Overexpression of orphan G-protein–coupled receptor, Gpr49, in human hepatocellular carcinomas with β-catenin mutations

Overexpression of Orphan G-Protein–Coupled Receptor, Gpr49, in Human Hepatocellular Carcinomas With ␤-Catenin Mutations Yoshiya Yamamoto,1,2 Michiie Sakamoto,1 Gen Fujii,1 Hitomi Tsuiji,1 Kengo Kanetaka,1 Masahiro Asaka,2 and Setsuo Hirohashi1 To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in ␤-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant ␤-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with ␤-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC. (HEPATOLOGY 2003;37:528-533.)

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epatocellular carcinoma (HCC) is one of the most common tumors worldwide, and its incidence is unlikely to be reduced in the near future in spite of much effort. In the geographic areas in which the incidence of HCC is high, it occurs most frequently after chronic liver disease resulting from hepatitis virus infection.1 Therefore, development of antiviral therapy is expected to contribute significantly to a decrease in Abbreviations: HCC, hepatocellular carcinoma; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; mRNA DD-PCR, mRNA differential display polymerase chain reaction; GAPDH, glyceraldehydephosphate dehydrogenase; TSHR, thyroid-stimulating hormone receptor; FSHR, follicle-stimulating hormone receptor; LHR, luteinizing hormone receptor. From the 1Pathology Division, National Cancer Center Research Institute, Tokyo, Japan; 2Third Department of Internal Medicine, Hokkaido University Faculty of Medicine, Sapporo, Japan. Received March 15, 2002; accepted August 11, 2002. Supported in part by a Grant-in-Aid for Research on Human Genome and Gene therapy and a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan. Y.Y. and K.K. are the recipients of Research Resident Fellowships from the Foundation for Promotion of Cancer Research in Japan. H.T. is a domestic research fellow supported by Japan Science and Technology Corporation. Address reprint requests to: Setsuo Hirohashi, M.D., Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 1040045, Japan. E-mail: [email protected]; fax: (81) 3-3248-2463. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3703-0008$30.00/0 doi:10.1053/jhep.2003.50029 528

the incidence of HCC. At the same time, it is very important to elucidate the molecular mechanisms of hepatocarcinogenesis and develop specific measures for prevention. Various genetic alterations in HCC have been reported, but much remains unknown. We have attempted to elucidate gene alterations in HCC by using mRNA differential display polymerase chain reaction (mRNA DD-PCR) to investigate the differences in mRNA expression in HCC cell lines. For example, we successfully cloned DRH1 as a novel molecule down-regulated in advanced HCC,2 although the direct role of DRH1 in the progression of HCC has not been elucidated. We report here that Gpr49,3,4 which belongs to the glycoprotein hormone receptor subfamily, is markedly up-regulated in HCCs carrying ␤-catenin mutations. It has recently been established that ␤-catenin is involved in carcinogenesis through activation of the Wnt-signaling pathway,5 and that several genes, such as c-myc and cyclin D1, are the downstream targets in this pathway.6-8 Our result suggests that Gpr49 is one of these downstream genes.

Materials and Methods Patients and Cell Lines. We analyzed 38 primary HCCs and their corresponding noncancerous liver tissues

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Table 1. Relationship Between Gpr49 mRNA Expression and Clinicopathologic Features and ␤-Catenin Mutation in 38 Patients With HCC

Features

Age (y) ⱕ 65 ⬎ 65 Sex Male Female Virus HBs Ag HCV Ab HBs Ag (⫺), HCV Ab (⫺) Serum AFP (ng/mL) ⱕ 100 ⬎ 100 Tumor size (cm) ⱕ3 ⬎3 Differentiation Well Moderate Poor Macroscopic type† 1 2 3 Massive Vascular tumor spread (and or intrahepatic metastasis) Present Absent Noncancerous liver tissue Normal CH LC ␤-catenin mutation in HCC Positive Negative

Gpr49 Overexpression* n (%)

P Value

.76 18 20

8 (44.4) 10 (50)

31 7

13 (41.9) 5 (71.4)

9 24 5

3 (33.3) 12 (50) 3 (60)

23 15

10 (43.5) 8 (53.3)

14 24

6 (42.9) 12 (50)

3 29 6

2 (66.6) 15 (51.7) 1 (17.7)

11 18 8 1

4 (36.4) 9 (50) 4 (50) 1 (100)

24 14

12 (50) 6 (42.9)

4 16 18

3 (75) 9 (56.3) 6 (33.3)

16 22

14 (87.5) 4 (18.2)

.22

.58

.74

.75

.23

.63

.74

.21

⬍.001

Abbreviations: HBs Ag, hepatitis B surface antigen; HCV Ab, hepatitis C antibody; AFP, alpha-fetoprotein. *Is defined as T/N ratio (Gpr49/GAPDH in T divided by Gpr49/GAPDH in N) over 3. †Is subclassified into single nodule (type 1), single nodule with extranodular growth (type 2), and confluent multinodule (type 3). Massive type means a large tumor that occupies one segment or more with unclear and irregular margin.

obtained from patients (mean age, 62.6 years; range, 20 to 76 years) who underwent surgical resection at the National Cancer Center Hospital, Japan, from July, 1998 to June, 1999. Normal liver tissues were obtained from patients with colorectal cancer with liver metastasis. Specimens were collected from surgically resected materials, and immediately treated and stored until use, as described previously.2 Identification of the HCCs, noncancerous liver tissues, and normal liver tissues was confirmed histologically by 2 independent pathologists. The main clinicopathologic features are presented in Table 1. The histopathologic grade of tumor

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differentiation was defined, as described previously.2 Macroscopic types of HCC were subclassified into single nodule (type 1), single nodule with extranodular growth (type 2), and confluent multinodule (type 3). In addition, the large HCC that occupies 1 segment or more with unclear and irregular margin was classified into massive type. Vascular tumor spread was defined to include both tumor thrombus in the portal vein and intrahepatic metastasis. In cases of multicentric HCC, the largest nodule was representatively used in real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The human HCC cell lines PLC/PRF/5 and HepG2 were obtained from the American Type Culture Collection. KYN-1,9 KYN-2,10 and KIM-111 were kindly provided by Dr. M. Kojiro (Kurume University, Kurume, Japan). Li7,12 Li21, Li24, tPH5T, and its corresponding nonneoplastic liver cell line tPH5CH13 and the mouse hepatocyte MHT were established in our laboratory. tPH5T, tPH5CH, and MHT were immortalized by introduction of SV40 large T antigen. All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 ␮g/mL streptomycin. RNA Preparation and mRNA DD-PCR. Total RNA was isolated with an acid guanidinium thiocyanatephenol-chloroform method.14 mRNA DD-PCR was performed on several HCC cell lines, as described previously.2 We obtained a fragment that was differentially expressed among HCC cell lines, and analyzed the nucleotide sequence with the BLAST programs of the National Center of Biotechnology Institute. Northern Blot Analysis. Total RNA (15 ␮g) was separated on a 1.0% agarose formaldehyde denaturing gel, transferred to Hybond-N⫹ (Amersham) by capillary blotting, and hybridized with DNA probes amplified by PCR for Gpr49 and glyceraldehydephosphate dehydrogenase (GAPDH), as described previously.2 Cloning of Gpr49. A cDNA library was constructed for the PLC/PRF/5 cell line and then screened, as described previously.2 The nucleotide sequence of positive clones was analyzed as described above. Real-Time Quantitative RT-PCR Analysis. We used the SYBR Green PCR Core Reagents kit (PerkinElmer Applied Biosystems), as described previously.15 cDNA of each tissue sample and cell line was prepared from DNase-treated total RNA by using oligo-dT primer and AMV Reverse Transcriptase XL (TaKaRa),2 as described previously. Using Primer Express software (Perkin-Elmer), we designed the following primers for Gpr49: 5⬘-CTCGTGGCCCCCTACTTC-3⬘ (GSP1: forward), 5⬘-ATGAATTTCAATGTGAAAACACGTT-3⬘ (GSP2: forward), 5⬘-GAGGATCTGGTGAGCCTGAGAA-3⬘ (GSP3: reverse), and 5⬘-CATAAGTGATGCTGGAG-

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Fig. 1. Cloning of full-length Gpr49. (A) Published Gpr49 cDNA (accession no. NM-003667; upper) and clone 141, obtained by mRNA DD-PCR and cDNA library screening (middle). The positions of 4 genespecific primers (GSPs) for RT-PCR analysis are shown (lower). A closed box indicates the region of the deduced open reading frame. (B) Agarose gel electrophoresis of the PCR product amplified by using GSP1 and GSP4. (C and D) Expression of Gpr49 in HCC cell lines and a noncancerous liver cell line. Northern blot analysis shows Gpr49 expression as a 4.4-kb band. (C) GAPDH is used as a control for the amount of RNA. Real-time quantitative RT-PCR analysis shows expression levels normalized to that of GAPDH mRNA in each sample (D). Lane 1, PLC/ PRF/5; Lane 2, KYN-2; Lane 3, Li7; Lane 4, KIM-1; Lane 5, HepG2; Lane 6, Li21; Lane 7, Li24; Lane 8, tPH5T; Lane 9, tPH5CH; Lane 10, KYN-1.

CTGGTAA-3⬘ (GSP4: reverse) (Fig. 1A), and for FEX,16 which is the mouse homologue of Gpr49, 5⬘-GAGTCAACCCAAGCCTTAGTATCC-3⬘ (GSP5: forward), and 5⬘-CATGGGACAAATGCAACTGAAG-3⬘ (GSP6: reverse). GSP2, GSP3, GSP5, and GSP6 were used for realtime quantitative RT-PCR. We used equivalent amount of cDNA sample which was derived from 50 ng total RNA for each PCR reaction. The inflextion of amplification of GAPDH was achieved at from 26 to 28 cycles in all samples. To standardize the amount of RNA and obtain normalized value of Gpr49 expression, we quantified the expression of GAPDH in each sample and the amount of expression of Gpr49 was divided by that of the GAPDH in each sample. To quantify the up-regulation of Gpr49 in HCC, we calculated the ratio of expression level in the tumor to that in the corresponding noncancerous liver tissue (T/N ratio; Gpr49/ GAPDH in T divided by Gpr49/GAPDH in N). We defined a T/N ratio ⬎ 3 to be overexpression. PCR and Sequencing of ␤-Catenin. PCR amplification of exon 3 of ␤-catenin, which contains 4 potential sites for phosphorylation of glycogen synthase kinase-3␤ (GSK-3␤), was carried out on the cDNAs from each sample. We used a ␤⫺catenin exon 2 forward primer (5⬘CCAGCGTGGACAATGGCTAC-3⬘), and a ␤⫺catenin exon 4 reverse primer (5⬘-TGAGCTCGAGTCATTGCATAC-3⬘). After confirmation of the PCR products by 2% agarose gel electrophoresis, we sequenced in both directions with the primers used for PCR.

Construction of Mutant ␤-Catenin and Retroviral Infection. Wild-type ␤-catenin cDNA containing the entire coding region was amplified by PCR and subcloned into pBluescript (Stratagene). Mutant ␤-catenin, with both Thr-41 and Ser-45 at the amino-terminal GSK-3␤ phosphorylation site changed to alanine residues, was constructed by using QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s recommendations. Subsequently, wild-type ␤-catenin and mutant ␤-catenin were subcloned into the retroviral expression vector pLNCX2 (Clontech). Recombinant retroviruses were generated by cotransfecting each above-mentioned construct and pCL-Eco17 into 293 cells with FuGENE 6 Transfection Reagents (Boehringer Mannheim, Indianapolis, IN). At 24 hours after transfection, the retrovirus-containing supernatant of the 293 cells was collected, filtered through a sterile 0.45-␮m filter, and transferred onto the MHT cells after addition of 8 ␮g/mL polybrane. At 24 hours after infection, infecting medium was replaced by 1,640 medium plus 10% FCS. At 48 hours after infection, cells were split into medium containing 500 ␮g/mL geneticin. Total RNA was extracted from resistant clones and analyzed by real-time quantitative RT-PCR. Statistical Analysis. We used an unpaired t test to analyze differences in Gpr49 expression levels between HCC and the corresponding noncancerous liver tissues, and the ␹2 test and Fisher’s exact test to analyze the association between Gpr49 expression and clinicopathologic parameters, with

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StatView (Version 5.0) software (Abacus Concepts, Berkeley, CA). P-values below .05 were judged to be significant.

Results Cloning of Full-Length Gpr49. Using mRNA DDPCR, we isolated clone 141, which was up-regulated in PLC/PRF/5 and HepG2. Because the sequence analysis of clone 141 gave little information, we screened a cDNA library and obtained a 2,324-bp clone. The 846-bp sequence at the 5⬘-end of this clone had 100% identity with orphan G-protein-coupled receptor (accession no. NM-003667) by BLAST search (Fig. 1A). This gene also is registered as Gpr49 in the Unigene database. The identity of the clone obtained by cDNA library screening with Gpr49 was confirmed by PCR amplification using GSP1 and GSP4 as primers (Fig. 1B). The reproducibility of Gpr49 expression in HCC cell lines was ascertained by Northern blot analysis and real-time quantitative RTPCR analysis using GSP2 and GSP3 (Fig. 1C, 1D). Gpr49 mRNA Expression in HCC. Real-time quantitative RT-PCR analysis was used to examine Gpr49 mRNA expression in surgical specimens of HCC. As shown in Figure 2, some HCCs showed marked up-regulation of Gpr49. By contrast, noncancerous liver and normal liver showed low levels of Gpr49 expression. The average expression level in HCC (Gpr49/GAPDH) was significantly higher than that in noncancerous liver (0.49 ⫾ 0.11 vs. 0.06 ⫾ 0.01; P ⫽ .0003). Overexpression (T/N ratio ⬎ 3) was found in 18 of 38 HCCs (47%). Clinicopathologically, Gpr49 overexpression in HCC of well to moderate differentiation was more frequent than in HCC of poor differentiation, 53% versus 18%, that in women was more frequent than in men, 71% versus 42%, and that in liver without cirrhosis was more frequent than in liver with cirrhosis, 60% versus 33%, although there was no statistical significance (Table 1). Strong Correlation of Gpr49 Overexpression With ␤-Catenin Mutations. To elucidate why Gpr49 was markedly up-regulated in some HCCs, we investigated the possibility of mutation in ␤-catenin, because we observed Gpr49 up-regulation in the PRF/PLC/5 and HepG2 cell lines, which show ␤-catenin accumulation in the cytoplasm and nucleus, respectively.18,19 Sequence analysis of exon 3 of ␤-catenin showed somatic mutation in 16 of 38 (42%) HCC samples (Fig. 2A). In cases 33 and 38, 2 mutations were found in each sample. In case 8, agarose gel electrophoresis after PCR amplification of exon 3 showed 2 separate bands, and sequence analysis found a 51-bp deletion. Fig. 3A and Fig. 3B show a representative electrophoretogram of a missense mutation and a summary of amino acid substitutions, respectively.

Fig. 2. Real-time quantitative RT-PCR analysis of HCC samples. (A) Gpr49 expression in 38 HCCs (black box), corresponding noncancerous livers (white box), and six normal livers (gray box). Expression levels are normalized to that of GAPDH mRNA in each sample. The number of the cases with Gpr49 overexpression is encircled. Asterisks show cases with somatic mutation of ␤-catenin. (B) Average expression levels of the Gpr49/GAPDH ratio in HCCs (T) compared with corresponding noncancerous livers (N). Bars, SE.

In most cases, amino acid substitutions occurred in residues 33, 37, 41, or 45; encoding serine or threonine; or in residues flanking them. As expected, overexpression of Gpr49 was frequently observed in HCC with mutation in exon 3 of ␤-catenin (14 of 16 cases, 87.5%), in contrast to HCC without ␤-catenin mutation (4 of 22 cases, 18.2%). Strong correlation between overexpression of Gpr49 and ␤-catenin mutation was observed (P ⫽ .001; Table 1). Clinicopathologically, the mutation of ␤-catenin was more frequently observed in HCV-related HCC (45.8%), well to moderately differentiated HCC (46.9%), and type 1 and 2 HCC (51.7%) than in HBVrelated HCC (22.2%), poorly differentiated HCC (16.7%), and type 3 HCC (12.5%), respectively, although there were no significant differences. Introduction of a ␤-Catenin Mutant Elevates mRNA for FEX, a Gpr49 Mouse Homologue, in MHT Cells. To investigate the effect of mutant ␤-catenin on the expression of the Gpr49 gene in hepatocyte, the

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Discussion

Fig. 3. Mutation analysis in exon 3 of the ␤-catenin gene. (A) Electrophoretogram indicating a mutation at codon 31 (TCT to TGT) in a sample from case 24. (B) Summary of amino acid substitutions. Eighteen mutations were found in 16 tumors. The encircled number is the case number. (C) Expression level of mRNA for FEX, a mouse homologue of Gpr49, in MHT cells infected with retrovirus carrying GFP, wild-type ␤-catenin, and mutant ␤-catenin. Data represent mean ⫾ SD of the results of 2 independent experiments carried out in triplicate. The numbers under the bars are the mean values.

mRNA expression level of FEX in MHT cells infected with retrovirus carrying GFP, wild-type ␤-catenin, or mutant ␤-catenin was analyzed by real-time quantitative RT-PCR. Infection with GFP was used as control, and showed that the infection efficiency exceeded 80% in each experiment (data not shown). Prospectively, mutant ␤-catenin elevated FEX mRNA in MHT cells over 3-fold higher than GFP or wild-type ␤-catenin (Fig. 3C).

We report the overexpression of Gpr49 in HCCs bearing ␤-catenin mutations. Gpr49 was originally isolated as an orphan G-protein-coupled 7-transmembrane receptor. Such receptors are members of the glycoprotein hormone receptor subfamily, which includes thyroid-stimulating hormone receptor (TSHR), follicle-stimulating hormone receptor (FSHR), and luteinizing hormone receptor (LHR).3,4 Although the ligand for Gpr49 has not yet been identified, its large extracellular domain with leucine-rich repeats suggests that the ligand is probably a larger glycoprotein than TSH, FSH, or LH. Compared with other members of the subfamily, Gpr49 characteristically shows wide tissue distribution. Expression of Gpr49 in the liver was very low, as reported previously, but expression was up-regulated markedly in HCC. Moreover, we could not detect any signal in fetal liver by Northern blot analysis (data not shown). Some G-protein-coupled receptors are critically involved in carcinogenesis.20 For example, a constitutively activating mutant of TSHR causes hyperfunctioning thyroid adenomas.21 In this subfamily, only Gpr49 has potential SH2- and SH3-interacting sequences in the C-terminal tail, so it may be able to link to additional signal transduction cascades involving mediators other than G proteins. Therefore, although we did not investigate any activity of Gpr49-related signaling, our observation of marked up-regulation of Gpr49 in HCC but not in noncancerous liver is important and suggests that Gpr49 may be involved in hepatocarcinogenesis. On the other hand, orphan G-protein-coupled receptors may be possible targets for new drugs.22 In fact, a number of drugs currently in use are agonists or antagonists of G-proteincoupled receptors. Because of its tumor-specific expression in liver, Gpr49 is a potential new therapeutic target in HCC if specific delivery of the drugs to liver could be achieved. We found no statistically significant association between Gpr49 overexpression and clinicopathologic features. However, it is notable that Gpr49 overexpression occurred more frequently in women than in men. Sex differences in Gpr49 expression may be related to the fact that Gpr49 is a member of a family of hormone receptors that includes gonadotropin receptors. Further study is needed to explain the different incidence of Gpr49 expression in association with tumor differentiation and the histologic status of noncancerous liver. ␤-Catenin is involved in both cadherin-mediated cellcell adhesion and Wnt-signaling. Recently it has been established that accumulated ␤-catenin can act as a coactivator of certain transcriptional factors, such as Tcf/LEF

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and CREB, subsequently resulting in the activation of downstream target genes, such as c-myc,7 cyclin D1,6 MDR1,23 and WISP1.8 Mutation of adenomatous polyposis coli (APC),7 axin,18 or ␤-catenin itself causes accumulation of ␤-catenin. We and others have reported the frequent occurrence of ␤-catenin mutations in human HCCs.24,25 In this study we observed mutation of ␤-catenin in 16 of 38 HCCs. This frequency is higher than in other previous reports. Our samples contained many HCV-related HCC (63.2%). ␤-catenin mutation in HCV-related HCC was more frequent than in HBVrelated HCC, 45.8% versus 22.2%. High frequency of ␤-catenin mutation could be partially explained by this fact.26 All HCC with mutation of ␤-catenin, except case 37, showed up-regulation of Gpr49. This observation suggests that Gpr49 is a new addition to the list of target genes, the expression of which is up-regulated by ␤-catenin mutation. Furthermore, introduction of mutant ␤-catenin to MHT cells caused up-regulation of the mouse homologue of Gpr49. Gpr49 may be the first gene that acts downstream of ␤-catenin to be identified in human HCC. The mutant ␤-catenin constructed in this study was able to more markedly induce the TopFlash reporter than wild type ␤-catenin (data not shown). The reasons for the up-regulation of Gpr49 by mutant but not wild-type ␤-catenin may be partially explained by the difference of transcriptional activity between the 2 proteins. Recently, it was suggested that the mutant ␤-catenin was not functionally equivalent to wild type ␤-catenin.26 Our findings are in accordance with this suggestion. Four HCCs with Gpr49 overexpression occurred without ␤-catenin mutation. Wnt-signaling pathways are composed of the complicated interaction of many molecules such as Axin1 and APC; therefore, further study is expected to clarify these mechanisms. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with ␤-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.

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