Steve F. Bronk

Mcl-1 Mediates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Resistance in Human Cholangiocarcinoma Cells

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-xL, 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-xL, 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.

Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells.

Interleukin 6 (IL‐6) contributes to the pathogenesis of cholangiocarcinoma by upregulating myeloid cell leukemia‐1 (Mcl‐1), a key antiapoptotic Bcl‐2 family member protein. IL‐6 can alter gene transcription via Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signal cascade. We examined this cascade in IL‐6 regulation of Mcl‐1 transcription in human cholangiocarcinoma cell lines. STAT3 was constitutively activated (i.e., tyrosine‐phosphorylated) in cholangiocarcinoma cells but not in nonmalignant cholangiocytes. Treatment with anti–IL‐6 antisera or the JAK inhibitor AG490 or transfection with dominant negative STAT3 diminished Mcl‐1 messenger RNA and protein levels. Likewise, these attempts to interrupt the STAT3 cascade also reduced Mcl‐1 promoter activity. Site‐directed mutagenesis of a putative STAT3 consensus binding sequence decreased Mcl‐1 promoter activity. Chromatin immunoprecipitation analysis demonstrated a direct binding of STAT3 to the putative STAT3 binding sequences in the Mcl‐1 promoter. Downregulation of Mcl‐1 by AG490 sensitized the cells to apoptosis mediated by tumor necrosis factor–related apoptosis‐inducing ligand. In conclusion, we have directly demonstrated a STAT3 regulatory element in the Mcl‐1 promoter. Downregulation of Mcl‐1 transcription by inhibiting this cascade is a potential strategy for the treatment of this cancer.(HEPATOLOGY 2005;42:1329–1338.)

miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma

It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR‐25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR‐25 expression, suggesting Hedgehog signaling stimulates miR‐25 production. Functionally, miR‐25 was shown to protect cells against TNF‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis. Correspondingly, antagonism of miR‐25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor‐4 (DR4) as a potential novel miR‐25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. Conclusion: These data implicate elevated miR‐25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR‐25 target DR4 provides a mechanism by which miR‐25 contributes to evasion of TRAIL‐induced cholangiocarcinoma apoptosis. (HEPATOLOGY 2012)

Tumor Necrosis Factor-related Apoptosis-inducing Ligand Activates a Lysosomal Pathway of Apoptosis That Is Regulated by Bcl-2 Proteins

The present studies were performed to determine whether lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity and to reconcile a role for lysosomes with prior observations that Bcl-2 family members regulate TRAIL-induced apoptosis. In KMCH cholangiocarcinoma cells stably expressing Mcl-1 small interference RNA (siRNA), treatment with TRAIL induced a redistribution of the cathepsin B from lysosomes to the cytosol. Pharmacological and small hairpin RNA-targeted inhibition of cathepsin B attenuated TRAIL-mediated apoptosis as assessed by morphological, biochemical, and clonogenic assays. Neither Bid siRNA nor Bak siRNA prevented cathepsin B release. In contrast, treatment of the cells with Bim siRNA or the JNK inhibitor SP600125 attenuated lysosomal permeabilization and cell death. Moreover, Bim and active Bax co-localized to lysosomes in TRAIL-treated cells in a JNK-dependent manner, and Bax siRNA reduced TRAIL-induced lysosomal permeabilization and cell death. Finally, BH3 domain peptides permeabilized isolated lysosomes in the presence of Bax. Collectively, these data suggest that TRAIL can trigger an apoptotic pathway that involves JNK-dependent activation of Bim, which in turn induces Bax-mediated permeabilization of lysosomes.

A Smac Mimetic Reduces TNF Related Apoptosis Inducing Ligand (TRAIL)-Induced Invasion and Metastasis of Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria‐derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584‐mediated loss of cellular inhibitor of apoptosis‐1 (cIAP‐1) and cIAP‐2, TRAIL failed to induce apoptosis in KMCH‐1, TFK‐1, and BDEneu CCA cells; a finding consistent with a downstream block in death signaling. Because cIAP‐1 and cIAP‐2 also promote nuclear factor kappa B (NF‐κB) activation by the canonical pathway, the effect of JP1584 on this signaling pathway was examined. Treatment with JP1584 inhibited TRAIL‐induced NF‐κB activation as well as TRAIL‐mediated up‐regulation of the NF‐κB target gene, matrix metalloproteinase 7 (MMP7). JP1584 also reduced TRAIL‐mediated CCA cell migration and invasion in vitro. Finally, in a syngeneic rat orthotopic CCA model, JP1584 administration reduced MMP7 messenger RNA levels and extrahepatic metastases.

A Hedgehog Survival Pathway in ‘Undead’ Lipotoxic Hepatocytes

BACKGROUND & AIMS Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro. METHODS Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3(-/-) mice. RESULTS Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3(-/-) cells during lipotoxic stress. CONCLUSIONS Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Protein-induced Lysosomal Translocation of Proapoptotic Effectors Is Mediated by Phosphofurin Acidic Cluster Sorting Protein-2 (PACS-2)

Background: Lysosome membrane permeabilization is required for TRAIL-induced apoptosis in liver cells. Results: TRAIL induces recruitment of PACS-2 to lysosomes where it interacts with Bim and Bax to permeabilize lysosomes and trigger apoptosis. Conclusion: The PACS-2, Bim, and Bax-containing PIXosome is a proapoptotic Bcl-2 scaffold. Significance: Understanding TRAIL-induced lysosome permeabilization may lead to new treatments for liver diseases. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.

BH3-only protein mimetic obatoclax sensitizes cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis.

Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer. [Mol Cancer Ther 2008;7(8):2339–47]

Hedgehog Inhibition Promotes a Switch from Type II to Type I Cell Death Receptor Signaling in Cancer Cells

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.

High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.