Erbin Influence Cancer Cell Proliferation Through Feedback Activation Of Akt-Skp2-p27 Signaling

BackgroundErbin(ErbB2 interacting protein) is a new combination of ErbB2 protein, was discovered in 2000 and named accordingly. Erbin belongs to LAP the new protein family member whose structure includes:a PDZ domain in C-terminal and 16 series LRR of the N-terminal domain. Erbin’s position is that through ErbB2 and c-Terminal PDZ domains specifically binding guide ErbB2 positioned in the basement membrane, maintenance of cellular polarity. Erbin as scaffold protein develops its function through its own domain interacts with other protein domains. Erbin and β-catenin interactions and come into the cytoskeleton network, participating in formation of adhesion of epithelial cells and desmosomes. The study found some maintenance of epithelial cell polarity of molecules involved in cell cycle regulation.In addition, Erbin connector protein, negative regulation of TGF-β/Smads and Ras/Raf-1/ERK as well as Nod2/NF-KB signaling pathways in a given condition. Thus, Erbin is a "multiple roles" of a polar molecule. So far,there is no commercialization of specific antibodies of Erbin. Erbin was preparation and purification of antibody early in our lab. Knockdown Erbin through downregulation p21 and p27 promote tumor cell proliferation and migration. We then discovered that Erbin can regulate the function of Akt-Skp2 pathway. These clues strongly suggest, Erbin may serve as an important regulatory factor in cancer related gene function. But how Erbin regulates Skp2 mediated cell proliferation, migration and tumor related gene function.There is interaction between Skp2 and the S phase kinase CyclinA-CDKI, by protein-protein interaction module, F-box series, "Linker" series which is connected in turn(such as leucine repeat domain). Research shows that the regulatory mechanism of Skp2 plays a dominant role in different kinds of cells in different, its dual regulation of expression by transcriptional and post transcriptional level.Regulate the expression of Skp2 and increase the stability of p27 inhibit the proliferation of cells. Study in prostate cancer, expression of Skp2 and tumor suppressor gene PTEN related protein, thus regulation of the tumor suppressor gene PTEN in expression of Skp2. The PTEN gene in human cancers is often losing, through inhibiting Akt signaling play a role of tumor suppressor gene. Therefore, due to the functional defect of PTEN gene inactivation or caused Akt signaling termination control tumors, Skp2 protein may be an effective therapeutic target. Skp2 closely related to the cell cycle regulation and development and prognosis of tumor, these are all through the multiple target protein of ubiquitin degradation.In the recent study, our results show the Skp2 is E3 ubiquitin ligase of Erbin. This suggests that possible interactions between Erbin and Skp2 and Skp2-mediated cell proliferation, migration and related genes function may depend on Erbin. ObjectiveExplore Erbin influence cancer cell proliferation through feedback activation of Akt-Skp2-p27 signaling Methods(1)in different experimental conditions to detect expression of Akt-Skp2-p27 signaling pathway in a variety of endogenous or exogenous protein in a variety of cells by Western Blot.(2)in different experimental conditions, the detection of Erbin, Skp2 positioning in the cell changes in HeLa cells by IF.(3) effect of silencing Erbin on Skp2 phosphorylation and acetylation.Whether there is interaction between Erbin and Skp2 by CO-IP.(4) the expression of Erbin and Skp2 in tumor tissues was detected in the Immunohistochemistry.(5)detection of cell proliferation or migration in HeLa cells by ATP,EDU, Colony formation, scratch test.(6) detection the proliferation of cancer cells in the body after Erbin lost by the establishment of a xenograft model. Results(1) downregulation of Erbin through p27 degradation promotes the stability of endogenous and exogenous Skp2. Akt-Skp2 feedback signal to regulate the expression of Erbin by Western Blot.(2) Erbin mainly located in the cell membrane and the junction of adjacent cells. Knockdown Erbin enhanced the stability of Skp2 protein, but did not affect the localization of Skp2.Akt activation induced by Skp2 increased cytoplasmic localization was not observed by IF.(3)knockdown Erbin promote Skp2 phosphorylation and acetylation; Erbin interact with Skp2 by CO-IP.(4)in breast cancer tissue, Erbin and negatively correlated with the expression of Skp2 by Immunohistochemistry.(5)Erbin lost promote cell proliferation.Erbin for regulating cell migration behavior depends on the Skp2 in HeLa cells by ATP,EDU, Colony formation, scratch test.(6) Erbin lost promotes cancer cell proliferation in the body by the establishment of a xenograft model. ConclusionThrough the study, we confirmed: Erbin expressed in a variety of epithelial tumors of imbalance. Erbin loss leads to cell cycle S accelerated, abnormal proliferation. To reveal the molecular mechanism of Erbin involved in the regulation of tumor cell G1/S conversion. Erbin loss promotes Akt-Skp2-p27 signal axis activation, the activation of Akt-Skp2 promotes the degradation of Erbin. Erbin is Skp2 degradation substrate and regulates Akt-dependent Skp2 protein stability and activity. In conclusion, this study reveals the negative regulation of Akt-Skp2-p27 molecule and suggested that Erbin in maintaining the new function of cell polarity.As a cell cycle of molecular brake, plays an important role in maintaining the normal progression of the cell cycle.