Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer.

Autophagy is a homeostatic, catabolic degradation process and cell fate essential regulatory mechanism. Protracted autophagy triggers cell death; its aberrant function is responsible for several malignancies. Panobinostat, a potent pan-deacetylase inhibitor, causes endoplasmic reticulum stress-induced cell death. The aim of this study was to investigate the role of autophagy in deacetylase inhibitor-triggered liver cancer cell death.HepG2 (p53wt) and Hep3B (p53 null) liver cancer cell lines were exposed to panobinostat. RT-qPCR and western blot confirmed autophagic factor modulation. Immuno-fluorescence, -precipitation and -histochemistry as well as transmission electron microscopy verified autophagosome formation. The cytotoxicity of panobinostat and autophagy modulators was detected using a real time cell viability assay.Panobinostat induced autophagy-related factor expression and aggregation. Map1LC3B and Beclin1 were significantly over-expressed in HepG2 xenografts in nude mice treated with panobinostat for 4 weeks. Subcellular distribution of Beclin1 increased with the appearance of autophagosomes-like aggregates. Cytosolic loss of p53, in HepG2, and p73, in Hep3B cells, and a corresponding gain of their nuclear level, together with modulation of DRAM1, were observed. Autophagosome aggregation was visible after 6 h of treatment. Treatment of cells stably expressing GFP-RFPtag Map1LC3B resulted in aggregation and a fluorescence switch, thus confirming autophagosome formation and maturation. Tamoxifen, an inducer of autophagy, caused only a block in cell proliferation; but in combination with panobinostat it resulted in cell death.Autophagy triggers cell demise in liver cancer. Its modulation by the combination of tamoxifen and panobinostat could be a new option for palliative treatment of hepatocellular carcinoma.

Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells.

HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1-/- HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV- HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection.

The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

Post-translational modifications of chromatin components are significantly involved in the regulation of tumor suppressor gene and oncogene expression. Connective tissue growth factor (CTGF) is an epigenetically regulated growth factor with functions in angiogenesis and cell-matrix interactions and plays a pivotal role in hepatocellular carcinoma (HCC). The pharmacologic inhibition of histone and protein deacetylases represents a new approach to interfere with pathways of apoptosis and angiogenesis. We investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on human HCC cell lines HepG2 (p53wt) and Hep3B (p53null) and in a subcutaneous xenograft model and explored the influence on angiogenesis. Specimens were characterized by quantitative real-time PCR. Protein was separated for western blotting against CTGF, VEGF, VEGF receptor-1 (VEGFR-1/FLT-1), VEGF receptor-2 (VEGFR-2/KDR), MAPK and phospho-MAPK. In vivo, HepG2 cells were xenografted to NMRI mice and treated with daily i.p. injections of 10 mg/kg panobinostat. After 1, 7 and 28 days, real-time PCR was performed. Immunohistochemistry and western blotting were examined after 28 days. An increased significant expression of CTGF was only seen after 24 h treatment with 0.1 µM panobinostat in HepG2 cells and Hep3B cells, whereas after 72 h treatment CTGF expression clearly decreased. In the xenografts, treatment with panobinostat showed a minimal CTGF expression after 1 day and 4 weeks, respectively. In vitro as well as in vivo, VEGF was not affected by panobinostat treatment at any time. In conclusion, panobinostat influences extracellular signaling cascades via CTGF-dependent pathways.

The pan-deacetylase inhibitor panobinostat suppresses the expression of oncogenic miRNAs in hepatocellular carcinoma cell lines.

Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa-miR-19a, hsa-miR-19b1 and the corresponding precursors were down-regulated by panobinostat in TP53(-/-) Hep3B and TP53(+/+) HepG2 cell lines; hsa-miR30a-5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT-qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance-based real-time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti-cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets.

Pathological impact of hepatitis B virus surface proteins on the liver is associated with the host genetic background.

BACKGROUND: While the immune pathogenesis caused by hepatitis B virus (HBV) infection has been studied extensively, little is known about direct pathogenic effects of HBV surface proteins. Here, we have investigated pathological cellular effects of HBV surface protein expression in the liver of transgenic mice with different genetic background. METHODS: The impact of HBV surface protein expression on the liver was studied in two mouse strains, BALB/c and C57BL/6. Histology and hydroxyproline assays were performed to investigate liver morphology and fibrosis. Gene expression and signaling were analyzed by microarray, qPCR and Western blotting. RESULTS: Expression of HBV surface proteins in the liver of transgenic mice induced activation of protein kinase-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation. Phosphorylation of eIF2α resulted in activation of the ER stress markers glucose regulated protein (GRP) 78 and pro-apoptotic C/EBP homologous protein (CHOP) in transgenic mice on BALB/c genetic background leading to stronger liver injury and fibrosis in comparison with transgenic mice on C57BL/6 background. Hepatic stellate cells represented the main collagen-producing liver cells in HBV transgenic mice. The key regulators of hepatocyte proliferation, transcription factors c-Jun and STAT3 were activated in HBV transgenic mice. Tumour incidence in transgenic mice was strain- and sex-dependent. CONCLUSIONS: Extent of liver injury, fibrosis, and tumour development induced by hepatic HBV surface protein expression considerably depends on host genetic background.

Endoplasmic reticulum stress plays a pivotal role in cell death mediated by the pan-deacetylase inhibitor panobinostat in human hepatocellular cancer cells.

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

The pan-deacetylase inhibitor panobinostat modulates the expression of epithelial-mesenchymal transition markers in hepatocellular carcinoma models.

Deacetylase inhibitors (DACis) represent a novel therapeutic option for human cancers by classically affecting proliferation or apoptosis. Since transdifferentiation and dedifferentiation play a key role in carcinogenesis, we investigated the epigenetic influence on the molecular differentiation status in human hepatocellular carcinoma (HCC) models. Markers of differentiation, including cytokeratin (Ck) 7, Ck8, Ck18, Ck19, Ck20, vimentin, sonic hedgehog homolog (SHH), smoothened (Smo), patched (Ptc), glioma-associated oncogene homolog 1 (Gli1), CD133, octamer-binding transcription factor 4 (Oct4) and ß-catenin, were examined in the human HCC cell lines HepG2 and Hep3B in vitro and in vivo (xenograft model) using quantitative real-time PCR and immunohistochemistry following treatment with the pan-DACi panobinostat (LBH589). Compared to untreated controls, treated HepG2 xenografts, and to a lesser extent cell lines, demonstrated a significant increase of differentiation markers Ck7 and Ck19 (classical cholangiocellular type) and Ck8 and Ck18 (classical HCC type), and a decreased level of dedifferentiation markers vimentin (mesenchymal) and SHH/Ptc (embryonic), paralleled with a more membranous expression of ß-catenin. These findings were dose-dependently correlated with tumor size, necrosis rate, microvessel density and mitosis/Ki-67-associated proliferation rate. Our results demonstrate that the differentiation status of human HCC cells is influenced by the pan-DACi panobinostat, indicating that this treatment may influence the epithelial-mesenchymal transition (EMT) status related to metastasis and aggressiveness.

Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition.

Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 µM gemcitabine. Surviving cells were collected after 1, 3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, ß-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by wound­healing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3­fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and ß-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC(50) from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proli-feration in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.

Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines.

Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate.

Selective induction of apoptosis by HMG-CoA reductase inhibitors in hepatoma cells and dependence on p53 expression.

HMG-CoA-reductase inhibitors (statins) are widely used drugs to interfere with cholesterol biosynthesis. Besides this usage, evidence is increasing that statins might also be useful in therapy of viral infections or cancer. We investigated the effects of fluva-, simva-, atorva-, rosuva- and lovastatin on the viability of primary mouse and human hepatocytes as well as mouse (Hepa1-6) and human (Huh7, HepG2) hepatoma cell lines. Our results show selective cytotoxic effects of fluva-, simva- and lovastatin on hepatoma cells in comparison to primary hepatocytes. Using human hepatoma cells we found significant reduction of cell viability and induction of apoptosis in HepG2 cells, while statins did not affect Huh7 cells at concentrations not toxic for primary hepatocytes. Stable knockdown of endogenous p53, which is overexpressed in Huh7 cells, was able to restore susceptibility of Huh7 cells towards statin-induced toxicity. The anti-tumor effect of statins did not depend on a lack of cholesterol production, but was restored by supplementation of mevalonate or geranyl-geranyl pyrophosphate, prerequisites for prenylation of small G proteins. In conclusion, statins display a selective apoptotic effect on human hepatoma cells, with fluva-, simva- and lovastatin being both, most selective for tumor cells and most effective in inducing tumor cell apoptosis. Additionally, our results implicate that anti-tumor activity of statins requires cell proliferation and is reduced by p53 overexpression.

New drugs, old fashioned ways: ER stress induced cell death.

Discovery of small molecules able to induce several cellular self-killing mechanisms improved cancer therapy in the last years. Research focused on canonical apoptotic (mitochondria or death receptor related) pathways to induce cell death in several hematologic and solid malignancies, showing that treatment with different synthetic and natural compounds reactivates the cell death machinery previously silenced in resistant cancer cells. Besides the canonical apoptotic pathways, alternative pathways of cell death induction have recently been rediscovered as potential new targets for cancer therapy. Under certain conditions, protein folding can be disturbed causing an accumulation of unfolded proteins inside the endoplasmic reticulum (ER). This situation leads to stress ER, involving the transcriptional and translational machinery to induce the expression and post-transcriptional modifications of many factors involved in ER stress response mediated cell death. In this scenario, some apoptotic players like caspase 4 or caspase 12 start to control cell fate by inducing downstream cell death proteins. Recently, inhibitors of protein deacetylases have been demonstrated to potently induce this alternative cell death pathway and will be reviewed here.

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC.

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1-3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

The pan-deacetylase inhibitor panobinostat inhibits growth of hepatocellular carcinoma models by alternative pathways of apoptosis.

Inhibition of deacetylases represents a new treatment option for human cancer diseases. We applied the novel and potent pan-deacetylase inhibitor panobinostat (LBH589) to human hepatocellular carcinoma models and investigated by which pathways tumor cell survival is influenced. HepG2 (p53wt) and Hep3B (p53null) responded to panobinostat treatment with a reduction of cell proliferation and a significant increase in apoptotic cell death at low micromolar concentrations. Apoptosis was neither mediated by the extrinsic nor the intrinsic pathway but quantitative RT-PCR showed an upregulation of CHOP, a marker of the unfolded protein response and endoplasmic reticulum stress with subsequent activation of caspase 12. Dependent on the p53 status, a transcriptional upregulation of p21(cip1/waf1), an increased phosphorylation of H2AX, and an activation of the MAPK pathway were observed. In a subcutaneous xenograft model, daily i.p. injections of 10 mg/kg panobinostat lead to a significant growth delay with prolonged overall survival, mediated by reduced tumor cell proliferation, increased apoptosis and reduced angiogenesis in tumor xenografts. Panobinostat increased the acetylation of histones H3 and H4. Panobinostat is a well tolerated new treatment option for HCC that activates alternative pathways of apoptosis, also in p53-deficient tumors.