Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC₅₀ of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.     

Prognostic value of neuron-specific enolase in patients with advanced and metastatic non-neuroendocrine non-small cell lung cancer

Background: Increased serum neuron-specific enolase (NSE) level was found in a substantial proportion (30–69%) of patients with non-small-cell lung cancer (NSCLC), but little was known about the clinical properties of NSE in NSCLC.Objective: We aimed to assess the level of serum NSE to predict prognosis and treatment response in patients with advanced or metastatic non-neuroendocrine NSCLC.Methods: We retrospectively analyzed 363 patients with advanced and metastatic NSCLC between January 2011 and October 2016. The serum NSE level was measured before initiation of treatment.Results: Patients with high NSE level (≥26.1 ng/ml) showed significantly shorter progression-free survival (PFS) (5.69 vs 8.09 months; P=0.02) and significantly shorter overall survival (OS) than patients with low NSE level (11.41 vs 24.31 months; P=0.01).NSE level was an independent prognostic factor for short PFS (univariate analysis, hazard ratio [HR] = 2.40 (1.71–3.38), P<0.001; multivariate analysis, [HR] = 1.81 (1.28–2.56), P=0.001) and OS (univariate analysis, [HR] = 2.40 (1.71–3.37), P<0.001; multivariate analysis, [HR] = 1.76 (1.24–2.50), P=0.002).Conclusion: The survival of NSCLC patients with high serum NSE level was shorter than that of NSCLC patients with low serum NSE levels. Serum NSE level was a predictor of treatment response and an independent prognostic factor.     

Histone Deacetylase-1 (HDAC1) Is a Molecular Switch between Neuronal Survival and Death

Both neuroprotective and neurotoxic roles have previously been described for histone deacetylase-1 (HDAC1). Here we report that HDAC1 expression is elevated in vulnerable brain regions of two mouse models of neurodegeneration, the R6/2 model of Huntington disease and the Ca²⁺/calmodulin-dependent protein kinase (CaMK)/p25 double-transgenic model of tauopathic degeneration, suggesting a role in promoting neuronal death. Indeed, elevating HDAC1 expression by ectopic expression promotes the death of otherwise healthy cerebellar granule neurons and cortical neurons in culture. The neurotoxic effect of HDAC1 requires interaction and cooperation with HDAC3, which has previously been shown to selectively induce the death of neurons. HDAC1-HDAC3 interaction is greatly elevated under conditions of neurodegeneration both in vitro and in vivo. Furthermore, the knockdown of HDAC3 suppresses HDAC1-induced neurotoxicity, and the knockdown of HDAC1 suppresses HDAC3 neurotoxicity. As described previously for HDAC3, the neurotoxic effect of HDAC1 is inhibited by treatment with IGF-1, the expression of Akt, or the inhibition of glycogen synthase kinase 3β (GSK3β). In addition to HDAC3, HDAC1 has been shown to interact with histone deacetylase-related protein (HDRP), a truncated form of HDAC9, whose expression is down-regulated during neuronal death. In contrast to HDAC3, the interaction between HDRP and HDAC1 protects neurons from death, an effect involving acquisition of the deacetylase activity of HDAC1 by HDRP. We find that elevated HDRP inhibits HDAC1-HDAC3 interaction and prevents the neurotoxic effect of either of these two proteins. Together, our results suggest that HDAC1 is a molecular switch between neuronal survival and death. Its interaction with HDRP promotes neuronal survival, whereas interaction with HDAC3 results in neuronal death.     

HDAC isoenzyme expression is deregulated in chronic lymphocytic leukemia B-cells and has a complex prognostic significance

Histone deacetylases (HDACs) play a crucial role in chromatin structure and, consequently, gene expression. Their deregulation has been reported in various cancers. We performed a complete and comprehensive study of the expression of 18 HDACs (including Sirtuin; SIRT) by real-time PCR in a cohort of 200 chronic lymphocytic leukemia (CLL) patients with a median follow-up of 77 mo, and compared it with the results obtained from normal B cells. We also compared HDAC expression at diagnosis and after relapse. We observed significant deregulation (mostly upregulation) of HDACs in CLL. In terms of clinical significance, only HDAC6 was significantly correlated with treatment-free survival (TFS), whereas HDAC3 and SIRT2, 3 and 6 were correlated with overall survival (OS). A multivariate Cox regression stepwise analysis indicated that HDAC6, 7 and 10 and SIRT3 were TFS independent predictors. Interestingly, poor prognosis was associated with an overexpression of HDAC7 and 10 but an underexpression of HDAC6 and SIRT3. Therefore, these factors were combined in a TFS score: patients with a score of 0–1–2, 3 and 4 had a median TFS of 107, 57 and 26 mo, respectively (HR = 4.03, p < 0.0001). For OS, SIRT5 and 6 allowed stratification into 3 groups, with a median OS of > 360, 237 and 94 mo (HR = 6.38, p < 0.0001). However, we could not find statistical differences in HDAC expression after relapse. These results, validated by a 5-fold cross-validation, highlight the complex impact of HDAC expression in CLL clinical course.     

Expression and Immune Responses to MAGE Antigens Predict Survival in Epithelial Ovarian Cancer

The MAGE cancer-testis antigens (CTA) are attractive candidates for immunotherapy. The aim of this study was to determine the frequency of expression, humoral immunity and prognostic significance of MAGE CTA in human epithelial ovarian cancer (EOC). mRNA or protein expression frequencies were determined for MAGE-A1, -A3, -A4, -A10 and -C1 (CT7) in tissue samples obtained from 400 patients with EOC. The presence of autologous antibodies against the MAGE antigens was determined from 285 serum samples. The relationships between MAGE expression, humoral immunity to MAGE antigens, and clinico-pathologic characteristics were studied. The individual frequencies of expression were as follows: A1: 15% (42/281), A3: 36% (131/390), A4: 47% (186/399), A10: 52% (204/395), C1: 16% (42/267). Strong concordant expression was noted with MAGE-A1:–A4, MAGE-A1:–C1 and MAGE-A4:–A10 (p<0.0005). Expression of MAGE-A1 or -A10 antigens resulted in poor progression free survival (PFS) (OR 1.44, CI 1.01–2.04, p = 0.044 and OR 1.3, CI 1.03–1.64, p = 0.03, respectively); whereas, MAGE-C1 expression was associated with improved PFS (OR 0.62, CI 0.42–0.92, p = 0.016). The improved PFS observed for MAGE-C1 expression, was diminished by co-expression of MAGE-A1 or -A10. Spontaneous humoral immunity to the MAGE antigens was present in 9% (27/285) of patients, and this predicted poor overall survival (log-rank test p = 0.0137). These findings indicate that MAGE-A1, MAGE-A4, MAGE-A3, and MAGE-A10 are priority attractive targets for polyvalent immunotherapy in ovarian cancer patients.     

Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC₅₀ of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.     

Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement

The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons.     

High Bone Sialoprotein (BSP) Expression Correlates with Increased Tumor Grade and Predicts a Poorer Prognosis of High-Grade Glioma Patients

Objectives

To investigate the expression and prognostic value of bone sialoprotein (BSP) in glioma patients.

Methods

We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model.

Results

Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001). Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS) and overall survival (OS) in both grade III and grade IV glioma patients [hazard ratio (HR) = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O⁶-methylguanine (O⁶-meG) DNA methyltransferase (MGMT) methylation and Karnofsky performance score (KPS) less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients.

Conclusion

High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.     

Preclinical anti-arthritic study and pharmacokinetic properties of a potent histone deacetylase inhibitor MPT0G009

The pathology of rheumatoid arthritis includes synoviocyte proliferation and inflammatory mediator expression, which may result from dysregulated epigenetic control by histone deacetylase (HDAC). Thus, HDAC inhibitors may be useful for treating inflammatory disease. This was a preclinical study of the HDAC inhibitor, MPT0G009. The IC₅₀ values of MPT0G009 for HDAC1, 2, 3, 6 and 8 enzymatic activities were significantly lower than those for the currently marketed HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat). In addition, MPT0G009 markedly inhibited cytokine secretion and macrophage colony-stimulating factor/receptor activator of nuclear factor kappa B ligand-induced osteoclastogenesis by macrophages (50 ng/ml each). These MPT0G009 effects on cytokine secretion and osteoclast formation were reduced by the overexpression of HDAC 1 (class I HDAC) and 6 (class II HDAC) in cells, suggesting that these effects were due to the inhibition of its activity. In an in vivo rat model, oral administration of MPT0G009 (25 mg/kg) significantly inhibited paw swelling and bone destruction. Furthermore, compared with SAHA, MPT0G009 exhibited longer half-life (9.53 h for oral administration) and higher oral bioavailability (13%) in rats. These results established the preclinical anti-arthritic efficacy and pharmacokinetic parameters of MPT0G009, which may provide a new therapeutic approach for treating inflammatory arthritis.     

Prognostic Significance of the Metabolic Marker Hexokinase-2 in Various Solid Tumors: A Meta-Analysis

ObjectiveRecently, numerous studies have reported that hexokinase-2 (HK2) is aberrantly expressed in cancer, indicating that HK2 plays a pivotal role in the development and progression of cancer. However, its prognostic significance in solid tumor remains unclear. Accordingly, we performed a meta-analysis to assess the prognostic value of HK2 in solid tumor.MethodsEligible studies were identified using PubMed, Embase, and Web of Science databases. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) or progression-free survival (PFS)/disease-free survival (DFS)/relapse-free survival (RFS) were estimated with random effects or fixed effects models, respectively. Subgroup analysis was also performed according to patients’ ethnicities, tumor types, detection methods, and analysis types.ResultsData from 21 included studies with 2532 patients were summarized. HK2 overexpression was significantly associated with worse OS (pooled HR = 1.90, 95% CI = 1.51–2.38, p < 0.001) and PFS (pooled HR = 2.91, 95% CI = 2.02–4.22, p < 0.001) in solid tumor. As to a specific form of cancer, the negative effect of HK2 on OS was observed in hepatocellular carcinoma (pooled HR = 2.06, 95% CI = 1.67–2.54, p < 0.001), gastric cancer (pooled HR = 1.72, 95% CI = 1.09–2.71, p = 0.020), colorectal cancer (pooled HR = 2.89, 95% CI = 1.62–5.16, p < 0.001), but not in pancreatic cancer (pooled HR = 1.13, 95% CI = 0.28–4.66, p = 0.864). No publication bias was found in the included studies for OS (Begg’s test, p = 0.325; Egger’s test, p = 0.441).ConclusionIn this meta-analysis, we identified that elevated HK2 expression was significantly associated with shorter OS and PFS in patients with solid tumor, but the association varies according to cancer type.     

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.     

“Ciliophagy”: The consumption of cilia components by autophagy

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/−, map1lc3b^−/−, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/−, map1lc3b^−/−, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

Identification of histone deacetylase 3 as a biomarker for tumor recurrence following liver transplantation in HBV-associated hepatocellular carcinoma

Background

Recent studies have shown that high expression levels of class I histone deacetylases (HDACs) correlate with malignant phenotype and poor prognosis in some human tumors. However, the expression patterns and prognostic role of class I HDAC isoforms in hepatocellular carcinoma (HCC) remain unclear.

Methodology/Principal Findings

The expression patterns and clinical significance of class I HDAC isoforms were assessed by immunohistochemistry in a cohort of 43 hepatitis B virus-associated HCC patients treated with liver transplantation. In addition, the effects of HDAC inhibition on HCC cell behavior were investigated by knockdown of the HDAC isoform with short interfering RNA. Class I HDACs were highly expressed in a subset of HCCs with positivity for HDAC1 in 51.2%, HDAC2 in 48.8%, and HDAC3 in 32.6% of cases. The expression levels of HDAC isoforms were significantly associated with the proliferation index of HCC. Kaplan-Meier curves showed that a high expression level of HDAC2 or HDAC3 implicated significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed HDAC3 overexpression was an unfavorable independent prognostic factor (P = 0.002; HR 3.907). In vitro, inhibition of HDAC2 and HDAC3, but not HDAC1, suppressed proliferation and the invasiveness of liver cancer cells.

Conclusions/Significance

Our findings demonstrate that HDAC3 plays a significant role in regulating tumor cell proliferation and invasion, and it could be served as a candidate biomarker for predicting the recurrence of hepatitis B virus-associated HCC following liver transplantation and a potential therapeutic target.