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.     

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

HDAC6 interacts with PTPN1 to enhance melanoma cells progression

Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity.     

Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6.PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6.PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC.phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.     

A regulatory circuit that involves HR23B and HDAC6 governs the biological response to HDAC inhibitors

Histone deacetylase (HDAC) is an emergent anticancer target, and HR23B is a biomarker for response to HDAC inhibitors. We show here that HR23B has impacts on two documented effects of HDAC inhibitors; HDAC inhibitors cause apoptosis in cells expressing high levels of HR23B, whereas in cells with low level expression, HDAC inhibitor treatment is frequently associated with autophagy. The mechanism responsible involves the interaction of HDAC6 with HR23B, which downregulates HR23B and thereby reduces the level of ubiquitinated substrates targeted to the proteasome, ultimately desensitising cells to apoptosis. Significantly, the ability of HDAC6 to downregulate HR23B occurs independently of its deacetylase activity. An analysis of the HDAC6 interactome identified HSP90 as a key effector of HDAC6 on HR23B levels. Our results define a regulatory mechanism that involves the interplay between HR23B and HDAC6 that influences the biological outcome of HDAC inhibitor treatment.     

2-Benzazolyl-4-Piperazin-1-Ylsulfonylbenzenecarbohydroxamic Acids as Novel Selective Histone Deacetylase-6 Inhibitors with Antiproliferative Activity

We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors, the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selectively inhibited HDAC6 (IC₅₀ = 0.1–1.0μM) over HDAC1 (IC₅₀ = 0.9–6μM) and moreover, also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells. The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is neglible as compared to controls. Molecular modeling studies uncovered that the MM-GBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing structural rationale for the HDAC6 selectivity.     

Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.     

组蛋白去乙酰化酶6(HDAC6)抑制剂分子水平高通量筛选模型的建立

旨在建立分子水平HDAC6小分子抑制剂的高通量筛选模型,用于新型HDAC6特异性小分子抑制剂的发现。建立HDAC6的昆虫表达系统,分离纯化HDAC6蛋白,利用底物Boc-Lys(Ac)-AMC对纯化的HDAC6进行测活,并对测活体系进行优化,以SAHA为阳性抑制剂,确定适合高通量筛选的酶及底物浓度,反应时间等。首先构建HDAC6昆虫真核细胞表达载体,转入昆虫细胞中表达,并利用GST亲和柱纯化获得较高纯度的GST-HDAC6融合蛋白;建立体外HDAC6分子测活方法,表明昆虫表达的GST-HDAC6融合蛋白具有去乙酰化酶活性,并通过对多种参数优化使得Z’因子达到0.60,表明分子水平的HDAC6小分子抑制剂高通量筛选体系成功建立。     

Depletion of HDAC6 Enhances Cisplatin-Induced DNA Damage and Apoptosis in Non-Small Cell Lung Cancer Cells

Histone deacetylase inhibitors (HDACi) are promising therapeutic agents which are currently used in combination with chemotherapeutic agents in clinical trials for cancer treatment including non-small cell lung cancer (NSCLC). However, the mechanisms underlying their anti-tumor activities remain elusive. Previous studies showed that inhibition of HDAC6 induces DNA damage and sensitizes transformed cells to anti-tumor agents such as etoposide and doxorubicin. Here, we showed that depletion of HDAC6 in two NSCLC cell lines, H292 and A549, sensitized cells to cisplatin, one of the first-line chemotherapeutic agents used to treat NSCLC. We suggested that depletion of HDAC6 increased cisplatin-induced cytotoxicity was due to the enhancement of apoptosis via activating ATR/Chk1 pathway. Furthermore, we showed that HDAC6 protein levels were positively correlated with cisplatin IC(50) in 15 NSCLC cell lines. Lastly, depletion of HDAC6 in H292 xenografts rendered decreased tumor weight and volume and exhibited increased basal apoptosis compared with the controls in a xenograft mouse model. In summary, our findings suggest that HDAC6 is positively associated with cisplatin resistance in NSCLC and reveal HDAC6 as a potential novel therapeutic target for platinum refractory NSCLC.     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

HDAC6 is required for invadopodia activity and invasion by breast tumor cells

Invasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that HDAC6 is required for two-dimensional matrix proteolysis. In addition, we demonstrate that HDAC6 acts as a tubulin and cortactin deacetylase. We also report that the inhibition of HDAC6 by siRNA or treatment with HDAC inhibitor TSA results in a decreased invasion capacity of a three-dimensional type I collagen matrix by MDA-MB-231 cells. These data identify HDAC6 as a critical component of the invasive apparatus of tumor cells, in both two- and three-dimensional matrices.     

Uropathogenic Escherichia coli invades host cells via an HDAC6-modulated microtubule-dependent pathway

Strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili that promote bacterial colonization and invasion of the bladder epithelium. Type 1 pilus-mediated interactions with host receptors, including alpha3beta1 integrin, trigger localized actin rearrangements that lead to internalization of adherent bacteria via a zipper-like mechanism. Here we report that type 1 pilus-mediated bacterial invasion of bladder cells also requires input from host microtubules and histone deacetylase 6 (HDAC6), a cytosolic enzyme that, by deacetylating alpha-tubulin, can alter the stability of microtubules along with the recruitment and directional trafficking of the kinesin-1 motor complex. We found that disruption of microtubules by nocodazole or vinblastine treatment, as well as microtubule stabilization by taxol, inhibited host cell invasion by UPEC, as did silencing of HDAC6 expression or pharmacological inhibition of HDAC6 activity. Invasion did not require two alternate HDAC6 substrates, Hsp90 and cortactin, but was dependent upon the kinesin-1 light chain KLC2 and an upstream activator of HDAC6, aurora A kinase. These results indicate that HDAC6 and microtubules act as vital regulatory elements during the invasion process, possibly via indirect effects on kinesin-1 and associated cargos.     

Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.     

Investigation of Decitabine Effects on HDAC3 and HDAC7 mRNA Expression in NALM-6 and HL-60 Cancer Cell Lines

Background:Decitabine is a potent anticancer hypomethylating agent and changes the gene expression through the gene''s promoter demethylation and also independently from DNA demethylation. So, the present study was designed to distinguish whether Decitabine, in addition to inhibitory effects on DNA methyltransferase, can change HDAC3 and HDAC7 mRNA expression in NALM-6 and HL-60 cancer cell lines.Methods:HL-60, NALM-6, and normal cells were cultured, and the Decitabine treatment dose was obtained (1 µM) through the MTT assay. Finally, HDAC3 and HDAC7 mRNA expression were measured by Real-Time PCR in HL-60 and NALM-6 cancerous cells before and after treatment. Furthermore, HDAC3 and HDAC7 mRNA expression in untreated HL-60 and NALM-6 cancerous cells were compared to normal cells.Results:Our results revealed that the expression of HDAC3 and HDAC7 in HL-60 and NALM-6 cells increases as compared to normal cells. After treatment of HL-60 and NALM-6 cells with Decitabine, HDAC3, and HDAC7 mRNA expression were decreased significantly.Conclusion:Our data confirmed that the effects of Decitabine are not limited to direct hypomethylation of DNMTs, but it can indirectly affect other epigenetic factors, such as HDACs activity, through converging pathways.Key Words: Decitabine, HDAC3, HDAC7, HL-60, NALM-6