Matthew W. Lawless

NF-κB regulation: The nuclear response

•  Introduction •  Activation of NF‐κB •  Regulators of NF‐κB activation •  Nuclear regulation of NF‐κB ‐  B cell lymphoma 3 (Bcl‐3) ‐  IκB‐ζ ‐  UXT, ZAS3 and PDLIM2 •  Post‐translational modification of NF‐κB complex in the nucleus ‐  Phosphorylation ‐  Acetylation and Deacetylation ‐  Sumoylation •  Nuclear regulators of NF‐κB: potential molecular chaperones •  Conclusion

Oxidative stress induced lung cancer and COPD: opportunities for epigenetic therapy

•  Oxidative stress, epigenetics, NSCLC and COPD ‐  Cigarette smoke mediated cellular proliferation ‐  Cigarette smoke mediated lung destruction ‐  DNA methylation and lung cancer ‐  Histone post‐translational modifications and lung cancer •  Linking epigenetics and oxidative stress to the development of COPD and NSCLC ‐  HATs and KATs in the developing lung ‐  HDACs and COPD ‐  Aberrant KAT/HDAC activity in NSCLC ‐  ATP‐dependent SWI/SNF chromatin remodelling complexes and NSCLC ‐  KATs/HDACs and oxidative stress pathways ‐  KEAP1‐NRF2‐ARE signalling pathway •  Epigenetics, COPD, NSCLC and oxidative stress pathways/genes ‐  NRF2 ‐  Heme oxygenase 1 ‐  Hypoxia‐inducible factor‐1α ‐  PGC‐1α ‐  Oxidative stress, epigenetics, glucocorticoids and COPD ‐  Oxidative stress, acetylation and its role in the regulation of NFκB ‐  Targeting epigenetic mechanisms in COPD and NSCLC •  Theophylline – a novel agonist of HDACs ‐  Current clinical trials involving epigenetic therapies in COPD and NSCLC •  Dietary HDAC inhibitors as therapeutic agents to target ‐ COPD or NSCLC? •  Sulforaphane •  Curcumin •  Epigallocatechin 3‐gallate (EGCG) •  Can dietary epigenetic inhibitors work in the clinical setting? •  Caveats •  Conclusions

Tauroursodeoxycholic acid inhibits apoptosis induced by Z alpha-1 antitrypsin via inhibition of bad†

Z alpha‐1 antitrypsin (AAT) deficiency is a genetic disease associated with accumulation of misfolded AAT in the endoplasmic reticulum (ER) of hepatocytes. ZAAT‐expressing cells display ER stress responses including nuclear factor kappaB activation and apoptosis. Using an in vitro model of ZAAT ER accumulation, we investigated the mechanism of ZAAT‐mediated ER‐induced apoptosis and evaluated methods to inhibit this process. Here we demonstrate that expression of ZAAT, but not normal MAAT, in HEK293 cells leads to cleavage and activation of caspase‐4 and induces apoptosis that is characterized by activation of caspase‐3 and caspase‐7 and DNA fragmentation. Similar effects are also induced using the ER agonist thapsigargin. A caspase‐4–specific short interfering RNA (siRNA) does not impair ZAAT‐induced caspase‐3/7 activation or cell death in these cells. However, inhibition studies performed using tauroursodeoxycholic acid (TUDCA) demonstrate its ability to inhibit caspase‐4 and caspase‐3/7 activation, mitochondrial cytochrome c release, and caspase‐3 cleavage induced by ZAAT and to promote cell survival. The mechanism by which TUDCA (tauroursodeoxycholic acid) promotes cell survival in ZAAT‐expressing cells involves phosphorylation and inactivation of the proapoptotic factor Bad. TUDCA is unable to rescue cells from apoptosis or phosphorylate Bad in the presence of LY294002, a selective P‐I‐3‐kinase inhibitor. Conclusion: These data show that caspase‐4 is not essential for ZAAT‐induced apoptosis in HEK293 cells and implicates P‐I‐3‐kinase and Bad as potential therapeutic targets for the liver disease associated with ZAAT deficiency. (HEPATOLOGY 2007.)

Defective bone morphogenic protein signaling underlies hepcidin deficiency in HFE hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common inherited iron overload disorder. The vast majority of patients carry the missense Cys282Tyr mutation of the HFE gene. Hepcidin, the central regulator of iron homeostasis, is deficient in HH, leading to unchecked iron absorption and subsequent iron overload. The bone morphogenic protein (BMP)/small mothers against decapentaplegic (Smad) signaling cascade is central to the regulation of hepcidin. Recent data from HH mice models indicate that this pathway may be defective in the absence of the HFE protein. Hepatic BMP/Smad signaling has not been characterized in a human HFE‐HH cohort to date. Hepatic expression of BMP/Smad‐related genes was examined in 20 HFE‐HH males with significant iron overload, and compared to seven male HFE wild‐type controls using quantitative real‐time reverse transcription polymerase chain reaction. Hepatic expression of BMP6 was appropriately elevated in HFE‐HH compared to controls (P = 0.02), likely related to iron overload. Despite this, no increased expression of the BMP target genes hepcidin and Id1 was observed, and diminished phosphorylation of Smad1/Smad5/Smad8 protein relative to iron burden was found upon immunohistochemical analysis, suggesting that impaired BMP signaling occurs in HFE‐HH. Furthermore, Smad6 and Smad7, inhibitors of BMP signaling, were up‐regulated in HFE‐HH compared to controls (P = 0.001 and P = 0.018, respectively). Conclusion: New data arising from this study suggest that impaired BMP signaling underlies the hepcidin deficiency of HFE‐HH. Moreover, the inhibitory Smads, Smad6, and Smad7 are identified as potential disruptors of this signal and, hence, contributors to the pathogenesis of this disease. (HEPATOLOGY 2010;)

Targeting histone deacetylases for the treatment of disease

•  Introduction •  HATs/HDACs and the pro‐inflammatory environment •  HATs/HDACs and endoplasmic reticulum (ER) stress •  The potential role of histone deacetylase inhibitors in the treatment of cancer ‐  Non‐small cell cancer (NSCLC) ‐  In vitro evidence for targeting HDACs in NSCLC ‐  Combinatorial therapies involving HDi in NSCLC ‐  Hepatocellular carcinoma (HCC) ‐  In vitro evidence for the use of HDi in liver cancer ‐  Combinatorial treatments involving HDi in hepatoma •  Potential role of histone deacetylase inhibitors in the treatment of diabetes ‐  Disease models, knockouts and assays ‐  Pancreatic islet protection using histone deacetylase inhibitors ‐  Additional in vitro evidence ‐  Stem cells, HDACs and histone deacetylase inhibitors •  The potential role of histone deacetylase inhibitors in the treatment of neurodegenerative conditions ‐  Neuronal traits are modulated by HDAC/REST complexes ‐  E2F, HDACs and neuronal survival mechanisms ‐  HDACs play important roles in stem cell neuronal differentiation •  Histone deacetylase inhibitors ‐  SAHA (vorinostat) ‐  Phenylbutyrate ‐  Valproic acid ‐  AN‐9 ‐  CI‐994 ‐  Romidepsin ‐  MS‐275 ‐  LBH589 (panobinostat) ‐  MGC D0103 •  Caveats ‐  Do HDAC inhibitors target genes or help chaperone activity as their primary response? •  Final comments

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

BackgroundHereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.ResultsVector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.ConclusionOur novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.

Pegylated interferon‐α induced hypoferremia is associated with the immediate response to treatment in hepatitis C

Pegylated interferon‐α (PEG‐IFN‐α) forms an integral part of the current treatment for hepatitis C virus (HCV) infection. PEG‐IFN‐α suppresses HCV production by augmenting the innate antiviral immune response. Recent studies have reported the induction of hepcidin, the iron regulatory hormone, by IFN‐α in vitro. As hepcidin plays an important role in innate immunity, we hypothesized that this finding may be of clinical relevance to HCV and investigated the changes in iron homeostasis during the first 24 hours of treatment. Blood samples were obtained from HCV patients immediately prior to and 6, 12, and 24 hours following the first dose of PEG‐IFN‐α/ribavirin (RBV). Samples were analyzed for hepcidin, cytokine, iron levels, and HCV viral load, and hepcidin messenger RNA (mRNA) expression was quantified in peripheral blood mononuclear cells. Hepcidin induction by IFN‐α was further analyzed in cell culture. In HCV patients a single dose of PEG‐IFN‐α/RBV resulted in a significant increase in serum hepcidin, peaking at 12 hours, coinciding with a 50% reduction in serum iron and transferrin saturation over the 24‐hour period. Patients with a ≥2 log decline in HCV viral load over the first 24 hours had significantly lower SI and TS levels at 12 and 24 hours. Moreover, 24‐hour SI levels were an independent predictor of the immediate HCV viral decline, an indicator of ultimate treatment outcome. In cell culture, a direct induction of hepcidin by IFN‐α was seen, controlled by the STAT3 transcription factor. Conclusion: Hepcidin induction occurs following the initiation of PEG‐IFN‐α treatment for HCV, and is mediated by way of STAT3 signaling. The subsequent hypoferremia was greatest in those with the most significant decline in viral load, identifying systemic iron withdrawal as a marker of immediate interferon‐α efficacy in HCV patients. (HEPATOLOGY 2012)

Variant in CD209 promoter is associated with severity of liver disease in chronic hepatitis C virus infection

CD209, a c-type lectin expressed by dendritic cells (DCs), acts as a pathogen recognition receptor. A single nucleotide polymorphism (SNP) in the promoter region of CD209 (-336 A/G; rs4804803) affects transcription and is associated with the severity of tuberculosis and dengue fever. Because CD209 binds hepatitis C virus (HCV) glycoprotein-E2, we investigated this SNP in the context of chronic HCV infection. A total of 131 Irish women who had received HCV-contaminated anti-D-immunoglobulin and 79 healthy control subjects were genotyped. We found no association between rs4804803 and the risk of HCV chronicity. However, of those with chronic infection, possession of at least one g-allele was associated with more advanced liver disease, with significantly higher liver fibrosis scores and levels of alanine transaminase (ALT) observed. We conclude that rs4804803, an SNP in the CD209 promoter, contributes to severity of liver disease in chronic HCV infection.

MicroRNAs and liver cancer associated with iron overload: Therapeutic targets unravelled

Primary liver cancer is a global disease that is on the increase. Hepatocellular carcinoma (HCC) accounts for most primary liver cancers and has a notably low survival rate, largely attributable to late diagnosis, resistance to treatment, tumour recurrence and metastasis. MicroRNAs (miRNAs/miRs) are regulatory RNAs that modulate protein synthesis. miRNAs are involved in several biological and pathological processes including the development and progression of HCC. Given the poor outcomes with current HCC treatments, miRNAs represent an important new target for therapeutic intervention. Several studies have demonstrated their role in HCC development and progression. While many risk factors underlie the development of HCC, one process commonly altered is iron homeostasis. Iron overload occurs in several liver diseases associated with the development of HCC including Hepatitis C infection and the importance of miRNAs in iron homeostasis and hepatic iron overload is well characterised. Aberrant miRNA expression in hepatic fibrosis and injury response have been reported, as have dysregulated miRNA expression patterns affecting cell cycle progression, evasion of apoptosis, invasion and metastasis. In 2009, miR-26a delivery was shown to prevent HCC progression, highlighting its therapeutic potential. Several studies have since investigated the clinical potential of other miRNAs with one drug, Miravirsen, currently in phase II clinical trials. miRNAs also have potential as biomarkers for the diagnosis of HCC and to evaluate treatment efficacy. Ongoing studies and clinical trials suggest miRNA-based treatments and diagnostic methods will have novel clinical applications for HCC in the coming years, yielding improved HCC survival rates and patient outcomes.

Stop feeding cancer: pro-inflammatory role of visceral adiposity in liver cancer.

Liver cancer is the fifth most common cancer in the world with an estimated over half a million new cases diagnosed every year. Due to the difficulty in early diagnosis and lack of treatment options, the prevalence of liver cancer continues to climb with a 5-year survival rate of between 6% and 11%. Coinciding with the rise of liver cancer, the prevalence of obesity has rapidly increased over the past two decades. Evidence from epidemiological studies demonstrates a higher risk of hepatocellular carcinoma (HCC) in obese individuals. Obesity is recognised as a low-grade inflammatory disease, this is of particular relevance as inflammation has been proposed as the seventh hallmark of cancer development with abdominal visceral adiposity considered as an important source of pro-inflammatory stimuli. Emerging evidence points towards the direct role of visceral adipose tissue rather than generalised body fat in carcinogenesis. Cytokines such as IL-6 and TNF-α secreted from visceral adipose tissue have been demonstrated to induce a chronic inflammatory condition predisposing the liver to a protumourigenic milieu. This review focuses on excess visceral adiposity rather than simple obesity; particularly adipokines and their implications for chronic inflammation, lipid accumulation, insulin resistance, Endoplasmic Reticulum (ER) stress and angiogenesis. Evidence of molecular signalling pathways that may give rise to the onset and progression of HCC in this context are depicted. Delineation of the pro-inflammatory role of visceral adiposity in liver cancer and its targeting will provide better rational and therapeutic approaches for HCC prevention and elimination. The concept of a central role for metabolism in cancer is the culmination of an effort that began with one of the 20th centurys leading biochemists and Nobel laureate of 1931, Otto Warburg.