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...

Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G 0 -G 1 arrest without inducing apoptosis. Notably, cetuximabsensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTENexpressing cell lines (14 F 5.0% versus 38.5 F 6.4% growth inhibition, mean F SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 F 4.3% versus 38.8 F 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.

The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4 -HDAC3-N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21. INTRODUCTIONThe acetylation of lysine residues in histones, and/or of transcription factors, is an important posttranslational mechanism of transcriptional regulation (Peterson and Laniel, 2004). Histone deacetylases (HDACs) catalyze the deacetylation of histone and nonhistone substrates, and in general act to repress transcription as part of larger corepressor complexes (Glozak et al., 2005;Yang and Gregoire, 2005). Eighteen mammalian HDACs have been identified to date, and they are grouped into four classes based on their respective homology to yeast deacetylases (Bolden et al., 2006). The class II HDACs can be further subdivided into class IIa (HDAC4, and class , based on the presence in class IIa members of conserved motifs in the N-terminal domain, and extended C terminal tails, that are essential in regulating their function (Yang and Gregoire, 2005).HDACs have emerged as critical regulators of cell growth, differentiation, and apoptotic programs (Bolden et al., 2006). A large body of literature indicates that HDAC inhibitors induce cell cycle arrest, differentiation, and apoptosis in colon cancer cell lines in vitro (Heerdt et al., 1994;Mariadason et al., 1997;Archer et al., 1998;Litvak et al., 1998;Mariadason et al.,...