Pathology – Research and Practice 210 (2014) 885–892
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Original Article
A low level of GPR37 is associated with human hepatocellular carcinoma progression and poor patient survival Fang Liu a,1 , Changlai Zhu a,1 , Xiaodong Huang b , Jing Cai b , Hua Wang b , Xinxiu Wang a , Song He b , Cheng Liu a , Xiaojing Yang a , Yixin Zhang b , Tianyi Zhang a,∗ a b
Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China Department of Pathology, Nantong University Cancer Hospital, Nantong, Jiangsu 226001, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 December 2013 Received in revised form 9 May 2014 Accepted 2 July 2014 Keywords: Human hepatocellular carcinoma (HCC) An orphan G protein-coupled receptor GPR37 Cell proliferation Pathogenesis
a b s t r a c t GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR). It has been reported that GPCRs play vital roles in the development and progression of cancer. To investigate the potential roles of GPR37 in hepatocellular carcinoma (HCC), expression of GPR37 was examined in human HCC samples. Immunohistochemistry and Western blot analyses were performed for GPR37 in 57 hepatocellular carcinoma samples. GPR37 expression was low in hepatocellular carcinoma as compared with the adjacent non-tumorous tissues. Clinicopathological analysis showed that GPR37 expression was significantly correlated with histological grade and the level of alpha fetal protein (AFP) (P = 0.000 and 0.002, respectively). The Kaplan–Meier survival curves revealed that decreasing GPR37 expression was associated with poor prognosis in HCC patients, while in vitro, following the release from serum starvation of HuH7 HCC cell, the expression of GPR37 was downregulated. In addition, the transient GPR37 knockdown by siRNA in HuH7 cells significantly decreased the apoptosis of hepatoma cells with activation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Our data suggest that GPR37 may play an important role in the pathogenesis of hepatocellular carcinoma by affecting the proliferation of H CC cells, and it could be a novel potential molecular therapy target for HCC. © 2014 Elsevier GmbH. All rights reserved.
Introduction Because of its high morbidity and poor prognosis, hepatocellular carcinoma (HCC) has long been known as one of the most lethal malignancies [1,2]. HCC is the third leading cause of cancer death, and its incidence in the world is increasing [3,4]. HCC carcinogenesis is a multistep process with many possible risk factors, including cirrhosis, hepatitis B and C viruses (HBV and HCV), aflatoxin exposure, alcoholic liver disease and possibly nonalcoholic liver disease, cigarette smoking and elevated endogenous testosterone in serum [5,6]. A large number of studies have shown that this molecular mechanism is involved in hepatocarcinogenesis [7–10]. Despite advances in therapy for HCC and modern surgical innovations, patient outcome has not substantially improved [11], and tumor mortality is very high. However, the molecular mechanism of HCC
∗ Corresponding author. Tel.: +86 051385511885. E-mail addresses: [email protected] (Y. Zhang), pro [email protected] (T. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.prp.2014.07.011 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
development and progression remains poorly understood. Hence, it is very important to identify novel and effective molecular markers which could provide some clues to understanding the mechanism of HCC and the development of new diagnostic strategies for treatment and prevention. GPR37 is a vertebrate G-protein-coupled orphan receptor [12], also known as the parkin-associated endothelin-like receptor (PaelR). Although GPR37 is predominantly expressed in the mammalian central nervous system, its expression and biological function still remain largely unknown [13]. The orphan G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, can mediate the extracellular environment to intracellular effectors of signal transduction [14], and regulate a variety of physiological and disease processes [15]. Many G protein-coupled receptors (GPCRs) have been found to play vital roles in the development and progression of cancer, including tumor growth and survival [16,17]. Therefore, we attempted to investigate the expression of GPR37 in the development and progression of HCC. The involvement of GPR37 in human tumor is not yet clearly understood. To solve this problem, GPR37 expression at protein levels in human hepatocellular carcinoma tissues and matched
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Table 1 GPR37 Ki67 expression and clinicopathological parameters in 57 HCC specimens. Parameters
Age (years) ≤45 >45 Gender Male Female Histological grade Well Mod Poor Metastasis Positive Negative Vein invasion Presence Absence Tumor size (cm) ≤5 >5 No. of tumor nodes Single Multiple/≥2 Capsular formation Presence Absence HBsAg (+) (−) Cirrhosis Positive Negative AFP (ng/ml) ≤50 >50
Total
GPR37
P
Low
High
24 33
14 23
10 10
45 12
31 6
17 19 21
Ki67
P
Low
High
0.271
12 17
12 16
0.562
14 6
0.189
20 9
25 3
0.059
5 12 20
12 7 1
0.000*
11 11 7
6 8 14
0.048*
8 49
4 33
4 16
0.284
5 24
3 25
0.373
14 43
7 30
7 13
0.153
9 20
5 23
0.199
27 30
18 19
9 11
0.506
14 15
13 15
0.55
35 22
25 12
10 10
0.155
16 13
19 9
0.239
40 17
25 12
15 5
0.394
21 8
19 9
0.465
43 14
27 10
16 4
0.402
21 6
22 8
0.409
45 12
28 3
17 9
0.321
21 8
24 4
0.183
27 30
23 14
4 16
0.002*
11 18
16 12
0.118
Abbreviations: HBsAg = hepatitis B surface antigen; AFP = alphafeto protein. Note. Statistical analyses were performed by the Pearson 2 test. * P < 0.05 was considered significant.
adjacent non-tumorous tissues was detected by Western blot and immunohistochemistry analyses. We also investigated its associations with clinicopathological characteristics and the prognostic implications. For the first time, our study found that GPR37 had an association with the proliferation of human hepatocellular carcinoma cells, which might be a valuable breakthrough point for experimental therapies of HCC.
Tissue samples were processed immediately after surgical removal. For histologic examination, all tumorous and surrounding non-tumorous tissue portions were fixed in formalin and embedded in paraffin. Protein was analyzed in eight snap-frozen tumorous and adjacent non-tumorous tissue samples stored at −80 ◦ C. Informed consent was obtained from all patients.
Immunohistochemistry Materials and methods Patients and tissue samples HCC tissues were obtained from 57 patients. All underwent hepatic surgical resection without postoperative systemic chemotherapy at the Surgery Department of the Affiliated Hospital of Nantong University. The diagnosis criteria for all patients in this study were confirmed by experienced pathologists through histological examination of H&E-stained biopsy sections; clinicopathologic and follow-up data were completely available. The main clinical and pathologic variables of the patients are shown in Table 1. Forty-five patients were men and 12 were women; their age ranged from 21 to 65 (mean = 47.19 years). Forty-three patients were positive for HBV surface antigen, 45 were positive for cirrhosis. Histological grades were classified as well differentiated (grade I; n = 17), moderately differentiated (grade II; n = 19), and poorly differentiated (grade III; n = 21). The followup time was 5 years, with a range of 1–80 months (median = 37 months). None of the patients had received postoperative adjuvant therapy.
Tissue sections (1 m) were cut, placed on APES-pretreated slides, deparaffinized, rehydrated through graded alcohol, and quenched in 3% hydrogen peroxide. Antigen retrieval was performed by microwave heating at high power (750 W) in 10 mM sodium citrate buffer (pH6.0) for three cycles of 5 min each. After blocking with normal serum for 1 h at room temperature, the sections were incubated overnight at 4 ◦ C with anti-human GPR37 rabbit polyclonal antibody (diluted 1:150; Cell Signaling Technology), anti-Ki67 mouse monoclonal antibody (diluted 1:100; clone 7B11; Zymed Laboratories, San Francisco, CA, USA). Negative control slides were also processed in parallel using a nonspecific immunoglobulin IgG (Sigma Chemical Co., St. Louis, MO) at the same concentration as the primary antibody. For the assessment of GPR37 and Ki-67, five high-power fields were randomly chosen, more than 500 cells were counted to determine the Labeling index (LI), which means that the percentage of immunostained cells is relative to the total number of cells [18]. The LI of GPR37 ranged from 7% to 87%. The mean percentage of positive cells was 47%. The LI of Ki-67 ranged from 4% to 71%. The mean percentage of positive cells was 43%. Tumors were scored as percentage of positive
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Fig. 1. GPR37 is low expressed in HCC. (A) Western blot analysis of whole-cell protein extracts prepared from eight representative paired samples of HCC tissues (T) and adjacent non-tumorous tissues (N). GAPDH was used as a control for protein load and integrity. (B) The bar chart demonstrates the ratio of GPR37 protein to GAPDH for the above by densitometry. The data are mean ± SEM (*P < 0.01, compared with adjacent non-tumorous tissues).
cells for each antigen. The GPR37 and Ki-67 immunostaining scores were calculated as both the percentage of positively stained tumor cells and the staining intensity. The percent positivity of GPR37 was scored as follows: 0 (70% positive tumor cells). The percent positivity of Ki67 was scored as: 0 (10% positive tumor cells); 1 (5–25% positive tumor cells); 2 (25–45% positive tumor cells); 3 (45–60%positive tumor cells); 4 (>60% positive tumor cells). Staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained), or “3” (strongly stained). The immunostaining score was calculated as the percentage positive score × the staining intensity score and ranged from 0 to12. A score of 0 was considered negative; 1–4 was considered weak; 5–9 was considered moderate; and 10–12 was considered strong. For statistical analysis, 0–4 were counted as low expression, while 5–12 were counted as overexpression [19].
100 g/ml streptomycin in 5% CO2 at 37 ◦ C. For cell cycle analysis, cells were fixed in 70% ethanol for 1 h at 4 ◦ C and then incubated with 1 mg/ml RNase A for 30 min at 37 ◦ C. Subsequently, cells were stained with propidiumiodide (50 g/mL PI) (Becton Dickinson, San Jose, CA) in PBS, 0.5% Tween-20, and analyzed with a Becton Dickinson flow cytometer BD FACScan (San Jose, CA) and Cell Quest acquisition and analysis programs. Cell proliferation assay Cell proliferation was measured using the CCK-8 (cell counting Kit-8) assay following the manufacturer’s instructions. Briefly, cells were seeded at 2 × 104 /well in a volume of 100 l in 96-well cell culture cluster (Corning Inc., Corning, NY) and grown overnight. Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) reagents were added to a subset of wells under different treatments and in cubated for 1 h at 37 ◦ C, after which absorbance was quantified on an Automated plate reader at a test wavelength of 490 nm at different times.
Western blot analysis Plasmid constructs siRNA and transfection Tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M Tris HCl pH7.5, 1%Triton X-100, 1% Nonidet p-40 (NP-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, leupeptin 10 g/ml, aprotinin 10 g/ml, and 1 mM PMSF, then centrifuged at 10,000 × g for 30 min to collect the supernatant liquid. Protein concentrations were determined with a Bio-Rad protein assay (BioRad, Hercules, CA, USA). The total cellular protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20. The antibodies used in this study included: anti-GPR37 (1:1000; Santa Cruz Biotechnology); GAPDH (1:1000; Sigma); anti-p27Kip1 (1:500; Santa Cruz Biotechnology); anti-PCNA (1:500; Santa Cruz Biotechnology); anti-cyclin D (1:500; Santa Cruz Biotechnology); anti-cyclin A (1:500; Santa Cruz Biotechnology). Blots were washed three times in PBS buffer each for 5 min, followed by incubation with the appropriate horseradish peroxidase-linked secondary antibodies. Then, the membrane was developed by the ECL detection systems. The experiments were carried out on three separate occasions.
The siRNA species for the GPR37 knockdown purchased from Biomics (Nantong, China) were synthesized to target the following cDNA sequences: GPR37-siRNA, sense =5 -GGUGGGAGCUCUAUUGUUAdTdT-3 and anti-sense = 5 UAACAAUGAGCUCCCACCdTdT-3 ; and negative control (NC), sense = 5 -UUCUCCGAACGUGUCACGUdTdT-3 and anti-sense = 5 ACGUGACACGUUCGGAGAAdTdT-3 . Cell transfection was performed with SuperFectin according to the manufacturer’s instructions. Apoptosis assay The apoptosis assays were performed at 72 h after the cells were transfected with 20 mM siGPR37 or NC. The Huh7 cells transfected with siGPR37 or NC were washed three times in ice-cold PBS, resuspended in 100 l of 1× Binding Buffer and incubated with Annexin V-FITC (Bestbio, China) for 15 min at 4 ◦ C in the dark, according to the manufacturer’s instructions. After staining, the cells were incubated with propidium iodide for 5 min at 4 ◦ C in the dark and then analyzed using a flow cytometer (Beckman, USA).
Cell culture and cell-cycle analysis
Statistical analysis
Huh7, a human hepatocarcinoma cell line, was obtained from our laboratory and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 u/ml penicillin, and
Statistical analysis was performed using the PASW statistics 18 software package. The association between Ki-67 and GPR37 expression and clinicopathological features was analyzed using the
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Fig. 2. The analysis of GPR37 expression in the primary hepatocellular carcinoma tissues with immunohistochemistry. (A) and (F) Immunostaining of an HCC tumor and the adjacent non-tumorous area. (B) and (G) Normal liver tissue distant from the tumor. (C) and (H) Well-differentiated HCC, strongly positive GPR37 expression. (D) and (I) Moderately differentiated HCC, weakly positive GPR37 expression. (E) and (J) poorly differentiated HCC, no GPR37 expression. N: adjacent non-tumor tissue; T: tumor tissue (A–E with 200× magnification; F–J with 400× magnification).
2 test. As the data were not normally distributed, Ki-67 and GPR37 expressions were analyzed by the Spearman rank correlation test. Overall survival curves were calculated with the Kaplan–Meier method and were analyzed with the log-rank test. A Cox proportional-hazards analysis was used in univariate and multivariate analyses to explore the effects of GPR37 expression and HCC clinicopathological variables on survival. The results were expressed as mean ± SD and analyzed using the Mann–Whitney U test. Differences were considered significant at P < 0.05. Results Expression of GPR37 protein in HCC, adjacent non-tumorous tissue The expression of GPR37 was examined with immunoblotting methods in eight paired adjacent non-tumorous tissue and HCC biopsy samples. The example of Western blot analysis is shown in Fig. 1. GPR37 expression was found to be lower in HCC tissue (T) than in adjacent non-tumorous tissues (N). The amount of GAPDH,
a housekeeping protein, was demonstrated to be rather constant among the samples.
Immunohistochemical analysis of GPR37 expression in HCC clinical samples and its relationship to clinicopathological parameters Expression of GPR37 was studied in 57 HCC specimens by immunohistochemical staining. Following the different histological grades, representative examples of reactivity for GPR37 are shown in Fig. 2. In the positive samples, GPR37 was detected in the plasma membrane of the cells (Fig. 2C). Its LIs ranged from 7% to 87%. The mean percentage of positive cell was 47%. GPR37 expression was also detected in normal liver tissues distant from the tumors (Fig. 2B). It was clear that the expression of GPR37 gradually decreased with histological grade from well-differentiated to poorly differentiated. Well-differentiated samples showed strongly positive GPR37 expression (Fig. 2C and H), moderately-differentiated samples showed weakly positive expression (Fig. 2D and I), and the most poorly differentiated
Fig. 3. Analysis of GPR37 and Ki67 expression. (A) Paraffin-embedded tissue sections were stained with antibodies against GPR37 and Ki67 counterstained with hematoxylin. (B, D) Low GPR37 expression in HCC tissues, whereas Ki67 staining showed the level was high in the same HCC tissues (SP × 400). N: adjacent non-tumorous tissues; T: tumor tissues (400× magnification). (B) The relationship between the Ki-67 proliferation index and GPR37 expression in HCC. Scatterplot of Ki67 versus GPR37 with regression line showing correlation using Spearman’s coefficient (P < 0.01).
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samples often showed no detectable GPR37 expression (Fig. 2E and J). The GPR37 expression with clinicopathologic variables is given in Table 1. Thirty-seven (64.9%) samples showed low GPR37 expression and 20 (35.1%) samples exhibited high GPR37 expression (Table 1). GPR37 expression was not significantly associated with age, gender, metastasis, vein invasion, tumor size, tumor nodes, capsular formation, HBsAg or cirrhosis, but it significantly correlated with histological grade (P = 0.000) and serum AFP (P = 0.002) (Table 1). The expression of GPR37 is associated with the proliferation marker Ki-67
Fig. 4. The Kaplan–Meier survival analysis of the primary HCC patients (n = 57) with high GPR37 expression (n = 20) and low GPR37 expression (n = 37) after surgical resection. Based on their GPR37 immunostaining scores, the HCC patients were divided into low-GPR37 expression (score < 5) and high-GPR37 expression (score ≥ 5) groups. The survival rate of the patients in the low-GPR37 group was significantly lower than that of the patients in the high-GPR37 group (P < 0.01 for the log-rank test).
We performed immunohistochemical staining to evaluate the expression of GPR37 and the proliferation marker Ki-67 in the same cohort of HCC tissues and adjacent non-tumorous tissues. Representative examples of reactivity for GPR37 and Ki-67 are shown in Fig. 3. There was low or no GPR37 expression (score < 5) in HCC tissues (Fig. 3A and B), while Ki67 staining showed a high level in the same HCC tissues (Fig. 3A and D). Ki-67 expression in HCC was scored as positive when expression was strong in the nuclei. Its LIs ranged from 4% to 71%. The mean percentage of positive cells was 43%. GPR37 and Ki-67 expressions in HCC were scored as positive (score ≥ 5). The association of Ki-67 and GPR37 expression with
Fig. 5. The expression of GPR37 and cell cycle-related molecules in proliferating HCC cells. (A) Flow cytometry quantification of cell cycle progression in Huh7 cells. Normal cells, cells that were serum starved for 72 h, and then addition of medium containing 10% FBS for the indicated time points. (B) Huh7 cells were serum starved for 72 h and upon serum releasing, cell lysates were prepared and analyzed by Western blot using antibodies directed against GPR37, p27Kip1 , cyclin A and cyclin D. GAPDH was used as a control for protein load and integrity. (C) The bar chart below demonstrates the ratio of GPR37, p27Kip1 , cyclin A and cyclin D protein to GAPDH for each time point by densitometry. The data are means ± SEM (n = 3, *,#,&,$ P < 0.01, compared with control: S72 h). S: serum starvation; R: serum release.
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clinicopathologic variables is summarized in Table 1. Furthermore, in most specimens, the proportion of GPR37-positive tumor cells was opposite to the proportion of Ki-67-positive tumor cells. A negative correlation between GPR37 expression and Ki-67 on the basis of proliferative activity was found (P < 0.01; Fig. 3B). Correlation between expression of GPR37 and survival rates in patients with HCC The prognostic value of GPR37 for overall survival in HCC patients was evaluated by comparing the patients with high and low GPR37 expression. Using the Kaplan–Meier analysis, low GPR37 expression was significantly associated with poor prognosis. The HCC patients with low GPR37 expression obviously had lower overall survival rates than those with high GPR37 expression (P < 0.01; Fig. 4). Expression of GPR37 in proliferating HCC cells Based on our results, we further detected the expression of GPR37 during cell-cycle progression in HCC cells. We analyzed the cell cycle after serum starvation and after refeeding with serum. Huh7 cells were arrested in the G1 phase by serum deprivation for 72 h, and the G1 phase increased from 48.43% to 79.12% (Fig. 5A). On serum addition, the cells were released from the G1 phase and re-entered the S phase. As expected, the expression of GPR37 was decreased as early as 8 h after serum stimulation in Huh7 cell (Fig. 5B and C). The expressions of GPR37 and p27Kip1 were high in the G1 phase and low in the S phase, while the expressions of cyclin A and cyclin D were inversely diminished in synchronization with the increase in the expression of GPR37. These results indicated that GPR37 was downregulated during the progression of HCC cell proliferation. Interference of GPR37 expression promotes cell proliferation in HCC cell lines To further clarify the potential effects of GPR37 in HCC cells proliferation, we used siRNA to inhibit GPR37 expression in the Huh7 cells. Small interfering RNA for interference GPR37 expression and Western blot was used for result verification (Fig. 6A). The growth of cells transfected with GPR37-siRNA was significantly increased as compared with control siRNA (Fig. 6B). The expression of PCNA, a cell proliferation marker, was also upregulated (Fig. 6C). In addition, GPR37 knockdown also caused the upregulation of cyclin A and cyclin D in Huh7 cells (Fig. 6C). Then, at this stage, we determined the effects of GPR37 on cell cycle progression. FACS analysis of cell cycle distribution discovered that GPR37 knockdown induced S phase arrest, with a concomitant reduction in the number of cells in G1 phase as compared with control siRNA (Fig. 6C), recommending that GPR37 can restrain G1-S transition of the cell cycle and thus cell growth. These data demonstrated that GPR37 may be involved in the regulation of the G1/S transition, which might participate in HCC cell proliferation. The absence of the GPR37 reduces apoptosis via activation of Akt kinase Finally, we examined the effect of GPR37 on the apoptosis of HCC cells. Since suppression of GPR37 expression promoted cancer cell growth, we monitored survival of GPR37-siRNA – transfected cells as well as control RNAi – transfected cells after detachment and replanting in dishes coated with polyhydroxyethylmethacrylate. Huh7 cells were transiently transfected with GPR37-siRNA or control siRNA for 72 h, the apoptosis of cells transfected with GPR37-siRNA was significantly decelerated as compared with
Fig. 6. GPR37 siRNA promotes HCC cell proliferation. (A) Huh7 cells were transiently transfected with GPR37-siRNA or control siRNA as described above for 72 h and immunoblot analysis of GPR37, and GAPDH was performed. (B) Cell Counting Kit (CCK)-8 assay showing that interference GPR37 expression promoted the proliferation of the Huh7 cells. Data show means ± S.D. of triplicates from one experiment representative of three experiments performed.* P < 0.05. (C) Huh7 cells transfected with GPR37-siRNA or control siRNA were lysated, then analyzed by Western blot using antibodies against GPR37, PCNA, p27Kip1 , cyclin A, cyclin D and GAPDH. (D) 72 h after transfection, the transfected cells, as described above, the analysis of DNA content by FACS staining with PI. Details of the experiments are given in “Materials and Methods”.
control siRNA cells (Fig. 7A). Therefore, these results show that inhibition of GPR37 in Huh7 cells promotes growth and decreases cell apoptosis. To further investigate the mechanisms of apoptosis in the GPR37-siRNA transfected HuH7 cells, we used Western blot to detect the potential impact of GPR37 knockdown on the apoptosis signaling pathway. As shown in Fig. 7B, the level of phosphorylation of Akt Ser473 signaling was increased in Huh7 cells inhibition of GPR37 expression. These results indicate that suppression of GPR37 disrupts cell survival via activation of the phosphatidylinositol 3kinase-Akt signaling pathway in Huh7 cells. Discussion In 2012, Chevalier showed that the expression of G-proteincoupled estrogen receptor (GPER/GPR30) was overexpressed in human seminoma and promoted seminoma cell proliferation [20]. Mingxia demonstrated that G protein-coupled receptor 87 (GPR87) promotes growth and metastasis of CD133 cancer stem-like cells in hepatocellular carcinoma [21]. Here, we found the possible association of GPR37 with HCC, a member of the G protein-coupled receptors family. Our results demonstrated that GPR37 expression in poorly differentiated HCC samples decreased significantly, which indicated that under-expression of GPR37 had a potential relation with the severity of malignancy of HCC. And we discovered
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Fig. 7. Under-expression of GPR37 in Huh7 Cells decreases cell apoptosis. (A) HepG2 cells transiently transfected with GPR37 siRNA or control siRNA and cultured in a humidified incubator. At 72 h post-infection, cells were harvested and measured for apoptosis using Annexin V-FITC apoptosis detection kit followed by flow cytometry analysis. (B) Cells were lysed and analyzed by Western blot. GAPDH was used as loading control.
that under-expression of GPR37 was closely related to proliferation of HCC. As a result, low expression of GPR37 correlated significantly with a poor prognosis. The survival rate of patients with low expression of GPR37 was lower than that of other patients, which suggested that the degree of expression of GPR37 may have an effect on the survival rate. The study found that the expression level of GPR37 was significantly lower than that of adjacent non-tumorous tissues. Consistent with these findings, immunohistological analysis also confirmed the different GPR37 expression levels in the samples of HCC and adjacent non-tumorous tissues. The majority of the welldifferentiated HCC samples were positive for GPR37 expression, but GPR37 expression was much weaker in the moderately and poorly differentiated tumor samples. GPR37 expression gradually decreased as histological grading increased, which indicates that GPR37 might be a cancer suppressor gene of HCC. The malignant transformation is a complex process. Low expression of GPR37 may represent an important mechanism in the development of HCC. In addition, we evaluated the correlation between GPR37 and clinicopathological parameters, as well as the prognosis of patients. Investigating a series of 57 HCC tissues, we found that decreased GPR37 expression significantly correlated with the histological grade (P = 0.000), the levels of serum AFP (P = 0.002), and poor survival (P < 0.01). We also investigated the correlation between GPR37 expression and Ki-67 immunoreactivity, which is a useful marker of tumor proliferative activity [22]. A negative correlation between GPR37 and Ki-67 was observed in HCC. Furthermore, our Kaplan–Meier survival analysis revealed that low expression of GPR37 significantly correlated with a poor prognosis in the HCC patients (P < 0.01). These results show that GPR37 may serve as a new predictor of prognosis in HCC. We followed the expression of GPR37 during cell-cycle progression in HCC cells and found that GPR37 expression was downregulated during the G1- to S-phase transition. Furthermore, we discovered that the expression of cyclins A and D was increased during cell-cycle progression, whereas p27kip1 was decreased. The p27kip1 is one of the most important cell cycle inhibitors, and its expression has the character of a tumor suppressor gene. Loss of the p27kip1 protein expression may result in tumor development and/or progression [23]. A lot of results demonstrate that the function of p27kip1 is determined by its localization. Cytoplasmic mislocalization of p27kip1 may show when its nuclear import is impaired or its export facilitated, and this is believed to affect the growth-inhibitory function of p27kip1 [24]. GPR37 was located in the plasma membrane. P27kip1 expression was noted throughout HuH7 cells, but mainly in the nucleus. After serum deprivation for 72 h, we found that GPR37 protein expression preferred to
accumulate in the plasma membrane, and p27kip1 protein expression preferred to accumulate in the nucleus. After serum release for 12 h, GPR37 as well as p27kip1 recovered its normal expression pattern (data not shown). Owing to these results, we hypothesized that GPR37 affected the synthesis of p27kip1 in HCC cells by causing its accumulation in the plasma membrane. These results indicated that GPR37 has suppressor potential. Consequently, to determine whether GPR37 plays a role in the proliferation of HCC, we altered the expression of GPR37 in HCC cells. When we knocked down the expression of GPR37 in Huh7 cells using siRNA, we observed a significant increase in the Sphase population. Also, CCK-8 assay of Huh7 cells treated with siRNA exhibited a significant increase of the proliferation rate as compared with the control siRNA, which further supported the assumption that GPR37 was a tumor suppressor of HCC. At the same time, we tested the expression of mitotic cyclins (cyclins A and D) and p27kip1 in GPR37-deleted cells and found that depletion of GPR37 up-regulated mitotic cyclins and down-regulated p27kip1 expression, suggesting that depletion of GPR37 causes degradation of p27kip1 . These result suggest a role of GPR37 in the proliferation of HCC through stabilizing p27kip1 . Apoptosis is cell suicide. Normally, cells in vivo could prevent abnormal cell proliferation mediated by programmed cell death, which also means apoptosis, while tumor cells could escape from the killing system of our body by rapid proliferation and migration. The GPR37 is a substrate of parkin [25,26], Parkin is a protein-ubiquitin ligase E3 involved in the ubiquitination and proteasome-mediated protein degradation [27]. Degradation of Pael-R is also dependent on the Parkin-mediated ubiquitinproteasome system. Since overexpression of Pael-R can cause protein in the endoplasmic reticulum to unfold, ultimately leading to apoptosis [26,28], we will next determine the potential role of GPR37 in the apoptosis of hepatoma cells. The experiments presented here suggested that interfering GPR37 expression could significantly decrease cell apoptosis as compared with control in Huh7 cells. Although the exact mechanism by which GPR37 is directly involved in regulating apoptosis remains to be determined, our results suggested that GPR37 might participate in the progression of HCC cell proliferation/survival by the activation of phosphatidylinositol3-kinase-Akt (PI3K-Akt) signaling pathway. Further study is required to determine how GPR37 activates the phosphatidylinositol3-kinase-Akt signaling pathway. In summary, our study demonstrates for the first time that GPR37 was decreased in HCC, suggesting a role in its progression. We have indicated that GPR37 was negatively correlated with cell proliferation and demonstrated that disorder of GPR37 expression might contribute to the deregulation of cell cycle and association
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with the pathogenesis of HCC, indicating that GPR37 is a very promising novel target for HCC prevention and treatment. However, the exact function of GPR37 remains unclear. Further research is required to clarify the specific molecular mechanisms of GPR37 in the pathogenesis of HCC and the role of low expression of GPR37 in HCC cell proliferation. Conflict of interest All the authors declare no conflict of interest. References [1] D.M. Parkin, P. Pisani, J. Ferlay, Estimates of the worldwide incidence of eighteen major cancers in 1985, Int. J. Cancer 54 (1993) 594–606. [2] P. Pisani, D.M. Parkin, F. Bray, J. Ferlay, Estimates of the worldwide mortality from 25 cancers in 1990, Int. J. Cancer 83 (1999) 18–29. [3] M.I.F. Shariff, I.J. Cox, A.I. Gomaa, S.A. Khan, W. Gedroyc, S.D. Taylor-Robinson, Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics, Expert Rev. Gastroenterol. Hepatol. 3 (2009) 353–367. [4] W.Y. Lau, E.C. Lai, Hepatocellular carcinoma: current management and recent advances, Hepatobiliary Pancreat. Dis. Int. 7 (2008) 237–257. [5] M.W. Yu, C.J. Chen, J.C. Luo, P.W. Brandt-Rauf, W.P. Carney, R.M. Santella, Correlations of chronic hepatitis B virus infection and cigarette smoking with elevated expression of neu oncoprotein in the development of hepatocellular carcinoma, Cancer Res. 54 (1994) 5106–5110. [6] A. Psyrri, N. Arkadopoulos, M. Vassilakopoulou, V. Smyrniotis, G. Dimitriadis, Pathways and targets in hepatocellular carcinoma, Expert Rev. Anticancer Ther. 12 (2012) 1347–1357. [7] J.M. Llovet, J. Bruix, Molecular targeted therapies in hepatocellular carcinoma, Hepatology 48 (2008) 1312–1327. [8] H.B. El-Serag, K.L. Rudolph, Hepatocellular carcinoma: epidemiology and molecular carcinogenesis, Gastroenterology 132 (2007) 2557–2576. [9] S. Ueda, Y. Basaki, M. Yoshie, K. Ogawa, S. Sakisaka, M. Kuwano, M. Ono, PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib, Cancer Res. 66 (2006) 5346–5353. [10] H.J. Baek, S.C. Lim, K. Kitisin, W. Jogunoori, Y. Tang, M.B. Marshall, B. Mishra, T.H. Kim, K.H. Cho, S.S. Kim, L. Mishra, Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis, Hepatology 48 (2008) 1128–1137. [11] Y. Ding, B. Chen, S. Wang, L. Zhao, J. Chen, L. Chen, R. Luo, Over expression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients, Int. J. Cancer 124 (2009) 653–658. [12] D. Marazziti, A. Gallo, E. Golini, R. Matteoni, G.P. Tocchini-Valentini, Molecular cloning and chromosomal localization of the mouse Gpr37 gene encoding an orphan G-protein-coupled peptide receptor expressed in brain and testis, Genomics 53 (1998) 315–324.
[13] J.H. Dunham, R.C. Meyer, E.L. Garcia, R.A. Hall, GPR37 surface expression enhancement via N-terminal truncation or protein–protein interactions, Biochemistry 48 (2009) 10286–10297. [14] M. Rezgaoui, U. Susens, A. Ignatov, M. Gelderblom, G. Glassmeier, I. Franke, J. Urny, Y. Imai, R. Takahashi, H.C. Schaller, The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37, J. Cell Sci. 119 (2006) 542–549. [15] J. Ito, M. Ito, H. Nambu, T. Fujikawa, K. Tanaka, H. Iwaasa, S. Tokita, Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice, Cell Tissue Res. 338 (2009) 257–269. [16] B.D. Spiegelberg, H.E. Hamm, Roles of G-protein-coupled receptor signaling in cancer biology and gene transcription, Curr. Opin. Genet. Dev. 17 (2007) 40–44. [17] S.M. Thomas, N.E. Bhola, Q. Zhang, S.C. Contrucci, A.L. Wentzel, M.L. Freilino, W.E. Gooding, J.M. Siegfried, D.C. Chan, J.R. Grandis, Cross-talk between G protein-coupled receptor and epidermal growth factor receptor signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma, Cancer Res. 66 (2006) 11831–11839. [18] X. Xu, H. Yamamoto, M. Sakon, M. Yasui, C.Y. Ngan, H. Fukunaga, T. Morita, M. Ogawa, H. Nagano, S. Nakamori, M. Sekimoto, N. Matsuura, M. Monden, Overexpression of CDC25A phosphatase is associated with hypergrowth activity and poor prognosis of human hepatocellular carcinomas, Clin. Cancer Res. 9 (2003) 1764–1772. [19] C. Yu, K. Chen, H. Zheng, X. Guo, W. Jia, M. Li, M. Zeng, J. Li, L. Song, Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis, Carcinogenesis 30 (2009) 894–901. [20] N. Chevalier, A. Vega, A. Bouskine, B. Siddeek, J.F. Michiels, D. Chevallier, P. Fenichel, GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation, PLoS ONE 7 (2012) e34672. [21] M. Yan, H. Li, M. Zhu, F. Zhao, L. Zhang, T. Chen, G. Jiang, H. Xie, Y. Cui, M. Yao, J. Li, G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133(+) cancer stem-like cells in hepatocellular carcinoma, PLOS ONE 8 (2013) e61056. [22] C.D. Mann, C.P. Neal, G. Garcea, M.M. Manson, A.R. Dennison, D.P. Berry, Prognostic molecular markers in hepatocellular carcinoma: a systematic review, Eur. J. Cancer 43 (2007) 979–992. [23] R.V. Lloyd, L.A. Erickson, L. Jin, E. Kulig, X. Qian, J.C. Cheville, B.W. Scheithauer, p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers, Am. J. Pathol. 154 (1999) 313–323. [24] G. Baldassarre, B. Belletti, P. Bruni, A. Boccia, F. Trapasso, F. Pentimalli, M.V. Barone, G. Chiappetta, M.T. Vento, S. Spiezia, A. Fusco, G. Viglietto, Over expressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells, J. Clin. Investig. 104 (1999) 865–874. [25] Y. Imai, M. Soda, H. Inoue, N. Hattori, Y. Mizuno, R. Takahashi, An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin, Cell 105 (2001) 891–902. [26] R. Takahashi, Y. Imai, Pael receptor, endoplasmic reticulum stress, and Parkinson’s disease, J. Neurol. 250 (Suppl. 3) (2003) III25–III29. [27] K.K. Dev, H. van der Putten, B. Sommer, G. Rovelli, Part I: parkin-associated proteins and Parkinson’s disease, Neuropharmacology 45 (2003) 1–13. [28] H.Q. Wang, Y. Imai, H. Inoue, A. Kataoka, S. Iita, N. Nukina, R. Takahashi, Pael-R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss, J. Neurochem. 107 (2008) 171–185.
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Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
A low level of GPR37 is associated with human hepatocellular carcinoma progression and poor patient survival Fang Liu a,1 , Changlai Zhu a,1 , Xiaodong Huang b , Jing Cai b , Hua Wang b , Xinxiu Wang a , Song He b , Cheng Liu a , Xiaojing Yang a , Yixin Zhang b , Tianyi Zhang a,∗ a b
Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China Department of Pathology, Nantong University Cancer Hospital, Nantong, Jiangsu 226001, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 December 2013 Received in revised form 9 May 2014 Accepted 2 July 2014 Keywords: Human hepatocellular carcinoma (HCC) An orphan G protein-coupled receptor GPR37 Cell proliferation Pathogenesis
a b s t r a c t GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR). It has been reported that GPCRs play vital roles in the development and progression of cancer. To investigate the potential roles of GPR37 in hepatocellular carcinoma (HCC), expression of GPR37 was examined in human HCC samples. Immunohistochemistry and Western blot analyses were performed for GPR37 in 57 hepatocellular carcinoma samples. GPR37 expression was low in hepatocellular carcinoma as compared with the adjacent non-tumorous tissues. Clinicopathological analysis showed that GPR37 expression was significantly correlated with histological grade and the level of alpha fetal protein (AFP) (P = 0.000 and 0.002, respectively). The Kaplan–Meier survival curves revealed that decreasing GPR37 expression was associated with poor prognosis in HCC patients, while in vitro, following the release from serum starvation of HuH7 HCC cell, the expression of GPR37 was downregulated. In addition, the transient GPR37 knockdown by siRNA in HuH7 cells significantly decreased the apoptosis of hepatoma cells with activation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Our data suggest that GPR37 may play an important role in the pathogenesis of hepatocellular carcinoma by affecting the proliferation of H CC cells, and it could be a novel potential molecular therapy target for HCC. © 2014 Elsevier GmbH. All rights reserved.
Introduction Because of its high morbidity and poor prognosis, hepatocellular carcinoma (HCC) has long been known as one of the most lethal malignancies [1,2]. HCC is the third leading cause of cancer death, and its incidence in the world is increasing [3,4]. HCC carcinogenesis is a multistep process with many possible risk factors, including cirrhosis, hepatitis B and C viruses (HBV and HCV), aflatoxin exposure, alcoholic liver disease and possibly nonalcoholic liver disease, cigarette smoking and elevated endogenous testosterone in serum [5,6]. A large number of studies have shown that this molecular mechanism is involved in hepatocarcinogenesis [7–10]. Despite advances in therapy for HCC and modern surgical innovations, patient outcome has not substantially improved [11], and tumor mortality is very high. However, the molecular mechanism of HCC
∗ Corresponding author. Tel.: +86 051385511885. E-mail addresses: [email protected] (Y. Zhang), pro [email protected] (T. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.prp.2014.07.011 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
development and progression remains poorly understood. Hence, it is very important to identify novel and effective molecular markers which could provide some clues to understanding the mechanism of HCC and the development of new diagnostic strategies for treatment and prevention. GPR37 is a vertebrate G-protein-coupled orphan receptor [12], also known as the parkin-associated endothelin-like receptor (PaelR). Although GPR37 is predominantly expressed in the mammalian central nervous system, its expression and biological function still remain largely unknown [13]. The orphan G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, can mediate the extracellular environment to intracellular effectors of signal transduction [14], and regulate a variety of physiological and disease processes [15]. Many G protein-coupled receptors (GPCRs) have been found to play vital roles in the development and progression of cancer, including tumor growth and survival [16,17]. Therefore, we attempted to investigate the expression of GPR37 in the development and progression of HCC. The involvement of GPR37 in human tumor is not yet clearly understood. To solve this problem, GPR37 expression at protein levels in human hepatocellular carcinoma tissues and matched
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Table 1 GPR37 Ki67 expression and clinicopathological parameters in 57 HCC specimens. Parameters
Age (years) ≤45 >45 Gender Male Female Histological grade Well Mod Poor Metastasis Positive Negative Vein invasion Presence Absence Tumor size (cm) ≤5 >5 No. of tumor nodes Single Multiple/≥2 Capsular formation Presence Absence HBsAg (+) (−) Cirrhosis Positive Negative AFP (ng/ml) ≤50 >50
Total
GPR37
P
Low
High
24 33
14 23
10 10
45 12
31 6
17 19 21
Ki67
P
Low
High
0.271
12 17
12 16
0.562
14 6
0.189
20 9
25 3
0.059
5 12 20
12 7 1
0.000*
11 11 7
6 8 14
0.048*
8 49
4 33
4 16
0.284
5 24
3 25
0.373
14 43
7 30
7 13
0.153
9 20
5 23
0.199
27 30
18 19
9 11
0.506
14 15
13 15
0.55
35 22
25 12
10 10
0.155
16 13
19 9
0.239
40 17
25 12
15 5
0.394
21 8
19 9
0.465
43 14
27 10
16 4
0.402
21 6
22 8
0.409
45 12
28 3
17 9
0.321
21 8
24 4
0.183
27 30
23 14
4 16
0.002*
11 18
16 12
0.118
Abbreviations: HBsAg = hepatitis B surface antigen; AFP = alphafeto protein. Note. Statistical analyses were performed by the Pearson 2 test. * P < 0.05 was considered significant.
adjacent non-tumorous tissues was detected by Western blot and immunohistochemistry analyses. We also investigated its associations with clinicopathological characteristics and the prognostic implications. For the first time, our study found that GPR37 had an association with the proliferation of human hepatocellular carcinoma cells, which might be a valuable breakthrough point for experimental therapies of HCC.
Tissue samples were processed immediately after surgical removal. For histologic examination, all tumorous and surrounding non-tumorous tissue portions were fixed in formalin and embedded in paraffin. Protein was analyzed in eight snap-frozen tumorous and adjacent non-tumorous tissue samples stored at −80 ◦ C. Informed consent was obtained from all patients.
Immunohistochemistry Materials and methods Patients and tissue samples HCC tissues were obtained from 57 patients. All underwent hepatic surgical resection without postoperative systemic chemotherapy at the Surgery Department of the Affiliated Hospital of Nantong University. The diagnosis criteria for all patients in this study were confirmed by experienced pathologists through histological examination of H&E-stained biopsy sections; clinicopathologic and follow-up data were completely available. The main clinical and pathologic variables of the patients are shown in Table 1. Forty-five patients were men and 12 were women; their age ranged from 21 to 65 (mean = 47.19 years). Forty-three patients were positive for HBV surface antigen, 45 were positive for cirrhosis. Histological grades were classified as well differentiated (grade I; n = 17), moderately differentiated (grade II; n = 19), and poorly differentiated (grade III; n = 21). The followup time was 5 years, with a range of 1–80 months (median = 37 months). None of the patients had received postoperative adjuvant therapy.
Tissue sections (1 m) were cut, placed on APES-pretreated slides, deparaffinized, rehydrated through graded alcohol, and quenched in 3% hydrogen peroxide. Antigen retrieval was performed by microwave heating at high power (750 W) in 10 mM sodium citrate buffer (pH6.0) for three cycles of 5 min each. After blocking with normal serum for 1 h at room temperature, the sections were incubated overnight at 4 ◦ C with anti-human GPR37 rabbit polyclonal antibody (diluted 1:150; Cell Signaling Technology), anti-Ki67 mouse monoclonal antibody (diluted 1:100; clone 7B11; Zymed Laboratories, San Francisco, CA, USA). Negative control slides were also processed in parallel using a nonspecific immunoglobulin IgG (Sigma Chemical Co., St. Louis, MO) at the same concentration as the primary antibody. For the assessment of GPR37 and Ki-67, five high-power fields were randomly chosen, more than 500 cells were counted to determine the Labeling index (LI), which means that the percentage of immunostained cells is relative to the total number of cells [18]. The LI of GPR37 ranged from 7% to 87%. The mean percentage of positive cells was 47%. The LI of Ki-67 ranged from 4% to 71%. The mean percentage of positive cells was 43%. Tumors were scored as percentage of positive
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Fig. 1. GPR37 is low expressed in HCC. (A) Western blot analysis of whole-cell protein extracts prepared from eight representative paired samples of HCC tissues (T) and adjacent non-tumorous tissues (N). GAPDH was used as a control for protein load and integrity. (B) The bar chart demonstrates the ratio of GPR37 protein to GAPDH for the above by densitometry. The data are mean ± SEM (*P < 0.01, compared with adjacent non-tumorous tissues).
cells for each antigen. The GPR37 and Ki-67 immunostaining scores were calculated as both the percentage of positively stained tumor cells and the staining intensity. The percent positivity of GPR37 was scored as follows: 0 (70% positive tumor cells). The percent positivity of Ki67 was scored as: 0 (10% positive tumor cells); 1 (5–25% positive tumor cells); 2 (25–45% positive tumor cells); 3 (45–60%positive tumor cells); 4 (>60% positive tumor cells). Staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained), or “3” (strongly stained). The immunostaining score was calculated as the percentage positive score × the staining intensity score and ranged from 0 to12. A score of 0 was considered negative; 1–4 was considered weak; 5–9 was considered moderate; and 10–12 was considered strong. For statistical analysis, 0–4 were counted as low expression, while 5–12 were counted as overexpression [19].
100 g/ml streptomycin in 5% CO2 at 37 ◦ C. For cell cycle analysis, cells were fixed in 70% ethanol for 1 h at 4 ◦ C and then incubated with 1 mg/ml RNase A for 30 min at 37 ◦ C. Subsequently, cells were stained with propidiumiodide (50 g/mL PI) (Becton Dickinson, San Jose, CA) in PBS, 0.5% Tween-20, and analyzed with a Becton Dickinson flow cytometer BD FACScan (San Jose, CA) and Cell Quest acquisition and analysis programs. Cell proliferation assay Cell proliferation was measured using the CCK-8 (cell counting Kit-8) assay following the manufacturer’s instructions. Briefly, cells were seeded at 2 × 104 /well in a volume of 100 l in 96-well cell culture cluster (Corning Inc., Corning, NY) and grown overnight. Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) reagents were added to a subset of wells under different treatments and in cubated for 1 h at 37 ◦ C, after which absorbance was quantified on an Automated plate reader at a test wavelength of 490 nm at different times.
Western blot analysis Plasmid constructs siRNA and transfection Tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M Tris HCl pH7.5, 1%Triton X-100, 1% Nonidet p-40 (NP-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, leupeptin 10 g/ml, aprotinin 10 g/ml, and 1 mM PMSF, then centrifuged at 10,000 × g for 30 min to collect the supernatant liquid. Protein concentrations were determined with a Bio-Rad protein assay (BioRad, Hercules, CA, USA). The total cellular protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20. The antibodies used in this study included: anti-GPR37 (1:1000; Santa Cruz Biotechnology); GAPDH (1:1000; Sigma); anti-p27Kip1 (1:500; Santa Cruz Biotechnology); anti-PCNA (1:500; Santa Cruz Biotechnology); anti-cyclin D (1:500; Santa Cruz Biotechnology); anti-cyclin A (1:500; Santa Cruz Biotechnology). Blots were washed three times in PBS buffer each for 5 min, followed by incubation with the appropriate horseradish peroxidase-linked secondary antibodies. Then, the membrane was developed by the ECL detection systems. The experiments were carried out on three separate occasions.
The siRNA species for the GPR37 knockdown purchased from Biomics (Nantong, China) were synthesized to target the following cDNA sequences: GPR37-siRNA, sense =5 -GGUGGGAGCUCUAUUGUUAdTdT-3 and anti-sense = 5 UAACAAUGAGCUCCCACCdTdT-3 ; and negative control (NC), sense = 5 -UUCUCCGAACGUGUCACGUdTdT-3 and anti-sense = 5 ACGUGACACGUUCGGAGAAdTdT-3 . Cell transfection was performed with SuperFectin according to the manufacturer’s instructions. Apoptosis assay The apoptosis assays were performed at 72 h after the cells were transfected with 20 mM siGPR37 or NC. The Huh7 cells transfected with siGPR37 or NC were washed three times in ice-cold PBS, resuspended in 100 l of 1× Binding Buffer and incubated with Annexin V-FITC (Bestbio, China) for 15 min at 4 ◦ C in the dark, according to the manufacturer’s instructions. After staining, the cells were incubated with propidium iodide for 5 min at 4 ◦ C in the dark and then analyzed using a flow cytometer (Beckman, USA).
Cell culture and cell-cycle analysis
Statistical analysis
Huh7, a human hepatocarcinoma cell line, was obtained from our laboratory and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 u/ml penicillin, and
Statistical analysis was performed using the PASW statistics 18 software package. The association between Ki-67 and GPR37 expression and clinicopathological features was analyzed using the
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Fig. 2. The analysis of GPR37 expression in the primary hepatocellular carcinoma tissues with immunohistochemistry. (A) and (F) Immunostaining of an HCC tumor and the adjacent non-tumorous area. (B) and (G) Normal liver tissue distant from the tumor. (C) and (H) Well-differentiated HCC, strongly positive GPR37 expression. (D) and (I) Moderately differentiated HCC, weakly positive GPR37 expression. (E) and (J) poorly differentiated HCC, no GPR37 expression. N: adjacent non-tumor tissue; T: tumor tissue (A–E with 200× magnification; F–J with 400× magnification).
2 test. As the data were not normally distributed, Ki-67 and GPR37 expressions were analyzed by the Spearman rank correlation test. Overall survival curves were calculated with the Kaplan–Meier method and were analyzed with the log-rank test. A Cox proportional-hazards analysis was used in univariate and multivariate analyses to explore the effects of GPR37 expression and HCC clinicopathological variables on survival. The results were expressed as mean ± SD and analyzed using the Mann–Whitney U test. Differences were considered significant at P < 0.05. Results Expression of GPR37 protein in HCC, adjacent non-tumorous tissue The expression of GPR37 was examined with immunoblotting methods in eight paired adjacent non-tumorous tissue and HCC biopsy samples. The example of Western blot analysis is shown in Fig. 1. GPR37 expression was found to be lower in HCC tissue (T) than in adjacent non-tumorous tissues (N). The amount of GAPDH,
a housekeeping protein, was demonstrated to be rather constant among the samples.
Immunohistochemical analysis of GPR37 expression in HCC clinical samples and its relationship to clinicopathological parameters Expression of GPR37 was studied in 57 HCC specimens by immunohistochemical staining. Following the different histological grades, representative examples of reactivity for GPR37 are shown in Fig. 2. In the positive samples, GPR37 was detected in the plasma membrane of the cells (Fig. 2C). Its LIs ranged from 7% to 87%. The mean percentage of positive cell was 47%. GPR37 expression was also detected in normal liver tissues distant from the tumors (Fig. 2B). It was clear that the expression of GPR37 gradually decreased with histological grade from well-differentiated to poorly differentiated. Well-differentiated samples showed strongly positive GPR37 expression (Fig. 2C and H), moderately-differentiated samples showed weakly positive expression (Fig. 2D and I), and the most poorly differentiated
Fig. 3. Analysis of GPR37 and Ki67 expression. (A) Paraffin-embedded tissue sections were stained with antibodies against GPR37 and Ki67 counterstained with hematoxylin. (B, D) Low GPR37 expression in HCC tissues, whereas Ki67 staining showed the level was high in the same HCC tissues (SP × 400). N: adjacent non-tumorous tissues; T: tumor tissues (400× magnification). (B) The relationship between the Ki-67 proliferation index and GPR37 expression in HCC. Scatterplot of Ki67 versus GPR37 with regression line showing correlation using Spearman’s coefficient (P < 0.01).
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samples often showed no detectable GPR37 expression (Fig. 2E and J). The GPR37 expression with clinicopathologic variables is given in Table 1. Thirty-seven (64.9%) samples showed low GPR37 expression and 20 (35.1%) samples exhibited high GPR37 expression (Table 1). GPR37 expression was not significantly associated with age, gender, metastasis, vein invasion, tumor size, tumor nodes, capsular formation, HBsAg or cirrhosis, but it significantly correlated with histological grade (P = 0.000) and serum AFP (P = 0.002) (Table 1). The expression of GPR37 is associated with the proliferation marker Ki-67
Fig. 4. The Kaplan–Meier survival analysis of the primary HCC patients (n = 57) with high GPR37 expression (n = 20) and low GPR37 expression (n = 37) after surgical resection. Based on their GPR37 immunostaining scores, the HCC patients were divided into low-GPR37 expression (score < 5) and high-GPR37 expression (score ≥ 5) groups. The survival rate of the patients in the low-GPR37 group was significantly lower than that of the patients in the high-GPR37 group (P < 0.01 for the log-rank test).
We performed immunohistochemical staining to evaluate the expression of GPR37 and the proliferation marker Ki-67 in the same cohort of HCC tissues and adjacent non-tumorous tissues. Representative examples of reactivity for GPR37 and Ki-67 are shown in Fig. 3. There was low or no GPR37 expression (score < 5) in HCC tissues (Fig. 3A and B), while Ki67 staining showed a high level in the same HCC tissues (Fig. 3A and D). Ki-67 expression in HCC was scored as positive when expression was strong in the nuclei. Its LIs ranged from 4% to 71%. The mean percentage of positive cells was 43%. GPR37 and Ki-67 expressions in HCC were scored as positive (score ≥ 5). The association of Ki-67 and GPR37 expression with
Fig. 5. The expression of GPR37 and cell cycle-related molecules in proliferating HCC cells. (A) Flow cytometry quantification of cell cycle progression in Huh7 cells. Normal cells, cells that were serum starved for 72 h, and then addition of medium containing 10% FBS for the indicated time points. (B) Huh7 cells were serum starved for 72 h and upon serum releasing, cell lysates were prepared and analyzed by Western blot using antibodies directed against GPR37, p27Kip1 , cyclin A and cyclin D. GAPDH was used as a control for protein load and integrity. (C) The bar chart below demonstrates the ratio of GPR37, p27Kip1 , cyclin A and cyclin D protein to GAPDH for each time point by densitometry. The data are means ± SEM (n = 3, *,#,&,$ P < 0.01, compared with control: S72 h). S: serum starvation; R: serum release.
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clinicopathologic variables is summarized in Table 1. Furthermore, in most specimens, the proportion of GPR37-positive tumor cells was opposite to the proportion of Ki-67-positive tumor cells. A negative correlation between GPR37 expression and Ki-67 on the basis of proliferative activity was found (P < 0.01; Fig. 3B). Correlation between expression of GPR37 and survival rates in patients with HCC The prognostic value of GPR37 for overall survival in HCC patients was evaluated by comparing the patients with high and low GPR37 expression. Using the Kaplan–Meier analysis, low GPR37 expression was significantly associated with poor prognosis. The HCC patients with low GPR37 expression obviously had lower overall survival rates than those with high GPR37 expression (P < 0.01; Fig. 4). Expression of GPR37 in proliferating HCC cells Based on our results, we further detected the expression of GPR37 during cell-cycle progression in HCC cells. We analyzed the cell cycle after serum starvation and after refeeding with serum. Huh7 cells were arrested in the G1 phase by serum deprivation for 72 h, and the G1 phase increased from 48.43% to 79.12% (Fig. 5A). On serum addition, the cells were released from the G1 phase and re-entered the S phase. As expected, the expression of GPR37 was decreased as early as 8 h after serum stimulation in Huh7 cell (Fig. 5B and C). The expressions of GPR37 and p27Kip1 were high in the G1 phase and low in the S phase, while the expressions of cyclin A and cyclin D were inversely diminished in synchronization with the increase in the expression of GPR37. These results indicated that GPR37 was downregulated during the progression of HCC cell proliferation. Interference of GPR37 expression promotes cell proliferation in HCC cell lines To further clarify the potential effects of GPR37 in HCC cells proliferation, we used siRNA to inhibit GPR37 expression in the Huh7 cells. Small interfering RNA for interference GPR37 expression and Western blot was used for result verification (Fig. 6A). The growth of cells transfected with GPR37-siRNA was significantly increased as compared with control siRNA (Fig. 6B). The expression of PCNA, a cell proliferation marker, was also upregulated (Fig. 6C). In addition, GPR37 knockdown also caused the upregulation of cyclin A and cyclin D in Huh7 cells (Fig. 6C). Then, at this stage, we determined the effects of GPR37 on cell cycle progression. FACS analysis of cell cycle distribution discovered that GPR37 knockdown induced S phase arrest, with a concomitant reduction in the number of cells in G1 phase as compared with control siRNA (Fig. 6C), recommending that GPR37 can restrain G1-S transition of the cell cycle and thus cell growth. These data demonstrated that GPR37 may be involved in the regulation of the G1/S transition, which might participate in HCC cell proliferation. The absence of the GPR37 reduces apoptosis via activation of Akt kinase Finally, we examined the effect of GPR37 on the apoptosis of HCC cells. Since suppression of GPR37 expression promoted cancer cell growth, we monitored survival of GPR37-siRNA – transfected cells as well as control RNAi – transfected cells after detachment and replanting in dishes coated with polyhydroxyethylmethacrylate. Huh7 cells were transiently transfected with GPR37-siRNA or control siRNA for 72 h, the apoptosis of cells transfected with GPR37-siRNA was significantly decelerated as compared with
Fig. 6. GPR37 siRNA promotes HCC cell proliferation. (A) Huh7 cells were transiently transfected with GPR37-siRNA or control siRNA as described above for 72 h and immunoblot analysis of GPR37, and GAPDH was performed. (B) Cell Counting Kit (CCK)-8 assay showing that interference GPR37 expression promoted the proliferation of the Huh7 cells. Data show means ± S.D. of triplicates from one experiment representative of three experiments performed.* P < 0.05. (C) Huh7 cells transfected with GPR37-siRNA or control siRNA were lysated, then analyzed by Western blot using antibodies against GPR37, PCNA, p27Kip1 , cyclin A, cyclin D and GAPDH. (D) 72 h after transfection, the transfected cells, as described above, the analysis of DNA content by FACS staining with PI. Details of the experiments are given in “Materials and Methods”.
control siRNA cells (Fig. 7A). Therefore, these results show that inhibition of GPR37 in Huh7 cells promotes growth and decreases cell apoptosis. To further investigate the mechanisms of apoptosis in the GPR37-siRNA transfected HuH7 cells, we used Western blot to detect the potential impact of GPR37 knockdown on the apoptosis signaling pathway. As shown in Fig. 7B, the level of phosphorylation of Akt Ser473 signaling was increased in Huh7 cells inhibition of GPR37 expression. These results indicate that suppression of GPR37 disrupts cell survival via activation of the phosphatidylinositol 3kinase-Akt signaling pathway in Huh7 cells. Discussion In 2012, Chevalier showed that the expression of G-proteincoupled estrogen receptor (GPER/GPR30) was overexpressed in human seminoma and promoted seminoma cell proliferation [20]. Mingxia demonstrated that G protein-coupled receptor 87 (GPR87) promotes growth and metastasis of CD133 cancer stem-like cells in hepatocellular carcinoma [21]. Here, we found the possible association of GPR37 with HCC, a member of the G protein-coupled receptors family. Our results demonstrated that GPR37 expression in poorly differentiated HCC samples decreased significantly, which indicated that under-expression of GPR37 had a potential relation with the severity of malignancy of HCC. And we discovered
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Fig. 7. Under-expression of GPR37 in Huh7 Cells decreases cell apoptosis. (A) HepG2 cells transiently transfected with GPR37 siRNA or control siRNA and cultured in a humidified incubator. At 72 h post-infection, cells were harvested and measured for apoptosis using Annexin V-FITC apoptosis detection kit followed by flow cytometry analysis. (B) Cells were lysed and analyzed by Western blot. GAPDH was used as loading control.
that under-expression of GPR37 was closely related to proliferation of HCC. As a result, low expression of GPR37 correlated significantly with a poor prognosis. The survival rate of patients with low expression of GPR37 was lower than that of other patients, which suggested that the degree of expression of GPR37 may have an effect on the survival rate. The study found that the expression level of GPR37 was significantly lower than that of adjacent non-tumorous tissues. Consistent with these findings, immunohistological analysis also confirmed the different GPR37 expression levels in the samples of HCC and adjacent non-tumorous tissues. The majority of the welldifferentiated HCC samples were positive for GPR37 expression, but GPR37 expression was much weaker in the moderately and poorly differentiated tumor samples. GPR37 expression gradually decreased as histological grading increased, which indicates that GPR37 might be a cancer suppressor gene of HCC. The malignant transformation is a complex process. Low expression of GPR37 may represent an important mechanism in the development of HCC. In addition, we evaluated the correlation between GPR37 and clinicopathological parameters, as well as the prognosis of patients. Investigating a series of 57 HCC tissues, we found that decreased GPR37 expression significantly correlated with the histological grade (P = 0.000), the levels of serum AFP (P = 0.002), and poor survival (P < 0.01). We also investigated the correlation between GPR37 expression and Ki-67 immunoreactivity, which is a useful marker of tumor proliferative activity [22]. A negative correlation between GPR37 and Ki-67 was observed in HCC. Furthermore, our Kaplan–Meier survival analysis revealed that low expression of GPR37 significantly correlated with a poor prognosis in the HCC patients (P < 0.01). These results show that GPR37 may serve as a new predictor of prognosis in HCC. We followed the expression of GPR37 during cell-cycle progression in HCC cells and found that GPR37 expression was downregulated during the G1- to S-phase transition. Furthermore, we discovered that the expression of cyclins A and D was increased during cell-cycle progression, whereas p27kip1 was decreased. The p27kip1 is one of the most important cell cycle inhibitors, and its expression has the character of a tumor suppressor gene. Loss of the p27kip1 protein expression may result in tumor development and/or progression [23]. A lot of results demonstrate that the function of p27kip1 is determined by its localization. Cytoplasmic mislocalization of p27kip1 may show when its nuclear import is impaired or its export facilitated, and this is believed to affect the growth-inhibitory function of p27kip1 [24]. GPR37 was located in the plasma membrane. P27kip1 expression was noted throughout HuH7 cells, but mainly in the nucleus. After serum deprivation for 72 h, we found that GPR37 protein expression preferred to
accumulate in the plasma membrane, and p27kip1 protein expression preferred to accumulate in the nucleus. After serum release for 12 h, GPR37 as well as p27kip1 recovered its normal expression pattern (data not shown). Owing to these results, we hypothesized that GPR37 affected the synthesis of p27kip1 in HCC cells by causing its accumulation in the plasma membrane. These results indicated that GPR37 has suppressor potential. Consequently, to determine whether GPR37 plays a role in the proliferation of HCC, we altered the expression of GPR37 in HCC cells. When we knocked down the expression of GPR37 in Huh7 cells using siRNA, we observed a significant increase in the Sphase population. Also, CCK-8 assay of Huh7 cells treated with siRNA exhibited a significant increase of the proliferation rate as compared with the control siRNA, which further supported the assumption that GPR37 was a tumor suppressor of HCC. At the same time, we tested the expression of mitotic cyclins (cyclins A and D) and p27kip1 in GPR37-deleted cells and found that depletion of GPR37 up-regulated mitotic cyclins and down-regulated p27kip1 expression, suggesting that depletion of GPR37 causes degradation of p27kip1 . These result suggest a role of GPR37 in the proliferation of HCC through stabilizing p27kip1 . Apoptosis is cell suicide. Normally, cells in vivo could prevent abnormal cell proliferation mediated by programmed cell death, which also means apoptosis, while tumor cells could escape from the killing system of our body by rapid proliferation and migration. The GPR37 is a substrate of parkin [25,26], Parkin is a protein-ubiquitin ligase E3 involved in the ubiquitination and proteasome-mediated protein degradation [27]. Degradation of Pael-R is also dependent on the Parkin-mediated ubiquitinproteasome system. Since overexpression of Pael-R can cause protein in the endoplasmic reticulum to unfold, ultimately leading to apoptosis [26,28], we will next determine the potential role of GPR37 in the apoptosis of hepatoma cells. The experiments presented here suggested that interfering GPR37 expression could significantly decrease cell apoptosis as compared with control in Huh7 cells. Although the exact mechanism by which GPR37 is directly involved in regulating apoptosis remains to be determined, our results suggested that GPR37 might participate in the progression of HCC cell proliferation/survival by the activation of phosphatidylinositol3-kinase-Akt (PI3K-Akt) signaling pathway. Further study is required to determine how GPR37 activates the phosphatidylinositol3-kinase-Akt signaling pathway. In summary, our study demonstrates for the first time that GPR37 was decreased in HCC, suggesting a role in its progression. We have indicated that GPR37 was negatively correlated with cell proliferation and demonstrated that disorder of GPR37 expression might contribute to the deregulation of cell cycle and association
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