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

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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.