Overexpressed LEF-1 proteins display different nuclear localization patterns of beta-catenin in normal versus tumor cells

Beta-catenin not only plays a role in cadherin-dependent cell adhesion, but also interacts with T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF-1) to affect gene expression. In this report, we describe the effects of exogenous LEF-1 and of treatment with leptomycin B (LMB), a specific inhibitor of CRM1-medicated nuclear export, on the nuclear localization and export of beta-catenin. Normal epithelial cells overexpressing LEF-1 accumulate nuclear beta-catenin in a LEF-1 concentration-dependent manner. Nuclear beta-catenin, once imported from the cytoplasm, is rapidly removed from the nucleus. Treatment with LMB results in dramatic retention of nuclear beta-catenin in normal epithelial cells transfected with LEF-1, and this effect is intensified by treatment of N-Acetyl-leucyl-leucyl-norleucinal together with LMB. Colon carcinoma cells containing an adenomatous polyposis coli mutation retain significant amounts of LEF-1 induced nuclear beta-catenin considerably after the time-point when beta-catenin disappears from the nuclei of LEF-1 transfected normal epithelial cells. beta-Catenin binds directly to CRM1, and overexpression of CRM1 reduces nuclear beta-catenin-mediated transactivation function.     

Tyrosine phosphorylation translocates beta-catenin from cell-->cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.     

Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators

beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.     

Wnt activation and alternative promoter repression of LEF1 in colon cancer

Alternative promoters within the LEF1 locus produce polypeptides of opposing biological activities. Promoter 1 produces full-length LEF-1 protein, which recruits beta-catenin to Wnt target genes. Promoter 2 produces a truncated form that cannot interact with beta-catenin and instead suppresses Wnt regulation of target genes. Here we show that promoter 1 is aberrantly activated in colon cancers because it is a direct target of the Wnt pathway. T-cell factor (TCF)-beta-catenin complexes bind to Wnt response elements in exon 1 and dynamically regulate chromatin acetylation and promoter 1 activity. Promoter 2 is delimited to the intron 2/exon 3 boundary and, like promoter 1, is also directly regulated by TCF-beta-catenin complexes. Promoter 2 is nevertheless silent in colon cancer because an upstream repressor selectively targets the basal promoter leading to destabilized TCF-beta-catenin binding. We conclude that the biological outcome of aberrant LEF1 activation in colon cancer is directed by differential promoter activation and repression.