GPCRs and cancer

Abstract

G-protein-coupled receptors (GPCRs), which represent the largest gene family in the human genome, play a crucial role in multiple physiological functions as well as in tumor growth and metastasis. For instance, various molecules like hormones, lipids, peptides and neurotransmitters exert their biological effects by binding to these seven-transmembrane receptors coupled to heterotrimeric G-proteins, which are highly specialized transducers able to modulate diverse signaling pathways. Furthermore, numerous responses mediated by GPCRs are not dependent on a single biochemical route, but result from the integration of an intricate network of transduction cascades involved in many physiological activities and tumor development. This review highlights the emerging information on the various responses mediated by a selected choice of GPCRs and the molecular mechanisms by which these receptors exert a primary action in cancer progression. These findings provide a broad overview on the biological activity elicited by GPCRs in tumor cells and contribute to the identification of novel pharmacological approaches for cancer patients.

Introduction

The seven-transmembrane G protein-coupled receptors (GPCRs), which belong to the largest superfamily of signal transduction proteins, regulate multiple biological functions coupling to a heterotrimeric G-protein associated with the inner surface of the plasma membrane¹. The heterotrimer that is composed of the Gα, Gβ, and Gγ subunits, binds to the guanine nucleotide GDP in its basal state. Upon activation by ligand binding, GDP is released and replaced by GTP, which leads to subunit dissociation into a βγ dimer and the GTP-bound α monomer² (Figure 1). On the basis of the sequence identity, the Gα subunit has been classified into four families: Gα_s, Gα_i, Gα_q, and Gα₁₂. Each Gα family can relay the GPCR signal stimulating different downstream effectors². Some GPCRs, such as the lysophosphatidic acid (LPA) receptors, can couple to more than one G protein triggering consequently diverse signaling cascades, whereas other GPCRs like sphingosine-1-phosphate (S1P) receptor 1 (S1P1) couple exclusively to one G protein^{3, 4}.

Figure 1.

Agonist binding to GPCRs promotes the dissociation of GDP bound to the Gα subunit and its replacement with GTP leading to the activation of the heterotrimeric G proteins and the subunit dissociation into a βγ dimer and the GTP-bound α monomer. Both subunits activate multiple downstream effectors which induce gene transcription and relevant biological responses. A cross-talk between several GPCRs and other membrane receptors as Epidermal Growth Factor Receptor (EGFR) contributes to the growth stimulation and invasion of cancer cells.

An increasing number of studies links aberrant GPCR expression and activation to numerous types of human malignancies^{5, 6} (Figure 1). For instance, several GPCRs are overexpressed in different tumors⁶ and GPCR variants can lead to increased cancer risk. In this regard, it should be mentioned that in genetic association studies melanocortin-1 receptor (MC1R) polymorphisms were associated with an enhanced threat of skin cancer⁷. In addition, an aberrant activation of GPCRs by high levels of ligands like LPA, S1P and chemokines was involved in cell transformation, proliferation, angiogenesis, metastasis and drug resistance⁶. Conversely, some members of GPCRs, such as the orexin receptor OX1R, were shown to mediate a pro-apoptotic action in various cancer cells⁸.

Cross-talk among different receptors including GPCRs triggers relevant biological functions in normal and neoplastic cells⁹. In this context, it has been reported that many GPCRs activate numerous signaling pathways interacting with other plasma membrane receptors⁹ (Figure 1). For example, the cross-talk between acetylcholine muscarinic receptors (mAChRs) and epidermal growth factor (EGFR) as well as platelet-derived growth factor (PDGFR) receptors leads to the activation of mitogenic pathways which mediate cell proliferation, differentiation and survival¹⁰. In addition, several GPCR ligands like bradikinin (BK), LPA, Gastrin-releasing peptide (GrP) and bombesin (BN) transactivate EGFR, then inducing stimulatory effects in different types of tumors⁶.

Currently, many agents targeting GPCRs, such as gonadotropin-releasing factor and somatostatin receptors, are used for cancer treatment on the basis of valuable experimental data and clinical benefits^{11, 12}. Moreover, various inhibitors of GPCRs are currently under evaluation in clinical trials as anticancer agents (http://www.clinicaltrials.gov/). This review recapitulates our current understanding regarding the mechanisms through which various GPCRs may contribute to tumor progression. On these bases, GPCRs may be considered as promising targets in novel pharmacological approaches for cancer patients.

GPCRs activated by bio-active lipids

GPCRs activated by the bio-active lipids LPA and S1P have been implicated in aberrant signaling in a wide range of tumors. LPA1, LPA2 and LPA3 represent the most widely expressed and well-characterized receptors for LPA and its analogues³. Upon binding to these receptors, LPA triggers a variety of signaling pathways engaging the heterotrimeric G proteins and their downstream effectors³. As a consequence, the transcriptional activation of multiple cancer-associated genes leads to cell survival and proliferation, migration, chemotaxis, vascular remodeling and angiogenesis¹³. Aberrant expressions and mutations of LPA receptors have been found in several types of tumors, suggesting their involvement in the growth advantage of cancer cells^{3, 14}, ¹⁵. For instance, LPA1 was inversely correlated in breast cancer tissues with the Nm23 metastases regulator¹⁶ and contributed to bone metastasis in breast cancer xenografts¹⁷. Furthermore, LPA induced migration in breast cancer cells by activating LPA1, which promoted the phosphorylation of nonmuscle myosin II (NM II) light chain through the activation of ROCK and RhoA activity¹⁸. In addition, the expression of LPA1, LPA2, and LPA3 in mammary epithelium of transgenic mice induced a high frequency of late-onset, estrogen receptor (ER)-positive, invasive and metastatic mammary cancer¹⁹. LPA stimulated also tumorigenesis and metastasis in ovarian malignancy²⁰. For instance, LPA exerted a growth factor-similar action and prevented apoptosis in ovarian cancer cells through redox-dependent activation of ERK, Akt and NF-κB-dependent signaling²¹. Recently, the LPA/LPA1-induced Rac activation as well as the integrity of SOS1/EPS8/ABI1 tri-complex were required for ovarian cancer metastasis²². Closely resembling the LPA-effects in human ovarian cancer cells, LPA induced metastasis of epithelial ovarian cancer in immuno-competent mice²³. As other GPCR ligands, LPA promoted stimulatory effects in different types of tumors by transactivating EGFR and triggering a functional cross-talk between its cognate receptors and EGFR-mediated signaling⁶. In this regard, it should be mentioned that EGFR activity was required for the activation of Gi-dependent cellular responses induced by LPA in ovarian cancer cells²⁴. Moreover, a cross-talk between LPA receptors and EGFR occurred in ovarian cancer cells as demonstrated by the increase of LPA production following ligand-dependent EGFR transactivation²⁵. In addition to breast and ovarian malignancies, LPA was involved in other types of tumors. In a murine xenograft model of lung adenocarcinoma, mesenchymal stem cells were recently shown to stimulate angiogenesis through a LPA1-dependent mechanism²⁶. Likewise, an engineered three-dimensional tumor xenograft model of non-small cell lung cancer (NSCLC) in nude mice regressed and lost vascularity in response to BrP-LPA, which acts as a LPA receptor antagonist and autotaxin inhibitor²⁷.

The bio-active lipid S1P has been involved in various aspects of tumor development, including cell proliferation, motility and invasiveness, apoptosis, differentiation, angiogenesis and inflammation²⁸. However, S1P can mediate both proliferative²⁹ and antiproliferative³⁰ effects in neoplastic cells. These opposite responses to S1P were attributed to the different activities exerted by its five receptors, which are coupled to distinct members of the G protein family and display a specific tissue expression pattern²⁸. In particular, S1P1 mediated pro-migratory effects³¹, whereas S1P2 inhibited cell migration³². An increased S1P1 expression, which was recently induced by the activator of transcription-3 (STAT3), up-regulated IL-6 and accelerated tumor growth and metastasis³³. In glioblastoma and in Wilms tumor cells S1P-dependent S1P1 signaling induced cell migration and invasion^{34, 35}, whereas in glioblastoma and melanoma cells S1P-dependent S1P2 pathway negatively directed migration and invasion^{32, 36}. Paralleling the aforementioned observations, S1P1 and S1P2 exerted opposite effects on tumor angiogenesis. For instance, S1P1 stimulated angiogenesis³⁷ and accordingly the administration of monoclonal anti-S1P antibody prevented tumor growth by inhibiting angiogenesis and motility, survival and proliferation³⁸. In addition, S1P1 was shown to be up-regulated in vessels at sites of tumor implantation, whereas S1P1 silencing resulted in the inhibition of tumor growth³⁹. In contrast to the Gi-dependent S1P1 stimulation of tumor angiogenesis, S1P2 mediated inhibitory effects on tumor angiogenesis through G12/Rho signaling⁴⁰. Together, these data suggest that the different action elicited by S1P1 and S1P2 may serve for novel pharmacological strategies based on therapeutics able to inhibit S1P1 and to activate S1P2 simultaneously.

GPCRs activated by peptides

The endothelin receptors (ET_AR and ET_BR) have been broadly involved in the regulation of mitogenesis, cell survival, angiogenesis, lymphangiogenesis, invasion and metastatic dissemination as well as epithelial-to-mesenchymal transition (EMT) in diverse types of malignancies⁴¹. Accordingly, high plasma endothelin-1 (ET-1) levels correlated with the tumor stage in cancer patients, suggesting that ET-1 can also serve as a prognostic marker^{41, 42}. Emerging data demonstrate that interfering with the ET receptors-dependent pathways may provide a significant chance for the development of novel anticancer strategies, in particular using ET_AR antagonists in combination with EGFR inhibitors as well as cytotoxic drugs⁴³. Nevertheless, antagonists of ET receptors, like atrasentan and zibotentan (ZD4054), used alone have also gained considerable interest in human clinical trials on the basis of their potential anticancer activity⁴³. For instance, the ET_AR blockade with the specific ET_AR antagonist zibotentan restored drug sensitivity to cytotoxic-induced apoptosis and inhibited ovarian cancer cell invasion⁴⁴.

The four receptors for the Gastrin-releasing peptide (GrP) were shown to be able to transactivate EGFR in lung, head and neck squamous tumor cells^{45, 46}. In addition, ligand-stimulation of GrP receptors contributed to the growth of several malignancies through the activation of diverse phospholipases and protein kinases⁴⁷. As GrP receptors were found overexpressed in a wide variety of tumors, their inhibition has been suggested as a promising objective in some malignancies⁴⁷. Hence, the use of antagonists of GrP receptors represents a potential approach to inhibit the GrP-dependent effects on tumor growth. In this regard, it was demonstrated that the anti-tumor activity of GrP antagonists involves different mechanisms as the reduction of EGFR and Her2 levels, the alteration of MAPK, PKC, pAkt, and COX-2 signaling, the attenuation of c-fos and c-jun expression, the modulation of wild-type and mutated forms of p53 along with an alteration of Bcl-2/BAX ratio, the inhibition of vascular endothelial growth factor (VEGF)⁴⁷. Radiolabeled GrP analogues represent another chance in targeting GrP receptors, thus they are currently considered promising radiopharmaceuticals for detection and treatment of different types of tumors^{48, 49}.

Protease-activated receptors (PARs) are a unique class of GPCRs that are activated by proteolytic cleavage of their extracellular domains⁵⁰. PAR-1 exerted a functional role in the growth, migration and metastasis in various tumors^{51, 52, 53}. For instance, its proteolytic activation by thrombin caused persistent activation of EGFR/ERK signaling, promoting thereafter breast carcinoma cell invasion⁵⁴. Moreover, PAR-1 negatively regulated the expression of the Maspin tumor-suppressor gene contributing to the metastatic phenotype of melanoma⁵⁵. It has been recently reported that metalloprotease-1 (MMP1) may function as a protease agonist of PAR-1 which then stimulates migration, invasion and angiogenesis in breast and ovarian malignancies^{56, 57}.

Activation of the canonical Wnt pathway occurs through the seven-transmembrane Frizzled (Fzd) family receptors and the co-receptors lipoprotein receptor-related protein (LRP) in order to initiate the β-catenin signaling cascade⁵⁸. The activated β-catenin translocates from the cytoplasm to the nucleus inducing the transcription of Wnt-responsive genes⁵⁸. Numerous studies have shown that the dysregulation of the canonical Wnt pathway may lead to cancer development and progression⁵⁸. In this regard, mutations of β-catenin, axin and other components of the Wnt pathway⁵⁹ as well as the activation of tissue-specific Wnt target genes^{60, 61} were found in a variety of human tumors. In addition, the non-canonical Wnt pathways that act independently of β-catenin promote the invasiveness and progression of tumors⁶². Several Fzd receptors are highly expressed in a variety of malignancies and involved in cancer cell growth, survival and invasion through both canonical and non-canonical Wnt pathways⁶. For instance, the pharmacological inhibition of Fzd7, which is frequently overexpressed in hepatocellular carcinoma (HCC), displayed anti-tumor properties by involving β-catenin and PKCδ signals⁶³. Fzd7 was also crucial through the canonical Wnt pathway for cell proliferation and invasiveness in triple negative breast cancer cells as well as for tumor formation in xenograft models⁶⁴. Moreover, the increased expression of Fzd4 through the up-regulation of β-catenin dependent Wnt signaling promoted in invasive glioma cells the acquisition of glioma stem cell-like properties and resistance to apoptosis⁶⁵. Next, a cross-talk between Wnt pathways and EGFR signaling occurred in multiple stages of cancer development⁵⁹.

The Hedgehog (Hh) signaling, which plays a key role in embryonic development, has been involved in the development of multiple malignancies⁶⁶. Hh ligands are secreted from different tissues at various stages of development and generate intracellular signaling by binding to and inactivating the Hh receptor Patched-1 (Ptch1), which relieves its catalytic inhibition of the GPCR-like signal transducer Smoothened (Smo). The activation of Smo triggers downstream events that culminate in the stimulation of the glioma-associated oncogene homologue (GLI) transcription factors, the up-regulation of target genes like cyclins, Bcl-2 and SNAIL and the production of VEGF and angiopoietins⁶⁶. Consequently, Hh signaling contributes to cancer cell proliferation and survival, angiogenesis and metastasis⁶⁶. In addition, mutations in components of the Hh pathway, such as Smo and Ptch1, lead to a constitutively activated Hh signaling in the absence of ligands in diverse cancer types, including basal cell carcinoma, medulloblastoma and non-small cell lung carcinoma^{66, 67, 68}. The overexpression of Hh ligands have been also identified in several tumors as a stimulating factor acting in an autocrine manner to induce cell proliferation and survival⁶⁹. Likewise, Hh ligands produced by stromal cells can promote tumor growth and survival in a paracrine manner⁶⁹. Conversely, tumor cells can produce Hh ligands which activate transduction pathways in stromal cells⁶⁹. Several small molecule antagonists for Smo have been developed with a promising preclinical efficacy in multiple tumors. For instance, the Smo inhibitor CUR61414 inhibited in mice skin the Hh signaling, blocked the induction of hair follicle anagen and shrank in basal cell carcinomas (BCCs), although in a phase I clinical study it did not show any activity in human superficial or nodular BCCs⁷⁰. Several other Smo antagonists, such as IPI-926, BMS-833923 and GDC-0449, are currently under evaluation in clinical trials as anticancer agents (http://www.clinicaltrials.gov/). In particular, GDC-0449 produced promising antitumor responses in a phase I study in patients with advanced BCCs as well as in a 26-year-old man with metastatic medulloblastoma which was unmanageable by conventional therapies⁷¹. However, the response of this patient to GDC-0449 treatment was only transient due to a mutation of Smo⁷¹. Recently, a number of Hh pathway antagonists targeting Smo mutants⁷² as well as inhibitors able to block both wild-type and Smo mutants have been identified⁷³. In addition to Smo antagonists, other inhibitors were used to block Hh signaling like the small molecule inhibitor of GLI1 and GLI2 transcription factors, GANT61, which induced colon carcinoma cell death in a higher extent respect to the conventional Smo inhibitor cyclopamin⁷⁴. The treatment with GANT61 reduced also the expression of the target gene Patched and decreased the viability of chronic lymphocytic leukemia cells⁷⁵. Recently, the systemic antifungal itraconazole which failed to bind to Smo at the same binding site of cyclopamine, showed a potent antagonism for the Hh signaling pathway associated with anti-tumor activity in a mouse medulloblastoma allograft model⁷⁶.

GPCRs activated by chemokines

Besides their functions in the immune system as mediators of leukocyte migration, chemokines and the cognate receptors play a critical role in tumor initiation and progression, including angiogenesis, attraction of leukocytes and induction of cell migration and homing in metastatic sites⁷⁷. The first described angiogenic chemokine, CXCL8/IL-8, which binds to CXCR1 and CXCR2⁷⁸, is secreted by a variety of normal and tumor cells exposed to pro-inflammatory cytokines like IL-1 and TNF-α⁷⁹. CXCR2 was associated to multiple signaling pathways involved in tumorigenesis, angiogenesis, proliferation and metastasis in several malignancies, including melanoma⁸⁰, lung⁸¹, pancreatic^{79, 82}, gastric⁸³, and ovarian⁵⁷ tumors. For instance, the overexpression of CXCR2 induced an aggressive phenotype of melanoma cells consisting with an enhanced proliferation, migration and tumor growth in mice⁸⁴.

The homeostatic chemokine stromal cell-derived factor-1, CXCL12/SDF-1, which regulates cardiac and neuronal development, stem cell motility and neovascularization, was also involved in diverse tumorigenic processes⁷⁷. The CXCL12/SDF-1 interaction with the widely expressed tumor cell surface receptor CXCR4 initiates divergent signaling pathways which can result in a variety of responses like chemotaxis, cell survival, proliferation and metastasis⁸⁰. CXCR4 is capable of orchestrating a complex signaling network, including the up-regulation of E-cadherin and c-myc as well as the modulation of molecules facilitating mammary epithelia cell transformation⁸⁵. Enhanced CXCR4 signaling was also involved in the resistance to endocrine therapy in breast cancer⁸⁶ and in the drug resistance of colon⁸⁷ and pancreatic cancer cells⁸⁸. Of note, CXCR4 expression and phosphorylation has been considered a negative prognostic marker in various types of cancer including acute myelogenous leukemia and B-acute lymphoblastic leukemia, breast and colon carcinomas, as it correlated with worse prognosis and decreased survival of patients^{86, 89, 90, 91, 92}. Increased levels of VEGF, the activation of nuclear factor kappa B (NF-ĸB) and some oncoproteins up-regulate CXCR4 expression, in particular during cancer progression^{93, 94} and under hypoxic conditions⁹⁵. CXCR4/SDF-1 contributes to tumor progression also through the activation of tumor-associated integrin and the production of matrix metalloproteases⁹³, as observed in human basal carcinoma cells⁹⁶ and oral squamous cell carcinomas⁹⁷. Recently, CXCR7/RDC1 has been identified as a novel receptor for CXCL12/SDF-1 and CXCL11⁹⁸, although its coupling to G-proteins remains controversial. CXCR7 is expressed in diverse cell types including malignant cells⁹⁸ as well as in tumor-associated blood vessels⁹⁹. CXCR7-dependent signals promote the growth of breast and lung tumors, enhance lung metastasis and tumor aggressiveness in prostate cancer^{99, 100}. Antagonists of CXCR7 prevented tumor growth in animal models, hence validating this receptor as a potential target for the development of novel anti-cancer therapeutics⁹⁸.

GPCRs activated by hormones

Numerous hormone-activated GPCRs are overexpressed in hormone-dependent and independent tumors and trigger multiple transduction pathways, which mediate relevant biological effects in diverse cancer cells. For instance, angiotensin II (Ang-II) and bradykinin (BK) receptors are overexpressed in prostate cancer^{101, 102} and mediate cell growth through Gα_q and/or Gα₁₃ which activate RhoA-dependent signaling¹⁰³. In this regard, it was shown that Rho is involved in the androgen-like activity of androgen receptor (AR) antagonists¹⁰⁴ and able to sensitize AR to low androgens levels¹⁰⁵. On the basis of these studies, it can be assumed that GPCRs may contribute to androgen-dependent and independent growth of prostate cancer¹⁰³. Recently, Ang-II exhibited in vitro and in vivo the potential to enhance the expression of AR in prostate cancer cells through angiotensin II type-1 receptor (AT1R)¹⁰⁶. In addition, Ang-II and BK receptors have been implicated in the development, growth, angiogenesis and metastasis in a wide number of tumors^{101, 102, 107, 108, 109, 110, 111}. For instance, Ang-II and BK stimulated DNA synthesis in pancreatic cancer cells¹¹². In the context of this malignancy, a cross-talk between insulin/insulin like growth factor-I (IGF-1) receptors and Ang-II and BK-activated GPCRs has been reported^{111, 112, 113, 114}. In particular, insulin induced the potentiation of Ang-II and BK-dependent signaling through the PI3K/Akt/mTOR transduction pathway¹¹³. Metformin, which is one of the most used drug in the treatment of type 2 diabetes, disrupted in pancreatic cancer cells the cross-talk between insulin receptor and GPCR signaling through the activation AMP kinase, which negatively regulated mTOR function¹¹³. Further supporting these observations, metformin prevented the growth of pancreatic cancer cells in xenograft models^{114, 115}. Cumulatively, these findings suggest that the cross-talk between insulin/IGF-1 receptors and GPCR-activated signaling can be considered as a mechanism involved in the development of certain tumors and a promising target for novel anti-cancer strategies.

As it concerns the renin-angiotensin system, an abundant generation of Ang-II stimulated by the angiotensin-converting enzyme (ACE) and the up-regulation of AT1R have been demonstrated in various tumors^{110, 116}. In this respect, ACE inhibitors and angiotensin II receptor blockers (ARBs) have recently acquired an increasing interest as chemopreventive agents^{110, 117}, ¹¹⁸. Of note, ARBs have been associated with reduced cancer occurrence in patients with essential hypertension and a longer exposure to ARBs has been related with major benefits in cancer patients¹¹⁹. Nevertheless, ARBs did not show the ability to reduce considerably cancer development in a meta-analysis of randomized controlled trials¹²⁰.

Among the GPCR family members, the gonadotropin-releasing hormone (GnRH) receptor is a well established target in the clinical practice of cancer treatment¹²¹. Several antagonist analogues of GnRH have been clinically tested and numerous orally active antagonists are under development¹²¹. The GnRH receptor is one of the smallest GPCRs as it lacks the characteristic intracellular carboxyl-terminal domain with a very short extracellular amino-terminus. GnRH receptors are expressed not only in the pituitary and in normal peripheral tissues¹²², but also in various tumor cells like melanoma, prostate and endometrial carcinomas, leiomyomas, leiomyosarcomas, breast cancer, choriocarcinoma, epithelial and stromal tumors of the ovary^{122, 123}. The activation of the peripheral GnRH receptor, which is coupled to the Gi protein in uterine leiomyosarcoma, ovarian and endometrial carcinomas, decreased intracellular cAMP levels leading to a down-regulation of gene transcription and antiproliferative effects in tumor cells¹²². Indeed, the repressive action of GnRH-I receptor on cell proliferation has been demonstrated in hormone-related tumors like prostate, breast, ovary and endometrium cancer¹²⁴. GnRH and the cognate receptors were also involved in the stimulation of motility and invasion in ovarian cancer cells^{125, 126}, however several studies suggested a protective role elicited by GnRH analogues against gonadal damage during chemotherapy in diverse types of tumors¹²². On the basis of these findings, GnRH analogues are used in many endocrine-dependent malignancies such as breast, endometrial, epithelial and stromal ovarian cancer. The antitumor activity of GnRH analogues was presumed to result from desensitization and/or decrease of GnRH receptors in the pituitary, with the consequent decline in gonadotropin secretion and gonadal hormone production. Nevertheless, GnRH analogues were shown to suppress directly the growth of endometrial, ovarian, breast and prostate tumors and uterine leiomyoma^{122, 127}, ¹²⁸. Likewise, the growth of prostate cancer cells in vitro and in tumor xenografts was inhibited by activating the GnRH receptor or by GnRH receptor blockade¹²⁹. In line with these observations, phase III trial data have demonstrated that GnRH agonists are effective and well tolerated in the treatment of hormone-sensitive prostate cancer¹³⁰. Recently, the possibility of using GnRH analogues to carry cytotoxic agents directly to cancer cells expressing GnRH receptors has been evaluated¹³¹. For instance, AN-152 conjugate which is made from doxorubicin being linked to [D-Lys6]GnRH agonist, reduced the proliferation of breast, ovarian and endometrial cancer cells in vitro and in xenografted nude mice^{132, 133}.

Estrogens influence many physiological processes, but are also implicated in the development or progression of various types of cancer¹³⁴. The multiple biological actions elicited by these hormones have traditionally been attributed to the classical nuclear estrogen receptor (ER)α and ERβ, which act as ligand-activated transcription factors¹³⁴. Surprisingly, a member of the GPCR family, GPR30/GPER, was recently shown to mediate the multifaceted actions of estrogens in different tissues including cancer cells¹³⁵. Importantly, GPER overexpression was associated with lower survival rates in endometrial and ovarian cancer patients and with an elevated risk of developing metastases in patients with breast cancer^{136, 137, 138}. GPER by transactivating EGFR triggers numerous transduction pathways including the intracellular cAMP, calcium mobilization, MAPK, PI3-K and phospholipase C activation in a variety of cell types¹³⁹. Moreover, it has been shown that the activation of the Gα_s protein by GPER is responsible for the estrogen, phyto- and xenoestrogens stimulation of adenylate cyclase and the ensuing increase in cAMP in breast cancer cells^{140, 141}. The signaling events upon GPER activation by both estrogens and notably ER antagonists can lead to gene transcription as well as to the growth and migration in diverse hormone-sensitive tumors like breast, endometrial and ovarian cancer^{142, 143, 144, 145, 146, 147, 148, 149}. Notably, GPER was also involved in the stimulatory effects elicited by estrogens and ER antagonists in cancer-associated fibroblasts^{147, 150}.

GPCRS activated by neurotransmitters

Emerging findings support the hypothesis that the development, progression and responsiveness to treatments in most tumors is strongly influenced by an imbalance in stimulatory and inhibitory neurotransmission¹⁵¹. The neurotransmitters adrenaline and noradrenaline act as powerful regulators of numerous cellular and tissue functions and can promote tumor growth and metastases through the β-adrenergic receptors (β-AR), which are Gs-protein coupled receptors^{152, 153, 154}. For instance, noradrenaline stimulates tumor progression in diverse types of malignancies activating β-AR which in turn induces the production of VEGF, interleukin-6 (IL-6) and matrix metalloproteinases^{153, 155}.

As for β-adrenergic compounds¹⁵⁶, the action of muscarinic acetylcholine receptors (mAchRs) on the proliferation of cancer cells is still questioned¹⁵⁷. In fact, these receptors interact with distinct G protein subunits triggering various cellular functions through specific downstream effectors. As it concerns M2 and M4 receptors, they interact with Gi proteins inhibiting adenylyl cyclase-dependent signaling. On the contrary, M1, M3 and M5 receptors coupled with Gq proteins activate phospholipase C, PKC and induce an increase of intracellular calcium¹⁵³. These mAchR subtypes can protect cells from the apoptosis subsequent to DNA damage, oxidative stress and mitochondrial dysfunction¹⁵⁸. Although muscarinic receptor expression was identified in cells derived from brain, breast, colon, lung, ovary, pancreas, prostate, skin, stomach and uterus malignancies¹⁵⁷, only for some of these receptors a functional role has been demonstrated. In ovarian cancer the expression of muscarinic receptors was associated with reduced survival¹⁵⁹, while in leukemia cells their activation resulted in increased intracellular calcium and up-regulation of the oncogene c-fos¹⁶⁰. Activation of M3 receptor by cholinergic agonists stimulated proliferation of primary astrocytoma cells in a ERK and NF-κB dependent manner¹⁵⁷. In breast cancer cells, M1 and M2 receptors were involved in angiogenesis and cell proliferation, whereas M3 receptor was associated only with cell growth¹⁶¹. Agonist binding to M3 receptor resulted in the activation of EGFR/MAPK transduction pathway, which then stimulated the proliferation in colon cancer cells¹⁶².

Somatostatin receptors (SSTRs), particularly SSTR subtype 2, were found highly expressed in many neoplastic cells and in tumoral blood vessels¹⁶³. SST analogues decreased tumor cell growth and angiogenesis as well as stimulated apoptosis in cancer cells¹⁶⁴. These findings contributed to develop various cytotoxic SST conjugates that displayed relevant anti-tumor abilities targeting selectively SSTR2-specific sites¹⁶⁵.

β-arrestins: novel transducers of GPCR signals

The sensitivity of GPCRs is regulated by G protein-coupled receptor kinases (GRKs) and β-arrestins families, that are known to exert a central role in GPCR endocytosis, intra-cellular trafficking, desensitization and resensitization^{166, 167}. As it concerns β-arrestins, they can also function as molecular mediators of G protein-independent signaling by activating a variety of transduction proteins like Src family kinases and components of the MAPK cascades^{166, 168}, ¹⁶⁹. On the basis of these findings, β-arrestins were included among the signaling factors mediating the action of diverse GPCRs in cancer¹⁷⁰. For instance, β-arrestin/Ral signaling was involved in the migration and invasion of breast cancer cells induced by LPA¹⁷⁰. In addition, prostaglandin E2 induced the association of prostaglandin E receptor 4 with β-arrestin 1 and c-Src, that formed a signaling complex able to induce the migration and metastasis of colorectal carcinoma cells¹⁷¹. In accordance with these data, β-arrestin 1 interacting with Src and ET_AR triggered EGFR transactivation and β-catenin phosphorylation, which stimulated invasion and metastasis in ovarian cancer cells¹⁷². Moreover, β-arrestin 1 forming a trimeric complex with ET_AR and axin contributed to the inactivation of glycogen synthase kinase (GSK)-3 and stabilization of β-catenin¹⁷². Collectively, these results indicate that β-arrestins exert an important role in GPCR-mediated signaling as well as a pivotal role in cancer invasion and metastasis. Novel pharmacological approaches targeting the β-arrestins pathway would provide further therapeutical opportunities in diverse types of tumors.

Orphan GPCRs and cancer

Relevant efforts were recently made in the deorphanization of the over 130 GPCRs for which ligands have not yet been identified. Some of these GPCRs have been linked to cancer development and progression on the basis of their overexpression and/or up-regulation by diverse factors^{173, 174, 175}. For instance, an elevated expression of the orphan G-protein-coupled receptor GPR49 was involved in the formation and proliferation of basal cell carcinoma¹⁷⁶, while GPR18 was found associated with melanoma metastases¹⁷⁷. In lung, cervix, skin, urinary bladder, testis, head and neck squamous cell carcinomas were detected high levels of GPR87^{178, 179} for which UDP-glucose, cysteinyl-leukotrienes and LPA exhibited binding properties¹⁸⁰. In breast and colon cancer cells, DNA damage has been recently found to regulate GPR87 expression in a p53-dependent manner¹⁷³. Taken together, these results suggest that GPR87 may elicit survival and anti-apoptotic actions, while its overexpression plays a pivotal role in the development and progression of diverse types of tumors. On the contrary, GPR56 inhibited prostate cancer progression and suppressed tumor growth and metastasis in melanoma xenografs¹⁸¹. Moreover, GPR56 inhibited VEGF production from melanoma cells and prevented melanoma angiogenesis and growth¹⁸². Accordingly, the expression of GPR56 has been found inversely correlated with melanoma malignancies, suggesting its potential role in cancer development and metastasis^{175, 182}.

Targeting deorphanized GPCRs also in combination with well-known anti-cancer agents would be expected to increase the effectiveness of the current therapeutical approaches. In this regard, extensive studies are required to completely decipher the biology of these receptors in order to provide the basis for the design and use of new drugs in different types of human tumors.

Concluding remarks

Despite GPCRs form the largest superfamily of cell surface receptors involved in signal transmission, in clinical practice only few anticancer compounds are currently used in order to interfere with GPCR-mediated signaling. Although the role played by GPCRs and their ligands in tumor pathophysiology is intricate, an increasing body of evidence has recently emerged linking indubitably these molecules to the development and progression of cancer. Consequently, GPCRs and their downstream-activated effectors represent a rich source of potential drug targets for innovative strategies in tumor prevention and treatment. Next, the identification of the transduction network maps connecting several GPCR-dependent signals with other transduction pathways will facilitate further investigations regarding the biological potential of these receptors, opening in the mean time a new valuable scenario for the discovery of novel anti-cancer therapeutics. Finally, the ongoing efforts to fully characterize the numerous orphan GPCRs will certainly lead in the near future to the identification of new targets toward innovative pharmacological strategies in cancer patients.