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