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E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832?M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF-LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF-LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a ß-catenin-dependent activation of the WNT pathway.Oncogene (2003) 22, 5716-5719. doi:10.1038/sj.onc.1206672 [ABSTRACT FROM AUTHOR]

Recent reports suggest that the ß-catenin-T-cell factor (Tcf) (BCT) signaling pathway is important in the progression of prostate cancer. Evidence suggests that the androgen receptor (AR) can repress BCT-mediated transcription both in prostate cancer and colon cancer cells (Chesire and Isaacs, 2002). In this study, we validate such findings and show that repression of BCT signaling is facilitated by competition between the AR and Tcf. Measurements of the Tcf transcriptional reporter (TOPFLASH) indicated that AR+DHT-mediated repression can inhibit BCT transcription in the presence of WT and exogenous activating ß-catenin (?1-130?bp). Transient transfections in SW480 cells (APCmut/mut) showed that this mode of repression is functionally independent of APC-mediated ß-catenin ubiquitination. Using a recently developed red flourescent protein (HcRed), we demonstrate novel observations about the nuclear distribution of Tcf. Furthermore, with the use of red (HcRed-AR and HcRed-Tcf) and green fusion proteins (ß-catenin-EGFP), we provide morphological evidence of a reciprocal balance of nuclear ß-catenin-EGFP (BC-EGFP). By cotransfecting in LNCaP prostate tumor cells and using quantitative imaging software, we demonstrated a 62.0% colocalization of HcRed-AR and BC-EGFP in the presence of DHT and 63.3% colocalization of HcRed-Tcf/BC-EGFP in the absence of DHT. Costaining for activated RNA Pol II (phosphoserine 2) and HcRed-Tcf suggested that Tcf foci contain transcriptional 'hotspots' validating that these sites have the capacity for transcriptional activity. Given this apparent androgen-dependent competition for nuclear BC-EGFP, we chose to assess our hypothesis by in vivo and in vitro binding assays. SW480 cells transiently transfected with an AR expression construct, treated with DHT and immunoprecipitated for Tcf showed less associated ß-catenin when compared to Tcf precipitates from untreated cells. Furthermore, by treating cells with DHT+Casodex, we were able to abrogate the androgen-sensitive AR/ß-catenin interaction, in addition to relieving transcriptional repression of the TOPFLASH reporter. In vitro binding assays, with increasing amounts of ARS35, resulted in decreased TcfS35 association with immunoprecipitated recombinant ß-catenin-HIS. These data suggest that in steady-state conditions, AR has the ability to compete out Tcf binding for ß-catenin. Finally, using SW480 cells, we show that AR-mediated repression of the BCT pathway has implications for cell cycle progression and in vitro growth. Using FACs analysis, we observed a 26.1% increase in accumulation of cells in the G1?phase of the cell cycle, while in vitro growth assays showed a 35% reduction in viable cells transfected with AR+DHT treatment. Together, our data strongly suggest that a reciprocal balance of nuclear ß-catenin facilitates AR-mediated repression of BCT-driven transcription and cell growth.Oncogene (2003) 22, 5602-5613. doi:10.1038/sj.onc.1206802 [ABSTRACT FROM AUTHOR]