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Downregulation of CD44v6 in colorectal carcinomas is associated with hypermethylation of the CD44 promoter region
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Experimental and Molecular Pathology 74 (2003) 262–266
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Downregulation of CD44v6 in colorectal carcinomas is associated with hypermethylation of the CD44 promoter region A. Stallmach,a,*,1 B.M. Wittig,a,1 K. Kremp,a R. Goebel,a S. Santourlidis,b M. Zeitz,c M. Menges,a J. Raedle,a S. Zeuzem,a and W.A. Schulzb a
Department of Internal Medicine II, Saarland University, 66421 Homburg/Saar, Germany b Department of Urology, Heinrich-Heine University, 040225 Du¨sseldorf, Germany c Medical Clinic I, Benjamin Franklin University Hospital, Free University, 12200 Berlin, Germany Received 4 February 2003
Abstract Overexpression of the cell adhesion protein CD44v6 has been demonstrated in colorectal cancer and other gastrointestinal tumors. While CD44v6 is upregulated in benign colorectal adenomas and well-differentiated colorectal cancer tissues, downregulation frequently occurs during disease progression. The mechanism of downregulation, however, is unknown. Therefore, we evaluated the methylation status of the CD44 promoter as a mechanism for decreased CD44v6 expression in advanced colorectal carcinomas. We demonstrated by methylationsensitive restriction enzyme digestion that the CpG islands of the CD44 promoter were methylated in 6/21 (28%) of benign colorectal adenomas. Interestingly, in colorectal carcinomas the frequency of promoter methylation was significantly increased (10/19; 53%) compared to 7/21 (33%) in the corresponding normal mucosa. Methylation seems to be associated with a more advanced cancer stage, but the trend did not reach statistical significance. In colorectal carcinomas with CD44 promoter methylation CD44v6 mRNA was detected by reverse transcription–polymerase chain reaction in 3/10 carcinomas, whereas in tumors without CD44 promoter methylation CD44v6 expression was observed in 8/9 (P ⱕ 0.05). These results demonstrated that methylation of the 5⬘CpG island of the CD44 gene is closely associated with decreased expression of CD44v6 in human colorectal carcinomas. © 2003 Elsevier Science (USA). All rights reserved. Keywords: CD44 variants; Colorectal carcinoma; Methylation; Promoter
Introduction Colorectal cancer is the second most frequent cause of cancer mortality in western countries. The prognosis of the patients mainly depends on the tumor stage at the time of surgery and is worse when development of metastasis has occurred. The molecular events leading to the metastatic phenotype of tumor cells and especially the role of cell adhesion molecules in this process are under intensive study. In this context, expressed variants of the glycoprotein CD44 are of special interest. CD44 was originally described as an integral lymphocyte homing receptor involved in
* Corresponding author. Fax: ⫹49-6841-162-3264. E-mail address: [email protected] (A. Stallmach). 1 These authors contributed equally.
endothelial transmigration and binding to lymph node stroma (Jalkanen et al., 1986, 1987). Various CD44 isoforms of higher molecular weight that contain additional extracellular domains inserted at a unique site of the molecule are expressed on epithelial tumors (Dougherty et al., 1991; Hofmann et al., 1991; Stamenkovic et al., 1991; Jackson et al., 1992). The genomic structure of the CD44 gene revealed at least 12 alternatively spliced exons spanning about 50 kb of DNA (Screaton et al., 1992). The significance of variant CD44 expression for the metastatic capacity of tumor cells was underlined by the demonstration that expression of a CD44 protein variant encoded by alternatively spliced CD44 exon v6 can confer metastatic potential to rat pancreas carcinoma cells (Gu¨nthert et al., 1991). It was therefore suggested that upregulation of CD44 variants might be associated with tumor spread and prognosis in
0014-4800/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4800(03)00025-X
A. Stallmach et al. / Experimental and Molecular Pathology 74 (2003) 262–266
cancer patients. Indeed, overexpression of CD44v6 has been demonstrated in colorectal cancer and other gastrointestinal tumors (Mulder et al., 1994; Finke et al., 1995; Imazeki et al., 1996; Wielenga et al., 1998; Menges et al., 2002). However, CD44v6 is mainly upregulated in benign colorectal adenomas and well-differentiated colorectal cancer tissue but not in metastatic disease or poorly differentiated colorectal cancer (Finke et al., 1995; Weg-Remers et al., 1998). Therefore, the question arises which molecular events contribute to downregulation of CD44v6 in advanced colorectal cancer. CpG methylation in promoter regions is known to be an epigenetic event that can cause gene silencing. This is accomplished through the binding of repressor complexes, including methyl-CpG-binding proteins (MeCPs) and histone deacetylases, which results in condensation of the chromatin structure and transcriptional silencing (Kass et al., 1997; Nan et al., 1998; Razin, 1998). Silencing of tumor suppressor genes or growth-inhibiting genes associated with CpG island methylation therefore can promote cancer development or progression (Bird, 1996; Karpf and Jones, 2002). In a variety of human cancers, CpG methylation has been described as a mechanism that inactivates several genes (Jarrard et al., 1996; Schulz, 1998), including GSTPI (Lee et al., 1997), MLHI (Raedle et al., 2001), and E-cadherin (Graff et al., 1997). Recently methylated CD44 promoter methylation was detetected by methylation-specific restriction enzyme digestion in the majority of primary prostate cancers. In the present work, we studied whether methylation of the CD44 promoter contributes to the decreased CD44v6 expression in advanced colorectal cancer investigating normal mucosa, adenomas, and cancers of the colon.
Material and methods Patients Twenty-one patients with colorectal adenomas and 19 patients with proven adenocarcinomas of the colorectum gave their consent to participate in this study. Tissue samples of colorectal adenomas or colorectal carcinomas, as well as and normal mucosa at least 10 cm distant from neoplastic tissue, were obtained during surgery or colonoscopy. Tissue specimens were snap-frozen in liquid nitrogen and stored at ⫺70°C until use. Grading and staging of the tumors according to the UICC classification was done by standard surgical pathology techniques. Histologically all of the investigated tumors were either adenocarcinomas or adenomas with different grades of differentiation. Preparation of cDNA and Southern blot analysis for CD44v6 expression analysis Preparation of mRNA from tissue samples was conducted using a commercially available mRNA purification
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Table 1 Oligonucleotide sequences of used primers Gene
Primer sequences (5⬘–3⬘)
GAPDH
5⬘GTG GGG CGC CCC AGG CAC CA 3⬘ 5⬘CTC CTT AAT TGT CAC GCA CGA TTC 3⬘
CD44 pan-CD44
CD44v6
(P1⫹) 5⬘CCT ACT GAT GAT GAC GTG AGC AGC G 3⬘ (P1⫺) 5⬘TCA GAT CCA TGA GTG GTA TGG GAC C 3⬘ (P6⫹) 5⬘GGC AAC TCC TAG TAG TAC AAC 3⬘ (P6⫺) 5⬘CAG CTG TCC CTG TTG TCG AAT 3⬘
kit (Pharmacia Biotech, Uppsala, Sweden). cDNA was synthesized with mRNA as template for an oligo(dT) primed reaction catalyzed by MMLV reverse transcriptase (Gibco/ BRL Carlsbad, CA) The quality of mRNA preparations and cDNA syntheses was checked including GAPDH-specific primers as an internal reverse transcription–polymerase chain reaction (RT–PCR) control. The GAPDH primer sequences (Table 1) were chosen according to the sequence of a full-length genomic DNA clone (Ercolani et al., 1988). The sense primer corresponded to positions 17–38 in exon 1 and the antisense primer to positions 2468 –2890 in exon 5 of the GAPDH gene. RT– PCR studies for CD44 were carried out using primers P1⫹ and P1⫺ (Table 1), sharing sequence homology with exons 5 and 15 and localized up- and downstream from the variable exons of the CD44 gene. Following RT–PCR DNA fragments were separated on agarose gels. Bands were visualized after Southern blotting by hybridization with a CD44v6-specific probe labeled by PCR with exon-specific primers (P6⫹, P6⫺) (Table 1) and digoxigenin-labeled dNTPs (Boehringer Mannheim, Mannheim, FRG). Following hybridization and stringent washing detection of filterbound digoxigenin probes was conducted by colorimetric detection with NBT/BCIP (Roche Boehringer, Mannheim, FRG). Analysis of CD44 promoter methylation Preparation of genomic DNA from tissue samples was performed using a commercially available DNA purification kit (Qiagen, Chatsworth, CA). Genomic DNA (0.5 g) was extensively digested with a methylation-sensitive restriction endonuclease HpaII or the nonsensitive isochizomer MspI, followed by ethanol coprecipitation with glycogen. Amplification was carried out on a thermocycler at 94°C for 45 s, 63°C for 2 min, and 72°C for 2 min for 40 cycles, followed by 72°C for 10 min. Primers yielding a 791-bp product were designed according to the published sequence of the 5⬘ region of CD44 (see Table 1). Twenty microliters of PCR product were loaded on nondenaturating polyacrylamide gels (5%) and bands were visualized after Southern blotting by hybridization with a CD44 promoter-specific probe.
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Probe was produced by PCR with promoter-specific primers (Table 1) and digoxigenin-labeled dNTPs (Boehringer Mannheim). Following stringent washings, detection of filter-bound digoxigenin probes was conducted by colorimetric detection with NBT/BCIP (Boehringer Mannheim). Genomic sequencing Bisulfite-modified DNA (100 ng) was amplified in a PCR analysis with human CD44 promoter-specific primers (see Table 1) recognizing methylated and modified unmethylated DNA. PCR conditions were as follows: 94°C for 5 min, followed by 35 cycles at 94°C, 50°C, and 72°C each for 1 min and a final extension for 10 min at 72°C. The PCR mixture contained 1 IU SuperTaq DNA polymerase (Sphaero Q, HT Biotechnology, Cambridge, United Kingdom), buffer containing 1.5 mM MgCl2 (Sphaero Q), 100 ng of each primer, and 0.2 mM dNTPs in a final volume of 50 l. Cloning of PCR fragments was performed with the pGEM-T Cloning kit (Promega, Madison, WI) according to the manufacturer’s protocol. Sequence analysis of clones was performed by dideoxynucleotide chain termination using a T7 sequence kit (Pharmacia, Uppsala, Sweden). Statistical analysis Statistical differences were calculated using Fisher’s Exact Test or chi-square-test and were considered to be significant at the P ⬍ 0.05 level.
Results Methylation of CpG islands is a frequent mechanism of gene silencing in human cancers. Sequence analysis of the 1582-bp flanking region of CD44 revealed a CpG-rich region that fulfills the criteria of a CpG island (GardinerGarden and Frommer, 1987; Shtivelman and Bishop, 1991). To test whether hypermethylation is involved in CD44 downregulation in colorectal carcinogenesis, we analyzed genomic DNA from normal mucosae, colorectal adenomas, and cancers by digestion with the methylation-sensitive restriction enzyme HpaII or its methylation-insensitive isoschizomer MspI (see Fig. 1). A total of 61 normal mucosa samples, benign adenomas, and primary colorectal cancers were examined for CD44 promoter methylation. Methylation of the CD44 promoter was observed in 7 of 21 samples of normal mucosa (33%) and 6 of 21 benign colorectal adenomas (29%). Interestingly, in 10/19 (53%) colorectal cancers an increased frequency of promoter methylation was observed (P ⱕ 0.05). Moreover, methylation seems to be associated with a more advanced stage of cancer (7 of 10 UICC III/IV tumors vs 3 of 9 UICC I/II tumors), although this did not reach statistical significance (P ⬎ 0.05). To test whether methylation of the CD44 promoter is associated with decreased CD44v6 expression, we analyzed
Fig. 1. Methylation analyses of the CpG island CD44 promoter in human colorectal cancer. Genomic DNA was extensively digested with the methylation-sensitive restriction enzyme HpaII (lane H) or its methylationinsensitive isoschizomer Msp1 (lane M). The PCR products were separated on 5% nondenaturating polyacrylamide gel. Bands were visualized after Southern blotting and hybridization with a CD44 promoter-specific probe. A 791 bp is only visible if all HpaII sites in the promoter region are methylated. (Lane 1) The LnCaP prostata carcinoma cell line with CD44 promoter methylation served as a positive control. (Lanes 2– 4) Patient 1 (colon carcinoma without CD44 promoter methylation), patient 2 (colon carcinoma with CD44 promoter methylation), and patient 3 (rectum carcinoma with CD44 promoter methylation).
CD44v6 transcript expression by RT–PCR using primers flanking the variable region. The primer were designed for detection of both standard and variant transcripts. In general, the expression of alternatively spliced variant CD44v6 in normal mucosa was low (2/21; 5%) compared to that of the common CD44 standard transcripts (21/21). In colorectal adenomas, expression of CD44v6 was significantly more frequent (52%) compared to normal mucosa (P ⬍ 0.01). Likewise, in colorectal carcinomas the CD44v6 frequency was also elevated (11/19; 58%) compared to normal mucosa (P ⬍ 0.001). In colorectal carcinomas with CD44 promoter methylation (n ⫽ 10) CD44v6 was positive in three carcinomas, whereas in tumors without CD44 promoter methylation (n ⫽ 9) CD44v6 expression was observed in 8 cases (P ⬍ 0.05). The results of this study can be summarized as follows: (a) 5⬘ CpG island methylation of the CD44 gene promoter is common in colorectal cancer and (b) methylation of this promoter region is associated with decreased CD44v6 expression in colorectal cancer.
Discussion In human cancer several mechanisms may contribute to loss of tumor suppressor gene function, including allelic loss in combination with mutations, homozygous deletions, CpG island methylation of promoter regions, abnormal splicing, and downregulation by growth factor or cytokinemediated activation of cell membrane receptors. In colorectal cancer epigenetic silencing by promoter methylation is a common event during carcinogenesis. In detail, methylation contributes to inactivation of the cell cycle regulator p16 (Herman et al., 1995), the growth suppressor ER (Issa et al., 1994), the angiogenesis inhibitor THBS1 (Ahuja et al., 1997), the metastasis suppressor TIMP3 (Cameron et al.,
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1999), the DNA repair gene MGMT (Esteller et al., 1999), and the mismatch repair gene MLHI (Kane et al., 1997); (Raedle et al., 2001). In sporadic colorectal cancer for example, methylation is frequently related to epigenetic inactivation of MLHI (Herman et al., 1998; Cunningham et al., 1998). In addition, other studies have shown that 5⬘ CpG island methylation of the CD44 gene promoter is also common in prostate cancer. The first substantial result of our study is the observation that CD44 promoter methylation is also a frequent event in colorectal cancer. Compared with a CD44 promoter methylation frequency of 28% in benign colorectal adenomas, more than 50% of the tested colorectal cancers demonstrated methylation of this region. Tumors with a methylated CD44 promoter region did not show different clinicopathological features, such as poor differentiation or anatomic location. However, CD44 promoter methylation seems to be associated with a more advanced stage of cancer, although this did not reach statistical significance. This support the finding that colorectal cancers acquire more and more genetic alterations during carcinogenesis and tumor progression. The second substantial result of our study is that methylation of the CD44 promoter region is associated with decreased CD44v6 expression in colorectal cancer. Expression of CD44v6 results from alternative splicing in colorectal cancer (Herrlich et al., 1995). However, little is known about regulatory factors that influence alternative splicing of cell adhesion molecules. Regulatory factors, like the family of arginine–serine-rich (SR) proteins are essential for spliceosome formation and can also influence splicing by selection of alternative splice sites. Tissue-specific differences in the activity of distinct SR proteins might be one mechanism leading to an alternative splicing of certain target genes (Liu et al., 1998; Screaton et al., 1995). In a recently described mammary tumor model, stage-specific changes in the expression of distinct SR splicing factors have also been associated with alternative splicing of CD44 (Stickeler et al., 1999). Although we observed this obvious correlation between the lacking CD44v6 expression and methylation of the CD44 promoter, we did not postulate that promoter methylation directly influence the alternative splicing of CD44v6 transcripts. Nevertheless, a reduced concentration of standard CD44 transcripts resulting from promoter methylation may lead to a decreased frequency of CD44v6 expression in advanced colorectal cancer as described by us and other groups (Mulder et al., 1994; Weg-Remers et al., 1998; Wielenga et al., 1998). In this context, quantification of standard CD44 in relation to the methylation status of CD44 promoter regions is of special interest in colorectal cancer. However, since immunohistological methods are not suitable to quantify CD44 expression this problem is unsolved. Quantification of CD44 standard molecules in colorectal cancer by ELISA technique indicated that in advanced tumors CD44 standard concentration is significant lower than in adenomas or localized cancers (own unpub-
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lished results). However, ELISA techniques cannot clearly distinguish between individual cell types in tissue samples. In conclusion, methylation of the CD44 promoter is common in colorectal cancers and associated with CD44 downregulation during colorectal carcinogenesis. A detailed understanding of the mechanism for CD44 promoter methylation may provide additional insights into the concept of progression of colorectal cancer. In addition, these results warrant further investigation whether CD44 promoter methylation might be an additional diagnostic marker for colorectal cancer progression. Acknowledgments This work has been supported in part by a grant from the Deutsche Forschungsgemeinschaft to A.S. (DFG STA 295/ 2-3). The authors thank the medical staff of the Department of Surgery (Head: Professor Dr. M. Schilling), Saarland University, for providing surgical specimens. References Ahuja, N., Mohan, A.L., Li, Q., Stolker, J.M., Herman, J.G., Hamilton, S.R., Baylin, S.B., Issa, J.P., 1997. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res. 57, 3370 –3374. Bird, A.P., 1996. The relationship of DNA methylation to cancer. Cancer Surv. 28, 87–101. Cameron, E.E., Bachman, K.E., Myohanen, S., Herman, J.G., Baylin, S.B., 1999. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107. Cunningham, J.M., Christensen, E.R., Tester, D.J., Kim, C.Y., Roche, P.C., Burgart, L.J., Thibodeau, S.N., 1998. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 58, 3455–3460. Dougherty, G.J., Landorp, P.M., Cooper, D.L., Humphries, R.K., 1991. Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte “homing” receptor expressed by hemopoietic cells. J. Exp. Med. 174, 1–5. Ercolani, L., Florence, B., Denaro, M., Alexander, M., 1988. Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J. Biol. Chem. 263, 15335–15341. Esteller M., Hamilton S.R., Burger P.C., Baylin S.B., Herman J.G. 1999. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res. 793–797. Finke, L.H., Terpe, H.J., Zo¨ rb, Q., Haensch, W., Schlag, P.M., 1995. Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins. Lancet 345, 583. Gardiner-Garden, M., Frommer, M., 1987. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282. Graff, J.R., Herman, J.G., Myohanen, S., Baylin, S.B., Vertino, P.M., 1997. Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation. J. Biol. Chem. 272, 22322–22329. Gu¨ nthert, U., Hofman, M., Rudy, W., Reber, S.M.Z., Haußmann, I., Matzku, S., Wenzel, A., Ponta, H., Herrlich, P., 1991. A new variant of glycoprotein CD44 confers metastatic capacity to rat carcinoma cells. Cell 65, 13–24. Herman, J.G., Merlo, A., Mao, L., Lapidus, R.G., Issa, J.P., Davidson, N.E., Sidransky, D., Baylin, S.B., 1995. Inactivation of the CDKN2/
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p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55, 4525– 4530. Herman, J.G., Umar, A., Polyak, K., Graff, J.R., Ahuja, N., Issa, J.P.J., Markowitz, S., Willson, J.K.V., Hamilton, S.R., Kinzler, K.W., 1998. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl. Acad. Sci. USA 95, 6870 – 6875. Herrlich, P., Pals, S., Ponta, H., 1995. CD44 in colon cancer. Eur. J. Cancer 31A, 1110 –1112. Hofmann, M., Rudy, W., Zo¨ ller, M., To¨ lg, C., Ponta, H., Herrlich, P., Gu¨ nthert, H., 1991. CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines. Cancer Res. 51, 5292–5297. Imazeki, F., Yokuzuka, O., Yamaguchi, T., Ohto, M., Isono, K., Omata, I., 1996. Expression of variant CD44 messenger RNA in colorectal adenomcarcinomas and adenomatous polyps in humans. Gastroenterology 110, 362–368. Issa, J.P., Ottaviano, Y.L., Celano, P., Hamilton, S.R., Davidson, N.E., Baylin, S.B., 1994. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat. Genet. 7, 536 –540. Jackson, D.G., Buckley, J., Bell, J.I., 1992. Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain. J. Biol. Chem. 267, 4732– 4739. Jalkanen, S., Reichert, R.A., Gallatin, W.M., Bargatze, R.F., Weissman, I.L., Butcher, E.C., 1986. Homing receptors and the control of lymphocyte migration. Immunol. Rev. 91, 39 – 60. Jalkanen, S., Bargatze, R.F., Toyos, J.D.L., Butcher, E.C., 1987. Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85–90kDa glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J. Cell. Biol. 105, 983–990. Jarrard, D.F., Bova, G.S., Isaacs, W.B., 1996. DNA methylation, molecular genetic, and linkage studies in prostate cancer. Prostate 6 (Suppl.), 36 – 44. Kane, M.F., Loda, M., Gaida, G.M., Lipman, J., Mishra, R., Goldman, H., Jessup, J.M., Kolodner, R., 1997. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 57, 808 – 811. Karpf, A.R., Jones, D.A., 2002. Reactivating the expression of methylation silenced genes in human cancer. Oncogene 12, 5496 –503. Kass, S.U., Pruss, D., Wolffe, A.P., 1997. How does DNA methylation repress transcription? Trends Genet. 13, 444 – 449. Lee, W.H., Isaacs, W.B., Bova, G.S., Nelson, W.G., 1997. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostate cancer biomarker. Cancer Epidemiol. Biomarkers Prev. 6, 443– 450.
Liu, H.X., Zhang, M., Krainer, A.R., 1998. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12, 1998 –2012. Menges, M., Goebel, R., Pueschel, W., Zeitz, M., Stallmach, A., 2002. Expression of CD44v5 and -v6 in Barrett’s carcinoma is not increased compared to nondysplastic Barrett’s mucosa. Exp. Mol. Pathol. 72, 207–212. Mulder, J.W.R., Kruyt, P.M., Sewnath, M., Oosting, J., Seldenrijk, C.A., Weidema, W.F., Offerhaus, G.J.A., Pals, S.T., 1994. Colorectal cancer prognosis of exon-v6-containing CD44 proteins. Lancet 344, 1470 – 1472. Nan, X., Ng, H.H., Johnson, C.A., Laherty, C.D., Turner, B.M., Eisenman, R.N., Bird, A., 1998. Transcriptional repression by the methyl-CpGbinding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386 –389. Raedle, J., Trojan, J., Brieger, A., Weber, N., Scha¨ fer, D., Plotz, G., Staib-Sebler, E., Kriener, S., Lorenz, M., Zeuzem, S., 2001. Bethesda Guidelines: relation to microsatellite instability and MLH1 promoter methylation in patients with colorectal cancer. Ann. Intern. Med. 135, 566 –576. Razin, A., 1998. CpG methylation, chromatin structure and gene silencing—a three-way connection. EMBO J. 17, 4905– 4908. Schulz, W.A., 1998. DNA methylation in urological malignancies. Int. J. Oncol. 13, 151–167. Screaton, G.R., Bell, M.V., Jackson, D.G., Cornelis, F.B., Gerth, U., JI, B., 1992. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. USA 89, 12160 –12164. Screaton, G.R., Ca´ ceras, J.F., Mayeda, A., Bell, M.V., Plebanski, M., Jackson, D.G., Bell, J.I., Krainer, A.R., 1995. Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J. 14, 4336 – 4349. Shtivelman, E., Bishop, M., 1991. Expression of CD44 is repressed in neuroblastoma cells. J. Mol. Cell. Biol. 11, 5446 –5453. Stamenkovic, I., Aruffo, A., Amiot, M., Seed, B., 1991. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 10, 343– 348. Stickeler, E., Kittrell, F., Medina, D., Berget, S.M., 1999. Stage-specific changes in SR splicing factors and alternative splicing in mammary tumorigenesis. Oncogene 18, 3574 –3582. Weg-Remers, S., Anders, M., von Lampe, B., Riecken, E.O., Schu¨ der, G., Feifel, G., Zeitz, M., Stallmach, A., 1998. Decreased expression of CD44 splicing variants in advanced colorectal carcinomas. Eur. J. Cancer 34, 1607–1611. Wielenga, V.J., van der Voort, R., Mulder, J.W., Kryt, P.M., Weidema, W.F., Oosting, J., Seldenrijk, C.A., van Krimpen, C., Offerhaus, G.J., Pals, S.T., 1998. CD44 splice variants as prognostic markers in colorectal cancer. Scand. J. Gastroenterol. 33, 82– 87.
Experimental and Molecular Pathology 74 (2003) 262–266
www.elsevier.com/locate/yexmp
Downregulation of CD44v6 in colorectal carcinomas is associated with hypermethylation of the CD44 promoter region A. Stallmach,a,*,1 B.M. Wittig,a,1 K. Kremp,a R. Goebel,a S. Santourlidis,b M. Zeitz,c M. Menges,a J. Raedle,a S. Zeuzem,a and W.A. Schulzb a
Department of Internal Medicine II, Saarland University, 66421 Homburg/Saar, Germany b Department of Urology, Heinrich-Heine University, 040225 Du¨sseldorf, Germany c Medical Clinic I, Benjamin Franklin University Hospital, Free University, 12200 Berlin, Germany Received 4 February 2003
Abstract Overexpression of the cell adhesion protein CD44v6 has been demonstrated in colorectal cancer and other gastrointestinal tumors. While CD44v6 is upregulated in benign colorectal adenomas and well-differentiated colorectal cancer tissues, downregulation frequently occurs during disease progression. The mechanism of downregulation, however, is unknown. Therefore, we evaluated the methylation status of the CD44 promoter as a mechanism for decreased CD44v6 expression in advanced colorectal carcinomas. We demonstrated by methylationsensitive restriction enzyme digestion that the CpG islands of the CD44 promoter were methylated in 6/21 (28%) of benign colorectal adenomas. Interestingly, in colorectal carcinomas the frequency of promoter methylation was significantly increased (10/19; 53%) compared to 7/21 (33%) in the corresponding normal mucosa. Methylation seems to be associated with a more advanced cancer stage, but the trend did not reach statistical significance. In colorectal carcinomas with CD44 promoter methylation CD44v6 mRNA was detected by reverse transcription–polymerase chain reaction in 3/10 carcinomas, whereas in tumors without CD44 promoter methylation CD44v6 expression was observed in 8/9 (P ⱕ 0.05). These results demonstrated that methylation of the 5⬘CpG island of the CD44 gene is closely associated with decreased expression of CD44v6 in human colorectal carcinomas. © 2003 Elsevier Science (USA). All rights reserved. Keywords: CD44 variants; Colorectal carcinoma; Methylation; Promoter
Introduction Colorectal cancer is the second most frequent cause of cancer mortality in western countries. The prognosis of the patients mainly depends on the tumor stage at the time of surgery and is worse when development of metastasis has occurred. The molecular events leading to the metastatic phenotype of tumor cells and especially the role of cell adhesion molecules in this process are under intensive study. In this context, expressed variants of the glycoprotein CD44 are of special interest. CD44 was originally described as an integral lymphocyte homing receptor involved in
* Corresponding author. Fax: ⫹49-6841-162-3264. E-mail address: [email protected] (A. Stallmach). 1 These authors contributed equally.
endothelial transmigration and binding to lymph node stroma (Jalkanen et al., 1986, 1987). Various CD44 isoforms of higher molecular weight that contain additional extracellular domains inserted at a unique site of the molecule are expressed on epithelial tumors (Dougherty et al., 1991; Hofmann et al., 1991; Stamenkovic et al., 1991; Jackson et al., 1992). The genomic structure of the CD44 gene revealed at least 12 alternatively spliced exons spanning about 50 kb of DNA (Screaton et al., 1992). The significance of variant CD44 expression for the metastatic capacity of tumor cells was underlined by the demonstration that expression of a CD44 protein variant encoded by alternatively spliced CD44 exon v6 can confer metastatic potential to rat pancreas carcinoma cells (Gu¨nthert et al., 1991). It was therefore suggested that upregulation of CD44 variants might be associated with tumor spread and prognosis in
0014-4800/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4800(03)00025-X
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cancer patients. Indeed, overexpression of CD44v6 has been demonstrated in colorectal cancer and other gastrointestinal tumors (Mulder et al., 1994; Finke et al., 1995; Imazeki et al., 1996; Wielenga et al., 1998; Menges et al., 2002). However, CD44v6 is mainly upregulated in benign colorectal adenomas and well-differentiated colorectal cancer tissue but not in metastatic disease or poorly differentiated colorectal cancer (Finke et al., 1995; Weg-Remers et al., 1998). Therefore, the question arises which molecular events contribute to downregulation of CD44v6 in advanced colorectal cancer. CpG methylation in promoter regions is known to be an epigenetic event that can cause gene silencing. This is accomplished through the binding of repressor complexes, including methyl-CpG-binding proteins (MeCPs) and histone deacetylases, which results in condensation of the chromatin structure and transcriptional silencing (Kass et al., 1997; Nan et al., 1998; Razin, 1998). Silencing of tumor suppressor genes or growth-inhibiting genes associated with CpG island methylation therefore can promote cancer development or progression (Bird, 1996; Karpf and Jones, 2002). In a variety of human cancers, CpG methylation has been described as a mechanism that inactivates several genes (Jarrard et al., 1996; Schulz, 1998), including GSTPI (Lee et al., 1997), MLHI (Raedle et al., 2001), and E-cadherin (Graff et al., 1997). Recently methylated CD44 promoter methylation was detetected by methylation-specific restriction enzyme digestion in the majority of primary prostate cancers. In the present work, we studied whether methylation of the CD44 promoter contributes to the decreased CD44v6 expression in advanced colorectal cancer investigating normal mucosa, adenomas, and cancers of the colon.
Material and methods Patients Twenty-one patients with colorectal adenomas and 19 patients with proven adenocarcinomas of the colorectum gave their consent to participate in this study. Tissue samples of colorectal adenomas or colorectal carcinomas, as well as and normal mucosa at least 10 cm distant from neoplastic tissue, were obtained during surgery or colonoscopy. Tissue specimens were snap-frozen in liquid nitrogen and stored at ⫺70°C until use. Grading and staging of the tumors according to the UICC classification was done by standard surgical pathology techniques. Histologically all of the investigated tumors were either adenocarcinomas or adenomas with different grades of differentiation. Preparation of cDNA and Southern blot analysis for CD44v6 expression analysis Preparation of mRNA from tissue samples was conducted using a commercially available mRNA purification
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Table 1 Oligonucleotide sequences of used primers Gene
Primer sequences (5⬘–3⬘)
GAPDH
5⬘GTG GGG CGC CCC AGG CAC CA 3⬘ 5⬘CTC CTT AAT TGT CAC GCA CGA TTC 3⬘
CD44 pan-CD44
CD44v6
(P1⫹) 5⬘CCT ACT GAT GAT GAC GTG AGC AGC G 3⬘ (P1⫺) 5⬘TCA GAT CCA TGA GTG GTA TGG GAC C 3⬘ (P6⫹) 5⬘GGC AAC TCC TAG TAG TAC AAC 3⬘ (P6⫺) 5⬘CAG CTG TCC CTG TTG TCG AAT 3⬘
kit (Pharmacia Biotech, Uppsala, Sweden). cDNA was synthesized with mRNA as template for an oligo(dT) primed reaction catalyzed by MMLV reverse transcriptase (Gibco/ BRL Carlsbad, CA) The quality of mRNA preparations and cDNA syntheses was checked including GAPDH-specific primers as an internal reverse transcription–polymerase chain reaction (RT–PCR) control. The GAPDH primer sequences (Table 1) were chosen according to the sequence of a full-length genomic DNA clone (Ercolani et al., 1988). The sense primer corresponded to positions 17–38 in exon 1 and the antisense primer to positions 2468 –2890 in exon 5 of the GAPDH gene. RT– PCR studies for CD44 were carried out using primers P1⫹ and P1⫺ (Table 1), sharing sequence homology with exons 5 and 15 and localized up- and downstream from the variable exons of the CD44 gene. Following RT–PCR DNA fragments were separated on agarose gels. Bands were visualized after Southern blotting by hybridization with a CD44v6-specific probe labeled by PCR with exon-specific primers (P6⫹, P6⫺) (Table 1) and digoxigenin-labeled dNTPs (Boehringer Mannheim, Mannheim, FRG). Following hybridization and stringent washing detection of filterbound digoxigenin probes was conducted by colorimetric detection with NBT/BCIP (Roche Boehringer, Mannheim, FRG). Analysis of CD44 promoter methylation Preparation of genomic DNA from tissue samples was performed using a commercially available DNA purification kit (Qiagen, Chatsworth, CA). Genomic DNA (0.5 g) was extensively digested with a methylation-sensitive restriction endonuclease HpaII or the nonsensitive isochizomer MspI, followed by ethanol coprecipitation with glycogen. Amplification was carried out on a thermocycler at 94°C for 45 s, 63°C for 2 min, and 72°C for 2 min for 40 cycles, followed by 72°C for 10 min. Primers yielding a 791-bp product were designed according to the published sequence of the 5⬘ region of CD44 (see Table 1). Twenty microliters of PCR product were loaded on nondenaturating polyacrylamide gels (5%) and bands were visualized after Southern blotting by hybridization with a CD44 promoter-specific probe.
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Probe was produced by PCR with promoter-specific primers (Table 1) and digoxigenin-labeled dNTPs (Boehringer Mannheim). Following stringent washings, detection of filter-bound digoxigenin probes was conducted by colorimetric detection with NBT/BCIP (Boehringer Mannheim). Genomic sequencing Bisulfite-modified DNA (100 ng) was amplified in a PCR analysis with human CD44 promoter-specific primers (see Table 1) recognizing methylated and modified unmethylated DNA. PCR conditions were as follows: 94°C for 5 min, followed by 35 cycles at 94°C, 50°C, and 72°C each for 1 min and a final extension for 10 min at 72°C. The PCR mixture contained 1 IU SuperTaq DNA polymerase (Sphaero Q, HT Biotechnology, Cambridge, United Kingdom), buffer containing 1.5 mM MgCl2 (Sphaero Q), 100 ng of each primer, and 0.2 mM dNTPs in a final volume of 50 l. Cloning of PCR fragments was performed with the pGEM-T Cloning kit (Promega, Madison, WI) according to the manufacturer’s protocol. Sequence analysis of clones was performed by dideoxynucleotide chain termination using a T7 sequence kit (Pharmacia, Uppsala, Sweden). Statistical analysis Statistical differences were calculated using Fisher’s Exact Test or chi-square-test and were considered to be significant at the P ⬍ 0.05 level.
Results Methylation of CpG islands is a frequent mechanism of gene silencing in human cancers. Sequence analysis of the 1582-bp flanking region of CD44 revealed a CpG-rich region that fulfills the criteria of a CpG island (GardinerGarden and Frommer, 1987; Shtivelman and Bishop, 1991). To test whether hypermethylation is involved in CD44 downregulation in colorectal carcinogenesis, we analyzed genomic DNA from normal mucosae, colorectal adenomas, and cancers by digestion with the methylation-sensitive restriction enzyme HpaII or its methylation-insensitive isoschizomer MspI (see Fig. 1). A total of 61 normal mucosa samples, benign adenomas, and primary colorectal cancers were examined for CD44 promoter methylation. Methylation of the CD44 promoter was observed in 7 of 21 samples of normal mucosa (33%) and 6 of 21 benign colorectal adenomas (29%). Interestingly, in 10/19 (53%) colorectal cancers an increased frequency of promoter methylation was observed (P ⱕ 0.05). Moreover, methylation seems to be associated with a more advanced stage of cancer (7 of 10 UICC III/IV tumors vs 3 of 9 UICC I/II tumors), although this did not reach statistical significance (P ⬎ 0.05). To test whether methylation of the CD44 promoter is associated with decreased CD44v6 expression, we analyzed
Fig. 1. Methylation analyses of the CpG island CD44 promoter in human colorectal cancer. Genomic DNA was extensively digested with the methylation-sensitive restriction enzyme HpaII (lane H) or its methylationinsensitive isoschizomer Msp1 (lane M). The PCR products were separated on 5% nondenaturating polyacrylamide gel. Bands were visualized after Southern blotting and hybridization with a CD44 promoter-specific probe. A 791 bp is only visible if all HpaII sites in the promoter region are methylated. (Lane 1) The LnCaP prostata carcinoma cell line with CD44 promoter methylation served as a positive control. (Lanes 2– 4) Patient 1 (colon carcinoma without CD44 promoter methylation), patient 2 (colon carcinoma with CD44 promoter methylation), and patient 3 (rectum carcinoma with CD44 promoter methylation).
CD44v6 transcript expression by RT–PCR using primers flanking the variable region. The primer were designed for detection of both standard and variant transcripts. In general, the expression of alternatively spliced variant CD44v6 in normal mucosa was low (2/21; 5%) compared to that of the common CD44 standard transcripts (21/21). In colorectal adenomas, expression of CD44v6 was significantly more frequent (52%) compared to normal mucosa (P ⬍ 0.01). Likewise, in colorectal carcinomas the CD44v6 frequency was also elevated (11/19; 58%) compared to normal mucosa (P ⬍ 0.001). In colorectal carcinomas with CD44 promoter methylation (n ⫽ 10) CD44v6 was positive in three carcinomas, whereas in tumors without CD44 promoter methylation (n ⫽ 9) CD44v6 expression was observed in 8 cases (P ⬍ 0.05). The results of this study can be summarized as follows: (a) 5⬘ CpG island methylation of the CD44 gene promoter is common in colorectal cancer and (b) methylation of this promoter region is associated with decreased CD44v6 expression in colorectal cancer.
Discussion In human cancer several mechanisms may contribute to loss of tumor suppressor gene function, including allelic loss in combination with mutations, homozygous deletions, CpG island methylation of promoter regions, abnormal splicing, and downregulation by growth factor or cytokinemediated activation of cell membrane receptors. In colorectal cancer epigenetic silencing by promoter methylation is a common event during carcinogenesis. In detail, methylation contributes to inactivation of the cell cycle regulator p16 (Herman et al., 1995), the growth suppressor ER (Issa et al., 1994), the angiogenesis inhibitor THBS1 (Ahuja et al., 1997), the metastasis suppressor TIMP3 (Cameron et al.,
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1999), the DNA repair gene MGMT (Esteller et al., 1999), and the mismatch repair gene MLHI (Kane et al., 1997); (Raedle et al., 2001). In sporadic colorectal cancer for example, methylation is frequently related to epigenetic inactivation of MLHI (Herman et al., 1998; Cunningham et al., 1998). In addition, other studies have shown that 5⬘ CpG island methylation of the CD44 gene promoter is also common in prostate cancer. The first substantial result of our study is the observation that CD44 promoter methylation is also a frequent event in colorectal cancer. Compared with a CD44 promoter methylation frequency of 28% in benign colorectal adenomas, more than 50% of the tested colorectal cancers demonstrated methylation of this region. Tumors with a methylated CD44 promoter region did not show different clinicopathological features, such as poor differentiation or anatomic location. However, CD44 promoter methylation seems to be associated with a more advanced stage of cancer, although this did not reach statistical significance. This support the finding that colorectal cancers acquire more and more genetic alterations during carcinogenesis and tumor progression. The second substantial result of our study is that methylation of the CD44 promoter region is associated with decreased CD44v6 expression in colorectal cancer. Expression of CD44v6 results from alternative splicing in colorectal cancer (Herrlich et al., 1995). However, little is known about regulatory factors that influence alternative splicing of cell adhesion molecules. Regulatory factors, like the family of arginine–serine-rich (SR) proteins are essential for spliceosome formation and can also influence splicing by selection of alternative splice sites. Tissue-specific differences in the activity of distinct SR proteins might be one mechanism leading to an alternative splicing of certain target genes (Liu et al., 1998; Screaton et al., 1995). In a recently described mammary tumor model, stage-specific changes in the expression of distinct SR splicing factors have also been associated with alternative splicing of CD44 (Stickeler et al., 1999). Although we observed this obvious correlation between the lacking CD44v6 expression and methylation of the CD44 promoter, we did not postulate that promoter methylation directly influence the alternative splicing of CD44v6 transcripts. Nevertheless, a reduced concentration of standard CD44 transcripts resulting from promoter methylation may lead to a decreased frequency of CD44v6 expression in advanced colorectal cancer as described by us and other groups (Mulder et al., 1994; Weg-Remers et al., 1998; Wielenga et al., 1998). In this context, quantification of standard CD44 in relation to the methylation status of CD44 promoter regions is of special interest in colorectal cancer. However, since immunohistological methods are not suitable to quantify CD44 expression this problem is unsolved. Quantification of CD44 standard molecules in colorectal cancer by ELISA technique indicated that in advanced tumors CD44 standard concentration is significant lower than in adenomas or localized cancers (own unpub-
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lished results). However, ELISA techniques cannot clearly distinguish between individual cell types in tissue samples. In conclusion, methylation of the CD44 promoter is common in colorectal cancers and associated with CD44 downregulation during colorectal carcinogenesis. A detailed understanding of the mechanism for CD44 promoter methylation may provide additional insights into the concept of progression of colorectal cancer. In addition, these results warrant further investigation whether CD44 promoter methylation might be an additional diagnostic marker for colorectal cancer progression. Acknowledgments This work has been supported in part by a grant from the Deutsche Forschungsgemeinschaft to A.S. (DFG STA 295/ 2-3). The authors thank the medical staff of the Department of Surgery (Head: Professor Dr. M. Schilling), Saarland University, for providing surgical specimens. References Ahuja, N., Mohan, A.L., Li, Q., Stolker, J.M., Herman, J.G., Hamilton, S.R., Baylin, S.B., Issa, J.P., 1997. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res. 57, 3370 –3374. Bird, A.P., 1996. The relationship of DNA methylation to cancer. Cancer Surv. 28, 87–101. Cameron, E.E., Bachman, K.E., Myohanen, S., Herman, J.G., Baylin, S.B., 1999. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107. Cunningham, J.M., Christensen, E.R., Tester, D.J., Kim, C.Y., Roche, P.C., Burgart, L.J., Thibodeau, S.N., 1998. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 58, 3455–3460. Dougherty, G.J., Landorp, P.M., Cooper, D.L., Humphries, R.K., 1991. Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte “homing” receptor expressed by hemopoietic cells. J. Exp. Med. 174, 1–5. Ercolani, L., Florence, B., Denaro, M., Alexander, M., 1988. Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J. Biol. Chem. 263, 15335–15341. Esteller M., Hamilton S.R., Burger P.C., Baylin S.B., Herman J.G. 1999. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res. 793–797. Finke, L.H., Terpe, H.J., Zo¨ rb, Q., Haensch, W., Schlag, P.M., 1995. Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins. Lancet 345, 583. Gardiner-Garden, M., Frommer, M., 1987. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282. Graff, J.R., Herman, J.G., Myohanen, S., Baylin, S.B., Vertino, P.M., 1997. Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation. J. Biol. Chem. 272, 22322–22329. Gu¨ nthert, U., Hofman, M., Rudy, W., Reber, S.M.Z., Haußmann, I., Matzku, S., Wenzel, A., Ponta, H., Herrlich, P., 1991. A new variant of glycoprotein CD44 confers metastatic capacity to rat carcinoma cells. Cell 65, 13–24. Herman, J.G., Merlo, A., Mao, L., Lapidus, R.G., Issa, J.P., Davidson, N.E., Sidransky, D., Baylin, S.B., 1995. Inactivation of the CDKN2/
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