Carcinogenesis vol.30 no.6 pp.977–981, 2009 doi:10.1093/carcin/bgp076 Advance Access publication April 3, 2009 Genetic variation in the transporter, SLC23A2, modifies the risk of HPV16-associated head and neck cancer

Alyce A.Chen1,2, Carmen J.Marsit3, Brock positive (3). HPV16-associated oropharyngeal HNSCC is also known C.Christensen3,4, E.Andre´s Houseman4, Michael to have distinct risk factors and, taken together, this suggests that the D.McClean5, Judith F.Smith6, Janine T.Bryan6, Marshall differing etiologies of this disease give rise to clinically, and poten- R.Posner7, Heather H.Nelson8,9 and Karl T.Kelsey3,4, tially molecularly, distinct diseases (3,4,7,8). We recently reported a significant interaction between HPV16 serology and citrus fruit in- 1Channing Laboratory, Brigham and Women’s Hospital, Boston, MA 02115, take in HNSCC (9). Among serologic HPV-negative persons, citrus USA, 2Division of Biological Sciences in Public Health, Harvard School of fruit consumption reduces risk of HNSCC, whereas among HPV- Public Health, Boston, MA 02115, USA, 3Department of Pathology and 4 positive individuals citrus fruit consumption is associated with an Laboratory Medicine and Department of Community Health, Center for increased risk for HNSCC. Environmental Health and Technology, Brown University, Providence, Given these risk profiles, we hypothesized that HPV infection, Downloaded from https://academic.oup.com/carcin/article/30/6/977/2476882 by guest on 24 September 2021 RI 02912, USA, 5Department of Environmental Health, Boston University School of Public Health, Boston, MA 02118, USA, 6Department of Vaccine marked by serology status, modifies the risk of HNSCC associated Basic Research, Merck and Co., West Point, PA 19486, USA, 7Head and Neck with normal variation in cancer-associated or dietary factors, Oncology Program, Dana-Farber Cancer Institute, Boston, MA 02115, USA, particularly citrus fruit intake. By identifying subgroups of geneti- 8Masonic Cancer Center and 9Department of Epidemiology and Community cally susceptible individuals, as well as delineating the significant Health, University of Minnesota, Minneapolis, MN 55455, USA differences seen in HPV-positive compared with HPV-negative dis- ease, we may be better able to characterize disease risk factors. We To whom correspondence should be addressed. Tel: þ1 401 863 6420; Fax: þ1 401 863 9008; initially utilized a standardized cancer genotyping platform developed Email: [email protected] by Illumina, based on the USA National Cancer Institute’s Cancer Genome Anatomy Project (the SNP500Cancer database) to identify Human papillomavirus (HPV) type 16 infection is an etiologic single-nucleotide polymorphisms (SNPs) differentially associated factor in a subset of head and neck squamous cell carcinomas with HPV-positive and negative HNSCC. Subsequently, additional (HNSCC). It is unknown if host genetic susceptibility modifies directed genotyping of the entire case–control study population for the HPV16–HNSCC association. DNA samples collected as part the candidate locus most associated with HNSCC revealed a signifi- of a Boston area case–control study of HNSCC were genotyped for cant interaction between a polymorphism in the vitamin C transporter single-nucleotide polymorphisms (SNPs) from the National Can- SLC23A2 and HPV16 antibody serology in HNSCC. Variation in cer Institute’s SNP500Cancer database. Analysis of demographic, SLC23A2 has been recently reported to be associated with non- phenotypic and genotypic data for 319 HNSCC cases and 495 Hodgkin lymphoma, colorectal adenocarcinoma and gastric cancer frequency-matched controls was performed using unconditional (10–12), but to our knowledge, this is the first report of variation in logistic regression. All reported P-values are two sided. We iden- SLC23A2 associated with HNSCC. tified a polymorphism in the sodium-dependent vitamin C trans- porter SLC23A2 that modifies the risk of HNSCC associated with HPV16 infection. Among those with a wild-type allele at Subjects and methods SLC23A2, the risk of HNSCC associated with HPV16-positive Study population serology was 5.0 (95% confidence interval (CI) 5 3.2–7.8). How- Details of this case–control study of HNSCC are described elsewhere (3,7). ever, among those with a homozygous variant genotype, the risk Briefly, cases were collected from Boston area hospitals from 1999 to 2003. of HNSCC associated with HPV16 was attenuated [odds ratio Controls were randomly selected from Massachusetts town books to match (OR) 5 2.8; 95% CI 5 1.2–6.2]. Further, when we tested whether cases on town of residence, gender and age (Table I). All case and control genotype modified the interaction between citrus exposure, subjects were provided written informed consent and the study was approved HPV16, and HNSCC, we found a dramatically increased risk of by institutional review boards of all participating institutions. Questionnaires HNSCC for those with a wild-type SLC23A2 allele, HPV16-posi- were administered to collect data on smoking, drinking, diet, family history, tive serology and high citrus intake (OR 5 7.4; 95% CI 5 3.6– medication history and demographics. Diet information was collected using 15.1). These results suggest that SLC23A2 genetic variation alters a semi-quantitative food frequency questionnaire assessing food consumption HPV16-associated HNSCC while also highlighting the important 5 years prior to diagnosis or date of enrollment. The food frequency question- naire has been previously validated (13,14). Fruit intake was calculated as the role of citrus exposure in this disease. amount consumed per day (or week or month if less frequent than once per day) based on subjects’ response for average year-round consumption. Citrus fruit consisted of grapefruit, grapefruit juice, orange and orange juice. A serv- ing was considered to be equivalent to one glass of juice, an orange, or half of Introduction a grapefruit. Low citrus intake was defined as ,0.67 servings per day and high Head and neck squamous cell carcinoma (HNSCC) is the sixth most citrus intake was defined as 0.67 servings per day, based on the median value in the control group. Subjects who completed less than half of the food fre- common cancer in men and 13th most common cancer in women in quency questionnaire or with missing citrus fruit intake data were eliminated the USA, with 47 500 incident cases in USA in 2007 (1). These from the analysis as were subjects who reported consuming ,800 calories (500 cancers arise from the epithelium of the oral cavity, oropharynx and calories if female) or .4200 calories (3500 calories if female). HPV16 larynx and are predicted to cause .11 000 deaths in the USA this year serology was determined using the HPV competitive Luminex immunoassay (1). Established risk factors for HNSCC include cigarette smoking, as described previously (15). use of smokeless tobacco, alcohol consumption and human papillo- DNA extraction and genotyping mavirus (HPV) (primarily HPV16) infection (2–5). HPV infection now accounts for 25% of head and neck cancer in many centers Genomic DNA was extracted from whole blood using standard protocols as described previously (16). To identify SNPs for further –environment in- in North America and Europe and is found primarily in tumors of the teraction testing, a subset of 138 cases (69 HPV16 serology positive and 69 oropharynx (3,6). Overall, 5 year survival of HNSCC approaches HPV16 serology negative) and 138 controls were genotyped on the Golden- 60%, but is markedly improved in those whose disease is HPV16 Gate SNP platform for 1536 loci on the SNP500Cancer array (17). Analysis of the initial genotyping screen was performed in the R software environment for Abbreviations: CI, confidence interval; HNSCC, head and neck squamous statistical computing (18) and employed an interaction model including vari- cell carcinoma; HPV, human papillomavirus; OR, odds ratio; SNP, single- ables for SNP genotype, smoking and drinking. The SLC23A2 polymorphism nucleotide polymorphism. rs4987219 was most significantly associated with case status. Subsequently,

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Table I. Demographic characteristics of cases and controls

Characteristics Cases (N 5 319), n (%) Controls (N 5 495), n (%) Risk of HNSCC

ORa (95% CI) P-valuea

Age (years) Mean ± SD 59.7 ± 12.0 61.0 ± 11.5 Gender Male 226 (71) 361 (73) Female 93 (29) 134 (27) Race White 295 (92) 459 (93) 1.00 Non-white 24 (8) 36 (7) 0.75 (0.40–1.41) 0.371 Education Below high school 46 (14) 36 (7) 1.00 Downloaded from https://academic.oup.com/carcin/article/30/6/977/2476882 by guest on 24 September 2021 High school diploma þ 273 (86) 459 (93) 0.67 (0.39–1.14) 0.136 Smoking (pack-years) Mean ± SD 35.1 ± 33.3 19.7 ± 25.0 0 62 (19) 165 (33) 1.00 1–20 66 (21) 139 (28) 1.09 (0.70–1.70) 0.719 20–45 82 (26) 111 (22) 1.57 (0.99–2.51) 0.056 45 109 (34) 80 (16) 3.24 (2.00–5.26) ,0.0001 Alcohol (drinks/week) Mean ± SD 24.4 ± 29.4 12.4 ± 20.2 ,3 62 (19) 154 (31) 1.00 3–8 67 (21) 151 (31) 1.15 (0.73–1.80) 0.557 8–25 82 (26) 127 (26) 1.51 (0.95–2.42) 0.084 25 108 (34) 63 (13) 3.88 (2.32–6.51) ,0.0001 Citrus fruits (servings/day) Mean ± SD 0.69 ± 0.74 0.79 ± 0.65 ,0.67 189 (59) 244 (49) 1.00 0.67 130 (41) 251 (51) 0.91 (0.66–1.25) 0.550 HPV16 antibody titer Negative 223 (70) 442 (89) 1.00 Positive 96 (30) 53 (11) 4.21 (2.81–6.29) ,0.0001 aORs and P-values estimated from unconditional logistic regression model adjusted for all variables listed in Table I.

we used Taqman allelic discrimination to genotype rs4987219 in the entire study population (96 HPV16 serology-positive cases, 223 HPV16 serology- negative cases and 495 controls) to confirm the initial results and to provide Table II. Associations of SLC23A2 SNP rs4987219 genotypes with HNSCC a larger sample size for examination of effect modification. For the 272 sub- risk jects that were genotyped on both platforms, we had a 99.3% concordance rate. Genotype Cases (N 5 319), Controls (N 5 495), ORa (95% CI) P-valuea Unconditional logistic regression in SAS 9.1 was utilized to examine the n (%) n (%) association between rs4987219 and HNSCC, while controlling for age, gender, race, education, smoking, drinking and HPV16 serology as previously reported GG 116 (36) 157 (32) 1.00 (7). In addition, we tested for statistical interaction with HPV16 serology using GC 145 (45) 250 (51) 0.81 (0.57–1.15) 0.240 a logistic model controlled for age, gender, race, education, smoking and CC 58 (18) 88 (18) 0.94 (0.60–1.50) 0.805 drinking. The significance of the interaction was tested using the Wald test. GG and GC 261 (82) 407 (82) 1.00 All reported P-values are two sided. Because HPV16 positive serology confers CC 58 (18) 88 (18) 1.07 (0.71–1.61) 0.755 a 10-fold risk of HNSCC of the pharynx and only a minimal, if any, increased risk of HNSCC of the larynx and oral cavity (7), we tested the association aORs and P-values estimated from unconditional logistic regression model between HPV16 serology and genotype, stratified by site of tumor. Further, as adjusted for age, gender, race, education, smoking, alcohol consumption and our previous report identified an interaction between HPV16 seropositivity and HPV16 serology. citrus consumption (9), we re-examined the statistical interaction between HPV16 serology and dietary citrus intake for each of two rs4987219 genotype groups: any wild-type allele (includes GG and GC genotypes) and homozygous CC genotype. Table III. Associations of SLC23A2 SNP rs4987219 genotypes and HPV16 serology with HNSCC risk Results Genotype HPV16 Cases Controls ORa (95% CI) Wald, Using the SNP500Cancer genotyping panel, we identified SNP serology (N 5 319), (N 5 495), P-valuea rs4987219, a non-coding SNP found in intron 8 of SLC23A2, one of n (%) n (%) two required sodium-dependent vitamin C transporters (reviewed in ref. 19), as a potential modifier of HPV-associated HNSCC. Upon GG and GC 0.105 genotyping the entire case–control population for rs4987219, we Negative 183 (57) 366 (74) 1.00 found that, although there was no association between this SNP and Positive 78 (24) 41 (8) 4.98 (3.17–7.84) CC case status (Table II), among HPV16 seropositives, homozygous var- Negative 40 (13) 76 (15) 1.27 (0.81–2.02) iant genotype decreased the risk of HNSCC (Table III). This effect Positive 18 (6) 12 (2) 2.78 (1.24–6.21) was not seen in subjects who were serologically HPV16 negative, indicating there is a potential interaction between rs4987219 and aORs and P-values estimated from unconditional logistic regression model HPV16 serology in HNSCC (Wald test, P 5 0.10, Table III). We have adjusted for age, gender, race, education, smoking and alcohol consumption. 978 Vitamin C transporter 2 and HPV16-associated head and neck cancer previously reported that positive antibody titer for HPV16 results in regulation of the transporters that, in turn, are regulated by substrate a .4-fold risk of HNSCC (7) and found this to be true in this subset of concentrations of ascorbate, by local and systemic inflammatory cy- the study population [odds ratio (OR) 4.2, 95% confidence interval tokines, by the presence of a dominant negative isoform of SVCT2, by (CI) 2.8, 6.3]. When considering the SNP in conjunction with HPV16 hormones and by kinase C signaling (27–32). The role of the serology, we observed the risk of HNSCC associated with HPV16 SLC23A2 SNP, rs4987219, in this complex environment has not been positivity was highest among those with a wild-type allele (OR 5.0, established. 95% CI 3.2, 7.8), and HNSCC risk was significantly attenuated among HPV16 infection initially occurs as the result of epithelial damage those who were homozygous variant (OR 2.8, 95% CI 1.2, 6.2). The and subsequent exposure and viral invasion of the basal epidermis. OR comparing HPV positive with HPV negative within the homozy- This results in the expression of viral transcription regulating genes, gous variant group is 2.1 (95% CI 0.8, 5.6), which is markedly ,5.0, alterations in cellular transcription factor regulation and viral onco- the corresponding OR for the wild-type/heterozygous. genes that alter cell cycle progression (reviewed in ref. 33). In our When we tested the association between HPV16 serology and ge- study, the association with HPV16 was established by a serological notype by site of tumor, the interaction between genotype and HPV marker of HPV16: antibodies specifically directed to the L1 viral was strongest for HNSCC of the pharynx (data not shown); however, capsid protein. While we have established the correlation between there was not sufficient power to test for a three-way interaction. serological presence of HPV16 antibodies and presence of HPV16 Downloaded from https://academic.oup.com/carcin/article/30/6/977/2476882 by guest on 24 September 2021 Building on our prior finding of an interaction between HPV16 se- DNA in HNSCC tumors (3), it is not known whether the SLC23A2 rology and citrus intake (9), we tested whether the effect of this in- loci can be directly or indirectly regulated via viral or altered cellular teraction on risk of HNSCC varied by SLC23A2 genotype (Figure 1). transcription factors or, if so, if the polymorphic variants could affect Dietary citrus intake modified the association of a positive serologic any such regulation. One could hypothesize that the sequence differ- response to HPV16 only in those who were wild-type or heterozygous ences resulting from SLC23A2 polymorphisms could influence tran- for the SLC23A2 genotype. HNSCC risk was .7-fold higher in the scription factor binding and regulation of vitamin C transporter stratum containing those with a SLC23A2 wild-type GG or heterozy- expression. In fact, rs4987219 is predicted to provide a CCAAT en- gous GC genotype, positive HPV16 serology and high citrus con- hancer binding protein beta site with the C allele that is not present sumption. HNSCC risk was markedly reduced among those with with the G allele (http://www.cbrc.jp/research/db/TFSEARCH.html). low citrus fruit exposure, homozygous SLC23A2 variant CC genotype Although no phenotype for any of the polymorphisms in SLC23A2 or negative HPV16 serology. This interaction was found to be statis- has been established, polymorphisms in intron 2 of SLC23A2 have tically significant (P 5 0.05). previously been associated with an increased risk of preterm delivery (34). These authors assert that the variant may be associated with Discussion diminished tensile strength of membranes and hence contribute to premature rupture of membranes and preterm infant delivery, al- We report that a SNP in intron 8 of SLC23A2 (rs4987219) alters though the data were not adjusted for dietary intake of ascorbate. susceptibility to HNSCC associated with HPV16. This result strongly These data suggest a possible novel mechanism of action of this suggests that vitamin C metabolism, manifested by an altered trans- variant in HNSCC; namely, the polymorphism may be associated with porter, modifies the likelihood or consequences of HPV16 infection a difference in the potential strength of the epithelial surface barrier, and subsequent associated HNSCC. Alternatively, it is possible that giving rise to a different potential for viral entry. the variant gene transporters have a differential exposure to HPV16, In another recent study, intronic SNPs in SLC23A2 were associated although it is unlikely that genotype of a SNP in a vitamin C trans- with an increased risk of non-Hodgkin lymphoma (10). Again, no porter would influence or be associated with HPV16 exposure. specific phenotype was established for the SNPs, but the authors The concentration of vitamin C (ascorbate) in the intracellular suggest one of the intronic SNPs studied may alter transcriptional environment is accomplished through passive and facilitated diffusion regulation of the gene, with the variant genotype causing decreased as well as via active transport of ascorbate through the sodium- binding of transcriptional enhancers. The authors also suggest that dependent vitamin C transporters (reviewed in ref. 19). The vitamin C, as a free radical scavenger, may be essential for preventing that facilitate this active transport, termed SVCT1 and SVCT2, are DNA damage from oxidative stress. Dietary intake of vitamin C was encoded by the genes SLC23A1 and SLC23A2, respectively. The not reported in this study nor was viral presence, although a viral SVCT1 protein is responsible for active transport of L-ascorbate from etiology for this disease has been suggested, and therefore the results the luminal surface of the gastrointestinal tract and kidney (20–22). In cannot be adjusted for this factor. contrast, SVCT2, which is expressed in most human tissues examined A case–control study of gastric cancer showed a significant asso- with the exception of lung and skeletal muscle, is thought to regulate ciation between a SNP in intron 2 of SLC23A2 and a borderline sig- intracellular levels of ascorbic acid for protection of the cell from nificant synonymous SNP in exon 12 (12). Additionally, a three SNP oxidative stress (23–25) and promote the maturation of type I collagen haplotype-containing SNPs located in intron 3 was significantly as- (26). Intracellular levels of vitamin C are tightly controlled through sociated with gastric cancer. There was no evidence of effect

20 any wild type allele (GG or GC) homozygous variant (CC) 15 *

10 * 5

OR (error bars = 95% CI) 0 low citrus high citrus low citrus high citrus

HPV16 negative HPV16 negative HPV16 positive HPV16 positive Dietary Citrus Intake and HPV16 Serology

Fig. 1. The ORs of dietary citrus intake and HPV16 serology for HNSCC. ORs are estimated from conditional logistic regression adjusted for age, gender, race, education, smoking and alcohol consumption. Low citrus intake is defined as ,0.67 servings per day. High citrus intake is 0.67 servings per day. Number of cases 5 319. Number of controls 5 495. P-value , 0.0001 when compared with the low citrus, HPV-negative reference group for that genotype. 979 A.A.Chen et al. modification by dietary intake of ascorbic acid while consumption of utor to this disease (i.e. HPV infection or tobacco and alcohol use), as fruits and juices was independently statistically significantly protec- there are biologically and clinically distinct pathways to carcinogen- tive of gastric cancer. These authors list several possible mechanisms esis of the head and neck that can be differentially effected by host through which ascorbic acid may play a role in gastric cancer pre- susceptibility. Such distinctions may point to novel modes of treat- vention, but there is no known mechanism for the polymorphisms ment and prevention of this disease. studied. A nested case–control study of colorectal adenoma looked at the Funding association of 11 SNPs in SLC23A2, including rs4987219, in 656 colorectal adenoma cases and 665 controls (11). None of the SNPs National Institutes of Health (CA100679, CA78609, T32 ES07155 were associated with cancer but a haplotype containing two SNPs, and T32 CA 09001); Friends of Dana-Farber Cancer Institute; Dan rs4987219 and a synonymous SNP in exon 11, was associated with and Gloria Schusterman Foundation. a decreased risk of colorectal adenoma. There was no association seen between vitamin C intake and disease. The authors suggest no mech- Acknowledgements anism but recommend further study of the region around the two Conflict of Interest Statement: J.T.B and J.F.S hold stock options employed by SNPs in the associated haplotype. Downloaded from https://academic.oup.com/carcin/article/30/6/977/2476882 by guest on 24 September 2021 The studies mentioned above, as well as our own investigation, Merck and Co., the company that developed and performed the CLIA testing and the makers of Gardasil vaccine. J.T.B is currently conducting research as have found that there are numerous low frequency haplotypes of the an employee of the company and J.F.S is currently conducting vaccine basic large and complex SLC23A2 gene (10,34). Fine mapping of this locus research at Merck. in a large number of individuals will be needed to further elucidate any allele that is associated with differential ascorbate absorption. References While the biologic mechanism underlying HNSCC disease in light of the modification of HPV16-associated cancer risk and citrus intake 1.Jemal,A. et al. (2008) Cancer statistics, 2008. CA Cancer J. 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