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G Protein‐Coupled Receptor GPR55 Promotes Colorectal Cancer and Has Opposing Effects to Cannabinoid Receptor 1

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G Protein‐Coupled Receptor GPR55 Promotes Colorectal Cancer and Has Opposing Effects to Cannabinoid Receptor 1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UCL Discovery G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1 Short title: GPR55 drives colorectal cancer Carina Hasenoehrl1, David Feuersinger1, Eva M Sturm1, Thomas Bärnthaler1, Ellen Heitzer2, Ricarda Graf2, Magdalena Grill1, Martin Pichler3, Stephan Beck4, Lee Butcher4, Dominique Thomas5, Nerea Ferreirós5, Rufina Schuligoi1, Caroline Schweiger6, Johannes Haybaeck6,7 and Rudolf Schicho1,8 * 1Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, ustria. 2Institute of Human Genetics, Medical University of Graz, Graz, Austria. 3Department of Internal Medicine, Division of Oncology, Medical University of Graz, Graz, Austria. 4UCL Cancer Institute, University College London, London, United )ingdom. 5Institute of Clinical Pharmacology, Goethe University, Frankfurt-Main, Germany. .Institute of Pathology, Medical University of Graz, Graz, ustria. 7Department of Pathology, Otto von Gueric,e University, Magdeburg, Germany. 12ioTechMed, Graz, Austria. *Corresponding author: Rudolf Schicho (Institute for Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4/I, 8010 Graz, Austria; phone: +433163807851; [email protected]) Key ords: GPR55; CB1; CNR1; colorectal carcinogenesis Abbreviations: AOM, azoxymethane; CB, Cannabinoid receptor; CD, cluster of differentiation; COX- 2, c0cloo80genase-2; CRC, colorectal cancer; DSS, de8tran sulfate sodium; F S, fetal 1ovine serum; GI, gastrointestinal; GPR55, G protein-coupled receptor 55; H SS, HankEs 1alanced salt solution; HEPES, 4-62-h0dro80eth0l)-1-piperazineethanesulfonic acid; IL, interleukin; LPI, L-F? l0sophosphatid0linositol; MDSC, m0eloid-derived suppressor cell; NF-G , nuclear factor Hkappa-light- chain-enhancerE of activated -cells; P S, phosphate-1uffered saline; PGF2α, prostaglandin F2alpha; RPIM, reads per kilo1ase per million mapped reads; STAT3, signal transducer and activator of transcription 3; TD 2, throm1o8ane 2 Article category5 Tumor immunology and microenvironment Novelty and Impact: Preclinical evidence suggests that activation of cannabinoid receptor 1 6C 1) has antitumor effects in colorectal cancer 6CRC). GPR55 shares endogenous and s0nthetic ligands with C 1 and was found to promote CRC in this study b0 mechanisms that involve modulation of tumor-promoting factors. Additionall0, differential regulation of DNA methylation and expression of C 1 and GPR55 was observed in human CRC patients, suggesting opposing roles of two cannabinoidresponsive receptors. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.31030 This article is protected by copyright. All rights reserved. International Journal of Cancer Page 2 of 29 Abstract The putative cannabinoid receptor GPR55 has 1een shown to play a tumor-promoting role in various cancers, and is involved in many ph0siological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 6C 1) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. 9sing azox0methane (ACM)- and de8tran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 pla0s a tumor-promoting role that involves alterations of leukoc0te populations, i.e. m0eloidderived suppressor cells and T l0mphoc0tes, within the tumor tissues. Concomitantl0, e8pression levels of CCD-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. 0 emplo0ing the experimental CRC models to C 1 knockout and C 1/GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to C 1. We report that GPR55 and C 1 mRNA expression are differentiall0 regulated in the e8perimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentiall0 regulated in human CRC tissue compared to control samples. Collectivel0, our data suggest that GPR55 and C 1 play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor. Introduction Despite benefits from earl0 screening, colorectal cancer (CRC) remains a leading cause among cancer-related deaths emphasizing the need for new treatment options.1 Recent studies indicate that in addition to traditional chemotherapy, immunotherap0 may become a valuable option of treatment against CRC.2 Knowledge on leukoc0tes, inflammator0 mediators and their receptors within the tumor microenvironment is therefore crucial in order to explore and design new immunotherapies. Although CRC can be traced back to a familial basis such as adenomatous pol0posis in some cases, the majority of incidences are sporadic cases and likel0 caused b0 environmental components and by inflammation of the colon 6reviewed in 3). For instance, c0cloox0genase 6CCD)-2derived mediators, transcription factors like STAT3 and NF-G , and c0tokines involved in intestinal inflammation, are now regarded as driving factors in colon carcinogenesis3, suggesting that molecules involved in gastrointestinal (GI) homeostasis are also critical in the development of GI cancer. 2 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved. In this context, the endocannabinoid s0stem is of great interest as it plays a prominent role in 4 physiological and pathoph0siological processes of the GI tract . Cannabinoid 6C ) receptors, in particular C 1, but also other cannabinoid-responsive G proteincoupled receptors 6GPCRs), such as the G proteincoupled receptor 55 (GPR55), are expressed in the GI tract 4,5 and they are fundamentall0 involved e.g. in the regulation of motilit0 and inflammation . In .,2 experimental models of colitis, C 1 and C 2 receptors exert anti-inflammator0 properties 8 while GPR55 has been linked to pro-inflammator0 processes . Genetic deletion of the C 1 min-6 9 receptor provokes a strong increase in intestinal tumors in pc mice . The role of GPR55 in GI carcinogenesis, however, has not been full0 elucidated so far. Activity of GPR55 has been shown to be modulated by the endocannabinoids anandamide 10 11 and virodhamine . In turn, C 1 ma0 regulate the signaling properties of GPR55 . Thus, 12 despite sharing little homolog0 with the classical C receptors , GPR55 can be considered part of an expanded endocannabinoid s0stem. Contrar0 to C receptors, GPR55 initiates 13 excitator0 rather than inhibitor0 effects by signaling through GF12/13 and Gq proteins , which 14 are known to promote carcinogenesis . Its structure is closel0 related to other cancer- 15 relevant GPCRs, such as GPR35, GPR92 and GPR23 . GPR55 expression has been demonstrated in several t0pes of tumor cell lines, such as breast, prostate, ovarian and colon 1.M18 cancer cells . In vivo, the receptor promotes carcinogenesis in a mouse model of skin 19 20 cancer . With the exception of cholangiocarcinoma , a majority of studies propose a pro- 1.M19 oncogenic role for GPR55 . Beside cancer cells, GPR55 is e8pressed in various t0pes of 8 21 22 leukoc0tes, such as macrophages , neutrophils , and l0mphoc0tes . Recentl0, we showed that the receptor drives intestinal inflammation via mechanisms involving leukoc0te 8 infiltration . We have further elucidated an involvement of GPR55 in migration and adhesion 18 of colon cancer cells and in liver metastasis . These effects were sensitive to the GPR55 11,18 antagonist CID16020046 . We have also found that levels of the endogenous ligand, L-F- 23 l0sophosphatid0linositol (LPI) were higher in blood samples of colon cancer patients than in 18 control individuals . Cther groups demonstrated an important role of the LPI-GPR55 axis in 24,25 breast cancer metastasis . ecause of its potentiall0 procarcinogenic behavior and its 8 actions within the endocannabinoid s0stem contrar0 to those of C 1 , we s0stemicall0 -/- explored the role of GPR55 in GPR55 knockout (GPR55 ) and wildt0pe mice using a mouse model of azox0methane (ACM)- and dextran sulfate sodium (DSS)-induced CRC that displa0s robust infiltration of inflammator0 cells, a feature not onl0 seen in colorectal tumors 3 connected to inflammator0 bowel diseases (7 D) but also in CRC not associated with 7 D . -/- -/- We also created C 1/GPR55 double knockout 6C 1 GPR55 ) mice and performed experiments in these mice. -/- Cur findings reveal that tumor burden in the colon of GPR55 mice was lower than in wild -/- types and this was in contrast to what we observed in C 1 knockouts (C 1 ) which displa0ed 3 This article is protected by copyright. All rights reserved. International Journal of Cancer Page 4 of 29 -/- -/- higher tumor burden in the colon. In the C 1 GPR55 mice, these effects were compensated as their tumor burden did not differ from that of their wildt0pe littermates. 9nexpectedl0, pharmacological manipulation of GPR55 in colon cancer cell lines failed to show effects on proliferation. However, in comparison with wildt0pe littermates, tumors of GPR55-/- mice revealed downregulation of CCD-2 and STAT3, increased influ8 of
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