We conducted this study to examine molecular correlates with β-catenin activation in colorectal cancer, using a large number of samples and robust DNA methylation detection methods. Discovering molecular correlates is important in cancer research because it may: 1) provide clues to pathogenesis; 2) propose or support the existence of a new molecular subtype; 3) alert investigators to potential confounding in association studies; and 4) suggest surrogate markers in clinical or research settings. We used quantitative DNA methylation assays (MethyLight), which are essential to reproducibly differentiate low-level methylation from high-level methylation [38
]. Compared to high-level methylation, low-level promoter methylation does not generally silence gene expression and, thus, can be regarded as biologic noise [38
]. Our resource of a large number of samples of colorectal cancer (relatively unbiased samples compared to retrospective or single hospital-based samples), derived from two large prospective cohorts, has enabled us to precisely estimate the frequency of colorectal cancers with specific molecular features (e.g., nuclear β-catenin expression, CIMP-high, MSI-high, and so on).
In particular, we sought to decipher the relationship between β-catenin and MSI/CIMP status. Molecular classification based on MSI and CIMP statuses is increasingly important [25
] because MSI and CIMP statuses reflect global genomic and epigenomic aberrations in colorectal cancer cells. Promoter CpG island methylation in tumor-suppressor genes has been shown to be an important mechanism in the development of various human malignancies, including colorectal cancer [1,44–47
]. We have demonstrated that nuclear and cytoplasmic β-catenin expressions (i.e., β-catenin activation) are inversely correlated with CIMP-high, independent of MSI status. Previous studies have shown an inverse relationship between β-catenin activation and MSI-high [24–26
]. We have further demonstrated that the inverse relation between MSI and β-catenin is indirect and is mediated by CIMP because MSI status is not correlated with β-catenin expression after stratification by CIMP status. Interestingly, APC
promoter methylation has been shown to be inversely correlated with features of CIMP in colorectal cancer [15
]. Together with our results, it is likely that not only APC promoter methylation but also overall WNT/β-catenin activation is inversely correlated with CIMP.
We assessed β-catenin activation status by immunohistochemistry because β-catenin localization reflects the status of β-catenin activation. Regardless of the mechanism of β-catenin activation (i.e., a mutation in APC
or other mechanisms), β-catenin accumulates in the cytoplasm and nucleus, then activates the WNTsignaling pathway. Although previous studies have shown positive correlations between “CTNNB1
(the β-catenin gene) mutations” and MSI-high in colorectal cancer [22,23
], these studies did not examine the frequencies of cytoplasmic or nuclear β-catenin localization (i.e., the overall frequencies of β-catenin activation). In fact, CTNNB1
mutation constitutes a mechanism of β-catenin/WNT activation in only a minority of colorectal cancers [19
]. Thus, β-catenin localization is a reasonable surrogate marker when one evaluates the overall frequency of β-catenin activation.
Interestingly, we have shown that COX-2 overexpression is correlated positively with cytoplasmic β-catenin expression, but not significantly with nuclear or membrane expression. A previous study also did not show a significant relationship between COX-2 expression and nuclear β-catenin [48
]. A possible link between COX-2 and the WNT signaling pathway has been suggested [10
]. COX-2 has been shown to activate β-catenin through prostaglandin E2
and G protein-coupled receptor EP2 [11
]. A recent study has also shown that β-catenin stabilizes COX-2 mRNA by interacting with AU-rich elements in a 3′ untranslated region [12
]. Considering these data, it may be possible that COX-2 and β-catenin may form a positive feedback loop. Our data support the role of cytoplasmic β-catenin in stabilizing COX-2 (PTGS2
) mRNA. That might be the reason why the relationship between COX-2 and β-catenin expression appears to be limited to cytoplasmic β-catenin.
We have analyzed β-catenin expression in each cellular compartment (cytoplasm, nucleus, and membrane), and we have also used the β-catenin scoring system based on the methods of Jass et al. [26
]. We have shown that separately analyzing cytoplasmic β-catenin expression and nuclear expression is also valuable because associations might be limited to expression in only one of the compartments (as shown by the association between COX-2 and cytoplasmic β-catenin in this study). Thus, molecular correlates with β-catenin expression in each compartment (in particular, cytoplasm and nucleus) can be valuable.
In conclusion, β-catenin activation is inversely associated with CIMP-high in colorectal cancer independent of MSI or BRAF status. Cytoplasmic β-catenin localization is correlated positively with COX-2 overexpression. Our results indicate that one should examine CIMP status when analyzing the WNT/β-catenin pathway in relation to clinical and/or other molecular variables because CIMP status reflects global epigenomic status in tumor cells and may be a confounding factor. The exact mechanisms of these molecular correlates in colorectal cancer need to be elucidated by additional studies.