G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers

Abstract

The GPR4 subfamily consists of four G protein-coupled receptors that share significant sequence homology. In addition to GPR4, this subfamily includes OGR1, TDAG8 and G2A. G2A has previously been shown to be a potent transforming oncogene for murine 3T3 cells. Here we show that GPR4 also malignantly transforms NIH3T3 cells and that TDAG8 malignantly transforms the normal mammary epithelial cell line NMuMG. Overexpression of GPR4 or TDAG8 in HEK293 cells led to transcriptional activation from SRE- and CRE-driven promoters, independent of exogenously added ligand. TDAG8 and GPR4 are also overexpressed in a range of human cancer tissues. Our results suggest that GPR4 and TDAG8 overexpression in human tumors plays a role in driving or maintaining tumor formation.

Main

Human cancers arise from genetic alterations that change the activities or expression levels of oncogenes and tumor suppressor genes. One important class of oncogenes encodes cell surface receptors. Cell surface receptors of the G protein-coupled receptor (GPCR) family transduce numerous extracellular signals into cells and play important roles in regulation of cell proliferation (Marinissen and Gutkind, 2001). Aberrant expression or mutations of GPCRs and associated G-proteins has been found in various cancers (Gutkind, 1998). For example, activating mutations of Gαs and Gαi have been found in a subset of endocrine tumors (Dumont et al., 1989; Gupta et al., 1992). Activating mutations of TSH receptors were detected in thyroid carcinomas (Russo et al., 1995). Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a GPCR that constitutively stimulates cell proliferation and angiogenesis (Bais et al., 1998) and may contribute to the formation of sarcoma in AIDS. Overexpression of GPCRs such as muscarinic acetylcholine receptors (Gutkind et al., 1991), the serotonin 1c receptor (Julius et al., 1989), the α1B-adrenergic receptor (Allen et al., 1991) and the thrombin receptor (Whitehead et al., 1995) has also been shown to have transforming and tumorigenic activities in cell culture and in mice.

The GPR4 subfamily consists of four closely related GPCRs (Marchese et al., 1999). In addition to GPR4/GPR6C.1 (An et al., 1995; Heiber et al., 1995; Mahadevan et al., 1995), this subfamily includes OGR1/GPR12A (An et al., 1995; Xu and Casey, 1996), TDAG8/GPR65 (Choi et al., 1996; Kyaw et al., 1998) and G2A (Weng et al., 1998). We investigated whether GPR4 and TDAG8 possess oncogenic activities since they are closely related to G2A, which has recently been shown to be a potent transforming oncogene (Zohn et al., 2000). When overexpressed in NIH3T3 cells by retrovirus-mediated infection, GPR4- and TDAG8-expressing cells exhibited a refractile and spindle-shaped phenotype not observed in cells expressing the control vector (Figure 1a). Foci were observed in GPR4- and TDAG8-expressing NIH3T3 cells transfected by calcium phosphate 3 weeks after reaching confluency. However, foci developed from GPR4 cells were well formed with a distinct boundary, while more diffuse foci were seen with TDAG8-expressing cells (Figure 1b). GPR4- and TDAG8-expressing cells proliferated rapidly in low serum concentrations of 0.1, 0.3 and 0.5%, resulting in significant increase in cell number when compared to vector control cells (Figure 1c). In fact, GPR4-expressing cells appeared to grow at a comparable rate to Ras-expressing 3T3 cells (Figure 1c). Therefore, overexpression of GPR4 and TDAG8 in NIH3T3 induced a full range of phenotypes characteristic of oncogenic transformation, such as refractile cell shape, foci formation and tolerance to low serum condition in vitro.

Figure 1
figure1

Oncogenicity of GPR4 and TDAG8. Full-length cDNA was obtained by PCR using gene-specific forward and reverse primers flanking the coding region of GPR4 (Accession No: NM_005582) and TDAG8 (Accession No: NM_003608). The PCR products were gel purified and cloned into pLPC retrovirus vector (Dr Scott Lowe, Cold Spring Harbor Laboratory). NIH3T3 cells were obtained from Cold Spring Harbor Laboratory. NMuMG cells were obtained from ATCC. Stable cell lines were generated by retrovirus-mediated infection as previously described (Serrano et al., 1997), followed with 2 μg/ml of puromycin selection. (a) Spindle-shaped phenotype of GPR4 and TDAG8-expressing NIH3T3 cells 1 week after retrovirus-mediated infection and puromycin selection. (b) Focus formation of GPR4 and TDAG8-expressing NIH3T3 cells. NIH3T3 cells (in DMEM+10% calf serum) were transfected with pLPC-GPR4, pLPC-TDAG8 and pLPC vector control using the calcium phosphate method (Profection Mammalian Transfections System, Pomega, Madison, WI, USA). After the cells had reached confluency (typically after 48 h), they were maintained in 5% calf serum. Foci were scored 3 weeks after confluency. (c) Cell proliferation assay. 2 × 105 3T3 cells (from Figure 1a) were grown in 10% calf serum in 12-well plates. Next day, the cells were rinsed with PBS and cultured in 0.1, 0.3 or 0.5% calf serum. After 48 h, the proliferation rate was determined using Celltiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer's instruction. Cell growth rate is expressed as absorbance at 490 nm. Data are representative of two independent transfections in triplicates. (d) For tumorigenicity assay, retrovirus-mediated and puromycin-selected stable 3T3 cells expressing GPR4 were harvested and re-suspended in MEM. 5 × 106 cells were injected subcutaneously into each of five athymic nude mice. Tumors were measured weekly with a caliper. (e) Transformed phenotype of retrovirus-mediated and puromycin-selected TDAG8-expressing NMuMG epithelial cells. (f) Tumorigenicity of TDAG8-NMuMG cells in nude mice. TDAG8-expressing NMuMG cells were obtained as described in (e) and injected into five athymic nude mice as described in (d). (g) The expression levels of GPR4 in retrovirus-mediated stable NIH3T3 cells and TDAG8 in retrovirus-mediated NMuMG cells, and in tumors (GPR4-3T3-Tu, TDAG8-NMuMG-Tu) obtained from nude mice were determined by RT–PCR. Total RNA was extracted using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA) and contaminated DNA was eliminated with DNase treatment (Ambion). First-strand cDNA was synthesized with random primers using a cDNA Cycle Kit (Invitrogen). PCR was carried out using gene-specific primers for GPR4 and TDAG8 to detect the full-length message (1 kb). β-actin was used as the control

To determine the oncogenic activity of GPR4 and TDAG8 in vivo, we injected retrovirus-infected stable NIH3T3 cells into athymic nude mice. All the five mice injected with GPR4-transformed cells developed tumors within 6 weeks (Figure 1d). In contrast, TDAG8-expressing NIH3T3 cells did not develop tumors even after 3 months (data not shown). However, TDAG8 overexpression caused a pronounced morphological change in a mouse epithelial cell line NMuMG (Figure 1e). Although NIH3T3 cells expressing TDAG8 did not induce tumor growth in nude mice, NMuMG expressing TDAG8 construct did (Figure 1f), suggesting that endogenous factors present in epithelial cells may help to promote TDAG8's oncogenicity. TDAG8-expressing cells induced smaller tumors (average size of less than 20 mm3) than GPR4-expressing cells (average size of 700 mm3), suggesting that TDAG8 may have weaker tumorigenic activity than GPR4. Tumor tissues obtained from nude mice confirmed the expression of GPR4 and TDAG8, respectively (Figure 1g).

The signals generated by a GPCR are transmitted mainly through specific compositions of the heterotrimeric G proteins to which it couples. Both α subunit and βγ dimer signal through the activation or inhibition of an expanding list of effectors. The Gs α subunit stimulates adenylyl cyclase, leading to cAMP response element (CRE)-dependent transcription via PKA phosphorylation of the CRE-binding (CREB) protein. Whereas the α subunits of Gi receptors inhibit adenylyl cyclase, their βγ subunits activate the MAP kinase (MAPK) in a protein kinase C (PKC)-independent, but Ras-dependent manner (Marinissen and Gutkind, 2001). The Gq family members activate phospholipase C, which increases intracellular calcium concentration and activates PKC, leading to transcription from the Nuclear Factor of Activated T cell (NFAT) and activation of MAPK (Marinissen and Gutkind, 2001). G12 and G13 appear to direct their effects on serum response element (SRE)-dependent transcription mainly through the small GTP-binding protein RhoA (Radhika and Dhanasekaran, 2001). To begin characterizing the signal transduction mechanisms of these oncogenic GPCRs, we used four different reporter gene constructs to delineate the signaling pathways of GPR4 and TDAG8. Heterologous expression of GPR4 and TDAG8 in HEK293 epithelial cells led to increased SRE-, NFAT- and CRE-driven transcription (Figure 2). GPR4 strongly activated all reporter genes, and TDAG8 activated SRE-, CREB-, and CRE-dependent transcriptions, indicating that multiple signaling pathways are engaged by these GPCRs. There is considerable synergism and cross-talk between various pathways, for example, MAPK and RhoA have been shown to link GPCRs to activation of SRE-dependent transcription, leading to cell proliferation and transformation (Fromm et al., 1997; Radhika and Dhanasekaran, 2001). Rho GTPases are believed to play important roles in cancer, including eliciting cell morphological changes (Ridley, 2004). In certain cell types, Gαs-coupled GPCRs have been shown to stimulate cell proliferation through the activation of CRE-driven transcription mediated by cyclic AMP (Stork and Schmitt, 2002). However, activation of the CRE-dependent transcription could also be the result of other signaling pathways implicated in cell proliferation independent of Gαs and cyclic AMP (Nathanson, 2000; Mayr et al., 2001). Thus, mitogenic inputs from multiple signaling pathways, including elevated SRE- and CRE-driven transcription, may contribute to tumorigenicity of GPR4- and TDAG8-overexpressing cells. More detailed elucidation of the signaling pathways and downstream events of these GPCRs will add valuable insights to the molecular and cellular mechanisms of GPCR-activated cell transformation. It is interesting to note that the transcriptional activities of these GPCRs were observed in the absence of the added ligand. This observation suggests that either these GPCRs are constitutively active in a ligand-independent manner, or the ligand(s) is present in the media, or the ligand(s) is released by the same cells in an autocrine fashion. The identity of the ligand(s) for these GPCRs has been a subject of several conflicting publications. Previously, bioactive lipids sphingosyl-phosphorylcholine (SPC), lysophosphatidylcholine (LPC) and psychosine were reported as high- and low-affinity ligands for GPR4 and TDAG8, respectively (Mahadevan et al., 1995; Im et al., 2001; Zhu et al., 2001). Recently, however, GPR4 was shown to be a receptor for protons, which elicits cAMP formation through Gs coupling (Ludwig et al., 2003). Our own data did not support the ligands being bioactive lipids, but rather favored protons being activators of GPR4 (An et al., unpublished observation). It is noted that tumor cells often exist in a hypoxic microenvironment with low extracellular pH (Torigoe et al., 2002; Subarsky and Hill, 2003). It is conceivable that overexpression of GPR4 and related receptors may provide tumor cells with advantages in hypoxic and acidic pH environment.

Figure 2
figure2

Activation of multiple signaling pathways by GPR4 and TDAG8. HEK293 cells were co-transfected with the pEF6 plasmid containing cDNAs for either GPR4 or TDAG8, together with one of the four reporter gene plasmids (Stratagene, La Jolla, CA, USA). These reporter gene constructs, pSRE-luc, pCRE-luc, pNFAT-luc, and pFA-CREB/pFR-luc, have firefly luciferase gene under the control of DNA-binding elements for either the Serum Response Factor (in pSRE-luc), cAMP Response Element Binding Protein (CREB) (in pCRE-luc), Nuclear Factor of Activated T cells (in pNFAT-luc), or the trans-reporter system with CREB as the transcription factor (in pFA-CREB/pFR-luc). The CRE-luciferase reporter is from the PathDetect cis-reporting system made by Stratagene. It contains tandem repeats of the cyclic AMP response element (CRE) in the promoter enhancer region of the luciferase reporter gene, which measures transcriptional activation from CRE. The CREB-trans reporter is from the PathDetect CREB Trans-Reporting system by Stratagene. This reporter gene measures more specifically the activation of protein kinase A that phosphorylates and activates CRE-binding protein (CREB). Transfection was done with LipofectAmine 2000 reagent in OPTI-MEM medium (Gibco-BRL, Gaithersberg, MA, USA) in 96-well multiwell plates. At 24 h after transfection, cells grown in OPTI-MEM medium were lysed and luciferase activities were measured on CLIPR (Molecular Devices, Sunnyvale, CA, USA) by using the Luciferase Assay Reagent from Promega (Madison, WI, USA). Transfection efficiency was normalized by co-transfection with the pTK-Renilla luciferase from Promega

Indeed, we have found GPR4, TDAG8 and G2A to be overexpressed in a range of human cancer tissues, suggesting that they might contribute to tumor development. We screened a panel of primary tumors for their overexpression on mRNA level, using quantitative fluorescence-based real-time PCR. As shown in Figure 3, GPR4 and TDAG8 are overexpressed more than fivefold over normal tissue controls in a significant portion of the tumors surveyed. GPR4 is overexpressed in 15% (n=60) of breast tumors, 29% (n=31) of ovarian tumors, 35% (n=49) of colon tumors, 13% (n=31) of liver tumors and 45% (n=31) of kidney tumors, but is not overexpressed in the lung and prostate tumors. Interestingly, TDAG8 shows a similar overexpression profile as GPR4 with 58% (n=31) overexpression in kidney tumors, 34% (n=32) overexpression in ovarian tumors, 17% (n=41) overexpression in colon tumors and 10% (n=48) overexpression in breast tumors. GPR4 and TDAG8 were overexpressed most frequently in kidney, colon and ovarian tumors, while G2A had the highest expression in breast and ovarian tumors. In all, 49% (n=57) of breast tumors and 53% (n=32) of ovarian tumors overexpressed G2A more than fivefold over normal controls. G2A is also overexpressed in about 20% of other tumor types including the colon, lung, liver and kidney. In the majority of cases, these receptors have unique distribution in tumors, but overlapping overexpression was observed, particularly in ovarian, colon and kidney tumors (Table 1). Their overlapping but distinct expression patterns suggest specific roles of each receptor in various tissues. Our current data provide evidence that GPR4 and related receptors are an emerging class of oncogenic GPCRs. As GPCRs represent the most attractive targets for small molecule drugs, antagonistic compounds may be developed as suitable tools to further assess the roles of GPR4 and related receptors in cancer.

Figure 3
figure3

Overexpression of G2A, GPR4 and TDGA8 in human cancers. Quantitative fluorescence-based real-time PCR was used to screen total RNA samples extracted from a panel of primary tumors. Breast and lung human tumor samples were obtained from the NCI Cooperative Human Tissue Network (CHTN) and Duke University. Prostate tumor samples were purchased from BioClinical Partners (Boston, MA, USA) or were obtained from ‘warm autopsies’ (Rubin et al., 2000). Ovarian tumor samples were obtained from CHTN. Colon tumor samples were obtained from CHTN and Dr Peggy Kemany of North Shore University Hospital. Total RNA was extracted using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Fluorogenic Taqman probes were designed using the PrimerExpress software (Applied Biosystems, Foster City, CA, USA) and synthesized by Operon Technologies. Absolute mRNA levels were determined by real-time reverse-transcription Taqman using ABI PRISM 7700 Sequence Detection System from Applied Biosystems (Foster City, CA, USA) through 40 cycles. Fluorogenic beta-actin probe was used as the standard. Each column contains data from 10 (or less) individual tumors. Filled red circles represent tumors that showed more than fivefold of overexpression when compared to normal tissues (filled green circles). Open blue circles represent data from tumors that showed less than fivefold of overexpression

Table 1 Distinct but overlapping overexpression patterns of GPR4, TDAG8 and G2A in human tumors

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Acknowledgements

We would like to thank Ken Nguyen and Lei-Hoon See for excellent technical support.

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Correspondence to Jianxin Yang.

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Keywords

  • human cancers
  • G protein-coupled receptors
  • GPR4
  • TDAG8
  • overexpression
  • tumorigenesis

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