Overexpression and enhanced signaling activity of the epithelial GPR35 isoform drives gastrointestinal tumorigenesis

G protein-coupled receptor 35 (GPR35) has been implicated in various physiological and pathological processes, including gastrointestinal diseases and cancer. However, the specific contributions of its isoforms to tumor development remain unclear. In this study, we investigated the role of GPR35 isoforms in gastrointestinal tumorigenesis, with a particular focus on the long isoform. We analyzed transcriptomic datasets, primary tissues, and cancer cell lines to determine GPR35 isoform expression patterns. Our findings reveal that the long isoform is predominantly expressed in epithelial cells of the gastrointestinal and biliary tract, whereas the short isoform is restricted to immune cells. Notably, GPR35-long expression was significantly elevated in colorectal cancer and cholangiocarcinoma compared to healthy tissues. Using immunohistochemistry and mass spectrometry, we confirmed that this isoform is translated into a distinct protein in tumor cells. To explore its functional impact, we overexpressed GPR35 isoforms in human cell lines and performed transcriptomic and metabolic analyses. GPR35-long overexpression promoted gene expression signatures associated with transport, wound healing, and metabolism. Complementary RNA-sequencing of Caco-2 cells further demonstrated that GPR35-long deletion disrupts key transcriptional programs, including pathways related to mitochondrial metabolism, cytokine signaling, and extracellular matrix remodeling. These results underscore the ligand-responsive role of GPR35-long in maintaining oncogenic transcriptional states in epithelial cells. Furthermore, metabolic assays demonstrated that GPR35-long enhances both mitochondrial and glycolytic ATP production, supporting increased cellular energy demands. Functional assays revealed that GPR35-long exhibits heightened constitutive and ligand-induced activity, leading to enhanced signaling through beta-arrestin. Importantly, the small molecule CID-2745687 effectively reduced GPR35-long-driven metabolic activity and proliferation, identifying it as a potential therapeutic candidate. Our findings establish GPR35-long as a key driver of gastrointestinal tumorigenesis and highlight its potential as a biomarker and therapeutic target in gastrointestinal malignancies.