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

胆管细胞癌(CCC)是一种恶性程度高、治疗效果差的肿瘤，起源于肝内外胆管。体内的CCC细胞矛盾地表达死亡配体肿瘤坏死因子相关的凋亡诱导配体(TRAIL)及其同源死亡受体，使其依赖强大的生存信号来规避TRAIL对细胞的死亡。由CCC细胞表达的一个众所周知的生存因子是抗凋亡B细胞淋巴瘤-2(Bcl2)家族蛋白髓系细胞白血病-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细胞的主要生存途径。与这一概念一致，申请人首次产生了初步数据，证明细胞周期调节Polo-like Kinase(PLK)蛋白家族的成员可能在CCC细胞中HH生存信号和Mcl-1表达之间发挥重要联系。此外，PLK1和PLK2蛋白在人的CCC样本中大量表达。这些初步数据导致了这一假设的中心假设，即通过PLK介导的Mcl-1正向调控的HH信号绕过了CCC细胞中TRAIL的细胞毒性。这项提案的具体目标将检验三个假设。首先，将检验HH信号调节CCC细胞中PLK蛋白的假设：a)通过调节PLK1/2的表达；b)通过将刺激性转录因子胶质瘤相关癌基因(GLI)1/2/3直接结合到PLK1/2基因的启动子区域。第二，通过抑制PLK/HH信号诱导CCC细胞死亡：a)通过使CCC细胞对TRAIL诱导的细胞凋亡敏感；b)通过促进Mcl-1的蛋白酶体降解。最后，将检验这一假设，即抑制PLK/HH在CCC啮齿动物模型中具有治疗作用：a)通过下调Mcl-1表达导致CCC细胞凋亡增加；以及b)通过导致肿瘤进展失败而改善动物存活率。为了解决这些假设，申请人已经熟练地在CCC细胞中使用TRAIL和Hedgehog信号，并与埃森大学医院合作，实现了CCC的同基因、原位、啮齿动物模型。这项提议在概念上是创新的，因为它可能确定CCC细胞生存信号的新机制，并有助于开发这种毁灭性疾病的特定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.