Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells

Abstract In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His→Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.     

帕比司他逆转前列腺癌细胞hepaCAM基因表达机制研究

目的:研究帕比司他(Panobinostat)逆转抑癌基因肝细胞粘附分子(hepatocyte cell adhesion molecule,hepaCAM)表达,协同hepaCAM抑制前列腺癌(prostate cancer,PCa)细胞生长。方法:不同浓度帕比司他作用于体外培养的PC3细胞,首先采用MTT法检测帕比司他对细胞增殖的影响,RT-PCR和Western blot法检测hepaCAM、组蛋白去乙酰化酶(histone deacetylases, HDACs)以及乙酰化组蛋白H3 赖氨酸9(Ac-H3K9)的表达变化。接着用不同因素处理细胞,MTT法检测细胞增殖活性,流式细胞术检测细胞周期改变,RT-PCR和Western blot法检测细胞周期调节因子cyclinD1 和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的基因表达。结果:帕比司他抑制PC3细胞生长与作用浓度增加和作用时间呈正相关, hepaCAM mRNA、蛋白以及细胞核中Ac-H3K9表达随帕比司他浓度升高而增高,HDAC1、 HDAC3、 HDAC4 mRNA和蛋白的表达随浓度升高而降低;单独过表达hepaCAM腺病毒和单独使用帕比司他组PC3细胞生长明显受到抑制且随作用时间延长抑制率增加,两者联用更为明显,差异有统计学意义(P < 0.05);与单独hepaCAM 腺病毒组和帕比司他组相比,两者联用S期细胞比例明显增高,差异有统计学意义(P < 0.05),且可进一步下调cyclinD1、 PCNA mRNA和蛋白的表达,差异有统计学意义(P < 0.05)。结论:帕比司他可通过抑制HDACs活性,加强PCa PC3细胞组蛋白H3 N-端的赖氨酸残基乙酰化,逆转hepaCAM表达;hepaCAM腺病毒和帕比司他联用可通过阻滞细胞周期于S期协同抑制PC3 细胞生长,作用机制可能与下调cyclinD1 和PCNA 的表达有关。这对揭示抑癌基因hepaCAM在肿瘤缺失的原因及为将帕比司他应用于临床治疗肿瘤提供了科学支持。     

Histone deacetylases inhibitor MS-275 suppresses human esophageal squamous cell carcinoma cell growth and progression via the PI3K/Akt/mTOR pathway

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor with low survival rate, so new therapies are urgently needed. Histone deacetylases (HDACs) play a critical role in tumorigenesis, and HDACs inhibition is a potential therapeutic target in ESSC. In our study, we evaluated the effect and molecular mechanism of MS-275 (an inhibitor of HDACs) on ESCC cells. We found that HDAC1 and HDAC2 were overexpressed in ESCC tissues and related with clinical pathological features of patients with ESCC. MS-275 markedly reduced HDAC1 and HDAC2 expression, whereas increased the level of AcH3 and AcH2B. MS-275 suppressed proliferation and clonogenicity of ESCC cells in a concentration-dependent manner. In addition, MS-275 induced apoptosis, arrested cell cycle, and inhibited migration, epithelial–mesenchymal transition, and sphere-forming ability of ESCC cells in vitro. Moreover, p-Akt1 and p-mTOR were downregulated by MS-275. Finally, MS-275 significantly inhibited tumor growth in vivo. Taken together, HDAC1 and HDAC2 are associated with the progression of ESCC, and MS-275 hinders the progression and stemness of ESCC cells by suppressing the PI3K/Akt/mTOR pathway. Our findings show that MS-275 inhibits ESCC cells growth in vitro and in vivo, which is a potential drug for the ESCC therapy.     

Inhibition of histone deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1

Histone deacetylases (HDACs) 1 and 2 share a high degree of homology and coexist within the same protein complexes. Despite their close association, each possesses unique functions. We show that the upregulation of HDAC2 in colorectal cancer occurred early at the polyp stage, was more robust and occurred more frequently than HDAC1. Similarly, while the expression of HDACs1 and 2 were increased in cervical dysplasia and invasive carcinoma, HDAC2 expression showed a clear demarcation of high-intensity staining at the transition region of dysplasia compared to HDAC1. Upon HDAC2 knockdown, cells displayed an increased number of cellular extensions reminiscent of cell differentiation. There was also an increase in apoptosis, associated with increased p21Cip1/WAF1 expression that was independent of p53. These results suggest that HDACs, especially HDAC2, are important enzymes involved in the early events of carcinogenesis, making them candidate markers for tumor progression and targets for cancer therapy.     

Histone deacetylase and DNA methyltransferase in human prostate cancer

CpG island hypermethylation and chromatin remodeling play important roles in repression of various genes during malignant transformation. We hypothesized that histone deacetylases (HDACs) and DNA methyltransferases (DNMTase) are associated with prostate cancer and we examined the enzyme activity, gene, and protein expression of HDAC1 and DNMT1 in cell lines and tissues. We found that DNMTase and HDACs activities were two- to threefold higher in cell lines compared to benign prostatic hyperplasia (BPH-1) cell line. Treatment of cells with 5-aza-2'-deoxycytidine decreased the activity of HDAC and DNMTase. The mRNA expression of these genes in BPH-1 cells and BPH tissues was lower than that in prostate cancer cells and tissues. HDAC1 and DNMT1 protein expression was higher in prostate cancer compared to BPH. This is the first report to demonstrate that DNMT1 and HDAC1 levels are up-regulated in prostate cancer compared to BPH, suggesting their roles in inactivation of various genes, by DNA-methylation-induced chromatin-remodeling, in prostate cancer.     

Distinct HDACs regulate the transcriptional response of human cyclin-dependent kinase inhibitor genes to Trichostatin A and 1alpha,25-dihydroxyvitamin D3

The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] converge via the interaction of un-liganded vitamin D receptor (VDR) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor (CDKI) genes of the INK4 and Cip/Kip family. We investigated the effects of the HDAC inhibitor Trichostatin A (TSA) and 1alpha,25(OH)2D3 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines. TSA induced the INK4-family genes p18 and p19, whereas the Cip/Kip family gene p21 was stimulated by 1alpha,25(OH)2D3. Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded VDR and repressed the basal level of CDKI genes, but their role in regulating CDKI gene expression by TSA and 1alpha,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1alpha,25(OH)2D3-dependent induction of the p21 gene, for which NCoR1 is essential. In contrast, TSA-mediated induction of the p18 gene was dependent on HDAC3 and HDAC4, but was opposed by NCoR1 and un-liganded VDR. This suggests that the attenuation of the response to TSA by NCoR1 or that to 1alpha,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.     

Distinct and redundant functions of histone deacetylases HDAC1 and HDAC2 in proliferation and tumorigenesis

Histone deacetylases (HDACs) are negative regulators of gene expression and have been implicated in tumorigenesis and tumor progression. Therefore, HDACs are promising targets for antitumor drugs. However, the relevant isoforms of the 18 members encompassing HDAC family have not been identified. Studies utilizing either gene targeting or knockdown approaches reveal both specific and redundant functions of the closely related class I deacetylases HDAC1 and HDAC2 in the control of proliferation and differentiation. Combined ablation of HDAC1 and HDAC2 in different cell types led to a severe proliferation defects or enhanced apoptosis supporting the idea that both enzymes are relevant targets for tumor therapy. In a recent study on the role of HDAC1 in teratoma formation we have reported a novel and surprising function of HDAC1 in tumorigenesis. In this tumor model HDAC1 attenuates proliferation during teratoma formation. In the present work we discuss new findings on redundant and unique functions of HDAC1 and HDAC2 as regulators of proliferation and tumorigenesis and potential implications for applications of HDAC inhibitors as therapeutic drugs.Key words: tumor therapy, HDAC inhibitor, teratoma, chromatin, epigenetics, proliferation, histone acetylation, tumorigenesis     

Proliferating cell nuclear antigen associates with histone deacetylase activity, integrating DNA replication and chromatin modification

Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G(2)-M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.     

HDAC1/2-mediated regulation of JNK and ERK phosphorylation in bovine mammary epithelial cells in response to TNF-α

Bovine mammary epithelial cells (MAC-Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC-T cells stimulated with tumor necrosis factor α (TNF-α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF-α-mediated inflammatory gene expression. Of note, we report that HDAC1/2-mediated inflammatory gene expression was partly regulated by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2-specific inhibitors may prove efficacious for the treatment of bovine mastitis.     

Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients.     

Distinct and redundant functions of histone deacetylases HDAC1 and HDAC2 in proliferation and tumorigenesis

Histone deacetylases (HDACs) are negative regulators of gene expression and have been implicated in tumorigenesis and tumor progression. Therefore, HDACs are promising targets for anti-tumor drugs. However, the relevant isoforms of the 18 members encompassing HDAC family have not been identified. Studies utilizing either gene targeting or knockdown approaches reveal both specific and redundant functions of the closely related class I deacetylases HDAC1 and HDAC2 in the control of proliferation and differentiation. Combined ablation of HDAC1 and HDAC2 in different cell types led to a severe proliferation defects or enhanced apoptosis supporting the idea that both enzymes are relevant targets for tumor therapy. In a recent study on the role of HDAC1 in teratoma formation we have reported a novel and surprising function of HDAC1 in tumorigenesis. In this tumor model HDAC1 attenuates proliferation during teratoma formation. In the present work we discuss new findings on redundant and unique functions of HDAC1 and HDAC2 as regulators of proliferation and tumorigenesis and potential implications for applications of HDAC inhibitors as therapeutic drugs.