Mohammad Nezhady Mohammad Ali

University of Montreal, Canada

Title: HCAR1 Nuclear Location Bias Drives Cancer Malignancy by Multiple Routes

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Biography

I am a cell and molecular biologist interested in signal transduction, specially GPCR signaling. Our lab was among the first ones, to show intracellular (Nuclear) localization of a GPCR in late 90s, and was a pioneer to show that these GPCRs could elicit differential signaling output compared to their plasma membrane counterpart. In this line, I am more specifically aimed to stablish location bias as an acceptable feature in GPCR biology. Ultimately, I endeavor to expand our knowledge about location bias and raise awareness in the community to consider more tailored drug development to target and deliver drug compounds to the organelle of interest where the effect is exerted. These types of approaches would advance our drug design toward more efficacy and less side effects, and potentially uncover previously missed valuable intracellular targets. 

Abstract

GPCR are virtually involved in all physiological processes. Medically speaking, they are the most important protein family as more than 40% of all marketed drugs target GPCRs. In this regard, signaling bias has greatly advanced our knowledge of GPCR biology leading to development of a new class of drugs, namely the allosteric modulators. Allosteric modulators offer a greater specificity, efficacy and less side effects. However, there is a new concept growing in the field called location bias, and this potentially could lead to more specific drugs with higher efficiency and lower unwanted effects. Unlike the classical concept which thought GPCRs are only present on the plasma membrane, the number of GPCRs detected inside the cell are growing. GPCRs are detected in all membranous cellular organelles including, endosomes, nucleus, ER, Golgi and mitochondria. Among intracellular organelles, GPCR are mostly detected in the nucleus. Additionally, it has been shown that nuclear GPCRs can perform functions distinct from their cell surface counterparts in vivo; and this phenomenon is termed location bias.

We investigated HCAR1, as a GPCR, and showed it has a nuclear localization and this localization pattern promotes cancer malignancy by multiple routes. HCAR1 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.

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 this nuclear localization pattern. 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 Gß? 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 that is the N-HCAR1 specifically promoting 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.

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. This is the location bias of HCAR1 driving cancer malignancy pointing to the importance of targeting specifically this pool of the receptor is necessary for potential therapeutic values. Our data also shows that GPCRs might have other functions rather than signaling receptor and potentially could be involved in protein complex formation, in this case DNA damage repair and translation. Understanding these mechanisms can provide targets and cues for therapeutic developments. This differential signaling output based on location bias in a GPCR emphasizes on the tailored drug development to reach and deliver the drug to the involved organelle.