The present investigation builds upon prior work showing that phytochemicals such as CHL and I3C can alter the spectrum of β-catenin mutations in DMH- and IQ-induced tumors [9
]. Under conditions of tumor promotion
, there was an increase in mutations affecting Ser37, Thr41 and Ser45 of β-catenin [11
]. The latter mutations have been reported in human cancers, but they occur less frequently in the colon tumors from rats exposed to chemical carcinogens in the absence of a tumor promoter [7
]. It is tempting to speculate that Ser37, Thr41 and Ser45 β-catenin mutations represent a genetic ‘fingerprint’ for cancers that have arisen due to initiating events coupled with a promotional stimulus, when cells normally deleted by apoptosis might survive to frank tumors. However, this would be an over-simplification. In PhIP-treated rats, promotion by a high-fat diet led to an increase in dysplastic aberrant crypt foci and colon tumors, but none of the mutations identified affected critical Ser/Thr residues in β-catenin [21
]. Conversely, a high frequency of codon 41 β-catenin mutation (Thr41Ile) was found in rats given DMH over a period of 20 weeks as part of a ‘complete carcinogen’ protocol, without a separate tumor promoting agent [10
]. Nonetheless, it is clear from published reports [9
] that the carcinogen and/or phytochemical exposure protocol can influence the final pattern of β-catenin mutations in colon tumors. Understanding how such patterns arise might provide better insight into the risk factors for human colorectal cancer development.
Previously, rat colon tumors with the highest levels of β-catenin, c-Myc and c-Jun proteins were found to contain codon 41 and 45 mutant β-catenins [9
]. These specific mutations in β-catenin were thought to more effectively stabilize β-catenin at the protein level, and thus provide for greater activation of β-catenin/Tcf target genes. In the present study, however, DMH-induced colon tumors and small intestine tumors had a wide range of β-catenin mRNA expression, and this was essentially independent of the β-catenin mutation status () or phytochemical exposure ( and data not shown). Transcript levels for three β-catenin/Tcf ‘downstream’ targets, namely c-myc, c-jun, and cyclin D1, were highly correlated with each other and with the β-catenin mRNA expression levels. The latter findings suggest a global dysregulation of gene expression, including transcriptional changes in the gene for β-catenin (Ctnnb1
It is well established that the progression to colorectal adenoma and carcinoma involves multiple genetic events and transcription changes in a broad array of genes [23
]. Indeed, β-catenin mRNA levels are known to be increased in human primary colorectal cancers and their liver metastases, compared with matched normal-looking tissue [5
]. A major regulator of β-catenin expression in human colon cancers is APC, but this is usually viewed from the perspective of β-catenin protein stabilization rather than transcriptional control of β-catenin mRNA levels. It is unclear what relationship, if any, exists between Apc mutation status and β-catenin mRNA expression in tumors. We did not examine Apc in the present investigation, because previous work showed that DMH- and IQ-induced tumors in the rat had a low frequency (<10%) of Apc mutations [11
]. Apc might be a candidate for further study, especially in DMH-induced tumors that were wild type for β-catenin and that expressed high levels of β-catenin mRNA (). It also remains to be determined whether β-catenin mRNA levels are altered in the tumors induced by other carcinogens, such as IQ and PhIP, and the mechanisms involved.
As indicated above, the regulation of β-catenin mRNA levels in tumors has not been well studied, and establishing what relationship might exist between β-catenin transcript and β-catenin protein expression may be critical to understanding its relevance to tumor development. In the present investigation, many of the tumors with the highest β-catenin mRNA levels had highly over-expressed β-catenin protein, and others with low or undetectable β-catenin mRNA also had low β-catenin protein expression, but there were exceptions (e.g. lanes 3 and 8 in ). We conclude that there is a general concordance between β-catenin mRNA and protein expression levels in DMH-induced tumors, but the correlation is not perfect. Other variables may be correlated with β-catenin transcript levels, such as tumor size, histopathology, or proliferative index, and additional work in this area appears to be warranted.
In summary, the present investigation has shown that small intestine tumors and colon tumors induced by DMH in the rat contain a wide range of expression of β-catenin mRNA. This was independent of β-catenin mutation status and treatment with CHL or I3C, but was highly correlated with c-myc, c-jun and cyclin D1 mRNA expression levels. These findings suggest the need for future studies on the mechanisms that regulate β-catenin at the transcriptional level, including further characterization of the transcription factors that bind to the promoter regions of human CTNNB1
, rat Ctnnb1
and mouse Catbn1