HCAR1 Nuclear Location Bias Drives Cancer Malignancy by Multiple Routes

Abstract

Introduction G-Protein Coupled Receptors (GPCR) are virtually involved in all physiological processes. HCAR1 (GPR81), as a GPCR, is endogenously activated by lactate and has been shown to promote cancer malignancy via higher level of glycolysis due to Warburg effect. Its expression level is highly elevated in many cancers and negatively correlates with patient's prognosis. However, its mechanism of action is not well understood. On the other hand, nuclear localization of several GPCRs have been described albeit it is unusual feature for them. Additionally, it has been shown that nuclear GPCRs can perform functions distinct from their cell surface counterparts in vivo. Here we demonstrate HCAR1 has a nuclear localization and this localization pattern promotes cancer malignancy by multiple routes. Methods and Results We determined HCAR1 nuclear localization pattern by cell fractionation, immunofluorescence confocal imaging and TEM. Site-directed mutagenesis showed ICL3 and phosphorylation of C-terminal domains are required for nuclear localization. We also demonstrated that this localization is ligand independent and there is a pool of nuclear HCAR1 (N-HCAR1) in the cells. We show N-HCAR1 induces intra-nuclear signaling through Gi and Gßγ by WB and ELISA leading to increased phosphorylation of nuclear ATK and ERK resulting in increased cancer cell survival and proliferation. Our ChIP-sequencing data shows N-HCAR1 binds to the genes regulating cell migration and we validated this in cellulo proving N-HCAR1 promotes migration. We identified N-HCAR1 interactome by Bio-ID coupled with mass spectrometry and found, it interacts with proteins involved in translation and DNA damage repair and our experimental data demonstrates that specifically the N-HCAR1 promotes both of those process in cellulo. Concordantly, our in vivo tumor xenografts and tail vein injections proves that tumors without N-HCAR1 have smaller size and tumor mass and lower metastatic rate as well. Conclusion Here we show an unusual localization of a GPCR in the nucleus and provide evidence that N-HCAR1 contributes to tumor malignancy by promoting classical GPCR signaling intra-nuclearly, directly regulating gene expression (opposed to signaling output) leading to increased cell migration, promoting translation and DNA damage repair, all hallmarks of cancer malignancy. The effect of N-HCAR1 is validate in vivo in tumor xenografts as well. Understanding these mechanisms can provide targets and cues for therapeutic developments.