Association Between the V109G Polymorphism of the P27 Gene and the Risk and Progression of Oral Squamous Cell Carcinoma

3996 Vol. 10, 3996–4002, June 15, 2004 Clinical Cancer Research

Featured Article Association between the V109G Polymorphism of the p27 Gene and the Risk and Progression of Oral Squamous Cell Carcinoma

Guojun Li,1 Erich M. Sturgis,1,2 Li-E. Wang,1 cavity cancer and possibly tumor progression. Larger stud- Robert M. Chamberlain,1 Margaret R. Spitz,1 ies with oral squamous cell carcinoma are needed to verify Adel K. El-Naggar,3 Waun K. Hong,4 and these findings. Qingyi Wei1 INTRODUCTION Departments of 1Epidemiology, 2Head and Neck Surgery, 3Pathology, and 4Thoracic and Head and Neck Medical Oncology, The University Squamous cell carcinoma of the head and neck (SCCHN), of Texas M. D. Anderson Cancer Center, Houston, Texas which includes cancers of the oral cavity, pharynx, and larynx, is a common malignancy with an estimated number of Ͼ500,000 new cases worldwide (1, 2). In the United States, ABSTRACT approximately 37,200 new SCCHN cases and 11,000 SCCHN Purpose: Abnormalities in p27 may alter cell cycle delay deaths occurred in 2003 (3). Survival rates vary depending on required for DNA repair after exposure to carcinogens. A tobacco and alcohol consumption, age, sex, ethnic background, coding exon 1 polymorphism at codon 109 (T3G) in p27 and geographic area (3). Many factors contribute to SCCHN, was identified and thought to have an effect on the functions including tobacco smoking (4, 5), alcohol use (5, 6), viral of its protein. We hypothesized that this p27 T109G poly- infection (7, 8), and genetic factors (8). Although smoking and morphism is associated with squamous cell carcinoma of the alcohol use play a major role in the etiology of SCCHN, only a head and neck (SCCHN) risk. fraction of smokers and drinkers develop SCCHN, suggesting Experimental Design: We tested this hypothesis in a that there is inter-individual variation in genetic susceptibility to hospital-based case-control study of 713 patients newly di- SCCHN in the general population. Identification of genetic agnosed with SCCHN and 1224 cancer-free controls fre- factors that modulate the risk of SCCHN could be of great value -quency matched to the cases by age (؎5 years), sex, and in identifying high-risk subgroups that could benefit from pri smoking status. All subjects were non-Hispanic whites. We mary prevention programs and management of predictable pa- genotyped for this p27 variant using genomic DNA from tient outcome. each subject. Although no major susceptibility genes for SCCHN have Results: Compared with the p27 109VV variant, the p27 been identified, gains and losses at several loci and altered 109GG variant was associated with a nonsignificantly in- expression of p53 and DNA repair genes suggest the involve- ment of altered oncogenes and tumor suppressor genes in %95 ;1.29 ؍ (creased risk of SCCHN [crude odds ratio (OR SCCHN tumorigenesis (9, 10). Alterations in genes involved in ;1.20 ؍ adjusted OR ;1.90–0.88 ؍ (confidence interval (CI -but the risk was statistically signifi- cell cycle control frequently result in deregulated cellular pro ,[1.77–0.81 ؍ CI 95% liferation; specifically, genes associated with the regulation of –1.00 ؍ CI %95 ,1.55 ؍ cant among men (adjusted OR ؍ CI %95 ,1.68 ؍ current alcohol users (adjusted OR ,(2.42 the G1 checkpoint in the cell cycle are frequently altered in 1.01–2.82), and patients with oral cavity cancer (adjusted cancer cells (11). For instance, cyclins and cyclin-dependent -The p27 109GG variant kinases (CDKs) form protein complexes to modulate cell pro .(3.04–1.03 ؍ CI %95 ,1.77 ؍ OR was also associated with oral tumor overall stage, suggesting liferation through the cell cycle control, and CDK inhibitors that it may play a role in tumor progression. inhibit kinase activities of the complexes and block transitions Conclusions: Our findings suggest that the p27 109GG of the cell cycle (12–14). Therefore, abnormalities in genes that variant genotype may not play a major role in the etiology of regulate cell proliferation may alter cell-cycle control and DNA SCCHN but may be associated with an increased risk in repair activities in response to DNA damage, especially p53 at-risk subgroups or subsets of SCCHN, particularly oral response to DNA damage (15, 16). In oral cancer, the functions of p53, p27, p16, and cyclin D1 are often altered through mutation, amplification, or deactivation (17). Therefore, indi- viduals carrying polymorphic CDK inhibitors that may affect its Received 1/15/04; revised 3/11/04; accepted 3/25/04. protein function are likely more susceptible to SCCHN devel- Grant support: NIH Grants ES 11740 (to Q. Wei) and in part by NIH opment. Grants CA 86390 and CA 97007 (to M. Spitz and W. Hong), CA 16672 The p27, a CDK inhibitor belonging to the ciprofloxacin/ and ES 07784 (to The University of Texas M. D. Anderson Cancer kinase inhibitor proteins family, which includes p21 (18) and Center), and CA 57730 (to R. Chamberlain). The costs of publication of this article were defrayed in part by the p57 (19, 20), is a putative tumor suppressor (21, 22). The p27 payment of page charges. This article must therefore be hereby marked maps to chromosome 12p13 and encodes for a 27-kDa protein advertisement in accordance with 18 U.S.C. Section 1734 solely to (23, 24). The p27, p21, and p57 share common sequence motifs indicate this fact. that mediate interaction between the CDK inhibitor and cyclin- Requests for reprints: Q. Wei, Department of Epidemiology, Unit 189, The University of Texas M. D. Anderson Cancer Center, 1515 Hol- CDK complexes (10, 25, 26). Because the expression level of combe Boulevard, Houston, TX 77030. Phone: (713) 792-3020; Fax: p27 correlates positively with cell differentiation (27), it is (713) 563-0999; E-mail: [email protected]. speculated that p27 is involved in pathways regulated by both Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 3997

mitogenic and antiproliferative extrinsic signals (25) and that our frequency matching purpose. The overall response rate loss of its function contributes to tumorigenesis. The expression among this hospital control group was approximately 75%. The of p27 is frequently reduced in cancers, including gastric, breast, other control population was genetically unrelated and were prostate, and non-small cell lung cancers (16), but p27 is rarely M. D. Anderson hospital visitors (n ϭ 622) accompanying other mutated in human malignancies (28). Previous studies indicate cancer patients to our outpatient clinics. We had first surveyed that reduced p27 expression correlates with poor clinical out- the potential control subjects with a short questionnaire to come (29, 30), invasiveness (31), poor prognosis (30, 32–38), determine their willingness to participate in research studies and tumor grade (32, 39, 40), and progression (32, 41) in human to obtain information about their smoking behavior and demo- malignancies. However, it is unknown whether genetic variants graphic factors. The response rate for this community control in p27 play a role in the carcinogenesis or progression of group was approximately 80%. SCCHN. We frequency matched the controls to the cases by age (Ϯ5 A total of 21 single nucleotide polymorphisms of p27 have years), sex, and smoking status (i.e., current, former, and never). been described (GenBank entry no. AF480891). Of these 21 The purpose of frequency matching was to minimize confound- single nucleotide polymorphisms, 11 have low allele frequency ing to evaluate the main effect of the p27 polymorphism. We (Ͻ5%) and 9 occur within the noncoding regions of p27. Only interviewed each eligible subject to obtain data on age, sex, one single nucleotide polymorphism (T3G) at codon 109 ethnicity, smoking status, and alcohol consumption (before the causes an amino acid substitution of glycine for valine. This p27 disease diagnosis for the cases and at the time of the interview V109G polymorphism may have an effect on p27 degradation in for the controls). Subjects who had smoked Ͼ100 cigarettes in vivo (42) and appears to be associated with the risk of prostate their lifetimes were categorized as ever smokers, and others carcinoma (42), particularly advanced prostate carcinoma (43). were never smokers. Ever smokers who had quit smoking Ͼ1 Because no reports have been published on an association year previously were categorized as former smokers, and the between the p27 V109G polymorphism and the risk of SCCHN other smokers were categorized as current smokers. Similarly, thus far, we hypothesized that the p27 V109G polymorphism subjects who had drunk alcoholic beverages at least once a week may contribute to the risk of SCCHN. In this study, we tested for Ͼ1 year previously were categorized as ever drinkers, and our hypothesis in a large, hospital-based case-control study of others were never drinkers. Ever drinkers who had quit drinking patients with incident SCCHN and cancer-free controls fre- Ͼ1 year previously were categorized as former drinkers, and the quency matched by age, sex, and smoking status. other drinkers were categorized as current drinkers. After the subjects provided their informed consent, each subject donated 30 ml of blood collected into heparinized tubes. MATERIALS AND METHODS The research protocol was approved by the M. D. Anderson Study Subjects. Between May 1995 and October 2003, Cancer Center and Kelsey-Seybold institutional review boards. 1043 patients with histologically confirmed SCCHN were re- PCR Restriction Fragment Length Polymorphism Anal- cruited at our Head and Neck Surgery Clinic. The overall stage ysis. We extracted genomic DNA from the buffy-coat fraction of SCCHN is defined from I to IV as follows (American Joint of the blood samples by using a Qiagen DNA blood mini kit Committee for Cancer Staging and End-Results reporting, (Qiagen Inc., Valencia, CA) according to the manufacturer’s

1992): overall stage I (T1N0M0), overall stage II (T2N0M0), instructions. We typed for the p27 V109G genotype by PCR and overall stage III (T3N0M0 or T1–3N1M0), and overall stage IV restriction digestion by using published primers and a modifi- (T4N0–1M0 or any TN2–3M0 or any T, and N, M1). T, N, and M cation of a method published previously (43). We performed the are defined as tumor stage (T1,T2,T3, and T4), nodal status (N0, PCRs with a PTC-200 DNA engine (Peltier thermal cycler; MJ ␮ N1,N2, and N3), and distant metastases (M0 and M1), respec- Research, Inc., Waltham, MA) in 10 l of PCR mixture. This

tively (here M0 for all cases in this study). Approximately 95% PCR mixture included approximately 20 ng of genomic DNA, of eligible incident cases agreed to participate in this study. 0.1 mM each deoxynucleoside triphosphate, 1ϫ PCR buffer (50 Patients with second SCCHN primary tumors, primary tumors mM KCl, 10 mM Tris HCl, and 0.1% Triton X-100), 1.5 mM

of the nasopharynx or sinonasal tract, primary tumors outside MgCl2, 0.5 units of Taq polymerase (Sigma-Aldrich Corp.; St. the upper aerodigestive tract, cervical metastases of unknown Louis, MO), and 2 pmol of each primer. The amplification origin, or histopathological diagnoses other than squamous cell conditions were 5 min for initial denaturation at 95°C followed carcinoma were excluded. Because genotype frequencies can by 35 cycles of 15 s at 94°C,30sat60°C, 1 min at 72°C, and vary between ethnic groups, only 716 non-Hispanic white pa- a final 5-min step at 72°C for final extension. PCR products tients were included in this analysis with primary tumors of the (454 bp) were digested with the restriction enzyme BglI (New oral cavity (n ϭ 220; 31%), oropharynx (n ϭ 325; 45%), England BioLabs, Beverly, MA) overnight at 37°C and sepa- hypopharynx (n ϭ 36; 5%), or larynx (n ϭ 135; 19%). The rated with 3% agarose gel containing ethidium bromide. The response rate among the patients with SCCHN was 81%. We genotype of codon 109 was determined by a Gly allele with also included 1229 cancer-free control subjects in this analysis, fragment lengths of 76, 116, and 262 bp and a Val allele with recruited from two populations during a similar period. One fragment lengths of 76 and 378 bp. We performed the PCRs and group were enrollees (n ϭ 607) in a multispecialty physician evaluated the results without knowing the case or control status practice, the Kelsey-Seybold Foundation, which has Ͼ20 clinics of the subjects. At least 10% of the samples were retested, and throughout the Houston, Texas metropolitan area. This control the results were 100% concordant. population is part of an ongoing lung cancer study, which Statistical Analysis. We first evaluated the differences in provided older men who were former and current smokers for the distributions of selected demographic variables, smoking,

alcohol consumption, and p27 genotype frequencies between Because the controls in this study were composed of sub- cases and controls by using the ␹2 test. We estimated the jects from two separate populations, we compared the genotype association between the p27 genotype and risk for SCCHN by distributions between the 607 hospital and 617 community computing the odds ratios (ORs) and their 95% confidence controls. The frequencies of the p27 VV, VG, and GG were intervals (CIs) by both univariate and multivariate logistic re- 56.8, 36.9, and 6.26, %, respectively, in the hospital controls and gression analyses. For logistic regression analysis, the p27 ge- 60.5, 35.0, and 4.54%, respectively, in community controls, and notype was also recoded as a dummy variable. We further the p27 G allele frequency was 0.247 and 0.220 for the hospital stratified the genotype data by subgroups of age, sex, smoking, and community controls, respectively; however, these differ- alcohol drinking, and tumor site and assessed any trend in risk ences were not statistically significant (␹2 ϭ 2.671, P ϭ 0.263) in multivariate logistic regression models. All of the statistical for the genotype distribution and ␹2 ϭ 2.55, P ϭ 0.110 for the analyses were performed with the SAS software (version 8e; allele frequency (Table 2). Therefore, to increase study power, SAS Institute Inc., Cary, NC). these two groups were combined and used as one control group (n ϭ 1224) in the final analysis, and the distributions of the RESULTS genotypes among the combined controls were in agreement with ϭ Three DNA samples from the 716 cases and five DNA the Hardy-Weinberg disequilibrium (P 0.895; Table 2). Al- samples from the 1,229 cancer-free control subjects failed to though the frequencies of the p27 109G homozygous allele and yield genotyping data on repeated experiments. Therefore, the variant genotype were greater among the cases (0.239 and final data analysis included 713 cases and 1,224 cancer-free 7.01%, respectively) than among the controls (0.234 and 5.39%, controls, as summarized in Table 1. All subjects were non- respectively), these differences were not statistically significant ϭ ϭ Hispanic whites. Because the cases and controls were frequency (P 0.720 for the allele frequency and P 0.274 for the matched by age (Ϯ5 years) and sex, there were no significant genotype frequency). In the logistic regression analysis, we differences in the distributions of age and sex between the cases assessed the association of p27 genotypes with risk of SCCHN and controls (P ϭ 0.316 for age and P ϭ 0.574 for sex). The using hospital, community, and all combined controls, respec- median age was 57 years for the cases (mean ϭ 56.9 years, tively (Table 2). Compared with the p27 109VV genotype, the range ϭ 18–90 years) and 58 years for the controls (mean ϭ adjusted OR associated with risk of SCCHN was 0.88 (0.70– ϭ 57.2 years, range ϭ 20–88 years). However, the frequency 1.11), 0.95 (0.75–1.21), and 0.92 (95% CI 0.75–1.12) for the matching by smoking status was imperfect. There were more p27 109VG genotype and 1.10 (0.69–1.69), 1.35 (0.82–2.24), ϭ current smokers and current drinkers among the cases than and 1.20 (95% CI 0.81–1.77) for the p27 109GG genotype for among the controls, and these differences were statistically using these three control groups, respectively. None of these significant (P Ͻ 0.001 for both smoking and drinking status). associations reached the level of statistical significance. Therefore, all of these variables were further adjusted for in the Associations between the p27 V109G polymorphism and multivariate regression analyses. the risk of SCCHN as stratified by the age, sex, smoking status, and alcohol use are shown in Table 3. The adjusted ORs for the p27 genotypes associated with risk of SCCHN at different sites were obtained from the comparison between all combined controls and each sub-site of SCCHN (Table 3). We observed Table 1 Frequency distribution of selected variables among patients with squamous cell carcinoma of the head and neck and significantly increased risks of SCCHN associated with the p27 cancer-free controls 109GG genotype among men (adjusted OR ϭ 1.55, 95% CI ϭ 1.00–2.42), current alcohol users (adjusted OR ϭ 1.68, 95% Cases Controls ϭ Variable (n ϭ 713) (n ϭ 1224) Pa CI 1.01–2.82), and patients with oral cavity cancer (adjusted OR ϭ 1.77, 95% CI ϭ 1.03–3.04) but not among the other Age, (mean, ϮSD in yrs) 56.9 Ϯ 11.84 57.2 Ϯ 11.65 0.663 Age group by yrs, (n, %) 0.316 subgroups (Table 3). We also observed a nonsignificantly ele- Յ45 104 14.6 195 15.9 vated risk associated with the p27 109GG genotype among 46–50 104 14.6 163 13.3 younger subjects (age Յ median, 58 years; adjusted OR ϭ 1.47, 51–55 122 17.1 180 14.7 95% CI ϭ 0.7–3.1), both current and never smokers (adjusted 56–60 107 15.0 182 14.9 ϭ ϭ ϭ ϭ 61–65 112 15.7 178 14.5 OR 1.75; 95% CI 0.9–3.4 and OR 1.67; 95% CI 65–70 78 10.9 175 14.3 0.76–3.64, respectively), and subjects with larynx cancer (ad- Ͼ70 86 12.1 151 12.3 justed OR ϭ 1.65, 95% CI ϭ 0.86–3.16). These nonsignifi- Sex (n, %) 0.574 cantly increased risks from stratified analyses could be attrib- Male 536 75.2 906 74.0 uted to relatively small numbers of the strata, and these findings Female 177 24.8 318 26.0 Smoking status (n,%) Ͻ0.001 need larger studies to be further verified. We did not find any Current 247 34.6 313 25.6 evidence of an interaction between the p27 variant genotypes Former 279 39.1 543 44.4 and smoking status or alcohol use on the risk of SCCHN in the Never 187 26.2 368 30.0 multivariate logistic regression models (data not shown), prob- Drinking status (n,%) Ͻ0.001 Current 364 51.1 533 43.6 ably because of insufficient statistical power. Former 193 27.1 321 26.2 Because the significantly elevated risk was confined to oral Never 156 21.8 370 30.2 cavity cancer, we also evaluated the association between the p27 a Two-sided ␹2 and Student t tests for differences between the cases polymorphism and overall stage of oral squamous cell carci- and controls. noma (Table 4). The overall stage has four levels from stage I to

Table 2 p27 genotype and allele frequencies among cases and controls and their association with the risk of SCCHNa Case Hospital Community All control Adjusted OR patients controlsb controlsb subjectsb Adjusted OR Adjusted OR using all (n ϭ 713) (n ϭ 607) (n ϭ 617) (n ϭ 1224) using hospital using community combined controls controls controls P27 Genotype No. % No. % No. % No. % (95% CI)c (95% CI)c (95% CI)c V109Gb VV(ref.) 422 59.2 345 56.8 373 60.5 718 58.7 1.00 1.00 1.00 VG 241 33.8 224 36.9 216 35.0 440 35.9 0.88 0.95 0.92 (0.70–1.11) (0.75–1.21) (0.75–1.12) GG 50 7.01 38 6.26 28 4.54 66 5.39 1.10 1.35 1.20 (0.69–1.69) (0.82–2.24) (0.81–1.77) G allele 0.239 0.247 0.220 0.234 a SCCHN, squamous cell carcinoma of the head and neck; OR, odd ratio; CI, confidence interval; ref., reference group; ref., reference group. b The ␹2 tests for comparisons with the following cases: P ϭ 0.476 for hospital controls, P ϭ 0.159 for community controls, and P ϭ 0.274 for difference in genotype distributions and P ϭ 0.633 for hospital controls, P ϭ 0.233 for community controls, and P ϭ 0.720 for differences in allele frequencies; the observed genotype frequency in the control subjects was in agreement with Hardy-Weinberg equilibrium (p2 ϩ 2pq ϩ q2 ϭ 1; P ϭ 0.839 for hospital controls, P ϭ 0.643 for community controls, and P ϭ 0.895 for all combined controls). c Adjusted for age, sex, smoking status, and alcohol use in a logistic regression model.

IV as described in the “Materials and Methods.” Each stage increased as the overall stage of oral cavity cancer increased in ϭ level was related to different tumor stages (T1–4), nodal status a dose-effect manner (trend test: P 0.025) but not for the (N0–3), and distant metastases (M0–1) We combined overall variant VG genotype (trend test: P ϭ 0.437) and the combined stage III and IV into one group because they represented an p27 GG/VG genotypes (trend test: P ϭ 0.164). advanced disease. The risk associated with the p27 V109G polymorphism increased as the overall stage of oral squamous cell carcinoma increased from I to III-IV. The association was DISCUSSION particularly pronounced in overall stage III-IV oral squamous In this study, we investigated the role of the V109G poly- cell carcinoma for p27 GG genotype (adjusted OR ϭ 1.99, 95% morphism in the CDK inhibitor gene p27 in the etiology of CI ϭ 1.00–3.95) but did not reach the significance level for p27 SCCHN. We observed a nonsignificantly increased risk of VG genotype and combined p27 GG/VG genotypes (adjusted SCCHN associated with the p27 variant 109GG genotype com- OR ϭ 1.29, 95% CI ϭ 0.86–1.95 and adjusted OR ϭ 1.39, 95% pared with the p27 109VV genotype, and the significantly CI ϭ 0.95–2.05, respectively; Table 4). Compared with the VV increased risk was mostly limited to male subjects and current genotype, the risk associated with the variant GG genotype was alcohol users. However, there was a significantly elevated risk

Table 3 Associations between the p27 genotypes and risk of SCCHNa stratified by selected variables No. of cases/controls, adjusted OR (95% CI)b VV (ref.) VG GG Total 422 of 718 1.00 241 of 440 0.92 (0.75–1.12) 50 of 66 1.20 (0.81–1.77) Age (yrs)c Յ58 236 of 381 1.00 129 of 235 0.86 (0.65–1.13) 28 of 31 1.34 (0.78–2.32) Ͼ58 186/337 1.00 112/205 1.01 (0.75–1.37) 22/35 1.15 (0.64–2.05) Sex Male 302 of 541 1.00 192 of 319 1.07 (0.85–1.35) 42 of 46 1.55 (1.00–2.42) Female 120 of 177 1.00 49 of 121 0.59 (0.39–0.89) 8 of 20 0.52 (0.22–1.24) Smoking status Current 133 of 184 1.00 90 of 111 1.04 (0.72–1.50) 24 of 18 1.75 (0.90–3.40) Former 178 of 312 1.00 88 of 198 0.78 (0.57–1.06) 13 of 33 0.69 (0.35–1.35) Never 111 of 222 1.00 63 of 131 0.97 (0.66–141) 13 of 15 1.67 (0.76–3.64) Drinking status Current 200 of 308 1.00 128 of 193 0.97 (0.73–1.30) 36 of 32 1.68 (1.01–2.82) Former 121 of 189 1.00 62 of 112 0.87 (0.59–1.28) 10 of 20 0.76 (0.35–1.69) Never 101 of 221 1.00 51 of 135 0.82 (0.55–1.23) 4 of 14 0.64 (0.20–1.99) Cancer sites Oral cavity 122 1.00 77 1.02 (0.75–1.40) 21 1.77 (1.03–3.04) Oropharynx 198 1.00 110 0.91 (0.70 1.19) 15 0.81 (0.45–1.46) Hypopharynx 21 1.00 13 1.00 (0.49–2.04) 1 0.50 (0.06–3.79) Larynx 81 1.00 41 0.84 (0.56–1.25) 13 1.65 (0.86–3.16) a SCCHN, squamous cell carcinoma of the head and neck; OR, odd ratio; CI, confidence interval; ref., reference group. b Adjusted for age, sex, smoking status, and alcohol status in a logistic regression model for the strata. c Stratified by the median of age among controls.

Table 4 Associations between the p27 genotypes and overall stage of squamous cell carcinoma of the oral cavity No. of case/controls, adjusted ORb (95% CI)c Overall stagea VV VG GG VGϩGG I 29 of 718 14 of 440 4 of 66 18 of 506 1.00 (ref.) 0.76 (0.40–1.47) 1.49 (0.50–4.41) 0.86 (0.47–1.56) II 30 of 718 16 of 440 5 of 66 21 of 506 1.00 (ref.) 0.85 (0.46–1.58) 1.71 (0.63–4.61) 0.96 (0.54–1.71) III-IV 58 of 718 46 of 440 12 of 66 58 of 506 1.00 (ref.) 1.29 (0.86–1.95) 1.99 (1.00–3.95) 1.39 (0.95–2.05) Trend test (P) 0.437 0.025 0.164 a Overall stage I, T1N0M0; overall stage II, T2N0M0; overall stage III, T3N0M0 or T1–3N1M0; and overall stage IV, T4N0–1M0 or any TN2–3M0 or any T, and N, M1. T, tumor stage (T1,T2,T3, and T4). N, nodal status (N0,N1,N2, and N3). M: distant metastases (M0 and M1; here M0 for all the cases in this study; American Joint Committee for Cancer Staging and End-Results reporting, 1992). b OR, odds ratio; CI, confidence interval; ref., reference group. c Adjusted for age, sex, smoking status, and alcohol status in a logistic regression model for the strata.

for oral cavity cancers associated with the p27 variant 109GG it is also possible that PGP9.5 might play a role in cancer growth genotype, and this risk was more pronounced in the advanced by regulating the level of nuclear p38jab1, leading to increased stage of oral cavity cancer; there was a dose-effect relationship degradation of p27 via its interaction and nuclear translocation between risk for the overall stage of oral cavity cancer and the with p38jab1 (48). Another possibility is that the p27 polymor- p27 homozygous 109GG variant geotype. To the best of our phism may be in linkage disequilibrium with other functional knowledge, this is the first large molecular epidemiological polymorphisms that affect either the expression or activity lev- study investigating the association between the p27 V109G els of enzymes involved in tumorigenesis of oral cavity (49). polymorphism and the risk of SCCHN. However, all these hypotheses remain to be tested in future Our findings suggest that although the p27 109G variant studies. genotypes (i.e., VG and GG) may not play a major role in the Thus far, only one published case-control study has exam- etiology of SCCHN, it may contribute to subsets of SCCHN and ined the association between the p27 V109G polymorphism and possibly tumor progression. These findings are biologically the risk of cancer (43), and no studies have examined the plausible. p27 is involved in cell cycle control, which is critical association between this polymorphism and the risk of SCCHN. for DNA repair in response to exposure to carcinogens, and In a case-control study of 96 cases and 106 controls in a therefore, p27 plays an important role in carcinogenesis. It is European-American population, Kibel et al. (43) examined possible that this p27 V109G polymorphism may interact with whether the p27 V109G polymorphism was associated with and modify other risk factors for SCCHN. Alternatively, the p27 advanced prostate cancer and found that the p27 109VV geno- variant allele may be functionally relevant or in linkage disequi- type was associated with a 95% increased risk of advanced librium with alleles at other susceptibility loci. prostate carcinoma (OR ϭ 1.95, 95% CI ϭ 1.09–3.47). The risk To date, there is no published study that provides direct was particularly increased in patients with androgen-indepen- information on potential functional relevance of this p27 poly- dent disease (OR ϭ 2.11, 95% CI ϭ 1.05–4.22) and in those morphism, which may cause structural change of p27 protein under the median age of diagnosis (OR ϭ 2.23, 95% CI ϭ and potentially alter interaction with other proteins. For in- 1.08–4.59). The same research group previously studied this stance, p38 encoded by the Jab1gene (44) interacts specifically polymorphism in 19 metastatic prostate carcinomas and found with p27, and overexpression of p38 in mammalian cells results that the distribution of the p27 genotypes was not in Hardy- in the translocation of p27 from the nucleus to the cytoplasm, Weinberg equilibrium (84% VV homozygous, 5% VG heterozy- causing a decrease in the amount of p27 in the cell by acceler- gous, and 11% GG homozygous; Ref. 50). Previously estimated ating its degradation through the ubiquitin/proteasome pathway frequencies of the T and G alleles were 0.73 and 0.27, respec- (45). Therefore, p38 is a specifically binding and negative tively, from 60 unrelated subjects (42) and 0.72 and 0.28, regulator of p27, and their interaction may explain the regulation respectively, from 105 controls (43). Consistently, our estimates of cell-cycle-dependent proteolytic machinery and selection of were 0.77 and 0.23, respectively, from the 1224 controls in our the key cell-cycle regulators for degradation. The p27 V109G study. The differences in these estimated frequencies of the T polymorphism, which results in a glycine for valine substitution, and G alleles could be attributed to the differences in the sample lies within the binding domain of p38jab1 that promotes the sizes and ethnic groups included in the studies. phosphorylation and cytoplasmic translocation of p27 for its We observed a significantly elevated risk of SCCHN subsequent degradation in the cytoplasm (46). Therefore, it is among current alcohol users, suggesting that a gene-environ- possible that the V allele may modify the affinity of p27 for p38 ment interaction may be involved in the development of by promoting p27 degradation, resulting in a decreased expres- SCCHN. However, the present study did not have sufficient sion of the p27 protein (46). In addition, PGP9.5 is one of the statistical power to detect such gene-environment interaction. If members of the ubiquitin COOH-terminal hydrolase (UCH) this may be true, there is a lack of a biological mechanism to family, UCH-L1, that has three human isozymes that may directly support this finding. Ethanol-mediated disturbance of involve in p38jab1 mediated-degradation of p27 (47). Therefore, the CDK system may play a role in cell cycle arrest and cell

growth (51, 52), which may explain the possible role of the p27 oral cavity cancer, quantitative measures of smoking and alco- polymorphism in alcohol-induced SCCHN, but this hypothesis hol exposure, and an emphasis on molecular changes in the needs to be further tested in future studies. It is also possible that target tissue. this borderline significant finding from a subgroup analysis could be because of chance. We found that the SCCHN risk associated with the p27 ACKNOWLEDGMENTS variant genotypes was significantly higher for patients with We thank Margaret Lung and Dr. Peggy Schuber for assistance in cancers of the oral cavity but not for those with cancers of the recruiting the subjects; Dr. Zhensheng Liu, Dr. Luo Wang, Jianzhong oropharynx, hypopharynx, or larynx, suggesting that oral cavity He, John I. Calderon, and Kejin Xu for laboratory assistance; Joanne Sider for manuscript preparation; and Katie Matias for scientific editing. carcinoma may have a different etiology in relation not only to environmental risk factors but also to genetic susceptibility. Although the finding of the p27 variant-associated cancer risk in REFERENCES a tissue-selective manner is unknown, this is consistent with the 1. Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide elevated risk associated with the p27 variant genotype in current mortality from 25 cancers in 1990. Int J Cancer 1999;83:18–29. alcohol drinkers described above. One study has demonstrated 2. Johnson NW, Ranasinghe AW, Warnakulasuriya KAAS. Potentially that among SCCHN sites, oropharyngeal cancers are strongly malignant lesions and conditions of mouth and oropharynx: natural associated with human papillomavirus type 16, oral cavity can- history-cellular and molecular markers of risk. 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