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Frequent Silencing of the GPC3 Gene in Ovarian Cancer Cell Lines

Laboratory of Biological Chemistry [H. L., P. J. M.], and Laboratory of Genetics [R. H., D. S.], Gerontology Research Center, National Institute on Aging, Baltimore, Maryland 21224, and Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287 [P. J. M.]

	ABSTRACT

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GPC3 encodes a glypican integral membrane protein and is mutated in the Simpson-Golabi-Behmel syndrome. Simpson-Golabi-Behmel syndrome, an X-linked condition, is characterized by pre- and postnatal overgrowth as well as by various other abnormalities, including increased risk of embryonal tumors. The GPC3 gene is located at Xq26, a region frequently deleted in advanced ovarian cancers. To determine whether GPC3 is a tumor suppressor in ovarian neoplasia, we studied its expression and mutational status in 13 ovarian cancer cell lines. No mutations were found in GPC3, but its expression was lost in four (31%) of the cell lines analyzed. In all of the cases where GPC3 expression was lost, the GPC3 promoter was hypermethylated, as demonstrated by Southern analysis. Expression of GPC3 was restored by treatment of the cells with the demethylating agent 5-aza-2'-deoxycytidine. A colony-forming assay confirmed that ectopic GPC3 expression inhibited the growth of ovarian cancer cell lines. Our results show that GPC3, a gene involved in the control of organ growth, is frequently inactivated in a subset of ovarian cancers and suggest that it may function as a tumor suppressor in the ovary.

	Introduction

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Twenty-five thousand new cases of ovarian cancer will be diagnosed this year in the United States, yet very little is known about the molecular determinants of this disease. Mutations in BRCA1, BRCA2, or mismatch repair genes can cause familial forms of the disease, but these syndromes affect only a small proportion of ovarian cancer patients (1) . Unfortunately, the genes involved in the familial syndromes have shed little light on the mechanisms of sporadic ovarian tumorigenesis. Several chromosomal abnormalities have been identified in sporadic ovarian cancers. The extent and location of LOH² depend on the subtype, but high frequencies of loss are generally observed at chromosomes 17p, 17q, and 22q (2 , 3) . Recently, it was reported that a region located at Xq26 is frequently deleted in advanced ovarian cancer (4) . GPC3, a gene located at Xq26, was recently shown to be mutated in Simpson-Golabi-Behmel syndrome, an overgrowth syndrome also involving multiple embryonal neoplasia (5) . The GPC3 gene occupies almost 1 Mb at Xq26 and makes this region relatively gene poor (6) . GPC3 is expressed ubiquitously in the embryo (5) , but shows an expression pattern restricted to the ovary and the colon in the adult.³ Moreover, GPC3 expression inhibits growth of mesotheliomas and MCF-7 breast cancer cells in vitro, probably by inducing apoptosis (7) . For these reasons, we decided to investigate GPC3 status in ovarian cancers.

	Materials and Methods

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Cell Lines.
Ovarian cancer cell lines ES-2, OV1063, MDAH 2774, SK-OV-3, HS571, CA-OV-3, and OVCAR-3 were obtained from the American Type Culture Collection. Cell lines AD 10, UCI101, UCI107, A222, and A224 were a gift from Dr. Michael Birrer (National Cancer Institute, Bethesda, MD). Cell line A2780 was kindly provided by Dr. V. Bohr (National Institute on Aging, Baltimore, MD). All cell lines were maintained in McCoy’s 5A medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), except lines AD 10, UCI101, UCI107, A222, and A224, which were maintained in RPMI 1640 (Life Technologies, Inc.), supplemented as above.

RT-PCR and Genomic PCR.
Total RNA was isolated using Trizol (Life Technologies, Inc.). Total RNA (10 µg) was used for reverse transcription using Superscript II (Life Technologies, Inc.). After synthesis, the cDNA mixture was diluted with one volume of TE [10 mM Tris-HCl (pH 7.5) and 1 mM EDTA], and 1–2 µl of the mixture was used as PCR template. Primers GPC-1a (5'-ctcctagctccctgcgaag) and GPC-6b (5'-tctccagtacttgtcaatctc) were used to amplify the 5' fragment of GPC3. The 3' fragment was amplified using primers GPC-9a (5'-gttactgcaatgtggtcatgc) and GPC-11b (5'-acatgtgctgggcaccag). Primers bact-1a (5'-tctacaatgagctgcgtgtg) and bact-4b (5'-catctcttgctcgaagtcc) were used for amplifying ß-actin as a control. GPC-EX1A (5'-aggtagctggcgaggaaac) and GPC-EX1B (5'-taggcacgctcaagggac) were used to amplify GPC3 exon 1 using 0.1 µg of genomic DNA. PCR conditions were as described (8) , using amplitaq DNA polymerase (Perkin-Elmer Corp.).

Methylation Analysis.
For Southern analysis, 10 µg of genomic DNA was digested with either EcoRI alone or a combination of EagI and EcoRI. The restriction digested DNA fragments were separated on a 0.8% agarose gel. After transfer, the membrane was hybridized with the radiolabeled 3' promoter fragment amplified by PCR using primers GPC3-pr8a (5'-tgagattcagtcacagtaagg) and GPC3-pr11b (5'-ttctggattggttctcgcac). Cell lines A224, ES-2, and OV-1063 were tested for restoration of expression by demethylase treatment (9) . Briefly, 50% confluent cells were treated with 0.5 µM 5-aza-2'-deoxycytidine (Sigma Chemical Co.), for either 2 or 4 days. The medium was replenished every 2 days. RNA isolation for RT-PCR was performed as described above.

Colony-forming Assay.
Cells were grown to 50–70% confluence in 100-mm Petri dishes and treated with a mixture of transit-100 (Panvera Co.) and pGPC3 or pCINEO plasmid DNA in serum free Optimem I media (Life Technologies, Inc.) for 5 h. At the end of the incubation, the cells were washed with PBS and grown in the regular medium for 48 h. The cells were then split into 1:5 or 1:10 ratio grown in media containing G418 (500 µg/µl) in 100-mm Petri dishes. About 7–9 days later, cells were stained with 0.2% crystal violet in 10% ethanol, and the visible colonies were counted and compared. Colony number for vector alone was normalized to 100%.

DNA Sequencing.
Two GPC3 cDNA fragments, 5' and 3', were PCR amplified separately using primers GPC-1a/GPC-6b and primers GPC-9a/GPC-11b, respectively. The PCR products were gel-purified using Spin-X centrifuge filter tubes (Costar), followed by ethanol precipitation. The purified fragments were used as DNA templates for direct sequencing using Thermosequenase (Amersham Corp.) and ³³P-labeled ddNTPs (Amersham Corp.). PCR primers (above) and internal primers GPC-4a (5'-caagcctgactccacaagc), GPC-13a (5'-gttgctcatgtagaacatgaag) and GPC-14a (5'-aactgaagcacattaaccagc) were used for sequencing. Exon 8 of GPC3 was amplified using primers GPC-EX8A (5'-tagtgttatactgaggctatg) and GPC-EX8B (5'-catggttagtcctctacttc) and sequenced using primer GPC-EX8A.

	Results

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GPC3 Expression in Epithelial Ovarian Cancer Cell Lines.
Because GPC3 is expressed in the normal adult ovary, we designed a RT-PCR strategy to investigate its expression in ovarian cancer cell lines. A 3' region of the GPC3 cDNA could be amplified from 9 of the 13 cell lines (Fig. 1) , whereas 4 cell lines (ES-2, OV1063, A222, and A224) did not show any amplification. The same result was obtained when using a 5' RT-PCR product.⁴ SK-OV-3 exhibited a smaller product (Fig. 1) that was shown, by sequencing, to be a nonspecific PCR artifact⁴ As a control, a PCR product corresponding to the housekeeping gene ß-actin was amplified from all of the lines, demonstrating the integrity of the cDNAs. Exon 1 (Fig. 1) , exon 3, and exon 8⁴ could be amplified from genomic DNA of all of the nonexpressing lines, verifying that the gene was not deleted or grossly rearranged.

View larger version (53K): [in this window] [in a new window]   Fig. 1. GPC3 expression in ovarian cancer cell lines. Top, the 0.9-kb 3' GPC3 fragment obtained by RT-PCR (arrow) in the indicated cell lines. As a positive control for cDNA integrity, a portion of ß-actin was amplified by RT-PCR (middle). Bottom, GPC3 exon1 as detected by genomic PCR for cell lines MDAH2774, ES-2, OV-1063, A222, and A224.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Hypermethylation of the GPC3 promoter. A, map of the Xq26, including the known genes. The GPC3 promoter is shown. The EagI restriction endonuclease is methylation-sensitive. B, the PCR product close to the 3' end (shown in A) was labeled and used as a probe for Southern analysis of the methylation status of ovarian cancer lines. After EcoRI or EagI and EcoRI restriction digests of genomic DNA of the indicated lines, hybridization was performed. The 8-kb band (corresponding to the methylated allele) and the 4.2-kb band (corresponding to the unmethylated allele) are shown by arrows.

View larger version (50K): [in this window] [in a new window]   Fig. 3. Reexpression of GPC3 after demethylation. The indicated cell lines were treated for 2 days with 0.5 µM 5-aza-2'-deoxycytidine. GPC3 expression was monitored by RT-PCR, as described in "Materials and Methods." An arrow shows the 3' GPC3 PCR fragment. The smaller DNA fragment amplified in OV-1063 after treatment is a nonspecific PCR product, which has been confirmed by sequencing. Untreated MDAH 2774 is included as a positive control.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Colony-forming assay. ES-2 or UCI101 cells were transfected with either the control plasmid pCINEO or a GPC3 expression plasmid (pGPC3). The number of colonies was reduced 80% in ES-2 cells when transfected with the GPC3 expression vector as compared with the pCINEO vector. The number of colonies of UCI 101, which expresses endogenous GPC3, was not significantly reduced by transfection with the GPC3 expression plasmid.

	Discussion

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Recently, it has become apparent that tumor suppressors can be inactivated in cancer by mutations, deletions, rearrangements, or hypermethylation (9, 10, 11, 12) . In this study, we show that GPC3 is silenced by hypermethylation of its promoter in 4 of 13 ovarian cancer cell lines. We believe GPC3 to be a good candidate for being the target gene of the LOH reported on Xq26. The LOH frequency of 30%, reported by Choi et al. (4) , is consistent with our finding that 31% of the cell lines have silenced GPC3. The minimum region of loss extends 5 cM centromeric to HPRT (4) and encompasses the GPC3 locus (5) . The only genes present in >2 Mb of DNA centromeric to HPRT are GPC3 and GPC4, another recently characterized glypican gene.⁵ GPC4, however, shows a ubiquitous pattern of expression in the adult. None of the other genes known to be in the region of loss are good candidates for being an ovarian tumor suppressor (Fig. 2A) . HDGF is an endothelial mitogen (13) , IGSF1 is an immunoglobulin domain-containing gene highly expressed in adult testis and fetal liver (14) , and OCRL is the gene mutated in Lowe’s syndrome, also known as oculocerebrorenal syndrome (15) .

A putative tumor suppressor gene on the X chromosome suggests several interesting mechanisms for tumorigenesis. It would seem unlikely to find a tumor suppressor gene on the X chromosome because men, with only one copy, would become highly susceptible to cancer compared with women. Negative growth-regulatory genes located on the X-chromosome would have to control the growth of female tissues, like the ovary.

Assuming that, in female cells, one allele of GPC3 is inactive as a result of X-inactivation, a single hit in the active allele would be sufficient to inactivate the gene. In that case, methylation, mutation, or LOH of the active allele could all contribute to the loss of function of the tumor suppressor gene. Because we observe no mutations and because the expression of GPC3 can be restored by demethylation, it seems that the most likely mechanisms in this case would be LOH or hypermethylation of the active allele. There is evidence that demethylation can lead to reactivation of X-inactivated genes (16) . On the other hand, Choi et al. have shown that the loss occurs in the inactive allele and suggested that the target gene at Xq26 may escape X-inactivation (4) . This seems to be in contradiction with results showing that GPC3 is X-inactivated in female cells (17) . It is possible that the GPC3 locus becomes demethylated early in tumorigenesis, which would explain why the loss at Xq26, a late event, can be found in the inactive allele. Resolving these possibilities will require LOH and GPC3 hypermethylation studies on a large panel of ovarian tumors. However, our finding that OV1063 contains only one, hypermethylated, allele is consistent with our hypothesis that both allelic loss and hypermethylation contribute to GPC3 inactivation.

The phenotype of Simpson-Golabi-Behmel syndrome patients suggests that GPC3 functions in regulating the balance between cell growth and cell death, as would be expected for a tumor suppressor gene. GPC3 belongs to a class of glypican-related integral membrane proteins that have been implicated in signal transduction (18) . Interestingly, GPC3 may interact directly with IGF2 (5) , a growth factor thought to be important in ovarian cancer (19) . In addition, it was recently shown that GPC3 induces apoptosis in MCF-7 cells and that it inhibits colony-forming ability in a cell line-specific manner (7) . More specifically, mesothelioma cell lines and MCF-7 breast cancer cells were inhibited by GPC3 transfections, but not colon line HT-29 or NIH 3T3 fibroblasts. This difference almost certainly reflects the differential activity of the pathway in different cells. Interestingly, mesotheliomas and ovarian cancers are related neoplasms because they both originate from the coelomic epithelial cell layer. We find that GPC3 inhibits growth of cells that have lost endogenous expression, strongly suggesting that the loss of expression is not a random event in tumorigenesis, but a selected event that favors ovarian cancer growth. Similar arguments have been made to interpret inhibition of colony-forming ability by p53 and p16 (20 , 21) . GPC3 may be involved in the regulation of apoptosis in the normal ovary. Lack of apoptosis leading to deregulated tissue homeostasis has emerged as an important mechanism for tumorigenesis (22) . The data presented here suggest a new pathway for ovarian carcinogenesis and may lead to novel screening, diagnosis, and treatment strategies.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Michael Birrer and Vilhelm Bohr for providing cell lines. We thank Drs. Colleen D. Hough, Michael T. Furlong, Ashani Weeraratna, and Bert Vogelstein for helpful comments and suggestions on the manuscript. We thank Cheryl A. Sherman-Baust for technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Laboratory of Biological Chemistry, Gerontology Research Center, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224. Phone: (410) 558-8506; Fax: (410) 558-8386.

² The abbreviations used are: LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR.

³ R. Huber, G. Pilia, and D. Schlessinger, unpublished observations.

⁴ H. Lin and P. J. Morin, unpublished observations.

⁵ R. Huber, R. Mazzarella, C. N. Chen, E. Chen, M. Ireland, S. Lindsay, G. Pilia, and L. Crisponi, submitted for publication.

Received 10/12/98. Accepted 1/ 4/99.

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