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

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

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Oncogene (2004) 23, 6299–6303

& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00

G protein-coupled receptors GPR4 and TDAG8 are oncogenic
and overexpressed in human cancers
Wun Chey Sin1, Yaoping Zhang1, Wendy Zhong2, Sree Adhikarakunnathu1, Scott Powers1,
Tim Hoey2, Songzhu An2 and Jianxin Yang*,1
Genomics Division, Tularik Inc., 266 East Pulaski Rd, Greenlawn, NY 11740, USA; 2Department of Biology, Tularik Inc.,
1120 Veterans Blvd, South San Francisco, CA 94080, USA

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 CREdriven 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.
Oncogene (2004) 23, 6299–6303. doi:10.1038/sj.onc.1207838
Published online 28 June 2004
Keywords: human cancers; G protein-coupled receptors;
GPR4; TDAG8; overexpression; tumorigenesis

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 Gas and
Gai 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 sarcomaassociated 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
*Correspondence: J Yang; E-mail: jyang@tularik.com
Received 24 February 2004; revised 20 April 2004; accepted 20 April
2004; published online 28 June 2004

GPCRs such as muscarinic acetylcholine receptors
(Gutkind et al., 1991), the serotonin 1c receptor (Julius
et al., 1989), the a1B-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, TDAG8expressing 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

Overexpression of oncogenic GPCRs in cancers
WC Sin et al


(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. TDAG8expressing 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 a subunit and
bg dimer signal through the activation or inhibition of
an expanding list of effectors. The Gs a subunit
stimulates adenylyl cyclase, leading to cAMP response
element (CRE)-dependent transcription via PKA phosphorylation of the CRE-binding (CREB) protein.

Overexpression of oncogenic GPCRs in cancers
WC Sin et al


Whereas the a subunits of Gi receptors inhibit adenylyl
cyclase, their bg 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, Gas-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 Gas 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 TDAG8overexpressing 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 ligandindependent 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 sphingosylphosphorylcholine (SPC), lysophosphatidylcholine
(LPC) and psychosine were reported as high- and lowaffinity 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
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 ¼ 60) of breast tumors, 29% (n ¼ 31) of

Figure 1 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 mg/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 þ 10% calf serum) were transfected with pLPC-GPR4, pLPCTDAG8 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). b-actin was used as the control

Overexpression of oncogenic GPCRs in cancers
WC Sin et al


Figure 2 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

ovarian tumors, 35% (n ¼ 49) of colon tumors, 13%
(n ¼ 31) of liver tumors and 45% (n ¼ 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 ¼ 31)
overexpression in kidney tumors, 34% (n ¼ 32) overexpression in ovarian tumors, 17% (n ¼ 41) overexpression in colon tumors and 10% (n ¼ 48) overexpression in

Figure 3 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

Overexpression of oncogenic GPCRs in cancers
WC Sin et al

Table 1 Distinct but overlapping overexpression patterns of GPR4, TDAG8 and G2A in human tumors
GPR4 only
TDAG8 only
G2A only
ALL three receptors
Total number of tumors















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 ¼ 57) of breast tumors
and 53% (n ¼ 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.
We would like to thank Ken Nguyen and Lei-Hoon See for
excellent technical support.

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