Preferential loss of a polymorphic RIZ allele in human hepatocellular carcinoma

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1 doi: / bjoc , vilble online t on Preferentil loss of polymorphic RIZ llele in humn heptocellulr crcinom W Fng 1, Z Pio 1, IM Buyse 1#, D Simon 2, JC Sheu 3, M Perucho 1 nd S Hung 1 1 Progrm in Oncogenes nd Tumor Suppressor Genes, The Burnhm Institute, L Joll, CA 92037; 2 Deprtment of Pthology, MCP-Hhnemnn School of Medicine, Phildelphi, PA 19109; 3 Deprtment of Internl Medicine, Ntionl Tiwn University Hospitl, No. 1, Chng-Te street, Tipei, Tiwn Summry The RIZ (PRDM2) locus commonly undergoes loss of heterozygosity (LOH) nd mps within the miniml deleted region on 1p36 in heptocellulr crcinom (HCC). Although peptide-ltering muttions of RIZ re rre in HCC, the RIZ1 product is commonly lost in HCC nd hs tumour suppressive ctivities. Here, we nlysed RIZ gene muttions nd LOH in HCC, brest cncer, fmilil melnom, colon cncer, nd stomch cncer. We found 7 polymorphisms but no muttions. By nlysing the Pro704-deletion polymorphism, we detected LOH of RIZ in 31 of 79 (39%) informtive HCC cses, 11 of 47 (23%) colon cncer cses, 8 of 43 (19%) brest cncer cses, 8 of 66 (12%) stomch cncer cses. Importntly, loss of the Pro704 + llele ws found in 74% of the 31 LOH positive HCC cses (P < 0.01), indicting preferentil loss nd hence stronger tumour suppressor role for this llele compred to the P704 llele. In ddition, the Pro704 + llele ws found to be more common in Asins (0.61) thn Cucsins (0.42) (P = ), suggesting n interesting link between gene polymorphisms nd potentil differences in tumour incidence between rcil groups. Keywords: RIZ (PRDM2); polymorphisms; heptom Inctivtion of tumour suppressor genes plys n importnt role in humn cncer formtion nd progression. The distl short rm of chromosome 1 or 1p36 is thought to hrbour severl tumour suppressor genes becuse loss of heterozygosity (LOH) in this region is common in lrge number of different types of humn cncers (Weith et l, 1996). One cndidte in this region is the retinoblstom protein-intercting zinc finger gene RIZ(PRDM2), which ws isolted in functionl screening for Rb-binding proteins (Buyse et l, 1995), lso independently isolted s DNA-binding protein MTB-Zf (Muros et l, 1996), GATA3 trnscription fctor binding protein G3B (Shpiro et l, 1995), nd coctivtor of oestrogen receptor (ER) (Abbondnz et l, 2000). The gene mps within the miniml deleted region on 1p36 in liver, brest nd fmilil colon cncers (Chdwick et l, 2000; Fng et l, 2000). RIZ is member of gene fmily which shres ~ 130 mino cid motif clled the PR (PRDI-BF1 nd RIZ) domin, lso termed SET (Suvr3-9, Enhncer-of-zeste, Trithorx); the fmily is known to ply n importnt role in chromtin-medited gene expression, development nd cncer (Hung et l, 1998). The PR/SET domin shows significnt homology with plnt protein lysine methyltrnsferse (MTse); the PR/SET domin of SUV39H1 cn methylte histone H3 nd plys n importnt role in chromtin condenstion during mitosis (Re et l, 2000). The PR/SET domin represents the ctlytic core motif nd ppers to define lrge fmily of protein lysine methyltrnsferses. The RIZ gene produces 2 products through lterntive promoters, RIZ1 tht contins the PR domin nd RIZ2 lcking the motif (Liu et l, 1997). Decresed or lost expression of RIZ1 mrna but not of RIZ2 is found in ll humn cncers exmined, including those of Received 21 June 2000 Revised 20 November 2000 Accepted 29 November 2000 Correspondence to: S Hung brest, liver, lung, colon, nd neuroendocrine tissues, suggesting tumour suppressor role for the RIZ1 product (He et l, 1998; Jing et l, 1999; Chdwick et l, 2000). In ddition, frequent frme shift muttions of RIZ pper selected in gstrointestinl, endometril, nd pncretic tumours ssocited with microstellite instbility (Chdwick et l, 2000; Pio et l, 2000; Skurd et l, 2001). Together, these findings show frequent inctivtion of RIZ1 in brod spectrum of humn cncers. Consistently, recombinnt denovirus-medited RIZ1 expression cn induce G2/M cell cycle rrest, poptosis, or both in severl tumour cell lines (He et l, 1998; Jing et l, 1999; Chdwick et l, 2000). To further determine the role of RIZ in HCC nd other 1p36-linked humn cncers, we here studied RIZ gene muttions nd LOH in HCC, brest cncer, fmilil melnom, colon cncer nd stomch cncer. The previously described Pro704-deletion polymorphism (Fng et l, 2000) ws nlysed for its potentil differentil involvement in crcinogenesis. The distribution of this polymorphism in Asin nd Cucsin popultions were lso determined. MATERIALS AND METHODS Tissue DNAs Blood DNAs of 6 ptients from 6 different melnom fmilies were exmined for peptide-ltering muttions in the entire coding region of RIZ1 by SSCP nlysis. The identifiction numbers of these DNAs were , , , , , nd These DNAs hve been described previously (Ble et l, 1989). 147 HCC cses were included in the study. Among these, 39 were collected t the Yonsei University Medicl Center, Seoul, # Present ddress: Deprtment of Moleculr nd Humn Genetics, Bylor DNA Dignostic Lbortory, Bylor College of Medicine, One Bylor Plz, Houston, TX

2 744 W Fng et l Kore, which hve been described previously (Pio et l, 1998, 1998b); 10 were collected t the University of Mimi nd University of Pittsburgh, which hve been described in Hmmond et l (1999) nd (Simon et l 1991); 98 were collected t the Ntionl Tiwn University Hospitl, which hve been described in (Lin et l, 1999). 40 brest cncer cses, 46 primry colon cncer cses, nd 86 stomch cncer cses were lso included in this study which were collected t the Yonsei University Medicl Center, Seoul, Kore. In ddition, 60 colon cncer cses, 39 stomch cncer cses, nd 41 brest cncer cses were obtined from the Connective Tissue Network t the University of Albm; the DNA from these tumours nd their djcent non-tumorl tissues were extrcted using stndrd procedures. DNAs from 30 norml Cucsin individuls were collected t the University of Leuven, Belgium. In ddition, DNAs from 13 norml Cucsin individuls were collected t McGill University, Montrel, Quebec (kind gift of Dr C Polychronkos of McGill University). SSCP, llelotyping, nd DNA sequencing nlysis PCR primers for SSCP nlysis of the PR domin region of RIZ1 including exons 2 7 were described previously. PCR primers for the remining prt of RIZ1 gene including exons 8 9 re listed in Tble 1. PCR rections were crried out in mixture of 20 µl contining 1.5 mm MgCl 2, 20 pmol primer, 0.2 mm ech datp, dgtp, dttp, 5 µm dctp, 1 µci α- 32 P-dCTP (3000 Ci mmol 1 ; NEN DuPont, Boston, MA), 25 ng of smple DNA, 1X PCR buffer nd 1.25 U Tq polymerse. After denturtion t 95 C for 4 min, DNA mplifiction ws performed in cycles consisting of denturtion t 95 C for 30 s, primer nneling t Tble 1 PCR primers for SSCP nlysis of RIZ gene Primers Coordintes Sequences (5 to 3 ) RP111 b intron 8 (622) CTT CTG CTT CCA TGT GCT RP CCC TCG TCT TCC AAC TC RP110 c 891 AGA AGA AGC CAG CAT GCC RP GCA CGG ATG AAG TTC TTT AA RP143 c 1413 GTC GTA GAA GAG AAT GGG RP CTC ACT ATT TGT GCT GCC RP GGC AGC ACA AAT AGT GAG RP ACT CCA TGC TGG TGA GTC RP29 c 2349 AGC ATG GAG TTT GTC TG RP GGT GTG GAC TCA ACA GT RP AGA TCC TGA CCT CGG TC RP GAC TGT GCG GTG GCA T RP CTT CCA GTG CAT CTC CAC A RP CTG GGT TTC AGA CCT TCA RP TGC TGC TGC ACA GGA TGT RP CGC TGG TGC TGC TGC A RP ATC TTT GTG TGT TCT GT RP TCG TGT AAA GCT CTT CAG RP GAG GAG TTA AAT GAT TC RP CTT CTT GTC ACT TGC AGA RP GGT GTC GAC AAT ATG CC RP GCT TTC TGT ACT AGC TG RP TGT CGT CGA ATA AGC TC RP TGT CCA CCT TTC TTA GA RP GCC GCC TTC AGC TGT CC RP TTC TGC TTG GAC CTG AAG RP TTG GGC AAG ACC AGA GC RP AAG CTG AGC AGA ATG A RP CCG ATA AGA ATG GCC AAA RP137 intron (5039) CCC ACC AGC TCC TGA GC RP CTA CAG CCT CCG CTT GGC G RP AGC AGC CAG AGT GTC TA The first nucleotide of the humn RIZ1 cdna coding region is designted 1. b These primers re locted within introns; the positions of the exon boundries re listed in brckets. c The PCR product of RP110+RP142 ws digested with Nsil before SSCP gel nlysis. The PCR product of RP143+RP118 ws digested with Hpll before SSCP gel nlysis. The PCR product of RP29+RP134 ws digested with EcoRI before SSCP gel nlysis.

Preferentil loss of polymorphic RIZ llele in HCC 745 55 60 C for 30 s, nd elongtion t 72 C for 30 s. GC-melt kit (Clontech, CA) ws used to PCR the GC rich exon 6.

3 Preferentil loss of polymorphic RIZ llele in HCC C for 30 s, nd elongtion t 72 C for 30 s. GC-melt kit (Clontech, CA) ws used to PCR the GC rich exon 6. Amplified DNA ws diluted 2-fold with stop solution (95% formmide, 20 mm EDTA, 0.05% xylene cynol nd 0.05% bromophenol blue). 3 microlitres of mplified product were loded onto MDE gel (FMC BioProducts, Rocklnd, ME 04841) for SSCP nlysis or 6% polycrylmide gel contining 5.6 M ure. For some PCR products, restriction digestion ws performed before loding. The gel ws dried on filter pper nd exposed to Kodk XAR-5 film. Allelic loss ws scored when bnd intensity of one llelic mrker ws significntly decresed (more thn 70% reduction) in tumour DNA s compred with tht in norml DNA. All vrints detected by SSCP nlysis were confirmed by DNA sequencing nlysis. Sttisticl nlysis We correlte the LOH frequency with severl clinicl spects, including sex, ge, tumour size, nd sttus of heptitis B or C virus infection. The sttisticl nlysis ws performed by the computer progrm STATISTICA (SttSoft, Tuls, OK, USA). The P vlues were obtined by Chi-squre test. RESULTS Lck of peptide ltering muttions in RIZ1 in fmilil melnom nd brest cncers A locus for fmilil cutneous mlignnt melnom-dysplstic nevus hs been mpped to the region between n nonymous DNA mrker (D1S47) nd the gene locus for prontrodiltin (NPPA) (Ble et l, 1989). To determine whether fmilil mlnom my crry peptide ltering muttions in RIZ which mps just distl to NPPA, we performed SSCP nlysis of DNAs from 6 fmilil melnom ptients. We did not detect muttions in the entire coding region of RIZ1 but found severl polymorphic lleles of RIZ s shown in Tble 2. 4 of these were silent nucleotide substitutions without ffecting mino cid sequences. 2 were mino-cid substitutions, D283E nd S450N. One ws deletion of proline t codon 704 tht hs previously been described (Fng et l, 2000). We lso scnned the PR domin region of RIZ1 in 61 brest cncer cses by SSCP nlysis. We did not find ny muttions besides the polymorphisms. LOH of RIZ in humn cncers nd preferentil deletion of the Pro704 + llele in HCC Becuse of the reltively high rte of heterozygosity of the Pro704-deletion polymorphism (~50%), we used it to study whether LOH t the RIZ locus my be common in humn cncers. We were lso interested to determine whether this polymorphism my show differentil linkge with humn cncers. A totl of 459 tumour cses were nlysed nd 235 of these were heterozygous for Pro704-deletion polymorphism s shown in Tble 3. The tumours studied included HCC, brest cncer, colon cncer nd stomch cncer. 58 of the 235 (25%) informtive cses showed LOH t the RIZ locus. LOH ws most common in HCC (31 of 79 or 39%), followed by colon crcinom (11 of 47 or 23%), brest cncer (8 of 43 or 19%), nd stomch cncer (8 of 66 or 12%). LOH of RIZ ppered more common in metsttic colon cncer (3/7) thn primry colon crcinom 8/40, but there ws no sttisticl significnce. No significnt ssocition of RIZ LOH with ny clinicl prmeters of these tumours ws observed, Notbly, the Pro704 + llele ws lost in 74% of the 31 LOHpositive HCC cses, indicting significnt preferentil loss of this llele compred to the Pro704 llele (P < 0.01) (Figure 1). This preferentil loss of Pro704 + llele did not correlte with ny clinicl prmeters of HCC, including gender, ge, tumour size, serum α-fetoprotein, nd sttus of heptitis B or C virus infection. A lrger smple size will be needed to determine whether preferentil loss of Pro704 + llele ws lso significnt in brest cncer Tble 2 RIZ polymorphic vrints Vrint forms PCR primers Het frequency R100 (CGA) to R100 (CGG) RP257 ND RP258 D283 (GAT) to E283 (GAA) RP % (n = 28) RP112 H364 (CAT) to H364 (CAC) RP % (n = 8) RP142 S450 (AGT) to N450 (AAT) RP % (n = 8) RP142 P704 (CCT) deletion RP % (n = 502) RP105 S1609 (TCG) to S1609 (TCA) RP % (n = 8) RP137 P1707 (CCG) to P1707 (CCA) RP95 N.D. RP100 ND = not determined N C N C N C N C N C N C N C Fig. 1 Preferentil loss of P704 + llele in humn HCC. Representtive utordiogrphs of LOH nlysis of the Pro704-deletion polymorphism. The HCC (C) nd mtched non-tumour tissue (N) re shown with cse numbers indicted on the top. Tumour cses 5 7 showed loss of the upper bnd representing the P704 + llele. Tble 3 LOH of RIZ locus in humn cncers P704 + del. P704 del. LOH rte HCC 23 8 (P < 0.01) 31/79 (39%) Colon cncer /47 (23%) Brest cncer 5 3 8/43 (19%) Stomch cncer 5 3 8/66 (12%) Totl /235 (25%) Tble 4 Distribution of the Pro704-deletion polymorphism in Koren HCC nd non-hcc ptients P704 + hom P704 hom Het Totl P704 freq HCC non-hcc Genomic DNAs of norml tissues from cncer ptients were used for the study.

4 746 W Fng et l (63% of 8 LOH positive cses), stomch cncer (63% of 8 LOH positive cses), nd colon cncer (55% of 11 LOH positive cses). Given the differentil involvement of the Pro704-deletion polymorphism with HCC, we next sked whether the Pro704 llele my be more enriched in HCC ptients reltive to non-hcc ptients. Becuse different rcil groups showed different llele frequency (see below), we limited our nlysis to Koren ptients. The Pro704 llele frequency in Koren HCC ptients (0.449) ws higher thn tht in non-hcc ptients (0.375) (Tble 4). But, this ws not sttisticlly significnt (P = 0.23). Distribution of the Pro704-deletion polymorphism in Cucsin nd Asin popultions Anlysis of DNAs from non-tumour tissues of 150 Cucsin cncer ptients from Americ showed tht Pro704 + llele (0.43) ws less common thn the Pro704 llele (Tble 5). This llele frequency ws found to be similr mong different groups of ptients with different cncers, indicting no preferentil ssocition of this llele with ny specific type of cncers. 43 norml Cucsins from Cnd nd Belgium were lso nlysed nd the Pro704 + llele (0.36) ws gin less common. The results did not show significnt vrition between Cucsin norml nd ptient popultions in the distribution of the Pro704-deletion polymorphism. The distribution of the Pro704-deletion polymorphism ws studied in 309 Asin cncer ptients using their norml tissue DNAs. The Pro704 + llele ws the more common llele (0.61). This is significntly different from Cucsin cncer ptient or norml popultions (P = ). DISCUSSION The RIZ1 product of the RIZ locus on 1p36 hs been suggested s cndidte tumour suppressor. Although it mps within the region thought to hrbour the fmilil melnom locus, our dt here suggest tht peptide-ltering muttions in RIZ my not be common in fmilil melnom. Whether other types of RIZ ltertions my be involved in this disese remins to be investigted. We showed tht LOH of RIZ ws common but tht peptideltering muttions in RIZ1 were rre in brest cncers. The pprent selection for RIZ2 expression in brest cncers my hve fvoured the strtegy of RIZ1 gene silencing rther thn ltering RIZ1 peptide sequences. This is similr to previous findings of lck of RIZ1 muttions in HCC where RIZ2 expression is uniformly present (Jing et l, 1999; Fng et l, 2000). These observtions suggest tht decresing RIZ1 expression my represent the more common wy of inctivting RIZ1, t lest in brest cncer nd HCC. Recent dt suggest tht decresed RIZ1 expression ws through DNA methyltion of promoter CpG islnd (Y. Du nd S.H., in preprtion). While the importnce of genetic muttions in cncer hs long been recognized, the pprecition of epigenetic inctivtion is more recent (Jones nd Lird, 1999; Bylin nd Hermn, 2000; Eng et l, 2000). Recent studies hve firmly estblished methyltion s one potentil hit in modified Knudson s two-hit model (Jones nd Lird, 1999). Thus, RIZ1 gene silencing nd LOH could together chieve the two-hit inctivtion of RIZ1. Humn HCC, colon crcinom, nd brest cncers re known to show LOH of 1p36 mrkers (Genurdi et l, 1989; Simon et l, 1991; Brdi et l, 1993; Kuroki et l, 1995). The miniml deleted regions in these cncers include the RIZ locus. Our results here confirm previous findings nd directly demonstrte LOH of RIZ in these cncers. To the best of our knowledge, there hs been no published report of LOH of 1p36 in stomch cncers. Here, we detected low frequency of LOH of RIZ in stomch cncer (12% in 66 stomch cncer cses), which indictes either minor role of RIZ or 1p36 in this cncer or simply bckground rte chromosoml instbilities. The preferentil loss of the Pro704 + llele in HCC suggests tht this llele my be stronger suppressor llele thn the Pro704 llele. This notion would predict tht individuls homozygous for the Pro704 llele would be more susceptible to tumour formtion thn those who re homozygous or heterozygous for the Pro704 + llele. At lest two observtions re consistent with this prediction. First, our dt did indicte trend towrd enrichment of the Pro704 llele in Koren HCC ptients (Tble 4). Tble 5 Distribution of the Pro704-deletion polymorphism P704 + hom P704 hom Het Totl Cucsin ptients (P704 + frequency = 0.43) US HCC US colon cncer US brest cncer US stomch cncer Totl 27 (17.5%) 49 (32.5%) 74 (50%) 150 Cucsin norml (P704 + frequency = 0.37) Cndin Belgin Totl 6 (14%) 18 (41.9) 19 (44.2%) 43 Asin ptients (P704 + frequency = 0.61) C. HCC b K. HCC K. brest cncer K. colon cncer K. stomch cncer Totl 109 (34.9%) 39 (13.1%) 161 (51.9%) 309 Genomic DNAs of norml tissues from cncer ptients were used for the study. b C = Chinese; K = Koren.

5 Preferentil loss of polymorphic RIZ llele in HCC 747 Second, the Pro704 llele is more enriched in Cucsin popultion compred to Asin popultion, which correltes with the known higher tumour incidence of Cucsins (see below). Nonetheless, this prediction needs to be further verified by future crefully controlled epidemiologicl studies. Also, the role of the Pro704 deletion polymorphism in non-hcc cncers will need to be further determined. Our dt show tht the Pro704 + llele of RIZ is significntly more common in cncer ptients of Koren nd Chinese thn in those of Cucsin. Becuse we did not observe significnt difference between cncer ptients nd norml individuls of Cucsin origin in the distribution of the Pro704 + llele, we conclude tht the llele frequency observed in Asin cncer ptients is lrgely representtive of norml popultions. In turn, we conclude tht the Pro704 + llele is more common in Asin thn Cucsin popultions. This conclusion revels n interesting link between stronger suppressor llele nd lower tumour incidence: Koren nd Chinese hve long been noted to hve lower overll tumour incidence thn Cucsins (Lndis et l, 1999; Registry, 1995). Although it remins n unresolved issue whether genetic fctors my exist to explin the disprity in tumour incidence between different rcil groups, our dt here suggests tht gene polymorphisms cn not be excluded. ACKNOWLEDGEMENTS This work ws support by NIH grnt R01-CA76146 to SH. We thnk Drs. A. Goldstein nd P. Tucker for genomic DNAs of fmilil melnom ptients nd C Polychronkos for genomic DNAs of norml individuls. REFERENCES Abbondnz C, Medici N, Nigro V, Rossi V, Gllo L, Piluso G, Belsito A, Roscigno A, Bontempo P, Puc AA, Molinri AM, Monchrmont B, Puc GAB nd Puc GA (2000) The retinoblstom-intercting zinc-finger protein RIZ is downstrem effector of estrogen ction. Proc Ntl Acd Sci USA 97: Ble SJ, Drcopoli NC, Tucker MA, Clrk WH, Jr, Frser MC, Stnger BZ, Green P, Donis-Keller H, Housmn DE nd Greene MH (1989) Mpping the gene for hereditry cutneous mlignnt melnom-dysplstic nevus to chromosome 1p. N Engl J Med 320: Brdi G, Pndis N, Fenger C, Kronborg O, Bomme L nd Heim S (1993) Deletion of 1p36 s primry chromosoml berrtion in intestinl tumorigenesis. Cncer Res 53: Bylin SB nd Hermn JG (2000) DNA hypermethyltion in tumorigenesis: epigenetics joins genetics. Trends Genet 16: Buyse IM, Sho G nd Hung S (1995) The retinoblstom protein binds to RIZ, zinc finger protein tht shres n epitope with the denovirus E1A protein. Proc Ntl Acd Sci USA 92: Chdwick RB, Jing G-L, Bennington GA, Yun B, Johnson CK, Stevens MW, Niemnn TH, Peltomki P, Hung S nd de l Chpelle A (2000) Cndidte tumor suppressor RIZ is frequently involved in colorectl crcinogenesis. Proc Ntl Acd Sci USA 97: Eng C, Hermn JG nd Bylin SB (2000) A bird s eye view of globl methyltion. Nt Genet 24: Fng W, Pio Z, Simon D, Sheu J-C nd Hung S (2000) Mpping of miniml deleted region in humn heptocellulr crcinom to 1p36.13 p36.23 nd muttionl nlysis of the RIZ (PRDM2) gene loclized to the region. Genes Chromosomes Cncer 28: Genurdi M, Tsihir H, Anderson DE nd Sunders GF (1989) Distl deletion of chromosome Ip in ductl crcinom of the brest. Am J Hum Genet 45: Hmmond C, Jeffers L, Crr BI nd Simon D (1999) Multiple genetic ltertions, 4q28, new suppressor region, nd potentil gender differences in humn heptocellulr crcinom. Heptology 29: He L, Yu JX, Liu L, Buyse IM, Wng M-S, Yng Q-C, Nkgwr A, Brodeur GM, Shi YE nd Hung S (1998) RIZ1, but not the lterntive RIZ2 product of the sme gene, is underexpressed in brest cncer, nd forced RIZ1 expression cuses G 2 -M cell cycle rrest nd/or poptosis. Cncer Res 58: Hung S, Sho G nd Liu L (1998) The PR domin of the Rb-binding zinc finger Protein RIZ1 is protein binding interfce nd is relted to the SET domin functioning in chromtin-medited gene expression. J Biol Chem 273: Jing G-L, Liu L, Buyse IM, Simon D nd Hung S (1999). Decresed RIZ1 expression but not RIZ2 in heptom nd suppression of heptom tumorigenicity by RIZ1. Int J Cncer 83: Jones PA nd Lird PW (1999) Cncer epigenetics comes of ge. Nture Genetics 21: Kuroki T, Fujiwr Y, Tsuchiy E, Nkmori S, Imok S, Knemtsu T nd Nkmur Y (1995) Accumultion of genetic chnges during development nd progression of heptocellulr crcinom: loss of heterozygosity of chromosome rm 1p occurs t n erly stge of heptocrcinogenesis. Genes Chromosomes nd Cncer 13: Lndis SH, Murry T, Bolden S nd Wingo PA (1999) Cncer sttistics, C: Cncer Journl for Clinicins 49: 8 31 Lin YW, Sheu JC, Hung GT, Lee HS, Chen CH, Wng JT, Lee PH nd Lu FJ (1999) Chromosoml bnormlity in heptocellulr crcinom by comprtive genomic hybridistion in Tiwn. Int J Cncer 35: Liu L, Sho G, Steele-Perkins G nd Hung S (1997) The retinoblstom intercting zinc finger gene RIZ produces PR domin lcking product through n internl promoter. J Biol Chem 272: Muros Y, Tkhshi K, Yoshizw M nd Shibhr S (1996) cdna cloning of novel protein contining two zinc-finger domins tht my function s trnscription fctor for the humn heme-oxygense-1 gene. Eur J Biochem 235: Pio Z, Prk C, Prk JH nd Kim H (1998) Allelotype nlysis of heptocellulr crcinom. Int J Cncer 75: Pio Z, Prk C, Prk JH nd Kim H (1998b) Deletion mpping of chromosome 4q in heptocellulr crcinom. Int J Cncer 79: Pio Z, Fng W, Mlkhosyn S, Kim H, Horii A, Perucho M nd Hung S (2000) Frequent frmeshift muttions of RIZ in humn gstrointestinl nd endometril crcinoms with microstellite instbility. Cncer Res 60: Re S, Elsenhber F, O Crroll D, Strhl B, Zu-Wen S, Mnfred S, Oprvil S, Mechtler K, Ponting C, Allis C nd Jenuwein T (2000) Regultion of chromtin structure by site-specific histone H3 methyltrnsferses. Nture 406: Registry CC (1995) Cncer Incidence nd Mortlity in Cliforni by Detiled Rce/Ethnicity, ( Skurd K, Furukw T, Kto Y, Kym T, Hung S nd Horii A (2001) RIZ, the retinoblstom protein intercting zinc finger gene, is mutted in geneticlly unstble cncers of the pncres, stomch, nd colorectum. Genes Chromosomes Cncer 30: Shpiro VS, Lee P nd Winoto A (1995) Identifiction nd cloning of the G3B cdna encoding 3 segment of protein binding to GATA-3. Gene 163: Simon D, Knowles BB nd Weith A (1991) Abnormlities of chromosome 1 nd loss of heterozygosity on 1p in primry heptoms. Oncogene 6: Weith A, Brodeur GM, Bruns GA, Mtise TC, Mischke D, Nizetic D, Seldin MF, vn Roy N nd Vnce J (1996) Report of the second interntionl workshop on humn chromosome 1 mpping Cytogenetics nd Cell Genetics 72:

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons