Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.     

Histone deacetylase 3 inhibits new tumor suppressor gene DTWD1 in gastric cancer

Cancer epigenetics plays an important role in the pathogenesis of many cancers including gastric cancer. Histone deacetylases (HDACs) emerge as exciting therapeutic targets for cancer treatment and prevention. In this study, we identified DTWD1 as one of the 122 genes upregulated after treatment of trichostatin A (TSA) in two gastric cancer cell lines. Moreover, DTWD1 was downregulated in gastric cancer cell lines and primary gastric carcinoma tissues. It was further identified as the new target of p53. Then we revealed that HDAC3 downregulated DTWD1 by disrupting the interaction of p53 with DTWD1 promoter. Furthermore, DTWD1 functioned as a tumor suppressor by downregulating cyclin B1 expression to inhibit proliferation. In summary, as the new p53 target gene, DTWD1 was downregulated in gastric cancer by HDAC3 and acted as a novel tumor suppressor gene. Specific inhibitors of HDAC3 might be a new approach for gastric cancer treatment by activating DTWD1 expression.     

HDAC inhibitors and immunotherapy; a double edged sword?

Epigenetic modifications, like histone acetylation, are essential for regulating gene expression within cells. Cancer cells acquire pathological epigenetic modifications resulting in gene expression patterns that facilitate and sustain tumorigenesis. Epigenetic manipulation therefore is emerging as a novel targeted therapy for cancer. Histone Acetylases (HATs) and Histone Deacetylases (HDACs) regulate histone acetylation and hence gene expression. Histone deacetylase (HDAC) inhibitors are well known to affect cancer cell viability and biology and are already in use for the treatment of cancer patients. Immunotherapy can lead to clinical benefit in selected cancer patients, especially in patients with limited disease after tumor debulking. HDAC inhibitors can potentially synergize with immunotherapy by elimination of tumor cells. The direct effects of HDAC inhibitors on immune cell function, however, remain largely unexplored. Initial data have suggested HDAC inhibitors to be predominantly immunosuppressive, but more recent reports have challenged this view. In this review we will discuss the effects of HDAC inhibitors on tumor cells and different immune cell subsets, synergistic interactions and possible mechanisms. Finally, we will address future challenges and potential application of HDAC inhibitors in immunocombination therapy of cancer.     

Naturally occurring benzoic acid derivatives retard cancer cell growth by inhibiting histone deacetylases (HDAC)

Histone deacetylases (HDACs), which modulate the expression of genes, are potential therapeutic targets in several cancers. Targeted inhibition of HDAC prevents the expression of oncogenes thereby help in the treatment of cancers. Hence, several pharmaceutical companies developed inhibitors of HDAC and tested them in preclinical models and in clinical trials. SAHA (suberanilohydroxamic acid) is one such HDAC inhibitor developed for treating breast and colorectal carcinomas. However, due to poor efficacy in clinical trials the utility of SAHA for treating cancers was discouraged. Similarly another HDAC inhibitor Trichostatin-A (TSA) also showed promising results in clinical trials but exhibited severe adverse effects, which dampened the interest of using this molecule for cancer treatment. Therefore, search for developing a potent HDAC inhibitor with minimal side effects still continues. Hence, in this study we have screened benzoic acid and benzoic acid derivatives with hydroxylic (-OH) groups and methoxy (-OCH3) groups for their efficacy to bind to the TSA binding site of HDAC using molecular docking studies. Molecules that showed much stronger affinity (than TSA) to HDAC were tested for inhibiting HDAC expressing cultured cancer cells. DHBA but not Dimethoxy Benzoic Acid (DMBA) inhibited HDAC activity, leading to cancer cell growth inhibition through the induction of ROS and cellular apoptosis mediated by Caspase-3. In addition, DHBA arrested cells in G2/M phase of the cell cycle and elevated the levels of sub-G0-G1 cell population. In summary, results of this study report that DHBA could be a strong HDAC inhibitor and inhibit cancer cell growth more effectively.     

Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment

Background

Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.

Methods

We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.

Results

Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC₅₀ 9 – 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

Conclusions

Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.     

Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy

High activity of histone deacetylases (HDACs) causes epigenetic alterations associated with malignant cell behaviour. Consequently, HDAC inhibitors have entered late-phase clinical trials as new antineoplastic drugs. However, little is known about expression and function of specific HDAC isoforms in human tumours including prostate cancer. We investigated the expression of class I HDACs in 192 prostate carcinomas by immunohistochemistry and correlated our findings to clinicopathological parameters including follow-up data. Class I HDAC isoforms were strongly expressed in the majority of the cases (HDAC1: 69.8%, HDAC2: 74%, HDAC3: 94.8%). High rates of HDAC1 and HDAC2 expression were significantly associated with tumour dedifferentiation. Strong expression of all HDACs was accompanied by enhanced tumour cell proliferation. In addition, HDAC2 was an independent prognostic marker in our prostate cancer cohort. In conclusion, we showed that the known effects of HDACs on differentiation and proliferation of cancer cells observed in vitro can also be confirmed in vivo. The class I HDAC isoforms 1, 2 and 3 are differentially expressed in prostate cancer, which might be important for upcoming studies on HDAC inhibitors in this tumour entity. Also, the highly significant prognostic value of HDAC2 clearly deserves further study.     

Defining the molecular action of HDAC inhibitors and synergism with androgen deprivation in ERG-positive prostate cancer

Gene fusions between prostate-specific, androgen responsive TMPRSS2 gene and oncogenic ETS factors, such as ERG, occur in up to 50% of all prostate cancers. We recently defined a gene signature that was characteristic to prostate cancers with ERG activation. This suggested epigenetic reprogramming, such as upregulation of histone deactylase 1 (HDAC1) gene and downregulation of its target genes. We then hypothesized that patients with ERG-positive prostate cancers may benefit from epigenetic therapy such as HDAC inhibition (HDACi), especially in combination with antiandrogens. Here, we exposed ERG-positive prostate cancer cell lines to HDAC inhibitors Trichostatin A (TSA), MS-275 and suberoylanilide hydroxamic acid (SAHA) with or without androgen deprivation. We explored the effects on cell phenotype, gene expression as well as ERG and androgen receptor (AR) signaling. When compared with 5 other prostate cell lines, ERG-positive VCaP and DuCap cells were extremely sensitive to HDACi, in particular TSA, showing synergy with concomitant androgen deprivation increasing apoptosis. Both of the HDAC inhibitors studied caused repression of the ERG-fusion gene, whereas the pan-HDAC inhibitor TSA prominently repressed the ERG-associated gene signature. Additionally, HDACi and flutamide caused retention of AR in the cytoplasm, indicating blockage of androgen signaling. Our results support the hypothesis that HDACi, especially in combination with androgen deprivation, is effective against TMPRSS2-ERG-fusion positive prostate cancer in vitro. Together with our previous in vivo observations of an "epigenetic reprogramming gene signature" in clinical ERG-positive prostate cancers, these studies provide mechanistic insights to ERG-associated tumorigenesis and suggest therapeutic paradigms to be tested in vivo.     

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells

Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors have emerged recently as promising chemotherapeutic agents. One such agent, suberoylanilide hydroxamic acid, is a potent inhibitor of HDACs that causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. Because of its low toxicity, suberoylanilide hydroxamic acid is currently in clinical trials for the treatment of cancer. HDAC inhibitors induce the expression of <2% of genes in cultured cells. In this study, we show that low micromolar concentrations of suberoylanilide hydroxamic acid induce the expression of 15-lipoxygenase-1 in human colorectal cancer cells. The expression of 15-lipoxygenase-1 correlates with suberoylanilide hydroxamic acid-induced increase in 13-S-hydroxyoctadecadienoic acid levels, growth inhibition, differentiation, and apoptosis observed with these cells. Furthermore, specific inhibition of 15-lipoxygenase-1 significantly reduced the suberoylanilide hydroxamic acid-induced effects. These novel findings are the first demonstration of a mechanistic link between the induction of 15-lipoxygenase-1 by a HDAC inhibitor and apoptosis in cancer cells. This result has important implications for the study of suberoylanilide hydroxamic acid and other HDAC inhibitors in the prevention and therapy of colorectal cancer and supports future investigations of the mechanisms by which HDAC inhibitors up-regulate 15-lipoxygenase-1.     

Drug-induced inactivation or gene silencing of class I histone deacetylases suppresses ovarian cancer cell growth: implications for therapy

There is an urgent need to develop new strategies to treat ovarian cancer, the most deadly gynecologic malignancy. Histone deacetylase (HDAC) inhibitors are emerging as novel therapeutic drugs in the treatment of a variety of cancers, including those resistant to standard chemotherapy. Since there are multiple HDAC isoforms, determining the precise role of individual HDAC isoenzymes in the growth and progression of ovarian cancer has the potential to influence the use of selective HDAC inhibitors as strategic therapeutic agents that elicit fewer undesirable side effects. Unfortunately, there is limited information about the expression of HDAC isoforms in human ovarian tissues. This report provides evidence for the first time that Class I HDACs are expressed at significantly higher levels in ovarian cancers in comparison to normal ovarian tissues, with no significant difference in Class II HDAC expression between the two groups. Furthermore, ovarian cancer cells are far more sensitive than normal ovarian cells to the potent HDAC inhibitor romidepsin (FK228), a drug that displays greater inhibitory selectivity for Class I HDACs over Class II isoforms. Using small interfering RNA (siRNA) methodology, we demonstrate that knocking down the gene expression of HDAC3 and other members of the Class I HDAC family suppresses ovarian cancer cell growth. Taken together, the present studies offer several novel findings that have direct relevance for the strategic use of inhibitors that target Class I HDACs, particularly HDAC3, in the treatment of ovarian cancer.     

Selective inhibition of histone deacetylase 2 silences progesterone receptor-mediated signaling

Several histone deacetylases (HDAC) are involved in estrogen receptor (ER)-mediated gene transactivation, and HDAC inhibitors have been reported to restore sensitivity to antihormonal therapy. The modulation of ER is the most promising approach to ER-expressing breast cancers. Recent studies further suggest a critical role of the progesterone receptor (PR) on ER signaling. Although HDAC inhibitors modulate ER, little is known about their effects on PR. We evaluated the roles of specific HDAC isoenzymes and their inhibition on both ER and PR signaling and their importance in response to endocrine therapy. The roles of individual HDAC isoenzymes on ER and PR expression and their functions were evaluated by depletion of select HDAC enzymes using siRNA or pharmacologic inhibition. Cotreatment of breast cancer cell lines with HDAC inhibitors and the antiestrogen, tamoxifen, resulted in synergistic antitumor activity with simultaneous depletion of both ER and PR. Selective inhibition of HDAC2, but not HDAC1 or HDAC6, was sufficient to potentiate tamoxifen-induced apoptosis in ER/PR-positive cells. Depletion of HDAC1 and HDAC6 was associated with down-regulation of ER but not PR. Only the selective depletion of HDAC2 siRNA down-regulated both ER and PR expression, and was sufficient to potentiate tamoxifen. Selective depletion of HDAC2 resulted in simultaneous depletion of ER and PR, and potentiated the effects of antihormonal therapy in ER-positive cells. A more effective pharmacologic inhibition of HDAC2 and evaluation of HDAC2 and PR as therapeutic targets or as predictive markers in hormonal therapy may be considered when combining HDAC inhibitors and hormonal therapy.     

Expression profile of class I histone deacetylases in human cancer tissues

Histone deacetylase (HDAC) activity is one of the widely used and well-established mechanisms for regulation of various genes in cancer. To identify which subtype of class I HDACs are overexpressed in cancers, we analyzed the expression of class I HDAC isotypes composed of HDAC1, 2, 3 and 8 in several cell lines and human cancer tissues, including cancer of the stomach, esophagus, colon, prostate, breast, ovary, lung, pancreas and thyroid. The results showed that >75% of human cancer tissues and their corresponding non-cancerous epithelium showed high expression of these class I HDACs. However, the immunoreactivity of HDAC8 in both prostatic cancer tissue and non-cancerous prostate glands was lower than that in other cancer tissues. Furthermore, 5-40% of cancer tissues overexpressed class I HDACs, when compared with normal epithelium. The results suggest the potential usefulness of HDAC inhibitors for the treatment of a wide variety of human cancers.     

Targeting histone deacetylase 6 mediates a dual anti‐melanoma effect: Enhanced antitumor immunity and impaired cell proliferation

The median survival for metastatic melanoma is in the realm of 8–16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan‐HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti‐melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti‐melanoma agents.     

Histone deacetylase is a target of valproic acid-mediated cellular differentiation

Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.     

Combination of HDAC and topoisomerase inhibitors in small cell lung cancer

Histone deacetylase (HDAC) inhibitors, including MGCD0103 and vorinostat, have led to tumor growth inhibition and apoptosis in vivo. However, with limited single-agent activity demonstrated in solid tumor trials, we examined the potential for enhanced effects in combination with topoisomerase I and II inhibitors, a staple for treatment in refractory small cell lung cancer (SCLC). SCLC cell lines were exposed to increasing concentrations of single-agent HDAC inhibitors and topoisomerase inhibitors, in various combinations, to assess for cell viability, additivity or synergy, and apoptosis. We found that MGCD0103 and vorinostat decreased cell viability by at least 60% and 80%, respectively. In the majority of cell lines, the strongest synergism was seen when vorinostat was followed by either etoposide or topotecan; concurrent therapy led to antagonism in most cell lines. Synergistic effects were seen when MGCD0103 was given concurrently or sequentially with both amrubicin and epirubicin. Enhanced additive effects leading to caspase activation were noted for the combination of MGCD0103 or vorinostat with a topoisomerase inhibitor vs. either agent alone. Thus, the combination of HDAC inhibitors and topoisomerase inhibitors showed enhanced cytotoxic effects in SCLC cell lines. Further evaluation in a clinical setting may be warranted.     

HDAC3 overexpression and colon cancer cell proliferation and differentiation

An immunohistochemical analysis of human colorectal adenocarcinomas showed that cancer cells express widely varying levels of HDAC3. The SW480 colon cancer cell line was found to express high levels of HDAC3 compared to other colon cancer cell lines. p21 was poorly induced in SW480 cells relative to the lower HDAC3-expressing HT-29 cells. RNAi-induced reduction of HDAC3 in SW480 cells increased their constitutive, butyrate-, TSA-, and TNF-alpha-induced expression of p21, but did not cause all the gene expression changes induced upon general histone deacetylase (HDAC) inhibition. SW480 cells with lower HDAC3 expression appeared to be poised for gene expression responses with increased histone H4-K12 acetylation, but not K5, K8, or K16 acetylation. Even though p21 was readily activated in HT29 cells, HDAC3 siRNA nonetheless stimulated p21 expression in these cells to a greater degree than HDAC1 and HDAC2 siRNA. SW480 cells with lower HDAC3 levels displayed an enhanced cell cycle arrest and growth inhibition by butyrate, but without changes in apoptosis or sensitivity to chemotherapeutic agents. As reported for other colon cancer cell lines, butyrate induced the rapid downregulation of the secretory cell differentiation markers mucin 2 and intestinal trefoil factor in SW480 cells. Interestingly, selective HDAC3 inhibition was sufficient to downregulate these genes. Our data support a central role for HDAC3 in regulating the cell proliferation and differentiation of colon cancer cells and suggest a potential mechanism by which colon cancers may become resistant to luminal butyrate.