Histone deacetylase 6-mediated downregulation of TMEM100 expedites the development and progression of non-small cell lung cancer.

The significance of epigenetic modulation, involving acetylation, methylation, as well as ubiquitination has been indicated in the regulation of gene expression and tumor progression. Here, we elucidated the role of histone deacetylase 6 (HDAC6) in regulating epithelial-mesenchymal transition (EMT)-mediated metastasis via mRNA in non-small cell lung cancer (NSCLC). Three microarrays associated with lung cancer metastasis or recurrence, GSE23361, GSE7880 and GSE162102, were downloaded from the GEO database. Transmembrane protein 100 (TMEM100) was revealed to be the only one mRNA that was significantly downregulated in three microarrays. TMEM100, poorly expressed in lung cancer tissues, was associated with poor prognosis of lung cancer patients. Moreover, TMEM100 transcription was regulated by HDAC6 which repressed TMEM100 expression by deacetylation modification on the TMEM100 promoter. Knockdown of HDAC6 or overexpression of TMEM100 in NSCLC cells significantly inhibited TGF-ß1-induced EMT and metastasis and suppressed the activation of Wnt/ß-catenin signaling pathway. Altogether, our study highlights HDAC6 as a lung cancer metastasis supporter through the suppression of TMEM100 and the induction of Wnt/ß-catenin signaling pathway.

The Role of HDAC6 in Autophagy and NLRP3 Inflammasome.

Autophagy fights against harmful stimuli and degrades cytosolic macromolecules, organelles, and intracellular pathogens. Autophagy dysfunction is associated with many diseases, including infectious and inflammatory diseases. Recent studies have identified the critical role of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasomes activation in the innate immune system, which mediates the secretion of proinflammatory cytokines IL-1ß/IL-18 and cleaves Gasdermin D to induce pyroptosis in response to pathogenic and sterile stimuli. Accumulating evidence has highlighted the crosstalk between autophagy and NLRP3 inflammasome in multifaceted ways to influence host defense and inflammation. However, the underlying mechanisms require further clarification. Histone deacetylase 6 (HDAC6) is a class IIb deacetylase among the 18 mammalian HDACs, which mainly localizes in the cytoplasm. It is involved in two functional deacetylase domains and a ubiquitin-binding zinc finger domain (ZnF-BUZ). Due to its unique structure, HDAC6 regulates various physiological processes, including autophagy and NLRP3 inflammasome, and may play a role in the crosstalk between them. In this review, we provide insight into the mechanisms by which HDAC6 regulates autophagy and NLRP3 inflammasome and we explored the possibility and challenges of HDAC6 in the crosstalk between autophagy and NLRP3 inflammasome. Finally, we discuss HDAC6 inhibitors as a potential therapeutic approach targeting either autophagy or NLRP3 inflammasome as an anti-inflammatory strategy, although further clarification is required regarding their crosstalk.

HDAC6 inhibitors sensitize non-mesenchymal triple-negative breast cancer cells to cysteine deprivation.

Triple-negative breast cancer (TNBC) is a highly malignant type of breast cancer and lacks effective therapy. Targeting cysteine-dependence is an emerging strategy to treat the mesenchymal TNBC. However, many TNBC cells are non-mesenchymal and unresponsive to cysteine deprivation. To overcome such resistance, three selective HDAC6 inhibitors (Tubacin, CAY10603, and Tubastatin A), identified by epigenetic compound library screening, can synergize with cysteine deprivation to induce cell death in the non-mesenchymal TNBC. Despite the efficacy of HDAC6 inhibitor, knockout of HDAC6 did not mimic the synthetic lethality induced by its inhibitors, indicating that HDAC6 is not the actual target of HDAC6 inhibitor in this context. Instead, transcriptomic profiling showed that tubacin triggers an extensive gene transcriptional program in combination with erastin, a cysteine transport blocker. Notably, the zinc-related gene response along with an increase of labile zinc was induced in cells by the combination treatment. The disturbance of zinc homeostasis was driven by PKCγ activation, which revealed that the PKCγ signaling pathway is required for HDAC6 inhibitor-mediated synthetic lethality. Overall, our study identifies a novel function of HDAC6 inhibitors that function as potent sensitizers of cysteine deprivation and are capable of abolishing cysteine-independence in non-mesenchymal TNBC.

Up-regulation of HDAC6 Results in Poor Prognosis and Chemoresistance in Patients With Advanced Ovarian High-grade Serous Carcinoma.

BACKGROUND: Ovarian high-grade serous carcinoma (HGSC) gradually acquires chemoresistance after recurrence. Our previous study on ovarian clear-cell carcinoma found histone deacetylase 6 (HDAC6) overexpression led to chemoresistance. This study aimed to evaluate HDAC6 as a predictor of chemoresistance and a therapeutic target for ovarian HGSC. PATIENTS AND METHODS: The clinical significance of HDAC6 as a predictor of prognosis and chemoresistance in HGSC was immunohistochemically evaluated. In addition, expression of programmed cell death ligand-1 (PD-L1), and hypoxia-inducible factor-1α (HIF1α) were analyzed using clinical samples from 88 patients with ovarian HGSC, and their clinicopathological characteristics were reviewed. RESULTS: Twenty-three patients had high HDAC6 expression, 10 positive PD-L1 expression, and 33 high HIF-1α expression. HDAC6 up-regulation was correlated with not undergoing interval debulking surgery (p<0.001), incomplete surgical resection (p=0.002), and frequent occurrence of stable disease/progressive disease according to the Response Evaluation Criteria in Solid Tumors (p=0.005) criteria. On Kaplan-Meier analysis, high HDAC6 expression was significantly associated with reduced progression-free (p=0.001) and overall (p=0.008) survival. On multivariate analysis, high HDAC6 expression (hazard ratio=1.65, 95% confidence interval 1.03-2.66; p=0.039) and surgery status were independent prognostic factors of progression-free survival. PD-L1 and HIF1α expression positively correlated with that of HDAC6. CONCLUSION: HDAC6 may become a potential therapeutic target in patients with ovarian HGSC since its up-regulation is considered to be associated with a poor prognosis in patients with this cancer.

SLAC2B-dependent microtubule acetylation regulates extracellular matrix-mediated intracellular TM4SF5 traffic to the plasma membranes.

Transmembrane 4 L six family member 5 (TM4SF5) translocates intracellularly and promotes cell migration, but how subcellular TM4SF5 traffic is regulated to guide cellular migration is unknown. We investigated the influences of the extracellular environment and intracellular signaling on the TM4SF5 traffic with regard to migration directionality. Cell adhesion to fibronectin (FN) but not poly-l-lysine enhanced the traffic velocity and straightness of the TM4SF5WT (but not palmitoylation-deficient mutant TM4SF5Pal- ) toward the leading edges, depending on tubulin acetylation. Acetylated-microtubules in SLAC2B-positive cells reached mostly the juxtanuclear regions, but reached-out toward the leading edges upon SLAC2B suppression. TM4SF5 expression caused SLAC2B not to be localized at the leading edges. TM4SF5 colocalization with HDAC6 depended on paxillin expression. The trimeric complex consisting of TM4SF5, HDAC6, and SLAC2B might, thus, be enriched at the perinuclear cytosols toward the leading edges. More TM4SF5WT translocation to the leading edges was possible when acetylated-microtubules reached the frontal edges following HDAC6 inhibition by paxillin presumably at new cell-FN adhesions, leading to persistent cell migration. Collectively, this study revealed that cell-FN adhesion and microtubule acetylation could control intracellular traffic of TM4SF5 vesicles to the leading edges via coordinated actions of paxillin, SLAC2B, and HDAC6, leading to TM4SF5-dependent cell migration.

HDAC6 in Diseases of Cognition and of Neurons.

Central nervous system (CNS) neurodegenerative diseases are characterized by faulty intracellular transport, cognition, and aggregate regulation. Traditionally, neuroprotection exerted by histone deacetylase (HDAC) inhibitors (HDACi) has been attributed to the ability of this drug class to promote histone acetylation. However, HDAC6 in the healthy CNS functions via distinct mechanisms, due largely to its cytoplasmic localization. Indeed, in healthy neurons, cytoplasmic HDAC6 regulates the acetylation of a variety of non-histone proteins that are linked to separate functions, i.e., intracellular transport, neurotransmitter release, and aggregate formation. These three HDAC6 activities could work independently or in synergy. Of particular interest, HDAC6 targets the synaptic protein Bruchpilot and neurotransmitter release. In pathological conditions, HDAC6 becomes abundant in the nucleus, with deleterious consequences for transcription regulation and synapses. Thus, HDAC6 plays a leading role in neuronal health or dysfunction. Here, we review recent findings and novel conclusions on the role of HDAC6 in neurodegeneration. Selective studies with pan-HDACi are also included. We propose that an early alteration of HDAC6 undermines synaptic transmission, while altering transport and aggregation, eventually leading to neurodegeneration.

Alleviation of depression-like behavior in a cystic fibrosis mouse model by Hdac6 depletion.

Cystic fibrosis (CF) patients experience heightened levels of anxiety and depression. Stress from dealing with chronic disease and rigorous treatment regimens certainly are primary contributors to these outcomes. We previously have demonstrated that microtubule alterations in CF are linked to a number of CF phenotypes including growth regulation and inflammatory responses to airway bacterial challenge. Deletion of histone deactelyase 6 (HDAC6), a cytosolic deacetylase that regulates tubulin acetylation, in CF mice restores growth and inflammatory phenotypes to wild type (WT) profiles. In this study, the hypothesis that Hdac6 depletion in CF mice would impact behaviors since Hda6 inhibition has been previously reported to have anti-depressive properties. Data demonstrate that CF mice exhibit reduced activity and reduced open arm time in an elevated plus maze test which can be consistent with anxiety-like behavior. CF mice also exhibit depression-like behaviors compared to WT mice in an age dependent manner. By eight weeks of age, CF mice exhibit significantly more immobile time in the tail-suspension test, however, Hdac6 depletion reverses the depressive phenotype. These data demonstrate that loss of CFTR function may predispose patients to experience depression and that this behavior is Hdac6 dependent.

Honokiol ameliorates angiotensin II-induced hypertension and endothelial dysfunction by inhibiting HDAC6-mediated cystathionine γ-lyase degradation.

Hypertension and endothelial dysfunction are associated with various cardiovascular diseases. Hydrogen sulphide (H2 S) produced by cystathionine γ-lyase (CSE) promotes vascular relaxation and lowers hypertension. Honokiol (HNK), a natural compound in the Magnolia plant, has been shown to retain multifunctional properties such as anti-oxidative and anti-inflammatory activities. However, a potential role of HNK in regulating CSE and hypertension remains largely unknown. Here, we aimed to demonstrate that HNK co-treatment attenuated the vasoconstriction, hypertension and H2 S reduction caused by angiotensin II (AngII), a well-established inducer of hypertension. We previously found that histone deacetylase 6 (HDAC6) mediates AngII-induced deacetylation of CSE, which facilitates its ubiquitination and proteasomal degradation. Our current results indicated that HNK increased endothelial CSE protein levels by enhancing its stability in a sirtuin-3-independent manner. Notably, HNK could increase CSE acetylation levels by inhibiting HDAC6 catalytic activity, thereby blocking the AngII-induced degradative ubiquitination of CSE. CSE acetylation and ubiquitination occurred mainly on the lysine 73 (K73) residue. Conversely, its mutant (K73R) was resistant to both acetylation and ubiquitination, exhibiting higher protein stability than that of wild-type CSE. Collectively, our findings suggested that HNK treatment protects CSE against HDAC6-mediated degradation and may constitute an alternative for preventing endothelial dysfunction and hypertensive disorders.

TLR4-TAK1-p38 MAPK pathway and HDAC6 regulate the expression of sigma-1 receptors in rat primary cultured microglia.

Microglia maintain brain homeostasis as the main immune cells in the central nervous system. Activation of sigma-1 receptor (Sig1R) plays neuroprotective and anti-inflammatory roles in microglia. Recent studies showed that Sig1R expression level has been reduced in the brain of the patients with neurodegenerative diseases including Alzheimer's disease. However, the mechanisms underlying the down regulation of the Sig1R has not been clear. Treatment of rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of Sig1R mRNA in a concentration and time-dependent manner. The effects of LPS were blocked by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) antagonist. Furthermore, inhibitors of transforming growth factor beta-activated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK) and histone deacetylase 6 (HDAC6) restored the LPS-induced downregulation of Sig1R. Thus, the current findings demonstrate that TLR4 activation leads to the downregulation of the Sig1R expression via TLR4-TAK1-p38 MAPK pathway and the inhibition of HDAC6 can increase Sig1R expression in microglia. The current findings suggest that downregulation of Sig1R may contribute to neuroinflammation-induced microglial dysfunction, regulation of microglial Sig1R may be novel therapeutic drug candidates for neurodegenerative and neuroinflammatory diseases.

Novel Selective Histone Deacetylase 6 (HDAC6) Inhibitors: A Patent Review (2016-2019).

BACKGROUND: Many human diseases are associated with dysregulation of HDACs. HDAC6 exhibits deacetylase activity not only to histone protein but also to non-histone proteins such as α- tubulin, HSP90, cortactin, and peroxiredoxin. These unique functions of HDAC6 have gained significant attention in the medicinal chemistry community in recent years. Thus a great deal of effort has devoted to developing selective HDAC6 inhibitors for therapy with the hope to minimize the side effects caused by pan-HDAC inhibition. OBJECTIVE: The review intends to analyze the structural feature of the scaffolds, to provide useful information for those who are interested in this field, as well as to spark the future design of the new inhibitors. METHODS: The primary tool used for patent searching is SciFinder. All patents are retrieved from the following websites: the World Intellectual Property Organization (WIPO®), the United States Patent Trademark Office (USPTO®), Espacenet®, and Google Patents. The years of patents covered in this review are between 2016 and 2019. RESULTS: Thirty-six patents from seventeen companies/academic institutes were classified into three categories based on the structure of ZBG: hydroxamic acid, 1,3,4-oxadiazole, and 1,2,4-oxadiazole. ZBG connects to the cap group through a linker. The cap group can tolerate different functional groups, including amide, urea, sulfonamide, sulfamide, etc. The cap group appears to modulate the selectivity of HDAC6 over other HDAC subtypes. CONCLUSION: Selectively targeting HDAC6 over other subtypes represents two fold advantages: it maximizes the pharmacological effects and minimizes the side effects seen in pan-HDAC inhibitors. Many small molecule selective HDAC6 inhibitors have advanced to clinical studies in recent years. We anticipate the approval of selective HDAC6 inhibitors as therapeutic agents in the near future.

Loss of HDAC6 alters gut microbiota and worsens obesity.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (Treg) cells. We asked whether mice that lack HDAC6 and have potent suppressive Treg cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4+ and CD8+ T-cell activation yet maintained the enhanced Treg cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.

HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit.

The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication.IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

HDAC6 regulates dental mesenchymal stem cells and osteoclast differentiation.

BACKGROUND: Dental and periodontal tissue development is a complicated process involving a finely regulated network of communication among various cell types. Understanding the mechanisms involved in regulating dental mesenchymal stem cells (MSCs) and osteoclast cell differentiation is critical. However, it is still unclear whether histone deacetylase HDAC6 is involved in dental MSCs fate determination and osteoclast differentiation. METHODS: We used shRNA and siRNA knockdown to explore the role of HDAC6 in dental MSCs odontogenic differentiation and osteoclasts maturation. RESULTS: Based on HDAC6 knockdown dental MSCs, our data suggest that HDAC6 knockdown significantly increases alkaline phosphate activity and mineralized nodules formation. Additionally, mRNA expression of odontogenic marker genes (OSX, OCN, and OPN) was induced by HDAC6 knockdown. By using HDAC6 siRNA, we knocked down HDAC6 in osteoclast precursor RAW 264.7 cells. Our data suggests that HDAC6 knockdown significantly inhibited osteoclasts differentiation. Additionally, mRNA expression of osteoclast marker genes Trap, Mmp9, and Ctsk was decreased by HDAC6 knockdown. CONCLUSIONS: Our study demonstrated that HDAC6 plays an important role in regulating dental MSCs and osteoclasts differentiation.

Gene expression regulation by heat-shock proteins: the cardinal roles of HSF1 and Hsp90.

The ability to permit gene expression is managed by a set of relatively well known regulatory mechanisms. Nonetheless, this property can also be acquired during a life span as a consequence of environmental stimuli. Interestingly, some acquired information can be passed to the next generation of individuals without modifying gene information, but instead by the manner in which cells read and process such information. Molecular chaperones are classically related to the proper preservation of protein folding and anti-aggregation properties, but one of them, heat-shock protein 90 (Hsp90), is a refined sensor of protein function facilitating the biological activity of properly folded client proteins that already have a preserved tertiary structure. Interestingly, Hsp90 can also function as a critical switch able to regulate biological responses due to its association with key client proteins such as histone deacetylases or DNA methylases. Thus, a growing amount of evidence has connected the action of Hsp90 to post-translational modifications of soluble nuclear factors, DNA, and histones, which epigenetically affect gene expression upon the onset of an unfriendly environment. This response is commanded by the activation of the transcription factor heat-shock factor 1 (HSF1). Even though numerous stresses of diverse nature are known to trigger the stress response by activation of HSF1, it is still unknown whether there are different types of molecular sensors for each type of stimulus. In the present review, we will discuss various aspects of the regulatory action of HSF1 and Hsp90 on transcriptional regulation, and how this regulation may affect genetic assimilation mechanisms and the health of individuals.

TGF-ß1 impairs mechanosensation of human osteoblasts via HDAC6-mediated shortening and distortion of primary cilia.

Transforming growth factor ß (TGF-ß) is a critical regulator of bone density owing to its multiple effects on cell growth and differentiation. Recently, we have shown that TGF-ß1 effectively blocks bone morphogenetic protein (BMP) induced maturation of osteoblasts by upregulating histone deacetylase (HDAC) activity. The current study aimed at investigating the effect of rhTGF-ß1 treatment on the expression of specific HDACs and their cellular effects, e.g., microtubule structures (primary cilia) and mechanosensation. Exposure to TGF-ß1 most significantly induced expression of HDAC6 both on gene and protein level. Being most abundant in the cytoplasm HDAC6 effectively deacetylates microtubule structures. Thus, TGF-ß1-induced expression of HDAC6 led to deformation and shortening of primary cilia as well as to reduced numbers of ciliated cells. Primary cilia are described to sense mechanical stimuli. Thus, fluid flow was applied to the cells, which stimulated osteoblast function (AP activity and matrix mineralization). Compromised primary cilia in TGF-ß1-treated cells were associated with reduced osteogenic function, despite exposure to fluid flow conditions. Chemical inhibition of HDAC6 with Tubacin restored primary cilium structure and length. These cells showed improved osteogenic function especially under fluid flow conditions. Summarizing our results, TGF-ß1 impairs human osteoblast maturation partially via HDAC6-mediated distortion and/or shortening of primary cilia. This knowledge opens up new treatment options for trauma patients with chronically elevated TGF-ß1-levels (e.g., diabetics), which frequently suffer from delayed fracture healing despite adequate mechanical stimulation. KEY MESSAGES: Exposure to TGF-ß1 induces expression of HDAC6 in human osteoblasts. TGF-ß1 exposed human osteoblasts show less and distorted primary cilia. TGF-ß1 exposed human osteoblasts are less sensitive towards mechanical stimulation. Mechanosensation can be recovered by HDAC6 inhibitor Tubacin in human osteoblasts.

Histone deacetylase 6 regulates cytokinesis and erythrocyte enucleation through deacetylation of formin protein mDia2.

The formin protein mDia2 plays a critical role in a number of cellular processes through its ability to promote nucleation and elongation of actin filaments. In erythroblasts, this includes control of cytokinesis and enucleation by regulating contractile actin ring formation. Here we report a novel mechanism of how mDia2 is regulated: through acetylation and deacetylation at lysine 970 in the formin homology 2 domain. Ectopic expression of an acetyl-mimic mDia2 mutant in mouse erythroblasts is sufficient to abolish contractile actin ring formation at the cleavage furrow and subsequent erythrocyte cytokinesis and enucleation. We also identified that class II histone deacetylase 6 deacetylates and subsequently activates mDia2. Knockdown or inhibition of histone deacetylase 6 impairs contractile actin ring formation, and expression of a non-acetyl-mimic mDia2 mutant restores the contractile actin ring and rescues the impairment of enucleation. In addition to revealing a new step in mDia2 regulation, this study may unveil a novel regulatory mechanism of formin-mediated actin assembly, since the K970 acetylation site is conserved among Dia proteins.

Requirement of HDAC6 for activation of Notch1 by TGF-ß1.

TGF-ß1 is enriched in the tumor microenvironment and acts as a key inducer of epithelial to mesenchymal transition (EMT) in lung cancer. The NOTCH signaling pathway is conserved across species and is an essential pathway for development, cell differentiation, and cancer biology. Dysregulation of Notch signaling is a common feature of non-small cell lung cancer (NSCLC) and is correlated with poor prognosis. Crosstalk exists between the NOTCH and TGF-ß signaling pathways in EMT. Herein we report that histone deacetylase 6 (HDAC6) modulates TGF-ß1-mediated activation of the Notch pathway. HDAC6, a primarily cytoplasmic deacetylase, mediates TGF-ß1-induced EMT in human lung cancer cells. Inhibition of HDAC6 with a small molecule inhibitor, namely tubacin or with siRNA attenuated TGF-ß1-induced Notch-1 signaling. We show that TGFß-1-induced EMT is accompanied by rapid HDAC6-dependent deacetylation of heat shock protein 90 (HSP90). Consistently, inhibition of HSP90 with its small molecule inhibitor 17AAG attenuated expression of TGF-ß1-induced Notch-1 target genes, HEY-1 and HES-1. These findings reveal a novel function of HDAC6 in EMT via mediating the TGF-ß-Notch signaling cascade, and support HDAC6 as a key regulator of TGFß-induced EMT in NSCLC. This work suggests that HDAC6 may be an attractive therapeutic target against tumor progression and metastasis.