Proteolytic cleavage of E-cadherin and beta-catenin by calpain in prostate and mammary tumor cells.

Abstract

The adherens junction, which includes E-cadherin and beta-catenin, maintains inter-cellular adhesion between adjacent epithelial cells and also mediates intracellular signals. Regulation of components of the adherens junction is important in cell survival and cell death. This dissertation reports a novel mechanism of E-cadherin inactivation through calpain-mediated cleavage of E-cadherin and generation of a stable 100-kDa fragment (E-cad<super>100 </super>). This fragment was generated in prostate and mammary tumor cells through PKC activation or calcium influx and lacked the beta-catenin binding domain. Mutagenesis revealed a six amino-acid sequence in the E-cadherin cytoplasmic domain that was required for cleavage. E-cad<super>100</super> fused to GFP did not associate or co-localize with endogenous beta-catenin in LNCaP cells. Because PKC activation induced aggregation-dependent survival of LNCaP cells that was dependent on functional E-cadherin, we hypothesized that overexpression of beta-catenin-binding deficient E-cad<super>100</super> might play an inhibitory role. Overexpression of E-cad<super>100</super> downregulated endogenous E-cadherin and potentiated PKC-induced cell death. These results demonstrated that calpain cleaves E-cadherin during PKC-induced apoptosis and overexpression of E-cad<super>100</super> potentiates cell death. Proteolysis of E-cadherin and elevated m-calpain expression were observed in localized and metastatic prostate cancer specimens compared to benign prostate tissue. Further analysis also detected a 75-kDa beta-catenin fragment (beta-cat<super> 75</super>), which lacked the N-terminal regulatory domain, in metastatic prostate cancer tissue as well as several prostate and breast cancer cell lines. Calcium influx induced the calpain-dependent generation of beta-cat<super> 75</super> that accumulated in nuclear and cytosolic compartments. Overexpression of a similar N-terminally truncated beta-catenin mutant activated TCF-dependent transcription. It is proposed that the loss of the N-terminal regulatory domain imposes greater stability and oncogenicity on beta-cat<super>75</super>, based on previous studies which demonstrated that similar N-terminal truncation mutants of beta-catenin are highly stable and tumorigenic. These results elucidate a role for calpain in regulation of E-cadherin and beta-catenin in both cell death and cell survival pathways. This dissertation describes a novel, calpain-mediated mechanism for the inactivation of E-cadherin and activation of beta-catenin in prostate and mammary tumor cells.