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D2s在上皮-间质转化（EMT）中的作用，EMT是一个涉及肿瘤转移的过程，由缺氧、转录因子或生长因子等多种因素启动。在运行拨款的第二个提案中详细的调查将涉及细致的细胞模型，致力于研究EMT。在多孔支架和/或3D胶原凝胶上培养的肿瘤细胞的详细形态学分析将通过各种生化，免疫荧光和免疫组织化学分析得到证实。体外实验将通过在CAM上的人类肿瘤异种移植以及在裸鼠上移植的肿瘤上的EMT蛋白标记物的分析来证实，这些已经从之前的项目中得到了证实。此外，PKD2在体内对乳腺癌细胞转移潜能的贡献将在免疫缺陷胸腺小鼠的新实验中描述。评估PKD2在EMT过程中的作用的研究将有助于拓宽我们对肿瘤发生其他方面对激酶的需求的认识，这是迄今为止未知的。提出的工作计划将深入了解PKD2参与由tgf - β、HSP90伴侣和缺氧启动和维持的EMT。回答PKD2是否通过控制肿瘤血管生成的相同分子机制（例如通过HIF-1 α）调节EMT，或者该激酶是否涉及其他有利于上皮癌细胞向间充质表型转变的潜在信号通路，将代表扩展提案的中心焦点。一些微阵列评估PKD2参与调节几个关键分子（NF- kappa B靶向，但不仅如此），据报道，这些分子发出信号诱导EMT，以及计划中的基于质谱的相互作用组在缺氧癌细胞上进行EMT，这不仅服务于当前的工作计划，而且还将有助于这项提议之外的研究。最后，hsp90抑制剂PU-H71（目前处于临床开发阶段）的使用将促进我们的研究结果，可用于针对PKD2稳定性的即时治疗方法，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.