Stabilization Of Pim-3by TCTP, Prevents Cell Apoptosis And Promotes Human Pancreatic Cancer Progression

[Background] Pim-3is a member of the proto-oncogene Pim family that expresses serine/threonine kinase activity. Previously study demonstrated that the Pim-3was aberrantly expressed in cancer cells but not in the normal cells of the pancreas. Pim-3can inactivate Bad by phosphorylating Bad at Ser112and maintain the expression of Bcl-XL and thus prevent apoptosis of human pancreatic cancer cells. Ablated Pim-3protein by RNA interference (Pim-3shRNA), the amount of phospho-Ser112Bad was markedly reduced, which resulted in a higher ratio of sub-Gl cell populations and cell cycle retardation, thereby led to reduced cell numbers in the cell line cultures. These observations imply that Pim-3might be a novel target for the treatment of refractory pancreatic cancer. However, the mechanism of Pim-3in pancreatic tumorigenesis is not clearly.[Purpose] Considering the critical role of Pim-3in tumor development and progression, defining regulatory mechanisms of Pim-3signaling networks is important. In order to identify potential novel regulators of Pim-3, we performed yeast two-hybrid screening, we observed that TCTP specifically interacts with Pim-3. In this study, we further investigate the relationship between TCTP and Pim-3and determine the role of the interaction between TCTP and Pim-3in pancreatic cancer development and its molecular mechanism. This finding will provide the theoretical basis for clinical diagnosis and prognosis assessment of human pancreatic cancer.[Methods] Using the yeast two-hybrid to screen the Pim-3interacting protein; co-immunoprecipitation assay was carried out to validate the specific interaction; a double-immunofluorescence analysis to detect TCTP and Pim-3co-localization; Western immunoblot analysis was performed to detect gene expresson in cell line; immunohistochemistry to detect the protein expression in pancreatic cancer sample; realtime-PCR to detect the mRNA expression; Cycloheximide pulse-chase experiments detect the protein degradation speeds. Cell Counting kit8(CCK-8) was used to evaluate cell proliferation in vitro. Cell cycle and cell apoptosis in vitro was analyzed by flow cytometry (FCM). Nude mouse xenograft assay were used to assess the tumorigenic ability of the stable cells in vivo. The χ2test was used to compare clinicopathologic characteristics of148patients with TCTP and Pim-3expression. Correlation between TCTP and Pim-3expression was evaluated with Spearmen correlation analysis.[Results]1. We identified that Pim-3interacted with TCTP protein by using yeast two-hybrid screening and co-immunoprecipitation assay.2. TCTP was enhanced expression and positively correlated with Pim-3in human pancreatic cancer cell lines and cancer tissues.3. Pim-3has no effect on the TCTP expression or phosphorylationlevel.4. TCTP enhanced the protein stability of Pim-3by blocking the Pim-3ubiquitin-proteasome degradation.5. Ablation of endogenous TCTP protein inhibits tumor growth in vitro and in vivo by arresting cell cycle progression and enhancing apoptosis.[Conclusion] We revealed, for the first time, TCTP enhances Pim-3stability by blocking the Pim-3ubiquitin-proteasome degradation, and thus, kinase activity to promote cell cycle progression and prevent apoptosis, in turn to promote human pancreatic cancer progression, which provided a new idea and experimental evidence for recognizing TCTP/Pim-3pathway as a target of human pancreatic cancer.