Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression

Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression British Journal of Cancer (2004) 91, 703 – 706 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $30.00 www.bjcancer.com Short Communication Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression ,1 1 1 1 1 S Scarpino , A Di Napoli , M Rapazzotti-Onelli , E Pilozzi and L Ruco Dipartimento di Diagnostica di Laboratorio e Patologia, II Facolta` di Medicina e Chirurgia, Ospedale Sant’Andrea, Via di Grottarossa 1035-1039, 00189 Universita` ‘La Sapienza’, Rome, Italy Hypomethylation has been reported to be responsible for the activation of several oncogenes. The possibility that hypomethylation is involved in the regulation of MET transcription was investigated through the analysis of the methylation status of one CpG island containing 43 CpGs in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre. Evidence of methylation was not found in any of the analysed CpG. British Journal of Cancer (2004) 91, 703–706. doi:10.1038/sj.bjc.6601988 www.bjcancer.com Published online 20 July 2004 & 2004 Cancer Research UK Keywords: DNA methylation; Met expression; papillary carcinoma of the thyroid The Met oncogene encodes a trans-membrane tyrosine kinase conditions. Fragments of fresh tissue were embedded in optimal identified as the receptor for hepatocyte growth factor (HGF) cryopreserving tissue (OCT) compound (Miles, Elkhart, IN, USA), (Giordano et al, 1989; Naldini et al, 1991). Immunohistochemical snap-frozen in liquid nitrogen, and stored at 801C until studies have demonstrated that Met protein is intensely expressed sectioning. Met protein was demonstrated with the DO-24 mouse in tumour cells of 495% cases of thyroid papillary carcinoma. In monoclonal antibody. tumour tissue, the levels of RNA transcripts for MET are 10–100- fold higher than in the surrounding normal thyroid (Di Renzo et al, DNA extraction 1992). Gene expression studies have demonstrated that MET gene is one of the 23 genes, which are significantly more expressed in For DNA extraction, 40 cryostat sections 10mm each were cut from papillary carcinoma (Huang et al, 2001). Mutations, amplifications six cases of papillary carcinoma of the thyroid (two follicular and or other alterations of the MET gene have not been recognised, four usual-type papillary carcinoma; female : male ratio was 5 : 1, raising the possibility that an aberrant transcriptional regulation and the mean age was 45 years), from the corresponding may play a critical role in gene activation. Evidence for activation peritumoral normal thyroid tissue, and from two cases of of oncogenes by specific gene demethylation in cancer has been hyperplastic goitre. They were added 1 ml of lysis solution reported in the body of several oncogenes including cMYC, c-JUN, containing 10ml Tris-HCl pH 8, 1 M,10ml EDTA 0.5 M,25ml SDS HOX11 and H-RAS (Vachtenheim et al, 1994; Watt et al, 2000). 20%, 20ml proteinase K 10 mg ml , and were incubated at 371C Since the MET promoter is a 697 bp 5 -untranslated region that overnight. DNA was extracted using phenol–chloroform method. contains a typical CpG island spanning, with a frequency of CpGs 10 times greater than in the total gene (Accession no. Z26936), in Bisulphite-PCR methylation analysis the present study, we have explored the possibility that an altered methylation status of the MET promoter is involved in the Sodium-bisulphite modification of genomic DNA and PCR were abnormal expression of Met protein in papillary carcinoma. performed according to Frommer’s method (Frommer et al, 1992). Bisulphite causes deamination of cytosine that is transformed into uracyl (thymine) unless the cytosine is methylated, in this case it remains as cytosine. Briefly, 8mg of genomic DNA was digested MATERIALS AND METHODS with 10 U of EcoR1 (PROMEGA) for 1 h at 421C. DNA was purified using phenol–chloroform–isoamylic alcohol, precipitated using Immunohistochemistry ethanol and sodium acetate and resuspended in water. It was Expression of Met protein was investigated in 137 cryopreserved denatured with 3 M NaOH for 20 min at 421C, treated with 3 M samples of thyroid tissue involved by various pathological sodium bisulphite (SIGMA-ALDRICH, St Louis, MO, USA) (pH 5) and 10 mM hydroquinone for 18 h at 551C. After treatment, DNA *Correspondence: Dr S Scarpino; E-mail: stefania.scarpino@uniroma1.it was purified using a Wizard DNA Clean-up kit (SIGMA-ALDRICH, This work has been supported from Associazione Italiana per la Ricerca St Louis, MO, USA) and desulphonated with 0.3 M NaOH and sul Cancro (AIRC) and from the CNR-MIUR project Diagnostica neutralised with 3 M ammonium acetate. To bisulphite-treated Molecolare in Oncologia DNA, 10mg of glycogen was added, precipitated with ethanol and Received 5 December 2003; revised 28 April 2004; accepted 17 May resuspended in 20ml sterile distilled water. PCR amplification was 2004; published online 20 July 2004 performed with 5ml of treated DNA. The sequence of interest in the Molecular and Cellular Pathology Molecular and Cellular Pathology Methylation in papillary carcinoma S Scarpino et al Table 1 Immunohistochemical expression of Met protein and methylation of MET promoter in papillary carcinoma and in other pathological conditions of the thyroid Met-positive MET promoter Histology No. cases Age mean7s.d. Sex F/M cases methylation Papillary carcinoma 61 41712 49/12 61 0/6 Peritumoral normal thyroid tissue 45 /+ 0/6 Insular carcinoma 3 42720 2/1 3 nd Anaplastic carcinoma 3 6772 3/0 3 nd Follicular carcinoma 4 3476 3/1 4 nd Oncocytic carcinoma 4 47713 4/0 2/4 nd Medullary carcinoma 1 51 0/1 0 nd Follicular adenoma 20 45714 14/6 4/20 nd Thyroiditis 3 4975 3/0 2/3 nd Nodular hyperplasia 38 52711 32/6 7/38 0/2 a b Frozen sections were immunostained with DO24 mouse monoclonal antibody. Methylation status of 43 CpGs of the Met promoter in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre was investigated. Sodium bisulphite modification of genomic DNA and PCR were performed according to Frommer’s method (see Materials and Methods). A weaker staining was present in the rim of peritumoral normal follicles with tall epithelium; whereas normal follicles with flat epithelium were not stained. nd ¼ not determined. bisulphite-treated DNA was amplified with bisulphite-specific 0 0 primers: (sense) 5 GGT TGT GTT AAT TTT AGA TT 3 , 0 0 (antisense) 5 ACT ACC CTA CCA ATA ACT CA 3 . The specific PCR product was 380 bp. Bisulphite sequencing Amplified bisulphite-PCR products were subcloned into TA vector system (Invitrogen, San Diego, CA, USA), according to the manufacturer’s instruction. Single colonies were amplified according to the manufacturer’s instructions. DNA sequence analysis was carried out by automated DNA sequencers (Applied Biosystems, Foster City, CA, USA) using Big Dye Terminator Version 1 (Applied Biosystems). In all, 10 independent clones per case were analysed. Bisulphite treatment efficiency was proven by the complete conversion of the C to T in all sequences analysed. RESULTS The pattern of expression of Met protein was investigated in frozen sections of 137 thyroid samples (Table 1). A marked reactivity for the protein was observed in tumour cells of 61 out of 61 cases of papillary carcinoma (Figure 1A); a much weaker staining was present in the rim of peritumoral normal follicles with tall epithelium, whereas normal follicles with flat epithelium were not stained. Met protein expression was investigated in frozen sections of 76 thyroid samples involved by pathological conditions other than papillary carcinoma. Among tumours, membrane staining for Met protein was observed in two out of three insular carcinomas, in two out of three undifferentiated carcinomas, and in one out of four Hu¨rthle cell tumours. A weak cytoplasm staining was observed in four follicular carcinomas, and in four out of 16 follicular adenomas. In non-neoplastic conditions, a marked expression of Met protein was observed in follicles embedded in a chronic inflammatory reaction (Figure 1B), whereas weak staining was observed in tall cell follicles of seven out of 38 hyperplastic goitres. The possibility that methylation is involved in the regulation of Figure 1 (A) Papillary carcinoma of the thyroid immunostained for Met MET transcription was investigated through the analysis of the protein with DO 24 monoclonal antibody. The tumour is intensely and methylation status of 43 CpGs in six cases of papillary carcinoma, diffusely positive; the peritumoral normal thyroid follicles are not stained in the corresponding normal thyroid tissue, and in two cases of (  100). (B) Chronic thyroiditis immunostained for Met protein. Only hyperplastic goitre (Figure 2). Evidence of methylation was not those follicles infiltrated and surrounded by inflammatory cells are stained found in any of the analysed CpG. (  250) (ABC-peroxidase, counterstained with haematoxylin). British Journal of Cancer (2004) 91(4), 703 – 706 & 2004 Cancer Research UK Methylation in papillary carcinoma S Scarpino et al Papillary carcinoma (T) and peritumoral normal thyriod tissue (N) 12 3 4 5 6 7 8 9 1011121314151617181920212223 24 25 26 27 28 29 30 31 32 33 34 35 36 3738 39 40 41 42 43 Case no. 1N 1T 2N 2T 3N 3T 4N 4T 5N 5T 6N 6T Nodular hyperplasia 12 3 4 5 6 7 8 9 10111213141516171819202122 2324 25 26 27 28 29 30 31 32 33 34 35 36 3738 39 40 41 42 43 Case no. Figure 2 Methylation status of 43 CpGs of the Met promoter in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre was investigated. Sodium-Bisulphite modification of genomic DNA and PCR were performed according to Frommer’s method (see Materials and Methods). In all, 10 colonies were analysed for each sample. Black and white areas represent methylated and unmethylated CpG sites, respectively. DISCUSSION (MT1G) (Huang et al, 2003) and the high-affinity cellular retinoic binding protein (CRABP1) (Huang et al, 2003). In the present study, we provide further evidence that Met protein Our findings strongly suggest that molecular mechanisms other is highly expressed in papillary carcinoma cells, whereas it is than hypomethylation of the gene are responsible for the high absent or poorly expressed in normal thyroid follicles; moreover, expression of Met protein in papillary carcinoma of the thyroid. So we demonstrate for the first time that the different patterns of far, it has been demonstrated that insertion of activated RAS and expression are not due to an altered methylation status of the MET RET in normal thyroid cells causes upregulation of MET promoter. transcription (Ivan et al, 1997). The frequent occurrence of RET The rationale for our study derives from previous reports rearrangements in papillary carcinoma (Elisei et al, 2001; Soares showing that hypomethylation is a kind of molecular mechanism et al, 2003) and the recent observation that a consistent number of leading to promoting high expression of oncogenes that encodes nonrearranged cases have an activating mutation of BRAF that also for some proteins with tyrosine kinase activity (Clark and Melki, cause signal transduction through the RET–RAS pathway 2002). They include several members of the Eph family of receptor (Fukushima et al, 2003) are consistent with the possibility that tyrosine kinases (RTK) (Dottori et al, 1999), the c-fms oncogene dysregulation of MET transcription is caused by the genetic that encodes for CSF 1R (Cui et al, 2001), and the erbB2/neu (Zhou transforming alterations specifically associated with this histotype. et al, 2001). In addition, it was recently shown that tumour hypoxia may cause Finally, in other studies on papillary carcinoma of the thyroid it an increased transcription of MET through the upregulation of the was shown that abnormal methylation may occur in tumour cells, hypoxia inducible factor-1 (HIF-1), which has two binding sites on and is probably responsible for loss or for decreased expression of the MET promoter (Pennacchietti et al, 2003). In a recent study, we several genes including TSH receptor (TSHR) (Xing et al, 2003a) have reported that HIF-1 is upregulated in tumour cells of most the Pendred syndrome gene SLC26A4 (Xing et al, 2003b), the Ras cases of papillary carcinoma and that histological alterations association domain family 1A gene (RASSF1A) (Schagdarsurengin suggestive of a hypoxic condition are frequently present in this et al, 2002), the metallothionein heavy metal binding protein gene specific tumour (Scarpino et al, 2004). REFERENCES Clark SJ, Melki J (2002) DNA methylation and gene silencing in cancer: met/hgf receptor gene in human thyroid carcinomas. Oncogene 7: which is the guilty party? Oncogene 21: 5380–5387 2549– 2553 Cui J, Yang DH, Bi XJ, Fan ZR (2001) Methylation status of c-fms oncogene Dottori M, Down M, Huttmann A, Fitzpatrick DR, Boyd AW (1999) Cloning in HCC and its relationship with clinical pathology. World J and characterization of EphA3 (Hek) gene promoter: DNA methylation Gastroenterol 7: 136–139 regulates expression in hematopoietic tumor cells. Blood 94: 2477– 2486 Di Renzo MF, Olivero M, Ferro S, Prat M, Bongarzone I, Pilotti S, Belfiore Elisei R, Romei C, Vorontsova T, Cosci B, Veremeychik V, Kuchinskaya E, A, Costantino A, Vigneri R, Pierotti MA (1992) Overexpression of the c- Basolo F, Demidchik EP, Miccoli P, Pinchera A, Pacini F (2001) RET/PTC & 2004 Cancer Research UK British Journal of Cancer (2004) 91(4), 703 – 706 Molecular and Cellular Pathology Molecular and Cellular Pathology Methylation in papillary carcinoma S Scarpino et al rearrangements in thyroid nodules: studies in irradiated and not Scarpino S, Cancellario d’Alena F, Di Napoli A, Pasquini A, Marzullo A, irradiated, malignant and benign thyroid lesions in children and adults. Ruco LP (2004) Papillary carcinoma of the thyroid: increased expression J Clin Endocrinol Metab 86: 3211 –3216 of Met protein is associated with upregulation of hypoxia inducible Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, factor-1 (HIF-1) in tumor cells. J Pathol 202: 352– 358 Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a Schagdarsurengin U, Gimm O, Hoang-Vu C, Dralle H, Pfeifer GP, positive display of 5-methylcytosine residues in individual DNA strands. Dammann R (2002) Frequent epigenetic silencing of the CpG island Proc Natl Acad Sci USA 89: 1827–1831 promoter of RASSF1A in thyroid carcinoma. Cancer Res 62: 3698– 3701 Fukushima T, Suzuki S, Mashiko M, Ohtake T, Endo Y, Takebayashi Y, Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, Maximo V, Sekikawa K, Hagiwara K, Takenoshita S (2003) BRAF mutations in Botelho T, Seruca R, Sobrinho-Simoes M (2003) BRAF mutations and papillary carcinomas of the thyroid. Oncogene 22: 6455–6457 RET/PTC rearrangements are alternative events in the etiopathogenesis Giordano S, Ponzetto C, Di Renzo MF, Cooper CS, Comoglio PM (1989) of PTC. Oncogene 22: 4578 –4580 Tyrosine kinase receptor indistinguishable from the c-met protein. Vachtenheim J, Horakova I, Novotna H (1994) Hypomethylation of CCGG Nature 339: 155–156 sites in the 3 region of H-ras protooncogene is frequent and is associated Huang Y, De La Chapelle A, Pellegata NS (2003) Hypermethylation, but not with H-ras allele loss in non-small cell lung cancer. Cancer Res. 54: LOH, is associated with the low expression of MT1G and CRABP1 in 1145– 1148 papillary carcinoma of the thyroid. Int J Cancer 104: 735–744 Watt PM, Kumar R, Kees UR (2000) Promoter demethylation accompanies Huang Y, Prasad M, Lemon WJ, Hampel H, Wright FA, Kornacker K, reactivation of the HOX11 proto-oncogene in leukemia. Genes Chromo- LiVolsi V, Frankel W, Kloos RT, Eng C, Pellegata NS, De La Chapelle A somes Cancer 29: 371 –377 (2001) Gene expression in papillary thyroid carcinoma reveals highly Xing M, Usadel H, Cohen Y, Tokumaru Y, Guo Z, Westra WB, Tong BC, consistent profiles. Proc Natl Acad Sci USA 98: 15044–15049 Tallini G, Udelsman R, Califano JA, Ladenson PW, Sidransky D (2003a) Ivan M, Bond JA, Prat M, Comoglio PM, Wynford-Thomas D (1997) Methylation of the thyroid-stimulating hormone receptor gene in Activated ras and ret oncogenes induce overexpression of c-met epithelial thyroid tumors: a marker of malignancy and a cause of gene (hepatocyte growth factor receptor) in human thyroid epithelial cells. silencing. Cancer Res 63: 2316–2321 Oncogene 14: 2417–2423 Xing M, Tokumaru Y, Wu G, Westra WB, Ladenson PW, Naldini L, Vigna E, Narsimhan RP, Gaudino G, Zarnegar R, Michalopoulos Sidransky D (2003b) Hypermethylation of the Pendred syndrome gene GK, Comoglio PM (1991) Hepatocyte growth factor (HGF) stimulates the SLC26A4 is an early event in thyroid tumorigenesis. Cancer Res 63: tyrosine kinase activity of the receptor encoded by the proto-oncogene c- 2312– 2315 Met. Oncogene 6: 501–504 Zhou H, Chen WD, Qin X, Lee K, Liu L, Markowitz SD, Gerson SL (2001) Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, Comoglio MMTV promoter hypomethylation is linked to spontaneous and MNU PM (2003) Hypoxia promotes invasive growth by transcriptional associated c-neu expression and mammary carcinogenesis in MMTV activation of the met protooncogene. Cancer Cell 3: 347–361 c-neu transgenic mice. Oncogene 20: 6009 –6017 British Journal of Cancer (2004) 91(4), 703 – 706 & 2004 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression

Loading next page...
 
/lp/springer-journals/papillary-carcinoma-of-the-thyroid-methylation-is-not-involved-in-the-dYjSEgNpN0

References (44)

Publisher
Springer Journals
Copyright
Copyright © 2004 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
ISSN
0007-0920
eISSN
1532-1827
DOI
10.1038/sj.bjc.6601988
Publisher site
See Article on Publisher Site

Abstract

British Journal of Cancer (2004) 91, 703 – 706 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $30.00 www.bjcancer.com Short Communication Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression ,1 1 1 1 1 S Scarpino , A Di Napoli , M Rapazzotti-Onelli , E Pilozzi and L Ruco Dipartimento di Diagnostica di Laboratorio e Patologia, II Facolta` di Medicina e Chirurgia, Ospedale Sant’Andrea, Via di Grottarossa 1035-1039, 00189 Universita` ‘La Sapienza’, Rome, Italy Hypomethylation has been reported to be responsible for the activation of several oncogenes. The possibility that hypomethylation is involved in the regulation of MET transcription was investigated through the analysis of the methylation status of one CpG island containing 43 CpGs in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre. Evidence of methylation was not found in any of the analysed CpG. British Journal of Cancer (2004) 91, 703–706. doi:10.1038/sj.bjc.6601988 www.bjcancer.com Published online 20 July 2004 & 2004 Cancer Research UK Keywords: DNA methylation; Met expression; papillary carcinoma of the thyroid The Met oncogene encodes a trans-membrane tyrosine kinase conditions. Fragments of fresh tissue were embedded in optimal identified as the receptor for hepatocyte growth factor (HGF) cryopreserving tissue (OCT) compound (Miles, Elkhart, IN, USA), (Giordano et al, 1989; Naldini et al, 1991). Immunohistochemical snap-frozen in liquid nitrogen, and stored at 801C until studies have demonstrated that Met protein is intensely expressed sectioning. Met protein was demonstrated with the DO-24 mouse in tumour cells of 495% cases of thyroid papillary carcinoma. In monoclonal antibody. tumour tissue, the levels of RNA transcripts for MET are 10–100- fold higher than in the surrounding normal thyroid (Di Renzo et al, DNA extraction 1992). Gene expression studies have demonstrated that MET gene is one of the 23 genes, which are significantly more expressed in For DNA extraction, 40 cryostat sections 10mm each were cut from papillary carcinoma (Huang et al, 2001). Mutations, amplifications six cases of papillary carcinoma of the thyroid (two follicular and or other alterations of the MET gene have not been recognised, four usual-type papillary carcinoma; female : male ratio was 5 : 1, raising the possibility that an aberrant transcriptional regulation and the mean age was 45 years), from the corresponding may play a critical role in gene activation. Evidence for activation peritumoral normal thyroid tissue, and from two cases of of oncogenes by specific gene demethylation in cancer has been hyperplastic goitre. They were added 1 ml of lysis solution reported in the body of several oncogenes including cMYC, c-JUN, containing 10ml Tris-HCl pH 8, 1 M,10ml EDTA 0.5 M,25ml SDS HOX11 and H-RAS (Vachtenheim et al, 1994; Watt et al, 2000). 20%, 20ml proteinase K 10 mg ml , and were incubated at 371C Since the MET promoter is a 697 bp 5 -untranslated region that overnight. DNA was extracted using phenol–chloroform method. contains a typical CpG island spanning, with a frequency of CpGs 10 times greater than in the total gene (Accession no. Z26936), in Bisulphite-PCR methylation analysis the present study, we have explored the possibility that an altered methylation status of the MET promoter is involved in the Sodium-bisulphite modification of genomic DNA and PCR were abnormal expression of Met protein in papillary carcinoma. performed according to Frommer’s method (Frommer et al, 1992). Bisulphite causes deamination of cytosine that is transformed into uracyl (thymine) unless the cytosine is methylated, in this case it remains as cytosine. Briefly, 8mg of genomic DNA was digested MATERIALS AND METHODS with 10 U of EcoR1 (PROMEGA) for 1 h at 421C. DNA was purified using phenol–chloroform–isoamylic alcohol, precipitated using Immunohistochemistry ethanol and sodium acetate and resuspended in water. It was Expression of Met protein was investigated in 137 cryopreserved denatured with 3 M NaOH for 20 min at 421C, treated with 3 M samples of thyroid tissue involved by various pathological sodium bisulphite (SIGMA-ALDRICH, St Louis, MO, USA) (pH 5) and 10 mM hydroquinone for 18 h at 551C. After treatment, DNA *Correspondence: Dr S Scarpino; E-mail: stefania.scarpino@uniroma1.it was purified using a Wizard DNA Clean-up kit (SIGMA-ALDRICH, This work has been supported from Associazione Italiana per la Ricerca St Louis, MO, USA) and desulphonated with 0.3 M NaOH and sul Cancro (AIRC) and from the CNR-MIUR project Diagnostica neutralised with 3 M ammonium acetate. To bisulphite-treated Molecolare in Oncologia DNA, 10mg of glycogen was added, precipitated with ethanol and Received 5 December 2003; revised 28 April 2004; accepted 17 May resuspended in 20ml sterile distilled water. PCR amplification was 2004; published online 20 July 2004 performed with 5ml of treated DNA. The sequence of interest in the Molecular and Cellular Pathology Molecular and Cellular Pathology Methylation in papillary carcinoma S Scarpino et al Table 1 Immunohistochemical expression of Met protein and methylation of MET promoter in papillary carcinoma and in other pathological conditions of the thyroid Met-positive MET promoter Histology No. cases Age mean7s.d. Sex F/M cases methylation Papillary carcinoma 61 41712 49/12 61 0/6 Peritumoral normal thyroid tissue 45 /+ 0/6 Insular carcinoma 3 42720 2/1 3 nd Anaplastic carcinoma 3 6772 3/0 3 nd Follicular carcinoma 4 3476 3/1 4 nd Oncocytic carcinoma 4 47713 4/0 2/4 nd Medullary carcinoma 1 51 0/1 0 nd Follicular adenoma 20 45714 14/6 4/20 nd Thyroiditis 3 4975 3/0 2/3 nd Nodular hyperplasia 38 52711 32/6 7/38 0/2 a b Frozen sections were immunostained with DO24 mouse monoclonal antibody. Methylation status of 43 CpGs of the Met promoter in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre was investigated. Sodium bisulphite modification of genomic DNA and PCR were performed according to Frommer’s method (see Materials and Methods). A weaker staining was present in the rim of peritumoral normal follicles with tall epithelium; whereas normal follicles with flat epithelium were not stained. nd ¼ not determined. bisulphite-treated DNA was amplified with bisulphite-specific 0 0 primers: (sense) 5 GGT TGT GTT AAT TTT AGA TT 3 , 0 0 (antisense) 5 ACT ACC CTA CCA ATA ACT CA 3 . The specific PCR product was 380 bp. Bisulphite sequencing Amplified bisulphite-PCR products were subcloned into TA vector system (Invitrogen, San Diego, CA, USA), according to the manufacturer’s instruction. Single colonies were amplified according to the manufacturer’s instructions. DNA sequence analysis was carried out by automated DNA sequencers (Applied Biosystems, Foster City, CA, USA) using Big Dye Terminator Version 1 (Applied Biosystems). In all, 10 independent clones per case were analysed. Bisulphite treatment efficiency was proven by the complete conversion of the C to T in all sequences analysed. RESULTS The pattern of expression of Met protein was investigated in frozen sections of 137 thyroid samples (Table 1). A marked reactivity for the protein was observed in tumour cells of 61 out of 61 cases of papillary carcinoma (Figure 1A); a much weaker staining was present in the rim of peritumoral normal follicles with tall epithelium, whereas normal follicles with flat epithelium were not stained. Met protein expression was investigated in frozen sections of 76 thyroid samples involved by pathological conditions other than papillary carcinoma. Among tumours, membrane staining for Met protein was observed in two out of three insular carcinomas, in two out of three undifferentiated carcinomas, and in one out of four Hu¨rthle cell tumours. A weak cytoplasm staining was observed in four follicular carcinomas, and in four out of 16 follicular adenomas. In non-neoplastic conditions, a marked expression of Met protein was observed in follicles embedded in a chronic inflammatory reaction (Figure 1B), whereas weak staining was observed in tall cell follicles of seven out of 38 hyperplastic goitres. The possibility that methylation is involved in the regulation of Figure 1 (A) Papillary carcinoma of the thyroid immunostained for Met MET transcription was investigated through the analysis of the protein with DO 24 monoclonal antibody. The tumour is intensely and methylation status of 43 CpGs in six cases of papillary carcinoma, diffusely positive; the peritumoral normal thyroid follicles are not stained in the corresponding normal thyroid tissue, and in two cases of (  100). (B) Chronic thyroiditis immunostained for Met protein. Only hyperplastic goitre (Figure 2). Evidence of methylation was not those follicles infiltrated and surrounded by inflammatory cells are stained found in any of the analysed CpG. (  250) (ABC-peroxidase, counterstained with haematoxylin). British Journal of Cancer (2004) 91(4), 703 – 706 & 2004 Cancer Research UK Methylation in papillary carcinoma S Scarpino et al Papillary carcinoma (T) and peritumoral normal thyriod tissue (N) 12 3 4 5 6 7 8 9 1011121314151617181920212223 24 25 26 27 28 29 30 31 32 33 34 35 36 3738 39 40 41 42 43 Case no. 1N 1T 2N 2T 3N 3T 4N 4T 5N 5T 6N 6T Nodular hyperplasia 12 3 4 5 6 7 8 9 10111213141516171819202122 2324 25 26 27 28 29 30 31 32 33 34 35 36 3738 39 40 41 42 43 Case no. Figure 2 Methylation status of 43 CpGs of the Met promoter in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre was investigated. Sodium-Bisulphite modification of genomic DNA and PCR were performed according to Frommer’s method (see Materials and Methods). In all, 10 colonies were analysed for each sample. Black and white areas represent methylated and unmethylated CpG sites, respectively. DISCUSSION (MT1G) (Huang et al, 2003) and the high-affinity cellular retinoic binding protein (CRABP1) (Huang et al, 2003). In the present study, we provide further evidence that Met protein Our findings strongly suggest that molecular mechanisms other is highly expressed in papillary carcinoma cells, whereas it is than hypomethylation of the gene are responsible for the high absent or poorly expressed in normal thyroid follicles; moreover, expression of Met protein in papillary carcinoma of the thyroid. So we demonstrate for the first time that the different patterns of far, it has been demonstrated that insertion of activated RAS and expression are not due to an altered methylation status of the MET RET in normal thyroid cells causes upregulation of MET promoter. transcription (Ivan et al, 1997). The frequent occurrence of RET The rationale for our study derives from previous reports rearrangements in papillary carcinoma (Elisei et al, 2001; Soares showing that hypomethylation is a kind of molecular mechanism et al, 2003) and the recent observation that a consistent number of leading to promoting high expression of oncogenes that encodes nonrearranged cases have an activating mutation of BRAF that also for some proteins with tyrosine kinase activity (Clark and Melki, cause signal transduction through the RET–RAS pathway 2002). They include several members of the Eph family of receptor (Fukushima et al, 2003) are consistent with the possibility that tyrosine kinases (RTK) (Dottori et al, 1999), the c-fms oncogene dysregulation of MET transcription is caused by the genetic that encodes for CSF 1R (Cui et al, 2001), and the erbB2/neu (Zhou transforming alterations specifically associated with this histotype. et al, 2001). In addition, it was recently shown that tumour hypoxia may cause Finally, in other studies on papillary carcinoma of the thyroid it an increased transcription of MET through the upregulation of the was shown that abnormal methylation may occur in tumour cells, hypoxia inducible factor-1 (HIF-1), which has two binding sites on and is probably responsible for loss or for decreased expression of the MET promoter (Pennacchietti et al, 2003). In a recent study, we several genes including TSH receptor (TSHR) (Xing et al, 2003a) have reported that HIF-1 is upregulated in tumour cells of most the Pendred syndrome gene SLC26A4 (Xing et al, 2003b), the Ras cases of papillary carcinoma and that histological alterations association domain family 1A gene (RASSF1A) (Schagdarsurengin suggestive of a hypoxic condition are frequently present in this et al, 2002), the metallothionein heavy metal binding protein gene specific tumour (Scarpino et al, 2004). REFERENCES Clark SJ, Melki J (2002) DNA methylation and gene silencing in cancer: met/hgf receptor gene in human thyroid carcinomas. Oncogene 7: which is the guilty party? Oncogene 21: 5380–5387 2549– 2553 Cui J, Yang DH, Bi XJ, Fan ZR (2001) Methylation status of c-fms oncogene Dottori M, Down M, Huttmann A, Fitzpatrick DR, Boyd AW (1999) Cloning in HCC and its relationship with clinical pathology. World J and characterization of EphA3 (Hek) gene promoter: DNA methylation Gastroenterol 7: 136–139 regulates expression in hematopoietic tumor cells. Blood 94: 2477– 2486 Di Renzo MF, Olivero M, Ferro S, Prat M, Bongarzone I, Pilotti S, Belfiore Elisei R, Romei C, Vorontsova T, Cosci B, Veremeychik V, Kuchinskaya E, A, Costantino A, Vigneri R, Pierotti MA (1992) Overexpression of the c- Basolo F, Demidchik EP, Miccoli P, Pinchera A, Pacini F (2001) RET/PTC & 2004 Cancer Research UK British Journal of Cancer (2004) 91(4), 703 – 706 Molecular and Cellular Pathology Molecular and Cellular Pathology Methylation in papillary carcinoma S Scarpino et al rearrangements in thyroid nodules: studies in irradiated and not Scarpino S, Cancellario d’Alena F, Di Napoli A, Pasquini A, Marzullo A, irradiated, malignant and benign thyroid lesions in children and adults. Ruco LP (2004) Papillary carcinoma of the thyroid: increased expression J Clin Endocrinol Metab 86: 3211 –3216 of Met protein is associated with upregulation of hypoxia inducible Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, factor-1 (HIF-1) in tumor cells. J Pathol 202: 352– 358 Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a Schagdarsurengin U, Gimm O, Hoang-Vu C, Dralle H, Pfeifer GP, positive display of 5-methylcytosine residues in individual DNA strands. Dammann R (2002) Frequent epigenetic silencing of the CpG island Proc Natl Acad Sci USA 89: 1827–1831 promoter of RASSF1A in thyroid carcinoma. Cancer Res 62: 3698– 3701 Fukushima T, Suzuki S, Mashiko M, Ohtake T, Endo Y, Takebayashi Y, Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, Maximo V, Sekikawa K, Hagiwara K, Takenoshita S (2003) BRAF mutations in Botelho T, Seruca R, Sobrinho-Simoes M (2003) BRAF mutations and papillary carcinomas of the thyroid. Oncogene 22: 6455–6457 RET/PTC rearrangements are alternative events in the etiopathogenesis Giordano S, Ponzetto C, Di Renzo MF, Cooper CS, Comoglio PM (1989) of PTC. Oncogene 22: 4578 –4580 Tyrosine kinase receptor indistinguishable from the c-met protein. Vachtenheim J, Horakova I, Novotna H (1994) Hypomethylation of CCGG Nature 339: 155–156 sites in the 3 region of H-ras protooncogene is frequent and is associated Huang Y, De La Chapelle A, Pellegata NS (2003) Hypermethylation, but not with H-ras allele loss in non-small cell lung cancer. Cancer Res. 54: LOH, is associated with the low expression of MT1G and CRABP1 in 1145– 1148 papillary carcinoma of the thyroid. Int J Cancer 104: 735–744 Watt PM, Kumar R, Kees UR (2000) Promoter demethylation accompanies Huang Y, Prasad M, Lemon WJ, Hampel H, Wright FA, Kornacker K, reactivation of the HOX11 proto-oncogene in leukemia. Genes Chromo- LiVolsi V, Frankel W, Kloos RT, Eng C, Pellegata NS, De La Chapelle A somes Cancer 29: 371 –377 (2001) Gene expression in papillary thyroid carcinoma reveals highly Xing M, Usadel H, Cohen Y, Tokumaru Y, Guo Z, Westra WB, Tong BC, consistent profiles. Proc Natl Acad Sci USA 98: 15044–15049 Tallini G, Udelsman R, Califano JA, Ladenson PW, Sidransky D (2003a) Ivan M, Bond JA, Prat M, Comoglio PM, Wynford-Thomas D (1997) Methylation of the thyroid-stimulating hormone receptor gene in Activated ras and ret oncogenes induce overexpression of c-met epithelial thyroid tumors: a marker of malignancy and a cause of gene (hepatocyte growth factor receptor) in human thyroid epithelial cells. silencing. Cancer Res 63: 2316–2321 Oncogene 14: 2417–2423 Xing M, Tokumaru Y, Wu G, Westra WB, Ladenson PW, Naldini L, Vigna E, Narsimhan RP, Gaudino G, Zarnegar R, Michalopoulos Sidransky D (2003b) Hypermethylation of the Pendred syndrome gene GK, Comoglio PM (1991) Hepatocyte growth factor (HGF) stimulates the SLC26A4 is an early event in thyroid tumorigenesis. Cancer Res 63: tyrosine kinase activity of the receptor encoded by the proto-oncogene c- 2312– 2315 Met. Oncogene 6: 501–504 Zhou H, Chen WD, Qin X, Lee K, Liu L, Markowitz SD, Gerson SL (2001) Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, Comoglio MMTV promoter hypomethylation is linked to spontaneous and MNU PM (2003) Hypoxia promotes invasive growth by transcriptional associated c-neu expression and mammary carcinogenesis in MMTV activation of the met protooncogene. Cancer Cell 3: 347–361 c-neu transgenic mice. Oncogene 20: 6009 –6017 British Journal of Cancer (2004) 91(4), 703 – 706 & 2004 Cancer Research UK

Journal

British Journal of CancerSpringer Journals

Published: Jul 20, 2004

There are no references for this article.