p 12 CDK 2-AP 1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo MouseModel of Head and Neck Cancer

Purpose:To test the potential of p12 (p12), a cell cycle regulator and cyclin-dependent kinase-2-associating protein commonly down-regulated in head and neck squamous cell carcinoma (f70%), as a gene therapy in inhibiting head and neck squamous cell carcinoma growth in vivo. Experimental Design:We addressed the effect of p12 expression on tumor growth by using a well-established squamous cell carcinomaVII/SF floor of mouth xenograft mouse model. The effect of therapy on tumor growth was determined for: (a) no treatment, (b) PBS, (c) vehicle (1,2-dioleoyloxy-3-trimethylammonium propane:cholesterol liposomes / 5% dextrose), (d) empty vector controls, and (e) p12-encoding vector experimental groups. Results: p12 gene therapy significantly induced antitumor effects as compared with controls, including (a) size and weight of p12-treated tumors decreased by 51% to 72% compared with all controls (P < 0.02), (b) tumor growth rate post-therapy was inhibited by 55% to 64% compared with empty vector controls (P < 0.0001), and (c) p12 expression was higher in p12treated than controls (P < 0.002) by two-tailed t test analyses. Mechanistically, p12 treatment affected cell turnover kinetics as assessed by apoptotic and cell proliferation indices. p12 therapy significantly increased terminal nucleotidyl transferase ^mediated nick end labeling (P < 0.05) and morphology-based apoptotic indices (P < 0.05) as well as significantly decreased Ki-67 cell proliferation indices (P < 0.001) compared with controls, resulting in a net cell turnover reduction in p12-treated tumors. Conclusions:We show that this novel therapeutic modality can significantly induce antitumor responses in vivo. These results support a role for p12 as a novel tumor growth suppressor gene therapy and suggest that optimization and/or combination with current therapies may hold considerable promise in preparation for clinical trials. Head and neck cancer affects approximately 38,000 new patients in the U.S. yearly, more than half a million patients worldwide, and despite improvements in surgery, chemotherapy, and radiation therapy, the overall survival has not improved in the past 30 years (1). For all stages combined, about 84% of patients diagnosed with head and neck cancer survive 1 year after diagnosis and the 5and 10-year relative survival rates are 57% and 45%, respectively (1). The localregional biology of head and neck squamous cell carcinomas (HNSCC) and the persistent poor treatment outcomes make this disease an ideal target for gene therapy, including tumor suppressor gene delivery (2). Recently, combinations of novel gene-based therapies with current therapeutic modalities have been explored, including tumor suppressor replacement gene therapy, suicide gene therapy, immunotherapy, and oncogene suppression (3, 4). Recently, p53 tumor suppressor replacement has been combined with chemotherapy and radiation therapy as a successful treatment for head and neck and other cancers in preclinical and clinical settings (4–6) and other tumor suppressor therapies such as p16 (p16) or retinoblastoma are currently in clinical development for head and neck after promising results from preclinical studies (7–9). Therefore, gene-based approaches are being examined for HNSCC treatment and of particular promise are combinations of current treatments with novel cell cycle–regulatory gene therapies. Abnormalities in various components of the cell cycle– regulatory machinery have been associated with tumorigenesis. Molecular alterations frequently associated with head and neck cancer include cyclin D1 amplification or overexpression (30-50%), decreased expression of tumor suppressor genes p53 (50%) and retinoblastoma (<10%), and decreased expression of cyclin-dependent kinase (CDK) inhibitors p27 (36%), p21 (46%), and p16 (69-80%) in SCC (10, 11). More recently, decreased expression of a novel CDK2 inhibitor, p12 (p12) has been associated with HNSCC (12, 13). www.aacrjournals. org Clin Cancer Res 2005;11(10) May15, 2005 3939 Authors’Affiliations: Laboratory of Head and Neck Cancer Research, School of Dentistry and Dental Research Institute, and Department of Head and Neck Surgery, University of California Los Angeles, Los Angeles, California Received10/12/04; revised 2/2/05; accepted 2/16/05. Grant support: RO1DE14857 (D.T.W.Wong), RO1DE14857S1 (M.L. Figueiredo), andT32DE007296-08 (Y. Kim). 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: David T.W. Wong, Laboratory of Head and Neck Cancer Research, Dental Research Institute, School of Dentistry, University of California Los Angeles, 10833 Le Conte Avenue, 73-017 CHS, Los Angeles, CA 90095. Phone: 310-206-3048; Fax: 310-794-7109; E-mail: dtww@ucla.edu. F2005 American Association for Cancer Research. Cancer Therapy: Preclinical Cancer Research. on January 29, 2018. © 2005 American Association for clincancerres.aacrjournals. org Downloaded from p12 is a growth suppressor gene identified and cloned from the Syrian hamster oral cancer model. The human p12 cDNA has been mapped to chromosome 12q24 and partially characterized (14); the full-length human p12 cDNA is 1.6 kb in length, encoding 115 amino acids (Mr 12,400). The rodent p12 protein has 97% identity with that of humans (14). p12 is also implicated in S phase–associated growth suppression, through binding with DNA polymerase a/primase and/or CDK2 (15, 16). Additionally, we have shown that the clinical behavior of p12 differs in normal and tumor oral mucosa, with reduced expression in 14.3% of oral dysplasias, and in 64% to 72% of HNSCC cases examined (12, 13). In combination, these findings have suggested that p12 loss or reduced expression might contribute to the multistep nature of oral carcinogenesis, and that its loss may be an event associated with tumor progression. Several in vitro studies have suggested a role for p12 in regulating cell growth. Ectopic expression of p12 into 7,12dimethylbenz(a)anthracene-induced hamster oral carcinoma HCPC-1 cells has been associated with growth suppression and a significant antiproliferative effect. Cells reverted to a normal cellular morphology, lost the ability to grow in soft agar, and displayed an increase in cell doubling time (17), consistent with a potential role for p12 as a tumor suppressor gene in oral keratinocytes. Suppression of cell growth in a similar manner was also observed when malignant human keratinocytes were transfected with p12 (16). Additionally, p12 transfection into HCPC-1 cells significantly increased the fraction of apoptotic cells in p12-transfected as compared with null vector or untransfected controls (18). Another study examining p12 expression levels in the hamster oral carcinogenesis model in vivo revealed that apoptotic cell indices were significantly reduced in malignant epithelium, perhaps reflecting the abrogation of critical pathway(s) governing cell death. Also, cell proliferation (proliferating cell nuclear antigen labeling index) was found to increase progressively through hamster oral malignant progression, whereas p12 expression levels progressively decreased through malignant progression (19). Finally, recent data using murine p12-targeted ES knockout clones (p12 / ) showed that in the absence of p12 expression, cellular proliferation is increased, with an increase in S phase and a decrease in G2-M phase populations, and apoptosis is significantly reduced with cisplatin treatment (20). In combination, these in vitro data support the role of p12 in regulating tumor cell phenotype and growth; however, specific examination of p12 antitumor effects had not been evaluated previously in vivo . Given the encouraging preclinical data using the tumor suppressors p16, p53, and retinoblastoma in gene therapy of head and neck cancer (4–9, 21), we hypothesized that p12 gene therapy would induce antitumor activity in vivo. In our present study, we assessed whether local expression of p12 altered tumor growth in a controlled model of