alvocidib and Cholangiocarcinoma

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Bile Duct Neoplasms; Carcinogenesis; Cell Line, Tumor; Cholangiocarcinoma; Chromatin; Cyclin-Dependent Kinase 9; DNA Polymerase II; Enhancer Elements, Genetic; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Mediator Complex; Mice; Mice, Transgenic; Phosphoproteins; Piperidines; Protein Binding; Signal Transduction; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Cholangiocarcinoma; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Flavonoids; Genes, bcl-2; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Piperidines; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha