ErbB <sub> To 2 </ Sub> And Its Binding Protein Erbin Role And Molecular Mechanism Of TRAIL-mediated Apoptosis Of Breast Cancer Cells

Tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) is a member of the TNF superfamily with the ability to selectively induce apoptosis in a wide variety of transformed cell lines of diverse origin,but not in the most of normal cells. However,some cancer cell lines are resistant to TRAIL cytotoxicity.In this study, two breast cancer cell lines(MDA-MB-231 and MCF-7) were used to examine the molecular mechanism of different sensitivity to rsTRAIL.Erbin is a new member of LAP family,with 16 LRRs and one PDZ domain,firstly identified as a binding partner for ErbB₂.Via its domains,Erbin could bind many proteins in cytoplasm. More and more studies indicate that Erbin plays an important role not only in receptor localization or cell signaling but also in the establishment and the maintenance of cell to cell and cell to basement membrane adhesion.Therefore,further studies on the role of Erbin in cell signal transduction,especially its interaction with ErbB₂,is significant for comprehensive understanding of carcinogenesis and apoptosis-based cancer therapy.We showed in the present study that the expression of ErbB₂ and Erbin were not changed in MCF-7 cells treated with TRAIL,which is not sensitive to TRAIL cytotoxicity,while PKCδinhibitor,rottlerin,sensitized MCF-7 cells to TRAIL cyototoxicity and down-regulated the expression of both ErbB₂ and Erbin in the presence of TRAIL or not.However,the expression of ErbB₂ was down-regulated, but the expression of Erbin was not changed in MDA-MB-231 cells in the presence of TRAIL only,since the cells are sensitive to TRAIL cytotoxicity.Further study demonstrated that the down-regulation of ErbB₂,but not Erbin,was arrested by the inhibitor of caspase-8,Z-IETD-FMK,in both MCF-7 and MDA-MB-231 cells treated with TRAIL and/or rottlerin,suggesting that caspase-8 down-regulated the expression level of ErbB₂ induced by TRAIL.Downstream signal study showed that NF-κB activity was inhibited by rsTRAIL in MDA-MB-231 cells and enhanced in MCF-7 cells,suggesting that NF-κB activity might be a determine factor for cell apoptosis and proliferation.These data indicated that TRAIL inhibited the proliferation signal such as NF-κB and ErbB₂,and activated the apoptosis signals,so the balance trended to apoptosis in MDA-MB-231 cells,while high activity of PKCδand NF-κB in MCF-7 cells induced by rsTRAIL enabled the cells insensitive to TRAIL cytotoxicity, and therefore,the combination of rottlerin and rsTRAIL inhibited the proliferation signal pathway and induced cell apoptosis.Furthermore,we demonstrated the suppression of Erbin expression by using RNAi technology facilitates the sensitivity of the cells to TRAIL,the expression of ErbB₂ was down-regulated,and ubiquitination of ErbB₂ was increased in the MCF-7 cells. Moreover,AKT signal pathway and NF-κB activation,downstream of ErbB₂,was also inhibited in the MCF-7 cells,in which the expression of Erbin was declined by siRNA technology,suggestiong that Erbin could sustain the stability of ErbB₂ by suppression ErbB₂ ubiquitination and it might be one of the reasons that MCF-7 cells are resistant to TRAIL cytotoxicity.In summary,we have reported for the first time that Erbin could regulate ErbB₂ expression by suppressing ErbB₂ ubiquitination.These results are significant implication for understanding the resistant mechanism to TRAIL in MCF-7 cells and the mechanism of apoptosis-induced by TRAIL.