The role of PKD2 in HSP90,- hypoxia- and TGF beta-mediated epithelial-to-mesenchymal transition and metastasis

该提案的重点是研究蛋白激酶D2 s对上皮-间质转化（EMT）的贡献，EMT是一个与肿瘤转移有关的过程，由缺氧，转录因子或生长因子等多种因素引发。在第二个提案中详细描述的研究将涉及致力于研究EMT的细致的细胞模型。在多孔支持物和/或3D胶原凝胶上培养的肿瘤细胞的详细形态学分析将与各种生物化学、免疫荧光和免疫组织化学分析相证实。体外实验将通过CAM上的人肿瘤异种移植物以及裸鼠中异种移植肿瘤上的EMT蛋白标志物分析来证实，这些已经从先前的项目中获得。此外，PKD 2对乳腺癌细胞转移潜力的体内贡献将在免疫缺陷无胸腺小鼠的新实验中描述。评估PKD 2在EMT过程中的作用的调查将有助于扩大我们对激酶在肿瘤发生的其他方面的需求的认识，迄今为止这种激酶还不清楚。拟议的工作计划将带来深入了解PKD 2参与EMT启动和维持TGF-β，HSP 90伴侣和缺氧。确定PKD 2是否通过与控制肿瘤血管生成相同的分子机制（例如通过HIF-1 α）调节EMT，或者该激酶是否涉及有利于上皮癌细胞向间充质表型转变的其他潜在信号传导途径，将代表扩展建议的中心焦点。几个微阵列评估PKD 2参与几个关键分子（NF-κ B B靶向，但不仅如此）的调节，这些分子被报道发出诱导EMT的信号，以及计划的对经历EMT的缺氧癌细胞的基于质谱的相互作用组将不仅服务于当前的工作计划，而且还将有助于该提议之外的研究。最后，使用HSP 90抑制剂PU-H71（目前在临床开发中）将促进我们的发现在靶向PKD 2稳定性的直接治疗方法中的可想象的用途，特别是在缺氧的人类肿瘤中。

The proposal focuses on the investigation of protein kinase D2s contribution to epithelial-to-mesenchymal transition (EMT), a process implicated in tumor metastasis and initiated by multiple factors such as hypoxia, transcription factors or growth factors. The investigations detailed in the second proposal of the running grant will engage meticulous cellular models dedicated to studying EMT. Detailed morphologic analysis of tumor cells cultivated on porous support and/or 3D collagen gels will be corroborated with various biochemical, immunofluorescence and immunohistochemical analysis. The in vitro experiments will be substantiated by human tumor xenograft on CAM as well as analysis of EMT protein markers on tumors xenografted in nude mice, already available from the previous project. Furthermore, the in vivo contribution of PKD2 to the metastatic potential of mammary cancer cells will be delineated in new experiments with immunodeficient athymic mice. Investigations assessing the role of PKD2 during EMT will contribute to the broadening of our knowledge with respect to the requirement of kinase for other aspects of tumorigenesis, unknown so far for this kinase. The proposed work program will bring insight to participation of PKD2 to EMT initiated and maintained by TGF-beta, HSP90 chaperone and hypoxia. Answering whether PKD2 regulates EMT via the same molecular mechanism/s that govern tumor angiogenesis (e.g. via HIF-1 alpha), or whether the kinase is implicated in other potential signaling pathways that favor the transition of epithelial cancer cells to a mesenchymal phenotype will represent a central focus of the extension proposal. Several microarrays assessing PKD2 involvement in the regulation of several crucial molecules (targeted by NF- kappa B, but not only) reported to emanate signals to induce EMT as well as the planned Mass spectrometry-based interactome on hypoxic cancer cells that undergo EMT will not only serve the current work program, but will also help investigations beyond this proposal. Finally, the use of HSP90-inhibitor PU-H71 (currently in clinical development) will promote our findings for a conceivable use in an immediate therapeutic approach targeting PKD2 stability in particularly hypoxic human tumors.