Cux1 regulation of the cyclin kinase inhibitor p27kip1 in polycystic kidney disease is attenuated by HDAC inhibitors

Polycystic kidney disease (PKD) is a common human genetic disease with severe medical consequences. Although it is appreciated that the cilium plays a central role in PKD, the underlying mechanism for PKD remains poorly understood and no effective treatment is available. In zebrafish, kidney cyst formation is closely associated with laterality defects and body curvature. To discover potential drug candidates and dissect signaling pathways that interact with ciliary signals, we performed a chemical modifier screen for the two phenotypes using zebrafish pkd2(hi4166) and ift172(hi2211) models. pkd2 is a causal gene for autosomal dominant PKD and ift172 is essential for building and maintaining the cilium. We identified trichostatin A (TSA), a pan-HDAC (histone deacetylase) inhibitor, as a compound that affected both body curvature and laterality. Further analysis verified that TSA inhibited cyst formation in pkd2 knockdown animals. Moreover, we demonstrated that inhibiting class I HDACs, either by valproic acid (VPA), a class I specific HDAC inhibitor structurally unrelated to TSA, or by knocking down hdac1, suppressed kidney cyst formation and body curvature caused by pkd2 deficiency. Finally, we show that VPA was able to reduce the progression of cyst formation and slow the decline of kidney function in a mouse ADPKD model. Together, these data suggest body curvature may be used as a surrogate marker for kidney cyst formation in large-scale high-throughput screens in zebrafish. More importantly, our results also reveal a critical role for HDACs in PKD pathogenesis and point to HDAC inhibitors as drug candidates for PKD treatment.

A Nuclear Function of β-Arrestin1 in GPCR Signaling: Regulation of Histone Acetylation and Gene Transcription

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a putative tumor suppressor whose expression can be increased by drug treatment. Glioblastoma is the most common central nervous system tumor, is associated with high morbidity and mortality, and responds poorly to surgical, chemical, and radiation therapy. The histone deacetylase inhibitors are under current consideration as therapeutic agents in treating glioblastoma. We investigated whether trichostatin A (TSA) would alter the expression of NAG-1 in glioblastoma cells. The DNA demethylating agent 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 expression and acted synergistically with 5-aza-dC to induce NAG-1 expression. TSA indirectly increases NAG-1 promoter activity and increases NAG-1 mRNA and protein expression in the T98G human glioblastoma cell line. TSA also increases the expression of transcription factors Sp-1 and Egr-1. Small interfering RNA experiments link NAG-1 expression to apoptosis induced by TSA. Reporter gene assays, specific inhibition by small interfering RNA transfections, and chromatin immunoprecipitation assays indicate that Egr-1 and Sp-1 mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA. TSA-induced NAG-1 expression involves multiple mechanisms at the transcriptional and post-transcriptional levels.

Regulation of tissue homeostasis is crucial to disease prevention; cell division, cell cycle arrest, differentiation and apoptosis have to be tightly controlled in order to maintain this homeostasis. Retinoic acid (RA) and the histone deacetylase inhibitors (HDACIs) have profound effects on these processes and thus may be critical regulators of homeostasis. Consequently, RA and/or histone deacetylase inhibitors are currently being tested in clinical trials for a variety of cancers. Unfortunately, little is known of the Regulation of tissue homeostasis is crucial to disease prevention; cell division, cell cycle arrest, differentiation and apoptosis have to be tightly controlled in order to maintain this homeostasis. Retinoic acid (RA) and the histone deacetylase inhibitors (HDACIs) have profound effects on these processes and thus may be critical regulators of homeostasis. Consequently, RA and/or histone deacetylase inhibitors are currently being tested in clinical trials for a variety of cancers. Unfortunately, little is known of the overall affect of these compounds on cellular gene expression. Therefore, we decided to compare the effects of all-trans retinoic acid (ATRA) and a particular HDACI – Trichostatin A (TSA) - on an embryonal carcinoma (EC) cell line (F9) using gene chip analysis. We have focused particular attention on those genes that may be differentially affected by these compounds. Within the parameters established for this study, only 116 of the 12,488 genes examined were similarly regulated by ATRA and TSA: 75 positively and 41 negatively. An additional 70 genes were affected by only one of the compounds and 19 genes were actually inversely regulated. The gene set inversely regulated by ATRA and TSA includes several important patterning genes as well as the crucial tumor suppressor/promoter, transforming growth factor beta 1 (TGF?1). Promoter analysis suggests a motif that may regulate one set of these genes. This study provides the first comprehensive comparison of global gene expression on EC cells as affected by ATRA and a HDAC inhibitor (TSA); reveals new targets for ATRA and HDAC inhibitors; identifies a new regulatory motif; demonstrates that ATRA and HDAC inhibitors do not always act synergistically on gene expression; and examines particular questions regarding their concurrent clinical application.ral important patterning genes as well as the crucial tumor suppressor/promoter, transforming growth factor beta 1 (TGF?1). Promoter analysis suggests a motif that may regulate one set of these genes. This study provides the first comprehensive comparison of global gene expression on EC cells as affected by ATRA and a HDAC inhibitor (TSA); reveals new targets for ATRA and HDAC inhibitors; identifies a new regulatory motif; demonstrates that ATRA and HDAC inhibitors do not always act synergistically on gene expression; and examines particular questions regarding their concurrent clinical application.

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Nuclear translocation of beta-catenin is a hallmark of Wnt signaling and is associated with various cancers. In addition to the canonical Wnt pathway activated by Wnt ligands, growth factors such as epidermal growth factor (EGF) also induce beta-catenin dissociation from the adherens junction complex, translocation into the nucleus, and activation of target genes such as c-myc. Here we report that EGF-induced beta-catenin nuclear localization and activation of c-myc are dependent on the deacetylase HDAC6. We show that EGF induces HDAC6 translocation to the caveolae membrane and association with beta-catenin. HDAC6 deacetylates beta-catenin at lysine 49, a site frequently mutated in anaplastic thyroid cancer, and inhibits beta-catenin phosphorylation at serine 45. HDAC6 inactivation blocks EGF-induced beta-catenin nuclear localization and decreases c-Myc expression, leading to inhibition of tumor cell proliferation. These results suggest that EGF-induced nuclear localization of beta-catenin is regulated by HDAC6-dependent deacetylation and provide a new mechanism by which HDAC inhibitors prevent tumor growth.

Mutation of Murine Rpl5 reveals a New Model for Diamond Blackfan Anemia Characterized By Defective Erythropoiesis

Dent disease type 1 is caused by changes in the chloride voltage-gated channel 5 (CLCN5) gene on chromosome X, resulting in the lack or dysfunction of chloride channel ClC-5. Individuals affected by Dent disease type 1 show proteinuria and hypercalciuria. Previously we found that lentiviral vector-mediated hCLCN5 cDNA supplementary therapy in ClC-5 null mice was effective only for three months following gene delivery, and the therapeutic effects disappeared four months after treatment, most likely due to immune responses to the ClC-5 proteins expressed in the treated cells. Here we tried two strategies to reduce possible immune responses: 1) confining the expression of ClC-5 expression to the tubular cells with tubule-specific Npt2a and Sglt2 promoters, and 2) performing gene therapy in newborn mutant mice whose immune system has not fully developed. We found that although Npt2a and Sglt2 promoters successfully drove ClC-5 expression in the kidneys of the mutant mice, the treatment did not ameliorate the phenotypes. However, gene delivery to the kidneys of newborn Clcn5 mutant mice enabled long-term transgene expression and phenotype improvement. Our data suggest that performing gene therapy on Dent disease affected subjects soon after birth could be a promising strategy to attenuate immune responses in Dent disease type 1 gene therapy.

The type III receptor tyrosine kinase, CD117, functions as a receptor for stem cell factor (SCF) and is encoded by the c-kit gene. During hematopoiesis, CD117 is normally expressed in hematopoietic stem cells, multipotent progenitors, common lymphoid progenitors, and early-stage thymocytes. Overexpression and/or activating mutations of c-kit have been demonstrated in acute myeloid leukemia (AML), early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), and B-cell acute lymphoblastic leukemia (B-ALL). It has been suggested that increased expression of CD117 is associated with stem-like phenotype and worse clinical outcomes in AML and T-ALL. The regulation of expression of c-kit in leukemia is still largely unknown. Here we report that transcription of c-kit in B-ALL is regulated by the Ikaros tumor suppressor protein and histone deacetylase HDAC1. Global genome-wide binding studies using ChIP-seq, demonstrate the occupancy of both Ikaros and HDAC1 at the promoter of the c-kit gene in B-ALL cells. Ikaros and HDAC1 binding to the c-kit promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). Overexpression of Ikaros via retroviral transduction results in reduced transcription of c-kit in B-ALL cells. Consistent with this, Ikaros knock-down with shRNA results in increased transcription of c-kit in B-ALL. These data suggest that Ikaros represses transcription of c-kit. Ikaros overexpression was associated with increased HDAC1 occupancy while Ikaros knock-down resulted in reduced HDAC1 binding to the promoter of the c-kit gene. We tested whether Ikaros-mediated transcriptional repression of c-kit requires HDAC1 activity. Results showed that inhibition of HDAC1 activity with a pan-histone deacetylase inhibitor, trichostatin (TSA), or a specific HDAC1 inhibitor, MS-275, abolishes Ikaros' ability to repress c-kit transcription in luciferase reporter assays. Molecular inhibition of HDAC1 with shRNA confirmed that HDAC1 activity is essential for Ikaros-mediated transcriptional repression of c-kit. A serial qChIP assay spanning the c-kit promoter was used to analyze the epigenetic changes that are associated with Ikaros and HDAC1 binding at the c-kit promoter. Results showed that increased Ikaros and HDAC1 occupancy at the c-kit promoter in B-ALL cells results in enrichment for the markers of the repressive chromatin, H3K9me3 and H3K27me3, as well as reduced occupancy of H3K9ac, a marker of active chromatin. In conclusion, the presented results show that the expression of c-kit in B-ALL is regulated at the transcriptional level by Ikaros and HDAC1 via chromatin remodeling. These data provide a novel insight into the role of Ikaros in both tumor suppression and transcriptional regulation of gene expression in B-cell acute lymphoblastic leukemia. Citation Format: Shriya Kane, Jonathon L. Payne, Mario Soliman, Chandrika Gowda, Meixan Xiang, Chunhua Song, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CD117 expression in B-cell acute lymphoblastic leukemia by Ikaros and histone deacetylase HDAC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1513.

SALL4 Is a Key Factor in HDAC Inhibitor Mediated Ex Vivo Expansion of Human Peripheral Blood Mobilized Stem/Progenitor CD34+CD90+ Cells

Cyclooxygenases (COXs), particularly inducible COX-2 catalyze the synthesis of pro-proliferative and pro-angiogenic prostaglandin E2 (PGE2) and NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is responsible for its metabolic inactivation. Down-regulation of 15-PGDH has been found in gastric, colorectal, breast and prostate cancers and is at least in part mediated by epigenetic silencing which we hypothesized also mediates 15-PGDH silencing observed in about half of all non-small cell lung cancers. Epigenetic strategies, in particular HDAC inhibitors have shown promise in combination with chemotherapy in the treatment of advanced non-small cell lung cancer. Therefore, we treated lung cancer cell lines with 5-aza-2′ deoxycytidine and/or the specific HDAC inhibitor, trichostatin A (TSA) in order to reverse epigenetic silencing and assess modulation of the Cox-2/15-PGDH axis. While 15-PGDH re-activation could be readily detected on the transcriptional level upon TSA but not DAC treatment, such changes could not be seen on the protein level questioning the functional significance of epigenetic regulation of 15-PGDH expression. However, TSA markedly induces COX-2 expression on both the transcriptional and translational level in all seven non-small cell lung cancer cell lines studied. The levels of COX-2 mRNA and protein were maximal at 24h after treatment with TSA and were also confirmed under hypoxic conditions better mimicking in vivo conditions. Five of these seven NSCLC cell lines indeed produced significantly more PGE2 than untreated cells did as determined by enzyme-linked immunosorbent assay under both normoxic and hypoxic conditions following TSA treatment, indicating that the induced COX-2 is functionally active. To confirm that PGE2 production was associated with the catalytic activity of COX-2, three NSCLC cell lines were selected and cultured in the presence of both TSA and indomethacin, a non-selective COX inhibitor and PGE2 levels were measured after 24 hours of co-treatment. Non-cytotoxic, 10uM concentration of indomethacin potently blocked PGE2 production induced by TSA, namely decreased PGE2 to the level of the control group in all three NSCLC cell lines examined. In conclusion, COX-2 is an inducible gene in NSCLC cells and appears to be transcriptionally regulated by a unique mechanism associated with histone acetylation. Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumor agents inhibiting cell proliferation and survival in tumor cells with very low toxicity toward normal cells. Induction of Cox-2 and as a result increased PGE2 production by HDAC inhibition could be an untoward side effect of clinical relevance. Our results suggest that combination therapy of TSA and indomethacin could be a potential strategy to overcome this undesirable effect of HDAC inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5432.

Introduction: Inflammation associated with acute lung injury (ALI) is caused by direct insult to the lungs or by sepsis, which is responsible for tissue damage and cell death. Although the therapeutic effectiveness of epigenetic modifiers can potentially limit lung inflammation, few studies have examined the combinatorial (Aza+TSA) use of a DNA methyl transferase (DNMT) inhibitor, Aza (5-Aza 2-deoxycytidine), and histone deacetylase (HDAC) inhibitor, TSA (Trichostatin A), on the recovery of cell death mechanisms in macrophages. Hypothesis: Therapy with Aza+TSA will reduce cell death by interfering with phosphorylation of JNK and ERK and thereby inhibit JMJD3 expression. Methods and Results: In an LPS-induced ALI mouse model, a single dose of Aza+TSA prevented lung inflammation and injury with significant reduction in mortality as compared with mice treated with either Aza or TSA alone (Thangavel J 2014). DNA ladder analysis shows a decrease in the DNA breaks in LPS-induced bone marrow derived macrophages (BMDMs) treated with Aza+TSA when compared to untreated LPS control (Fig. A) . There is also a reduction in phosphorylation of JNK and ERK in LPS-induced BMDMs when treated together compared to untreated or treated with either Aza or TSA alone. This reduction is caused by the decrease in JMJD3 expression when STAT3 was pulled down after the combined treatment of Aza+TSA, emphasizing the involvement of STAT3-JMJD3 signaling in inflammation associated cell death mechanism (Fig. B) . Conclusions: LPS acts on macrophages through activation of the MAPK pathway followed by the modulation of STAT3-JMJD3 associated with cell death. Administration of DNMT and HDAC inhibitor together interrupts STAT3-mediated decrease in the JMJD3 signaling pathway and cell death in BMDM. !--[endif]-->

The Polycystic Kidney Disease-1 Gene Is a Target for p53-mediated Transcriptional Repression

The antineoplastic activity of HDAC inhibitors is an unquestionable property of these compounds, but recent studies in tumor cells have revealed the potential of HDAC inhibitors (e.g., suberoylanilide hydroxamic acid SAHA, valproic acid VPA) to cause acquisition of HDAC inhibitor resistance. We report that trichostatin A (TSA), an HDAC inhibitor structurally related to SAHA, causes the acquisition of multidrug resistance transporter-independent and irreversible 3-fold resistance to TSA in MLH1-deficient (absent MLH1 protein expression) but not in MLH1-proficient (expressing MLH1 protein) HCT116 colon tumor cells. This MLH1-deficient subline selected for TSA resistance by stepwise exposures to increasing TSA concentrations exhibited failure in the accumulation of acetylated histones, in p21 induction, and in apoptosis activation. These are cellular responses normally seen in tumor cells treated with HDAC inhibitors. Whereas the absence of acetyl-histone accumulation did not correlate with altered HDAC activity, the absence of apoptosis correlated with reduced expression of (pro-apoptotic) Bax. This TSA-resistant subline was cross-resistant to SAHA and VPA but not to 'classic' non-HDAC inhibitor-type anticancer agents such as docetaxel and doxorubicin. These herein presented results expand on a previous study reporting HDAC inhibitor resistance acquisition by SAHA which was independent of the MLH1 expression status. Taken together, the present study identifies TSA, besides SAHA and VPA, as another potential causative of HDAC inhibitor resistance acquisition specifically in MLH1-deficient HCT116 colon tumor cells, and it reveals a possible function of MLH1 protein in protecting colon tumor cells from resistance acquisition by TSA.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.