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. 2007 May 1;10(3):778–788. doi: 10.1111/j.1582-4934.2006.tb00437.x

Analysis of p16 expression and allelic imbalance / loss of heterozygosity of 9p21 in cutaneous squamous cell carcinomas

Sarah E Gray a, Elaine Kay a, Mary Leader a, M Mabruk a,b,*
PMCID: PMC3933159  PMID: 16989737

Abstract

Deletions of the short arm of chromosome 9 have been reported in different types of malignancies. This chromosomal region contains a number of known tumour suppressor genes, including the p16INK4A (CDKN2A), p15INK4B and MTAP tumour suppressor genes located at 9p21. In this study twenty-two paraffin embedded invasive cutaneous SCC were examined for allelic imbalance/ loss of heterozygosity (AI/LOH) of the 9p region (in particular 9p21), and for p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells and analysed for AI/LOH by using six fluorescently labelled microsatellite markers that map to the 9p region. P16 protein expression was examined by immunohistochemistry. At each of the six microsatellite markers the majority of SCC analysed showed AI/LOH. Overall both AI/LOH within the CDKN2A locus and absence of p16 protein expression were frequent among the cutaneous SCC analysed, suggesting that p16 inactivation may play a role in cutaneous SCC development. The majority of the SCC analysed also had AI/LOH of the marker within the MTAP gene, and at markers flanking the CDKN2A gene; thus further investigation as to a possible role for these genes in the development of cutaneous SCC is warranted.

Keywords: cutaneous SCC, AI/LOH, p16, p15, MTAP

References

  • 1.Stott FJ, Bates S, James MC, McConnell BB, Starborg M, Brookes S, Palmero I, Ryan K, Hara E, Vousden KH, Peters G. The alternative product from the human CDKN2A locus, p14 (ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 1998;17:5001–14. doi: 10.1093/emboj/17.17.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Medema RH, Herrera RE, Lam F, Weinberg RA. Growth suppression by p16ink4 requires functional retinoblastoma protein. Proc Natl Acad Sci USA. 1995;92:6289–93. doi: 10.1073/pnas.92.14.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sherr C. The INK4a/ARF network in tumour suppression. Nat Reviews; Mol Cell Biol. 2001;2:731–7. doi: 10.1038/35096061. [DOI] [PubMed] [Google Scholar]
  • 4.Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, Johnson BE, Skolnick MH. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994;264:436–40. doi: 10.1126/science.8153634. [DOI] [PubMed] [Google Scholar]
  • 5.Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–7. doi: 10.1038/366704a0. [DOI] [PubMed] [Google Scholar]
  • 6.Quelle DE, Zindy F, Ashmun RA. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell. 1995;83:993–10. doi: 10.1016/0092-8674(95)90214-7. [DOI] [PubMed] [Google Scholar]
  • 7.MacKie RM, Andrew N, Lanyon WG, Connor JM. CDKN2A germline mutations in UK patients with familial melanoma and multiple primary melanomas. J Invest Dermatol. 1998;111:269–72. doi: 10.1046/j.1523-1747.1998.00267.x. [DOI] [PubMed] [Google Scholar]
  • 8.Reed JA, Loganzo F, Jr, Shea C, Walker GJ, Flores JF, Glendening JM, Bogdany JK, Shiel MJ, Haluska FG, Fountain JW. Loss of expression of the p16/cyclin-dependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression. Cancer Res. 1995;55:2713–8. [PubMed] [Google Scholar]
  • 9.Gorgoulis VG, Koutroumbi EN, Kotsinas A, Zacharatos P, Markopoulos C, Giannikos L, Kyriakou V, Voulgaris Z, Gogas I, Kittas C. Alterations of p16-pRb pathway and chromosome locus 9p21-22 in sporadic invasive breast carcinomas. Mol Med. 1998;4:807–22. [PMC free article] [PubMed] [Google Scholar]
  • 10.Muzeau F, Flejou JF, Thomas G, Hamelin R. Loss of heterozygosity on chromosome 9 and p16 (MTS1, CDKN2) gene mutations in esophageal cancers. Int J Cancer. 1997;72:27–30. doi: 10.1002/(sici)1097-0215(19970703)72:1<27::aid-ijc3>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]
  • 11.Jares P, Fernandez PL, Nadal A, Cazorla M, Hernandez L, Pinyol M, Hernandez S, Traserra J, Cardesa A, Campo E. p16MTS1/CDK4I mutations and concomitant loss of heterozygosity at 9p21-23 are frequent events in squamous cell carcinoma of the larynx. Oncogene. 1997;15:1445–53. doi: 10.1038/sj.onc.1201309. [DOI] [PubMed] [Google Scholar]
  • 12.Viswanathan M, Tsuchida N, Shanmugam G. Promoter hypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cell carcinoma. Int J Cancer. 2003;105:41–6. doi: 10.1002/ijc.11028. [DOI] [PubMed] [Google Scholar]
  • 13.Yakushiji T, Uzawa K, Shibahara T, Noma H, Tanzawa H. Over-expression of DNA methyltransferases and CDKN2A gene methylation status in squamous cell carcinoma of the oral cavity. Int J Oncol. 2003;22:1201–7. [PubMed] [Google Scholar]
  • 14.Sanchez-Cespedes M, Decker PA, Doffek KM, Esteller M, Westra WH, Alawi EA, Herman JG, Demeure MJ, Sidransky D, Ahrendt SA. Increased loss of chromosome 9p21 but not p16 inactivation in primary non-small cell lung cancer from smokers. Cancer Res. 2001;61:2092–6. [PubMed] [Google Scholar]
  • 15.Hashiguchi Y, Tsuda H, Yamamoto K, Inoue T, Ishiko O, Ogita S. Combined analysis of p53 and RB pathways in epithelial ovarian cancer. Hum Pathol. 2001;32:988–96. doi: 10.1053/hupa.2001.27115. [DOI] [PubMed] [Google Scholar]
  • 16.Enders GH, Koh J, Missero C, Rustgi A. Harlow E. P16 inhibition of transformed and primary squamous epithelial cells. Oncogene. 1996;12:1239–45. [PubMed] [Google Scholar]
  • 17.Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA. Role of the INK4a locus in tumor suppression and cell mortality. Cell. 1996;85:27–37. doi: 10.1016/s0092-8674(00)81079-x. [DOI] [PubMed] [Google Scholar]
  • 18.Soufir N, Moles JP, Vilmer C, Moch C, Verola O, Rivet J, Tesniere A, Dubertret L, Basset-Seguin N. P16 UV mutations in human skin epithelial tumors. Oncogene. 1999;18:5477–81. doi: 10.1038/sj.onc.1202915. [DOI] [PubMed] [Google Scholar]
  • 19.Kubo Y, Urano Y, Matsumoto K, Ahsa K, Arase S. Mutations of the INK4a locus in squamous cell carcinomas of human skin. Biochem Biophys Res. Commun. 1997;232:38–41. doi: 10.1006/bbrc.1997.6217. [DOI] [PubMed] [Google Scholar]
  • 20.Saridaki Z, Liloglou T, Zafiropoulos A, Koumantaki E, Zoras O, Spandidos DA. Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin. Br J Dermatol. 2003;148:638–48. doi: 10.1046/j.1365-2133.2003.05230.x. [DOI] [PubMed] [Google Scholar]
  • 21.Kreimer-Erlacher H, Seidl H, Back B, Cerroni I, Kerl H, Wolf P. High frequency of Ultraviolet mutations at the INK4a-ARF locus in squamous cell carcinomas from Psoralen-Plus-Ultraviolet-A-Treated Psoriasis Patients. J Invest Dermatol. 2003;120:676–82. doi: 10.1046/j.1523-1747.2003.12085.x. [DOI] [PubMed] [Google Scholar]
  • 22.Hannon GJ, Beach D. p15 (INK4B) is a potential effector of TGF-beta-induced cell cycle arrest. Nature. 1994;371:257–61. doi: 10.1038/371257a0. [DOI] [PubMed] [Google Scholar]
  • 23.Tanaka H, Shimada Y, Imamura M, Shibagaki I, Ishizaki K. Multiple types of aberrations in the p16 (INK4a) and the p15 (INK4b) genes in 30 esophageal squamous-cell-carcinoma cell lines. Int J Cancer. 1997;70:437–42. doi: 10.1002/(sici)1097-0215(19970207)70:4<437::aid-ijc11>3.0.co;2-c. [DOI] [PubMed] [Google Scholar]
  • 24.Roesler JM, Livingston EH, Srivatsan E. Hang P, Wang MB. Deletion of P15 (MTS2) in head and neck squamous cell carcinomas. J Surg Res. 1998;77:50–4. doi: 10.1006/jsre.1998.5337. [DOI] [PubMed] [Google Scholar]
  • 25.Ichikawa Y, Yoshida S, Koyama Y, Hirai M, Ishikawa T, Nishida M, Tsunoda H, Kubo T, Miwa M, Uchida K. Inactivation of p16/CDKN2 and p15/MTS2 genes in different histological types and clinical stages of primary ovarian tumors. Int J Cancer. 1996;69:466–70. doi: 10.1002/(SICI)1097-0215(19961220)69:6<466::AID-IJC8>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  • 26.Stadler WM, Olopade OI. The 9p21 region in bladder cancer cell lines: large homozygous deletion inactivate the CDKN2, CDKN2B and MTAP genes. Urol Res. 1996;24:239–44. doi: 10.1007/BF00295899. [DOI] [PubMed] [Google Scholar]
  • 27.Wong TS, Man MW, Lam AK, Wei WI, Kwong YL, Yuen AP. The study of p16 and p15 gene methylation in head and neck squamous cell carcinoma and their quantitative evaluation in plasma by real-time PCR. Eur J Cancer. 2003;39:1881–7. doi: 10.1016/s0959-8049(03)00428-3. [DOI] [PubMed] [Google Scholar]
  • 28.Furonaka O, Takeshima Y, Awaya H, Ishida H, Kohno N, Inai K. Aberrant methylation of p14 (ARF), p15 (INK4b) and p16 (INK4a) genes and location of the primary site in pulmonary squamous cell carcinoma. Pathol Int. 2004;54:549–55. doi: 10.1111/j.1440-1827.2004.01663.x. [DOI] [PubMed] [Google Scholar]
  • 29.Shintani S, Nakahara Y, Mihara M, Ueyama Y, Matsumura T. Inactivation of the p14 (ARF), p15 (INK4B) and p16 (INK4A) genes is a frequent event in human oral squamous cell carcinomas. Oral Oncol. 2001;37:498–04. doi: 10.1016/s1368-8375(00)00142-1. [DOI] [PubMed] [Google Scholar]
  • 30.Subhi A, Diegelman P, Porter C, Tang B, Lu Z, Markham M, Kruger W. Methylthioadenosine phosphorylase regulates omithine decarboxylase by production of downstream metabolites. J Biol Chem. 2003;278:49868–73. doi: 10.1074/jbc.M308451200. [DOI] [PubMed] [Google Scholar]
  • 31.Behrmann I, Wallner S, Komyod W, Heinrich P, Schuierer M, Buettner R, Bosserhoff A. Characterization of methylthioadenosin phosphorylase (MTAP) expression in malignant melanoma. Am J Pathol. 2003;163:683–90. doi: 10.1016/S0002-9440(10)63695-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Christopher S, Diegelman P, Porter K, Kruger W. Methylthioadenosine phosphorylase, a gene frequently codeleted with p16cdkN2a/ARF, acts as a tumor suppressor in a breast cancer cell line. Cancer Res. 2002;62:6639–44. [PubMed] [Google Scholar]
  • 33.Schmid M, Malicki D, Nobori T, Rosenbach M, Campbell K, Carson D, Carrera C. Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC) Oncogene. 1998;17:2669–75. doi: 10.1038/sj.onc.1202205. [DOI] [PubMed] [Google Scholar]
  • 34.Garcia-Castellano JM, Villanueva A, Healey JH, Beart RW, Van Tornout JM, Jones PA. Methylthioadenosine phosphorylase gene deletions are common in osteosarcoma. Clin Cancer Res. 2002;8:782–7. [PubMed] [Google Scholar]
  • 35.Butler D, Collins C, Mabruk M, Barry WC, Leader MB, Kay EW. Deletion of the FHIT gene in neoplastic and invasive cervical lesions is related to high-risk HPV infection but is independent of histopathological features. J Pathol. 2000;192:502–10. doi: 10.1002/1096-9896(2000)9999:9999<::AID-PATH718>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  • 36.Zauber NP, Sabbath-Solitare M, Marotta SP. Comparison of allelic ratios from paired blood and paraffin embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity. Molec Diagn. 1999;4:29–35. doi: 10.1016/s1084-8592(99)80047-2. [DOI] [PubMed] [Google Scholar]
  • 37.Hecker KH, Roux KH. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques. 1996;20:478–85. doi: 10.2144/19962003478. [DOI] [PubMed] [Google Scholar]
  • 38.Cawkwell L, Bell SM, Lewis FA, Dixon MF, Taylor GR, Quirke P. Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology. Br J Cancer. 1993;67:1262–7. doi: 10.1038/bjc.1993.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Quinn AG, Campbell C, Healy E, Rees JL. Chromosome 9 allele loss occurs in both basal and squamous cell carcinomas of the skin. J Invest Dermatol. 1994;102:300–3. doi: 10.1111/1523-1747.ep12371786. [DOI] [PubMed] [Google Scholar]
  • 40.Mortier L, Marchetti P, Delaporte E, Martin DL, Thomas P, Piette F, Formstecher P, Polakowska R, Danze PM. Progression of actinic keratosis to squamous cell carcinoma of the skin correlates with deletion of the 9p21 region encoding the p16 (INK4a) tumor suppressor. Cancer Lett. 2002;176:205–14. doi: 10.1016/s0304-3835(01)00757-1. [DOI] [PubMed] [Google Scholar]
  • 41.Nobori T, Takabayashi K, Tran P. Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers. Proc Natl Adcad Sci USA. 1996;93:6203–8. doi: 10.1073/pnas.93.12.6203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Schraml P, Struckmann K, Bednar R, Fu W, Gasser T, Wilber K, Kononen J, Sauter G, Mihatsch MJ, Moc H. CDKNA2A mutation analysis, protein expression, and deletion mapping of chromosome 9p in conventional clear-cell renal carcinomas: evidence for a second tumor suppressor gene proximal to CDKN2A. Am J Pathol. 2001;158:593–601. doi: 10.1016/s0002-9440(10)64001-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM, Jones PA. Methylation of the 5’CpG island of the p16/CDKN2 tumour suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995;55:4531–5. [PubMed] [Google Scholar]
  • 44.Kanao H, Enomoto T, Ueda Y. Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett. 2004;213:31–7. doi: 10.1016/j.canlet.2004.03.030. [DOI] [PubMed] [Google Scholar]
  • 45.Klaes R, Friedrich T, Spitkovsky D, Cerroni I, Kerl H, Wolf P. Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer. 2001;92:276–84. doi: 10.1002/ijc.1174. [DOI] [PubMed] [Google Scholar]
  • 46.Sano T, Oyama T, Kashiwabara K. Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions. Am J Pathol. 1998;153:1741–8. doi: 10.1016/S0002-9440(10)65689-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Brown VL, Harwood CA, Crook T. p16INK4a and p14ARF tumor suppressor genes are commonly inactivated in cutaneous squamous cell carcinoma. J Invest Dermatol. 2004;122:1284–92. doi: 10.1111/j.0022-202X.2004.22501.x. [DOI] [PubMed] [Google Scholar]
  • 48.Hodges A, Smoller BR. Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin. Mod Pathol. 2002;15:1121–5. doi: 10.1097/01.MP.0000032536.48264.D1. [DOI] [PubMed] [Google Scholar]
  • 49.Rehman I, Quinn AG, Takata M. Low frequency of allelic loss in skin tumours from immunosuppressed individuals. Br J Cancer. 1997;76:757–9. doi: 10.1038/bjc.1997.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Salama ME, Mahmood MN, Qureshi HS. p16INK4a expression in actinic keratosis and Bowen’s disease. Br J Dermatol. 2003;149:1006–12. doi: 10.1111/j.1365-2133.2003.05654.x. [DOI] [PubMed] [Google Scholar]

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