Mott, Justin L.

Abstract Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor‐mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor‐induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. © 2013 American Physiological Society. Compr Physiol 3:977‐1010, 2013.

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary neoplasm with limited therapeutic options. Thus, new therapeutic strategies are needed to advance our treatment armamentarium for this devastating disease. MicroRNAs represent a new class of endogenous nucleic acids regulating development, cell death, and carcinogenesis. Recent studies have indicated that the miR-106b∼25 cluster, which is upregulated in cancer, has potential antiapoptotic properties. Meanwhile, we have demonstrated that Hedgehog (Hh) signaling is activated in CCA and also has a strong antiapoptotic effect. Based on this information, we posited that Hh signaling may regulate mir-106b∼25 expression. Due to the dependence of CCA on antiapoptotic signals, we conducted a study to determine the role of miR-106b∼25 in CCA apoptosis and unveil its regulatory mechanisms. Experimental procedures: Immortalized benign and malignant human cholangiocyte cell lines were used for the experiments. MicroRNA expression was analyzed using real-time quantitative RT-PCR (qRT-PCR) assays. The effect of the microRNAs was studied using Lipofectamine-based transfection with precursor microRNAs or locked-nucleic acid antagonists (LNAs) to respectively potentiate or decrease microRNAs expression. Functional apoptosis studies employed DAPI staining with fluorescent microscopic analysis of nuclear morphology, or biochemical measurement of caspase-3/7 activity. Western blotting and luciferase assays were conducted to identify potential messenger RNA targets. Results: Expression of miR-106b∼25 was enhanced in malignant vs. benign cholangiocyte cell lines. Treatment with the known antagonist of Hh signaling, cyclopamine, resulted in downregulation of the cluster. Because CCA cells express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in vivo, we next examined TRAIL-induced apoptosis in CCA cells. TRAIL-mediated apoptosis was attenuated by transfection with miR-25 and enhanced by LNAs. Computational analysis identified the TRAIL death receptor 4 (DR4) as a potential target of this cluster. Indeed, we observed downregulation of DR4 cellular protein levels during overexpression of miR-106b∼25 and upregulation of DR4 upon cluster inhibition with LNAs. The 3’untranslated region (UTR) of the DR4 mRNA was cloned into a luciferase reporter, and the potential direct regulation of DR4 by miR-106b∼25 examined. Indeed, the regulation of DR4 by miR-106b∼25 was confirmed by this luciferase assay demonstrating a direct inhibitory effect of the miR-106b∼25 polycistron on the DR4 3’UTR. Conclusions: The miR-106b∼25 cluster is overexpressed in malignant cholangiocyte cell lines, likely by a Hh signaling pathway. miR-106b∼25 inhibits apoptosis, at least in part, via downregulation of DR4. LNA targeted inhibition of miR-106b∼25 sensitizes CCA cells to apoptosis and may represent a new therapeutic strategy for this cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2011-199

Abstract Background and Aims: Cholangiocarcinomas (CCA) are highly desmoplastic tumors containing abundant cancer associated myofibroblasts (CAF). An inverse association between the abundance of CAF and survival has been demonstrated in CCA, suggesting that CAF play a pivotal role in CCA development and progression. The CAF phenotype represents an ‘activated cell state’ and as such may be uniquely susceptible to apoptosis. BH3 mimetics induce apoptosis in cells ‘primed’ for cell death, representing a therapeutic strategy for cancer. Herein we examine whether CAF display a selective sensitivity to the BH3 mimetic ABT-263 and whether ABT-263 induced ablation of CAF reduces tumor growth and progression. Methods: We employed human and rat CAF, myofibroblastic human LX2 cells, human and rat control fibroblasts, the rat cholangiocarcinoma cell line BDEneu and human CCA cell lines for these studies. In-vivo experiments were conducted using a syngeneic, orthotopic rat CCA model. Results: We observed a significant and dose dependent induction of apoptosis by ABT-263 in human and rat primary CAF as well as activated LX2 cells as compared to rat BDEneu and human CCA cell lines. Comparison of Bcl-2 protein expression in quiescent fibroblasts versus CAF demonstrated a consistent upregulation of Bax in the CAF, likely explaining their enhanced sensitivity to pro-death stimuli. Indeed, ABT-263 treatment of CAF was associated with Bax activation as compared to human CCA cell lines. Finally, administration of ABT-263 decreased tumor size and metastasis in a human-like in-vivo rat model of CCA. Analysis of the rat tumors revealed a decrease in Tenascin C positive tumor stroma and an increase of TUNEL-positive apoptotic stromal cells. Conclusions: CCA associated CAF appear to be ‘primed’ for cell death, possibly due to overexpression of the pro-apoptotic Bcl-2 protein Bax. Consequently CAF display increased sensitivity to the BH3 mimetic ABT-263, which selectively depletes them from a rat in-vivo model of CCA, thereby reducing tumor growth and metastasis. Thus targeting CCA tumor stroma by inducing apoptosis in CAF may represent a new therapeutic approach in human CCA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4905. doi:1538-7445.AM2012-4905

Abstract [Background and Aim] TRAIL is a promising therapeutic agent for human malignancies that can activate apoptosis via mitochondria-independent (Type I) or mitochodria-dependent (Type II) death receptor signaling. However, many malignant cells are resistant to TRAIL cytotoxicity and fail to trigger Type I or Type II killing. Using cholangiocarcinoma cells, we identified a role for Hedgehog signaling via GLI transcription factors in the regulation of TRAIL cytotoxicity, and provide new mechanistic insights whereby Hedghog inhibition increases apoptosis. [Materials and Methods] The human cholangiocarcinoma cell lines KMCH, HuCCT-1, and Mz-ChA-1 were employed. Hedgehog signaling was inhibited with cyclopamine or an shRNA construct targeting smootherned (SMO). Type II TRAIL signaling was inhibited with shRNA constructs for Bid or Bim and siRNA for Bax and Bak. Antiapoptotic mediators including Inhibitor of Apoptosis Proteins (IAPs) and Bcl-2 family proteins were examined by immunoblot analysis and/or real time RT-PCR. Apoptosis was quantified morphologically by DAPI and fluorescence microscopy, and biochemically by caspase-3/7 activity. GLI transcription factor binding to the XIAP promoter was examined by EMSA and chromatin immuneprecipitation (ChIP) assay. [Results] Pharmacologic or genetic inhibition of Hedgehog signaling down-regulates both XIAP and cIAP-1 mRNA and protein expression. Knockdown of XIAP, but not cIAP-1, by small-interfering RNA markedly sensitized cells to TRAIL-induced apoptosis, suggesting XIAP is the functional target. Putative GLI-binding sites were identified in the XIAP promoter, and EMSA and ChIP assays demonstrated direct binding by GLI proteins. Sensitization to TRAIL-induced apoptosis was not altered by knockdown of the BH3-only proteins Bim and Bid, or by knockdown of Bax plus Bak, implicating a mitochondria-independent Type I pathway of apoptosis. [Conclusion] These data provide evidence for up-regulation of XIAP by an oncogenic Hedgehog pathway and demonstrate that Hedgehog inhibition sensitizes cells to TRAIL via decreasing XIAP levels. Hedgehog inhibition thereby promotes Type I death receptor signaling and sensitizes cells to apoptosis, which suggests Hedgehog and XIAP inhibition may be a therapeutic approach for TRAIL-resistant neoplasms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2011-4105

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH 2 -terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.

Abstract 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]