The role of polo-like kinases1/2 as mediators of hedgehog survival signaling in cholangiocarcinoma cells

胆管癌是一种起源于肝内外胆管的高度恶性且难治的肿瘤。CCC细胞在体内矛盾地表达死亡配体肿瘤坏死因子相关凋亡诱导配体（TRAIL）及其同源死亡受体，使得这种癌症依赖于有效的存活信号来规避TRAIL引起的细胞死亡。由CCC细胞表达的众所周知的存活因子是抗凋亡B细胞-淋巴瘤-2（Bcl-2）家族蛋白髓样细胞白血病-1（Mcl-1）。也有越来越多的证据表明，重新激活的发育刺猬（Hh）信号通路是CCC细胞中的主要存活通路。与这一概念一致，申请人已经产生了初步数据，首次证明细胞周期调节polo样激酶（PLK）蛋白家族的成员可能在CCC细胞中作为Hh存活信号传导和Mcl-1表达之间的重要联系。此外，PLK 1和PLK 2蛋白在人CCC样品中大量表达。这些初步的数据导致了该提议的中心假设，即Hh信号通过PLK介导的Mcl-1的正调控规避CCC细胞中的TRAIL细胞毒性。本提案的具体目标将检验三个假设。首先，将检验Hh信号传导调节CCC细胞中PLK蛋白的假设：a）通过调节PLK 1/2的表达; B）通过刺猬转录因子胶质瘤相关癌基因（GLI）1/2/3与PLK 1/2基因的启动子区的直接结合。其次，将检验PLK/Hh信号传导的抑制诱导CCC细胞死亡的假设：a）通过使CCC细胞对TRAIL诱导的细胞凋亡敏感;和B）通过促进Mcl-1的蛋白酶体降解。最后，将检验PLK/Hh抑制在CCC的啮齿动物模型中是治疗性的假设：a）通过下调Mcl-1表达导致CCC细胞凋亡增加;和B）通过导致肿瘤进展失败并提高动物存活率。为了解决这些假设，申请人已经熟练于CCC细胞中的TRAIL以及hedgehog信号传导，并且合作地在埃森大学医院实施了CCC的同基因、原位啮齿动物模型。该提案在概念上具有创新性，因为它可能会识别CCC细胞存活信号的新机制，并有助于制定这种毁灭性疾病的特异性治疗和化学预防策略。

Cholangiocarcinoma (CCC) is a highly malignant and poorly treatable neoplasm originating from the intra- and extrahepatic bile ducts. CCC cells in vivo paradoxically express the death ligand tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its cognate death receptors, making this cancer dependent upon potent survival signals to circumvent cell death by TRAIL. A well known survival factor expressed by CCC cells is the anti-apoptotic B cell-lymphoma-2 (Bcl-2) family protein myeloid cell leukemia-1 (Mcl-1). There is also rising evidence that the reactivated developmental hedgehog (Hh) signaling pathway is a major survival pathway in CCC cells. Consistent with this concept, the applicant has generated preliminary data demonstrating for the first time that members of the cell cycle regulatory polo-like kinase (PLK) protein family may function as an important link between Hh survival signaling and Mcl-1 expression in CCC cells. In addition, PLK1 and PLK2 proteins are abundantly expressed in human CCC samples. These preliminary data lead to the central hypothesis of this proposal that Hh signaling via PLK-mediated positive regulation of Mcl-1 circumvents TRAIL cytotoxicity in CCC cells. The specific aims of this proposal will test three hypotheses. First, the hypothesis will be tested that Hh signaling regulates PLK proteins in CCC cells: a) by modulating the expression of PLK1/2; b) by direct binding of the hedgehog transcription factors GLIoma-associated oncogene (GLI)1/2/3 to the promoter regions of the PLK1/2 genes. Second, the hypothesis will be tested that inhibition of PLK/Hh signaling induces CCC cell death: a) by sensitizing CCC cells to TRAIL-induced apoptosis; and b) by promoting proteasomal degradation of Mcl-1. Finally, the hypothesis will be tested that PLK/Hh inhibition is therapeutic in a rodent model of CCC: a) by downregulation of Mcl-1 expression leading to increased CCC cell apoptosis; and b) by resulting in failure of tumor progression with improved animal survival. To address these hypotheses, the applicant has become adept at TRAIL as well as hedgehog signaling in CCC cells, and, in cooperation, implemented a syngeneic, orthotopic, rodent model of CCC at the University Hospital of Essen. The proposal is conceptually innovative as it might identifiy new mechanisms for CCC cell survival signaling and help develop strategies for the specific treatment and chemoprevention of this devastating disease.