Carcinogenesis vol.34 no.12 pp.2861–2869, 2013
doi:10.1093/carcin/bgt229
Advance Access publication June 26, 2013
GPR48, a poor prognostic factor, promotes tumor metastasis and activates β-catenin/
TCF signaling in colorectal cancer
Jinhua Wu†, Na Xie†, Ke Xie1,†, Jun Zeng, Lin Cheng,
Yunlong Lei, Yuan Liu, Linhong Song2, Dandan Dong2,
Yi Chen3, Rui Zeng4, Edouard C.Nice5, Canhua Huang*
and Yuquan Wei
The State Key Laboratory of Biotherapy, West China Hospital, Sichuan
University, Chengdu 610041, P. R. China, 1Department of Oncology
and 2Department of Pathology, Sichuan Provincial People’s Hospital,
Chengdu 610041, P. R. China, 3Department of Gastrointestinal Surgery and
4
Department of Cardiology, West China Hospital, School of Clinic Medicine,
Sichuan University, Chengdu 610041, P. R. China and 5Department of
Biochemistry and Molecular Biology, Monash University, Clayton, Victoria
3800, Australia
*To whom correspondence should be addressed. Tel: +86 132 5837 0346;
Fax: +86 28 8516 4060;
Email: [email protected]
G-protein-coupled receptor 48 (GPR48) is an orphan receptor belonging to the G-protein-coupled receptors family, which
plays an important role in the development of various organs
and cancer development and progression such as gastric cancer
and colorectal cancer (CRC). However, the prognostic value of
GPR48 expression in patients with CRC has not been reported.
In this study, we observed that GPR48 was overexpressed in primary CRC and metastatic lymph nodes and closely correlated
with tumor invasion and metastasis. Multivariate analysis indicated that high GPR48 expression was a poor prognostic factor
for overall survival in CRC patients. In vitro and in vivo assays
demonstrated that enforced expression of GPR48 contributed to
enhance migration and invasion of cancer cells and tumor metastasis. In addition, we found that GPR48 increased nuclear β-catenin
accumulation, T-cell factor 4 (TCF4) transcription activity, and
expression of its target genes including Cyclin D1 and c-Myc in
CRC cells. Correlation analysis showed that GPR48 expression in
CRC tissues was positively associated with β-catenin expression.
Upregulation of GPR48 resulted in increased phosphorylation of
glycogen synthase kinase 3β, Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) in CRC cells, while inhibition of PI3K/
Akt and mitogen-activated protein kinase /ERK1/2 pathways was
sufficient to abolish the effect of GPR48 on β-catenin/TCF signaling. Taken together, GPR48 could serve as both a prognostic
biomarker and a therapeutic target for resectable CRC patients.
Introduction
Colorectal cancer (CRC) is the third most common malignancy
and fourth most common cause of cancer mortality worldwide (1).
Despite improvements in surveillance and clinical treatment over
the past decades, almost 50% of CRC patients eventually develop
recurrent disease and distant metastasis after curative resection
(2). Metastasis of tumor cells to distant organs is the main cause
of treatment failure and the high mortality rate (3). The 5-year
survival rate for patients with regional and distal metastases is
only ~60 and 10%, respectively (4). Hence, the identification
Abbreviations: APC, adenomatous polyposis coli; CI, confidence interval;
CRC, colorectal cancer; EGFR, epidermal growth factor receptor; ERK,
extracellular signal-regulated kinase; GPR48, G-protein-coupled receptor 48;
GSK-3β, glycogen synthase kinase 3β; HR, hazards ratio; LGRs, Leucine-rich
repeat-containing G-protein-coupled receptors; MAPK, mitogen-activated
protein kinase; OS, overall survival; SD, standard deviation; TCF, T-cell factor.
†
These authors contributed equally to this work.
of proteins associated with CRC metastasis would contribute
to understanding the mechanisms involved in CRC malignancy,
leading to the development of novel biomarkers and therapeutic
targets for CRC (5).
Leucine-rich repeat-containing G-protein-coupled receptors
(LGRs) belong to the G-protein-coupled receptors superfamily
and are characterized by the presence of a seven-transmembrane
domain and a large extracellular domain with a long N-terminal
segment containing leucine-rich repeats motifs that are involved
in receptor–ligand interactions (6–8). LGRs can be classified
into three subtypes, including three known glycoprotein hormone receptors (LH, FSH and TSH), LGR4-6 and LGR7-8 (9).
G-protein-coupled receptor 48 (GPR48), also known as LGR4, is
closely related to LGR5 and LGR6 with ~50% amino acid identity
between them (10) and is widely expressed in diverse tissues at both
the embryonic and adult stages (11). Substantial evidence demonstrates that GPR48 plays an important role in the development of
multiple organs including the male reproductive tract, eyelid, hair
follicle, ocular anterior segment and mouse intestinal crypt (9,11–
15). During eyelid development, GPR48 can promote epithelial
cell proliferation and migration through activation of the epidermal growth factor receptor (EGFR) pathway (16). Recent studies
showed that GPR48 was overexpressed in several types of cancer
including colorectal and gastric cancer (17,18). Upregulation of
GPR48 mediated by p27Kip1 contributed to enhance colorectal carcinoma cell invasiveness and metastasis (17). In addition, Steffen
et al. (18) found that GPR48 was differentially expressed in gastric
cancer and closely associated with node involvement. However,
the prognostic value of GPR48 expression in patients with CRC
has not been reported.
It has been shown that GPR48 and LGR5/GPR49 function as the
cognate receptors of R-spondin to potentiate Wnt/β-catenin signaling (19,20). Conditional deletion of GPR48 and GPR49 genes
in the mouse gut impairs Wnt target gene expression and results
in the rapid demise of intestinal crypts, thus phenocopying Wnt
pathway inhibition (15). β-Catenin serves as a central downstream
effector of the Wnt signaling pathway and plays a key role in the
regulation of growth and development (21). In addition, activation of β-catenin signaling could control the ability of melanomas
to metastasize to lung, bowel and spleen (22). In the absence of
Wnt ligand, levels of cytoplasmic β-catenin are tightly regulated
by a multiprotein complex of adenomatous polyposis coli (APC),
axin, casein kinase 1 and glycogen synthase kinase 3β (GSK-3β),
which facilitates phosphorylation and proteasomal degradation of
β-catenin (21). However, mutational loss of APC function, or less
commonly, stabilizing mutations of β-catenin, mutations of axin
2 or dysregulation of Wnt ligands results in loss of β-catenin degradation in cells (23). Consequently, β-catenin that accumulates
in the cytoplasm is translocated to the nucleus, where it binds to
the T-cell factor (TCF)/lymphoid enhancer factor and thereby activates transcription of its target genes such as cyclin D1 and c-Myc
(1,21). In addition to the Wnt-dependent regulation of β-catenin, it
has been reported that certain growth stimuli or oncogenes can stabilize β-catenin through phosphorylation of GSK-3β at Ser9 mediated by extracellular signal-regulated kinase 1/2 (ERK1/2), AKT or
PKC (24–26).
Our previous proteomics study showed that GPR48 expression was
much higher in the metastatic colon cancer cell line SW620 than in
the matched primary colon cancer cell line SW480 (27). In this study,
we have investigated the predictive values of GPR48 for the prognosis
of CRC patients. GPR48 overexpression in CRC is associated with
adverse clinical outcome, supporting its potential roles as a prognostic
biomarker and a therapeutic target.
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Materials and methods
Clinical specimens
All colorectal carcinomas, corresponding adjacent normal tissues and metastatic lymph nodes were obtained from the patients who underwent surgical
resections during 2006 at the Sichuan Provincial People’s Hospital (Chengdu,
China). A total of 113 patients were involved in the study. Fresh frozen tumor
tissues and the corresponding normal tissue were randomly selected from five
patients for western blot analysis. Formalin-fixed paraffin-embedded tissues
from the other 108 patients including 65 paired normal mucosa were used
for immunohistochemical analysis. Tumor stage was determined according
to the TNM classification system of the International Union against Cancer
(UICC) (28). Histological differentiation was determined according to the
criteria of World Health Organization (29). During the follow-up period, 47
(43.5%) patients died of CRC, and 10 (9.26%) patients were lost to follow-up
because of an incorrect address in the register or other reasons. This study was
approved by the Institutional Review Board (IRB) of West China Hospital,
Sichuan University and informed consent for tissue procurement was obtained
from all patients, or their relatives, before study initiation.
Plasmid constructions and cell transfection
GPR48-plasmid encoding full-length human GPR48 and GPR48 control plasmid
were purchased from Gene Copoeia (Guangzhou, China). For GPR48 overexpression, the GPR48 plasmid and empty vector were separately transfected into
HCT116 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according
to the manufacturer’s instructions and the stable transfectants were selected in
the presence of 600 μg/ml G418 (Amresco, Solon, OH). Knockdown vectors
for GPR48 (pGPU6/GFP/Neo-sh-GPR48) were constructed by introduction
of 355-CAGTACCCAGTGAAGCCAT-373 (NM_018490.2) into the pGPU6/
GFP/Neo vectors by GenePharma Co. (Shanghai, China). pGPU6/GFP/Neosh-NC (control shRNA) that targets a sequence not found in the human, mouse
or rat genome databases (5′-GTTCTCCGAACGTGTCACGT-3′) was used as a
negative control. The GPR48 shRNA and control shRNA were transfected into
HCT116, LoVo and HEK293T cells using Lipofectamine 2000 according to the
manufacturer’s instructions. Construct-carrying clones were selected in G418containing medium beginning 48 h after transfection.
Immunohistochemistry and evaluation of immunohistochemical staining
Tissues were formalin fixed and paraffin embedded, and serial 4 µm thickness
sections were taken for immunohistochemistry analysis using a EnVision™
Detection System, Peroxidase/DAB, Rabbit/Mouse (K5007; DAKO) according to the manufacturer’s instructions. The protocol for immunohistochemical
staining has been described previously (30). Immunostaining was considered
negative if <10% of the tumor cells were stained. The immunohistochemistry
staining was evaluated independently by two board-certified clinical pathologists (L.S. and D.D.) blinded to the clinical parameters. CRC was considered
positive for GPR48 staining when >10% of tumor cells. Staining for GPR48
was assessed using a scoring method as described previously (31,32). Briefly,
the sections were scored using a four-tier scale according to percentage of
positive cells and staining intensity: (−) or 0, tissue specimens without staining (0–10%); (+) or 1, tissue specimens with weak staining (10–25%); (++)
or 2, tissue specimens with moderate staining (25–50%) and (+++) or 3, tissue specimens with strong staining (>50%) (31,32). (−) and (+) were defined
as low expression, and (++) and (+++) were defined as high expression (or
overexpression). Any discrepancy between the two evaluators was resolved by
reevaluation and careful discussion until agreement was reached.
Statistical analysis
Values are presented as mean ± standard deviation (SD). Unpaired t-test or
Pearson’s correlation test was used to compare quantitative variables; Pearson
chi test or Fisher’s exact test was applied to compare qualitative variables;
Patients’ survival curve was plotted using the Kaplan–Meier method, and the
log-rank test was used to determine the significant difference among groups; the
Cox regression model was used to perform multivariate analysis. Analysis was
performed using SPSS 13.0 for Windows (SPSS, Chicago, IL). P < 0.05 was
considered statistically significant.
Additional methods
Detailed methodology is described in Supplementary Materials and methods,
available at Carcinogenesis Online.
Results
GPR48 is overexpressed in human CRC
In our previous proteomic studies, the expression of GPR48 in a cell
line (SW620) generated from a lymph node metastasis was found
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to be much higher than that observed in the primary CRC cell line
(SW480) from the same patient (27). Herein, we confirmed that the
expression of GPR48 was markedly increased in highly metastatic
cell lines (SW620 and KM12L4A) compared with poorly metastatic
cell lines (SW480 and KM12C) at both the mRNA and protein levels
(Figure 1A and B). In addition, we observed that GPR48 expression
in a series of CRC cell lines (SW480, SW620, KM12L4A, KM12C,
LoVo and HCT116) was significantly higher than that in a human normal colorectal epithelial cell line (NCM460) (33) at both mRNA and
protein levels (P < 0.001) (Figure 1A and B), indicating a differential
expression pattern of GPR48 in CRC cells.
To investigate the expression pattern of GPR48 in human CRC, paired
non-tumor and tumor tissues from frozen tissue samples were analyzed by immunoblot analysis. The results showed that the expression
of GPR48 protein in CRC tissues was ~5 times higher than that in the
normal tissues (Figure 1C; P < 0.001). Upregulation of GPR48 expression was further confirmed in 108 paraffin-embedded, archival primary
CRC tissues and 65 normal colorectal mucosa tissues by immunohistochemical analysis, respectively (the clinicopathological parameters
are summarized in Supplementary Table 1, available at Carcinogenesis
Online). As shown in Figure 1D and Supplementary Figure 1A, available at Carcinogenesis Online, most of the normal colorectal mucosa
tissues exhibited low membrane and cytoplasmic GPR48 expression in
crypt epithelial cells. In CRC tissues, GPR48 expression was observed
in the membrane and cytoplasm of tumor cells (Figure 1D). GPR48 was
expressed at higher levels in 75% (81/108) of CRC tissues compared
with those in 29.2% (19/65) of normal mucosa tissues, and the difference was statistically significant (Figure 1E; P < 0.001). In addition, we
compared the intensity of staining for GPR48 in normal mucosa and
tumor tissues, and the results showed that GPR48 expression was significantly higher in tumor cells than in normal mucosal epithelial cells
(Supplementary Table 2, available at Carcinogenesis Online). These
data suggest that GPR48 is overexpressed in human CRC.
GPR48 is correlated with several clinicopathologic features of
CRC patients
We next investigated the relationship between GPR48 expression and
the clinicopathological features of 108 CRC patients (Table I). GPR48
expression was significantly correlated with tumor stage (P = 0.010) and
lymph node status (P = 0.010) (Table I). However, no significant correlation was found between GPR48 expression and other clinicopathological variables studied such as patient age (P = 0.149), gender (P = 1.000),
tumor location (P = 0.315), tumor differentiation (P = 1.000), and
depth of invasion (P = 0.098) (Table I; P > 0.05). In addition, we found
that GPR48 expression in moderately and poorly differentiated CRC
was higher than that in well-differentiated CRC (85.2 versus 41.4%;
Figure 2A). Taken together, these results show that high expression of
GPR48 was associated with a more aggressive biological behavior.
GPR48 is a poor prognostic factor for patients with CRC
The median follow-up time of 108 patients was 42 months (Figure 2B).
Kaplan–Meier survival analysis revealed a close correlation between
GPR48 expression and overall survival (OS) (Figure 2C; log-rank P
= 0.002). The 5-year OS rates of patients with high GPR48 expression
(40.7%) were significantly lower than those with low GPR48 expression
(73.1%). The univariate analysis of prognostic markers of CRC is
summarized in Table II. GPR48 expression (P = 0.003, hazards ratio
(HR): 3.365, 95% confidence interval (CI): 1.511–7.496) and lymph node
status (P < 0.001, HR: 4.533, 95% CI: 2.487–8.263) were prognostic
factors for OS by univariate analysis. Factors showing significance
by univariate analysis were adopted in multivariate Cox proportional
hazards analysis. Multivariate analysis revealed that GPR48 expression
(P = 0.015, HR: 2.740, 95% CI: 1.219–6.158) and lymph node status
(P < 0.001, HR: 4.080, 95% CI: 2.225–7.483) were independent prognostic
factors that could affect the OS of CRC patients (Table II). Furthermore,
we evaluated the correlation between GPR48 and Ki67, a marker of cell
proliferation (34). The result showed that expression of Ki67 was unrelated
to GPR48 expression (R2 = 0.022, P = 0.174; Figure 2D).
Prognostic role of GPR48 in human colorectal cancer
Fig. 1. GPR48 is overexpressed in human CRC cell lines/tissues. (A) Reverse transcription–polymerase chain reaction analysis of GPR48 mRNA from normal
colon cell line (NCM460) and CRC cell lines (SW480, SW620, KM12L4A, KM12C, LoVo and HCT116 cell). The dashed line representing the median. Error
bars represented triplicate experiments ± SD (P value calculated by Student’s t-test). (B) Immunoblot analysis of GPR48 protein from the indicated cell lines.
Expression levels of GPR48 were normalized to those of β-actin. Error bars represented triplicate experiments ± SD (P value calculated by Student’s t-test).
(C) Immunoblot analysis of expression of GPR48 protein in human CRC tissues (T) and adjacent normal mucosa tissues (N) from the same patient. The bar
graph represents relative intensity against β-actin (right). Bars correspond to mean ± SD, ***P < 0.001. (D) Immunohistochemical staining of GPR48 in tumor
and corresponding colorectal mucosa. Magnification: ×200; scale bar, 100 μm. (E) The box plot compares the expression levels of GPR48 in normal colorectal
mucosa and CRC tissues.
GPR48 is upregulated in the invasive front and metastatic lymph
nodes of CRC
When analyzing the levels of GPR48 expression in CRC tissues by
immunohistochemistry, we found an interesting phenomenon where
GPR48 expression was markedly upregulated at the tumor invasive
front compared with the tumor center (88.9 versus 45.4%; P < 0.001)
(Figure 2E and Supplementary Table 3, available at Carcinogenesis
Online). In addition, overexpression of GPR48 was observed in tumor
budding ahead of the invasive front (Supplementary Figure 1B, available
at Carcinogenesis Online, right). GPR48 expression at the tumor invasive front was significantly associated with the degree of tumor budding
(P = 0.014; Supplementary Table 4, available at Carcinogenesis Online).
Next, we performed immunohistochemical analysis of GPR48
expression on 25 matched primary CRC and metastatic lymph nodes
from individual patients. The results showed that GPR48 expression
was much higher in metastatic lymph nodes than in matched primary
tumors (92 versus 76%; P < 0.001; Figure 2F). These findings suggest
that GPR48 expression might be involved in invasion and metastasis
of human CRC.
Overexpression of GPR48 promotes migration and invasion of CRC
cells and tumor metastasis
To investigate the role of GPR48 in the aggressive phenotype of
CRC cells, HCT116 cells stably expressing full-length GPR48
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J.Wu et al.
Table I. Relationship between the clinicopathological features and the
expression of GPR48 in colorectal cancer
Clinicopathological
features
GPR48 expression
P value
Low
High
No. of patients (%)
No. of patients (%)
Age (years)
<65
17 (30.9)
≥65
10 (18.9)
Gender
Male
16 (23.4)
Female
11 (25.0)
Tumor location
Colon
10 (20.4)
Rectum
17 (28.8)
Tumor stage
I–II
21 (34.4)
III
6 (12.8)
Tumor differentiation
Well/moderate
26 (25.5)
Poor
1 (16.7)
Depth of invasion
T2
3 (60.0)
T3–T4
24 (23.3)
Lymph node metastasis
N0
21 (34.4)
N1N2
6 (12.8)
38 (69.1)
43 (81.1)
0.149
48 (76.6)
33 (75.0)
1.000
39 (79.6)
42 (71.2)
0.315
40 (65.6)
41 (87.2)
0.010*
76 (74.5)
5 (83.3)
1.000
2 (40.0)
79 (76.7)
0.098
40 (65.6)
41 (87.2)
0.010*
*P < 0.05 was considered statistically significant. The P values of chi-square
test.
(HCT116/GPR48 cells) or the empty vector (HCT116/vector cells)
were established and subjected to in vitro migration and invasion
assays. In the wound-healing assay, the number of HCT116/GPR48
cells migrating into the wound area was much higher than control
cells (Figure 3A). Consistent with this observation, the number of
HCT116/GPR48 cells migrating into the lower chamber was 10-fold
higher than that of control cells in a transwell chamber migration
assay (Figure 3B). A similar result was observed in HCT116/
GPR48 shRNA and HCT116/control shRNA cells (Supplementary
Figure 2A, available at Carcinogenesis Online). A Matrigel invasion
assay was used to evaluate the effects of GPR48 on invasion of
CRC cells. The results revealed a 19-fold increase in invasive
capacity of HCT116/GPR48 cells compared with the control cells
(Figure 3C). A tail vein metastatic assay was performed in nude mice
to examine the in vivo metastatic potential of HCT116/GPR48 and
HCT116/vector cells. As shown in Figure 3D, the number of lung
metastatic lesions derived from HCT116/GPR48 cells was markedly
increased compared with control cells. The average number of both
the micrometastases and macrometastases in mice lungs following
injection of HCT116/GPR48 cells was >2-fold greater than when
injected with control cells as determined by H&E staining (P <
0.01; Figure 3D). Additionally, we also assessed the effect of GPR48
on proliferation and found that tumor cell growth in an in vitro
colony formation assay and in vivo xenograft tumor model was not
influenced by overexpression of GPR48 (Supplementary Figure 2B
and C, available at Carcinogenesis Online). Collectively, these
results show that GPR48 could promote migration and invasion of
CRC cells and tumor metastasis but did not enhance cell proliferation
and tumor growth.
GPR48 upregulates the expression of β-catenin in vitro and in vivo
It has recently been reported that GPR48 is involved in regulation of Wnt/β-catenin signaling in HEK293T cells (20). We sought
to determine whether this also applied to CRC cells. Firstly, we
measured total and nuclear protein levels of β-catenin in HCT116/
GPR48 and HCT116/vector cells. Immunoblot analysis showed
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that total and nuclear β-catenin was increased 2- to 3-fold in
HCT116/GPR48 cells compared with control cells (Figure 4A
and B). Deletion of GPR48 in LoVo, HCT116 and HEK293T
cells decreased total β-catenin protein levels (Figure 4A and
Supplementary Figure 3A, available at Carcinogenesis Online).
Subsequently, the subcellular distribution of β-catenin in HCT116/
GPR48 cells and HCT116/vector cells was examined by immunofluorescence staining. We observed that β-catenin was mainly
distributed in the cytoplasm and the nucleus of HCT116/GPR48
cells compared with the cytoplasm and membrane of HCT116/vector cells (Supplementary Figure 3B, available at Carcinogenesis
Online), indicating that GPR48 could induce nuclear β-catenin
accumulation in CRC cells.
To determine whether β-catenin expression is upregulated in vivo
by GPR48, primary CRC tissues from 34 patients were stained for
β-catenin. As shown in Figure 4C, β-catenin protein in CRC tissues
was mainly accumulated in the cytoplasm and nuclear compartment,
whereas moderate membranous and weak cytoplasmic β-catenin
staining was observed in noncancerous mucosa. The expression of
β-catenin in the cell membrane as appeared normal, whereas the
expression in cytoplasm and nuclei was ectopic. High β-catenin
expression in the cell membrane, cytoplasm and nuclei was observed
in 18 (52.9%), 24 (70.6%) and 9 (26.5%) cases of CRC, respectively (Supplementary Table 5, available at Carcinogenesis Online).
Correlation analysis showed that expression of GPR48 was positively
related to β-catenin expression (R2 = 0.166; P = 0.017; Supplementary
Figure 3C, available at Carcinogenesis Online). These data suggest
that GPR48 expression was associated with activation of β-catenin
in CRC.
GPR48 activates β-catenin/TCF signaling pathway and increases
expression of its downstream target genes in CRC cells
Next, we determined whether overexpression of GPR48 affected
β-catenin signaling in CRC cells using the luciferase-based
TOPflash/FOPflash assay. TCF4 transcription activity was increased
1.5- to 2.7-fold in HCT116/GPR48 and HEK293T/GPR48 cells
compared with control cells (Figure 4D). In contrast, knockdown of
GPR48 resulted in reduced TCF4 transcriptional activity in LoVo/
GPR48 shRNA and HEK293T/GPR48 shRNA cells relative to that
in control cells (Figure 4E). In addition, we examined the expression
of β-catenin target genes by reverse transcription–polymerase chain
reaction. Expression levels of the β-catenin target genes including Cyclin D1 and c-Myc were much higher in HCT116/GPR48
cells than that in the corresponding control cells (Supplementary
Figure 3D, available at Carcinogenesis Online). These results demonstrate that GPR48 could activate β-catenin/TCF signaling and
increase expression of its target genes (Cyclin D1 and c-Myc) in
CRC cells.
GPR48-mediated β-catenin/TCF signaling is associated with
activation of PI3K/Akt and mitogen-activated protein kinase/
ERK1/2 pathway in CRC cells
GPR48 could activate EGFR and EGFR downstream signaling pathways, including mitogen-activated protein kinase (MAPK)/ERK1/2
and STAT3 pathways (16,35). It is known that ERK and AKT can
phosphorylate GSK-3β at Ser9, resulting in phosphorylation and degradation of β-catenin (24–26). To better understand the mechanism
underlying by which GPR48 activates β-catenin/TCF signaling, we
next examined whether EGFR downstream pathways including PI3K/
Akt and MAPK/ERK1/2 pathways are activated by GPR48 in CRC
cells. Immunoblot analysis showed that upregulation of GPR48 in
HCT116/GPR48 cells was associated with increased phosphorylation of GSK-3β (at Ser9), ERK1/2 and Akt, whereas total GSK-3β,
ERK1/2 and Akt levels were unchanged (Figure 4F). In contrast,
depletion of GPR48 in LoVo and HEK293T cells led to decreased
phosphorylation levels of GSK-3β, ERK1/2 and Akt (Figure 4F
and Supplementary Figure 3E, available at Carcinogenesis Online),
Prognostic role of GPR48 in human colorectal cancer
Fig. 2. GPR48 is correlated with shorter OS of CRC patients and is upregulated in the invasive front and metastatic lymph nodes of CRC. (A) Immunohistochemical
staining of GPR48 expression in normal mucosa, well, moderately and poorly differentiated CRC tissues. Magnification: ×200; scale bar, 100 μm (upper). (B) The
median follow-up time of these 108 patients was 42 months. (C) Kaplan–Meier survival curves of CRC patients with low (n = 27) and high (n = 81) GPR48
expression, black indicates high and gray indicates low GPR48 expression. P values were calculated by the log-rank test. **P < 0.01. (D) Correlation between Ki67
and GPR48 in 85 CRC samples. Pearson’s correlation coefficient between GPR48 and Ki67 was −0.149, R2 = 0.022, P = 0.174. (E) Increased expression of GPR48
at the invasive front of human CRC samples compared with tumor center. Magnification: ×100. (F) Immunostaining for GPR48 in a primary CRC and its lymph node
metastasis from the same patient (left). Box plot compares the distribution of GPR48 expression between primary CRC and corresponding matched metastatic lymph
nodes (right).
suggesting that GPR48 could activate MAPK/ERK1/2 and PI3K/
AKT pathways in CRC cells.
To investigate whether two pathways participate in activation of
β-catenin/TCF signaling in CRC cells, pharmacological inhibitors
of PI3K (LY294002) or/and MEK (U0126) were used in TCF4
transcriptional activity assays. As shown in Figure 4G (left), treatment
of HCT116/GPR48 cells with PI3K inhibitor LY294002 (10 μmol/l)
and MEK inhibitor U0126 (10 μmol/l) alone, or in combination,
markedly decreased GPR48-induced TCF4 transcriptional activity,
suggesting that PI3K/Akt and MAPK/ERK1/2 pathways were
required for GPR48-mediated β-catenin signaling. To further confirm
that GSK-3β, a downstream target of PI3K/AKT and MAPK/ERK1/2
pathways, is involved in GPR48-mediated β-catenin signaling,
we cotransfected HCT116 cells with GPR48 cDNA and SuperTop
TCF4 luciferase reporter plasmid and treated these with LiCl, a
GSK-3β inhibitor. The results showed that GPR48 and LiCl alone
or in combination increased TCF4 transcriptional activity compared
with HCT116/vector cells (Supplementary Figure 3F, available at
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J.Wu et al.
Carcinogenesis Online). These findings suggest that GPR48 activates
β-catenin/TCF signaling via regulation of GSK-3β phosphorylation
through MAPK/ERK1/2 and PI3K/Akt pathways in CRC cells.
Table II. Univariate and multivariate analysis for OS
Factor
OS
Univariate analysis
Multivariate analysis
P value
HR
Age, years (<65
0.856
versus ≥65)
Gender (male
0.867
versus female)
Tumor location
0.540
(colon versus
rectum)
Tumor
0.625
differentiation
(well/moderate
versus poor)
Depth of invasion 0.222
(T2 versus T3T4)
LN metastasis
<0.001
(N0 versus N1N2)
GPR48 (low
0.003
versus high)
95% CI
P value
NA
NA
NA
NA
NA
4.080
2.225–7.483
<0.001
2.740
1.219–6.158
0.015
NA, not adopted. Multivariate analysis and Cox proportional hazards
regression model were used. Variables were adopted for their prognostic
significance by univariate analysis with forward stepwise selection (forward,
likelihood ratio). Variables were adopted for their prognostic significance by
univariate analysis (P< 0.05).
Discussion
G-protein-coupled receptors, the largest family of cell-surface molecules with key roles in signal transmission, are emerging as crucial
players in tumor growth and metastasis (36). Recent studies have
highlighted the role of GPR48, a member of the G-protein-coupled
receptor family, in carcinogenesis and development of CRC and gastric carcinoma (17,18), but the correlation between GPR48 expression
and clinical outcomes in human CRC has not been fully investigated.
Here, we show that GPR48 is highly expressed in primary CRC and
correlates with the poor prognosis of patients with CRC.
LGRs, including GPR48, GPR49 and LGR6, have previously
been reported to be involved in many different physiological and
pathophysiological processes (18). GPR49, a marker of cancer
stem cells, was found to be overexpressed in several types of cancer
including hepatocellular carcinoma, ovarian cancer and CRC, and
its overexpression was associated with lymph node metastasis, liver
metastasis and poor prognosis for CRC patients (37,38). Although
GPR48 shares ~50% sequence homology with GPR49, few studies
have been reported on the potential of GPR48 as a marker for the
prognosis of patients with cancer. Recent studies have shown that
GPR48 expression is upregulated in gastric cancer at the transcription
and translation levels (10,18). In addition, Gao et al. found that GPR48
mRNA expression was increased in CRC tissues compared with normal
tissues and was associated with tumor differentiation and lymphatic
involvement (17). In this study, GPR48 protein was frequently
upregulated in primary CRC tissues, and its high expression closely
correlated with advanced tumor stage and lymph node metastases,
suggesting that GPR48 is involved in CRC development. In log-rank
test, we found that CRC patients with low GPR48 expression lived
longer than those with high GPR48 expression. Multivariate analysis
indicated that GPR48 served as an independent prognostic marker for
OS in patients with CRC. In contrast, Steffen et al. did not find that
Fig. 3. GPR48 overexpression promotes cells migration, invasion and tumor metastasis. (A) Wound-healing assay for HCT116-vector control and HCT116GPR48 cells. Photographs were taken at 72 h postwounding. Dashed lines indicated the original wound boundaries. (B) Cell migration assays for HCT116-vector
and HCT116-GPR48 cells. Original magnification, ×100. Results were presented as the ratios of migrated HCT116-GPR48 cells relative to those of the control
cells. Data were representative of three experiments. Bars correspond to mean ± SD, ***P < 0.001. (C) Cell invasion assays for HCT116-vector and HCT116GPR48 cells. Original magnification, ×100. ***P < 0.001. (D) H&E staining of lung sections in mice-bearing HCT116 xenografts. Quantification of the lung
micrometastases or macrometastases of vector (left) and GPR48-(right) expressing cells. **P < 0.01.
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Prognostic role of GPR48 in human colorectal cancer
Fig. 4. GPR48 induces nuclear β-catenin accumulation and activates β-catenin/TCF signaling in CRC cells via regulation of PI3K/Akt and MAPK/ERK1/2
signaling pathways. (A) Immunoblot analysis of β-catenin expression in HCT116/vector and HCT116/GPR48 or LoVo/control shRNA and LoVo/GPR48 shRNA
cells. β-Actin was used as a loading control. (B) Cytoplasmic and nuclear fractions of HCT116/vector and HCT116/GPR48 cells were subjected to sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and then immunoblotted with anti-β-catenin, β-actin and histone-3 (right). β-Actin was used as a cytoplasmic
protein loading control, and histone-3 was used for nuclear protein loading control. (C) Immunohistochemical staining of GPR48 and β-catenin in the sample of
primary CRC (lower) and adjacent normal tissues (upper). Original magnification, ×200. (D) Luciferase assay for HCT116 and HEK293T cells expressing empty
vector or GPR48 vector, which were transfected with the Super TOP Flash TCF4 reporter construct along with a mutant control FOP Flash reporter construct.
Luciferase reporter activity was measured after 48 h. Results were presented relative to FOP Flash luciferase activity. Mean values ± SD from three separate
experiments were shown. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test. (E) Luciferase assay for LoVo and HEK293T cells expressing control shRNA
or GPR48 shRNA. (F) Expression levels of GSK-3β, p-GSK-3β, Akt, p-Akt, ERK1/2 and p-ERK1/2 protein in HCT116 cells and LoVo cells determined by
immunoblot blotting. β-Actin was used as a loading control. (G) Luciferase assay for HCT116/vector and HCT116/GPR48 cells (left). Cells were then treated
with Ly294002/Ly (10 μmol/l) and U0126/U0 (10 μmol/l) alone, or in combination, for an additional 24 h. Luciferase reporter activity was measured after 48 h.
**P < 0.01; ***P < 0.001, Student’s t-test. (H) A model to illustrate the regulation of β-catenin signaling by GPR48 in CRC cells. In the absence of Wnt protein
(left panel), β-catenin associates with the APC/Axin/GSK-3β-complex and is phosphorylated by GSK-3β, which leads to its ubiquitylation and degradation by
the proteasome. Overexpression of GPR48 leads to the transactivation of EGFR (data not shown) and the subsequent activation of PI3K/Akt and MAPK/ERK1/2
pathways (right panel), which could inhibit GSK-3β activity, thereby blocking the proteosomal degradation of β-catenin. Accumulated β-catenin then translocates
into the nucleus and activates Wnt target genes in concert with TCF/LEF cofactors, including Cyclin D1 and c-Myc.
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J.Wu et al.
GPR48 expression had positive predictive values for OS of gastric
cancer patients (18). This discrepancy might be due to the different
cancer types and the different cutoff values used (they used negative
or positive GPR48 expression, whereas we used high or low GPR48
expression) for distinguishing differential subgroups.
Cancer cells undergo a series of events during metastasis, including
invasion, entry into the circulatory system, arrest at a distant site, proliferation and induction of angiogenesis (39). Lymph node metastasis
is one of the most important factors that influences the survival of
surgical patients with CRC (40). Positive correlation between GPR48
expression and lymph node metastasis has been reported for human
gastric cancer and CRC (17,18). In our study, we found that GPR48
expression in lymph node metastasis was much higher than that in
matched primary CRC, suggesting that GPR48 might be a potential
marker of lymph node metastasis. The invasive front of a tumor is a
region containing useful prognostic information, presumably because
the most invasive cells are located there (41). In this study, we found
that GPR48 was overexpressed at the invasive front of tumors, indicating that GPR48 might play a role in acquisition of migrating and
invading cell phenotype that is a prerequisite for malignancy. Previous
studies have reported that tumor budding often occurs ahead of the
defined invasive edge in CRC tissues and the presence of budding
was significantly correlated with a higher rate of lymph node metastasis and poor prognosis of patients (42,43). We found that GPR48
was overexpressed in tumor budding and its expression at the invasive front correlated with grade of tumor budding, which further confirmed that GPR48 expression was associated with more aggressive
tumor phenotype. In vitro and in vivo study further validated the role
of GPR48 in CRC invasion and metastasis. In line with these results,
depletion of endogenous GPR48 in HeLa and Lewis lung carcinoma
cells markedly diminished their invasive and metastatic activities
(17). These data, in concert with the present findings, further support a critical role of GPR48 in regulating invasion and metastasis of
CRC. However, so far, the relevance of GPR48 expression in CRC
metastasis to distant organs such as liver and lung has not been investigated and the precise mechanism by which GPR48 promotes tumor
invasion and metastasis remains unclear: further studies are needed
to reveal this.
The Wnt/β-catenin signaling pathway plays major roles in stem
cell biology, organogenesis, tissue homeostasis and cancer (44,45).
In the canonical Wnt signaling pathway, Wnt ligands bind to Frizzled
and low-density lipoprotein-related receptors 5 and 6, which in turn
inhibits phosphorylation of β-catenin by disrupting the destruction
complex consisting of the APC, axin and GSK-3β proteins, resulting
in β-catenin accumulation in the nucleus and activation of the transcription of Wnt target genes such as cyclin D1 and c-Myc (1,21).
Mutations in APC, β-catenin or axin result in increased β-catenin
levels and activation of Wnt signaling and have been found in numerous human cancers including colorectal, gastric and ovarian cancer
(46). However, mutations of Wnt pathway components are not the
only factors that regulate activation of β-catenin signaling. Recent
data suggest that GPR48 and GPR49 function as the cognate receptor of R-spondin to potentiate Wnt/β-catenin signaling by enhancing
Wnt-induced LRP6 phosphorylation (19,20). Conditional deletion of
GPR48 and GPR49 genes in the mouse gut impairs Wnt target gene
expression and results in Wnt pathway inhibition (15). In this study,
our data show that GPR48 induces nuclear β-catenin accumulation
and augments β-catenin/TCF signaling accompanied by an increased
expression of its target genes (including cyclin D1 and c-Myc) in
CRC cells with mutations in either APC or β-catenin. It is noteworthy
that in human CRC tissues, GPR48 was found to be associated with
activation of β-catenin, which further confirmed that GPR48 was a
positive regulator of β-catenin signaling in CRC.
Although recent data suggest that GPR48 could mediate Wnt/βcatenin signaling in HEK293T cells, the precise mechanism by which
GPR48 increases β-catenin protein levels and modulates β-catenin/
TCF pathway is still unclear. Mustata et al. found that downregulation of Wnt target genes caused by GPR48 knockout could be partially rescued by addition of LiCl but not by Wnt agonists Wnt3a
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and R-spondin, indicating that GPR48 may be involved in interaction between the Wnt and PI3K/Akt pathways (14). Our present data
indicate that overexpression of GPR48 could increase phosphorylation levels of GSK-3β (at Ser9), Akt and ERK1/2, which can regulate phosphorylation and degradation of β-catenin (47). It has been
reported that in response to certain growth stimuli and oncogenes,
activated Akt and ERK1/2 can phosphorylate GSK-3β at Ser9, leading
to inactivation of GSK-3β, upregulation of β-catenin and activation of
Wnt/β-catenin signaling (24–26). Our data suggest that GPR48 activates β-catenin signaling in a similar manner. Either PI3K inhibitor or
MEK inhibitor treatment could block activation of β-catenin signaling in CRC cells, whereas LiCl treatment further stimulated β-catenin
signaling in the presence of GPR48, suggesting that GPR48 activates
β-catenin signaling at least partially through the inhibition of GSK3β in a PI3K- and ERK1/2-dependent manner. However, it remains to
be elucidated whether other signaling pathways are also involved in
GPR48-induced β-catenin signaling activation.
In summary, we observed that GPR48 was overexpressed in human
CRC and this overexpression was associated with a more aggressive
biological behavior. We report for the first time that GPR48 expression is associated with unfavorable prognosis in CRC patients postoperatively. Furthermore, our data demonstrate that GPR48 activates
β-catenin signaling in CRC by regulating the phosphorylation of
GSK-3β via PI3K/AKT and MAPK/ERK1/2 pathways. These results
highlight the importance of GPR48 for a better understanding of CRC
pathogenesis and its potential implication in the management of CRC,
as a prognostic factor or a future therapeutic target.
Supplementary material
Supplementary Tables 1–5 and Figures 1–3 can be found at http://
carcin.oxfordjournals.org/
Funding
National 973 Basic Research Program of China (2013CB911300,
2012CB518900,
2011CB910703);
the
National
Science
and
Technology
Major
Project
(2011ZX09302-001-01,
2012ZX09501001-003); Doctoral Fund of Ministry of Education of
China (20120181110024); the National Natural Science Foundation
of China (81072022, 81172173, 81225015).
Conflict of Interest Statement: None declared.
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Received January 27, 2013; revised May 6, 2013; accepted June 8, 2013
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