GPR68, or ovarian cancer G protein-coupled receptor 1, responds to extracellular acidity and is highly expressed in head and neck squamous cell carcinoma (HNSCC) as well as normal esophageal tissue. To study the role of GPR68 in oral dysplasia, wild-type and GPR68−/− mice were treated with 4-Nitroquinoline N-oxide (4NQO) in drinking water for 11–13 weeks, followed by normal water for 11–12 weeks. 4NQO treatment resulted in 45 percent of GPR68−/− mice developing severe dysplasia or squamous cell carcinoma compared to only 10.5 percent of GPR68+/+ mice. This correlated with increased frequencies of regulatory T cells in the spleens of male GPR68−/− mice. Dysplastic regions of the tongue had increased CD31 staining compared to normal regions in both GPR68−/− and GPR68+/+ mice, suggesting that angiogenesis was GPR68-independent. RNA knockdown studies using HNSCC cell lines demonstrated no direct effect of GPR68 on survival or growth. Overall, we demonstrate that GPR68-deficiency worsens the severity of chemical-induced oral dysplasia, suggesting a protective role for this gene in tumorigenesis.

CD4 T cells contribute to host defense against pathogens, but are also implicated in autoimmunity and allergies when directed against innocuous antigens. Since Th17 cells are upregulated in several autoimmune diseases, identifying genes involved in their activation, proliferation or survival may lead to novel therapies for inflammatory diseases. We previously demonstrated that the aryl hydrocarbon receptor increases expression of the proton sensing receptor GPR68 in human CD4 T cells. Since T cell clonal expansion is associated with a metabolic shift towards anaerobic glycolysis resulting in extracellular acidification, we tested the hypothesis that GPR68 is required for optimal T cell proliferation and survival following activation. To delete GPR68 in T cells, GPR68-floxed mice were crossed with a strain expressing Cre-recombinase under the CD4 promoter. Purified CD4 T cells from CD4-Cre+ and Cre-negative littermate controls were activated in vitro with anti-CD3 and Th17-inducing cytokines (IL-1b, IL-6, IL-23, TGF-b) for 1–5 days. In the control (Cre-negative) group, T cell numbers increased over the first 2 days following activation and then stabilized from days 3–5. In the GPR68-deficient group (Cre+), T cell numbers significantly decreased after day 2, demonstrating a role for GPR68 in promoting CD4 T cell survival. Nevertheless, IL-17 production was GPR68-independent, demonstrating that GPR68 did not regulate Th17 differentiation. GPR68 was especially critical for survival when low doses of anti-CD3 were used (<2.5ug/mL), but not at a high dose (10ug/mL). Overall, our data demonstrate that GPR68 increases the sensitivity of CD4 T cells to survival signals induced by CD3 stimulation.