VRK1 Signaling Pathway in the Context of the Proliferation Phenotype in Head and Neck Squamous Cell Carcinoma Claudio R. Santos, 1 Marı ´a Rodrı ´guez-Pinilla, 2 Francisco M. Vega, 1 Jose ´ L. Rodrı ´guez-Peralto, 3 Sandra Blanco, 1 Ana Sevilla, 1 Alberto Valbuena, 1 Teresa Herna ´ ndez, 1 Andre ´ J. van Wijnen, 4 Fengzhi Li, 5 Enrique de Alava, 1 Montserrat Sa ´ nchez-Ce ´ spedes, 2 and Pedro A. Lazo 1 1 Instituto de Biologı ´a Molecular y Celular del Ca ´ ncer, Consejo Superior de Investigaciones Cientı ´ficas, Universidad de Salamanca, Spain; 2 Programa de Patologı ´a Molecular, Centro Nacional de Investigaciones Oncolo ´ gicas; 3 Departamento de Patologı ´a, Hospital Universitario 12 de Octubre, Madrid, Spain; 4 Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts; and 5 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, State University of New York at Buffalo, Buffalo, New York Abstract The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in the context of the p53 pathway and the proliferation phenotype in a series of 73 head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also correlated positively with several proteins associated with proliferation, such as cyclin-dependent kinase 2 (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, survivin, and Ki67. The level of VRK1 protein behaves like a proliferation marker in this series of head and neck squamous cell carcinomas. To identify a possible regulatory role for VRK1 and because it regulates gene transcription, the promoters of two genes were studied, CDK2 and SURVIVIN , whose proteins correlated positively with VRK1. VRK1 increases the activity of both the CDK2 and SURVIVIN gene promoters. The expression of VRK1 was analyzed in the context of regulators of the G 1 -S transition. VRK1 protein levels increase in response to E2F1 and are reduced by retinoblastoma and p16. These data suggest that VRK1 might play a role in cell cycle regulation and is likely to represent the beginning of a new control mechanism of cell cycle, particularly late in the G 1 -S phase. (Mol Cancer Res 2006;4(3):177 – 85) Introduction Head and neck squamous cell carcinoma (HNSCC) constitutes the sixth most common type of cancer worldwide. In the United States, it accounts for f3% of new cases and 2% of the deaths annually. Pathogenically, the development of these tumors has been associated with the mutagenic role of tobacco carcinogens (1), which can induce specific mutations in different genes, including p53 (2-5). The strong association with carcinogens suggests that pathways related with the cellular response to genotoxic damage might be implicated in HNSCC; in addition, there might be a genetic susceptibility affecting genes that are implicated in DNA repair processes or metabolism of carcinogens (6). There are also ethnic differences in its incidence, with the Black population presenting higher rates, whereas the Hispanic and Asian populations have lower rates (1). A role for certain types of human papillomaviruses has also been suggested (7, 8). The vaccinia-related kinase 1 (VRK1) protein belongs to a new family of serine/threonine kinases in the human kinome (9, 10). This family has three members in mammals, but Drosophila and Caenorhabditis elegans have only one homo- logue gene. The inactivation of the C. elegans homologue causes embryonic lethality (11). Inactivation of the distant homologue in yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae , has been shown to be implicated in the response to DNA damage (12, 13). VRK1 is highly expressed in human tumor cell lines (14) and murine embryos (15), suggesting it might be associated with cell proliferation. This association was Received 10/19/05; revised 1/31/06; accepted 2/9/06. Grant support: Fondo de Investigacio ´n Sanitaria grant FIS02/0585 (P.A. Lazo); Ministerio de Educacio ´n y Ciencia grant SAF2004-02900 (P.A. Lazo); Junta de Castilla y Leo ´n grants SAN/SA-01/04, SAN/SA-04/05, and CSI05A05 (P.A. Lazo); Fundacio ´n de Investigacio ´n Medica MM (P.A. Lazo); Ministerio de Ciencia y Tecnologı ´a grant SAF2002-01595 (M. Sa ´nchez-Ce ´spedes); Comunidad de Madrid grant CAM 08.1/0032/2003 (M. Sa ´nchez-Ce ´spedes); and Fundacio ´n Cientı ´fica de la Asociacio ´ n Espan ˜ola contra el Ca ´ncer (F.M. Vega), Fundac ¸a ˜o para a Cie ˆncia e a Tecnologia Portugal (C.R. Santos), Ministerio de Educacio ´ n y Ciencia (S. Blanco and A. Valbuena), and Consejo Superior de Investigaciones Cientificas predoctoral fellowships (A. Sevilla). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Pedro A. Lazo, Instituto de Biologı ´a Molecular y Celular del Ca ´ncer, Centro de Investigacio ´n del Ca ´ncer, Consejo Superior de Inves- tigaciones Cientificas, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain. Phone: 34-923-294-804; Fax: 34-923-294-795. E-mail: plazozbi@usal.es Copyright D 2006 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-05-0212 Mol Cancer Res 2006;4(3). March 2006 177 Downloaded from http://aacrjournals.org/mcr/article-pdf/4/3/177/3139584/177.pdf by guest on 20 February 2023