GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System.

A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FALCOscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)-stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.

Proton-sensing G protein-coupled receptors: detectors of tumor acidosis and candidate drug targets.

Cells in tumor microenvironments (TMEs) use several mechanisms to sense their low pH (<7.0), including via proton-sensing G protein-coupled receptors (psGPCRs): GPR4, GPR65/TDAG8, GPR68/OGR1 and GPR132/G2A. Numerous cancers have increased expression of psGPCRs. The psGPCRs may contribute to features of the malignant phenotype via actions on specific cell-types in the TME and thereby promote tumor survival and growth. Here, we review data regarding psGPCR expression in tumors and cancer cells, impact of psGPCRs on survival in solid tumors and a bioinformatics approach to infer psGPCR expression in cell types in the TME. New tools are needed to help define contributions of psGPCRs in tumor biology and to identify potentially novel therapeutic agents for a variety of cancers.

GPCRs in pancreatic adenocarcinoma: Contributors to tumour biology and novel therapeutic targets.

Pancreatic cancer has one of the highest mortality rates (5-year survival ~9%) among cancers. Pancreatic adenocarcinoma (PAAD) is the most common (>80%) and the most lethal type of pancreatic cancer. A need exists for new approaches to treat pancreatic adenocarcinoma. GPCRs, the largest family of cell-surface receptors and drug targets, account for ~35% of approved drugs. Recent studies have revealed roles for GPCRs in PAAD cells and cells in the tumour micro-environment. This review assesses current information regarding GPCRs in PAAD by summarizing omics data for GPCRs expression in PAAD. The PAAD "GPCRome" includes GPCRs with approved agents, thereby offering potential for their repurposing/repositioning. We then reviewed the evidence for functional roles of specific GPCRs in PAAD. We also highlight gaps in understanding the contribution of GPCRs to PAAD biology and identify several GPCRs that may be novel therapeutic targets for future work in search of GPCR-targeted drugs to treat PAAD tumours.

Detection and Quantification of GPCR mRNA: An Assessment and Implications of Data from High-Content Methods.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and targets for approved drugs. The analysis of GPCR expression is, thus, important for drug discovery and typically involves messenger RNA (mRNA)-based methods. We compared transcriptomic complementary DNA (cDNA) (Affymetrix) microarrays, RNA sequencing (RNA-seq), and quantitative polymerase chain reaction (qPCR)-based TaqMan arrays for their ability to detect and quantify expression of endoGPCRs (nonchemosensory GPCRs with endogenous agonists). In human pancreatic cancer-associated fibroblasts, RNA-seq and TaqMan arrays yielded closely correlated values for GPCR number (∼100) and expression levels, as validated by independent qPCR. By contrast, the microarrays failed to identify ∼30 such GPCRs and generated data poorly correlated with results from those methods. RNA-seq and TaqMan arrays also yielded comparable results for GPCRs in human cardiac fibroblasts, pancreatic stellate cells, cancer cell lines, and pulmonary arterial smooth muscle cells. The magnitude of mRNA expression for several Gq/11-coupled GPCRs predicted cytosolic calcium increase and cell migration by cognate agonists. RNA-seq also revealed splice variants for endoGPCRs. Thus, RNA-seq and qPCR-based arrays are much better suited than transcriptomic cDNA microarrays for assessing GPCR expression and can yield results predictive of functional responses, findings that have implications for GPCR biology and drug discovery.

GPCRomics: An Approach to Discover GPCR Drug Targets.

G protein-coupled receptors (GPCRs) are targets for ∼35% of approved drugs but only ∼15% of the ∼800 human GPCRs are currently such targets. GPCRomics, the use of unbiased, hypothesis-generating methods [e.g., RNA-sequencing (RNA-seq)], with tissues and cell types to identify and quantify GPCR expression, has led to the discovery of previously unrecognized GPCRs that contribute to functional responses and pathophysiology and that may be therapeutic targets. The combination of GPCR expression data with validation studies (e.g., signaling and functional activities) provides opportunities for the discovery of disease-relevant GPCR targets and therapeutics. Here, we review insights from GPCRomic approaches, gaps in knowledge, and future directions by which GPCRomics can advance GPCR biology and the discovery of new GPCR-targeted drugs.

GPR68: An Emerging Drug Target in Cancer.

GPR68 (or ovarian cancer G protein-coupled receptor 1, OGR1) is a proton-sensing G-protein-coupled receptor (GPCR) that responds to extracellular acidity and regulates a variety of cellular functions. Acidosis is considered a defining hallmark of the tumor microenvironment (TME). GPR68 expression is highly upregulated in numerous types of cancer. Emerging evidence has revealed that GPR68 may play crucial roles in tumor biology, including tumorigenesis, tumor growth, and metastasis. This review summarizes current knowledge regarding GPR68-its expression, regulation, signaling pathways, physiological roles, and functions it regulates in human cancers (including prostate, colon and pancreatic cancer, melanoma, medulloblastoma, and myelodysplastic syndrome). The findings provide evidence for GPR68 as a potentially novel therapeutic target but in addition, we note challenges in developing drugs that target GPR68.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets.

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.

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

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.