JC Virus T-Antigen in Colorectal Cancer Is Associated with p53 Expression and Chromosomal Instability, Independent of CpG Island Methylator Phenotype

Neoplasia. 2009 January; 11(1): 87–95.

PMCID: PMC2606122

JC Virus T-Antigen in Colorectal Cancer Is Associated with p53 Expression and Chromosomal Instability, Independent of CpG Island Methylator Phenotype¹

Katsuhiko Nosho,^*² Kaori Shima,^*² Shoko Kure,^*² Natsumi Irahara,^* Yoshifumi Baba,^* Li Chen,^† Gregory J Kirkner,^‡ Charles S Fuchs,^*^‡ and Shuji Ogino^*^†^§

^*Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

^†Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA

^‡Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA

^§Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA

Address all correspondence to: Shuji Ogino, MD, PhD, Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, 44 Binney St, Room JF-215C, Boston, MA 02115. E-mail: shuji_ogino/at/dfci.harvard.edu

²K.N., K.S., and S.K. contributed equally.

Received September 17, 2008; Revised October 16, 2008; Accepted October 16, 2008.

This article has been cited by other articles in PMC.

Abstract

JC virus has a transforming gene encoding JC virus T-antigen (JCVT). JCVT may inactivate wild-type p53, cause chromosomal instability (CIN), and stabilize β-catenin. A link between JCVT and CpG island methylator phenotype (CIMP) has been suggested. However, no large-scale study has examined the relations of JCVT with molecular alterations, clinical outcome, or prognosis in colon cancer. We detected JCVT expression (by immunohistochemistry) in 271 (35%) of 766 colorectal cancers. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) and eight other loci (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, WRN) by MethyLight. We examined loss of heterozygosity in 2p, 5q, 17q, and 18q. JCVT was significantly associated with p53 expression (P < .0001), p21 loss (P < .0001), CIN (≥2 chromosomal segments with LOH; P < .0001), nuclear β-catenin (P = .006), LINE-1 hypomethylation (P = .002), and inversely with CIMP-high (P = .0005) and microsatellite instability (MSI) (P < .0001), but not with PIK3CA mutation. In multivariate logistic regression analysis, the associations of JCVT with p53 [adjusted odds ratio (OR), 8.45; P < .0001], CIN (adjusted OR, 2.53; P = .003), cyclin D1 (adjusted OR, 1.57; P = .02), LINE-1 hypomethylation (adjusted OR, 1.97 for a 30% decline as a unit; P = .03), BRAF mutation (adjusted OR, 2.20; P = .04), and family history of colorectal cancer (adjusted OR, 0.64; P = .04) remained statistically significant. However, JCVT was no longer significantly associated with CIMP, MSI, β-catenin, or cyclooxygenase-2 expression in multivariate analysis. JCVT was unrelated with patient survival. In conclusion, JCVT expression in colorectal cancer is independently associated with p53 expression and CIN, which may lead to uncontrolled cell proliferation.

Introduction

The Polyomavirus family includes the simian virus 40 (SV40), JC virus, and BK virus. JC virus is a 5.12-kb, double-stranded, circular, negatively supercoiled DNA virus that commonly infects human cells [1,2]. It has a transforming gene encoding JC virus T-antigen (JCVT), which is believed to mediate the oncogenic potential of the virus [1–10]. Previous studies have suggested the link between JCVT and various human cancers [1,3–9,11,12], including colon cancers [5–7,9,12]. JC virus T-antigen has been reported to bind and inactivate wild type p53 [5,11] and cause chromosomal instability (CIN) [5–7], as well as the stabilization of β-catenin [8,9]. In addition, a previous study has suggested a link between JCVT and promoter methylation leading to the CpG island methylator phenotype (CIMP) in colorectal cancer [6].

CpG island methylator phenotype is characterized by a widespread, concordant CpG island methylation pattern [13–16]. CpG island methylator phenotype-high in colorectal cancer has been associated with older age, female sex, proximal tumor location, BRAF mutation, microsatellite instability (MSI), wild type TP53, inactive WNT/β-catenin, high-level long interspersed nucleotide element 1 (LINE-1) methylation, and stable chromosomes [17–30]. However, to our knowledge, no large-scale study has been conducted to examine the relationship of JCVT with genetic/epigenetic alterations or clinical outcome in colorectal cancer.

In this study, using a large number (n = 766) of stage I to IV colorectal cancers in two independent cohort studies, we examined tumoral JCVT expression in relation to clinical, pathologic, and molecular features in colorectal cancers. We have found that JCVT expression is significantly associated with p53 expression and CIN but not independently with CIMP or patient survival.

Materials and Methods

Study Group

We used the databases of two large prospective cohort studies; the Nurses' Health Study (n = 121,700 women observed since 1976) [31,32], and the Health Professional Follow-up Study (n = 51,500 men observed since 1986) [32]. Data on height and weight were obtained by biennial questionnaire. A subset of the cohort participants developed colorectal cancers during prospective follow-up. Previous studies on the Nurses' Health Study and Health Professionals Follow-up Study have described baseline characteristics of cohort participants and incident colorectal cancer cases and confirmed that our colorectal cancers were a good representative as a population-based sample [31,32]. Data on tumor location and stage were obtained through medical record review. We collected paraffin-embedded tissue blocks from hospitals where cohort participants with colorectal cancers had undergone resections of primary tumors. On the basis of availability of adequate tissue specimens and results, a total of 766 colorectal cancers were included. Written informed consent was obtained from all study subjects. Among our cohort studies, there was no significant difference in the demographic features between cases with tissue available and those without available tissue [32]. This current analysis represents a new analysis of JCVT on the existing colorectal cancer database that have been previously characterized for CIMP, MSI, p53, KRAS, or BRAF [33], which is analogous to novel studies using the well-described cell lines or animal models. In any of our previous studies, we have not examined JCVT expression or the relations between JCVT and other molecular events. This study represents a unique novel study about the following: 1) a large sample size analyzed for JCVT; 2) the validated set of CIMP-specific methylation markers; and 3) a number of other molecular events analyzed, including eight CpG islands other than the CIMP-specific markers, MSI, CIN, KRAS, BRAF, PIK3CA, p53, LINE-1 methylation, cyclin D1, p21, cyclooxygenase 2 (COX-2), and β-catenin. Tissue collection and analyses were approved by the Harvard School of Public Health and Brigham and Women's Hospital Institutional Review Boards.

Histopathologic Evaluations

Hematoxylin and eosin-stained tissue sections were examined by a pathologist (S.O.) unaware of other data. The tumor grade was categorized as low (≥50% gland formation) or high (<50% gland formation). The presence and extent of extracellular mucin were categorized as 0% (no mucin) or =1% of the tumor volume. The presence and extent of signet ring cells were categorized as 0% (no signet ring cells) or ≥1% of the tumor volume.

Sequencing of KRAS, BRAF, and PIK3CA and Analyses for MSI and LOH

Genomic DNA was extracted from dissected tumor tissue sections, and whole-genome amplification was performed by polymerase chain reaction (PCR) using random 15-mer primers [34]. Polymerase chain reaction and Pyrosequencing targeted for KRAS (codons 12 and 13), BRAF (codon 600), and PIK3CA (exons 9 and 20) were performed as previously described [34]. Microsatellite instability analysis was performed, using 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67, and D18S487) [33]. Microsatellite instability-high was defined as the presence of instability in ≥30% of the markers. Microsatellite instability-low was defined as instability in <30% of the markers, and microsatellite stable (MSS) tumors were defined as tumors without an unstable marker.

For loss of heterozygosity (LOH) analysis using microsatellite markers (D2S123, D5S346, D17S250, D18S55, D18S56, D18S67, and D18S487), we duplicated the PCR in each sample to exclude allele dropouts of one of two alleles. Loss of heterozygosity at each locus was defined as ≥40% reduction of one of two allele peaks in tumor DNA relative to normal DNA. We obtained informative CIN results in 514 tumors (67%). The overall CIN score was defined as the number of chromosomal segments (among 2p, 5q, 17q, and 18q) that were positive for LOH.

Real-time PCR (MethyLight) for Quantitative DNA Methylation Analysis

Sodium bisulfite treatment on genomic DNA and subsequent real-time PCR (MethyLight) [35] were validated and performed as previously described [36]. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] [24,33], all of which were selected from screening of 195 CpG islands [24,33]. CpG island methylator phenotype-high was defined as the presence of ≥6 of 8 methylated promoters, CIMP-low as the presence of 1/8 to 5/8 methylated promoters, and CIMP-0 as the absence (0/8) of methylated promoters, according to the previously established criteria [33]. In addition, we quantified DNA methylation in eight other CpG islands (not in the CIMP panel), including CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN. Primers and probes were previously described [24], except for HIC1, IGFBP3, p14, and WRN: HIC1-F, 5′-TTC GTT ACG GTA GTC GTT GTT TTC-3′ (GenBank no. L41919, nucleotide nos. 43–66); HIC1-R, 5′-GAA AAC TAT CAA CCC TCG ATC GA-3′ (nucleotide nos. 94–116); HIC1-probe, 6FAM-5′-TCG CGC GGT CGT CGT TCG-3′-BHQ-1 (nucleotide nos. 72–89); IGFBP3-F, 5′-GTT TCG GGC GTG AGT ACG A-3′ (GenBank no. M35878, nucleotide nos. 1692–1710); IGFBP3-R, 5′-GAA TCG ACG CAA ACA CGA CTA C-3′ (nucleotide nos. 1789–1810) and IGFBP3-probe, 6FAM-TCG GT T GT T TAG GGC GAA GTA CGG G-BHQ-1 (nucleotide nos. 1760–1784; bisulfite-converted nucleotides are highlighted by bold face and italics); P14 (CDKN2A/ARF)-F, 5′-T TG GAG GCG GCG AGA ATA T-3′ (GenBank no. L41934, nucleotide nos. 238–256); P14-R, 5′-CCC CGT AAA CCG CGA AAT A-3′ (nucleotide nos. 332–350); P14-probe, 6FAM-5′-CGG TTC GTC GCG AGT GAG GGT T-3′ -BHQ-1 (nucleotide nos. 299–320); WRN-F, 5′-GTA TCG TTC GCG GCG TTT AT-3′ (GenBank no. AY442327, nucleotide nos. 1827–1846); WRN-R, 5′-ACG AAA CCG ATA TCC GAA ATC A -3′ (nucleotide nos. 1887–1908) and WRN-probe, 6FAM-TTT TTT TTG CGG TCG TTG CGG G-BHQ-1 (nucleotide nos. 1855–1876). The PCR condition for all markers was initial denaturation at 95°C for 10 minutes followed by 45 cycles at 95°C for 15 seconds and 60°C for 1 minute [37].

Pyrosequencing to Measure LINE-1 Methylation

To accurately quantify relatively high methylation levels in LINE-1 repetitive elements, we used Pyrosequencing as previously described [29]. The LINE-1 methylation level measured by Pyrosequencing has been shown to correlate well with overall 5-methylcytosine level (i.e., genome-wide DNA methylation level) in tumor cells [38,39].

Immunohistochemistry for p53, p21, β-catenin, COX2, Cyclin D1, and JCVT

Tissue microarrays (TMAs) were constructed as previously described [32]. Methods of immunohistochemical procedures and interpretations were previously described for p53 [40], p21 (CDKN1A) [41,42], β-catenin [25], COX-2 [32], and cyclin D1 [43].

For JCVT, antigen retrieval was performed, and deparaffinized tissue sections in Antigen Retrieval Citra Solution (Biogenex Laboratories, San Ramon, CA) were treated with microwave in a pressure cooker for 20 minutes. Tissue sections were incubated with 3% H₂O₂ (10 minutes) to block endogenous peroxidase (Dako Cytomation, Carpinteria, CA). Primary antibody against JCVT [mouse monoclonal anti-SV40 Tantigen (clone PAb416), 1:60 dilution; Oncogene Research Products, San Diego, CA] was applied, and the slides were maintained overnight at room temperature. Next, we applied an antimouse IgG antibody (Vector Laboratories, Burlingame, CA) for 30 minutes, followed by an avidin-biotin complex conjugate (Vector Laboratories) for 30 minutes, diaminobenzidine (5 minutes) and methyl-green counterstain. Nuclear JCVT expression was recorded as no expression, weak expression, or moderate/strong expression (Figure 1). JC virus T-antigen positivity (i.e., expression) was defined as the presence of at least weak nuclear staining. Appropriate positive and negative controls were included in each run of immunohistochemistry. All immunohistochemically stained slides were interpreted by one of the investigators (JCVT, cyclin D1, and β-catenin by K.N.; p53, p21, and COX-2 by S.O.) unaware of other data. A random selection of 147 cases was examined for JCVT by a second observer (Y.B.) unaware of other data, and concordance between the two observers was 0.87 (κ = 0.74, P < .0001), indicating substantial agreement.

Figure 1

JC virus T-antigen expression in colorectal cancer. (A) No overexpression of JCVT in cancer cells (arrows). (B) Overexpression of JCVT in nuclei of cancer cells (block arrows).

Statistical Analysis

All statistical analyses used SAS program (Version 9.1; SAS Institute, Cary, NC). All P values were two-sided, and statistical significance was set at P ≤ .05; however, P values were conservatively interpreted, considering multiple hypotheses testing. For categorical data, the χ² test (or Fisher exact test when any expected cell count was less than 5) was performed and odds ratio (OR) with 95% confidence interval (CI) was computed. To compare mean LINE-1 methylation levels, the t test assuming unequal variances was performed. The κ coefficient was calculated to assess an agreement between the two interpreters in immunohistochemistry. To assess independent relations of JCVT with a number of variables, a multivariate logistic regression analysis was performed. Odds ratio was adjusted for age (<65 vs ≥65 years), sex, tumor location (proximal vs distal), body mass index (BMI, ≥30 vs <30 kg/m²), tumor stage (I–II vs III–IV), grade (low vs high), family history (present vs absent), mucin (present vs absent), signet ring cells (present vs absent), CIMP status (high vs low/0), MSI status (high vs low/MSS), LINE-1 methylation (as a continuous variable), β-catenin, COX-2, cyclin D1, p53, p21, BRAF, KRAS, and PIK3CA.

For survival analysis, Kaplan-Meier analysis was performed to assess survival time distributions according to JCVT status, and logrank test was performed. We also constructed a multivariate, stage-matched conditional Cox proportional hazard model to compute hazard ratios (HRs) according to tumoral JCVT status, adjusted for age, sex, year of diagnosis, tumor location, stage, grade, CIMP, MSI, KRAS, BRAF, PIK3CA, β-catenin, COX-2, cyclin D1 p53, p21, and LINE-1 methylation. In addition, a univariate Cox proportional hazard model was used to assess the main effect of JCVT on patient mortality. For the analyses of colorectal cancer-specific mortality, death because of colorectal cancer was the primary end point and deaths because of other causes were censored. The proportionality of hazards assumption was satisfied by evaluating time-dependent variables, which were the cross product of the JCVT variable and survival time (P = .30 for colon cancer-specific mortality; P = .48 for overall mortality). To adjust for potential confounding, age, year of diagnosis, and LINE-1 methylation were used as continuous variables, and all of the other covariates were used as categorical variables. An interaction was assessed by including the cross product of the JCVT variable and another variable of interest in a multivariate Cox model, and the likelihood ratio test was performed.

Results

JC Virus T-Antigen Expression in Colorectal Cancers

Among the 766 colorectal cancers assessed by immunohistochemistry, 271 (35%) tumors overexpressed JCVT. Table 1 summarizes the frequencies of JCVT expression according to various clinical and pathologic features. JC virus T-antigen expression was inversely associated with proximal location (P = .002), high grade (P = .046), and mucinous component (P = .0004).

Table 1

Frequency of JCVT Expression in Colorectal Cancer.

Relationship of JCVT with CIMP and MSI Status

We determined CIMP status using MethyLight assays on a panel of eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) [24,33]. Table 2 summarizes the frequencies of JCVT expression according to CIMP and other molecular features in colorectal cancer. JC virus T-antigen expression was inversely associated with CIMP-high (≥6/8 methylated promoters; OR, 0.40; 95% CI, 0.24–0.68, compared to CIMP-0; P = .0005) and MSI-high (OR, 0.25; 95% CI, 0.14–0.44, compared to MSS; P < .0001). To examine the combined effect of MSI and CIMP on JCVT expression, we classified tumors into four subtypes according to CIMP and MSI status (Table 2). JC virus T-antigen overexpression was significantly less common in CIMP-high MSI-high [14% (10/70), P < .0001) and CIMP-low/0 MSI-high [13% (4/31), P = .002] than in CIMP-low/0 MSI-low/MSS tumors [42% (238/573)].

Table 2

Frequency of JCVT Expression in Colorectal Cancer According to Various Molecular Features.

JC Virus T-Antigen and Other Molecular Changes

We determined the CIN score in MSS/MSI-low tumors as the number of chromosomal segments (among 2p, 5q, 17q, and 18q) positive for LOH. JC virus T-antigen was significantly associated with high CIN score (≥2+; OR, 3.02; 95% CI, 1.82–5.00, compared to CIN score 0; P < .0001; Table 2). JC virus T-antigen expression was also significantly associated with p53 expression (P < .0001), loss of p21 expression (P < .0001), nuclear β-catenin expression (P = .006), and COX-2 expression (P = .02). In contrast, JCVT expression was not significantly associated with alterations in KRAS, BRAF, PIK3CA, or cyclin D1.

JC Virus T-Antigen and Methylation in Individual CpG Islands

Because JCVT has been implicated in CpG island methylation [6], we examined the relationship of JCVT with methylation in 16 individual CpG islands, including the 8 CIMP-specific promoters (Table 3). In the univariate analysis, JCVT was inversely associated with methylation in CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, HIC1, MINT31, p14, and WRN. However, after adjusting for CIMP, all of these associations were markedly attenuated, and no association was considered to be highly significant, given multiple hypotheses testing. These results suggest that none of these 16 methylation markers were directly related with JCVT.

Table 3

Frequency of JCVT in Colorectal Cancer According to Methylation Marker Status.

JC Virus T-Antigen Is Independently Associated with p53 Expression and CIN

We performed multivariate logistic regression analysis, to examine which variables were independently associated with JCVT (Table 4). JC virus T-antigen was significantly associated with p53 expression (multivariate OR, 8.45; 95% CI, 5.72–12.5; P < .0001) and high CIN score (multivariate OR, 2.53; 95% CI, 1.38–4.62; P = .003) and inversely with high tumor grade (multivariate OR, 0.44; 95% CI, 0.24–0.79; P = .006). JC virus T-antigen might also be associated with cyclin D1 expression, LINE-1 hypomethylation, BRAF mutation, and signet ring cells and inversely with family history of colorectal cancer (all P values between 0.05 and 0.01); however, given multiple hypotheses testing, any of these associations might be a chance event.

Table 4

Multivariate Analysis of the Relations with JCVT in Colorectal Cancer.

JC Virus T-Antigen and Patient Survival

We assessed the influence of JCVT expression on survival of patients with stage I to IV colorectal cancers. We have previously shown that clinical outcome data in our two independent cohort studies are valid and reliable to detect significant molecular predictors of patient survival [44–46]. In the Kaplan-Meier analysis, JCVT expression was not significantly associated with patient survival (log-rank, P = .31 for colorectal cancer-specific mortality; log-rank, P = .67 for overall mortality). We performed Cox regression analysis to assess mortalities according to JCVT status (Table 5). For both cancer-specific and overall mortalities, JCVT was not significantly related with patient outcome in univariate analysis, stage-matched analysis, or multivariate analysis. When we limited cases to only colon cancers, JCVT remained unrelated with patient outcome. We examined whether JCVT was associated with patient mortality in any of the strata of clinical or molecular variables (such as age, sex, tumor stage, location, CIMP, MSI, BRAF, LINE-1, etc.). However, there was no evidence for the significant relation between JCVT and clinical outcome in any of the strata, and there was no evidence for significant interaction between JCVT and any of the variables in survival analysis (data not shown).

Table 5

JC Virus T-Antigen Expression and Patient Mortality in Colorectal Cancer.

Discussion

We conducted this study to examine the relations of JCVT expression with clinical, pathologic, and molecular characteristics and patient survival in colorectal cancers. Molecular correlates with JCVT may be important for a better understanding of genetic and epigenetic alterations during the colorectal carcinogenic process. We have found that JCVT is independently associated with p53 expression and CIN. In contrast, JCVT is inversely related with the CIMP and MSI in univariate analysis but not in multivariate analysis. Our data support the hypothesis that JCVT may affect p53 expression and CIN rather than CIMP and MSI in colorectal cancer.

Studying molecular changes is important in cancer research [47–64], and classification of colorectal cancer based on MSI and CIMP is increasingly important because it reflects genomic and epigenomic alterations, respectively, in tumor cells and largely determines clinical, pathologic, and molecular characteristics [65]. To measure DNA methylation, we used real-time PCR (MethyLight Technology) for DNA methylation in eight CIMP-specific loci [33] as well as in eight other CpG islands. We also used Pyrosequencing to measure LINE-1 methylation, which has been correlated well with cellular 5-methylcytosine level (i.e., genome-wide DNA methylation level) [29,38,39]. Our resource of a large number of colorectal cancers derived from the two prospective cohort studies has enabled us to precisely estimate the frequency of colorectal cancers with a specific molecular feature (such as JCVT expression, CIMP-high, p53 expression, etc.). The large number of cases has also provided a sufficient power in our multivariate logistic regression analysis and survival analysis.

Previous studies have reported the relationship of JC virus with CIN and LOH [6,7]. Introduction of JC virus into a diploid cell line can lead to CIN [7]. In addition, JCVT is strongly associated with LOH in colorectal cancers [6]. In the current study, we have shown that JCVT is associated with CIN and LINE-1 hypomethylation, independent of other variables. These data collectively support a possible role of JCVT in the development of CIN and genome-wide DNA hypomethylation in colorectal cancers.

Regarding the relation between JCVT and p53, a previous study has reported that JCVT can bind and inactivate both p53 and phospho-RB proteins [11]. Especially, JCVT may affect other regulatory mechanisms for p53, which have been implicated in cancer development [9]. Together with our current data of the independent association between JCVT and p53 expression, accumulating data suggest that JCVT may dysregulate the p53 pathways, which can lead to uncontrolled proliferation of colorectal cancer cells.

We have demonstrated that JCVT expression is associated with p53 expression in colorectal cancer. One possible explanation for this phenomenon is that there might be some cases with poor antigenicity of JCVT and p53. Poor quality of tissue for immunohistochemistry would have yielded a false-negative result in either the JCVT or p53 immunoassay, driving the overall relationship between JCVT and p53 expression toward a concordant pattern. However, we have shown that JCVT expression is inversely associated with p21 expression, which cannot be explained by the presence of poor-quality specimens. p21 has been known to be induced by wild-type p53, and indeed, p53 expression (a surrogate of p53 mutation) was inversely associated with p21 expression in our cohorts. These results imply that the strong relation between JCVT and p53 expression we have observed is not simply caused by the presence of poor-quality specimens, leaving a possibility of a molecular interaction or other molecular correlates between JCVT and p53. Interestingly, the relation between JCVT and p21 loss did not persist after adjusting for p53, suggesting that the link between JCVT and p21 loss was mediated by p53 expression.

Previous studies have reported that methylation of host cell gene is not unique to JC virus and occurs with other oncogenic viruses [55,57,61,66]. Associations have been shown between methylation of multiple genes and Epstein-Barr virus in gastric cancer [55,57] and between promoter methylation and hepatitis B virus/hepatitis C virus in hepatocellular cancer [61,66]. A significant association has been found between the presence of SV40 T-antigen and methylation of multiple genes in non-Hodgkin lymphomas and mesotheliomas [67,68]. In addition, a previous study has reported that JCVT may induce CIMP in colorectal cancers through multiple mechanisms of epigenetic alterations [6]. However, our data do not support the relation of JCVT with CIMP (determined by the validated panel of eight CIMP-specific promoters [24,33]) or methylation in any of the 16 CpG islands we examined, using a large number of colorectal cancers. We have shown an “inverse association” between JCVT and CIMP in univariate analysis (P = .0005), which became insignificant in multivariate analysis, indicating no independent association between JCVT and CIMP. In addition, none of the 16 CpG islands seemed to be specifically related with JCVT after adjusting for CIMP. This discrepancy is likely caused by the differences in the sample sizes (n = 100 in reference [6] vs n = 766 in our current study), the methylation markers examined (MLH1, APC, CDKN2A, p14, PTEN, TIMP3, RUNX3, HIC1, and RARB in reference [6] vs CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, and eight other CpG islands in our current study), the methods to detect DNA methylation (nonquantitative methylation-specific PCR in reference [6] vs quantitative MethyLight in our current study) and the criteria for methylator type or CIMP-high (no clear definition in reference [6] vs the presence of ≥6 of 8 methylated CIMP-specific promoters in our current study), and the statistical methods (no multivariate analysis in reference [6] vs both multivariate and univariate analyses performed in our current study). Considering that there is considerable heterogeneity of tumors with regard to CpG island methylation and that CpG islands are methylated in a different manner, the difference in the methylation markers between the two studies may explain discrepancies, at least in part. However, some CpG islands (MLH1, RUNX3, CDKN2A, p14, and HIC1) were used in both studies, and results on the same markers seemed to be discordant. We have conducted rigorous statistical analysis for each marker and performed multivariate analysis to assess independent associations and significant confounding. In addition, we have comprehensively examined the relation between JCVT and CIMP using the validated CIMP marker panel [24,33] and a large number (n = 766) of colorectal cancers with robust statistics. Our results suggest that JCVT may not contribute to CIMP in colorectal cancer. On the basis of our current results and data in the literature, Figure 2 represents hypothetical relations with JCVT in colorectal cancer.

Figure 2

Hypothetical relations with JCVT expression in colorectal cancer. The thick lines with p53 and CIN imply the particularly tight relations with JCVT. Data are based on this current study and references [25,26,29,30,42].

In conclusion, using a large number of colorectal cancers, we have shown that JCVT is independently associated with p53 expression and CIN. Conversely, JCVT seems to be unrelated with CIMP, MSI, or patient outcome. Our data suggest that JCVT may contribute to CIN and dysregulation of the p53 pathways, which may lead to uncontrolled proliferation of colorectal cancer cells.

Acknowledgments

The authors thank the Nurses' Health Study and Health Professionals Follow-up Study cohort participants who have generously agreed to provide the biological specimens and information through responses to questionnaires. The authors thank Frank Speizer, Walter Willett, Susan Hankinson, Graham Colditz, Meir Stampfer, and many other staff members who implemented and have maintained the cohort studies.

No conflicts of interest exist.

Abbreviations


BMI	body mass index

CI	confidence interval

CIMP	CpG island methylator phenotype

CIN	chromosomal instability

COX-2	cyclooxygenase-2 (PTGS2)

HR	hazard ratio

JCVT	JC virus T-antigen

LINE-1	long interspersed nucleotide element 1

LOH	loss of heterozygosity

MSI	microsatellite instability

MSS	microsatellite stable

OR	odds rati

Footnotes

¹This work was supported by US National Institutes of Health (NIH) grants P01 CA87969, P01 CA55075, P50 CA127003, and K07 CA122826 (to S.O.) and in part by grants from the Bennett Family Fund and from the Entertainment Industry Foundation through the National Colorectal Cancer Research Alliance. K.N. was supported by a fellowship grant from the Japan Society for Promotion of Science. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1. Shin SK, Li MS, Fuerst F, Hotchkiss E, Meyer R, Kim IT, Goel A, Boland CR. Oncogenic T-antigen of JC virus is present frequently in human gastric cancers. Cancer. 2006;107:481–488. [PubMed]

2. Ricciardiello L, Laghi L, Ramamirtham P, Chang CL, Chang DK, Randolph AE, Boland CR. JC virus DNA sequences are frequently present in the human upper and lower gastrointestinal tract. Gastroenterology. 2000;119:1228–1235. [PubMed]

3. Krynska B, Del Valle L, Croul S, Gordon J, Katsetos CD, Carbone M, Giordano A, Khalili K. Detection of human neurotropic JC virus DNA sequence and expression of the viral oncogenic protein in pediatric medulloblastomas. Proc Natl Acad Sci USA. 1999;96:11519–11524. [PubMed]

4. Del Valle L, Gordon J, Assimakopoulou M, Enam S, Geddes JF, Varakis JN, Katsetos CD, Croul S, Khalili K. Detection of JC virus DNA sequences and expression of the viral regulatory protein T-antigen in tumors of the central nervous system. Cancer Res. 2001;61:4287–4293. [PubMed]

5. Niv Y, Goel A, Boland CR. JC virus and colorectal cancer: a possible trigger in the chromosomal instability pathways. Curr Opin Gastroenterol. 2005;21:85–89. [PubMed]

6. Goel A, Li MS, Nagasaka T, Shin SK, Fuerst F, Ricciardiello L, Wasserman L, Boland CR. Association of JC virus T-antigen expression with the methylator phenotype in sporadic colorectal cancers. Gastroenterology. 2006;130:1950–1961. [PubMed]

7. Ricciardiello L, Baglioni M, Giovannini C, Pariali M, Cenacchi G, Ripalti A, Landini MP, Sawa H, Nagashima K, Frisque RJ, et al. Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus. Cancer Res. 2003;63:7256–7262. [PubMed]

8. Gan DD, Khalili K. Interaction between JCV large T-antigen and beta-catenin. Oncogene. 2004;23:483–490. [PubMed]

9. Enam S, Del Valle L, Lara C, Gan DD, Ortiz-Hidalgo C, Palazzo JP, Khalili K. Association of human polyomavirus JCV with colon cancer: evidence for interaction of viral T-antigen and beta-catenin. Cancer Res. 2002;62:7093–7101. [PubMed]

10. Shimazu T, Komatsu Y, Nakayama KI, Fukazawa H, Horinouchi S, Yoshida M. Regulation of SV40 large T-antigen stability by reversible acetylation. Oncogene. 2006;25:7391–7400. [PubMed]

11. Ludlow JW. Interactions between SV40 large-tumor antigen and the growth suppressor proteins pRB and p53. FASEB J. 1993;7:866–871. [PubMed]

12. Enam S, Gan DD, White MK, Del Valle L, Khalili K. Regulation of human neurotropic JCV in colon cancer cells. Anticancer Res. 2006;26:833–841. [PubMed]

13. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA. 1999;96:8681–8686. [PubMed]

14. Issa JP, Shen L, Toyota M. CIMP, at last. Gastroenterology. 2005;129:1121–1124. [PubMed]

15. Grady WM. CIMP and colon cancer gets more complicated. Gut. 2007;56:1498–1500. [PMC free article] [PubMed]

16. Teodoridis JM, Hardie C, Brown R. CpG island methylator phenotype (CIMP) in cancer: causes and implications. Cancer Lett. 2008;268:177–186. [PubMed]

17. Whitehall VL, Wynter CV, Walsh MD, Simms LA, Purdie D, Pandeya N, Young J, Meltzer SJ, Leggett BA, Jass JR. Morphological and molecular heterogeneity within nonmicrosatellite instability-high colorectal cancer. Cancer Res. 2002;62:6011–6014. [PubMed]

18. Hawkins N, Norrie M, Cheong K, Mokany E, Ku SL, Meagher A, O'Connor T, Ward R. CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability. Gastroenterology. 2002;122:1376–1387. [PubMed]

19. van Rijnsoever M, Grieu F, Elsaleh H, Joseph D, Iacopetta B. Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands. Gut. 2002;51:797–802. [PMC free article] [PubMed]

20. Kambara T, Simms LA, Whitehall VLJ, Spring KJ, Wynter CVA, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut. 2004;53:1137–1144. [PMC free article] [PubMed]

21. Nagasaka T, Sasamoto H, Notohara K, Cullings HM, Takeda M, Kimura K, Kambara T, MacPhee DG, Young J, Leggett BA, et al. Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation. J Clin Oncol. 2004;22:4584–4594. [PubMed]

22. Samowitz W, Albertsen H, Herrick J, Levin TR, Sweeney C, Murtaugh MA, Wolff RK, Slattery ML. Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology. 2005;129:837–845. [PubMed]

23. Ogino S, Cantor M, Kawasaki T, Brahmandam M, Kirkner G, Weisenberger DJ, Campan M, Laird PW, Loda M, Fuchs CS. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies. Gut. 2006;55:1000–1006. [PMC free article] [PubMed]

24. Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–793. [PubMed]

25. Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Dehari R, Meyerhardt JA, Fuchs CS, Ogino S. Correlation of beta-catenin localization with cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in colorectal cancer. Neoplasia. 2007;9:569–577. [PMC free article] [PubMed]

26. Goel A, Nagasaka T, Arnold CN, Inoue T, Hamilton C, Niedzwiecki D, Compton C, Mayer RJ, Goldberg R, Bertagnolli MM, et al. The CpG island methylator phenotype and chromosomal instability are inversely correlated in sporadic colorectal cancer. Gastroenterology. 2007;132:127–138. [PubMed]

27. Samowitz WS, Slattery ML, Sweeney C, Herrick J, Wolff RK, Albertsen H. APC mutations and other genetic and epigenetic changes in colon cancer. Mol Cancer Res. 2007;5:165–170. [PubMed]

28. Iacopetta B, Grieu F, Li W, Ruszkiewicz A, Caruso M, Moore J, Watanabe G, Kawakami K. APC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2006;119:2272–2278. [PubMed]

29. Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs CS. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122:2767–2773. [PMC free article] [PubMed]

30. Ogino S, Kawasaki T, Kirkner GJ, Ohnishi M, Fuchs CS. 18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high. BMC Cancer. 2007;7:72. [PMC free article] [PubMed]

31. Colditz GA, Hankinson SE. The Nurses' Health Study: lifestyle and health among women. Nat Rev Cancer. 2005;5:388–396. [PubMed]

32. Chan AT, Ogino S, Fuchs CS. Aspirin and the risk of colorectal cancer in relation to the expression of COX-2. N Engl J Med. 2007;356:2131–2142. [PubMed]

33. Ogino S, Kawasaki T, Kirkner GJ, Kraft P, Loda M, Fuchs CS. Evaluation of markers for CpG island methylator phenotype (CIMP) in colorectal cancer by a large population-based sample. J Mol Diagn. 2007;9:305–314. [PubMed]

34. Nosho K, Kawasaki T, Ohnishi M, Suemoto Y, Kirkner GJ, Zepf D, Yan L, Longtine JA, Fuchs CS, Ogino S. PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations. Neoplasia. 2008;10:534–541. [PMC free article] [PubMed]

35. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 2000;28:E32. [PMC free article] [PubMed]

36. Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D, Makrigiorgos GM, Weisenberger DJ, Laird PW, Loda M, et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn. 2006;8:209–217. [PubMed]

37. Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs CS, Ogino S. IGFBP3 promoter methylation in colorectal cancer: relationship with microsatellite instability, CpG island methylator phenotype, and p53. Neoplasia. 2007;9:1091–1098. [PMC free article] [PubMed]

38. Estecio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, et al. LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS ONE. 2007;2:e399. [PMC free article] [PubMed]

39. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38. [PMC free article] [PubMed]

40. Ogino S, Kawasaki T, Kirkner GJ, Yamaji T, Loda M, Fuchs CS. Loss of nuclear p27 (CDKN1B/KIP1) in colorectal cancer is correlated with microsatellite instability and CIMP. Mod Pathol. 2007;20:15–22. [PubMed]

41. Ogino S, Meyerhardt JA, Cantor M, Brahmandam M, Clark JW, Namgyal C, Kawasaki T, Kinsella K, Michelini AL, Enzinger PC, et al. Molecular alterations in tumors and response to combination chemotherapy with gefitinib for advanced colorectal cancer. Clin Cancer Res. 2005;11:6650–6656. [PubMed]

42. Ogino S, Kawasaki T, Kirkner GJ, Ogawa A, Dorfman I, Loda M, Fuchs CS. Down-regulation of p21 (CDKN1A/CIP1) is inversely associated with microsatellite instability and CpG island methylator phenotype (CIMP) in colorectal cancer. J Pathol. 2006;210:147–154. [PubMed]

43. Nosho K, Kawasaki T, Chan AT, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs CS, Ogino S. Cyclin D1 is frequently overexpressed in microsatellite unstable colorectal cancer, independent of CpG island methylator phenotype. Histopathology. 2008;53:588–598. [PMC free article] [PubMed]

44. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, Giovannucci EL, Fuchs CS. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. (in press)

45. Ogino S, Nosho K, Kirkner GJ, Shima K, Irahara N, Kure S, Chan AT, Engelman JA, Kraft P, Cantley LC, et al. PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. J Clin Oncol. (in press)

46. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci EL, Fuchs CS. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. (in press) (published online on 2 Oct 2008. doi:2010.1136/Gut2008.155473)

47. Sasaki T, Kitadai Y, Nakamura T, Kim JS, Tsan RZ, Kuwai T, Langley RR, Fan D, Kim SJ, Fidler IJ. Inhibition of epidermal growth factor receptor and vascular endothelial growth factor receptor phosphorylation on tumor-associated endothelial cells leads to treatment of orthotopic human colon cancer in nude mice. Neoplasia. 2007;9:1066–1077. [PMC free article] [PubMed]

48. Chung YL, Troy H, Kristeleit R, Aherne W, Jackson LE, Atadja P, Griffiths JR, Judson IR, Workman P, Leach MO, et al. Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of a novel histone deacetylase inhibitor, LAQ824, in human colon carcinoma cells and xenografts. Neoplasia. 2008;10:303–313. [PMC free article] [PubMed]

49. Ahlquist T, Bottillo I, Danielsen SA, Meling GI, Rognum TO, Lind GE, Dallapiccola B, Lothe RA. RAS signaling in colorectal carcinomas through alteration of RAS, RAF, NF1, and/or RASSF1A. Neoplasia. 2008;10:680–686. [PMC free article] [PubMed]

50. Derks S, Postma C, Carvalho B, van den Bosch SM, Moerkerk PT, Herman JG, Weijenberg MP, de Bruine AP, Meijer GA, van Engeland M. Integrated analysis of chromosomal, microsatellite and epigenetic instability in colorectal cancer identifies specific associations between promoter methylation of pivotal tumour suppressor and DNA repair genes and specific chromosomal alterations. Carcinogenesis. 2008;29:434–439. [PubMed]

51. Derouet M, Wu X, May L, Hoon Yoo B, Sasazuki T, Shirasawa S, Rak J, Rosen KV. Acquisition of anoikis resistance promotes the emergence of oncogenic K-ras mutations in colorectal cancer cells and stimulates their tumorigenicity in vivo. Neoplasia. 2007;9:536–545. [PMC free article] [PubMed]

52. Ueno K, Hiura M, Suehiro Y, Hazama S, Hirata H, Oka M, Imai K, Dahiya R, Hinoda Y. Frizzled-7 as a potential therapeutic target in colorectal cancer. Neoplasia. 2008;10:697–705. [PMC free article] [PubMed]

53. Henkhaus RS, Roy UK, Cavallo-Medved D, Sloane BF, Gerner EW, Ignatenko NA. Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells. Neoplasia. 2008;10:140–148. [PMC free article] [PubMed]

54. Tsareva SA, Moriggl R, Corvinus FM, Wiederanders B, Schutz A, Kovacic B, Friedrich K. Signal transducer and activator of transcription 3 activation promotes invasive growth of colon carcinomas through matrix metalloproteinase induction. Neoplasia. 2007;9:279–291. [PMC free article] [PubMed]

55. Kang GH, Lee S, Kim WH, Lee HW, Kim JC, Rhyu MG, Ro JY. Epstein-Barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma. Am J Pathol. 2002;160:787–794. [PubMed]

56. Cengel KA, Voong KR, Chandrasekaran S, Maggiorella L, Brunner TB, Stanbridge E, Kao GD, McKenna WG, Bernhard EJ. Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells. Neoplasia. 2007;9:341–348. [PMC free article] [PubMed]

57. Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M, Hosokawa M, Shinomura Y, Imai K, Tokino T. Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus. Cancer. 2006;106:1467–1479. [PubMed]

58. Rocken C, Neumann K, Carl-McGrath S, Lage H, Ebert MP, Dierkes J, Jacobi CA, Kalmuk S, Neuhaus P, Neumann U. The gene polymorphism of the angiotensin I-converting enzyme correlates with tumor size and patient survival in colorectal cancer patients. Neoplasia. 2007;9:716–722. [PMC free article] [PubMed]

59. Xiong H, Zhang ZG, Tian XQ, Sun DF, Liang QC, Zhang YJ, Lu R, Chen YX, Fang JY. Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells. Neoplasia. 2008;10:287–297. [PMC free article] [PubMed]

60. Campos AC, Molognoni F, Melo FH, Galdieri LC, Carneiro CR, D'Almeida V, Correa M, Jasiulionis MG. Oxidative stress modulates DNA methylation during melanocyte anchorage blockade associated with malignant transformation. Neoplasia. 2007;9:1111–1121. [PMC free article] [PubMed]

61. Zhang C, Guo X, Jiang G, Zhang L, Yang Y, Shen F, Wu M, Wei L. CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma. Int J Cancer. 2008;123:998–1004. [PubMed]

62. Chilukamarri L, Hancock AL, Malik S, Zabkiewicz J, Baker JA, Greenhough A, Dallosso AR, Huang TH, Royer-Pokora B, Brown KW, et al. Hypomethylation and aberrant expression of the glioma pathogenesis-related 1 gene in Wilms tumors. Neoplasia. 2007;9:970–978. [PMC free article] [PubMed]

63. Kuester D, Dar AA, Moskaluk CC, Krueger S, Meyer F, Hartig R, Stolte M, Malfertheiner P, Lippert H, Roessner A, et al. Early involvement of death-associated protein kinase promoter hypermethylation in the carcinogenesis of Barrett's esophageal adenocarcinoma and its association with clinical progression. Neoplasia. 2007;9:236–245. [PMC free article] [PubMed]

64. Litkouhi B, Kwong J, Lo CM, Smedley JG JG, III, McClane BA, Aponte M, Gao Z, Sarno JL, Hinners J, Welch WR, et al. Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin. Neoplasia. 2007;9:304–314. [PMC free article] [PubMed]

65. Ogino S, Goel A. Molecular classification and correlates in colorectal cancer. J Mol Diagn. 2008;10:13–27. [PubMed]

66. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol. 2003;163:1101–1107. [PubMed]

67. Suzuki M, Toyooka S, Shivapurkar N, Shigematsu H, Miyajima K, Takahashi T, Stastny V, Zern AL, Fujisawa T, Pass HI, et al. Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection. Oncogene. 2005;24:1302–1308. [PubMed]

68. Shivapurkar N, Takahashi T, Reddy J, Zheng Y, Stastny V, Collins R, Toyooka S, Suzuki M, Parikh G, Asplund S, et al. Presence of simian virus 40 DNA sequences in human lymphoid and hematopoietic malignancies and their relationship to aberrant promoter methylation of multiple genes. Cancer Res. 2004;64:3757–3760. [PubMed]

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