Histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones and contribute to transcriptional repression. In addition, the HDAC inhibitors induce apoptosis in cancer cells through alterations in histone acetylation and activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Lysophosphatidic acid (LPA) is a growth factor that promotes survival of cancer cells through activation of G protein-coupled receptors. Here we show that HDAC inhibitors can induce apoptosis through activation of the TRAIL apoptotic pathway, and LPA prevented HDAC inhibitor-induced apoptosis and increased TRAIL receptor DR4 (death receptor 4) protein expression. This was associated with increased HDAC1 recruitment to the DR4 promoter following LPA treatment and a reduction in HDAC inhibitor-induced histone acetylation in the DR4 promoter. In addition, LPA induces HDAC enzyme activity in a dose- and time-dependent manner, and this is associated with HDAC1 activation and increased binding of HDAC1 to HDAC2. Reducing the expression of HDAC1 significantly lowered LPA-induced HDAC activity and increased histone acetylation. LPA induction of HDAC activity was blocked by the LPA receptor antagonist, Ki16425, or by inhibiting receptor activation with pertussis toxin. Reducing the expression of the LPA receptor LPA(1) also blocked LPA-induced HDAC activation. In addition, LPA reduced histone acetyltransferase enzymatic activity. Finally, LPA attenuated the ability of the HDAC inhibitor to reduce HDAC activity. Thus, LPA enhances survival of cancer cells by increasing HDAC activity and reducing histone acetylation.

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin.

Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here, we report that patients with CTCL show increased IL15 in a clinical stage-dependent manner. Mechanistically, we show that ZEB1 is a transcriptional repressor of IL15 in T cells and that hypermethylation of the ZEB1 binding region within the IL15 promoter, as seen in patients with CTCL, prevents ZEB1 binding and causes increased transcription of IL15 Using a transgenic mouse model of IL15, we provide evidence that overexpression of IL15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL15 in T cells inhibits an HDAC1-mediated negative autoregulatory loop, resulting in the upregulation of HDAC1 and HDAC6 and transcriptional induction of the onco-miR-21. Interruption of IL15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides preclinical evidence supporting a hierarchical model of oncogenic signaling in CTCL.

To date, CTCL pathogenesis remains unknown, and there are no curative therapies. Our findings not only demonstrate a critical role for IL15-mediated inflammation in cutaneous T-cell lymphomagenesis, but also uncover a new oncogenic regulatory loop in CTCL involving IL15, HDAC1, HDAC6, and miR-21 that shows differential sensitivity to isotype-specific HDAC inhibitors. Cancer Discov; 6(9); 986-1005. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

Transcription factors belonging to the Krüppel-like zinc finger family of proteins participate in the regulation of cell differentiation and development. Although many of these proteins have been identified, little is known about their structure and function. We recently purified ZNF224, a new Krüppel-like zinc finger protein, that contains a Krüppel-associated box (KRAB) domain at the NH2 terminus, and 19 Cys2-His2 zinc-finger domains at the COOH terminus. Using chromatin immunoprecipitation and transient transfection assays, we demonstrate that ZNF224 binds in vivo to the distal promoter of the aldolase A gene and represses its transcription. The results of transient co-transfection experiments show that ZNF224-mediated transcription repression requires the 45-amino acid long KRAB A domain. The ability of KRAB-containing ZNF224 protein to repress transcription depends on specific interaction with the KAP-1 co-repressor molecule. Finally, using selective treatment with the HDAC1 inhibitor trichostatin A, we demonstrate that ZNF224-mediated repression requires histone deacetylases.

Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R(1692-1731) mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R(1692-1731) mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

Hepatitis B virus (HBV) X protein (HBx) is considered to play a role in the development of hepatocellular carcinoma (HCC) during HBV infection. HCC was shown to be more prevalent in men than in women. Estrogen, which exerts its biological function through estrogen receptor (ER), can inhibit HBV replication. ERDelta5, an ERalpha variant lacking exon 5, was found to be preferentially expressed in patients with HCC compared with patients with normal livers. Here, we report the biological role of ERDelta5 and a novel link between HBx and ERalpha signaling in hepatoma cells. ERDelta5 interacts with ERalpha in vitro and in vivo and functions as a dominant negative receptor. Both ERalpha and ERDelta5 associate with HBx. HBx decreases ERalpha-dependent transcriptional activity, and HBx and ERDelta5 have additive effect on suppression of ERalpha transactivation. The HBx deletion mutant that lacks the ERalpha-binding site abolishes the HBx repression of ERalpha. HBx, ERalpha and histone deacetylase 1 (HDAC1) form a ternary complex. Trichostatin A, a specific inhibitor of HDAC enzyme, can restore the transcriptional activity of ERalpha inhibited by HBx. Our data suggest that HBx and ERDelta5 may play a negative role in ERalpha signaling and that ERalpha agonists may be developed for HCC therapy.

Omentin-1, a new adipokine released from adipose tissue, is associated with several key aspects of metabolic syndrome such as insulin sensitivity. However, it is not known whether omentin-1 affects cancer cell growth. In this study, we studied the influence of omentin-1 on two types of human hepatocellular carcinoma cells: HepG2 and HuH-7 cells. Cell viability assay showed that omentin-1 (1 and 2 μg/ml) significantly inhibited the proliferation of HepG2 and HuH-7 cells. Both annexin+PI staining and TUNEL assay showed that omentin-1 induced apoptosis in these cells. Moreover, omentin-1 treatment upregulated protein levels of p53 and p21, a main transcriptional target of p53. Interestingly, omentin-1 did not affect p53 mRNA level. Further mechanism study showed that omentin-1 upregulated p53 protein level through decreasing p53 deacetylation and thereby increasing the stability of p53 protein. Using small interfering RNA (siRNA)-mediated knockdown, we found that Sirt1 deacetylase, but not histone deacetylase 1 (HDAC1), was required for the effect of omentin-1 on p53 deacetylation and cancer cell proliferation. In omentin-1 treated HepG2 cells, the bax/bcl-2 protein ratio was increased, while the caspase-3 signaling pathway was also activated. Omentin-1 triggered JNK signaling but not p38 and ERK1/2 signaling pathways. Collectively, our data suggests that the novel adipokine omentin-1 may contribute to the therapeutic strategy for hepatocellular carcinoma.

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.

Cancer cells accumulate genetic and epigenetic changes that alter gene expression to drive tumorigenesis. Epigenetic silencing of tumor suppressor, cell cycle, differentiation and DNA repair genes contributes to neoplastic transformation. The ING (inhibitor of growth) proteins (ING1-ING5) have emerged as a versatile family of growth regulators, phospholipid effectors, histone mark sensors and core components of HDAC1/2 - and several HAT chromatin-modifying complexes. This review will describe the characteristic pathways by which ING family proteins differentially affect the Hallmarks of Cancer and highlight the various epigenetic mechanisms by which they regulate gene expression. Finally, we will discuss their potentials as biomarkers and therapeutic targets in epigenetic treatment strategies.

The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.

The current therapy on allergic inflammation is unsatisfactory. Probiotics improve the immunity in the body. This study aims to test a hypothesis that administration with Clostridium butyricum (C. butyricum) enforces the effect of specific immunotherapy (SIT) on intestinal allergic inflammation. In this study, an ovalbumin (OVA) specific allergic inflammation mouse model was created. The mice were treated with SIT or/and C. butyricum. The results showed that the intestinal allergic inflammation was only moderately alleviated by SIT, which was significantly enforced by a combination with C. butyricum; treating with C. butyricum alone did not show much inhibitory efficacy. The increase in the frequency of the interleukin (IL)-10-producing OVA-specific B cell (OVAsBC) was observed in mice in parallel to the inhibitory effect on the intestinal allergic inflammation. The in vitro treatment of the OVAsBCs with OVA increased the histone deacetylase-1 (HDAC1) phosphorylation, modulated the transcription of the Bcl6 gene, and triggered the OVAsBCs to differentiate to the IgE-producing plasma cells. Exposure to both OVA and butyrate sodium in the culture increased the expression of IL-10 in OVAsBCs. In conclusion, administration with C. butyricum enforces the inhibitory effect of SIT on allergic inflammation in the mouse intestine.

OBJECTIVE

Histone deacetylase inhibitors (HDACi) have recently emerged as efficacious therapies that target epigenetic mechanisms in hematologic malignancies. One such hematologic malignancy, B-cell acute lymphoblastic leukemia (B-ALL), may be highly dependent on epigenetic regulation for leukemia development and maintenance, and thus sensitive to small-molecule inhibitors that target epigenetic mechanisms.

METHODS

A panel of B-ALL cell lines was tested for sensitivity to HDACi with varying isoform sensitivity. Isoform-specific shRNAs were used as further validation of HDACs as relevant therapeutic targets in B-ALL. Mouse xenografts of B-cell malignancy-derived cell lines and a pediatric B-ALL were used to demonstrate pharmacologic efficacy.

RESULTS

Nonselective HDAC inhibitors were cytotoxic to a panel of B-ALL cell lines as well as to xenografted human leukemia patient samples. Assessment of isoform-specific HDACi indicated that targeting HDAC1-3 with class I HDAC-specific inhibitors was sufficient to inhibit growth of B-ALL cell lines. Furthermore, shRNA-mediated knockdown of HDAC1 or HDAC2 resulted in growth inhibition in these cells. We then assessed a compound that specifically inhibits only HDAC1 and HDAC2. This compound suppressed growth and induced apoptosis in B-ALL cell lines in vitro and in vivo, whereas it was far less effective against other B-cell-derived malignancies.

CONCLUSIONS

Here, we show that HDAC inhibitors are a potential therapeutic option for B-ALL, and that a more specific inhibitor of HDAC1 and HDAC2 could be therapeutically useful for patients with B-ALL.

The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) γ. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPARγ and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.

OBJECTIVE

To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.

RESULTS

We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.

CONCLUSIONS

Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.

In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamster dhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on the dhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression of dhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G(0), recruitment of p130 to E2F-4-DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repress dhfr promoter activity in quiescent cells.

BACKGROUND

Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)).

METHODS

We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry.

RESULTS

Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P>> 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression.

CONCLUSIONS

Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma

UNASSIGNED

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.

Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF-kappaB binding site was found in the ING2 promoter region. We confirmed that NF-kappaB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. ING2 regulation of MMP13 expression was confirmed in both ING2 overexpression and knock down experiments. MMP13 expression was further induced by coexpression of ING2 with HDAC1 or with mSin3A, suggesting that the ING2-HDAC1-mSin3A complex members regulates expression of MMP13. In vitro invasion assay was performed to determine functional significance of ING2 upregulation. ING2 overexpressed cells exhibited greater invasive potential. Taken together, upregulation of ING2 was associated with colon cancer and MMP13-dependent cellular invasion, indicating that ING2 expression might be involved with cancer invasion and metastasis.

Previously we showed that spatial and developmental modulation of ARNT (HIF1β) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) - the most highly expressed EGFR ligand in human keratinocytes - whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation of the AREG-EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results suggest that ARNT controls AREG expression and the downstream EGFR-ERK pathway in keratinocytes, at least in part, by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.

The molecular pathway leading to pituitary tumorigenesis is still largely unknown and is one of the challenges of the endocrine oncology. The development of pituitary adenomas in HMGA2 transgenic mice and the finding of HMGA2 amplification and overexpression in human pituitary adenomas led us to investigate the mechanism by which the HMGA2 gene plays a crucial role in pituitary oncogenesis. This mechanism has been recently described by our group: it entails the acetylation of E2F1, and its consequent enhanced activity, following the displacement of HDAC1 from the pRB/E2F1 inhibitory complex. Based on the mating between HMGA2 transgenic and E2F1 knockout mice, the activation of E2F1 appears to be the main mechanism of the onset of HMGA2-induced pituitary adenoma development. Nevertheless, other events may be also involved in this process, and are discussed here.

Previously, we reported that BRCA1 inhibits progesterone receptor (PR) activity and blocks progesterone-stimulated gene expression and cell proliferation. In the present manuscript, we studied the mechanism of BRCA1 inhibition of PR activity, using c-Myc as a model progesterone-regulated promoter. Here, we found that BRCA1 has little or no effect on PR ligand-binding affinity. However, BRCA1 overexpression inhibited the R5020-induced recruitment of PR to the c-Myc and mouse mammary tumor virus progesterone response elements (PREs) and blocked R5020-stimulated c-Myc expression, whereas BRCA1 underexpression did the opposite. In EMSAs, BRCA1 overexpression blocked the R5020-induced complex formation between PR and several radiolabeled PRE-containing oligonucleotides, and in vitro-translated BRCA1 blocked the interaction of full-length PR-A or a fragment containing the DNA-binding domain of PR with a radiolabeled PRE oligonucleotide. In further studies, BRCA1 overexpression inhibited the recruitment of coactivators (steroid receptor coactivator 1 and amplified in breast cancer 1) and enhanced the recruitment of a corepressor (histone deacetylase 1) to the c-Myc PRE, whereas BRCA1 knockdown increased the abundance of AIB1 and decreased the abundance of HDAC1 at the c-Myc PRE. These findings suggest that BRCA1 inhibits progestin-stimulated PR activity, in part, by preventing PR from binding to the PRE and by promoting the formation of a corepressor complex rather than a coactivator complex.

We analysed the in vitro effects of a new hydroxamate derivative, ITF2357, on AML cells. ITF2357 potently induced histone acetylation. ITF2357 0.1 microM blocked proliferation and induced apoptosis in AML1/ETO-positive Kasumi-1 cells, while AML1/ETO-negative HL60, THP1 and NB4 cell lines were sensitive only to 1 microM ITF2357. Apoptosis was induced by 0.1 microM ITF2357 in AML1/ETO-positive primary blasts and U937-A/E cells induced to express AML1/ETO, but not in U937-A/E cells non-expressing AML1/ETO. In Kasumi-1 cells 0.1 microM ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. ITF2357 0.1 microM also determined DNMT1 efflux from, and p300 influx to, the nucleus. Moreover, 0.1 microM ITF2357 determined local H4 acetylation and release of DNMT1, HDAC1 and AML1/ETO, paralleled by recruitment of p300 to the IL-3 gene promoter. ITF2357 treatment, however, did not induce re-expression of IL-3 gene. Accordingly, the methylation level of IL-3 promoter, as well as of several other genes, was unmodified. In conclusion, ITF2357 emerged as an anti-leukaemic agent very potent on AML cells, and on AML1/ETO-positive cells in particular. More relevantly, clearly emerged from our results that ITF2357 could be an ideal agent to treat AML subtypes presenting AML1/ETO fusion protein which determine HDAC involvement in leukaemogenesis.

The tumor suppressor, microRNA-34 (miR-34), a transcriptional target of TP53, functions in a positive feedback loop to activate TP53. Although miR-34 can inhibit cancer cells carrying TP53 mutations, this feedback to TP53 may be a prerequisite for full miR-34 function and may restrict its therapeutic application to patients with intact TP53. To investigate the functional relationships between TP53 and miR-34, and that of other TP53-regulated miRNAs including miR-215/192, we have used a panel of isogenic cancer cell lines that differ only with respect to their endogenous TP53 status. miR-34-induced inhibition of cancer cell growth is the same in TP53-positive and TP53-negative cells. In contrast, miR-215/192 functions through TP53. In the absence of TP53, miR-34, but not miR-215/192, is sufficient to induce an upregulation of the cell cycle-dependent kinase inhibitor p21(CIP1/WAF1). We identify histone deacetylase 1 (HDAC1) as a direct target of miR-34 and demonstrate that repression of HDAC1 leads to an induction of p21(CIP1/WAF1) and mimics the miR-34 cellular phenotype. Depletion of p21(CIP1/WAF1) specifically interferes with the ability of miR-34 to inhibit cancer cell proliferation. The data suggest that miR-34 controls a tumor suppressor pathway previously reserved for TP53 and provides an attractive therapeutic strategy for cancer patients irrespective of TP53 status.

High-fiber diets decrease the incidence of colorectal cancers, and SCFA, derived from dietary fiber, are involved in the regulation of cell growth, differentiation, and apoptosis of the colonic epithelium. The mediators of these effects remain poorly defined. Krüppel-like factor-4 (KLF4/GKLF) is a zinc-finger transcription factor that exhibits some physiologic properties similar to those of SCFA in the colon. The present study was undertaken to examine the role of KLF4 in the butyrate-mediated effect in colon cancer HT-29 cells. Butyrate induced KLF4 mRNA expression and stimulated KLF4 promoter activity in a dose- and time-dependent manner in HT-29 cells. Similar effects were observed in SCFA possessing different carbon lengths (C3-C7), but not in branched isobutyric acid, indicating that the stimulatory properties of SCFA were related to fatty acid structure. Transfection studies using 5' deletion and mutant constructs of the KLF4 promoter demonstrated that the butyrate-responsive element was located at a putative stimulatory protein (Sp)1-binding site. Electrophoretic mobility shift assays using an oligonucleotide containing a consensus Sp1-binding element revealed a DNA-protein complex that was enhanced by butyrate treatment and supershifted by the Sp1 antiserum. Furthermore, the effects of butyrate on cell growth and KLF4 mRNA expression were the same as those of trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC1). Overexpression of HDAC1 significantly attenuated transcriptional activation of the KLF4 promoter by butyrate or TSA. These results suggest that KLF4 may function as one of the downstream effectors of butyrate that mediates its growth arrest effect in the colon. Moreover, transactivation of KLF4 by butyrate appears to be mediated through interaction with a Sp1-binding domain on the promoter and is also likely to involve histone acetylation.