Inhibitors of histone deacetylases (HDACs) induce growth arrest, differentiation, and apoptosis of colon cancer cell lines in vitro and have demonstrated anti-cancer efficacy in clinical trials. Whereas a role for HDAC1 and -2 in mediating components of the HDAC inhibitor response has been reported, the role of HDAC3 is unknown. Here we demonstrate increased protein expression of HDAC3 in human colon tumors and in duodenal adenomas from Apc1638 N/؉ mice. HDAC3 was also maximally expressed in proliferating crypt cells in normal intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 and other Class I HDACs inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. However, the magnitude of the effects elicited by silencing of individual Class I HDACs was significantly less than that induced by HDAC inhibitors. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression. These findings also demonstrate that multiple Class I HDACs are involved in repressing p21 and suggest that the growthinhibitory and apoptotic effects induced by HDAC inhibitors are probably mediated through the inhibition of multiple HDACs.Acetylation of DNA-bound core histones and sequence-specific transcription factors is a fundamental mechanism of transcriptional regulation. Histone acetylation is typically associated with increased transcription (1) and is regulated by two opposing classes of enzymes: histone acetyltransferases, which add acetyl groups to specific amino acids of the histone protein, and histone deacetylases (HDACs), 2 which catalyze their removal. A second mechanism by which HDACs may regulate gene transcription is by regulating acetylation of DNA sequence-specific transcription factors. Examples include p53, E2F, and Sp3, where deacetylation has been linked to reduced DNA binding or transcriptional activity (2-4). Through these mechanisms, HDACs are emerging as critical regulators of cell growth, differentiation, and apoptotic programs. We and others have demonstrated that inhibitors of HDACs, such as sodium butyrate, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and valproic acid, induce cell cycle arrest, differentiation, and apoptosis in colon cancer cell lines in vitro (5-10). These observations suggest a physiological role for transcriptional repression mediated by HDACs in maintaining cell proliferation and survival and inhibiting differentiation. Correspondingly, the deregulation of HDAC-mediated transcriptional repression has been linked to tumorigenesis. The up-regulated e...

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G 1 -phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at ؊82 and ؊69 relative to the transcription start site. We also found that a TATA element at ؊46 and two overlapping consensus Sp1 sites at ؊60 and ؊55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.Butyrate is one of the most abundant short chain fatty acids in the large intestine, generated by bacterial fermentation of dietary fibers (1). Butyrate shows potent effects on growth arrest and differentiation in vitro in various malignant tumor cell lines, such as breast cancer cells, hepatoma cells, and others (2-5). In colorectal cancer cells, butyrate inhibits cell growth and induces differentiation marker proteins such as alkaline phosphatase and carcinoembryonic antigen (6 -9). Furthermore, butyrate arrests the cell cycle progression at the G 1 phase (9) and decreases c-myc oncogene expression in human colon cancer cell lines (9, 10). However, the precise mechanism of growth suppression by butyrate in colon cancer cells has not been clarified. WAF1/Cip1 protein potently inhibits the various G 1 cyclindependent kinases activities (11-13) by suppressing the phosphorylation of retinoblastoma (RB) protein, thereby supposedly inhibiting the G 1 -S phase transition (11,14). Besides its role as a kinase inhibitor, it has been reported recently that WAF1/ Cip1 at low doses assembles kinase complexes and promotes a kinase activity (15). Furthermore, the transcription of the WAF1/Cip1 gene is directly activated by wild-type p53 protein (16). Thus, WAF1/Cip1 could play a key role as a downstream mediator of the p53-induced cell growth arrest.Several studies have already shown the p53-independent induction of WAF1/Cip1 by serum, transforming growth factor ␤, and other differentiation-inducers (17)(18)(19)(20). In addition, butyrate has been reported to induce WAF1/Cip1 mRNA independently of p53 during differentiation of hematopoietic cells, hepatoma cells, and colon cancer cells in vitro (18,21). Butyrate can also dephosphorylate the retinoblastoma protein in mouse fibroblasts (22). To investigate the mechanism of butyrateinduced growth arrest, we used a human colon cancer cell line WiDr harboring a point mutation in p53 at codon 273 (23) and examined the effect of butyrate on t...

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Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with high potency in inducing di erentiation of cultured murine erythroleukemia cells. We have recently demonstrated that SAHA induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclindependent kinase inhibitor, p21 WAF1/CIP1 , via a p53-independent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated gene activation. Treatment of human breast cancer cell line MCF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activation. Moreover, SAHA activated the p21 promoter primarily through two Sp1 sites located at 782 and 769 relative to the transcription start site. Furthermore, Sp1 and Sp3 proteins were the major factors binding to the Sp1 site of the p21 promoter. However, SAHA did not alter their DNA binding activities, suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Sp1 and Sp3. Further studies using the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GAL4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promoter driven by ®ve GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein was suppressive in the absence of SAHA treatment. Collectively, our results suggest that SAHA activates the p21 promoter through the Sp1 sites, and that both Sp1 and Sp3 proteins can mediate SAHA-induced gene activation.

Tumors with mutant RAS are often dependent on extracellular signal–regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.

Trichostatin A (TSA), a specific histone deacetylase inhibitor, induces histone hyperacetylation and modulates the expression of some genes. We examined the effects of TSA on MG63 cells. TSA induced growth arrest and expression of the p21/WAF1/Cip1 protein. A close correlation between the level of histone acetylation and induction of the p21/WAF1/Cip1 protein was detected. Using several mutant p21/WAF1/Cip1 promoter fragments, mutation of either of two Sp1 sites at -82 or -69 of the p21/WAF1/Cip1 promoter reduced the responsiveness to TSA. This finding indicates that TSA activates the p21/WAF1/Cip1 promoter through the Sp1 sites in a p53-independent manner.

Purpose: MAPK-pathway is one of the most important pathways for a new anticancer drug development. We performed a high-throughput screening for compounds that induce expressions of p15INK4b, and finally identified a novel MEK1/2 inhibitor JTP-74057 (GSK1120212) being evaluated in clinical trials. We characterized its antitumor activities in vitro and in vivo. Experimental design: The MEK inhibitory activity was evaluated using recombinant proteins, and its specificity was confirmed against multiple kinases. Several colorectal cancer cell lines were used for proliferation assay, apoptosis assay, and xenograft model. Results: JTP-74057 strongly inhibited MEK1/2 kinase activities, but did not inhibit other 98 kinase activities. Treatment with JTP-74057 resulted in growth inhibition accompanied with upregulation of p15INK4b and/or p27KIP1 in most colorectal cancer cell lines, which we tested. Daily oral administration of JTP-74057 for 14 days suppressed tumor growth both of HT-29 and COLO205 xenografts in nude mice. Interestingly, the tumor regression was observed only in COLO205 xenografts, and COLO205 was also much more sensitive to JTP-74057 for inducing apoptosis than HT-29 in vitro. Treatment with an Akt inhibitor enhanced the JTP-74057-induced apoptosis in HT-29 cells. Finally, JTP-74057 exhibited an additive or a synergistic effect in combination with the standard-of-care agents, 5-fluorouracil, oxaliplatin or SN-38. Conclusions: JTP-74057, a highly specific and potent MEK1/2 inhibitor, exerts favorable antitumor activities in vitro and in vivo. Sensitivity to JTP-74057-induced apoptosis might be an important factor for the estimation of in vivo efficacy, and the treatment with an Akt inhibitor enhanced the induction of apoptosis. These results suggest the usefulness of JTP-74057 in therapeutic applications for colorectal cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2011-3585