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Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma

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Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma Research Article Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma Mark R. Morris,1,2 Dean Gentle,1,2 Mahera Abdulrahman,2 Esther N. Maina,2 Kunal Gupta,2 Rosamonde E. Banks,3 Michael S. Wiesener,4 Takeshi Kishida,5 Masahiro Yao,5 Bin Teh,6 Farida Latif,1,2 and Eamonn R. Maher1,2 1Cancer Research UK Renal Molecular Oncology Group; 2Section of Medical and Molecular Genetics, Department of Pediatrics and Child Health, University of Birmingham, Birmingham, United Kingdom; 3Cancer Research UK Clinical Centre, St. James’s University Hospital, Leeds, United Kingdom; 4Department of Nephrology and Medical Intensive Care, University Clinic Charite´, Berlin, Germany; 5Yokohama City University School of Medicine, Yokohama, Japan; and 6Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Michigan Abstract cause hereditary type 1 papillary RCC (HPRC1) and von Hippel- Following treatment with a demethylating agent, 5 of 11 renal Lindau disease, respectively (2, 3). HPRC1-associated MET cell carcinoma (RCC) cell lines showed increased expression of mutations activate MET signaling to promote cell growth and hepatocyte growth factor (HGF) activator inhibitor type 2 motility (4–7). The VHL tumor suppressor gene is inactivated by (HAI-2/SPINT2/Bikunin), a Kunitz-type protease inhibitor somatic mutation or promoter methylation in most sporadic clear that regulates HGF activity. As activating mutations in the cell RCC (8–11). However, somatic MET-activating mutations are MET proto-oncogene (the HGF receptor) cause familial RCC, uncommon (<10%) in sporadic papillary RCC and are not a feature we investigated whether HAI-2/SPINT2 might act as a RCC of sporadic clear cell RCC (3). Thus, despite reports of increased tumor suppressor gene. We found that transcriptional expression of hepatocyte growth factor (HGF) and MET in RCC and silencing of HAI-2 in RCC cell lines was associated with synergy between the effect of VHL inactivation and increased MET promoter region methylation and HAI-2/SPINT2 protein signaling (12), direct genetic evidence for importance of the HGF/ MET signaling in the pathogenesis of RCC is only present in a small expression was down-regulated in 30% of sporadic RCC. Furthermore, methylation-specific PCR analysis revealed minority of cases. promoter region methylation in 30% (19 of 64) of clear cell Methylation of CpG dinucleotides in the promoter regions of RCC and 40% (15 of 38) of papillary RCC, whereas mutation tumor suppressor genes producing transcriptional silencing plays a analysis (in 39 RCC cell lines and primary tumors) revealed a major role in many human cancers (13–16). The frequency of missense substitution (P111S) in one RCC cell line. Restora- tumor suppressor gene inactivation by de novo methylation varies tion of HAI-2/SPINT2 expression in a RCC cell line reduced between tumor suppressor genes and between cancers. Thus, the in vitro colony formation, but the P111S mutant had no 3p21.3 renal tumor suppressor gene, RASSF1A, is usually inacti- significant effect. Increased cell motility associated with vated by epigenetic silencing and only rarely by somatic mutations HAI-2/SPINT2 inactivation was abrogated by treatment with (17–22), whereas the VHL tumor suppressor gene is inactivated more commonly by somatic mutation than by epigenetic silencing extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phospholipase C-g inhibitors, but (8–11). For certain cancers, notably colorectal cancer, a subset of not by an inhibitor of atypical protein kinase C. These findings tumors may show extensive tumor suppressor gene promoter are consistent with frequent epigenetic inactivation of HAI-2/ methylation (23). However, in a methylation profile analysis of RCC, SPINT2, causing loss of RCC tumor suppressor activity and we found promoter methylation of only a minority of tumor implicate abnormalities of the MET pathway in clear cell and suppressor genes that were known to be inactivated in other tumor papillary sporadic RCC. This information provides opportu- types (24, 25). This observation prompted us to investigate whether nities to develop novel targeted approaches to the treatment gene expression profiling of RCC cell lines treated with the of RCC. (Cancer Res 2005; 65(11): 4598-606) demethylated agent 5-azacytidine might identify novel RCC tumor suppressor genes (26–28). During our investigations, we found silencing or down-regulation of HAI-2/SPINT2 expression in a Introduction number of RCC cell lines. HAI-2/SPINT2 (also known as bikunin) Renal cell carcinoma (RCC) is a heterogeneous disorder with the encodes Kunitz-type protease inhibitor that regulates HGF activity. majority (f75%) of cases classified as clear cell (conventional) and Thus, HGF is secreted as an inactive proform and needs to be the next most frequent subtype is papillary RCC (f15% of all cases; activated by HGF activator enzyme to initiate MET signaling. HAI- ref. 1). Germline activating mutations in the MET proto-oncogene 2/SPINT2 is an endogenous inhibitor of HGF activator enzyme and and inactivating mutations in the VHL tumor suppressor gene has been implicated in the pathogenesis of ovarian and hepatocellular carcinoma (29–31). In view of these findings, we proceeded to investigate whether epigenetic inactivation of SPINT2 Note: The present address for M.S. Wiesener is Interdisciplinary Center for Clinical Research (IZKF), University of Erlangen, Nuremberg, Germany. Supplementary data for might play a role in renal tumorigenesis. this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Eamonn R. Maher, Section of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Edgbaston, Materials and Methods B15 2TT Birmingham, United Kingdom. Phone: 44-121-627-2741; Fax: 44-121-414-2538; E-mail: [email protected]. Patients and samples. DNA from a total of 102 primary RCCs were I2005 American Association for Cancer Research. analyzed and subdivided into 64 clear cell and 38 papillary RCCs. Protein Cancer Res 2005; 65: (11). June 1, 2005 4598 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on April 13, 2017. © 2005 American Association for Cancer Research. Epigenetic Inactivation of HAI-2/SPINT2 in Cancer lysates from 31 additional RCC tumors (29 clear cell RCC and 2 nonclear Mutation analysis of primary tumors and cell lines. Intron-exon cell RCC) was also analyzed. Local ethics committees approved the boundaries were determined by matching the cDNA sequence for HAI-2/ collection of samples and informed consent was obtained from each SPINT2 with the working draft sequence of the human genome, the UCSC patient. assembly 5 (http://genome.ucsc.edu/). Mutation screening was done by Cell lines, 5-azadeoxycytidine treatment, and microarray analysis. direct sequencing on an ABI 3730 automated sequencer. The sequences of RCC cell lines KTCL 26, RCC4, UMRC2, UMRC3, SKRC18, SKRC39, SKRC45, the primers used along with the annealing temperature are available upon SKRC47, SKRC54, 786-0, and Caki-1 were routinely maintained in DMEM request. (Invitrogen, San Diego, CA) supplemented with 10% FCS at 37jC, 5% CO2. Immunoblotting. Protein extraction and blotting was done essentially The demethylating agent 5-azadeoxycytidine (Sigma, Gillingham, Dorset, as described previously (33). Sections of frozen tissue were fractionated, United Kingdom) was freshly prepared in double-distilled H2O and filter weighed, and homogenized into 20-fold excess of extraction buffer [7 mol/L sterilized. Cell lines were plated in 75 cm2 flasks in DMEM supplemented urea/10% glycerol/10 mmol/L Tris-HCl (pH 6.8)/1% SDS/5 mmol/L DTT/0.5 with 10% FCS at differing densities, depending upon their replication factor, mmol/L phenylmethylsulfonyl fluoride (PMSF) and 1 mg/L of aprotinin, to ensure that both control and 5-azadeoxycytidine–treated lines reached pepstatin, and leupeptin] with an electric homogenizer (Ultra-Turrax; IKA, f75% confluence at the point of RNA extraction. Twenty-four hours later, Staufen, Germany). Extracts were quantified with the Bio-Rad detergent- cells were treated with 5 Amol/L 5-azadeoxycytidine. The medium was compatible protein assay (Bio-Rad, Hemel Hempstead, Hertfordshire, changed 24 hours after treatment and then changed again after 72 hours. United Kingdom). Protein lysates from cell line clones and mixed RNA was prepared 5 days after treatment using RNABee (AMS Biotechno- populations were prepared with NETS lysis buffer containing 3 mmol/L logy, Abingdonoxon, United Kingdom). RNA extracted from KTCL26, PMSF, 20 Ag/mL aprotinin, and 10 Ag/mL leupeptin (all chemicals from SKRC39, SKRC45, and SKRC47 F 5-azadeoxycytidine was analyzed by Sigma-Aldrich, Gillingham, Dorset, United Kingdom). Twenty micrograms microarray as previously described (32). of each extract were resolved on polyacrylamide gels. Proteins were HAI-2/SPINT2 expression was detected by reverse transcription-PCR transferred onto Immobilon P (Millipore, Bedford, MA) for 2 hours and (RT-PCR) using the following primers: 5V-CAGCATCCACGACTTCTGCCTG- probed with anti–HAI-2 goat antibody (R&D Systems, Minneapolis, MN) at 3V and 5V-GGCGGTGCAGTATTCTTCATAG-3V. 0.2 Ag/mL. Signals were detected with horseradish peroxidase–conjugated Expression of GAPDH was used as a control. The GAPDH primers were antigoat antibody diluted 1/2,000 (DAKO,
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