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
quantitative MethyLight in our current study) and the criteria for methylator type or CIMP-high (no clear definition in reference [6
the presence of ≥6 of 8 methylated CIMP-specific promoters in our current study), and the statistical methods (no multivariate analysis in reference [6
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, 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.