The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Compared with PSCs and PFs, CAFs have increased expression of GPR68 (a proton-sensing GPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Co-culture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.-Wiley, S. Z., Sriram, K., Liang, W., Chang, S. E., French, R., McCann, T., Sicklick, J., Nishihara, H., Lowy, A. M., Insel, P. A. GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors in the human genome, are also the largest class of targets of approved drugs. Are the optimal GPCRs (in terms of efficacy and safety) currently targeted therapeutically? Especially given the large number (∼120) of orphan GPCRs (which lack known physiologic agonists), it is likely that previously unrecognized GPCRs, especially orphan receptors, regulate cell function and can be therapeutic targets. Knowledge is limited regarding the diversity and identity of GPCRs that are activated by endogenous ligands and that native cells express. Here, we review approaches to define GPCR expression in tissues and cells and results from studies using these approaches. We identify problems with the available data and suggest future ways to identify and validate the physiologic and therapeutic roles of previously unrecognized GPCRs. We propose that a particularly useful approach to identify functionally important GPCRs with therapeutic potential will be to focus on receptors that show selective increases in expression in diseased cells from patients and experimental animals.

Pancreatic ductal adenocarcinoma (PDAC) has a unique tumor microenvironment that is characterized by a dense fibrotic stroma (desmoplasia) that is generated by pancreatic cancer associated fibroblasts (PCAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach we identified GPR68, a proton sensing GPCR, has much higher expression in PCAFs compared to both PFs and PSCs. GPR68 also had increased expression in PDAC tumors compared to normal pancreas and its expression was increased in PSCs by co‐culture with PDAC cells or incubation with TNFα. GPR68 activation in PCAFs enhanced expression of interleukin‐6 (IL‐6) via a cAMP/PKA/CREB signaling pathway and GPR68 knockdown with siRNA diminished IL‐6 production by PCAFs and an enhancement in proliferation of PDAC cells by PCAF conditioned media. Use of the allosteric modulator compound ogerin enhanced pH‐dependent increase in cAMP. Furthermore, a pilot screening of 96 GPCR specific compounds tested with GPR68 overexpressing HEK cells identified five compounds that decrease intracellular cAMP at pH6.4, indicating that they may be inhibitors of GPR68. We conclude that PDAC cells induce expression of GPR68 in PCAF, resulting in increased IL‐6 production by PCAFs and enhanced PDAC cell proliferation. This PCAF‐expressed GPR68 thus contributes to PDAC cell‐PCAF interaction and may be a novel therapeutic target for pancreatic cancer, a type of cancer for which new therapies are an important unmet medical need.Support or Funding InformationThis work was supported by National Institutes of Health (NIH), National Cancer Institute (NCI) Therapeutic Training Grant 5T32CA121938, NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017, and by an American Society for Pharmacology and Experimental Therapeutics (ASPET)‐David Lehr Research Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

G protein-coupled receptors (GPCRs) are the largest class of signaling receptors in humans and other species and in addition, the most widely targeted class for FDA-approved therapeutics. However, GPCRs are not commonly targeted in cancers other than endocrine tumors. We hypothesize that GPCRs may have been “missed” as targets in cancer, perhaps in part because they have not been frequently identified as driver mutations; however, GPCRs may have unappreciated utility as therapeutic targets in a variety of tumors. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA-seq), mining of publicly available datasets (e.g., The Cancer Genome Atlas, TCGA), and validation studies (e.g., protein and functional analyses) to assess GPCRs in a variety of human tumors, cancer cells and stromal cells (cancer-associated fibroblasts, CAFs). A major focus of our efforts has been on pancreatic ductal adenocarcinoma (PDAC) cells/tumors and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express >70 different GPCRs, including many orphan GPCRs (receptors without known physiologic agonists) and that numerous GPCRs are expressed at much higher levels than in normal precursor cells (pancreatic ductal epithelial cells for PDAC cells and pancreatic stellate cells and pancreatic fibroblasts for PCAFs). We find that a cluster of GPCRs is overexpressed in PDAC tumors. Two such receptors are orphan GPCRs, Orphan 1 and Orphan A, neither of which is mutated but each has high expression, respectively, in PCAFs and PDAC cells. Orphan 1 and Orphan A have at least 2-fold higher expression in >90% of PDAC tumors in TCGA compared to that in normal pancreatic tissue (compiled from the GTEX database). Higher expression of orphan A is associated with decreased survival and remission, while Orphan 1 expression correlates with that of numerous fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells while transfection of Orphan A increases DNA synthesis of normal pancreatic ductal epithelial cells. Taken together, these and other results suggest that in addition to their role in endocrine tumors, GPCRs represent previously unrecognized contributors to cancer through actions on tumor cells and stromal cells. These data suggest the possibility that highly expressed GPCRs (such as Orphan A) may function as oncogenes. Thus, GPCRs may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are an important, unmet need. Note: This abstract was not presented at the meeting. Citation Format: Paul A. Insel, Krishna Sriram, Shu Z. Wiley, Thalia McCann, Randall P. French, Andrew M. Lowy. G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1139. doi:10.1158/1538-7445.AM2017-1139