Elucidation of Epigenetic Inactivation of SMAD8 in Cancer Using Targeted Expressed Gene Display

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[CANCER RESEARCH 64, 1639–1646, March 1, 2004] Elucidation of Epigenetic Inactivation of SMAD8 in Cancer Using Targeted Expressed Gene Display

Kuang-hung Cheng,1,2 Jose F. Ponte,1 and Sam Thiagalingam1,2,3 Departments of 1Medicine (Genetics Program, Pulmonary Center and Cancer Research Center), 2Pathology and Laboratory Medicine, and 3Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts

ABSTRACT signaling and Smad2 and Smad3, which are TGF-␤/actin pathway restricted. The SMAD8 gene, which displays a high degree of homol- To address the challenge of identifying related members of a large ogy to the SMAD1 and SMAD5 genes, was originally described as family of genes, their variants and their patterns of expression, we have MADH6 in human, often referred to as SMAD9 and currently listed as developed a novel technique known as targeted expressed gene display. Here, we demonstrate the general application of this technique by ana- MADH9 (HUGO ID) in EnsEMBL at the genomic location 35220321 lyzing the SMAD genes and report that the loss of SMAD8 expression is to 35292902 bp on chromosome 13 (1–3). The R-Smads are activated associated with multiple types of cancers, including 31% of both breast directly via phosphorylation by type I (RI) receptors after the forma- and colon cancers. Epigenetic silencing of SMAD8 expression by DNA tion of a complex consisting of the ligand-bound heteromeric RI/RII hypermethylation in cancers directly correlates with loss of SMAD8 ex- receptors. Phosphorylated R-Smads interact with the second class of pression. The SMAD8 alteration in a third of breast and colon cancers Smads known as the common mediator Smad (Co-Smad) to form a makes it a significant novel tumor marker as well as a potential thera- heteromeric complex (4). Smad4 is the only member of this class of peutic target. The utility of targeted expressed gene display for the anal- Smads known in mammals. The third class of Smads includes Smad6 ysis of highly homologous gene families as demonstrated by its application and Smad7, which were identified as anti-Smads or inhibitory Smads to the SMAD genes suggests that it is an efficient tool for the identification (I-Smad) because of their ability to act as inhibitors of the signaling of novel members, simultaneous analysis of differential expression patterns, and initial discovery of alterations of expressed genes. pathway (5–7). Because the signaling pathways mediated by the members of the TGF-␤ family are implicated in a number of biological processes, INTRODUCTION including cell differentiation, cell proliferation, determination of cell Methods such as reverse transcription-PCR (RT-PCR), cDNA sub- fate during embryogenesis, cell adhesion, cell death, angiogenesis, traction, differential display, representational difference analysis, se- metastasis, and immunosuppression, it is conceivable that genetic or rial analysis of gene expression, and microarrays have been widely epigenetic anomalies leading to altered expression patterns of various used in the identification of novel transcripts as well as in the assess- Smad molecules could contribute to different aspects of neoplastic ment of their levels of expression in development and various cellular progression (2, 8–11). Although there has been significant progress in processes and diseases, including cancer. Despite the usefulness of elucidating the association between genetic alterations in the SMAD4 these techniques in the overall assessment of genes that are highly gene and cancer, the nature of defects involving the other Smads has divergent at the DNA sequence, accurate and high-throughput evalu- been elusive (12–17). A study aimed at analyzing the expression of ation and discovery of related members of a gene family have re- Smads in 14 colon tumors compared with normal tissue at the protein mained a challenge. These methods have generally been unable to level using immunohistochemistry suggested that the levels were discriminate between different members of the gene families with increased for Smad2, Smad3, or Smad5, only occasionally increased consistency because of the inherent redundancy in DNA sequence for Smad1 or Smad8, and there were no apparent differences in among these unique genes and transcripts. A novel method described Smad4 (18). The apparent lack of genetic alterations in the majority of here, targeted expressed gene display (TEGD), validated using the SMAD genes analyzed thus far in cancer provides compelling support SMAD family of genes as the prototype enabling one to overcome this for the potential role of epigenetic alterations, whereby abnormalities dilemma when gene family members contain at least two regions of in signaling could occur at the level of regulation of gene expression homology separated by a divergent region of variable length. or processing of the transcripts (19–21). Our analysis of the SMAD The discovery of the Smad family of signal transducer proteins as genes provides evidence for the exploitation of the novel TEGD mediators of tumor growth factor (TGF)-␤ and TGF-␤-like cytokine- method described in this article in the initial determination of the mediated signaling from the cell membrane to the nucleus has revo- potential for an epigenetic mode of inactivation of the SMAD genes in lutionized the understanding of the molecular basis of the signaling cancer. On the basis of these observations, we predict that the effec- and inactivation of TGF-␤/BMP pathways in cancer (1). To date, tive utilization of the method described here will find wide use not eight human homologues of the SMAD genes have been identified and only in the discovery of novel members of a family of genes and are classified into three distinct classes based on their structures and splice variants of a specific gene but also for the simultaneous analysis biological functions (1, 2). The first category consists of pathway- of the transcript levels of individual genes or their spliced variants in restricted or receptor-regulated Smads (R-Smads): Smad1, Smad5, various diseases and during development. and Smad8, which are involved in bone morphogenetic protein (BMP) MATERIALS AND METHODS Received 8/27/03; revised 12/17/03; accepted 1/5/04. Grant support: Evans Medical Foundation and the Department of Defense Career Cell Culture, RNA Isolation, and cDNA Synthesis. Cancer cell lines Development Award DAMD 17-01-1-0160 (to S. Thiagalingam). J. F. Ponte was sup- were purchased from American Type Culture Collection or the Coriell Cell ported by the NIH Training Grant T32HL07035. S. Thiagalingam is a Dolphin Trust investigator supported by The Medical Foundation. Repository and culture conditions were followed as suggested by the provider. The costs of publication of this article were defrayed in part by the payment of page Tumor samples, some of the cell lines, and their derivatives or nucleic acids charges. This article must therefore be hereby marked advertisement in accordance with isolated from the samples used in this study were obtained from Subra 18 U.S.C. Section 1734 solely to indicate this fact. Kugathasan (Medical College of Wisconsin), Peter Thomas (Boston University Requests for reprints: Sam Thiagalingam, Genetics Program, Boston University School of Medicine, 715 Albany Street, L320, Boston, MA 02118. E-mail: samthia@ School of Medicine), Douglas Faller (Boston University School of Medicine), bu.edu. Ramon Parsons (Columbia University), and Kornelia Polyak (Dana-Farber 1639

Cancer Institute). RNA isolation and cDNA synthesis from the cell lines and DNA Sequencing. DNA sequence analysis was performed using the Geno- tumor samples were carried out using previously described procedures (22). myxLR analyzer (Beckman Coulter). The cycle sequencing procedure used in Degenerate RT-PCR of the SMAD Genes. On the basis of the amino acid these studies used 33P dideoxynucleotide triphosphates (ddNTPs) (Amersham) sequences of the human Smads 1–8, regions that are identical and conserved along with the ThermoSequanase kit (USB, Cincinnati, OH) as described (MH1 and MH2) among the Smads were mapped out (1, 2). The residues previously (22). targeted for primer design were localized to the MH1 and MH2 domains, and Genomic DNA Isolation. Genomic DNA from cell lines and tumors was the intervening linker regions were highly divergent enabling the generation of isolated using the DNeasy Tissue kit (Qiagen) according to the manufacturer’s PCR products that are of unique size to correspond to specific Smad homo- instructions. logues. The forward and reverse primers were designed based on the mainte- Homozygous Deletion Analysis of SMAD8. Radiolabeled microsatellite nance of codon degeneracy and the representation of the various amino acids markers, D13S927 and D13S928, that are localized to the beginning and end, at a given position among the known Smad family members as determined respectively, of the SMAD8 gene in its genomic contig and gene-specific from the sequence alignment of the various homologues. All primers were primers localized to the 5Ј-untranslated region (SMAD8 UTR-F: 5Ј-GAAA- obtained from Integrated DNA Technologies (Coralville, IA). CATGTGAGGAACAGCAGC-3Ј; and SMAD8 UTR-R: 5Ј-CGAGACAGCG- The SMAD family-specific degenerate primers used for TEGD are as GCTGCAGCAGCG-3Ј) and encompassing the first exon (SMAD8 EX-1F: follows: SmadXF2(5Ј-primer), TNTKBMGVTGGCCNGAYYTBM; SmadXR1 Ј Ј Ј (3Ј-primer), CCAVCCYTTSRCRAARCTBAT (codes for mixing of bases to 5 -GCCTGGTTCTGTTGCTCAGGCTG-3 ; and SMAD8 EX-1R: 5 -GTGT- Ј generate degeneracy: R ϭ A,G; Y ϭ C,T; M ϭ A,C; K ϭ G,T; S ϭ C,G; TCCTGTGGCATTCAGGC-3 )oftheSMAD8 gene were used in PCR am- W ϭ A,T; H ϭ A,C,T; V ϭ A,C,G; D ϭ A,G,T; and N ϭ A,C,G,T). The plifications and gel electrophoretic analysis to determine deletion of this SmadXF2 primer is inclusive of amino acid residues with a consensus RWPDL genomic region (13). in all of the Smads. The nucleotide positions for the RWPDL in the different Analysis of Gene Expression Using Semiquantitative RT-PCR. Total SMADs are in parentheses: SMAD1 (271–292); SMAD2 (414–435); SMAD3 RNA prepared from samples was used for cDNA synthesis, and PCR ampli- (276–297); SMAD4 (294–315); SMAD5 (279–300); SMAD6 (690–711); fication was done essentially as described previously (22). The gene specific SMAD7 (486–507); and SMAD8 (285–306). The SmadXR1 primer is inclusive primer pairs used in the analysis of the indicated specific SMAD genes and the of amino acid residues with a consensus SFVKGW in all of the Smads. The ␤-actin gene used for standardization to normalize the abundance of the nucleotide positions for the SFVKGW in the different Smads are in parenthe- various transcripts analyzed are described in Table 1. ses: SMAD1 (1242–1253); SMAD2 (1248–1259); SMAD3 (1133–1154); The relative abundance of the various SMAD gene-specific PCR products SMAD4 (1509–1530); SMAD5 (1242–1263); SMAD6 (1398–1419); SMAD7 was normalized to ␤-actin or other unaffected SMADs by comparative abun- (1194–1215); and SMAD8 (1137–1158). dance of the products using densitometry. A 20-␮l PCR reaction mixture contained 67 mM Tris-HCl (pH 8.8), 16.6 Processing of Genomic DNA for the Evaluation of Methylation Status. ␤ mM ammonium sulfate, 6.7 mM magnesium chloride, 1 mM -mercaptoetha- For bisulfite sequencing and the methylation-specific PCR (MSP) assay, ␮ nol, 6% DMSO, 100 M each of dATP, dGTP, dCTP, dTTP, and radioactive genomic DNA was isolated from cell lines and primary tumors using the ␮ ␣32 ␮ ␮ ␮ dCTP [0.25 lof P- dCTP (10 Ci/ l); Amersham] for labeling, 20 M Qiagen DNeasy Tissue kit. Genomic DNA was subjected to a deamination each of the primers, 50 ng of cDNA template, and 2.5 units of Platinum Taq reaction by incubation with sodium bisulfite essentially as described previ- (Invitrogen). An initial denaturation at 94°C for 2 min was followed by 30 ously (23). In brief, 0.5–2 ␮g of genomic DNA were denatured with 2 M NaOH cycles, each carried out at 94°C for 30 s, 57°C for 1 min, and 70°C for 1 min for 10 min, followed by bisulfite modification by treatment with freshly and 20 s, and one final extension cycle at 70°C for 10 min. prepared 10 mM hydroquinone and 3 M sodium bisulfite (pH 5.0; Sigma), TEGD Gel Electrophoresis and Recovery of DNA Bands. The samples which converts unmethylated cytosines to uracil but does not change methy- from the degenerate RT-PCR of the SMAD genes were loaded onto a 4.5% denaturing polyacrylamide gel after a 2 min denaturation step at 95°C. Elec- lated cytosines. Each reaction was overlaid with mineral oil and incubated at trophoresis was performed in a GenomyxLR analyzer (Beckman Coulter) for 50°C for 16–20 h. After treatment, the modified DNA was purified using a 4.5 h at 80 W with constant power (voltage not to exceed 2500 V). The gel was Wizard DNA purification kit (Promega, Madison, WI), followed by desulfon- dried and autoradiography performed on the gel. DNA bands of interest on the ation by treating with 3 M NaOH. The ethanol precipitated purified DNA pellet gel were oriented using the autoradiogram, cut out of the gel, and isolated by was dissolved in 30 ␮l of distilled water. soaking the gel slice in 1ϫ 10 mM Tris (pH 8)-1 mM EDTA buffer, freezing Bisulfite Sequencing. The promoter region of the SMAD8 gene containing at Ϫ80°C for 30 min, heating at 60°C for 5 min, and spinning at high speed to CpG islands was first PCR amplified from bisulfite modified DNA (50–100 separate gel fragments from the aqueous-phase containing DNA. The DNA ng) using gene-specific primers (5Ј-GAAATATGTGAGGAATAGTAGTT- fragments were ethanol precipitated and isolated using conventional methods TAG-3Ј and 5Ј-CCACTCATCCCTCCCCCACCCAAATC-3Ј), and the prod- and TA cloned into pCR2.1 (Invitrogen) for sequencing. uct was gel purified. Genomic sequencing of the SMAD8 gene-specific PCR

Table 1 Primers used for semiquantitative reverse transcription-PCR Annealing Major product Gene Primer sequencea temperature size (bp) SMAD1 F: 5Ј-CCACTGGAATGCTGTGAGTTTCC-3Ј 58°C 950 R: 5Ј-GTAAGCTCATAGACTGTCTCAAATCC-3Ј SMAD2 F: 5Ј-GGTAAGAACATGTCCATCTTGCC-3Ј (22) 58°C 950 R: 5Ј-CATGGGACTTGATTGGTGAAGC-3Ј (22) SMAD3 F: 5Ј-CGGGCCATGGAGCTGTGTGAGTTCG-3Ј 62°C 700 R: 5Ј-CGGGTCAACTGGTAGACAGCCTC-3Ј SMAD4 F: 5Ј-GGACAATATGTCTATTACGAATAC-3Ј (22) 57°C 1720 R: 5Ј-TTTATAAACAGGATTGTATTTTGTAGTCC-3Ј (22) SMAD5 F: 5Ј-GTATCAACCCATACCACTATAAGAG-3Ј 55°C 1010 R: 5Ј-CAGAGGGGAGCCCATCTGAGTAAG-3Ј SMAD6 F: 5Ј-CGACTTTGGCGAAGTCGTGTG-3Ј 60°C 1130 R: 5Ј-GGATGCCGAAGCCGATCTTGC-3Ј SMAD7 F: 5Ј-GGTGCGAGGTGCCAAATGTCACC-3Ј 58°C 910 R: 5Ј-GATGAACTGGCGGGTGTAGCAC-3Ј SMAD8 F: 5Ј-CTCTTATGCACTCCACCACCCCCATC-3Ј 58°C 980 R: 5Ј-CTTAAGACATGACTGTTAAGACACTG-3Ј ␤-Actin F: 5Ј-ACACTGTGCCCATCTACGAGG-3Ј 60°C 600 R: 5Ј-AGGGGCCGGACTCGTCATACT-3Ј a F, forward; R, reverse. 1640

Fig. 1. Targeted expressed gene display (TEGD) and tissue-wide expression of SMAD genes. A, schematic representation of TEGD for the SMAD family of genes. MH1 and MH2 indicate highly homologous regions in the amino acid as well as the DNA sequence among the various Smad gene family members. The forward and reverse primers for PCR amplification of the cDNA were designed in the conserved regions as indicated. The radiolabeled PCR products were analyzed by denaturing acrylamide gel electrophoresis. A typical signature banding pattern of the various SMADs is indicated in the bottom panel. B, TEGD analysis of the SMAD family of genes in various tissue types. PCR products for SMADs using degenerate primers were analyzed by TEGD. Lanes 1–17 correspond to PCR products generated using cDNA templates from brain, lung, stomach, heart, liver, spleen, kidney, colon, bone marrow, small intestine, trachea, prostate, uterus, thymus, testis, skeletal muscle, and mammary gland, respectively. The lines on the right hand panel point to distinct PCR products. The approximate size of PCR products in bp is indicated on the left panel. The positions of various SMAD genes and their variants as identified from sequence analysis are indicated on the right panel. C, reverse transcription-PCR analysis of SMAD genes. Semiquantitative reverse transcription-PCR analysis of the indicated SMAD genes and SMADs 1 and 2 (data not shown) was carried out as described under “Materials and Methods.” The cDNA template was derived from total RNA from normal tissues of brain, lung, heart, liver, bone marrow, kidney, spleen, thymus, prostate, testis, uterus, small intestine, mammary gland, skeletal muscle, stomach, and colon, Lanes 1–16, respectively.

product was accomplished by using the DNA sequencing primer, 5Ј-GTAAG- RESULTS TAGGGTTTTTTGGT-3Ј, along with 33P ddNTPs and the ThermoSequanase kit (USB, Cincinnati, OH) as described previously (22). TEGD and Signature Banding Pattern (SBP) of the SMADs. MSP. The methylation status of the SMAD8 promoter region was also Members of the SMAD family of genes have highly homologous analyzed by MSP with the use of primers designed for the amplification of amino acid sequences at their N- and COOH-terminal regions (MH1 defined CpG islands containing DNA sequences of either unmethylated or and MH2 domains, respectively), which are separated by a highly methylated DNA using bisulfite-treated genomic DNA as the template (23). divergent linker region rich in proline, serine, and threonine (1, 2). Sequences of the forward (F) and reverse (R) MSP primers to distinguish These regions may have arisen from divergence because of functional between the methylated (M) and unmethylated (U) genomic DNA used specificities from an ancestral unit of activity that has maintained Ј Ј in this study were as follows: 5 -GATGTGAGGTGATTTATGTAGT-3 some degree of evolutionary conservation at the protein level. The (SMAD8U-F) and 5Ј-CACAACAACCTACAACTCAATTCCCT-3Ј (SMAD8U- examination of the MH domains from various SMAD genes indicated R), and 5-GACGCGAGGCGATTTACG-3Ј (SMAD8M-F) and 5Ј-CGACCACG- TACGCGAAAACTCGCG-3Ј (SMAD8M-R). PCR conditions were as follows: that there are either identical or conserved amino acid residues at 94°C for 2 min, 35 cycles of 94°C for 30 s, 58°C for 1 min, 70°C for 1 min, defined positions, which is consistent with critical structural features followed by a final extension at 70°C for 10 min. A 10-␮l sample of each PCR required for the function of these proteins. The sequence conservation product was mixed with 1ϫ loading buffer and analyzed by electrophoresis on a at the amino acid level is also reflected at the DNA sequence level. nondenaturing 8% polyacrylamide gel and visualized by staining with ethidium Although the delineation of the alterations in SMADs is essential for bromide. the comprehension of the molecular basis of various defective pro- 5؅-Aza-2؅ Deoxycytidine and Trichostatin A (TSA) Treatment. HTB129, cesses, the analysis of defects in individual members in this type of MDAMB468, MDAMB231, CaCo2, H441, CCL230, and HT29 cells were family of genes poses a formidable task for efficient detection in a Ј Ј incubated in culture medium with and without 5 -Aza-2 deoxycytidine high-throughput platform. Success in identifying alterations in SMAD (Sigma) at a concentration of 1–5 ␮M for 7 days or with 300 nM TSA for 24 h. genes could be expected to provide critical information necessary for To assess the effect of a combination of 5Ј-Aza-2Ј deoxycytidine and TSA, cells were exposed sequentially for 7 days to 5Ј-Aza-2Ј deoxycytidine and then deciphering the molecular basis of their functions. The fact that the to TSA for an additional 24 h. Total RNA was isolated and SMAD8 expression SMAD genes contain two distinct highly conserved regions separated was determined by RT-PCR using the primers SMAD8-1F: 5Ј-CAGCTCAG- by a highly variable intervening linker region allowed us to develop a CCTCCTGGCCAAG-3Ј and SMAD8-1R: 5Ј-GAGGAAGCCTGGAATGT- novel screening strategy to simultaneously analyze all of the known CTC-3Ј. members of this family (Fig. 1A). 1641

Fig. 2. Analysis of SMAD expression in cancer. A, targeted expressed gene display analysis of SMAD genes in cancer. PCR products of SMADs generated using degenerate primers as described under Fig. 1 were obtained from different cancers and analyzed by targeted expressed gene display. The cDNA templates used in reactions analyzed on lanes NC, NB, and NS are from normal cells from colon, breast, and stomach tissues; C1-7, B1-7, and S1-4 are from colon, breast, and gastric cancers, respectively. The arrows point to distinct PCR products that were absent compared with the normal control. The positions of various SMAD genes and their variants as identified from sequence analysis are indicated on the right panel. B, SMAD8 expression in cancer cell lines and tumors. Total RNA prepared from cell lines and tumors from the lung, breast and colon cancers were analyzed by reverse transcription-PCR (Lanes 1–14). Lane 1 in each of the different reverse transcription-PCR panels corresponds to the normal sample of the indicated tissue type. SMAD8␣, SMAD8␤, and SMAD8␥ are three of the major differentially spliced forms of SMAD8, which correspond to transcripts that are full-length, that exhibit deletion of exon 2, and that exhibit deletions of exons 2 and 3, respectively. Analysis of the ␤-actin gene is used for normalization and quantitation of the expression of SMAD8.

We have designed degenerate oligonucleotide primers correspond- tative RT-PCR (Fig. 1C). The expression patterns of the various ing to the conserved regions of the SMAD family of genes based on SMADs detected by TEGD remained consistent with the semiquanti- the preservation of codon degeneracy and conserved amino acids at a tative RT-PCR results. Almost all of the SMADs were expressed in all given position among the known Smads for PCR amplification of the of the tissue types that we have analyzed. However, SMAD8 expres- cDNA templates. PCR amplification in the presence of radiolabeled sion was lost in the liver and was decreased to barely detectable levels nucleotides and the subsequent analysis of the products using dena- in the bone marrow and uterus (Fig. 1C). These results indicated to us turing PAGE revealed distinct bands on the gel (Fig. 1, A and B). We that TEGD could be used as a tool for initial diagnostic high-through- recovered the distinct bands corresponding to the PCR products put evaluations to determine SMAD gene expression patterns simul- generated using SMAD-specific degenerate primers and sequenced taneously and with a high degree of efficiency. Thus, TEGD can be them. The bands corresponding to the 1200-, 960-, 840-, 680-, and regarded as a highly improved alternate method that may substitute 570-bp PCR products were found to be identical to the cDNA se- for the traditional multiplex PCR technique not only because of the quences for SMAD4, SMAD1 and SMAD5, SMAD2, SMAD3 and increased level of sensitivity with TEGD and ability to discriminate SMAD8, and SMAD6 and SMAD7, respectively, as predicted from between genes that are closely related at their DNA sequence but also their estimated sizes and sequences (1, 2; Fig. 1B). These results because of the low level of cDNA template required for the analysis. suggested to us that once the SBP of the TEGD is optimized and established such as in this case with the SMAD family of genes, repeat Differentially Spliced Variants of the SMADs. TEGD also en- analysis of gene expression in tissues, or other samples of unknown abled us to identify the various differentially spliced forms of the origin could be easily adopted for a routine high-throughput analysis. SMAD2, SMAD3, SMAD5, and SMAD8 genes (Fig. 1, B and C; data Although we generated and analyzed radiolabeled nucleotide incor- not shown). Alternatively spliced variants of SMAD2 with a deletion porated PCR products in these initial studies, one could also achieve of exon3 (SMAD2⌬exon3; SMAD2␤), SMAD3 with deletions of both the same results using fluorescently or radioactively end-labeled prim- exons 3 and 7 (SMAD3⌬exon3 ⌬exon7; SMAD3␤), SMAD5 with a ers for PCR amplification. deletion of exon3 (SMAD5⌬exon3; SMAD5␤), and SMAD8 with Validation of SMAD Expression Patterns Determined from deletions of either exon3 (SMAD8⌬exon3; SMAD8␤) or both exons 2 TEGD. We confirmed the presence or absence of SMAD expression and3(SMAD8⌬exon2⌬exon3; SMAD8␥) were detected in our anal- determined from TEGD using gene specific primers by semiquanti- ysis. Although one of these variants (SMAD2⌬exon3; SMAD2␤) has been previously reported, the experimental evidence for the existence Table 2 Altered expression of SMAD8 in cancers of the others is presented for the first time in this study (24). However, our study did not verify the existence of two previously reported Samples with loss of SMAD8 Cancer Total no. of samples expression (%) alternatively spliced forms with deletions at the 3Ј-ends, potentially Breast 35 11/35 (31) because of the placement of the TEGD primers inside the affected Colon 41 13/41 (31) sequence of these alternatively spliced forms (25, 26). The encoded Esophagus 4 0/4 (0) Head & Neck 4 2/4 (50) proteins of Smad2, Smad5, and Smad8 resulting from full-length and Lung 19 1/19 (5) variant transcripts that have been described also exhibit differences in Ovary 2 1/2 (50) Pancreas 3 2/3 (65) their biochemical properties (23–25). The overall significance of the Prostate 4 3/4 (75) described and predicted novel spliced forms of SMADs reported both Stomach 4 2/4 (50) here and elsewhere in disease phenotypes, including cancer, requires 1642

Fig. 3. Epigenetic gene silencing of SMAD8 because of altered DNA methylation. A, schematic drawing of the landscape of CpG island methylation patterns in the region of genomic DNA from upstream promoter region of the SMAD8 gene in the 5Ј-untranslated region (5Ј-UTR). Exon I corresponds to the first exon of the SMAD8 gene [MADH9-002 (Vega_transcript ID)]. The flag represents the ATG corresponding to the first methi- onine of the predicted peptide. Vertical lines indicate CpG islands in the DNA sequence. E represents unmethylated cytosines, whereas F represents methylated cytosines as determined by bisulfite sequencing. The circles above the horizon- tal line indicate the methylation pattern observed in the CpG islands of the cell lines that express SMAD8. The circles below the line indicate the methylation pattern of the CpG islands from samples, which lacked SMAD8 expression. The nucleotide sequence of the DNA within the dotted lines is shown with the asterisks, indicating CpG islands. B, bisulfite sequence analysis of the indicated CpG islands of the promoter region of the SMAD8 gene in the cell lines that are either proficient (ϩϩ)or deficient (Ϫ)inSMAD8 expression. Cell lines proficient for SMAD8 expression have no bands in the C lane indicative of conversion of unmethylated cytosines to uracil upon bisulfite treatment. C, methylation-specific PCR (MSP) analysis of the various cancers that have lost or retained SMAD8 expression. The methylation-specific PCR products in lanes U and M indicate the presence of unmethylated and methylated templates, respectively. Pla- cental DNA and in vitro methylated DNA serve as negative and positive controls, respectively.

further study (Refs. 24–26; University of California at Santa Cruz, SMAD3 expression in colon cancer will be dealt with in greater detail Santa Cruz, CA genome browser).4 elsewhere (Cheng and Thiagalingam, unpublished results). TEGD in the Analysis of SMADs in Cancer. The ability to Molecular Mechanism for the Silencing of SMAD8 Expression. simultaneously probe multiple members of a gene family using TEGD From our analysis, loss of expression of the SMAD8 gene was esti- prompted us to apply this technique to analyze differential expression mated to occur in nearly one-third of both breast and colon cancers, patterns of the various SMADs in cancer to validate its use for which are two of the leading causes of cancer deaths in women and in diagnostic screening (Fig. 2A). We were able to use the SBPs estab- general, respectively (Fig. 2B; Table 2). Hence, we investigated lished within the normal tissues to determine the retention or loss of potential mechanisms for the loss of SMAD8 gene expression in specific DNA bands corresponding to the defined full-length and cancer because of the prevalence of this alteration, which is similar to variant transcripts (Figs. 1B and 2A). The TEGD analysis of the or even more frequent than known tumor markers. We examined SMAD genes in cancers leads us to conclude that there is a significant whether genetic alterations such as chromosomal deletions affecting level of loss in the expression of SMAD3 and SMAD8 in colon cancer the SMAD8 gene could lead to the loss of its expression by homozy- and of SMAD8 in breast and other cancers (Table 2). These initial gous deletion analyses. We used microsatellite markers corresponding observations were additionally validated by analyzing the expression to the SMAD8 gene based on the genomic contig as well as by patterns of the SMAD8 gene more carefully using gene specific genomic PCR using primers that amplified the genomic region cor- primers and semiquantitative RT-PCR (Fig. 2B). These results con- responding to the 5Ј-untranslated region and the first exon of the firmed the TEGD data and provided the first clues to suggest that the SMAD8 gene. These experiments indicated that gross genomic dele- SMAD8 gene is a critical target for loss of function due to down- tions are apparently not the major mechanism of SMAD8 inactivation regulation of gene expression in 31% of breast and colon cancers in the affected cancers (data not shown). Therefore, we considered (Table 2). The analysis of establishing the significance of the loss of epigenetic silencing as an alternate mechanism for SMAD8 gene silencing. 4 Internet address: http://genome.ucsc.edu. The genomic sequence of the translated SMAD8 gene (EnsEMBL 1643

Gene ID: ENSG 00000120693; Vega_transcript ID; MADH9-002; exons: 6; transcript length: 2036 bp; chromosome 13: nucleotides 35220321–35251966) and the upstream region was inspected for the presence of CpG islands that may be the targets of DNA hypermethylation and associated chromatin modification effects for their involve- ment in the silencing of SMAD8 gene expression. Several CpG islands in the upstream promoter of the SMAD8 gene were tested as likely candidate regions that could be critical for differential DNA methylation patterns coinciding with the loss of SMAD8 expression (data not shown, Fig. 3). DNA sequence analysis of the bisulfite-treated genomic DNA revealed that CpG islands localized to nucleotides 35292323 to 35292369 of the promoter region (exon 1 of the untranslated hypothetical transcript MADH9-001 (Vega_transcript ID: OT- THUMT00013001062); chromosome 13q12-14 on the reverse strand between RB and BRCA2)oftheSMAD8 gene are only methylated in cancers that exhibit loss of expression (UCSC genome browser; Fig. 3, A and B).4 MSP was carried out using primers designed to these corresponding differentially methylated regions, additionally confirm- ing that the SMAD8 gene is silenced in cancers because of DNA hypermethylation affecting CpG islands in the promoter of the SMAD8 gene (Fig. 3C). DNA Hypermethylation and SMAD8 Expression in Cancer. To directly determine the physiological significance, the role(s) of apparent epigenetic DNA methylation by itself or in combination with histone acetylation/deacetylation on differential regulation of SMAD8 expression in cancers was examined. We chose six cell lines derived from breast, colon, and lung cancers (HTB129, HT29, CaCo2, CCL253, MDAMB468, and H441) that exhibited loss of SMAD8 Fig. 4. The effects of DNA demethylation and inhibition of histone deacetylases on expression and one cell line (MDAMB231) that retained SMAD8 SMAD8 gene expression. The indicated cell lines were treated with 5Ј-aza-2Ј-deoxycyti- Ј Ј dine (5-AZA-dC) for 7 days or with trichostatin A (TSA) for 24 h. To assess the effect of expression as a control and examined the effects of 5 -aza-2 -deoxy- both 5-AZA-dC and TSA simultaneously, cells were exposed sequentially for 7 days to cytidine (a DNA demethylating agent) and/or TSA (an inhibitor of 5-AZA-dC and subsequently to TSA for an additional 24 h. Total RNA and genomic DNA histone deacetylases) on SMAD8 expression. A substantial increase in were isolated and SMAD8 expression and DNA hypermethylation were determined by RT-PCR (A) and MSP (B) analysis, respectively. Demethylation of the regulatory CpG SMAD8 expression was observed with 5Ј-aza-2Ј-deoxycytidine treat- sequences that were analyzed by methylation-specific PCR was additionally confirmed by ment in all of the cell lines, which were previously determined to bisulfite-treated DNA sequencing (data not shown). MDAMB231 cells were used as the exhibit DNA hypermethylation-mediated gene silencing of SMAD8 positive control. (Fig. 4A). TSA by itself caused only a slight increase in the levels of the transcript in two of the tested cell lines (CaCo2 and CCL253) but variable length. TEGD provides a distinct advantage over techniques had no effect on the majority of the tested cell lines. However, there such as differential display, a comparable methodology that has been was a slight up-regulation of SMAD8 expression in the presence of adopted for the simultaneous analysis of multiple genes because of the both drugs (Fig. 4A). MSP analysis and bisulfite-modified DNA latter’s inability to detect differential gene expression patterns of sequence analysis of the target CpG islands in the promoter region of targeted and defined genes. Furthermore, even an improved version of the SMAD8 gene that were differentially methylated in affected and differential display designed to analyze related genes (e.g., kinases) control cell lines revealed that demethylation due to 5Ј-aza-2Ј-deoxy- fell short of efficiently establishing distinct expression patterns of the cytidine treatment accompanies a corresponding increase in SMAD8 related genes and failed to identify novel but related genes with gene expression (Figs. 4, A and B; data not shown). These observa- different functional roles (27–30). tions strongly support the notion that the loss of SMAD8 expression in On the other hand, with TEGD, once a SBP of the TEGD is cancers is primarily mediated by hypermethylation of cis-regulatory optimized and established with an array of different normal tissues CpG islands of the gene. such as in this case with the SMAD family of genes, repeat analysis of gene expression of samples of unknown origin could be easily carried DISCUSSION out in a routine high throughput manner (Figs. 1 and 2). We believe that the TEGD technique should sufficiently address the dilemma of The analysis of highly homologous members of a family of genes efficient simultaneous expression pattern analysis of related genes to detect and establish differential gene expression patterns as well as with relatively minute amounts of samples in clinical and investiga- the genetic or epigenetic alterations responsible for cancer and other tional research settings. The development of an algorithm to predict diseases with limited amounts of clinical sample has remained a the suitability of the applications of TEGD based on the presence of formidable task. Efficient methods to simultaneously analyze the two distinct homologous regions separated by an intervening variable closely related yet functionally divergent genes belonging to families region that would enable the establishment of SBPs from the available would not only be important in accurate diagnosis and prognostic sequences of already identified genes or expressed tag sequences is in evaluation of a disease but could also be exploited for the identifica- progress. We believe that TEGD has the potential to advance the tion of pharmacogenetic targets to customize therapy. We propose that ability to probe gene families for genetic and epigenetic defects to a the TEGD technique described in this article can be effectively used new level of sophistication and could be adopted for routine use in the to analyze families of genes that contain at least two stretches of future. The application of the TEGD technique to simultaneously conserved regions, which are separated by a divergent linker region of analyze multiple members of the SMAD family of genes has not only 1644

Fig. 5. A model for the Smad8 connection to cancer. Bone morphogenetic protein (BMP) signaling is initiated by the association between the BMPs and type I (RI) and type II (RII) heteromeric tetrameric receptors. It is followed by the phosphorylation of the type I receptor (RI) kinase that in turn phosphorylates the receptor-regulated Smads (R-Smad), such as Smad8 and initiates the signaling events. The phosphorylated Smad8 forms a heteromeric complex with the common-mediator Smad, Smad4, and is translocated into the nucleus. In the nucleus, the Smad8/Smad4 hetero-oligomer either by itself or by associating with heterologous Smad-interacting DNA binding proteins or other cofactors, could mediate specific transcriptional ac- tivation or repression responses. The inhibitory Smads (I-Smad) such as Smad6 and Smad7 are able to compete with the R-Smads by stably binding the RI kinase or by preventing association of R-Smads with the common-mediator Smad, effectively blocking the signaling cascade. There are numerous other signaling pathways such as the Ras-MEK pathway that could also modulate the end effects by establishing cross-talk among the different pathway members. BMP signaling is implicated in tumor suppression, bone homeostasis, angiogenesis, and metastasis.

validated the enormous advantage of the technique as an initial ostasis by enhancing osteoclastic bone resorption, leading to osteo- diagnostic tool but also has illustrated an efficient way to identify lytic lesions within the bone (37–40). novel genes that are closely related at the level of their nucleotide Although inactivation of the SMAD2 and SMAD4 genes due to sequence, to identify splice variants of a gene as well as to detect their intrageneic mutations and homozygous deletions has been reported in altered expression patterns. ϳ20% of colorectal cancers, evidence for genetic or epigenetic inac- Our survey of the various SMAD genes using the novel TEGD tivation of other SMAD gene targets at significant levels had remained technique described in this article enabled us to obtain the first clues elusive until this study (2, 22). The loss of expression of SMAD8 in in identifying the SMAD8 gene as an important target for loss of ϳ31% of colon cancers is more significant than any other Smad expression in multiple types of cancers, including ϳ31% of breast and alteration known to date. Determination of whether the affected cells colon cancers (Table 2). This level of alteration is even more frequent play a critical role in tumorigenesis by a mechanism similar to that in than that of the SMAD4 gene, the most frequent target for genetic breast cancer requires further study. Interestingly, the presence of inactivation of the known Smad signaling genes in colon cancer. It is germ-line mutations in the BMP receptor 1A in juvenile polyposis, also more frequent than the HER/neu gene amplification, the most which increase the risk of developing gastrointestinal cancers, sug- celebrated tumor marker for breast cancer, which occurs in ϳ20–30% gests that inactivation of BMP signaling may play a critical role in of breast cancer cases (31). The failure of a previous study to provide colon cancer (41, 42). Although the elucidation of BMP-mediated evidence for an association between the levels of Smad8 expression signaling pathways in which Smad8 is a critical mediator is still in its and colon cancer could be largely because of the unavailability of an infancy, these studies clearly provide the incentive for additional antibody with a high level of specificity to Smad8 at the time of the investigations that may help gain a better understanding of the effects study (18). of Smad8 inactivation in cancer and could pave the way for the The data presented in this article also provides the first direct exploration of its potential use in diagnosis, prognosis, and for tar- evidence that silencing of gene expression via DNA hypermethylation geting in the development of therapeutic modalities. of the SMAD8 gene could be an important event in tumorigenesis of several cancers, including one-third of breast and colon cancers. It is interesting to note that Smad8 is apparently the major target for loss ACKNOWLEDGMENTS of function among the SMAD genes in breast cancer and is an We thank our colleagues, Subra Kugathasan (Medical College of Wiscon- R-Smad, which becomes phosphorylated during BMP signaling sin), Peter Thomas (Boston University School of Medicine), Douglas Faller events and modulates BMP-responsive genes, including those that (Boston University School of Medicine), Ramon Parsons (Columbia Univer- may affect bone homeostasis (Refs. 32–36; Fig. 5). Additionally, sity), and Kornelia Polyak (Dana-Farber Cancer Institute) for generously Smad signaling events via the BMP cytokines are also implicated in donating reagents and cell lines and Andrea Russo and the anonymous review- other signaling events that regulate biological processes, including ers for critical reading of the manuscript. cell differentiation, proliferation, determination of cell fate during embryogenesis, cell adhesion, cell death, angiogenesis, metastasis, and immunosuppression (1, 2; Fig. 5). Although it is intriguing that REFERENCES metastasis to bone is often associated with advanced stage breast and 1. Thiagalingam, S., Cheng, K-H., Foy, R. L., Lee, H. J., Chinnappan, D., and Ponte, other cancers, additional studies would be required to understand J. F. TGF-␤ and its Smad connection to cancer. Curr. Genom., 3: 449–476, 2002. 2. Massague, J. TGF-␤ signal transduction. Annu. Rev. Biochem., 67: 753–791, 1998. whether metastatic breast cancer cells defective in Smad8 signaling 3. Watanabe, T. K., Suzuki, M., Omori, Y., Hishigaki, H., Horie, M., Kanemoto, N., could be responsible for causing an imbalance in normal bone home- Fujiwara, T., Nakamura, Y., and Takahashi, E. Cloning and characterization of a 1645

novel member of the human Mad gene family (MADH6). Genomics, 42: 446–451, 21. Jones, P. A., and Laird, P. W. Cancer epigenetics comes of age. Nat. Genet., 21: 1997. 163–167, 1999. 4. Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, 22. Thiagalingam, S. Molecular detection of Smad4/Smad2 alterations in colorectal J. L. T␤RI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2- tumors: Colorectal Cancer Methods and Protocols. In: S. M. Powell (ed.), Methods in Smad4 complex formation and signaling. J. Biol. Chem., 272: 27678–27685, 1997. Molecular Medicine, Vol. 50, pp. 149–165. New Jersey: Humana Press, Inc., 2001. 5. Nakao, A., Afrakhte, M., Moren, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, 23. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. Methy- S., Kawabata, M., Heldin, N. E., Heldin, C. H., and ten Dijke, P. Identification of lation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Smad7, a TGF-␤-inducible antagonist of TGF-␤ signalling. Nature (Lond.), 389: Natl. Acad. Sci. USA, 93: 9821–9826, 1996. 631–635, 1997. 24. Yagi, K., Goto, D., Hamamoto, T., Takenoshita, S., Kato, M., and Miyazono, K. 6. Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J., Kawabata, M., and Alternatively spliced variant of Smad2 lacking exon 3. Comparison with wild-type Miyazono, K. Smad6 inhibits signaling by the TGF-␤ superfamily. Nature (Lond.), Smad2 and Smad3. J. Biol. Chem., 274: 703–709, 1999. 389: 622–626, 1997. 25. Jiang, Y., Liang, H., Guo, W., Kottickal, L. V., and Nagarajan, L. Differential 7. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., Richardson, expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D. The stem cells and leukemia. Blood, 95: 3945–3950, 2000. MAD-related protein Smad7 associates with the TGF-␤ receptor and functions as an 26. Nishita, M., Ueno, N., and Shibuya, H. Smad8B, a Smad8 splice variant lacking the antagonist of TGF-␤ signaling. Cell, 89: 1165–1173, 1997. SSXS site that inhibits Smad8-mediated signalling. Genes Cells, 4: 583–591, 1999. 8. Massague, J., and Wotton, D. Transcriptional control by the TGF-␤/Smad signaling 27. Liang, P. A decade of differential display. Biotechniques, 33, 338–344, 346, 2002. system. EMBO J., 19: 1745–1754, 2000. 28. Okada, M., Yamaga, S., Yasuda, S., Weissman, S. M., and Yasukochi, Y. Differential 9. de Caestecker, M. P., Piek, E., and Roberts, A. B. Role of transforming growth factor display of protein tyrosine kinase cDNAs from human fetal and adult brains. Bio- ␤ signaling in cancer. J. Natl. Cancer Inst. (Bethesda), 92: 1388–1402, 2000. techniques, 32: 856, 858, 860, 863–865, 2002. 10. Massague, J., Blain, S. W., and Lo, R. S. TGF-␤ signaling in growth control, cancer, 29. Bosch, I., Melichar, H., and Pardee, A. B. Identification of differentially expressed and heritable disorders. Cell, 103: 295–309, 2000. genes from limited amounts of RNA. Nucleic Acids Res., 28: e27, 2000. 11. Letterio, J. J., and Roberts, A. B. Regulation of immune responses by TGF-␤. Annu. 30. Ranamukhaarachchi, D. G., Rajeevan, M. S., Vernon, S. D., and Unger, E. R. Rev. Immunol., 16: 137–161, 1998. Modifying differential display polymerase chain reaction to detect relative changes in 12. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., gene expression profiles. Anal. Biochem., 306: 343–346, 2002. Zborowska, E., Kinzler, K. W., Vogestein, B., and Wilson, J. K. V. Inactivation of the 31. Zujewski, J. A. “Build quality in”-HER2 testing in the real world. J. Natl. Cancer Inst. type II TGF-␤ receptor in colon cancer cells with microsatellite instability. Science (Bethesda), 94: 788–789, 2002. (Wash. DC), 268: 1336–1338, 1995. 32. Hogan, B. L. M. Bone morphogenetic proteins: multifunctional regulators of verte- 13. Thiagalingam, S., Lengauer, C., Leach, F. S., Schutte, M., Hahn, S. A., Overhauser, brate development. Genes Dev., 10: 1580–1594, 1996. J., Willson, J. K. V., Markowitz, S., Hamilton, S. R., Kern, S. E., Kinzler, K. W., and 33. Hogan, B. L. M. Morphogenesis. Cell, 96: 225–233, 1999. Vogelstein, B. Evaluation of candidate tumour suppressor genes on chromosome 18 34. Massague, J., and Chen, Y. G. Controlling TGF-␤ signaling. Genes Dev., 14: in colorectal cancers. Nat. Genet., 13: 343–346, 1996. 627–644, 2000. 14. Eppert, K., Scherer, S. W., Ozcelik, H., Pirone, R., Hoodless, P., Kim, H., Tsui, L-C., 35. Attisano, L., and Wrana, J. L. Smads as transcriptional co-modulators. Curr. Opin. Bapat, B., Gallinger, S., Andrulis, I. L., Thomsen, G. H., Wrana, J. L., and Attisano, Cell Biol., 12: 235–243, 2000. L. MADR2 maps to 18q21 and encodes a TGF-␤ regulated MAD related protein that 36. Miyazono, K., Kusanagi, K., and Inoue, H. Divergence and convergence of TGF-␤/ is mutated in colorectal carcinoma. Cell, 86: 543–552, 1996. BMP signaling. J. Cell. Physiol., 187: 265–276, 2001. 15. Riggins, J. G., Thiagalingam, S., Rozenblum, E., Weinstein, C. L., Kern, S. E., 37. Coleman, R. E., Smith, P., and Rubens, R. D. Clinical course and prognostic factors Hamilton, S. R., Willson, J. K. V., Markowitz, S., Kinzler, K. W., and Vogelstein, B. following bone recurrence from breast cancer. Br. J. Cancer, 77: 336–340, 1998. MAD-related genes in the human. Nat. Genet., 13: 347–349, 1996. 38. Coleman, R. E., and Rubens, R. D. The clinical course of bone metastases from breast 16. Schutte, M., Hruban, R. H., Hedrick, L., Cho, K. R., Nadasdy, G. M., Weinstein, cancer. Br. J. Cancer, 55: 61–66, 1987. C. L., Bova, G. S., Isaacs, W. B., Cairns, P., Nawroz, H., Sidransky, D., Casero, R. A., 39. Reddi, A. H., Roodman, D., Freeman, C., and Mohla, S. Mechanisms of tumor Jr., Meltzer, P. S., Hahn, S. A., and Kern, S. E. DPC4 gene in various tumor types. metastasis to the bone: challenges and opportunities. J. Bone Miner. Res., 18: Cancer Res., 56: 2527–2530, 1996. 190–194, 2003. 17. Riggins, J. G., Kinzler, K. W., Vogelstein, B., and Thiagalingam, S. Frequency of 40. Reddi, A. H. Role of morphogenetic proteins in skeletal tissue engineering and Smad gene mutations in human cancers. Cancer Res., 57: 2578–2580, 1997. regeneration. Nat. Biotechnol., 16: 247–252, 1998. 18. Korchynskyi, O., Landstrom, M., Stoika, R., Funa, K., Heldin, C. H., ten Dijke, P., 41. Howe, J. R., Howe, J. R., Bair, J. L., Sayed, M. G., Anderson, M. E., Mitros, F. A., and Souchelnytskyi, S. Expression of Smad proteins in human colorectal cancer. Int. Petersen, G. M., Velculescu, V. E., Traverso, G., and Vogelstein, B. Germline J. Cancer, 82: 197–202, 1999. mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile 19. Thiagalingam, S., Cheng, K-H., Lee, H. J., Mineva, N., Thiagalingam, A., and Ponte, polyposis. Nat. Genet., 28: 184–187, 2001. J. F. Histone deacetylases: Unique players in shaping the epigenetic histone code. 42. Sayed, M. G., Ahmed, A. F., Ringold, J. R., Anderson, M. E., Bair, J. L., Mitros, Ann. N. Y. Acad. Sci., 983: 84–100, 2003. F. A., Lynch, H. T., Tinley, S. T., Petersen, G. M., Giardiello, F. M., Vogelstein, B., 20. Baylin, S. B. Tying it all together: epigenetics, genetics, cell cycle, and cancer. and Howe, J. R. Germline SMAD4 or BMPR1A mutations and phenotype of juvenile Science (Wash. DC), 277: 1948–1949, 1997. polyposis. Ann. Surg. Oncol., 9: 901–906, 2002.

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Kuang-hung Cheng, Jose F. Ponte and Sam Thiagalingam

Cancer Res 2004;64:1639-1646.

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