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Illuminating the Onco-GPCRome: Novel G protein–coupled receptor-driven oncocrine networks and targets for cancer immunotherapy

  • Victoria Wu
    Affiliations
    Department of Pharmacology, UCSD Moores Cancer Center, La Jolla, California 92093
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  • Huwate Yeerna
    Affiliations
    Department of Medicine, UCSD Moores Cancer Center, La Jolla, California 92093
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  • Author Footnotes
    1 Present address: Oncology Science Unit, MSD K.K., Tokyo 102-8667, Japan.
    Nijiro Nohata
    Footnotes
    1 Present address: Oncology Science Unit, MSD K.K., Tokyo 102-8667, Japan.
    Affiliations
    Department of Pharmacology, UCSD Moores Cancer Center, La Jolla, California 92093
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  • Joshua Chiou
    Affiliations
    Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, California 92093
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  • Olivier Harismendy
    Affiliations
    Department of Medicine, UCSD Moores Cancer Center, La Jolla, California 92093

    Department of Medicine, UCSD Moores Cancer Center, La Jolla, California 92093
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  • Author Footnotes
    2 Supported by an Alexander Von Humboldt post-doctoral fellowship.
    ,
    Author Footnotes
    3 Supported by the Cell Networks Excellence Initiative of the Germany Research Foundation (DFG) and a Michael J. Fox Foundation Research Grant.
    Francesco Raimondi
    Footnotes
    2 Supported by an Alexander Von Humboldt post-doctoral fellowship.
    3 Supported by the Cell Networks Excellence Initiative of the Germany Research Foundation (DFG) and a Michael J. Fox Foundation Research Grant.
    Affiliations
    CellNetworks, Bioquant, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

    Biochemie Zentrum Heidelberg (BZH), Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
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  • Author Footnotes
    4 Supported by JSPS KAKENHI Grant 17K08264, the PRIME JP17gm5910013, and the LEAP JP17gm0010004 from the Japan Agency for Medical Research and Development (AMED).
    Asuka Inoue
    Footnotes
    4 Supported by JSPS KAKENHI Grant 17K08264, the PRIME JP17gm5910013, and the LEAP JP17gm0010004 from the Japan Agency for Medical Research and Development (AMED).
    Affiliations
    Graduate School of Pharmaceutical Science, Tohoku University, Sendai, Miyagi 980-8578, Japan
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  • Author Footnotes
    3 Supported by the Cell Networks Excellence Initiative of the Germany Research Foundation (DFG) and a Michael J. Fox Foundation Research Grant.
    ,
    Author Footnotes
    5 Part of the Germany Research Foundation SFB/TPR186 Molecular Switches in the Spatio-Temporal Control of Cellular Signal Transmission and the BMBF German Network for Bioinformatics (de.NBI).
    Robert B. Russell
    Footnotes
    3 Supported by the Cell Networks Excellence Initiative of the Germany Research Foundation (DFG) and a Michael J. Fox Foundation Research Grant.
    5 Part of the Germany Research Foundation SFB/TPR186 Molecular Switches in the Spatio-Temporal Control of Cellular Signal Transmission and the BMBF German Network for Bioinformatics (de.NBI).
    Affiliations
    CellNetworks, Bioquant, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

    Biochemie Zentrum Heidelberg (BZH), Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

    Division of Biomedical Informatics, University of California San Diego School of Medicine, La Jolla, California 92093
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  • Pablo Tamayo
    Affiliations
    Department of Medicine, UCSD Moores Cancer Center, La Jolla, California 92093
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  • J. Silvio Gutkind
    Correspondence
    To whom correspondence should be addressed. Tel.: 858-534-5980
    Affiliations
    Department of Pharmacology, UCSD Moores Cancer Center, La Jolla, California 92093
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  • Author Footnotes
    1 Present address: Oncology Science Unit, MSD K.K., Tokyo 102-8667, Japan.
    2 Supported by an Alexander Von Humboldt post-doctoral fellowship.
    3 Supported by the Cell Networks Excellence Initiative of the Germany Research Foundation (DFG) and a Michael J. Fox Foundation Research Grant.
    4 Supported by JSPS KAKENHI Grant 17K08264, the PRIME JP17gm5910013, and the LEAP JP17gm0010004 from the Japan Agency for Medical Research and Development (AMED).
    5 Part of the Germany Research Foundation SFB/TPR186 Molecular Switches in the Spatio-Temporal Control of Cellular Signal Transmission and the BMBF German Network for Bioinformatics (de.NBI).
Open AccessPublished:June 05, 2019DOI:https://doi.org/10.1074/jbc.REV119.005601
      G protein–coupled receptors (GPCRs) are the largest gene family of cell membrane–associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established. The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception (e.g. vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits. Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development. However, the study of the role of GPCRs in tumor biology has only just begun to make headway. Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion. Indeed, GPCRs are widely dysregulated in cancer and yet are underexploited in oncology. We present here a comprehensive analysis of GPCR gene expression, copy number variation, and mutational signatures in 33 cancer types. We also highlight the emerging role of GPCRs as part of oncocrine networks promoting tumor growth, dissemination, and immune evasion, and we stress the potential benefits of targeting GPCRs and their signaling circuits in the new era of precision medicine and cancer immunotherapies.

      Introduction

      The G protein–coupled receptor (GPCR)
      abbreviations used are: GPCR
      G protein–coupled receptor
      7TM
      7 transmembrane
      CNV
      copy number variation
      COAD
      colon adenocarcinoma
      COX
      cyclooxygenase
      DC
      dendritic cell
      DLBC
      diffuse large B-cell lymphoma
      GBM
      glioblastoma multiforme
      GEF
      guanine nucleotide exchange factor
      GISTIC
      Genomic Identification of Significant Targets in Cancer
      GOF
      gain–of–function
      KSHV
      Kaposi's sarcoma herpesvirus
      LOF
      loss–of–function
      MDSC
      myeloid-derived suppressor cell
      NGF
      nerve growth factor
      NK
      natural killer cell
      NSAID
      nonsteroidal anti-inflammatory drug
      OV
      ovarian serous cystadenocarcinoma
      PAAD
      pancreatic adenocarcinoma
      PD-1
      programmed cell death protein 1
      PGE2
      prostaglandin E2
      PKA
      protein kinase A
      SHH
      Sonic hedgehog
      SKCM
      skin cutaneous melanoma
      SMO
      Smoothened
      STAD
      stomach adenocarcinoma
      TAM
      tumor-associated macrophage
      TCGA
      Cancer Genome Atlas
      Treg
      regulatory T cell
      UVM
      uveal melanoma
      VEGF
      vascular endothelial growth factor
      FDA
      Food and Drug Administration
      mAChR
      muscarinic acetylcholine receptor
      FZD
      Frizzled
      TSHR
      thyroid-stimulating hormone receptor
      PAR
      protease-activated receptor
      GRP
      gastrin-releasing peptide
      NMB
      neuromedin B
      SCLC
      small cell lung cancer
      CTCL
      cutaneous T-cell lymphoma
      FAK
      focal adhesion kinase
      MAPK
      mitogen-activated protein kinase
      DAG
      diacylglycerol
      mTOR
      mammalian target of rapamycin
      LPA
      lysophosphatidic acid
      S1P
      sphingosine 1-phosphate
      BCC
      basal cell carcinoma
      YAP
      Yes-associated protein
      ERK
      for extracellular signal-regulated kinase
      PI3K
      phosphatidylinositol 3-kinase
      GI
      gastrointestinal
      IL
      interleukin
      MHC
      major histocompatibility complex
      EGFR
      epidermal growth factor receptor.
      family of proteins includes over 800 members and comprises ∼4% of the encoded human genome, making it the largest gene family involved in signal transduction (
      • Fredriksson R.
      • Lagerström M.C.
      • Lundin L.G.
      • Schiöth H.B.
      The G-protein–coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.
      ,
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ). Common to all GPCRs is the 7-transmembrane domain structure, which has an extracellular N terminus and an intracellular C terminus. The importance of the multiple biological roles GPCRs is reflected in the range of key physiological processes that they regulate, including vision, olfaction, neurotransmission, hormone and enzyme release, immune response, hemostasis, cardiac response and blood pressure regulation, epithelial cell renewal, stem cell fate decisions, tissue development, and homeostasis. In fact, dysfunction of GPCRs contributes to some of the most prevalent human diseases, which is reflected by the 475 currently approved drugs that target 108 unique GPCRs and represent 34% of all FDA-approved drugs (https://www.centerwatch.com/drug-information/fda-approved-drugs
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      (
      • Hauser A.S.
      • Attwood M.M.
      • Rask-Andersen M.
      • Schiöth H.B.
      • Gloriam D.E.
      Trends in GPCR drug discovery: new agents, targets and indications.
      ,
      • Santos R.
      • Ursu O.
      • Gaulton A.
      • Bento A.P.
      • Donadi R.S.
      • Bologa C.G.
      • Karlsson A.
      • Al-Lazikani B.
      • Hersey A.
      • Oprea T.I.
      • Overington J.P.
      A comprehensive map of molecular drug targets.
      ). Although drugs for GPCRs represent ∼34% of the global therapeutic drug market (
      • Hauser A.S.
      • Attwood M.M.
      • Rask-Andersen M.
      • Schiöth H.B.
      • Gloriam D.E.
      Trends in GPCR drug discovery: new agents, targets and indications.
      ,
      • Santos R.
      • Ursu O.
      • Gaulton A.
      • Bento A.P.
      • Donadi R.S.
      • Bologa C.G.
      • Karlsson A.
      • Al-Lazikani B.
      • Hersey A.
      • Oprea T.I.
      • Overington J.P.
      A comprehensive map of molecular drug targets.
      ), only a handful of these are drugs for oncology; of the current FDA-approved anti-cancer drugs, only eight of these target GPCRs, as described in detail below. GPCRs have been a longstanding topic of interest in the Journal of Biological Chemistry, and here we will expand on the impact of GPCRs in cancer biology. This review will summarize the current knowledge of how GPCRs are altered in cancer and how these aberrations can contribute to cancer initiation and progression. We also bring forth an emerging role of GPCRs as part of autocrine and paracrine signaling processes, which we refer to collectively as oncocrine networks that drive tumor formation, growth, and immune evasion. We also highlight the potential benefits of targeting GPCRs in the new era of precision cancer immunotherapies.

      Historical perspective

      The first evidence demonstrating a role for GPCRs in tumorigenesis came over 30 years ago in 1986 when studies illustrated that the GPCR encoded by the Mas1 gene (MAS1) produced tumors in nude mice (
      • Young D.
      • Waitches G.
      • Birchmeier C.
      • Fasano O.
      • Wigler M.
      Isolation and characterization of a new cellular oncogene encoding a protein with multiple potential transmembrane domains.
      ). This finding was largely underappreciated, likely because in contrast to most oncogenes discovered at the time, these receptors did not harbor activating mutations, similarly to the behavior of WT 5HT1c receptors (HTR1C) that resulted in NIH3T3 cell transformation (
      • Julius D.
      • Livelli T.J.
      • Jessell T.M.
      • Axel R.
      Ectopic expression of the serotonin 1c receptor and the triggering of malignant transformation.
      ). Further work, however, revealed that WT GPCRs can become tumorigenic in a ligand-dependent fashion. This was best demonstrated in 1991 in studies depicting the oncogenic transforming ability of mAChRs in NIH3T3 cells only in combination with the agonist, carbachol, and exclusively for Gαq-coupled mAChR subtypes (M1, M3, and M5, gene names CHRM1, CHRM3, and CHRM5, respectively) (
      • Gutkind J.S.
      • Novotny E.A.
      • Brann M.R.
      • Robbins K.C.
      Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes.
      ). With this, these studies brought to light the possibility of G protein–dependent oncogenic roles for GPCRs when activated by locally produced or circulating ligands and raised the possibility that activating mutations in key conserved GPCR residues could result in transforming potential even without agonist stimulation.
      These early studies introduced GPCRs as a new class of receptors capable of oncogenic transformation. Aligned with this possibility, mutational alteration of α1B adrenergic receptor (ADRA1B) can lead to transformation, providing an enhanced ability for tumor generation in nude mice (
      • Allen L.F.
      • Lefkowitz R.J.
      • Caron M.G.
      • Cotecchia S.
      G-protein–coupled receptor genes as protooncogenes: constitutively activating mutation of the α1B-adrenergic receptor enhances mitogenesis and tumorigenicity.
      ). The identification of activating mutations in the thyrotropin receptor gene (TSHR) in hyperfunctioning thyroid adenomas provided the first evidence that mutant GPCRs can initiate a neoplastic disease (
      • Parma J.
      • Duprez L.
      • Van Sande J.
      • Cochaux P.
      • Gervy C.
      • Mockel J.
      • Dumont J.
      • Vassart G.
      Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas [see comments].
      ). Downstream of the receptor, somatic mutations that impair the GTPase activity of Gαs conferred constitutive activation of adenylyl cyclase, leading to development of hyperfunctioning thyroid adenomas and pituitary tumors (
      • Landis C.A.
      • Masters S.B.
      • Spada A.
      • Pace A.M.
      • Bourne H.R.
      • Vallar L.
      GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.
      ,
      • Lyons J.
      • Landis C.A.
      • Harsh G.
      • Vallar L.
      • Grünewald K.
      • Feichtinger H.
      • Duh Q.Y.
      • Clark O.H.
      • Kawasaki E.
      • Bourne H.R.
      Two G protein oncogenes in human endocrine tumors.
      • Esapa C.
      • Foster S.
      • Johnson S.
      • Jameson J.L.
      • Kendall-Taylor P.
      • Harris P.E.
      G protein and thyrotropin receptor mutations in thyroid neoplasia.
      ). Although these lesions are benign in nature, and hence often neglected in cancer biology, recent studies demonstrated similar activating mutations in the Gαs-encoding gene (GNAS oncogene) in multiple cancer types, including pancreatic and colorectal cancer (
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G proteins and G-protein–coupled receptors in cancer.
      ,
      • Wu J.
      • Matthaei H.
      • Maitra A.
      • Dal Molin M.
      • Wood L.D.
      • Eshleman J.R.
      • Goggins M.
      • Canto M.I.
      • Schulick R.D.
      • Edil B.H.
      • Wolfgang C.L.
      • Klein A.P.
      • Diaz Jr., L.A.
      • Allen P.J.
      • Schmidt C.M.
      • et al.
      Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development.
      • Parish A.J.
      • Nguyen V.
      • Goodman A.M.
      • Murugesan K.
      • Frampton G.M.
      • Kurzrock R.
      GNAS, GNAQ, and GNA11 alterations in patients with diverse cancers.
      ). In addition, our systematic analysis of the transforming potential of G proteins revealed that the genes encoding the Gαq/11 (GNAQ and GNA11) and Gα12/13 (GNA12 and GNA13) G protein α subunits harbor transforming potential (
      • Kalinec G.
      • Nazarali A.J.
      • Hermouet S.
      • Xu N.
      • Gutkind J.S.
      Mutated α subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.
      ,
      • Fromm C.
      • Coso O.A.
      • Montaner S.
      • Xu N.
      • Gutkind J.S.
      The small GTP-binding protein Rho links G protein–coupled receptors and Gα12 to the serum response element and to cellular transformation.
      • Xu N.
      • Bradley L.
      • Ambdukar I.
      • Gutkind J.S.
      A mutant α subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells.
      ), thus contributing to the more recent discovery of multiple G protein–driven cancers (see below).
      Remarkably, many human cancer-associated viruses utilize GPCR signaling for their life cycle. These include Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) (
      • Arvanitakis L.
      • Geras-Raaka E.
      • Varma A.
      • Gershengorn M.C.
      • Cesarman E.
      Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.
      ,
      • Bais C.
      • Santomasso B.
      • Coso O.
      • Arvanitakis L.
      • Raaka E.G.
      • Gutkind J.S.
      • Asch A.S.
      • Cesarman E.
      • Mesri E.A.
      • Gershengorn M.C.
      G-protein–coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.
      ), human cytomegalovirus (
      • Chee M.S.
      • Satchwell S.C.
      • Preddie E.
      • Weston K.M.
      • Barrell B.G.
      Human cytomegalovirus encodes three G protein–coupled receptor homologues.
      ,
      • Maussang D.
      • Verzijl D.
      • van Walsum M.
      • Leurs R.
      • Holl J.
      • Pleskoff O.
      • Michel D.
      • van Dongen G.A.
      • Smit M.J.
      Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis.
      ), and Epstein-Barr virus (
      • Paulsen S.J.
      • Rosenkilde M.M.
      • Eugen-Olsen J.
      • Kledal T.N.
      Epstein-Barr virus-encoded BILF1 is a constitutively active G protein–coupled receptor.
      ), which encode receptors in their genomes that resemble human chemokine receptors, and deploy them to recruit immune cells and exploit the immune system for viral dissemination (reviewed in Ref.
      • Sodhi A.
      • Montaner S.
      • Gutkind J.S.
      Viral hijacking of G-protein–coupled-receptor signalling networks.
      ). Specifically, the discovery that the GPCR encoded by KSHV/HHV8, often referred to as vGPCR or ORF74, initiates Kaposi's sarcomagenesis provided the first link between GPCRs and virally-associated human malignancies (
      • Bais C.
      • Santomasso B.
      • Coso O.
      • Arvanitakis L.
      • Raaka E.G.
      • Gutkind J.S.
      • Asch A.S.
      • Cesarman E.
      • Mesri E.A.
      • Gershengorn M.C.
      G-protein–coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.
      ,
      • Montaner S.
      • Sodhi A.
      • Molinolo A.
      • Bugge T.H.
      • Sawai E.T.
      • He Y.
      • Li Y.
      • Ray P.E.
      • Gutkind J.S.
      Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes.
      ). Viral GPCRs can signal through Gα proteins independent of ligand activation, and they take advantage of this “constitutive activation” to promote tumorigenesis and aid in tumor survival, growth, and metastasis (
      • Sodhi A.
      • Montaner S.
      • Gutkind J.S.
      Viral hijacking of G-protein–coupled-receptor signalling networks.
      ,
      • Zhang J.
      • Feng H.
      • Xu S.
      • Feng P.
      Hijacking GPCRs by viral pathogens and tumor.
      ). The dawn of these studies opened a new door to establish the link between GPCRs and cancers.
      Despite this large body of information, GPCRs were generally not thought to represent traditional “genetic drivers” in cancer, thus pursuing GPCRs in oncology was neglected for some time. In the past decade, however, studies bloomed linking GPCRs to many cancers and mechanisms of tumorigenesis, metastasis, and immune evasion. The goal of the comprehensive expression, mutation, and copy number alteration omics information presented in this review is to shed light on understudied GPCRs and G proteins in different cancers and, for leading experts in studying particular cancers, to direct more attention in considering GPCRs as potential therapeutic targets.

      Canonical and noncanonical G protein and GPCR signaling

      As a result of the use of alignment tools and gene ontology, 342 functional nonolfactory human GPCRs (
      • Fredriksson R.
      • Lagerström M.C.
      • Lundin L.G.
      • Schiöth H.B.
      The G-protein–coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.
      ) have been documented and further classified into major receptor families based on sequence similarity and function (
      • Kolakowski Jr., L.F.
      GCRDb: a G-protein–coupled receptor database.
      ,
      • Schiöth H.B.
      • Fredriksson R.
      The GRAFS classification system of G-protein coupled receptors in comparative perspective.
      ) (http://gpcrdb.org/). To date, over 600 inactivating and almost 100 activating mutations in GPCRs have been identified, which are responsible for more than 30 different human diseases (
      • Thompson M.D.
      • Hendy G.N.
      • Percy M.E.
      • Bichet D.G.
      • Cole D.E.
      G protein–coupled receptor mutations and human genetic disease.
      ). Consequently, GPCRs have remained a long-standing interest as pharmacological targets.
      GPCRs bind a wide variety of agonists, including ions, amines, purines, lipids, peptides, and proteins. Upon agonist binding, a conformational change is induced in the extracellular loops of the transmembrane region for ligand binding and in the intracellular loops (primarily in the second, third, and fourth loops), which promotes receptor activation and G protein coupling (
      • Rasmussen S.G.
      • DeVree B.T.
      • Zou Y.
      • Kruse A.C.
      • Chung K.Y.
      • Kobilka T.S.
      • Thian F.S.
      • Chae P.S.
      • Pardon E.
      • Calinski D.
      • Mathiesen J.M.
      • Shah S.T.
      • Lyons J.A.
      • Caffrey M.
      • Gellman S.H.
      • et al.
      Crystal structure of the β2 adrenergic receptor-Gs protein complex.
      ,
      • Hilger D.
      • Masureel M.
      • Kobilka B.K.
      Structure and dynamics of GPCR signaling complexes.
      • Wootten D.
      • Christopoulos A.
      • Marti-Solano M.
      • Babu M.M.
      • Sexton P.M.
      Mechanisms of signalling and biased agonism in G protein–coupled receptors.
      ). The basic signaling unit of a GPCR system includes five main components: the receptor; the trimeric αβγ G protein; an effector; RGSs (regulators of G protein signaling) that accelerate GTP hydrolysis and inactivate G proteins; and arrestins that control receptor fate and signal modulation (
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ). Once activated, the receptor binds the heterotrimeric G proteins, which promotes the release of GDP from the Gα subunit and the exchange for GTP and the functional dissociation of the GTP-bound α subunit from βγ dimers (
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ,
      • Hilger D.
      • Masureel M.
      • Kobilka B.K.
      Structure and dynamics of GPCR signaling complexes.
      ). Both parts remain attached to the plasma membrane but free from the GPCR to interact with downstream signaling proteins.
      A defining feature of GPCRs is the ability to activate one or multiple Gα proteins, which can be subdivided into four major families based on sequence similarity: Gαs, Gαi, Gαq/11, and Gα12/13 (Fig. 1). As reviewed previously (
      • O'Hayre M.
      • Degese M.S.
      • Gutkind J.S.
      Novel insights into G protein and G protein–coupled receptor signaling in cancer.
      ,
      • Dorsam R.T.
      • Gutkind J.S.
      G-protein–coupled receptors and cancer.
      ), Gαs activates adenylyl cyclases to catalyze the conversion of ATP to cAMP, which is produced as a second messenger and activates protein kinase A (PKA) and in some cells guanine nucleotide exchange factors (GEFs) for the small GTPase RAP1. Members of the Gαi family primarily inhibit cAMP production, activate a variety of phospholipases and phosphodiesterases, and promote the opening of several ion channels. The Gαq/11 family converts phosphatidylinositol 4,5-bisphosphate to DAG and inositol 1,4,5-trisphosphate to activate PKC and elevates intracellular Ca2+ levels. In a noncanonical fashion, Gαq/11 also stimulates Rho GEFs thereby stimulating Rho GTPases (
      • Chikumi H.
      • Vázquez-Prado J.
      • Servitja J.M.
      • Miyazaki H.
      • Gutkind J.S.
      Potent activation of RhoA by Gαq and Gq-coupled receptors.
      ,
      • Vaqué J.P.
      • Dorsam R.T.
      • Feng X.
      • Iglesias-Bartolome R.
      • Forsthoefel D.J.
      • Chen Q.
      • Debant A.
      • Seeger M.A.
      • Ksander B.R.
      • Teramoto H.
      • Gutkind J.S.
      A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein–coupled receptors.
      ), whereas DAG activates Ras-GEFs (
      • Chen X.
      • Wu Q.
      • Depeille P.
      • Chen P.
      • Thornton S.
      • Kalirai H.
      • Coupland S.E.
      • Roose J.P.
      • Bastian B.C.
      RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma.
      ). Gα12/13 signaling involves a family of RhoGEFs harboring an RGS domain by which they associate with active Gα12/13 and stimulate Rho GTPase (reviewed in Ref.
      • Vázquez-Prado J.
      • Basile J.
      • Gutkind J.S.
      Modular architecture and novel protein–protein interactions regulating the RGS-containing Rho guanine nucleotide exchange factors.
      ). In turn, as depicted in Fig. 1, the coordinated activation of second messenger systems and Rho and Ras GTPases will result in the stimulation of multiple kinase cascades regulating key cellular functions. These include one or more members of the mitogen-activated protein kinases (MAPK) (e.g. ERK1 and ERK2, JNK1–3, p38α-δ, and ERK5, AKT, and mTOR), second messenger–regulated kinases (e.g. PKA, PKC, PKD, PKG, and CAMKs) and phosphatases (e.g. calcineurin), and multiple kinases regulated by Rho (e.g. ROCK, LIMK, PKN, Citron kinase, PAKs, and MLKs) and Ras (e.g. BRAF, ARAF, and CRAF) GTPases, which in turn regulate nuclear events contributing to normal and malignant cell growth (reviewed in Refs.
      • O'Hayre M.
      • Degese M.S.
      • Gutkind J.S.
      Novel insights into G protein and G protein–coupled receptor signaling in cancer.
      ,
      • Dorsam R.T.
      • Gutkind J.S.
      G-protein–coupled receptors and cancer.
      ). In addition, Gαs-coupled receptors activate and Gα12/13-, Gαi-, and Gαq/11-coupled receptors inhibit LATS1/2 kinases, which are key components of the recently described Hippo kinase cascade (
      • Yu F.X.
      • Zhao B.
      • Panupinthu N.
      • Jewell J.L.
      • Lian I.
      • Wang L.H.
      • Zhao J.
      • Yuan H.
      • Tumaneng K.
      • Li H.
      • Fu X.D.
      • Mills G.B.
      • Guan K.L.
      Regulation of the Hippo-YAP pathway by G-protein–coupled receptor signaling.
      ). LATS kinases phosphorylate and inhibit the transcription coactivator Yes-associated protein (YAP) and its related protein, TAZ, thereby causing their cytoplasmic retention and degradation (
      • Pan D.
      The Hippo signaling pathway in development and cancer.
      ). By inhibiting LATS1/2, Gαq- and Gα12/13-coupled GPCRs stimulate the ability of YAP/TAZ to promote the expression growth and anti-apoptotic genes (
      • Yu F.X.
      • Zhao B.
      • Panupinthu N.
      • Jewell J.L.
      • Lian I.
      • Wang L.H.
      • Zhao J.
      • Yuan H.
      • Tumaneng K.
      • Li H.
      • Fu X.D.
      • Mills G.B.
      • Guan K.L.
      Regulation of the Hippo-YAP pathway by G-protein–coupled receptor signaling.
      ). See below for exciting new information on how oncogenic Gαq proteins regulate the Hippo pathway and its therapeutic potential for Gαq-driven malignancies.
      Figure thumbnail gr1
      Figure 1GPCR signaling. Agonist-activated GPCRs promote the dissociation of GDP bound to the α subunit of heterotrimeric G proteins and its replacement by GTP. Gα and Gβγ subunits can then activate numerous downstream effectors. The 16 human G protein α subunits can be divided into the four subfamilies, and a single GPCR can couple to one or more families of Gα subunits. Downstream effectors regulated by their targets include a variety of second messenger systems (red), GEFs (yellow), and Rho and Ras GTPases (green), which will result in the stimulation of multiple kinase cascades (blue) regulating key cellular functions. These include members of the MAPK, AKT, and mTOR, second messenger regulated kinases and phosphatases, and multiple kinases regulated by Rho and Ras GTPases. In addition, Gαs-coupled receptors inhibit and Gα12/13-, Gαi-, and Gαq/11-coupled receptors activate the transcription coactivator YAP and its related protein TAZ, the most downstream targets of the Hippo kinase cascade, as well as β-catenin and the Shh pathway, among others. Ultimately, these large numbers of effector molecules can have multiple effects in the cytosol and nucleus to regulate gene expression, cell metabolism, migration, proliferation, and survival by GPCRs, which can contribute to normal and malignant cell growth. See text for details.
      Once functionally dissociated from the Gα protein, Gβγ dimers also play a central signaling role, first described in the context of ion channel regulation. For example, Gβγ can inhibit some voltage-activated Ca2+ channels and activate G protein–activated inwardly rectifying K channels (GIRKs) (
      • Dascal N.
      • Kahanovitch U.
      The roles of Gβγ and Gα in gating and regulation of GIRK channels.
      ). In the context of cancer signaling, Gβγ dimers were initially shown to mediate the activation of ERK downstream from GPCRs linked to Gαi (
      • Crespo P.
      • Xu N.
      • Simonds W.F.
      • Gutkind J.S.
      Ras-dependent activation of MAP kinase pathway mediated by G-protein βγ subunits.
      ). We now know that Gβγ stimulates PLCβ, adenylyl cyclases, PI3Ks (primarily PI3Kγ and PI3Kβ in cells lacking PI3Kγ expression) and GEFs stimulating the small GTPase Rac, such as PREX1 (
      • O'Hayre M.
      • Degese M.S.
      • Gutkind J.S.
      Novel insights into G protein and G protein–coupled receptor signaling in cancer.
      ). By doing so, Gβγ signaling contributes to the prosurvival and migratory activity of many GPCRs, with emphasis on chemokine receptors involved in cancer metastasis, such as CXCR4 (see below and Ref.
      • Sosa M.S.
      • Lopez-Haber C.
      • Yang C.
      • Wang H.
      • Lemmon M.A.
      • Busillo J.M.
      • Luo J.
      • Benovic J.L.
      • Klein-Szanto A.
      • Yagi H.
      • Gutkind J.S.
      • Parsons R.E.
      • Kazanietz M.G.
      Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.
      ).
      Ultimately, the signaling pathways stimulated by each GPCR depends on its G-protein–coupling specificity, which can be distinct for each ligand (often referred to as “biased agonism”), the intensity and duration of receptor activation, and the level of expression of each G protein subunit and the repertoire of signaling molecules expressed in each cell type. The most proximal signaling pathways stimulated by each G protein subunit are summarized in Fig. 1.
      In addition to canonical signaling through heterotrimeric G proteins, some classes of GPCRs can initiate G protein–independent signal transduction. For example, some GPCRs also initiate intracellular signaling by engaging the scaffolding activity of β-arrestins, particularly for the activation of ERK and JNK3 (
      • Lefkowitz R.J.
      • Shenoy S.K.
      Transduction of receptor signals by β-arrestins.
      ). However, it is possible that G proteins may be required to initiate signal transduction, with β-arrestins playing a more important modulatory role in signal transmission, by shaping and fine-tuning dynamic GPCR responses (
      • Gutkind J.S.
      • Kostenis E.
      Arrestins as rheostats of GPCR signalling.
      ).
      G protein–independent signaling is well-exemplified by the Frizzled (FZD) family of receptors. In this case, the FZD ligand, WNT, stimulates a signal transduction cascade that results in β-catenin activation through the protein disheveled (DVl), which plays a key role in embryonic development and cancer. WNT proteins bind FZD and a single-pass transmembrane molecule, low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6), leading to the dimerization of the two receptors (
      • Nusse R.
      • Clevers H.
      Wnt/β-catenin signaling, disease, and emerging therapeutic modalities.
      ). The resulting conformational changes cause the phosphorylation of the cytoplasmic tail of LRP at multiple residues and the recruitment of GSK3β bound to scaffold protein Axin, whereas FZD associates with Dishevelled. This complex formation prevents the persistent phosphorylation and consequent degradation of β-catenin bound to its degradation complex, which includes Axin, the tumor suppressor APC, the kinases GSK-3α/β and CK1, and the E3-ubiquitin ligase β-TrCP, thereby stabilizing β-catenin and promoting its nuclear-signaling activity (
      • Nusse R.
      • Clevers H.
      Wnt/β-catenin signaling, disease, and emerging therapeutic modalities.
      ). The WNT/β-catenin pathway is frequently dysregulated in cancer, with particularly high incidence in colorectal cancer (
      • Kinzler K.W.
      • Nilbert M.C.
      • Su L.K.
      • Vogelstein B.
      • Bryan T.M.
      • Levy D.B.
      • Smith K.J.
      • Preisinger A.C.
      • Hedge P.
      • McKechnie D.
      • et al.
      Identification of FAP locus genes from chromosome 5q21.
      ).
      An interesting aspect of WNT signaling is that FZDs are persistently ubiquitinated and down-regulated by the transmembrane proteins ZNRF3 and RNF43, and that this negative effect can be circumvented by secreted proteins of the R-spondin family that bind ZNRF3/RNF43 together with the GPCRs LRG4 and LGR5, suppressing ZNRF3/RNF43 function and leading to enhanced WNT signaling indirectly (
      • Nusse R.
      • Clevers H.
      Wnt/β-catenin signaling, disease, and emerging therapeutic modalities.
      ). However, as recently reviewed, FZD can also activate G proteins of the Gαi, Gαq, and Gα13 families, mediating many of the responses initiated by WNT exposure (
      • Schulte G.
      • Wright S.C.
      Frizzleds as GPCRs–more conventional than we thought!.
      ).
      Another GPCR involved in development and cancer, particularly in basal cell carcinoma, is smoothened (SMO), which acts in the sonic hedgehog (SHH) pathway primarily by regulating the activity of the GLI transcription factor by a not fully understood mechanism in mammalian cells (
      • Lee R.T.
      • Zhao Z.
      • Ingham P.W.
      Hedgehog signalling.
      ). Traditionally, this effect was considered to be G protein–independent, but GLI activation requires the inhibition of PKA, and growing evidence suggests that this aspect may require the activation of Gαi proteins or the inhibition of Gαs or its coupled receptors (reviewed in Ref.
      • Kinzler K.W.
      • Nilbert M.C.
      • Su L.K.
      • Vogelstein B.
      • Bryan T.M.
      • Levy D.B.
      • Smith K.J.
      • Preisinger A.C.
      • Hedge P.
      • McKechnie D.
      • et al.
      Identification of FAP locus genes from chromosome 5q21.
      ). How G protein signaling by FZD is coordinated in space and time with canonical β-catenin signaling, and how SMO regulates G protein–independent and G protein–regulated pathways to activate GLI and other signaling events in the context of cancer stemness and metastasis is an active area of current investigation (
      • Schulte G.
      • Wright S.C.
      Frizzleds as GPCRs–more conventional than we thought!.
      ,
      • Lee R.T.
      • Zhao Z.
      • Ingham P.W.
      Hedgehog signalling.
      • Zhan T.
      • Rindtorff N.
      • Boutros M.
      Wnt signaling in cancer.
      ). Its full elucidation may have important implications for the design of new pharmacological interventions in cancers that involve persistent G protein–independent and/or -dependent WNT and SHH signaling.

      Mutational landscape of G proteins and GPCRs in cancer

      The Cancer Genome Atlas (TCGA) is a comprehensive, publicly available database launched by the National Institutes of Health, which includes large-scale genome sequencing analyses through multiple omics platforms for a variety of cancer types (
      • Hoadley K.A.
      • Yau C.
      • Hinoue T.
      • Wolf D.M.
      • Lazar A.J.
      • Drill E.
      • Shen R.
      • Taylor A.M.
      • Cherniack A.D.
      • Thorsson V.
      • Akbani R.
      • Bowlby R.
      • Wong C.K.
      • Wiznerowicz M.
      • Sanchez-Vega F.
      • et al.
      Cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer.
      ). In addition to this, the TCGA database also includes array-based DNA methylation sequencing for methylation profiling and reverse-phase protein array for large-scale protein expression profiling. These platforms can add a multidimensional view to the landscape of GPCRs and G proteins in cancer. Here, we built on our prior cancer genome-wide study (
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G proteins and G-protein–coupled receptors in cancer.
      ), performing an in-depth omics analysis of the mutational landscape of 33 cohorts of cancer patients in TCGA by new bioinformatics approaches (Table S1B).
      The power of this analysis revealed that 20% of all human tumors sequenced contained mutations in genes encoding GPCRs. In particular, we used MutSig2CV, a now widely used computational biology tool that takes mutations discovered by DNA sequencing to illuminate genes that are statistically more frequently mutated relative to the background mutation rate of individual lesions (
      • Lawrence M.S.
      • Stojanov P.
      • Polak P.
      • Kryukov G.V.
      • Cibulskis K.
      • Sivachenko A.
      • Carter S.L.
      • Stewart C.
      • Mermel C.H.
      • Roberts S.A.
      • Kiezun A.
      • Hammerman P.S.
      • McKenna A.
      • Drier Y.
      • Zou L.
      • et al.
      Mutational heterogeneity in cancer and the search for new cancer-associated genes.
      ). Many G proteins and GPCRs were found to be mutated. For visualizing the data, we used a very stringent criterion (MutSig2CV q-value <0.25) to identify the most statistically significant mutated genes in each cancer type. An unexpected observation was that among all cancer cohorts, cancers arising in the gastrointestinal (GI) tract, including colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD), and pancreatic adenocarcinoma (PAAD) displayed the highest number of significantly mutated GPCRs and G proteins (Fig. 2 and Tables S2, A and B). This may be independent of the mutational burden of these tumors, which are lower than that of other typical highly-mutated cancers such as melanoma and lung cancer, for example (
      • Alexandrov L.B.
      • Nik-Zainal S.
      • Wedge D.C.
      • Aparicio S.A.
      • Behjati S.
      • Biankin A.V.
      • Bignell G.R.
      • Bolli N.
      • Borg A.
      • Børresen-Dale A.L.
      • Boyault S.
      • Burkhardt B.
      • Butler A.P.
      • Caldas C.
      • Davies H.R.
      • et al.
      Signatures of mutational processes in human cancer.
      ). However, the phenotypic and biological outcome of these mutations remains largely unknown, and thus these findings provide a wealth of information for the development of hypothesis-driven approaches to investigate their cancer relevance.
      Figure thumbnail gr2
      Figure 2Top significant mutations of GPCRs and G proteins in cancer. From MutSig2CV analysis, the proportion of TCGA cohorts (sample number) with highly-significant (MutSig2CV q-value <0.25) mutations in genes encoding GPCRs (black) and G proteins (red) are shown. The statistically significant mutated genes for each cohort are plotted outside of the pie; cohorts are colored based on number of significant genes.
      In addition to our analysis of the most statistically significant mutated and genomically altered G proteins and GPCRs in cancer (q < 0.25), we have compiled the frequency of mutations of all G proteins and GPCR genes for each cancer type investigated in TCGA (Table S6). We expect that this color-coded table will provide easy access and visualization of the cancers in which G proteins and GPCRs of interest are most frequently mutated. We generated this table using the more recent and robust Multi-Center Mutation Calling in Multiple Cancers (MC3) Project TCGA PanCancer 2018 dataset (
      • Ellrott K.
      • Bailey M.H.
      • Saksena G.
      • Covington K.R.
      • Kandoth C.
      • Stewart C.
      • Hess J.
      • Ma S.
      • Chiotti K.E.
      • McLellan M.
      • Sofia H.J.
      • Hutter C.
      • Getz G.
      • Wheeler D.
      • Ding L.
      • MC3 Working Group, and Cancer Genome Atlas Research Network
      Scalable open science approach for mutation calling of tumor exomes using multiple genomic pipelines.
      ). This database includes mutation-calling algorithms that account for variance and batch effects to enable more precise cross-tumor–type analyses (
      • Ellrott K.
      • Bailey M.H.
      • Saksena G.
      • Covington K.R.
      • Kandoth C.
      • Stewart C.
      • Hess J.
      • Ma S.
      • Chiotti K.E.
      • McLellan M.
      • Sofia H.J.
      • Hutter C.
      • Getz G.
      • Wheeler D.
      • Ding L.
      • MC3 Working Group, and Cancer Genome Atlas Research Network
      Scalable open science approach for mutation calling of tumor exomes using multiple genomic pipelines.
      ). We have also provided a direct link for each gene to their corresponding page in cBioPortal Cancer Genomics portal (http://www.cbioportal.org/)8 (
      • Gao J.
      • Aksoy B.A.
      • Dogrusoz U.
      • Dresdner G.
      • Gross B.
      • Sumer S.O.
      • Sun Y.
      • Jacobsen A.
      • Sinha R.
      • Larsson E.
      • Cerami E.
      • Sander C.
      • Schultz N.
      Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.
      ,
      • Cerami E.
      • Gao J.
      • Dogrusoz U.
      • Gross B.E.
      • Sumer S.O.
      • Aksoy B.A.
      • Jacobsen A.
      • Byrne C.J.
      • Heuer M.L.
      • Larsson E.
      • Antipin Y.
      • Reva B.
      • Goldberg A.P.
      • Sander C.
      • Schultz N.
      The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
      ) for the visualization, analysis, and download of mutational information. The cBioPortal for Cancer Genomics is a web resource for dissecting and visualizing multidimensional cancer genomics data. These data include information about somatic mutations, copy number alterations, mRNA expression, DNA methylation, and transcript and protein abundance from multiple cancer omics studies (
      • Gao J.
      • Aksoy B.A.
      • Dogrusoz U.
      • Dresdner G.
      • Gross B.
      • Sumer S.O.
      • Sun Y.
      • Jacobsen A.
      • Sinha R.
      • Larsson E.
      • Cerami E.
      • Sander C.
      • Schultz N.
      Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.
      ). Please note that the percentage of mutated samples may vary with our analysis, as cBioPortal analysis uses different instances of the TCGA PanCancer dataset from 2013–2018 (
      • Cancer Genome Atlas Research Network
      • Weinstein J.N.
      • Collisson E.A.
      • Mills G.B.
      • Shaw K.R.
      • Ozenberger B.A.
      • Ellrott K.
      • Shmulevich I.
      • Sander C.
      • Stuart J.M.
      The cancer genome atlas pan-cancer analysis project.
      ). We encourage our colleagues to follow the corresponding links to gain easy access to the following: (a) “Cancer Types Summary,” in which all genomics alterations are displayed for all cancer types; (b) “Mutations,” which provide a visual representation of the most frequently mutated and altered residues and a downloadable list of samples that includes their corresponding protein change mutations, mutation type, and CNV type; (c) “Survival,” which shows the overall survival (length of time that the patients are alive) of cancer patients harboring genomic alterations versus those without (although, we recommend to perform this analysis for each particular cancer type of interest); and (d) “Expression,” which provides a graphical representation of the mRNA expression level of each sample in every cancer type, together with their mutational status.

      Significantly mutated G proteins in cancer

      Whereas the contribution of each GPCR mutation in cancer is still under evaluation, the recent discovery of hot spot mutations in G proteins as oncogenic drivers in multiple highly prevalent cancer types has accelerated tremendously the research in this field. Indeed, many G protein genes (GNAS, GNA11, GNAQ, and GNA13) are part of the current ∼400 gene panels of cancer-associated genes sequenced routinely by clinical oncology services in many cancer centers and by all large cancer genomic testing providers and institutional genomics cores. Among them, the summary of our MutSig2CV analysis revealed that GNAS is the most highly mutated G protein in human cancer (Table S2B). From this analysis, GNAS is significantly mutated in COAD (6.19%), PAAD (5.09%), and STAD (7.52%). As described above, GNAS is a known oncogene that was first described in growth hormone–secreting pituitary adenomas and has since been found to be mutated in a number of neoplasms, predominantly at the codon 201 hotspot (
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G proteins and G-protein–coupled receptors in cancer.
      ,
      • Vallar L.
      • Spada A.
      • Giannattasio G.
      Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas.
      ). Mutations occurring at arginine 201 of GNAS activate adenylate cyclase and lead to constitutive cAMP signaling by reducing the rate of GTP hydrolysis of the active GTP-bound Gαs, as well as by adopting an active-like conformation even when bound to GDP (
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G proteins and G-protein–coupled receptors in cancer.
      ,
      • Hu Q.
      • Shokat K.M.
      Disease-causing mutations in the G protein Gαs subvert the roles of GDP and GTP.
      ). In COAD, a synergistic effect with the MAPK pathway is likely, as GNAS is co-mutated with KRAS in a large portion of adenomas and carcinomas. Similarly, GNAS mutations are found in ∼50% of low-grade appendiceal mucinous neoplasms (
      • Alakus H.
      • Babicky M.L.
      • Ghosh P.
      • Yost S.
      • Jepsen K.
      • Dai Y.
      • Arias A.
      • Samuels M.L.
      • Mose E.S.
      • Schwab R.B.
      • Peterson M.R.
      • Lowy A.M.
      • Frazer K.A.
      • Harismendy O.
      Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin.
      ) and are highly prevalent in a subset of pancreatic tumors, including intraductal papillary mucinous neoplasms and adenocarcinomas (
      • Wu J.
      • Matthaei H.
      • Maitra A.
      • Dal Molin M.
      • Wood L.D.
      • Eshleman J.R.
      • Goggins M.
      • Canto M.I.
      • Schulick R.D.
      • Edil B.H.
      • Wolfgang C.L.
      • Klein A.P.
      • Diaz Jr., L.A.
      • Allen P.J.
      • Schmidt C.M.
      • et al.
      Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development.
      ). In this regard, recent mouse models revealed that GNAS and KRAS mutations are necessary and sufficient to initiate this particular subtype of pancreatic adenocarcinomas (
      • Patra K.C.
      • Kato Y.
      • Mizukami Y.
      • Widholz S.
      • Boukhali M.
      • Revenco I.
      • Grossman E.A.
      • Ji F.
      • Sadreyev R.I.
      • Liss A.S.
      • Screaton R.A.
      • Sakamoto K.
      • Ryan D.P.
      • Mino-Kenudson M.
      • Castillo C.F.
      • et al.
      Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism.
      ,
      • Ideno N.
      • Yamaguchi H.
      • Ghosh B.
      • Gupta S.
      • Okumura T.
      • Steffen D.J.
      • Fisher C.G.
      • Wood L.D.
      • Singhi A.D.
      • Nakamura M.
      • Gutkind J.S.
      • Maitra A.
      GNAS(R201C) induces pancreatic cystic neoplasms in mice that express activated KRAS by inhibiting YAP1 signaling.
      ).
      Emerging studies have begun to explain the functional impact of GNAS mutations. In 1991, GNAS mutations were discovered in McCune-Albright syndrome and pituitary tumors (
      • Weinstein L.S.
      • Shenker A.
      • Gejman P.V.
      • Merino M.J.
      • Friedman E.
      • Spiegel A.M.
      Activating mutations of the stimulatory G protein in the McCune-Albright syndrome [see comments].
      ). In cancer, GNAS has been linked to pro-inflammatory functions, which could mimic the impact of chronic inflammation on tumor development. Gαs is well-documented to mediate the effects of inflammatory mediators like cyclooxygenase (COX) 2-derived prostaglandins. Its inflammatory role in cancer is best shown in colon neoplasia where COX2-derived prostaglandin E2 (PGE2) enhances colon cancer progression via activation of PI3K and AKT and relieving the inhibitory phosphorylation of β-catenin as part of Gαs oncogenic signaling (
      • Castellone M.D.
      • Teramoto H.
      • Williams B.O.
      • Druey K.M.
      • Gutkind J.S.
      Prostaglandin E2 promotes colon cancer cell growth through a Gs–axin–β-catenin signaling axis.
      ). Activating mutations in GNAS have also been found in gastric adenocarcinomas, leading to activation of the Wnt/β-catenin signaling pathway (
      • Nomura R.
      • Saito T.
      • Mitomi H.
      • Hidaka Y.
      • Lee S.Y.
      • Watanabe S.
      • Yao T.
      GNAS mutation as an alternative mechanism of activation of the Wnt/β-catenin signaling pathway in gastric adenocarcinoma of the fundic gland type.
      ).
      Mutations in GNAQ and GNA11 are most relevant in uveal melanoma (UVM) incidence, as 93% of patients harbor mutations in these genes encoding constitutively active Gαq family members (
      • Van Raamsdonk C.D.
      • Griewank K.G.
      • Crosby M.B.
      • Garrido M.C.
      • Vemula S.
      • Wiesner T.
      • Obenauf A.C.
      • Wackernagel W.
      • Green G.
      • Bouvier N.
      • Sozen M.M.
      • Baimukanova G.
      • Roy R.
      • Heguy A.
      • Dolgalev I.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ,
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ). All cancer mutations in Gαq or Gα11 occur at either glutamine 209 or, in a smaller proportion, arginine 183 (Gln-209 and Arg-183, respectively; Arg-183 is the identical position to Arg-201 in Gαs) (
      • Van Raamsdonk C.D.
      • Griewank K.G.
      • Crosby M.B.
      • Garrido M.C.
      • Vemula S.
      • Wiesner T.
      • Obenauf A.C.
      • Wackernagel W.
      • Green G.
      • Bouvier N.
      • Sozen M.M.
      • Baimukanova G.
      • Roy R.
      • Heguy A.
      • Dolgalev I.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      ,
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ). Mutations affecting Gln-209 in GNAQ or GNA11 are present in most primary UVM lesions and their metastases (
      • Van Raamsdonk C.D.
      • Bezrookove V.
      • Green G.
      • Bauer J.
      • Gaugler L.
      • O'Brien J.M.
      • Simpson E.M.
      • Barsh G.S.
      • Bastian B.C.
      Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
      ). Mutated residues impair GTPase activity (diminish GTP hydrolysis), which ultimately leads to prolonged signaling. Although initial studies supported a role of ERK signaling in UVM development, targeting this pathway did not improve the survival of UVM metastatic patients (
      • Carvajal R.D.
      • Piperno-Neumann S.
      • Kapiteijn E.
      • Chapman P.B.
      • Frank S.
      • Joshua A.M.
      • Piulats J.M.
      • Wolter P.
      • Cocquyt V.
      • Chmielowski B.
      • Evans T.R.J.
      • Gastaud L.
      • Linette G.
      • Berking C.
      • Schachter J.
      • et al.
      Selumetinib in combination with dacarbazine in patients with metastatic uveal melanoma: a phase III, multicenter, randomized trial (SUMIT).
      ). Instead, our genome-wide RNAi screens revealed that the noncanonical activation of RhoGEFs, specifically TRIO, by Gαq mediates UVM progression (
      • Feng X.
      • Degese M.S.
      • Iglesias-Bartolome R.
      • Vaque J.P.
      • Molinolo A.A.
      • Rodrigues M.
      • Zaidi M.R.
      • Ksander B.R.
      • Merlino G.
      • Sodhi A.
      • Chen Q.
      • Gutkind J.S.
      Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.
      ). Furthermore, we discovered that the activation of YAP, the most downstream target of the Hippo pathway, by the novel TRIO–RHO signaling arm is essential for UVM, thus identifying a druggable target downstream from mutated Gαq (
      • Feng X.
      • Degese M.S.
      • Iglesias-Bartolome R.
      • Vaque J.P.
      • Molinolo A.A.
      • Rodrigues M.
      • Zaidi M.R.
      • Ksander B.R.
      • Merlino G.
      • Sodhi A.
      • Chen Q.
      • Gutkind J.S.
      Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.
      ).
      GNAQ mutations are also associated with a smaller proportion of skin cutaneous melanoma (SKCM) and have been recently described in vascular tumors, such as hemangiomas and angiosarcomas (
      • Parish A.J.
      • Nguyen V.
      • Goodman A.M.
      • Murugesan K.
      • Frampton G.M.
      • Kurzrock R.
      GNAS, GNAQ, and GNA11 alterations in patients with diverse cancers.
      ,
      • Ayturk U.M.
      • Couto J.A.
      • Hann S.
      • Mulliken J.B.
      • Williams K.L.
      • Huang A.Y.
      • Fishman S.J.
      • Boyd T.K.
      • Kozakewich H.P.
      • Bischoff J.
      • Greene A.K.
      • Warman M.L.
      Somatic activating mutations in GNAQ and GNA11 are associated with congenital hemangioma.
      ). GNAQ R183Q mutations are also specifically responsible for a frequent congenital neurocutaneous disorder characterized by port wine skin lesions that are vascularly-derived, which is known as Sturge-Weber syndrome (
      • Shirley M.D.
      • Tang H.
      • Gallione C.J.
      • Baugher J.D.
      • Frelin L.P.
      • Cohen B.
      • North P.E.
      • Marchuk D.A.
      • Comi A.M.
      • Pevsner J.
      Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ.
      ). Thus, mutations in GNAQ appear to be responsible for numerous disease conditions for which there are no current targeted therapeutic options.
      Mutations in GNA13 have been characterized in both liquid and solid tumors and are present at high frequency in bladder carcinoma. In addition, recent genome-wide sequencing efforts have unveiled the presence of frequent mutations in GNA13 in lymphomas, specifically Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL) (
      • O'Hayre M.
      • Inoue A.
      • Kufareva I.
      • Wang Z.
      • Mikelis C.M.
      • Drummond R.A.
      • Avino S.
      • Finkel K.
      • Kalim K.W.
      • DiPasquale G.
      • Guo F.
      • Aoki J.
      • Zheng Y.
      • Lionakis M.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.
      ,
      • Love C.
      • Sun Z.
      • Jima D.
      • Li G.
      • Zhang J.
      • Miles R.
      • Richards K.L.
      • Dunphy C.H.
      • Choi W.W.
      • Srivastava G.
      • Lugar P.L.
      • Rizzieri D.A.
      • Lagoo A.S.
      • Bernal-Mizrachi L.
      • Mann K.P.
      • et al.
      The genetic landscape of mutations in Burkitt lymphoma.
      • Lohr J.G.
      • Stojanov P.
      • Lawrence M.S.
      • Auclair D.
      • Chapuy B.
      • Sougnez C.
      • Cruz-Gordillo P.
      • Knoechel B.
      • Asmann Y.W.
      • Slager S.L.
      • Novak A.J.
      • Dogan A.
      • Ansell S.M.
      • Link B.K.
      • Zou L.
      • et al.
      Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.
      ). These mutations in GNA13 as well as in RhoA, a downstream target of Gα13, have been shown to be inhibitory in nature, suggesting a tumor-suppressive role for Gα13 and RhoA in Burkitt's lymphoma and DLBCL (
      • O'Hayre M.
      • Inoue A.
      • Kufareva I.
      • Wang Z.
      • Mikelis C.M.
      • Drummond R.A.
      • Avino S.
      • Finkel K.
      • Kalim K.W.
      • DiPasquale G.
      • Guo F.
      • Aoki J.
      • Zheng Y.
      • Lionakis M.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.
      ). In this case, loss–of–function (LOF) mutations rather than gain–of–function (GOF) mutations underlie the oncogenic activity of GNA13, likely by disrupting the normal differentiation program of B cells (
      • O'Hayre M.
      • Inoue A.
      • Kufareva I.
      • Wang Z.
      • Mikelis C.M.
      • Drummond R.A.
      • Avino S.
      • Finkel K.
      • Kalim K.W.
      • DiPasquale G.
      • Guo F.
      • Aoki J.
      • Zheng Y.
      • Lionakis M.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.
      ). In contrast, WT GNA13 overexpression has been implicated in many solid tumors, such as in gastric cancer (
      • Zhang J.X.
      • Yun M.
      • Xu Y.
      • Chen J.W.
      • Weng H.W.
      • Zheng Z.S.
      • Chen C.
      • Xie D.
      • Ye S.
      GNA13 as a prognostic factor and mediator of gastric cancer progression.
      ), nasopharyngeal carcinoma (
      • Liu S.C.
      • Jen Y.M.
      • Jiang S.S.
      • Chang J.L.
      • Hsiung C.A.
      • Wang C.H.
      • Juang J.L.
      G(α)12-mediated pathway promotes invasiveness of nasopharyngeal carcinoma by modulating actin cytoskeleton reorganization.
      ), prostate cancer (
      • Kelly P.
      • Stemmle L.N.
      • Madden J.F.
      • Fields T.A.
      • Daaka Y.
      • Casey P.J.
      A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion.
      ), and breast cancer (
      • Kelly P.
      • Moeller B.J.
      • Juneja J.
      • Booden M.A.
      • Der C.J.
      • Daaka Y.
      • Dewhirst M.W.
      • Fields T.A.
      • Casey P.J.
      The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis.
      ). Furthermore, GNA13 levels modulate drug resistance and tumor-initiation phenotypes in patient-derived head and neck squamous cell carcinoma cells in vitro and in vivo (
      • Rasheed S.A.K.
      • Leong H.S.
      • Lakshmanan M.
      • Raju A.
      • Dadlani D.
      • Chong F.T.
      • Shannon N.B.
      • Rajarethinam R.
      • Skanthakumar T.
      • Tan E.Y.
      • Hwang J.S.G.
      • Lim K.H.
      • Tan D.S.
      • Ceppi P.
      • Wang M.
      • et al.
      GNA13 expression promotes drug resistance and tumor-initiating phenotypes in squamous cell cancers.
      ). In this case, GNA13 or GNA12 overexpression may enhance the proliferative and pro-migratory function of multiple GPCRs that converge to activate these G protein α subunits. A causal role of excessive Gα12 signaling may be elucidated by a use of a recently developed Gα12-coupled chemogenetic designer GPCR (Designer Receptors Exclusively Activated by Designer Drugs (DREADD)) (
      • Inoue A.
      • Raimondi F.
      • Kadji F.M.N.
      • Singh G.
      • Kishi T.
      • Uwamizu A.
      • Ono Y.
      • Shinjo Y.
      • Ishida S.
      • Arang N.
      • Kawakami K.
      • Gutkind J.S.
      • Aoki J.
      • Russell R.B.
      Illuminating G-protein–coupling selectivity of GPCRs.
      ).
      Mutations in Gβ subunits are infrequent, and yet activating mutations in Gβ1 and Gβ2 (GNB1 and GNB2, respectively) has been identified in myeloid and B-cell neoplasms, which act as an oncogenic driver and confer resistance to kinase inhibitors targeting typically mutated kinases in these malignancies, including BCR–ABL, BRAF, and JAK2 (
      • Yoda A.
      • Adelmant G.
      • Tamburini J.
      • Chapuy B.
      • Shindoh N.
      • Yoda Y.
      • Weigert O.
      • Kopp N.
      • Wu S.C.
      • Kim S.S.
      • Liu H.
      • Tivey T.
      • Christie A.L.
      • Elpek K.G.
      • Card J.
      • et al.
      Mutations in G protein β subunits promote transformation and kinase inhibitor resistance.
      ). Certainly, this information suggests that other Gβ subunit mutations may also harbor tumorigenic potential.

      Mutated oncoGPCRome

      The most frequently mutated GPCRs in each cancer type are depicted in Fig. 2 and are listed in Table S2A with the corresponding statistical significance (q-value) and frequency. As mentioned above, the high frequency of GPCR mutations specifically in tumors arising from the gastrointestinal tract is intriguing as it likely reflects their ability to stimulate organ-specific growth-promoting pathways in these cancers. Although a discussion of each specific GPCR is beyond the goals of our review, we will discuss new emerging concepts and specific cases that may exemplify the challenges and opportunities for future exploration in this area and its potential for drug discovery.
      Whether mutations in GPCRs result in GOF or LOF, or represent passenger mutations with little impact on cancer progression, in most cases is still unknown. A complicating factor is that most GPCRs do not harbor hotspot mutations, meaning that mutations in each GPCR do not occur with high frequency in a single or limited numbers of codons, and in addition, each tumor exhibits a different repertoire of mutated GPCRs. To address this daunting question, we have recently developed new bioinformatics approaches analyzing GPCR mutations in the context of multiple sequence alignments (MSA) defining the conserved seven-transmembrane (7TM) domain, as well as considering 3D structures and interaction partners (
      • Raimondi F.
      • Inoue A.
      • Kadji F.M.N
      • Shuai N.
      • Gonzalez J.C.
      • Singh G.
      • Alonso de la Vega A.
      • Sotillo R.
      • Fischer B.
      • Aoki J.
      • Gutkind J.S.
      • Russell R.B.
      Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm.
      ). We have used this approach to model the most significantly mutated GPCRs (Table S2A). Remarkably, visualization of the most mutated 7TM positions on a representative GPCR 3D structure revealed that most mutations occur in “hotspot structural motifs” rather than being randomly distributed (Fig. 3 and Table S3). This includes frequent mutations in the DRY arginine motif, which is as important for class A GPCR activation as it is responsible for the intramolecular polar contacts that keep the receptor inactive until ligand binding (
      • Rosenbaum D.M.
      • Rasmussen S.G.
      • Kobilka B.K.
      The structure and function of G-protein–coupled receptors.
      ). Other structural mutation hotspots are found at or nearby highly-conserved GPCR regions, including the ligand and G protein–binding sites, as well as the NPXXY and other conserved motifs that regulate in an allosteric way receptor's activation (
      • Thal D.M.
      • Glukhova A.
      • Sexton P.M.
      • Christopoulos A.
      Structural insights into G-protein–coupled receptor allostery.
      ). Collectively, this supports that most cancer-associated mutations in GPCRs occur in “structural hotspots,” similar to other oncogenes and tumor suppressor genes, a property that could have not been predicted from the analysis of individual GPCRs.
      Figure thumbnail gr3
      Figure 3Significantly mutated genes in 7TM positions. 3D “putty” drawing of most mutated 7TM positions in significantly mutated genes from the TCGA database is shown. A prototypical GPCR structure (i.e. ADRB2, Protein Data Bank code 3NYA) is used for representation. Cartoon diameter and coloring (blue to red) are directly proportional to the number of unique samples carrying mutations at given 7TM positions. To identify these, mutated receptor sequences were aligned (using PFAM 7tm_1 Hidden Markov Model), and Ballesteros/Weinstein numberings were assigned (see ). Conserved functional motives are highlighted and labeled.
      Although the functional impact of these alterations may need to be investigated for each GPCR, our recent computational analysis of cancer genomes indicates that most Gαi-linked GPCRs exhibit DRY mutations that are inhibitory in nature (inhibit function), which typically occur mutually exclusively with GNAS9-activating mutations (
      • Raimondi F.
      • Inoue A.
      • Kadji F.M.N
      • Shuai N.
      • Gonzalez J.C.
      • Singh G.
      • Alonso de la Vega A.
      • Sotillo R.
      • Fischer B.
      • Aoki J.
      • Gutkind J.S.
      • Russell R.B.
      Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm.
      ). This suggests the exciting possibility that mutations in Gαi–GPCRs may mimic GNAS mutants leading to higher cAMP activity to drive tumorigenesis (
      • Raimondi F.
      • Inoue A.
      • Kadji F.M.N
      • Shuai N.
      • Gonzalez J.C.
      • Singh G.
      • Alonso de la Vega A.
      • Sotillo R.
      • Fischer B.
      • Aoki J.
      • Gutkind J.S.
      • Russell R.B.
      Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm.
      ).
      A particular challenge when analyzing the potential impact of cancer mutations is that longer genes exhibit a higher number of mutations, which would achieve statistical significance (MutSig2CV analysis) only when higher than the background mutation rate of individual lesions. This is well-exemplified by GPR98, which is the most frequently mutated GPCR across all cancer types and, concomitantly, is the GPCR with the highest number of amino acids. GPR98 is an adhesion receptor, and its ligand and physiological functions are currently poorly understood. GPR98 mutations are known to cause febrile seizures and one form of Usher syndrome, the most common genetic cause of combined blindness and deafness (
      • Nakayama J.
      • Fu Y.H.
      • Clark A.M.
      • Nakahara S.
      • Hamano K.
      • Iwasaki N.
      • Matsui A.
      • Arinami T.
      • Ptácek L.J.
      A nonsense mutation of the MASS1 gene in a family with febrile and afebrile seizures.
      ,
      • Mathur P.
      • Yang J.
      Usher syndrome: hearing loss, retinal degeneration and associated abnormalities.
      ). GPR98 has been shown to have significant association with glioblastoma (GBM) (
      • Sadeque A.
      • Serão N.V.
      • Southey B.R.
      • Delfino K.R.
      • Rodriguez-Zas S.L.
      Identification and characterization of alternative exon usage linked glioblastoma multiforme survival.
      ) and lymphoblastic leukemia (
      • Nugent A.
      • Proia R.L.
      The role of G protein–coupled receptors in lymphoid malignancies.
      ), and the evaluation of the impact of GPR98 mutations in cancer warrants further investigation. The family of metabotropic glutamate GPCRs, GRM1–8, are also frequently mutated in many cancer cohorts. Mutations of GRM1, GRM5, and GRM3 have been shown in breast cancer and melanoma (
      • Pollock P.M.
      • Cohen-Solal K.
      • Sood R.
      • Namkoong J.
      • Martino J.J.
      • Koganti A.
      • Zhu H.
      • Robbins C.
      • Makalowska I.
      • Shin S.S.
      • Marin Y.
      • Roberts K.G.
      • Yudt L.M.
      • Chen A.
      • Cheng J.
      • et al.
      Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.
      ,
      • Choi K.Y.
      • Chang K.
      • Pickel J.M.
      • Badger 2nd, J.D.
      • Roche K.W.
      Expression of the metabotropic glutamate receptor 5 (mGluR5) induces melanoma in transgenic mice.
      • Speyer C.L.
      • Smith J.S.
      • Banda M.
      • DeVries J.A.
      • Mekani T.
      • Gorski D.H.
      Metabotropic glutamate receptor-1: a potential therapeutic target for the treatment of breast cancer.
      ). In addition, their transforming potential and increased secretion of their ligand, glutamate, by the tumor microenvironment makes the GRM receptor family an intriguing area of study.
      The analysis of the mutational landscape of GPCRs suggest that COAD harbors the highest incidence of significantly mutated receptors. Among them, thyroid-stimulating hormone receptor (TSHR) was the most frequently mutated GPCR, involving ∼14% of COAD patients. Mutations in the P2Y purinoceptor 13 (P2RY13) gene were the most statistically significant in this cancer type and occurred in ∼5% of COAD patients. P2RY13 encodes for a purine receptor and has been shown to be overexpressed in acute myeloid leukemia samples but not involved in other nonhematologic malignancies (
      • Maiga A.
      • Lemieux S.
      • Pabst C.
      • Lavallée V.P.
      • Bouvier M.
      • Sauvageau G.
      • Hébert J.
      Transcriptome analysis of G protein–coupled receptors in distinct genetic subgroups of acute myeloid leukemia: identification of potential disease-specific targets.
      ). On a related note, mucosal biopsies from the colon of Crohn's disease and ulcerative colitis patients have shown abnormalities in P2RY13, which may suggest a role for the receptor in GI inflammatory diseases (
      • Rybaczyk L.
      • Rozmiarek A.
      • Circle K.
      • Grants I.
      • Needleman B.
      • Wunderlich J.E.
      • Huang K.
      • Christofi F.L.
      New bioinformatics approach to analyze gene expressions and signaling pathways reveals unique purine gene dysregulation profiles that distinguish between CD and UC.
      ). The importance of TSHR-activating mutations in human neoplasia was first demonstrated in thyroid adenomas (
      • Parma J.
      • Duprez L.
      • Van Sande J.
      • Cochaux P.
      • Gervy C.
      • Mockel J.
      • Dumont J.
      • Vassart G.
      Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas [see comments].
      ) and are also found in some thyroid carcinomas. However, the roles of both TSHR and P2Y13 in COAD remain largely unexplored.
      Recently, analysis of hotspot mutations in oncogenes uncovered a mutation in cysteinyl leukotriene receptor 2 (CYSLTR2) in a UVM cohort. This GOF mutation results in an L129Q substitution and leads to the Gαq-coupled receptor to be constitutively active (
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • Zhang J.Q.
      • Walczak E.G.
      • Kazmi M.A.
      • Taylor B.S.
      • Huber T.
      • Chi P.
      • Sakmar T.P.
      • Chen Y.
      Recurrent activating mutations of G-protein–coupled receptor CYSLTR2 in uveal melanoma.
      ). This mutant protein is insensitive to leukotriene stimulation, constitutively activates Gαq, and can promote tumorigenesis in melanocytes in vivo (
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • Zhang J.Q.
      • Walczak E.G.
      • Kazmi M.A.
      • Taylor B.S.
      • Huber T.
      • Chi P.
      • Sakmar T.P.
      • Chen Y.
      Recurrent activating mutations of G-protein–coupled receptor CYSLTR2 in uveal melanoma.
      ). According to MutSig2CV analysis, CysLT2 is the most frequently mutated GPCR (3.75%) in UVM. While representing a small fraction of all UVM cases, these mutations in CYSLTR2 are mutually exclusive with known drivers in UVM (GNA11 and GNAQ) (
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • Zhang J.Q.
      • Walczak E.G.
      • Kazmi M.A.
      • Taylor B.S.
      • Huber T.
      • Chi P.
      • Sakmar T.P.
      • Chen Y.
      Recurrent activating mutations of G-protein–coupled receptor CYSLTR2 in uveal melanoma.
      ). Therefore, CYSLTR2 mutations promote persistent Gαq activation substituting for GNA11 and GNAQ mutations to drive aberrant Gαq signaling in UVM. This receptor is also mutated in COAD at a distinct amino acid, and hence its consequences (GOF or LOF) are still unknown. Recently, small molecules have been discovered and utilized against WT CysLT2, but development of higher-affinity molecules or antibodies that can stabilize the mutated receptor in its inactive state will be required to explore the therapeutic benefit of targeting CysLT2 in UVM.
      Our current analysis also identified many adhesion receptors and class A GPCRs that are mutated with high frequency in cancer. The former includes GPR98, BAI3, ADGRL1, CELSR1, GPR125, GPR110, GPR112, and GPR126, which can now be prioritized for their individual analysis. A recent comprehensive mutagenesis screen in ADGRL1 revealed that many cancer-associated mutations result in GOF alterations and persistent activity (
      • Nazarko O.
      • Kibrom A.
      • Winkler J.
      • Leon K.
      • Stoveken H.
      • Salzman G.
      • Merdas K.
      • Lu Y.
      • Narkhede P.
      • Tall G.
      • Promel S.
      • Araç D.
      A comprehensive mutagenesis screen of the adhesion GPCR latrophilin-1/ADGRL1.
      ).
      Among the typical class A GPCRs, some of the more frequently mutated genes are muscarinic receptors M2 and M3 (CHRM2 and CHRM3), multiple P2Y receptors, serotonin receptors (HTR1E, HTR1F, HTR2A, and HTR7), and adenosine receptors (ADORA3), among others, all of which could be activated by locally produced ligands as well. Notable mutated GPCRs also include the PAR2 receptor (F2RL1), which is often amplified and will be discussed below, as well as multiple orphan GPCRs whose coupling specificity and biological activity is still largely unknown. Given the emerging studies supporting the notion that aberrant GPCR activity leads to tumor initiation and progression, we expect that the emerging mutational information will guide new cancer-relevant studies addressing each of these frequently mutated GPCRs. Given that many ligands of GPCRs may be produced in significantly higher amounts in the hypoxic, metabolic, and acidic tumor microenvironment, the tumorigenic synergism between ligand availability and activating mutations in receptors should also be explored.

      Gene copy number alterations and G protein and GPCR expression in cancer

      In addition to mutations, alterations in gene expression and copy number of G protein and GPCR genes have been detected. Determining the contribution of such alterations to cancer initiation and progression remains a significant challenge, yet it may be critical both for the discovery of driver oncogenic processes and for the development of targeted therapeutics. Indeed, aberrant expression of many WT G proteins and GPCRs can contribute to cancer growth even if not mutated, often as part of oncocrine signaling networks (see below).
      Somatic alterations are acquired at random during cell division, and some of these participate in tumorigenesis or tumor growth. Here, we used GISTIC (Genomic Identification of Significant Targets in Cancer), an algorithm that identifies genes targeted by somatic CNVs that may contribute to tumorigenesis by evaluating the frequency and amplitude of observed events (
      • Mermel C.H.
      • Schumacher S.E.
      • Hill B.
      • Meyerson M.L.
      • Beroukhim R.
      • Getz G.
      GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers.
      ). To illuminate the most relevant GPCR candidates in tumorigenesis, we also filtered the large list of CNVs for those that correlated with mRNA expression. Our analysis revealed that 28 out of 33 TCGA cancer cohorts included alterations of GPCR and G protein that are significantly correlated with mRNA expression of the corresponding genes (R > 0.33) (Tables S4, A and B, and S5, and Fig. 4).
      Figure thumbnail gr4
      Figure 4Top significant CNVs of GPCRs and G proteins in cancer. From GISTIC analysis, the proportion of TCGA cohorts (sample number) with highly significant (GISTIC q-value <0.05 and mRNA correlation >0.333) CNVs in genes encoding GPCRs (black) and G proteins (red) are shown. The significant genes for each cohort are plotted outside of the cohort pie; cohorts are colored based on the number of significant genes, and amplification is denoted by red highlighting, and deletion is denoted by a blue highlighting.
      Among the G proteins, copy number gain in GNA12 is remarkably significant in ovarian cancer (OV). This cancer type is characterized by few driver mutations and by the accumulation of high concentrations of LPA in ascites fluids, which may work through Gα12 to promote growth and metastasis (reviewed in Ref.
      • Fang X.
      • Gaudette D.
      • Furui T.
      • Mao M.
      • Estrella V.
      • Eder A.
      • Pustilnik T.
      • Sasagawa T.
      • Lapushin R.
      • Yu S.
      • Jaffe R.B.
      • Wiener J.R.
      • Erickson J.R.
      • Mills G.B.
      Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer.
      ). Similarly, GNAI1 (encoding Gαi1) is significantly amplified in breast-invasive carcinoma (BRCA), a cancer type in which many Gαi-coupled GPCRs, including CXCR4, are well-established as metastatic drivers (see below). The significance of other genomic alterations in G proteins, including copy number gains in Gβ subunits (GNAB1, GNAB2, GNAB3, and GNAB5) and Gγ (GNG4, GNG5, GNG7, GNG12, and GNGT1) in multiple cancers likely reflect the broad signaling capacity of Gβγ dimers (see Fig. 1).
      Testicular germ cell tumor displayed the most genomic alterations in genes encoding GPCRs, which included mostly orphan, taste, and adhesion receptors. In contrast, F2RL1, the gene encoding -activated receptor (PAR) 2, was the most significantly altered gene in OV. PAR2 is a protease-activated receptor and is expressed in many organs. The ability of proteases to degrade extracellular matrices and to activate PARs render them important in the facilitation of tumor growth and metastasis (
      • Shi X.
      • Gangadharan B.
      • Brass L.F.
      • Ruf W.
      • Mueller B.M.
      Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis.
      ,
      • López-Otín C.
      • Matrisian L.M.
      Emerging roles of proteases in tumour suppression.
      ). Overexpression of F2RL1 has been linked to some of the most diagnosed cancers, including lung, breast, colon, and pancreatic cancers (
      • Shi X.
      • Gangadharan B.
      • Brass L.F.
      • Ruf W.
      • Mueller B.M.
      Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis.
      ,
      • Su S.
      • Li Y.
      • Luo Y.
      • Sheng Y.
      • Su Y.
      • Padia R.N.
      • Pan Z.K.
      • Dong Z.
      • Huang S.
      Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration.
      ,
      • Darmoul D.
      • Gratio V.
      • Devaud H.
      • Laburthe M.
      Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation.
      ). Functionally, PAR2 has also been linked to cancer cell migration and stimulates vascular endothelial growth factor (VEGF) production for angiogenesis (
      • Dutra-Oliveira A.
      • Monteiro R.Q.
      • Mariano-Oliveira A.
      Protease-activated receptor-2 (PAR2) mediates VEGF production through the ERK1/2 pathway in human glioblastoma cell lines.
      ,
      • Rasmussen J.G.
      • Riis S.E.
      • Frøbert O.
      • Yang S.
      • Kastrup J.
      • Zachar V.
      • Simonsen U.
      • Fink T.
      Activation of protease-activated receptor 2 induces VEGF independently of HIF-1.
      ). Another unexpected observation was that most kidney cancers (KIPAN) exhibit highly-significant copy number gains in genes for multiple chemokine receptors (CCR2, CCR5, CCR6, CCR9, CX3CR1, and CXCR6) and histamine receptors (HRH2), among others. The frizzled family of GPCRs and LPA receptors (in particular LPAR6) were also genetically altered in multiple cancer types. Overall, although gene copy gains and losses may reflect cancer-associated genomic instability, most cancers exhibit a very specific pattern of copy number variations in G protein and GPCR genes, whose biological relevance can now be examined.

      G proteins and GPCRs as tumor suppressor genes?

      An interesting observation of the pattern of genomic alterations is that many cancers lose one or both copies of specific G protein and GPCR genes. This raises the possibility that certain G protein/GPCRs may act as tumor suppressors rather than oncogenes. Indeed, as described above, GNA13 is significantly mutated in diffuse B-cell lymphoma and Burkitt's lymphoma, and detailed experimental analysis revealed that in all cases this involves LOF mutations, resulting in the inability of B cells to undergo terminal differentiation and hence increasing their uncontrolled growth (
      • O'Hayre M.
      • Inoue A.
      • Kufareva I.
      • Wang Z.
      • Mikelis C.M.
      • Drummond R.A.
      • Avino S.
      • Finkel K.
      • Kalim K.W.
      • DiPasquale G.
      • Guo F.
      • Aoki J.
      • Zheng Y.
      • Lionakis M.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.
      ). Analysis of candidate Gα13-coupled GPCRs identified inactivating mutations in P2RY8 and LOF mutations in RHOA (
      • O'Hayre M.
      • Inoue A.
      • Kufareva I.
      • Wang Z.
      • Mikelis C.M.
      • Drummond R.A.
      • Avino S.
      • Finkel K.
      • Kalim K.W.
      • DiPasquale G.
      • Guo F.
      • Aoki J.
      • Zheng Y.
      • Lionakis M.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.
      ,
      • Muppidi J.R.
      • Schmitz R.
      • Green J.A.
      • Xiao W.
      • Larsen A.B.
      • Braun S.E.
      • An J.
      • Xu Y.
      • Rosenwald A.
      • Ott G.
      • Gascoyne R.D.
      • Rimsza L.M.
      • Campo E.
      • Jaffe E.S.
      • Delabie J.
      • et al.
      Loss of signalling via Gα13 in germinal centre B-cell–derived lymphoma.
      ). These mutations appear to be mutually exclusive, supporting the notion that in these B-cell malignancies P2RY8–GNA13–RHOA are part of a tumor-suppressive axis.
      Surprisingly, while conducting conditional gene knockout studies of Gαs in the skin, we observed that mice develop massive basal cell carcinomas (BCC) in only 2 weeks after Gnas excision (
      • Iglesias-Bartolome R.
      • Torres D.
      • Marone R.
      • Feng X.
      • Martin D.
      • Simaan M.
      • Chen M.
      • Weinstein L.S.
      • Taylor S.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivation of a Gα(s)–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis.
      ). This involved a widespread activation of the SHH pathway in Gnas−/− tumor lesions (
      • Iglesias-Bartolome R.
      • Torres D.
      • Marone R.
      • Feng X.
      • Martin D.
      • Simaan M.
      • Chen M.
      • Weinstein L.S.
      • Taylor S.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivation of a Gα(s)–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis.
      ), thus phenocopying the effects of LOF mutations in PATCHED (PTCH) or GOF mutations in SMO, which are the best known BCC tumor suppressor and oncogenes, respectively. Activation of the SHH pathway is also typical of a subset of medulloblastomas, a childhood malignancy (
      • Cowley G.S.
      • Weir B.A.
      • Vazquez F.
      • Tamayo P.
      • Scott J.A.
      • Rusin S.
      • East-Seletsky A.
      • Ali L.D.
      • Gerath W.F.
      • Pantel S.E.
      • Lizotte P.H.
      • Jiang G.
      • Hsiao J.
      • Tsherniak A.
      • Dwinell E.
      • et al.
      Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies.
      ). Remarkably, homozygous GNAS gene loss was identified in a group of SHH subtype medulloblastomas that does not harbor mutations in PTCH or SMO (
      • Cowley G.S.
      • Weir B.A.
      • Vazquez F.
      • Tamayo P.
      • Scott J.A.
      • Rusin S.
      • East-Seletsky A.
      • Ali L.D.
      • Gerath W.F.
      • Pantel S.E.
      • Lizotte P.H.
      • Jiang G.
      • Hsiao J.
      • Tsherniak A.
      • Dwinell E.
      • et al.
      Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies.
      ). In these particular cancers that express SHH pathway components, Gαs and its downstream target PKA act as tumor suppressors by preventing the activation of GLI transcription factors (
      • Iglesias-Bartolome R.
      • Torres D.
      • Marone R.
      • Feng X.
      • Martin D.
      • Simaan M.
      • Chen M.
      • Weinstein L.S.
      • Taylor S.S.
      • Molinolo A.A.
      • Gutkind J.S.
      Inactivation of a Gα(s)–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis.
      ), whereas in most GI tissues, GNAS and PKA signaling act as tumor promoters. The former implies that certain yet to be identified Gαs-coupled receptors may exert a tumor-suppressive function in BCC. The latter raises the possibility that in GI cancers, Gαi-coupled GPCRs may act as tumor suppressors and hence that their LOF mutations might be pro-tumorigenic. This is aligned with the large number of Gαi-coupled GPCRs that are mutated in GI tumors; however, whether they exhibit GOF or LOF mutations has not yet been tested formally. These particular predictions are of high clinical relevance, as overactive cAMP/PKA activity in many GI tumors could be counteracted therapeutically by stimulating locally expressed Gαi–GPCRs, whereas BCCs and SHH-subtype medulloblastomas may be treated by raising cAMP using phosphodiesterase inhibitors (as proposed in Ref.
      • Cowley G.S.
      • Weir B.A.
      • Vazquez F.
      • Tamayo P.
      • Scott J.A.
      • Rusin S.
      • East-Seletsky A.
      • Ali L.D.
      • Gerath W.F.
      • Pantel S.E.
      • Lizotte P.H.
      • Jiang G.
      • Hsiao J.
      • Tsherniak A.
      • Dwinell E.
      • et al.
      Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies.
      ) or by stimulating locally (or systemically) Gαs–GPCRs expressed in these tissues. These exciting possibilities will likely be explored in the near future.

      pan-Cancer GPCRs expression

      In addition to mutations, normal GPCRs can play a key role in cancer progression, and they can be targeted pharmacologically for therapeutic purposes. A typical problem when analyzing gene expression changes in cancer is that often both normal and cancerous tissues are heterogeneous, including multiple cell types. Hence, relative changes (fold changes and over- and underexpression) may reflect cellular heterogeneity more than the progression from a normal cell to its distinct cancer states. For example, comparison of GPCRs expressed in cutaneous melanoma with normal skin may grossly overestimate the relative changes in expression between normal and cancerous melanocytes, as the normal skin includes a very limited number of melanocytes. Moreover, although fold changes can provide useful information, this takes attention away from GPCRs that may exert important functions for cancer transformation through increased local ligand secretion or aberrant downstream signaling activity. A recent study has documented relative changes in GPCR expression in cancer (
      • Insel P.A.
      • Sriram K.
      • Wiley S.Z.
      • Wilderman A.
      • Katakia T.
      • McCann T.
      • Yokouchi H.
      • Zhang L.
      • Corriden R.
      • Liu D.
      • Feigin M.E.
      • French R.P.
      • Lowy A.M.
      • Murray F.
      GPCRomics: GPCR expression in cancer cells and tumors identifies new, potential biomarkers and therapeutic targets.
      ). Instead, we focus here on illuminating absolute expression levels of each GPCR and provide visual representations to gauge absolute GPCR levels. Certainly, a limitation of this analysis is that the precise cells that express each GPCR within the tumors, such as cancer and tumor stromal cells (e.g. cancer associated fibroblasts, blood vessels, and immune infiltrating cells), will need to be established in future efforts, for example by the use of modern single cell sequencing approaches. Nonetheless, we expect that we can gain an unprecedented new perspective on GPCR expression patterns in human malignancies by utilizing information gained from this analysis.
      Specifically, as shown in Fig. 5, an intriguing area of study is the expression of orphan GPCRs in cancer. The endogenous ligands of more than 140 of these receptors remain unidentified and/or poorly understood, thus, their natural function is currently largely unknown (
      • Wacker D.
      • Stevens R.C.
      • Roth B.L.
      How ligands illuminate GPCR molecular pharmacology.
      ). Nevertheless, according to our pan-cancer analysis, orphan GPCRs are differentially expressed across cancer types, and they may exert multiple functions during cancer progression (Fig. S1M). For example, since a decrease in extracellular pH is a major tumor-promoting factor in the tumor microenvironment, an intriguing area of research is the group of proton-sensing GPCRs: GPR132, GPR65, GPR68, and GPR4, which are highly expressed in a large range of human cancers. Both GPR4 and TDAG8 (GPR65) have been shown to be overexpressed in many cancers and can cause malignant transformation of cells in vitro (
      • Sin W.C.
      • Zhang Y.
      • Zhong W.
      • Adhikarakunnathu S.
      • Powers S.
      • Hoey T.
      • An S.
      • Yang J.
      G protein–coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers.
      ). GPR132 (also known as G2A) was previously shown to have tumor suppressor properties, as it prevents oncogenic transformations of pre-B cells by the BCR–ABL oncogene, similar to the role of GNA13 in these cell types (
      • Lin P.
      • Ye R.D.
      The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis.
      ). However, GPR132 has been shown to be highly transforming in fibroblasts (
      • Whitehead I.P.
      • Zohn I.E.
      • Der C.J.
      Rho GTPase-dependent transformation by G protein–coupled receptors.
      ). Thus, proton-sensing GPCRs may display tumor-promoting or -suppressive functions depending on the cancer cell of origin and may also display pro-tumorigenic activity when activated in the tumor stroma (
      • Insel P.A.
      • Sriram K.
      • Wiley S.Z.
      • Wilderman A.
      • Katakia T.
      • McCann T.
      • Yokouchi H.
      • Zhang L.
      • Corriden R.
      • Liu D.
      • Feigin M.E.
      • French R.P.
      • Lowy A.M.
      • Murray F.
      GPCRomics: GPCR expression in cancer cells and tumors identifies new, potential biomarkers and therapeutic targets.
      ). Interestingly, in our recent G protein–coupling predictor trained by a large experimental dataset, orphan GPCRs tend to show a higher proportion of coupling toward Gα12/13 than other GPCR classes (
      • Inoue A.
      • Raimondi F.
      • Kadji F.M.N.
      • Singh G.
      • Kishi T.
      • Uwamizu A.
      • Ono Y.
      • Shinjo Y.
      • Ishida S.
      • Arang N.
      • Kawakami K.
      • Gutkind J.S.
      • Aoki J.
      • Russell R.B.
      Illuminating G-protein–coupling selectivity of GPCRs.
      ) further suggesting potential importance of orphan GPCRs in cancers that involve aberrant Gα12/13 signaling.
      Figure thumbnail gr5
      Figure 5Expression of class A orphan receptors in cancer. Gene expression for class A orphan GPCRs from the UCSC TCGA PanCan Cohort RNA-seq dataset is shown. Expression values are summarized by defining transcripts per million (TPM), which normalizes for both gene length and sequencing depth. Expression values are log2(TPM + 0.001) averaged within the primary tumor samples of each cancer. GPCRs are clustered based on similarity across cancer types.
      The leucine-rich repeat-containing GPCRs (LGR) LGRs 4–8 are known for their role in development, bone formation, and remodeling, but LGR4 and LGR5 are also up-regulated in several cancer types (
      • Barker N.
      • Clevers H.
      Leucine-rich repeat-containing G-protein–coupled receptors as markers of adult stem cells.
      ). These receptors are expressed in multiple tissue-resident stem cells, and their overexpression may reflect the expansion of this cellular compartment as well as the establishment of cancer stem cell niches (
      • Barker N.
      • Clevers H.
      Leucine-rich repeat-containing G-protein–coupled receptors as markers of adult stem cells.
      ). Overexpression of LGR4 and LGR5 in colon and ovarian tumors most likely enhances cell proliferation and metastasis (
      • Tanese K.
      • Fukuma M.
      • Yamada T.
      • Mori T.
      • Yoshikawa T.
      • Watanabe W.
      • Ishiko A.
      • Amagai M.
      • Nishikawa T.
      • Sakamoto M.
      G-protein–coupled receptor GPR49 is up-regulated in basal cell carcinoma and promotes cell proliferation and tumor formation.
      ,
      • McClanahan T.
      • Koseoglu S.
      • Smith K.
      • Grein J.
      • Gustafson E.
      • Black S.
      • Kirschmeier P.
      • Samatar A.A.
      Identification of overexpression of orphan G protein–coupled receptor GPR49 in human colon and ovarian primary tumors.
      ). Interestingly, many class A orphan GPCRs are rarely expressed across cancer types. These include the MAS oncogene, which can explain the limitations in analyzing its role in human cancer despite its initial identification during transfection experiments several decades ago. Others are expressed in a single cancer (e.g. GPR22 in pheochromocytoma and paraganglioma) or a few cancers (e.g. GPR17 and GPR37L1 that are expressed only in GBM and brain lower grade glioma), whereas others are expressed in most cancers, such as OPN3 and LGR4. These studies de-orphaning GPCRs and uncovering the function of additional overexpressed GPCRs may provide promising candidates for therapeutic intervention in cancer.
      The pan-cancer expression of each GPCR class is depicted in Fig. S1, A–N. We hope that this information will be useful for hypothesis generation in our large community of scientists working in the field of GPCRs in academia and industry. Although this review will not provide a comprehensive analysis of each GPCR, a few concepts may be worth discussing. For example, expression of the purinergic P2Y11 and adenosine A2A receptors is widespread in all cancers, whereas GBM tumors express high levels of ADORA1, ADORA2, and ADORA3, all of which can be activated by adenosine in the tumor microenvironment. Multiple lipid receptors for S1P (S1P1–3) and LPA (LPA1, LPA2, and LPA6) are widely expressed as well. These receptors are intriguing because ligands for these receptors have been shown to accumulate in the tumor microenvironment (
      • Nagahashi M.
      • Ramachandran S.
      • Kim E.Y.
      • Allegood J.C.
      • Rashid O.M.
      • Yamada A.
      • Zhao R.
      • Milstien S.
      • Zhou H.
      • Spiegel S.
      • Takabe K.
      Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis.
      ,
      • Xu Y.
      • Fang X.J.
      • Casey G.
      • Mills G.B.
      Lysophospholipids activate ovarian and breast cancer cells.
      ). Conserved residues in these receptors also display a high mutational rate, which suggests that they may play vital roles in receptor signaling initiation, termination, and coupling specificity (
      • O'Hayre M.
      • Vázquez-Prado J.
      • Kufareva I.
      • Stawiski E.W.
      • Handel T.M.
      • Seshagiri S.
      • Gutkind J.S.
      The emerging mutational landscape of G proteins and G-protein–coupled receptors in cancer.
      ).
      This is also highly relevant for the 17 known GPCRs that specifically recognize intermediates or (by)products of cellular metabolism, which are often involved in nutrient sensing (
      • Husted A.S.
      • Trauelsen M.
      • Rudenko O.
      • Hjorth S.A.
      • Schwartz T.W.
      GPCR-mediated signaling of metabolites.
      ). These include receptors sensing amino acids and amino acid metabolites (GPR142, CasSR, GPR35, TAAR1, and FOPR1/2), bile acid (TGR5/GPBAR1), triglyceride metabolites (e.g. FFA1/GPR40, FFA4/GPR120, and GPR119), products of the intermediary metabolism and small carboxylic metabolites such as acetate and propionate (FFA2/GPR43 and FFA3/GPR41), butyrate (FFA2/GPR43, FFA3/GPR41, and HCA2/GPR109A), β-hydroxybutyrate (HCA2/GPR109A), β-hydroxyoctanoate (HCA3/GPR109B), lactate (HCA1/GPR81), succinate (GPR91), and capric acid (GPR84) receptors, as well as gut microbiota-derived products (e.g. short-chain fatty acids, such as acetate, propionate, and butyrate) (reviewed in Ref.
      • Husted A.S.
      • Trauelsen M.
      • Rudenko O.
      • Hjorth S.A.
      • Schwartz T.W.
      GPCR-mediated signaling of metabolites.
      ). These receptors are highly expressed in multiple organs of the digestive tract and immune cells (
      • Offermanns S.
      Free fatty acid (FFA) and hydroxy carboxylic acid (HCA) receptors.
      ), and they may be persistently activated in the tumor microenvironment due to the high metabolic rate that characterizes most solid tumors.
      The EP4 (PTGER4) and EP2 (PTGER2) receptors for the typical inflammatory mediator PGE2 (see below) are also widely expressed, whereas EP3 (PTGER3) is mainly expressed in kidney cancer. PGE2 plays a critical role in epithelial regeneration following tissue injury and cancer growth, which occurs via PI3K/Akt and β-catenin pathways (
      • Castellone M.D.
      • Teramoto H.
      • Williams B.O.
      • Druey K.M.
      • Gutkind J.S.
      Prostaglandin E2 promotes colon cancer cell growth through a Gs–axin–β-catenin signaling axis.
      ,
      • North T.E.
      • Goessling W.
      • Walkley C.R.
      • Lengerke C.
      • Kopani K.R.
      • Lord A.M.
      • Weber G.J.
      • Bowman T.V.
      • Jang I.H.
      • Grosser T.
      • Fitzgerald G.A.
      • Daley G.Q.
      • Orkin S.H.
      • Zon L.I.
      Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.
      ). COX2 overexpression and enhanced PGE2 production is most notable in colorectal cancer, and COX2 blockade can help explain the cancer chemopreventive activity of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) (
      • Chan A.T.
      • Ogino S.
      • Fuchs C.S.
      Aspirin and the risk of colorectal cancer in relation to the expression of COX-2.
      ). However, direct roles for PGE2 in tumorigenesis have been demonstrated for many other human malignancies, including breast, lung, liver, and gastric cancers, among others. For example, in laboratory models of breast and gastric cancers, COX2 overexpression and alterations in Wnt signaling both led to increased tumorigenesis (
      • Chen E.P.
      • Smyth E.M.
      COX-2 and PGE2-dependent immunomodulation in breast cancer.
      ,
      • Itadani H.
      • Oshima H.
      • Oshima M.
      • Kotani H.
      Mouse gastric tumor models with prostaglandin E2 pathway activation show similar gene expression profiles to intestinal-type human gastric cancer.
      ). Moreover, EP3 has been shown to be involved in angiogenesis in lung cancer cell lines by increasing VEGF and metalloproteinase-9 (MMP-9) expression (
      • Amano H.
      • Ito Y.
      • Suzuki T.
      • Kato S.
      • Matsui Y.
      • Ogawa F.
      • Murata T.
      • Sugimoto Y.
      • Senior R.
      • Kitasato H.
      • Hayashi I.
      • Satoh Y.
      • Narumiya S.
      • Majima M.
      Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis.
      ).
      Among the class of GPCRs for proteins (Fig. S1E), which includes chemokine receptors, CXCR4 is the most widely expressed. This may include many cancers that express CXCR4 under hypoxic conditions, as well as in blood vessels and immune cells (see below) (
      • Staller P.
      • Sulitkova J.
      • Lisztwan J.
      • Moch H.
      • Oakeley E.J.
      • Krek W.
      Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL.
      ,
      • Liu Y.L.
      • Yu J.M.
      • Song X.R.
      • Wang X.W.
      • Xing L.G.
      • Gao B.B.
      Regulation of the chemokine receptor CXCR4 and metastasis by hypoxia-inducible factor in non small cell lung cancer cell lines.
      • Schioppa T.
      • Uranchimeg B.
      • Saccani A.
      • Biswas S.K.
      • Doni A.
      • Rapisarda A.
      • Bernasconi S.
      • Saccani S.
      • Nebuloni M.
      • Vago L.
      • Mantovani A.
      • Melillo G.
      • Sica A.
      Regulation of the chemokine receptor CXCR4 by hypoxia.
      ). Other chemokine receptors that are highly expressed in immune cells (see below) were less well-represented, suggesting a more limited impact of immune infiltrating cells to the overall mRNA expression patterns in our pan-cancer analysis. The analysis of GPCRs activated by peptides (Fig. S1F) show a clear widespread expression in genes for thrombin PAR1 (F2R) and PAR2 (F2RL1) receptors and endothelin receptors (EDNRB), the latter with particularly higher expression in SKCM and uveal (UM) melanomas. HRH1, encoding H1 histamine receptor, is the most widely expressed aminergic GPCR (Fig. S1G), whereas M1 muscarinic receptors (CHRM1) and β1-adrenergic receptors (ADRB1) are highly expressed in prostate cancer, the latter receptor being of unexpected importance for the most highly prevalent cancer among males (see below). Another interesting finding was the high level of expression of dopamine receptor 2 (DRD2) in a well-defined set of cancers, including GBM, considering that a new family of antagonists for this receptor has exhibited encouraging anti-tumor activity in multiple cancer types (
      • Arrillaga-Romany I.
      • Chi A.S.
      • Allen J.E.
      • Oster W.
      • Wen P.Y.
      • Batchelor T.T.
      A phase 2 study of the first imipridone ONC201, a selective DRD2 antagonist for oncology, administered every three weeks in recurrent glioblastoma.
      ,
      • Kline C.L.B.
      • Ralff M.D.
      • Lulla A.R.
      • Wagner J.M.
      • Abbosh P.H.
      • Dicker D.T.
      • Allen J.E.
      • El-Deiry W.S.
      Role of dopamine receptors in the anticancer activity of ONC201.
      ).
      Interestingly, from our analysis of Frizzled GPCRs, SMO is widely expressed in most cancers, beyond its initial main role in BCC. This might be due to SMO being expressed in cancer stromal cells that are present in most solid tumors (Fig. S1G) (
      • Xie J.
      • Murone M.
      • Luoh S.M.
      • Ryan A.
      • Gu Q.
      • Zhang C.
      • Bonifas J.M.
      • Lam C.W.
      • Hynes M.
      • Goddard A.
      • Rosenthal A.
      • Epstein Jr., E.H.
      • de Sauvage F.J.
      Activating smoothened mutations in sporadic basal-cell carcinoma.
      ,
      • Walter K.
      • Omura N.
      • Hong S.M.
      • Griffith M.
      • Vincent A.
      • Borges M.
      • Goggins M.
      Overexpression of smoothened activates the sonic hedgehog signaling pathway in pancreatic cancer-associated fibroblasts.
      ). There is also widespread expression of FZD6 and a more cancer-restricted expression of FZD1 and FZD4 (Fig. S1H).
      Intriguingly, analysis of the sensory GPCRs revealed a high level of expression of the taste receptor, TAS1R3, across most cancer types, which has not been previously investigated (Fig. S1J).
      The adhesion GPCR family has mainly been studied in immunological and developmental functions, but they have recently been linked to cancer (Fig. S1M). For example, EMR2 (ADGRE2) is overexpressed in human breast cancer, and increased nuclear expression of EMR2 is negatively correlated with tumor grade (
      • Davies J.Q.
      • Lin H.H.
      • Stacey M.
      • Yona S.
      • Chang G.W.
      • Gordon S.
      • Hamann J.
      • Campo L.
      • Han C.
      • Chan P.
      • Fox S.B.
      Leukocyte adhesion-GPCR EMR2 is aberrantly expressed in human breast carcinomas and is associated with patient survival.
      ). Additionally, CD97 (ADGRE5) and GPR56 (ADGRG1) are the highest expressed adhesion GPCRs across all cancers, but they have only been studied in the context of melanoma, gastric, esophageal, and thyroid cancers (
      • Aust G.
      • Eichler W.
      • Laue S.
      • Lehmann I.
      • Heldin N.E.
      • Lotz O.
      • Scherbaum W.A.
      • Dralle H.
      • Hoang-Vu C.
      CD97: a dedifferentiation marker in human thyroid carcinomas.
      ,
      • Yang L.
      • Chen G.
      • Mohanty S.
      • Scott G.
      • Fazal F.
      • Rahman A.
      • Begum S.
      • Hynes R.O.
      • Xu L.
      GPR56 regulates VEGF production and angiogenesis during melanoma progression.
      • Xu L.
      • Begum S.
      • Hearn J.D.
      • Hynes R.O.
      GPR56, an atypical G protein–coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.
      ). Additionally, GPR65 (TDAG8) and GPR133 (ADGRD1) have also been associated with human cancers and linked to tumor promotion (
      • Sin W.C.
      • Zhang Y.
      • Zhong W.
      • Adhikarakunnathu S.
      • Powers S.
      • Hoey T.
      • An S.
      • Yang J.
      G protein–coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers.
      ,
      • Bayin N.S.
      • Frenster J.D.
      • Kane J.R.
      • Rubenstein J.
      • Modrek A.S.
      • Baitalmal R.
      • Dolgalev I.
      • Rudzenski K.
      • Scarabottolo L.
      • Crespi D.
      • Redaelli L.
      • Snuderl M.
      • Golfinos J.G.
      • Doyle W.
      • Pacione D.
      • et al.
      GPR133 (ADGRD1), an adhesion G-protein–coupled receptor, is necessary for glioblastoma growth.
      ), but the role of this highly-expressed family of GPCRs in tumor initiation and metastasis is still not fully understood.
      Overall, we expect that the emerging pan-cancer information on GPCR expression will ignite new interest on their study in human malignancies.

      GPCRs in metastasis and angiogenesis

      Metastasis is one of the cancer hallmarks, in which tumor cells can acquire the ability to migrate and disseminate from the tumor to distant tissues. Cancer cells spread from the primary organ to secondary sites through lymphatic vessels and blood and are the result of a sequential, highly-organized, and organ-selective process. The precise mechanisms determining the directional migration and invasion of tumor cells into specific organs remain to be fully established, but chemokine receptors, all of which are GPCRs, have been the most popular place to look (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ,
      • Balkwill F.
      Cancer and the chemokine network.
      ). Chemokines are small, cytokine-like proteins that induce directional migration for immune cells through interaction with GPCRs. Chemokines are secreted by multiple organs and act in a coordinated fashion with cell-surface proteins to direct homing of immune cells to specific anatomical sites (
      • Stein J.V.
      • Nombela-Arrieta C.
      Chemokine control of lymphocyte trafficking: a general overview.
      ,
      • Ransohoff R.M.
      Chemokines and chemokine receptors: standing at the crossroads of immunobiology and neurobiology.
      ). To serve a similar purpose, tumor cells can hijack chemokine receptor networks and migrate toward specific chemokines, facilitating metastasis to other organs, primarily the liver, lungs, brain, lymph nodes, and bone marrow (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ). In addition, the tumor microenvironment includes chemokines that can enhance the motility and survival of cancer cells in an autocrine and paracrine fashion, a process that we refer as oncocrine signaling.
      There are 23 distinct chemokine receptors in humans, and they are divided into four classes according to the type of chemokine with which they interact (CC, CXC, CX3C, or XC) (
      • Balkwill F.
      Cancer and the chemokine network.
      ,
      • Zlotnik A.
      • Yoshie O.
      Chemokines: a new classification system and their role in immunity.
      ,
      • Murphy P.M.
      • Baggiolini M.
      • Charo I.F.
      • Hébert C.A.
      • Horuk R.
      • Matsushima K.
      • Miller L.H.
      • Oppenheim J.J.
      • Power C.A.
      International union of pharmacology. XXII. Nomenclature for chemokine receptors.
      ). CXCR4 and CCR7 represent the best-studied chemokine receptors driving cancer metastasis, as they play active roles in tumor growth, invasion, angiogenesis, metastasis, and cancer relapse and therapeutic resistance (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ,
      • Müller A.
      • Homey B.
      • Soto H.
      • Ge N.
      • Catron D.
      • Buchanan M.E.
      • McClanahan T.
      • Murphy E.
      • Yuan W.
      • Wagner S.N.
      • Barrera J.L.
      • Mohar A.
      • Verástegui E.
      • Zlotnik A.
      Involvement of chemokine receptors in breast cancer metastasis.
      ,
      • Li Y.M.
      • Pan Y.
      • Wei Y.
      • Cheng X.
      • Zhou B.P.
      • Tan M.
      • Zhou X.
      • Xia W.
      • Hortobagyi G.N.
      • Yu D.
      • Hung M.C.
      Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis.
      ). CXCL12, the chemokine for CXCR4, is highly expressed in multiple tissues, mainly lungs, liver, and bone marrow, and it is also secreted by tumor and stromal cells (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ,
      • Müller A.
      • Homey B.
      • Soto H.
      • Ge N.
      • Catron D.
      • Buchanan M.E.
      • McClanahan T.
      • Murphy E.
      • Yuan W.
      • Wagner S.N.
      • Barrera J.L.
      • Mohar A.
      • Verástegui E.
      • Zlotnik A.
      Involvement of chemokine receptors in breast cancer metastasis.
      ). CXCL12 expression levels are highest in these common sites of metastasis, which could recruit cancer cells to these distant organs. CCR7 binds the ligands, CCL21 and CCL19, and guides the migration of lymphocytes and dendritic cells (DC) to lymph nodes (
      • Förster R.
      • Davalos-Misslitz A.C.
      • Rot A.
      CCR7 and its ligands: balancing immunity and tolerance.
      ). Expression of CCR7 in tumor cells has emerged as an important predictor of lymph node metastasis and poor prognosis in cervical cancer, gastric carcinoma, and breast cancer, among others (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ).
      CCR7 and CXCR4 are the main receptors typically present on metastasizing cells, but there are other chemokine receptors that may dictate a more organ-specific metastasis. For example, the small intestine is an organ that expresses high levels of CCL25 physiologically to guide CCR9+ lymphocytes to this tissue. Because melanoma, breast cancer, and ovarian cancer express high levels of CCR9, this receptor may play a pivotal role in the preferential metastasis of these tumors to the small intestine (
      • Amersi F.F.
      • Terando A.M.
      • Goto Y.
      • Scolyer R.A.
      • Thompson J.F.
      • Tran A.N.
      • Faries M.B.
      • Morton D.L.
      • Hoon D.S.
      Activation of CCR9/CCL25 in cutaneous melanoma mediates preferential metastasis to the small intestine.
      ,
      • Johnson-Holiday C.
      • Singh R.
      • Johnson E.
      • Singh S.
      • Stockard C.R.
      • Grizzle W.E.
      • Lillard Jr., J.W.
      CCL25 mediates migration, invasion and matrix metalloproteinase expression by breast cancer cells in a CCR9-dependent fashion.
      • Singh R.
      • Stockard C.R.
      • Grizzle W.E.
      • Lillard Jr., J.W.
      • Singh S.
      Expression and histopathological correlation of CCR9 and CCL25 in ovarian cancer.
      ). Additionally, malignant melanomas express high levels of CCR10, a receptor that guides leukocytes to the skin, and consequently, CCR10 expression in melanoma may drive metastasis to the skin (
      • Homey B.
      • Wang W.
      • Soto H.
      • Buchanan M.E.
      • Wiesenborn A.
      • Catron D.
      • Müller A.
      • McClanahan T.K.
      • Dieu-Nosjean M.C.
      • Orozco R.
      • Ruzicka T.
      • Lehmann P.
      • Oldham E.
      • Zlotnik A.
      Cutting edge: the orphan chemokine receptor G protein–coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC).
      ,
      • Simonetti O.
      • Goteri G.
      • Lucarini G.
      • Filosa A.
      • Pieramici T.
      • Rubini C.
      • Biagini G.
      • Offidani A.
      Potential role of CCL27 and CCR10 expression in melanoma progression and immune escape.
      ). CXCR3 and CXCR5 have both been shown to play a role in lymph node metastasis (
      • López-Giral S.
      • Quintana N.E.
      • Cabrerizo M.
      • Alfonso-Pérez M.
      • Sala-Valdés M.
      • De Soria V.G.
      • Fernández-Ranada J.M.
      • Fernández-Ruiz E.
      • Muñoz C.
      Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination.
      ,
      • Kawada K.
      • Hosogi H.
      • Sonoshita M.
      • Sakashita H.
      • Manabe T.
      • Shimahara Y.
      • Sakai Y.
      • Takabayashi A.
      • Oshima M.
      • Taketo M.M.
      Chemokine receptor CXCR3 promotes colon cancer metastasis to lymph nodes.
      ).
      With increasing nutrients and oxygen demands by the tumor cell, solid tumors produce angiogenic factors that promote the migration and proliferation of endothelial cells to form new vessels. Many of these factors exert their functions through GPCRs expressed on endothelial cells, including thrombin, prostaglandins, S1P, and many chemokines. In addition, chemokines, like CCL2, CCL5, and CXCL8/IL-8, can recruit leukocytes and macrophages to the tumor site, which leads to production of VEGF and other angiogenic factors that contribute to the growth of tumor-associated blood vessels (
      • Zlotnik A.
      • Burkhardt A.M.
      • Homey B.
      Homeostatic chemokine receptors and organ-specific metastasis.
      ,
      • Balkwill F.
      Cancer and the chemokine network.
      ). Production of inflammatory cytokines can also promote new vessel formation by elevating COX2 expression, and in turn prostaglandin E2 (PGE2) increases the expression of VEGF, CXCL8, and CXCL5 by tumor cells (
      • Põld M.
      • Zhu L.X.
      • Sharma S.
      • Burdick M.D.
      • Lin Y.
      • Lee P.P.
      • Põld A.
      • Luo J.
      • Krysan K.
      • Dohadwala M.
      • Mao J.T.
      • Batra R.K.
      • Strieter R.M.
      • Dubinett S.M.
      Cyclooxygenase-2–dependent expression of angiogenic CXC chemokines ENA-78/CXC ligand (CXCL) 5 and interleukin-8/CXCL8 in human non-small cell lung cancer.
      ,
      • Wang D.
      • Wang H.
      • Brown J.
      • Daikoku T.
      • Ning W.
      • Shi Q.
      • Richmond A.
      • Strieter R.
      • Dey S.K.
      • DuBois R.N.
      CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer.
      ).
      In the case of thrombin, this serine proteinase plays a vital role in regulating hemostasis by converting fibrinogen into fibrin to stimulate platelet aggregation and coagulation (
      • Offermanns S.
      • Laugwitz K.L.
      • Spicher K.
      • Schultz G.
      G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.
      ). Thrombin carries out its effects through the PAR family of receptors, which exhibit the unique property of harboring a tethered ligand within the receptor that becomes exposed upon cleavage of the N-terminal extracellular region by thrombin (
      • Coughlin S.R.
      How the protease thrombin talks to cells.
      ). Thrombin promotes angiogenesis by increasing metalloproteinases and decreasing the ability of endothelial cells to adhere to the extracellular matrix (
      • Duhamel-Clérin E.
      • Orvain C.
      • Lanza F.
      • Cazenave J.P.
      • Klein-Soyer C.
      Thrombin receptor-mediated increase of two matrix metalloproteinases, MMP-1 and MMP-3, in human endothelial cells.
      ,
      • Herbert J.M.
      • Dupuy E.
      • Laplace M.C.
      • Zini J.M.
      • Bar Shavit R.
      • Tobelem G.
      Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway.
      ), while promoting the expression and activity of the VEGF receptor, VEGFR-2, through Gα13 and its target RhoA (
      • Sivaraj K.K.
      • Takefuji M.
      • Schmidt I.
      • Adams R.H.
      • Offermanns S.
      • Wettschureck N.
      G13 controls angiogenesis through regulation of VEGFR-2 expression.
      ). However, S1P1 stimulates endothelial cell proliferation, survival, and migration and also regulates sprouting angiogenesis through cross-talk with VEGFR-2 and enhanced tissue hypoxia and VEGF production (
      • Ryu Y.
      • Takuwa N.
      • Sugimoto N.
      • Sakurada S.
      • Usui S.
      • Okamoto H.
      • Matsui O.
      • Takuwa Y.
      Sphingosine-1-phosphate, a platelet-derived lysophospholipid mediator, negatively regulates cellular Rac activity and cell migration in vascular smooth muscle cells.
      ,
      • Lee M.J.
      • Thangada S.
      • Claffey K.P.
      • Ancellin N.
      • Liu C.H.
      • Kluk M.
      • Volpi M.
      • Sha'afi R.I.
      • Hla T.
      Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.
      • Jung B.
      • Obinata H.
      • Galvani S.
      • Mendelson K.
      • Ding B.S.
      • Skoura A.
      • Kinzel B.
      • Brinkmann V.
      • Rafii S.
      • Evans T.
      • Hla T.
      Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development.
      ). The effects of S1P on angiogenesis largely depend on the GPCR it binds (S1P1–5). S1P stimulates angiogenesis mainly through S1P1 and S1P3, and it mediates endothelial cell migration and formation of capillary structures through Gαi (or more likely its associated Gβγ subunits) activation of the small GTPase Rac1 (
      • Ishii I.
      • Fukushima N.
      • Ye X.
      • Chun J.
      Lysophospholipid receptors: signaling and biology.
      ). In contrast, S1P2 has been shown to be involved in the suppression of angiogenesis most likely through the inhibition of Rac and cell migration (
      • Du W.
      • Takuwa N.
      • Yoshioka K.
      • Okamoto Y.
      • Gonda K.
      • Sugihara K.
      • Fukamizu A.
      • Asano M.
      • Takuwa Y.
      S1P(2), the G protein–coupled receptor for sphingosine-1-phosphate, negatively regulates tumor angiogenesis and tumor growth in vivo in mice.
      ). Altogether, GPCRs participate in angiogenesis either by promoting the proliferation, migration, and sprouting of endothelial cells or by the release of pro-angiogenic factors for new blood vessel formation, thereby increasing the blood supply to the growing tumors.

      Key role for GPCRs in cancer immunology

      In the last few years, cancer immunotherapy became one of the most exciting breakthroughs in cancer treatment. Recent revolutionary discoveries have highlighted the importance of the tumor microenvironment and its associated immune cells in cancer development and therapeutic resistance. Tumors can deploy multiple mechanisms to avoid immune recognition and an anti-tumor immune response, including the recruitment of myeloid-derived suppressor cells (MDSC) and conditioning of the surrounding microenvironment to become highly immune-suppressive by expressing cytokines, such as IL-6, IL-10, and transforming growth factor β (
      • Veglia F.
      • Perego M.
      • Gabrilovich D.
      Myeloid-derived suppressor cells coming of age.
      ). This can lead to the accumulation of suppressive regulatory T cells (Tregs) and the polarization of macrophages toward an immune-suppressive phenotype, which is often referred to as M2 or tumor-associated macrophage (TAM) phenotype (
      • Devaud C.
      • John L.B.
      • Westwood J.A.
      • Darcy P.K.
      • Kershaw M.H.
      Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy.
      ). A key emerging mechanism of tumor immunosuppression involves the induction of T-cell exhaustion through activation of T-cell checkpoints, including programmed death 1 (PD-1). Its ligand, programmed death-ligand 1 (PD-L1), is expressed by macrophages and some cancer cells, which can restrain T-cell activation and induce immunosuppresion (
      • Sharma P.
      • Allison J.P.
      The future of immune checkpoint therapy.
      ,
      • Wei S.C.
      • Duffy C.R.
      • Allison J.P.
      Fundamental mechanisms of immune checkpoint blockade therapy.
      • Thommen D.S.
      • Schumacher T.N.
      T cell dysfunction in cancer.
      ). Together, these conditions contribute to the suppression of cytotoxic CD8+ T lymphocyte recruitment, survival, and function, and ultimately to the loss of an effective anti-tumor immune response. Although the aberrant function and dysregulated expression of GPCRs is now beginning to be linked directly to the tumor itself, the role of GPCRs on immune cells infiltrating tumors is still not fully understood and grossly underappreciated. Given the diversity of GPCRs and the variety of GPCR families, current studies have only scratched the surface of delineating GPCRs on immune cells in cancer. The importance of studying GPCRs in the context of cancer immunology is reflected by the multiple roles that this receptor family plays in inflammation, orchestrating immune cell trafficking and regulating the tumor microenvironment, as summarized in Fig. 6. A crucial first step in anti-tumor immunity is the migration of cytotoxic cells recognizing tumor antigens to the tumor, and this is mediated largely by chemokine receptors.
      Figure thumbnail gr6
      Figure 6Function of GPCRs in cancer. Top, GPCRs contribute to both tumor promotion, angiogenesis, metastasis, and immune evasive functions in the tumor microenvironment. Multiple GPCR agonists released by the tumors or accumulating in the tumor microenvironment promote angiogenesis by stimulating GPCRs on endothelial cells. GPCRs play multiple roles in cell communication between tumors cells, tumor stroma, endothelial cells, and blood vessels and immune cells, as well as in response to neurotransmitters released as a consequence of tumor-induced axonogenesis and tumor innervation as part of autocrine and paracrine (oncocrine) signaling networks that drive tumorigenesis. GPCRs present on tumor cells assist in extravasation and migration of circulating tumor cells to promote metastasis to distant organ destinations. Bottom, chemokine receptors recruit a variety of immune cells to the primary tumor and release agents that both promote and suppress immune functions. Immune-suppressive cells promote tumor growth by inhibiting functions of cytotoxic immune cells or secreting hypoxic and anti-inflammatory molecules to sculpt the suppressive tumor microenvironment. Anti-tumor immune cells that are recruited to the tumor secrete highly cytotoxic molecules for tumor cell destruction. See text for details. (Abbreviations used are as follows: ROS, reactive oxygen species; iNOS, inducible nitric-oxide synthase; ARG1, arginase 1; EMT, epithelial to mesenchymal transition; ADCC, antibody-dependent cellular cytotoxicity.

      GPCRs orchestrate immune cell migration and recruitment

      In the 1960s, it became clear that chemoattractants can bind and act directly on lymphocytes, and from there, GPCRs in the immune system became mostly known for their ability to steer cell migration toward chemokine gradients (
      • Ward P.A.
      • Cochrane C.G.
      • Mueller-Eberhard H.J.
      The role of serum complement in chemotaxis of leukocytes in vitro.
      ,
      • Ward P.A.
      • Lepow I.H.
      • Newman L.J.
      Bacterial factors chemotactic for polymorphonuclear leukocytes.
      ). Cytotoxic immune cells, including natural killer (NK) cells and CD8 T cells that are specific for tumor antigens, are guided to the tumor where they secrete cytotoxic molecules to induce tumor cell death.
      CXCR3 on CD8 T cells and NK cells binds the ligands CXCL9 and CXCLl0 to migrate into tumors (reviewed in Ref.
      • Nagarsheth N.
      • Wicha M.S.
      • Zou W.
      Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy.
      ). Indeed, increased CXCL9 and CXLC10 and tumor-infiltrating CD8 T cells correlates with improved survival and decreased cancer metastasis (
      • Zhang L.
      • Conejo-Garcia J.R.
      • Katsaros D.
      • Gimotty P.A.
      • Massobrio M.
      • Regnani G.
      • Makrigiannakis A.
      • Gray H.
      • Schlienger K.
      • Liebman M.N.
      • Rubin S.C.
      • Coukos G.
      Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer.
      ,
      • Pagès F.
      • Berger A.
      • Camus M.
      • Sanchez-Cabo F.
      • Costes A.
      • Molidor R.
      • Mlecnik B.
      • Kirilovsky A.
      • Nilsson M.
      • Damotte D.
      • Meatchi T.
      • Bruneval P.
      • Cugnenc P.H.
      • Trajanoski Z.
      • Fridman W.H.
      • Galon J.
      Effector memory T cells, early metastasis, and survival in colorectal cancer.
      ). Furthermore, CXCL10, which is also known as interferon-induced protein 10 (IP10), as well as CXCL9 and CXCL11 are known to be induced by interferon α, β, and γ and are part of the “interferon gene signature” that is often used to predict a favorable response to anti-PD-1 treatment (
      • Ayers M.
      • Lunceford J.
      • Nebozhyn M.
      • Murphy E.
      • Loboda A.
      • Kaufman D.R.
      • Albright A.
      • Cheng J.D.
      • Kang S.P.
      • Shankaran V.
      • Piha-Paul S.A.
      • Yearley J.
      • Seiwert T.Y.
      • Ribas A.
      • McClanahan T.K.
      IFN-γ–related mRNA profile predicts clinical response to PD-1 blockade.
      ). This provides direct evidence highlighting the importance of chemokine and chemokine receptors in the new era of immunotherapies.
      The activation of these antigen-experienced cytotoxic CD8+ T cells is driven by DCs that capture cancer cell antigens on their major histocompatibility (MHC) I or MHCII molecules and present them to naïve T cells to drive effector T-cell activation, bridging the innate and adaptive immune system (
      • Chen D.S.
      • Mellman I.
      Oncology meets immunology: the cancer-immunity cycle.
      ). DCs are antigen-presenting cells that traffic to draining lymph nodes for stimulation and activation of T cells, and their mobilization is largely driven by CCR7 and its ligands CCL19 and CCL21 (
      • Randolph G.J.
      • Angeli V.
      • Swartz M.A.
      Dendritic-cell trafficking to lymph nodes through lymphatic vessels.
      ). The endocytosis of apoptotic cells has been shown to induce CCR7 expression and subsequent migration of DCs, and CCR7-mediated activation of Rac1 and Rac2 may have redundant functions in migration to the lymph nodes, as shown by the absence of DC mobilization in Rac1- and Rac2-deficient mice (
      • Kerksiek K.M.
      • Niedergang F.
      • Chavrier P.
      • Busch D.H.
      • Brocker T.
      Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo.
      ). Dendritic cells play a crucial role in immunosurveillance for elimination of cancer cells.
      In addition to DC mobilization and recruitment of cytotoxic immune cells to the tumor, chemokine receptors also participate in promoting tumorigenesis by mediating the recruitment of immunosuppressive immune cells, specifically Treg cells and MDSCs (
      • Nagarsheth N.
      • Wicha M.S.
      • Zou W.
      Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy.
      ). Treg cells express CCR4 and migrate to the tumor in response to CCL22 produced by macrophages and tumor cells (
      • Curiel T.J.
      • Coukos G.
      • Zou L.
      • Alvarez X.
      • Cheng P.
      • Mottram P.
      • Evdemon-Hogan M.
      • Conejo-Garcia J.R.
      • Zhang L.
      • Burow M.
      • Zhu Y.
      • Wei S.
      • Kryczek I.
      • Daniel B.
      • Gordon A.
      • et al.
      Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.
      ). Blockade of this receptor with a therapeutic mAb (mogamulizumab) or small molecule inhibitors is under current evaluation for cancer treatment when combined with immune-checkpoint inhibitors (
      • Marin-Acevedo J.A.
      • Dholaria B.
      • Soyano A.E.
      • Knutson K.L.
      • Chumsri S.
      • Lou Y.
      Next generation of immune checkpoint therapy in cancer: new developments and challenges.
      ). The hypoxic regions of the tumor microenvironment also generate CCL28, which can recruit CCR10-expressing Tregs (
      • Facciabene A.
      • Peng X.
      • Hagemann I.S.
      • Balint K.
      • Barchetti A.
      • Wang L.P.
      • Gimotty P.A.
      • Gilks C.B.
      • Lal P.
      • Zhang L.
      • Coukos G.
      Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T(reg) cells.
      ). The immunosuppressive microenvironment of the bone marrow has been linked to high frequencies of Tregs. Tregs can be mobilized from the bone marrow into the periphery by granulocyte colony-stimulating factor (G-CSF), which promotes the degradation of CXCLl2, a ligand for CXCR4 (
      • Zou L.
      • Barnett B.
      • Safah H.
      • Larussa V.F.
      • Evdemon-Hogan M.
      • Mottram P.
      • Wei S.
      • David O.
      • Curiel T.J.
      • Zou W.
      Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals.
      ). CXCL12 can lead to higher numbers of Tregs in the bone marrow, promoting an immunosuppressive environment that favors the establishment of metastatic niches, which may help explain why many cancers often metastasize to the bone marrow.
      Chemokine receptors also direct MDSCs to the tumor thus favoring an immunosuppressive, tumor-promoting environment. Monocytic MDSCs, including TAMs, are recruited to the tumor by CCL2, CXCL5, and CXCL12 acting on CCR2, CXCR2, and CXCR4 (
      • Kitamura T.
      • Qian B.Z.
      • Soong D.
      • Cassetta L.
      • Noy R.
      • Sugano G.
      • Kato Y.
      • Li J.
      • Pollard J.W.
      CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages.
      ). MDSCs have been also shown to secrete ligands for CCR5 to direct the migration of CCR5+ Tregs, although various studies have also shown that CCR5 guides MDSCs themselves as well (
      • Schlecker E.
      • Stojanovic A.
      • Eisen C.
      • Quack C.
      • Falk C.S.
      • Umansky V.
      • Cerwenka A.
      Tumor-infiltrating monocytic myeloid–derived suppressor cells mediate CCR5-dependent recruitment of regulatory T cells favoring tumor growth.
      ,
      • Yang L.
      • Wang B.
      • Qin J.
      • Zhou H.
      • Majumdar A.P.N.
      • Peng F.
      Blockade of CCR5-mediated myeloid derived suppressor cell accumulation enhances anti-PD1 efficacy in gastric cancer.
      • Umansky V.
      • Blattner C.
      • Gebhardt C.
      • Utikal J.
      CCR5 in recruitment and activation of myeloid-derived suppressor cells in melanoma.
      ). Moreover, CXCL8 and CXCL1 are often secreted by most solid tumors and by certain subsets of Tregs and act on granulocytic MDSCs that express CXCR1 and CXCR2, including neutrophils, to promote their recruitment to tumors. Because neutrophils secrete tumor- and angiogenesis-promoting molecules, CXCL8 is generally thought to contribute to the immunosuppressive environment leading to tumor angiogenesis and progression (
      • Nagarsheth N.
      • Wicha M.S.
      • Zou W.
      Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy.
      ,
      • Waugh D.J.
      • Wilson C.
      The interleukin-8 pathway in cancer.
      ).
      In summary, a variety of chemokines dictate the recruitment of different immunosuppressive immune cells into the tumor microenvironment, and through these processes, tumors can evolve to avoid detection and destruction by the innate and adaptive immune system. This can, in turn, provide an opportunity to disable the immune evasive mechanisms by targeting chemokine receptors with an increasing repertoire of small molecule inhibitors and negative allosteric modulators (
      • van der Westhuizen E.T.
      • Valant C.
      • Sexton P.M.
      • Christopoulos A.
      Endogenous allosteric modulators of G protein-coupled receptors.
      ) and/or with blocking antibodies. These therapeutic strategies are already in use or under clinical evaluation in multiple chronic inflammatory diseases, and their study in the context of cancer immunotherapy will likely represent one of the most exciting areas of future exploration in the GPCR targeting field.

      Modulation of immunosuppressive GPCRs by the tumor microenvironment

      The immunosuppressive and hypoxic nature of the tumor microenvironment can also largely influence the function of cytotoxic immune cells and the success of cancer immunotherapies. A driving force behind the malignancy and morbidity of cancer is its ability to proliferate unrestrained, by creating an immunosuppressive environment favoring tumor growth. The nucleoside adenosine is a potent physiologic and pharmacologic regulator that is released from injured and necrotic cells by extracellular breakdown of ATP by the action of the ectonucleotidases CD39 and CD73 (reviewed in Ref.
      • Young A.
      • Mittal D.
      • Stagg J.
      • Smyth M.J.
      Targeting cancer-derived adenosine: new therapeutic approaches.
      ). Typical extracellular adenosine levels are low, but at injury sites with tissue breakdown and hypoxia, the adenosine levels can rise from nanomolar to micromolar concentrations. Extracellular adenosine can signal through four GPCRs: A1, A2A, A2B, and A3 adenosine receptors (ADORA1, ADORA2A, ADORA2B, and ADORA3, respectively) (
      • Cekic C.
      • Linden J.
      Purinergic regulation of the immune system.
      ). A1 and A3 receptors signal through Gαi and lead to decreased cAMP. Activation of A2A and A2B receptors, which are expressed on immune and endothelial cells, leads to signaling through Gαs proteins, and A2B can also signal through Gαq (
      • Borea P.A.
      • Gessi S.
      • Merighi S.
      • Vincenzi F.
      • Varani K.
      Pharmacology of adenosine receptors: the state of the art.
      ). Of the four adenosine receptors, A2A receptor (encoded by the ADORA2A gene) is the predominantly expressed subtype in most immune cells. In general, stimulation of the A2A receptor provides an immunosuppressive signal in T cells (
      • Cekic C.
      • Linden J.
      Purinergic regulation of the immune system.
      ), NK cells (
      • Young A.
      • Ngiow S.F.
      • Gao Y.
      • Patch A.M.
      • Barkauskas D.S.
      • Messaoudene M.
      • Lin G.
      • Coudert J.D.
      • Stannard K.A.
      • Zitvogel L.
      • Degli-Esposti M.A.
      • Vivier E.
      • Waddell N.
      • Linden J.
      • Huntington N.D.
      • et al.
      A2AR adenosine signaling suppresses natural killer cell maturation in the tumor microenvironment.
      ), DCs (
      • Novitskiy S.V.
      • Ryzhov S.
      • Zaynagetdinov R.
      • Goldstein A.E.
      • Huang Y.
      • Tikhomirov O.Y.
      • Blackburn M.R.
      • Biaggioni I.
      • Carbone D.P.
      • Feoktistov I.
      • Dikov M.M.
      Adenosine receptors in regulation of dendritic cell differentiation and function.
      ), and neutrophils (
      • Visser S.S.
      • Theron A.J.
      • Ramafi G.
      • Ker J.A.
      • Anderson R.
      Apparent involvement of the A(2A) subtype adenosine receptor in the anti-inflammatory interactions of CGS 21680, cyclopentyladenosine, and IB-MECA with human neutrophils.
      ). A2A receptor stimulation interferes with trafficking of T cells and NK cells by desensitizing chemokine receptors and reducing levels of pro-inflammatory cytokines (
      • Leone R.D.
      • Lo Y.C.
      • Powell J.D.
      A2aR antagonists: next generation checkpoint blockade for cancer immunotherapy.
      ). Blocking the adenosine-generating pathway has shown tumor regression in breast cancer, colorectal cancer, and melanoma (reviewed in Refs.
      • Young A.
      • Mittal D.
      • Stagg J.
      • Smyth M.J.
      Targeting cancer-derived adenosine: new therapeutic approaches.
      ,
      • Ohta A.
      A metabolic immune checkpoint: adenosine in tumor microenvironment.
      ), and small molecule inhibitors of A2A receptor as well as blocking antibodies anti-CD73 and anti-CD39 are under current evaluation for combination cancer immunotherapies (
      • Young A.
      • Mittal D.
      • Stagg J.
      • Smyth M.J.
      Targeting cancer-derived adenosine: new therapeutic approaches.
      ,
      • Antonioli L.
      • Yegutkin G.G.
      • Pacher P.
      • Blandizzi C.
      • Haskó G.
      Anti-CD73 in cancer immunotherapy: awakening new opportunities.
      ). Although these immunotherapies aim to boost immune cell activity in the immunosuppressive tumor microenvironment, it is also important to consider the effects of tumor-driven inflammation, largely driven by prostaglandins and prostaglandin receptors.

      GPCRs link inflammation to cancer immune evasion

      Inflammation occurs as the immune system responds to infection and injury to beneficially remove the offending factors and restore tissue structure and physiological function. However, with subsequent tissue injury, cells that have sustained DNA damage or mutagenic assault will continue to proliferate in microenvironments rich in inflammatory cells and growth/survival factors that support their growth. Prostaglandins are a group of physiologically-active lipid compounds found in almost every tissue in humans and animals, and they play a key role in the generation of an inflammatory response (
      • Ricciotti E.
      • FitzGerald G.A.
      Prostaglandins and inflammation.
      ). They are enzymatically derived from arachidonic acid by the COX isoforms, COX1 and COX2, and are powerful vasodilators (
      • Sugimoto Y.
      • Narumiya S.
      Prostaglandin E receptors.
      ). PGE2 is the most abundant prostaglandin produced in cancers, and the prostanoid receptor family, which are GPCRs, includes the following: E prostanoid receptor 1 (EP1, PTGER1), EP2 (PTGER2), EP3 (PTGER3), and EP4 (PTGER4). Of these, EP1 is coupled to Gαq; EP3 is coupled to Gαi, and both EP2 and EP4 are coupled to Gαs (
      • Sugimoto Y.
      • Narumiya S.
      Prostaglandin E receptors.
      ). PGE2 binding to different EP receptors can regulate the function of many immune cell types, including macrophages, DCs, T cells, and B cells, as will be discussed here.
      PGE2 produced by cancer cells has been linked to increased expression of FOXP3 in Treg cells, promoting the immune-suppressive activity of Tregs (
      • Baratelli F.
      • Lin Y.
      • Zhu L.
      • Yang S.C.
      • Heuzé-Vourc'h N.
      • Zeng G.
      • Reckamp K.
      • Dohadwala M.
      • Sharma S.
      • Dubinett S.M.
      Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells.
      ). In addition to Tregs, PGE2 has also been linked to increased recruitment of MDSCs (
      • Obermajer N.
      • Kalinski P.
      Key role of the positive feedback between PGE(2) and COX2 in the biology of myeloid-derived suppressor cells.
      ), decreased CD8 T-cell activation (
      • Iñiguez M.A.
      • Punzón C.
      • Fresno M.
      Induction of cyclooxygenase-2 on activated T lymphocytes: regulation of T-cell activation by cyclooxygenase-2 inhibitors.
      ,
      • Sreeramkumar V.
      • Fresno M.
      • Cuesta N.
      Prostaglandin E2 and T cells: friends or foes?.
      ), and increased expression of inhibitory markers, like PD-1 (
      • Obermajer N.
      • Kalinski P.
      Key role of the positive feedback between PGE(2) and COX2 in the biology of myeloid-derived suppressor cells.
      ,
      • Iñiguez M.A.
      • Punzón C.
      • Fresno M.
      Induction of cyclooxygenase-2 on activated T lymphocytes: regulation of T-cell activation by cyclooxygenase-2 inhibitors.
      ,
      • Wang J.
      • Zhang L.
      • Kang D.
      • Yang D.
      • Tang Y.
      Activation of PGE2/EP2 and PGE2/EP4 signaling pathways positively regulate the level of PD-1 in infiltrating CD8(+) T cells in patients with lung cancer.
      ). PGE2 alters the differentiation, maturation, and cytokine secretion of DCs by up-regulating CD25 and indoleamine-pyrrole 2,3-dioxygenase and decreased expression of CD80, CD86, and MHCI maturation markers (
      • von Bergwelt-Baildon M.S.
      • Popov A.
      • Saric T.
      • Chemnitz J.
      • Classen S.
      • Stoffel M.S.
      • Fiore F.
      • Roth U.
      • Beyer M.
      • Debey S.
      • Wickenhauser C.
      • Hanisch F.G.
      • Schultze J.L.
      CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition.
      ). Recently, NSAIDs that block COX2 and/or COX1 and COX2 were found to have beneficial effects on reducing the risk of developing esophageal, stomach, skin, and breast cancers, in addition to their best-established function in preventing colorectal cancer (
      • Drew D.A.
      • Cao Y.
      • Chan A.T.
      Aspirin and colorectal cancer: the promise of precision chemoprevention.
      ,
      • Fischer S.M.
      • Hawk E.T.
      • Lubet R.A.
      Coxibs and other nonsteroidal anti-inflammatory drugs in animal models of cancer chemoprevention.