Epigenetic Silencing of Novel Tumor Suppressors in Malignant Melanoma

Research Article

Viswanathan Muthusamy,1 Sekhar Duraisamy,2 C. Matthew Bradbury,1 Cara Hobbs,1 David P. Curley,1 Betsy Nelson,1 and Marcus Bosenberg1

1Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont and 2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts

Abstract infrequent in the genome but are associated with the promoter regions of nearly half of all known human genes (10). Examination Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on of familial cancer genes has revealed that tumor-specific hyper- survival, emphasizing the need for a better understanding of methylation may act as a second hit by preferentially targeting the genetic, epigenetic, and phenotypic changes in melanoma the wild-type allele of tumor suppressors, whereas the promoter region of the mutant allele is not affected by methylation (11). formation and progression. We identified 17 genes that were de novo Dnmt1 not previously known to be silenced by methylation in Inhibition of methylation by down-regulation has melanoma using a microarray-based screen following treat- been shown to reduce tumor formation in several settings (12–14). Decreased Dnmt1 function completely suppressed polyp formation ment of melanoma cell lines with the DNA methylation ApcMin/+ inhibitor 5-Aza-2¶-deoxycytidine. Eight of these genes have in mice, showing that aberrant hypermethylation events not been previously shown to undergo DNA methylation in any are necessary to form and maintain tumors (12, 13, 15). However, in QPCT, CYP1B1 LXN other cases, genomic hypomethylation induced by down-regulation form of cancer. Three of the genes, , and , Dnmt1 are densely methylated in >95% of uncultured melanoma of increased the rate of specific malignancies, including tumor samples. Reexpression of either of two of the silenced lymphoma (16). genes, HOXB13 and SYK, resulted in reduced colony formation Epigenetic inactivation of individual tumor suppressors by DNA in vitro and diminished tumor formation in vivo, indicating methylation has also been shown in malignant melanoma (17–22). A high incidence of methylation in uncultured melanoma tissue that these genes function as tumor suppressors in melanoma. RARB RASSF1A (Cancer Res 2006; 66(23): 11187-93) samples has been reported for (70%), (55%), PYCARD (50%), MGMT (34%), DAPK (19%), and APC (19%), which may play tumor suppressor roles in several tumor settings Introduction (summarized in Supplementary Table S1; refs. 19–22). Other tumor Melanoma is the most lethal form of skin cancer with an expected suppressor genes confirmed to be methylated in melanoma, albeit 3 62,190 new cases and 7,910 deaths in the United States in 2006. at a lower incidence, are the familial melanoma gene CDKN2A Several genetic changes associated with melanoma have been (10%) and in CDKN1B (9%) and PRDX2 (8%; refs. 17, 18, 23). INK4A identified (1). The CDKN2A locus at 9p21 that encodes the p16 A useful attribute of epigenetic silencing is that it is amenable to ARF and p14 tumor suppressor genes is mutated in f20% of familial reversal by methylation inhibitors. We have exploited this property melanoma and is inactivated in the majority of spontaneous in a microarray-based assay to screen for genes that are melanomas (2, 3). Activation of mitogen-activated protein kinase reexpressed following treatment of melanoma cells with 5-Aza-2¶- signaling also seems to occur in the vast majority of melanomas, deoxycytidine (5AzadC). We show DNA methylation and silencing either by activating mutations in the BRAF serine/threonine kinase of 17 genes in melanoma cell lines and in uncultured melanoma (f70% of melanomas) or by mutation of NRAS (f20% of tumor samples. DNA methylation has not been previously shown in melanomas; refs. 4, 5). BRAF is also mutated in f70% of benign eight of these genes in any form of cancer. In addition, we show melanocytic nevi, suggesting that additional genetic or epigenetic tumor suppressor properties in melanoma for two of the genes, hits are needed for progression to malignancy (6). Widespread HOXB13 and SYK. chromosomal instability is another characteristic feature of melanoma, with frequent losses of chromosomal regions 6q, 8p, Materials and Methods 9p, 10, 13, and 21q and gains of 6p, 7, 8q, 11q13, 17q, and 20q (7). Epigenetic changes may also result in inactivation of tumor Tissue specimens, primary cells, and cell lines. Melanoma tissue samples were collected in accord with Institutional Review Board–approved suppressors and progression to malignancy (8, 9). DNA methylation protocols at the Memorial Sloan-Kettering Cancer Center (New York, NY) is one form of epigenetic change and involves the covalent addition and Dana-Farber Cancer Institute (Boston, MA). All the cell lines used in the of a methyl group to cytosine residues in CpG dinucleotides by study (MelJuSo, UACC 903, C8161, Neo6/C8161, WM1205 Lu, WM35, Roth, DNA methyltransferases. CpG-rich sequences (CpG islands) are Carney, and WM455) were propagated in DMEM-F12 (Invitrogen) supplemented with 5% fetal bovine serum and nonessential amino acids (Invitrogen). Primary cultured human foreskin melanocytes were grown in Note: Supplementary data for this article are available at Cancer Research Online Medium 254 supplemented with human melanocyte growth serum (http://cancerres.aacrjournals.org/). (Cascade Biologics). Stable MelJuSo cell lines expressing HOXB13 and SYK Requests for reprints: Marcus Bosenberg, Department of Pathology, University of were generated by reverse transcription-PCR (RT-PCR) amplification of Vermont College of Medicine, 323 Health Science Research Facility, 149 Beaumont Avenue, Burlington, VT 05405. Phone: 802-656-0309; Fax: 802-656-8892; E-mail: [email protected]. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1274 3 Cancer Facts and Figures 2006, American Cancer Society, http://www.cancer.org. www.aacrjournals.org 11187 Cancer Res 2006; 66: (23). December 1, 2006

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Cancer Research full-length coding sequence from primary melanocytes and subcloning into the pTRE expression vector (Clontech). The inserts were sequenced to ensure an absence of introduced mutations. Stable transfectants were produced by transfection with LipofectAMINE (Invitrogen) and selected by growth in medium containing G418 (800 Ag/mL; Invitrogen). Colonies were ring cloned, expanded, and analyzed for transgene expression using quantitative RT-PCR. 5AzadC treatment and microarray analysis. Cells were grown to 50% confluence in 100-mm culture plates and treated with 5 Amol/L 5AzadC (Sigma) dissolved in growth medium. Fresh 5AzadC medium was added every 24 hours until the end of the assay (96 hours). RNA and DNA were isolated from a batch of 5AzadC-treated cells every 24 hours starting with 0-hour controls. RNA was isolated following 0 and 48 hours of 5AzadC treatment of six melanoma cell lines (MelJuSo, UACC 903, C8161, Neo6 C8161, WM1205, and WM35) and used for the reexpression microarray analysis. Total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Preparation of double-stranded cDNA and in vitro transcription was as per manufacturer’s recommendations. Biotinylated cRNA obtained from in vitro transcription was fragmented and hybridized on Human Genome U133A (HG-U133A) array as per manufacturer’s recommendations (Affymetrix). Microarray Suite 5.0 (MAS 5.0; Affymetrix) software was used to analyze the HG-U133A arrays and to determine the statistical significance of a particular probe set. Probe set signal calls with Ps >0.01 were not further evaluated. All the arrays were normalized to a target signal intensity value of 500. Baseline analyses were done using 0-hour treatment array signals as a baseline for the respective 48-hour treatment. The data were further processed using customized programs written for conditional formatting analyses to select for genes whose expression on 5AzadC treatment was significantly altered in more than one cell line. These genes were examined for presence of CpG islands in their promoter regions using the National Center for Biotechnology Information MapViewer and the European Molecular Biology Laboratory CpGPlot program.4 Criteria used to identify prospective methylated genes included (a) up-regulation of expression (>4-fold) on 5AzadC treatment in at least one melanoma cell line, (b) significant expression (MAS 5.0 score of present) in cultured melanocytes but no significant changes in expression (<2-fold) of the gene on 5AzadC treatment (in melanocytes), (c) down-regulation of expression of the gene in untreated melanoma cell lines compared with primary melanocytes (>4-fold down-regulation in at least two melanoma cell lines), and (d) presence of a CpG island in the promoter region. Bisulfite sequencing and quantitative PCR. DNA was isolated from cells and tissues using standard phenol-chloroform extraction. Bisulfite modification was done as described previously (24) on DNA from melanocytes, melanoma cell lines before and after 48 hours of treatment with 5AzadC, and DNA from melanoma tissues. PCRs were carried out using 50 ng of bisulfite-modified DNA in a 30 AL volume with a 0.2 Amol/L primer concentration (primer sequences and detailed PCR conditions are provided in the Supplementary Table S2). PCR products were purified using the QIAquick Gel Extraction kit (Qiagen) and directly sequenced on the ABI 3100-Avant Automated DNA Sequencer. Particular CpG sites were scored as positive if the C:T peak ratio exceeded 1:3 (at least 25% methylation). Real- time quantitative PCR was done for validation of microarray expression data of selected candidate genes by using 2.5 AL of 100-fold diluted cDNA template and 0.2 Amol/L gene-specific primers in a 25 AL PCR using JumpStart SYBR Green kit (Sigma) according to the manufacturer’s instructions in an ABI 7700. The reactions were done in duplicate, CT values obtained were normalized to glyceraldehyde-3-phosphate dehydrogenase C levels, and quantification was done using the comparative T method. Western blotting. Western blotting experiments were done by separation of 15 Ag of cell lysate per sample on SDS-PAGE, transfer to Immobilon-P membranes (Millipore), blocking with 0.1 mol/L PBS containing 0.2% Tween 20 and 5% nonfat milk, and incubation with Figure 1. Expression changes induced by 5AzadC treatment in melanoma cell antisera to HOXB13 (F-9), SYK (4D10; both from Santa Cruz Biotechnology, lines. A, changes in expression of selected candidate genes on 48-hour 5AzadC Santa Cruz, CA), or actin (AC-40; Sigma). Following washes and incubation treatment validated by quantitative RT-PCR analysis. B, expression of the genes in untreated melanoma cell lines relative to primary melanocytes by quantitative RT-PCR. C, microarray profile data of genes showing significant expression changes on 48-hour 5AzadC treatment. Genes were ordered by 4 http://www.ebi.ac.uk/emboss/cpgplot/. median expression change relative to corresponding untreated cells.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. DNA Methylation in Malignant Melanoma with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies following 5AzadC treatment (Supplementary Fig. S4). We chose to (Amersham Biosciences), Enhanced Chemiluminescence Advance Detection evaluate two candidate genes SYK and HOXB13 further because of System (Amersham Biosciences) was used for chemiluminescent detection. their loss of expression in most melanoma cell lines relative to In vitro proliferation assays and tumor formation in nude mice. For melanocytes (Fig. 1B). in vitro growth curve assays, three replicates of 10,000 cells each of the Tumor suppressor properties of SYK in malignant melano- vector and HOXB13-orSYK-transfected cells were seeded in six-well cell culture plates for each time point. The cell were trypsinized, diluted, and ma. We found that SYK mRNA expression was markedly reduced in counted on a flow cytometer (Beckman Coulter) at 24-hour intervals all nine melanoma cell lines compared with primary cultured A starting at 0 hour and ending at 120 hours. Colony formation assays were human melanocytes (Fig. 4 ), and 5AzadC treatment resulted in done by plating three replicates each of 1,000, 500, or 250 cells of the vector >2-fold up-regulation of SYK in a subset (four of eight) of cell lines and HOXB13-orSYK-transfected cells in six-well cell culture plates. with SYK methylation (Fig. 3B). Immunoblotting showed complete Colonies were allowed to form for 2 weeks and stained with 0.005% crystal absence of protein expression in all but one cell line (Fig. 4B). SYK 6 violet. For xenografting experiments, 0.5 Â 10 cells of the vector or mRNA expression was reduced >4-fold in 77% of the primary HOXB13 SYK -or -transfected cells were injected s.c. into flank skin of nude melanoma tissue samples (10 of 13; Fig. 4C). Promoter region mice. Four injections were carried out per condition. Tumor dimensions methylation of SYK was detected in 89% of the melanoma cell lines were measured once every week, and the mice were sacrificed at 10 weeks and 30% of primary tumors (Fig. 2A-C). To examine if SYK has a after injection when end point measurements of tumor weights were obtained. Statistical significance of the end point data of the in vitro and tumor suppressor role in melanoma, we analyzed phenotypic in vivo assays was evaluated using the Student’s paired t test. changes resulting from stable reexpression of SYK in a SYK- negative melanoma cell line (MelJuSo). Expression of SYK at comparable levels with those seen in primary melanocytes Results (Supplementary Fig. S5) resulted in reduced cell proliferation Identification of candidate genes silenced by DNA methyl- (Fig. 6B) and markedly reduced colony formation in vitro (Fig. 6B). ation in melanoma. To determine DNA methylation-related In s.c. xenografting experiments with nude mice, the average tumor expression changes, we treated six human melanoma cell lines with 5AzadC and compared expression changes at 48 hours after treatment relative to untreated melanoma cell lines. Expression of 150 genes was increased by >4-fold on 5AzadC treatment in at least one of the six melanoma cell lines. This represents a small fraction (0.8%) of the 18,400 transcripts represented on the microarray. Eighty percent (120 of 150) of the genes with altered expression were associated with promoter region CpG islands, suggesting that these genes might be directly methylated in the melanoma cell lines (Fig. 1). We selected a subset of 25 candidate genes for further validation by bisulfite sequencing using the additional criterion of >4-fold down-regulation of expression of the gene in at least two untreated melanoma cell lines compared with primary melanocytes. We found that 68% (17 of 25) of these genes were densely methylated in the promoter region in at least 1 of 9 melanoma cell lines or 20 melanoma tumor tissue samples (Fig. 2A and B). No DNA methylation was detected in primary human melanocytes at any of the loci. DNA methylation of specific genes occurred at similar frequencies in cell lines and tumor tissues and was present both in melanoma cell lines and melanoma tissue samples for all genes tested (Fig. 2C). Several of the melanoma cell lines (7 of 9) and melanoma tumor specimens (7 of 20) exhibited DNA methylation of >50% of the genes tested. The methylation status of each potential CpG methylation site for all 17 genes in all melanoma cell lines, primary melanocytes, and melanoma tumor samples is depicted in Supplementary Fig. S3. Identification of methylation-targeted tumor suppressor candidate genes in melanoma. A very high incidence of promoter methylation (>75%) was observed in CYP1B1, QPCT, LXN, COL1A2, and GDF15, and an incidence of 50% to 60% was observed in PCSK1, BST2, and DNAJC15 in melanoma tumor samples (Fig. 2B and C; Supplementary Table S1). The genes CDKN1C, MFAP2, SYK, HOXB13, PTGS2, and WFDC1 were methylated in 20% to 35% and CDH8, DAL1, and LRRC1 in 5% to 10% melanoma tumor tissues. Figure 2. Methylation status of candidate melanoma-associated genes. A, methylation status of candidate genes in nine melanoma cell lines. Using quantitative RT-PCR, we confirmed that promoter methyl- Black shading box, presence of methylation in the promoter region CpG island of ation of several of the above candidate genes was accompanied by the gene. Asterisk, additional cell lines used in the bisulfite sequencing analysis reduced expression relative to melanocytes and that this loss of originally not included in the microarray screening. B, methylation status of the candidate tumor suppressor genes in a panel of 20 melanoma tumor tissues. expression could be reversed in the majority of cases by 5AzadC C, a comparison of frequency of methylation of the candidate genes in treatment (Figs. 1A and C and 3). Demethylation was also induced tumors versus cell lines. www.aacrjournals.org 11189 Cancer Res 2006; 66: (23). December 1, 2006

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Cancer Research size was reduced by >8-fold in SYK-expressing clones compared Discussion B with vector controls (Fig. 6 ). Several genes have been analyzed for promoter methylation in HOXB13 Tumor suppressor properties of in malignant melanoma using candidate gene-based approaches (17–22). We melanoma. We found that HOXB13 mRNA expression was have used a microarray-based strategy to identify 17 genes that are A markedly reduced (>16-fold) in all the melanoma cell lines (Fig. 5 ); not previously known to be silenced by DNA methylation in however, one of the cell lines had retained protein expression malignant melanoma. Eight of these genes have not been B (Fig. 5 ), which may indicate increased protein stability in that previously shown to undergo DNA methylation in any form of cell line. In the melanoma tumor samples, we observed >4-fold cancer. Several of these genes are candidate tumor suppressors in reduction of HOXB13 mRNA compared with melanocytes in >60% melanoma. Reexpression of either of two of the silenced genes, (8 of 13) of the samples (Fig. 5C). Promoter region methylation was HOXB13 and SYK, resulted in reduced colony formation in vitro detected in 33% of the melanoma cell lines (Fig. 2A) and 20% of and diminished tumor formation in vivo, indicating that these the tumor samples (Fig. 2B) tested. Reexpression of HOXB13 genes function as tumor suppressors in melanoma. occurred on treatment with 5AzadC in two of three cell lines with QPCT was methylated in all melanomas and encodes a promoter region methylation (Fig. 3A). We stably expressed glutaminyl cyclase that converts precursor glutaminyl peptides to HOXB13 in a HOXB13-deficient cell line (MelJuSo) at similar levels their bioactive pyroglutaminyl peptide forms (25, 26). LXN was to that seen in primary melanocytes (Supplementary Fig. S5) to methylated in 95% of melanomas and encodes a global inhibitor of characterize the possible tumor suppressor function of HOXB13 mammalian carboxypeptidases and may function to limit prostate in melanoma cells. Cell proliferation and colony formation were tumor aggressiveness by inhibiting carboxypeptidase 4 (27, 28). reduced in HOXB13-transfected clones compared with vector COL1A2, which was found to be methylated in 80% of melanomas, controls (Fig. 6A). HOXB13-transfected MelJuSo lines exhibited >4- has been found to be frequently hypermethylated in several human fold reduction in tumor size relative to vector controls in malignancies, including breast cancer, hepatocellular carcinomas, xenografting experiments in immunodeficient mice (Fig. 6A and and colorectal cancer (29). Expression of COL1A2 in a tumorigenic C). These results indicate that HOXB13 is a tumor suppressor cell line led to increased adhesion, slower growth, and reduced in melanoma and is targeted for silencing by promoter hyper- colony formation in soft agar, features that are suggestive of a methylation. tumor-suppressive role for COL1A2 (29).

Figure 3. Analysis of expression changes following 5AzadC treatment by quantitative RT-PCR analysis. Shaded circles on top of the columns, presence of methylation in the promoter region of the gene in the corresponding cell line.

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chemotherapeutic drugs may contribute to the drug resistance phenotype commonly observed in malignant melanoma. The SYK cytoplasmic tyrosine kinase plays a role in coupling activated immune receptors to downstream signaling effectors (35). It is expressed in normal breast epithelial tissue and has tumor suppressor properties in breast cancer cells and may affect mitotic progression (36, 37). Promoter hypermethylation of SYK occurs frequently in breast tumors and cell lines and is associated with loss of expression that could be restored on treatment with 5AzadC (38). Similar findings confirming the role of SYK as a tumor suppressor in melanoma have recently been described (39). We now show that promoter hypermethylation is one of the mechanisms that results in loss of SYK expression in melanoma. HOXB13 is a member of the highly conserved HOX transcription factors that regulate differentiation and pattern formation during embryogenesis (40). HOXB13 has been found to negatively regulate wound healing possibly by down-regulating hyaluronic acid

Figure 4. SYK expression in melanoma cell lines and melanoma tumor samples. A, quantitative RT-PCR analysis of SYK mRNA expression in melanoma cell lines compared with primary human melanocytes. B, Western blot of SYK expression in melanoma cell lines. C, quantitative RT-PCR analysis of SYK mRNA expression in tumor samples compared with melanocytes.

Interestingly, two genes involved in the metabolic modification of chemotherapeutic agents were identified with the microarray screen: CYP1B1, which was universally methylated, and DNAJC15, which was methylated in 50% of melanomas. CYP1B1 is a member of the cytochrome P450 family of monooxygenases and has a wide range of substrates, including estrogen, androgens, and chemotherapeutic drugs (30). Methylation of CYP1B1 is associated with a poor prognosis in breast cancer (31). DNAJC15 (also known as DNAJD1 MCJ and ) is inactivated by promoter hypermethylation Figure 5. HOXB13 expression in melanoma cell lines and melanoma tumor in ovarian carcinoma, pediatric brain tumors, and Wilm’s tumor samples. A, quantitative RT-PCR analysis of HOXB13 mRNA expression in (32–34). Loss of DNAJC15 confers resistance to various chemo- melanoma cell lines compared with primary human melanocytes. B, Western blot of HOXB13 expression in melanoma cell lines. C, quantitative RT-PCR therapeutic agents used in the treatment of ovarian cancers (32). analysis of HOXB13 mRNA expression in tumor samples compared with Thus, inactivation of genes involved in metabolic activation of melanocytes. www.aacrjournals.org 11191 Cancer Res 2006; 66: (23). December 1, 2006

Figure 6. A, tumor suppressor characteristics of HOXB13 in vitro and in vivo.a, in vitro proliferation assay of HOXB13-transfected MelJuSo clones compared with vector controls (pTRE). The results are an average of three replicates counted on a flow cytometer (P 120 hours < 0.01). b, representative plates of a colony formation assay comparing vector and HOXB13-transfected MelJuSo clones. c, tumor growth of s.c. xenografts of vector and HOXB13- transfected clones in nude mice. The results represent average of four s.c. injections (P 10 weeks < 0.05). d, representative tumors formed by vector controls and HOXB13-transfected MelJuSo cell line. B, tumor suppressor characteristics of SYK in vitro and in vivo.a, in vitro proliferation assay of SYK-transfected MelJuSo clones compared with vector controls. The results are an average of three replicates counted on a flow cytometer (P 120 hours < 0.01). b, representative plates of a colony formation assay comparing vector and SYK-transfected MelJuSo clones. c, tumor growth of s.c. xenografts of vector and SYK-transfected clones in nude mice showing tumor kinetics over a 10-week period. The results represent average of four s.c. injections (P 10 weeks < 0.05). C, average weights of tumors formed by vector control and HOXB13, SYK-transfected clones at 10 weeks following xenografting. The results represent an average of eight tumors each of vector, HOXB13- transfected clones and four tumors of SYK-transfected clone (P < 0.01).

(41, 42). HOXB13 was found to be down-regulated in prostate assays for CYP1B1, QPCT,andLXN on blood or serum of and colorectal cancer cells, where it has an antiproliferative role melanoma patients could be used as possible staging markers as (43, 44). More recently, HOXB13 was found to be epigenetically has recently been described using the lower frequency markers inactivated in a subset of renal cell carcinomas and had growth- RARB (70%) and RASSF1A (55%; ref. 46). inhibitory effects in vitro (45). We have shown that HOXB13 has tumor-suppressive properties in melanoma and is frequently Acknowledgments inactivated by promoter region hypermethylation. CYP1B1 QPCT Received 4/10/2006; revised 8/24/2006; accepted 9/11/2006. The high rates of methylation of (100%), (100%), Grant support: J. Walter Juckett Postdoctoral Fellowship (V. Muthusamy); K08 and LXN (95%) in uncultured metastatic melanoma tumor samples CA89124, R01 CA112054, and a University of Vermont New Research Initiative Grant suggest that these loci would be ideal markers for a variety of (M. Bosenberg); and P30CA22435 and P20 RR16462 (Vermont Cancer Center DNA Analysis Core Facility). melanoma clinical trials. In particular, QPCT could be used as a The costs of publication of this article were defrayed in part by the payment of page marker for successful induction of demethylation (as per charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supplementary Fig. S4) in melanoma patients being treated with We thank Dr. Carlos Cordon-Cardo for providing several of the melanoma tissue demethylating agents. Alternatively, methylation-specific PCR samples and Dr. Nicholas Heintz for reviewing the article and insightful comments.

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www.aacrjournals.org 11193 Cancer Res 2006; 66: (23). December 1, 2006

Viswanathan Muthusamy, Sekhar Duraisamy, C. Matthew Bradbury, et al.

Cancer Res 2006;66:11187-11193.

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