Our decision to pursue detailed studies of the means by which mutated β-cat proteins contribute to neoplastic transformation was motivated by recent evidence that defects in β-cat regulation are found in nearly all colon cancers and a subset of other cancers (9
). In normal epithelial cells, the bulk of β-cat is complexed at the plasma membrane with the E-cad cell adhesion molecule (4
). The abundance of β-cat in the cytosol and nucleus is regulated by a multiprotein complex containing the GSK3β and APC proteins (24
). Activation of the Wnt pathway inhibits GSK3β, thus inhibiting β-cat degradation. In colon cancer, regulation of the cytosolic and nuclear pools of β-cat is most often disrupted as a result of APC inactivation (25
). In some colon and other cancers, missense mutations or deletions of presumptive GSK3β phosphorylation sites in the β-cat N terminus render the protein resistant to regulation by the GSK3β/APC/axin complex (9
). Regardless of the underlying cause, the consequences appear to be increased levels of β-cat in the cytosol and nucleus, constitutive interaction of β-cat with Tcf/Lef transcription factors, and activation of Tcf/Lef-regulated genes (25
Here, we have shown that mutated β-cat proteins, harboring either a human cancer-derived missense mutation in a presumptive GSK3β phosphorylation site (S33Y) or deletions of up to the first 132 N-terminal amino acids will induce neoplastic transformation of RK3E, an E1A-immortalized epithelial cell line derived from neonatal rat kidney. The reason why mutated β-cat proteins transformed RK3E cells but not the NIH 3T3, IEC-18, or 1811 lines is not known. In contrast to mutated β-cat, activated Ras proteins readily transform NIH 3T3 and IEC-18. Further work is required to determine whether the transforming activity of mutated β-cat in RK3E is attributable to the presence of the adenovirus E1A protein, the specific constellation of other gene defects in the line, or another aspect of the phenotype of RK3E, such as its particular cell of origin. NIH 3T3 cells may not be transformed by mutated β-cat, because they lack critical targets of β-cat action, such as Lef-1 or certain Tcfs (13
Mutated β-cat proteins capable of transforming RK3E cells were able to activate a Tcf reporter gene, although some N-terminally truncated β-cat mutants (e.g., ΔN132) had only a modest ability to activate Tcf transcription in our transient-transfection assay. The reduced transcriptional activity of N-terminally truncated forms, particularly the ΔN132 form, may be attributable, at least in part, to the fact that the N-terminal region of β-cat plays a role in activation of Tcf transcription (13
). It is also possible that the transient-transfection assay of Tcf transcriptional activity does not entirely reflect the biological activity of the β-cat mutants. The apparent common theme we observed among β-cat proteins with transforming activity was that the mutated proteins accumulated to higher levels in the cytosol than did wild-type β-cat. As suggested previously, this characteristic is most probably due to the inability of APC and GSK3β to regulate N-terminally mutated forms of β-cat. Wild-type β-cat fails to transform RK3E even when overexpressed, because cells with wild-type APC and GSK3β function can appropriately regulate the abundance of the wild-type β-cat protein. Our observation that the S33Y mutant form of β-cat accumulated to higher levels in the cytosol in transient-transfection assays and was more active in the focus formation assay than were the N-terminally truncated forms is consistent with the fact that β-cat proteins with single-amino-acid substitutions and single-amino-acid deletions in the GSK3β consensus are more frequently found in colon cancer than are proteins with larger N-terminal truncations (25
). Nonetheless, after expansion of the β-cat-transformed foci into stable cell lines, regardless of whether transformation of the line was initiated by the S33Y mutant β-cat protein or an N-terminal truncation (e.g., ΔN47 or ΔN132), the stably transformed RK3E lines expressed high levels of the mutant β-cat protein and displayed essentially uniform growth and tumorigenicity properties. These findings are consistent with the notion that other secondary genetic and epigenetic changes collaborate with the mutated β-cat proteins to induce the full neoplastic phenotype in RK3E. The reduced transcriptional and focus-forming activities of the N-terminally truncated forms of β-cat compared to the S33Y mutant may be of consequence only in initiation of RK3E neoplastic transformation, not in its maintenance.
Studies of the β-cat domains required for the transforming activity of the S33Y mutant protein revealed that armadillo repeats 3 to 8 (aa 218 to 467) and the C-terminal 85 aa were particularly critical, although deletion of N-terminal aa 48 to 217 also had a clear effect on the activity of the S33Y mutant protein. The requirement of armadillo repeats 3 to 8 implies that interaction of mutated β-cat with Tcf/Lef transcription factors is required for transformation. The C-terminal 85 aa of β-cat have previously been implicated in transcriptional activation (13
), as have sequences at the N terminus (13
), indicating that β-cat transformation is probably dependent on the transcriptional activation of Tcf/Lef-regulated genes. Other data support the view that activation of Tcf/Lef transcription is critical in β-cat-induced transformation. First, all cell lines stably transformed by mutated β-cat proteins displayed markedly elevated Tcf transcription activity, ranging from 30- to 700-fold higher than that of parental RK3E or K-Ras-transformed RK3E. Second, the polyclonal RK3E/Tcf-4ΔN31 line with stable expression of a dominant negative Tcf-4 mutant protein was resistant to transformation by mutated β-cat but could be transformed by activated K-Ras. Finally, recent studies indicated that a chimeric protein in which Lef-1 sequences are fused to potent transcriptional activation domains will transform chicken embryo fibroblasts (3
The identity of Tcf/Lef-regulated genes in mammalian cells, particularly of genes responsible for neoplastic transformation, is poorly understood. Recently, He et al. (12
) found that c-MYC
expression was suppressed in a colorectal cancer cell line with an endogenous APC
gene defect, following induction of an exogenous APC
gene. The critical elements in the c-MYC
promoter responsible for APC
-mediated suppression included Tcf-4 binding sites. Wild-type APC suppressed the activity of heterologous reporter gene constructs containing the Tcf-4 regulatory elements from the c-MYC
promoter, and mutated β-cat strongly activated gene expression from constructs containing the regulatory elements. Thus, the data of He et al. (13
) imply that c-MYC
is a critical downstream target of the APC/β-cat/Tcf pathway in cancer cells.
Our studies failed to provide evidence that c-myc was a critical target in β-cat-mediated neoplastic transformation of RK3E. In transient-transfection assays in RK3E, we found that mutated β-cat proteins could modestly increase c-myc gene expression, although the time course of c-myc activation was delayed relative to accumulation of mutant β-cat proteins and Tcf activation. More significantly, roughly half of the RK3E lines stably transformed by mutant β-cat had no detectable increase in c-myc gene expression relative to control RK3E lines, even though all β-cat-transformed lines had markedly elevated, constitutive Tcf transcription activity. Expression of c-myc was increased, relative to that in parental RK3E or RK3E/Kras cells, in several transformed lines with the highest Tcf transcriptional activity. Nevertheless, a clear correlation between Tcf transcriptional activity and c-myc expression was not observed in the β-cat-transformed lines. As such, it is possible that increased c-myc expression in some RK3E lines simply reflects the transformed phenotype. Additional data on the role of c-myc in β-cat transformation was obtained in studies in which a polyclonal RK3E line with stable expression of a dominant negative c-Myc mutant protein (i.e., RK3E/MycΔ106–143) could still be transformed by mutated β-cat proteins. The presence of the E1A protein in the RK3E cells may have substituted for c-Myc in neoplastic transformation by β-cat. However, while E1A might substitute for the functional requirement for c-Myc in β-cat transformation of RK3E, if the c-myc gene were truly a direct transcriptional target of the Tcf/β-cat complex, then we should have observed uniformly elevated c-myc gene expression levels in all of the β-cat-transformed RK3E lines. Because we failed to obtain such results, our findings imply that c-myc is not a common downstream target of the APC/β-cat/Tcf pathway in all neoplastic cells. The studies described here illustrate the value and utility of the RK3E system for evaluation of effector proteins and candidate target genes in β-cat transformation of epithelial cells. Future research with the RK3E system should offer additional insights into the means through which defects in β-cat regulation contribute to cancer.