Epigenetic Suppression of Histone Deacetylase 4 Boosts T Cell Homing via CXCR3 in Lung Cancer: A Step Toward Ultrasound-Guided Immunotherapy.

The expression levels of histone deacetylase 2 (HDAC2), eukaryotic initiation factor 5 (eIF5), and eukaryotic initiation factor 6 (eIF6), and relationship between HDAC2 and eIF5 or eIF6 in lung cancer tissues were investigated, in order to charify the relationship between HDAC2 and the prognosis of lung cancer patients and its influence on the expression of eIF5 and eIF6. The expression of HDAC2, eIF5, and eIF6 in lung cancer tissues was detected by quantitative reverse transcription polymerase chain reaction. The expression correlation between HDAC2 and eIF5 or eIF6 was tested using a t test. The correlation between HDAC2 and eIF5 or eIF6 was analyzed using the TCGA database. The identified cells were constructed with small interfering siRNA and HDAC2 overexpression plasmid. The proliferation and migration ability of the identified cells was investigated by CCK8 and Transwell assays, respectively. HDAC2, eIF5, and eIF6 were overexpressed in lung cancer tissues, and HDAC2 expression level was negatively correlated with the prognosis of lung cancer patients. HDAC2 expression level was positively correlated with eIF5 and eIF6 expression levels. HDAC2 could regulate the expression of eIF5 and eIF6. The regulation of proliferation and invasion of lung cancer cells by HDAC2 depended on eIF5 and eIF6. HDAC2, eIF5, and eIF6 were closely related with lung cancer tumorigenesis, which might be potential biological markers and therapeutic targets for lung cancer.

Objective To investigate the expression of histone deacetylase 1 (HDAC1) gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor. Methods HDAC1 small interfering RNA (siRNA) and recombinant expression vector pcDNA3.1-HDAC1 were transfected with A549 lung cancer cells, respectively. Stable transfected cell lines were screened by G418 selection. reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression levels of HDAC1 mRNA and HDAC1 protein in the cell; cell proliferation was measured by cell counting kit-8 (CCK-8). Clone formation experiment was used to observe the effect of up-regulation and down-regulation of HDAC1 expression on the radiation sensitivity of A549 cells, cell apoptosis was detected by transferase-mediated dUTP nick-end labelling (TUNEL) method. Nude mice experiment of transplantable tumor was applied for in vivo detection of the influence of up-regulated and down-regulation of HDAC1 expression combined with X-ray irradiation in the inhibition of lung cancer growth. Results The expression of HDAC1 gene and protein were significantly lower to 1.86±0.09 and 2.41±0.10 (P=0.015, 0.006) in HDAC1 cell line A549, and the HDAC1 up-regulation group were significantly higher to 3.65±0.14 and 5.41±0.12 (P=0.012, 0.008); The radiation sensitivity of lung cancer A549 cells was increased in the down-regulation group of HDAC1 (P=0.025), and the radiation sensitivity of lung cancer A549 cells was decreased (P=0.009)in the HDAC1 up-regulation group. Down-regulation of HDAC1 inhibited the proliferation of A549 lung cancer, increased the rate of apoptosis to (42.17±3.19)% (P=0.008), proliferation of A549 cells was significantly enhanced in HDAC1 up-regulated group, associated with reduced percentage of apoptotic cells (9.11±1.46)% (P=0.006); Average volume in the nude mice of HDAC1 down-regulated group (547.37±89.68) mm3 were significantly smaller than the control group (1 238.44±212.36) mm3 (P=0.008); furthermore, mean volume of tumor was greater in the HDAC1 up-regulated group than in the control group (1 681.27±195.37) mm3 (P=0.006). After 20 Gy γ irradiation, the average volume of HDAC1 down-regulated group were significantly decreased (214.15±54.26) mm3 (P=0.011). Compared with that before irradiation, the average volume of the tumor in the nude mice was not significantly changed after irradiation (P=0.264). Conclusion Down-regulated expression of HDAC1 can inhibit the growth of human lung cancer A549 cells in nude mice, enhance the radiation sensitivity of tumor cells, whereas up-regulated expression of HDAC1 can produce tumor radiation resistance. Key words: Lung cancer; A549 cell; Histone deacetylase 1; Transplantable tumor; Nude mice

Objective To detect the expression of histone deacetylase 6 (HDAC6) and its significance in non-small cell lung cancer (NSCLC).Methods The expression of HDAC6 was measured by SP staining in 70 cases of NSCLC and 18 cases of normal lung tissues.Results Positive rate of HDAC6 immunostaining in NSCLC was 58.57％,which was significantly higher than 16.67％ in normal lung tissues(P ＜0.01 ).No correlation was found between HDAC6 expression and the age,sex,differentiated level,histological type ( P ＞0.05).But the expression level of HDAC6 was closely related to clinical stage and lymph node metastasis in NSCLC patients ( P ＜ 0.05 ).Conclusion HDAC6 over expression may be related to the pathogenesis and development of NSCLC. Key words: Carcinoma, non-small-cell lung; Immunohistochemistry; Histone deacetylase 6

The immunohistochemical expression and potential prognostic value of HDAC6 and AR in invasive breast cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

The prefrontal cortex (PFC), a region responsible for high-order cognitive functions, such as decision-making, attention and working memory, is highly influenced by stress and corticosteroid stress hormones. Recently it has been shown that acute stress affects PFC functions by potentiating glutamatergic transmission via a mechanism dependent on glucocorticoid receptor (GR) and its downstream target, serum and glucocorticoid-inducible kinase (SGK). To identify the key regulators of stress responses, we examined the role of histone deacetylase 6 (HDAC6), a unique member of the HDAC family that could regulate the GR chaperone protein heat shock protein 90 (HSP90), in the synaptic action of acute stress in PFC. We found that HDAC6 inhibition or knockdown blocked the enhancement of glutamatergic transmission and glutamate receptor trafficking by acute stress in vivo or corticosterone treatment in vitro. In addition, HDAC6 inhibition blocked the up-regulation of SGK in animals exposed to acute stress. HSP90 inhibition or knockdown produced a similar blockade of the acute stress-induced enhancement of glutamatergic signalling. These findings have identified HDAC6 as a key molecule gating the effects of acute stress on synaptic functions in the PFC.

A Centrosomal Cdc20-APC Pathway Controls Dendrite Morphogenesis in Postmitotic Neurons

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.

Histone deacetylase 6 modulates autophagy through HIF-1α/BNIP3 signaling in lung cancer.

Histone deacetylase 5 (HDAC5) belongs to class II HDAC subfamily and is reported to be increased in the kidneys of diabetic patients and animals. However, little is known about its function and the exact mechanism in diabetic kidney disease (DKD). Here, we found that HDAC5 was located in renal glomeruli and tubular cells, and significantly upregulated in diabetic mice and UUO mice, especially in renal tubular cells and interstitium. Knockdown of HDAC5 ameliorated high glucose-induced epithelial–mesenchymal transition (EMT) of HK2 cells, indicated in the increased E-cadherin and decreased α-SMA, via the downregulation of TGF-β1. Furthermore, HDAC5 expression was regulated by PI3K/Akt signaling pathway and inhibition of PI3K/Akt pathway by LY294002 treatment or Akt phosphorylation mutation reduced HDAC5 and TGF-β1 expression in vitro high glucose-cultured HK2 cells. Again, high glucose stimulation downregulated total m6A RNA methylation level of HK2 cells. Then, m6A demethylase inhibitor MA2 treatment decreased Akt phosphorylation, HDAC5, and TGF-β1 expression in high glucose-cultured HK2 cells. In addition, m6A modification-associated methylase METTL3 and METTL14 were decreased by high glucose at the levels of mRNA and protein. METTL14 not METTL3 overexpression led to PI3K/Akt pathway inactivation in high glucose-treated HK2 cells by enhancing PTEN, followed by HDAC5 and TGF-β1 expression downregulation. Finally, in vivo HDACs inhibitor TSA treatment alleviated extracellular matrix accumulation in kidneys of diabetic mice, accompanied with HDAC5, TGF-β1, and α-SMA expression downregulation. These above data suggest that METTL14-regulated PI3K/Akt signaling pathway via PTEN affected HDAC5-mediated EMT of renal tubular cells in diabetic kidney disease.

Objective To evaluate the role of autophagy in cerebral ischemia-reperfusion (I/R) injury in diabetic mice and the relationship with histone deacetylase 3 (HDAC3)/Bmal1 signaling pathway. Methods Healthy clean-grade male C57BL/6 mice were used in the study.Diabetes mellitus was induced by intraperitoneal injection of streptozotocin.Thirty-six mice with diabetes mellitus after being fed for 8 weeks were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), I/R group and I/R plus HDAC3 inhibitor group (group I/R-H). Cerebral I/R was induced by middle cerebral artery occlusion for 1 h, followed by 24-h reperfusion in anesthetized mice.Specific HDAC3 inhibitor RGFP966 10 mg/kg was subcutaneously injected at 30 min before establishing the model in group I/R-H.Brain tissues were obtained at 24 h of reperfusion for microscopic examination and for determination of cerebral infarct size (by TTC), cell apoptosis (by TUNEL), activities of superoxide dismutase (SOD) and reactive oxygen species (ROS) and malondialdehyde (MDA) content (by colorimetric assay), expression of autophagy-related protein Beclin-1 and LC3B (by immunofluorescence), and expression of HDAC3, Bmal1, GSK-3β and p62 (by Western blot). Apoptosis index (AI) was calculated. Results Compared with group S, the cerebral infarct size was significantly increased, the activities of SOD and ROS and content of MDA in brain tissues were decreased, the expression of Bmal1, p-GSK-3β and HDAC3 was down-regulated, and AI was increased in group I/R (P<0.05). Compared with group I/R, the cerebral infarct size was significantly increased, the activities of SOD and ROS and content of MDA in brain tissues were increased, the expression of Bmal1, p-GSK-3β, Beclin-1 and LC3B was up-regulated, AI was decreased, and the expression of HDAC3 and p62 was down-regulated in group I/R-H (P<0.05). Conclusion HDAC3/Bmal1 signaling pathway exerts endogenous protective effect through activating autophagy and increasing the antioxidant capacity following cerebral I/R in diabetic mice. Key words: HDAC3; Bmal1; Autophagy; Diabetic; Cerebral ischemia reperfusion injury

Histone deacetylase5 (HDAC5), as a member of the class IIa family of HDACs, is frequently dysregulated in human malignancies. However, little is known regarding the specific role of HDAC5 in lung cancer. We aimed to evaluate HDAC5 expression in human lung cancer and to determine the effects of HDAC5 on lung cancer cells. First, the expression levels of both HDAC5 protein and mRNA were evaluated in lung cancer tissues and cell lines by western blot analysis and RT‑qPCR, and the results suggested that HDAC5 was significantly upregulated in human lung cancer tissues and cell lines. To address the effects of HDAC5 on the biological behavior of human lung adenocarcinoma cells, we generated human lung cancer A549 cell lines in which HDAC5 was either overexpressed or depleted. The results indicated that overexpression of HDAC5 significantly promoted the proliferation and invasion, and inhibited the apoptosis of A549 cells. On the contrary, HDAC5 knockdown largely decreased the proliferation and invasion and enhanced the apoptosis of A549 cells. Furthermore, we demonstrated that HDAC5 overexpression promoted the expression of DLL4, Six1, Notch1 and Twist1 in A549 cells. Downregulation of HDAC5 caused a significant inhibition of the expression of DLL4, Six1, Notch1 and Twist1 in A549 cells. Taken together, our data demonstrated that HDAC5 displayed a significant upregulation in lung cancer, and elevated HDAC5 might be involved in the potentiation of proliferation and invasion of lung cancer cells, as well as the inhibition of lung cancer cell apoptosis by the upregulation of DLL4, Six1, Notch1 and Twist1. The present study may provide an evidence for the potential application of HDAC5 inhibitors in the therapy of lung cancer.

BackgroundThe leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown.MethodsThe clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT–PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells.ResultsHDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth.ConclusionsHDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Characterisation of Histone Deacetylase (HDAC) Expression Profiles in Acute Myeloid Leukaemia: A Basis for the Development of Targeted Therapy Using Histone Deacetylase Inhibitors.

Histone deacetylase 1 mRNA expression in lung cancer