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Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. Here we report that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer.[Keywords: AKAP; Aurora; HDAC; histone code; mitosis] Supplemental material is available at http://www.genesdev.org.

Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately À1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the À88/ +54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the À88/À66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1A expression. This occurs due to decreased protein translation without any change in the level of HIF-1A mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1A expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression. (Cancer Res 2006; 66(6): 3197-204)

Gold nanoparticles (AuNPs) have generated interest as both imaging and therapeutic agents. AuNPs are attractive for imaging applications since they are nontoxic and provide nearly three times greater X-ray attenuation per unit weight than iodine. As therapeutic agents, AuNPs can sensitize tumor cells to ionizing radiation. To create a nanoplatform that could simultaneously exhibit long circulation times, achieve appreciable tumor accumulation, generate computed tomography (CT) image contrast, and serve as a radiosensitizer, gold-loaded polymeric micelles (GPMs) were prepared. Specifically, 1.9 nm AuNPs were encapsulated within the hydrophobic core of micelles formed with the amphiphilic diblock copolymer poly(ethylene glycol)-b-poly(ε-capralactone). GPMs were produced with low polydispersity and mean hydrodynamic diameters ranging from 25 to 150 nm. Following intravenous injection, GPMs provided blood pool contrast for up to 24 h and improved the delineation of tumor margins via CT. Thus, GPM-enhanced CT imaging was used to guide radiation therapy delivered via a small animal radiation research platform. In combination with the radiosensitizing capabilities of gold, tumor-bearing mice exhibited a 1.7-fold improvement in the median survival time, compared with mice receiving radiation alone. It is envisioned that translation of these capabilities to human cancer patients could guide and enhance the efficacy of radiation therapy.

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of ␥-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of ␥-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of ␥-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.Glioblastoma multiforme, the most common primary adult brain tumor, has a dismal prognosis. Even with aggressive surgery, radiotherapy, and chemotherapy, the median survival for patients with glioblastomas is under one year (1, 2). The phosphatidylinositol-3-OH kinase (PI3K) 3 signaling pathway is commonly activated in these tumors, often by virtue of PTEN gene mutation but possibly also by epidermal growth factor receptor expression (3, 4). PTEN encodes a phosphatase that dephosphorylates phosphatidylinositol-3,4,5 triphosphate to convert it to phosphatidylinositol-4,5 bisphosphate. Therefore, inactivation of PTEN leads to increased levels of phosphatidylinositol-3,4,5 triphosphate and increased Akt activation (5). Conversely, restoration of PTEN leads to inhibition of Akt. Chakravarti et al. (3) found significantly reduced survival times in patients whose tumors showed PI3K pathway activation. These patients were treated with a combination of surgery with postoperative radiation as the only adjuvant therapy, which suggested that this pathway might play an important role in radiation resistance.One of the factors implicated in radioresistance is activation of the Ras/PI3K/Akt pathway (6, 7). Data from numerous investigators show that inhibition of this pathway leads to radiosensitization, not just of glioblastomas but also carcinoma of the colon, bladder, prostate, head and neck, and cervix (6 -15).The precise mechanism by which the PI3K/Akt pathw...

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.,...

Blood tests to detect circulating tumor cells (CTC) offer great potential to monitor disease status, gauge prognosis, and guide treatment decisions for patients with cancer. For patients with brain tumors, such as aggressive glioblastoma multiforme, CTC assays are needed that do not rely on expression of cancer cell surface biomarkers like epithelial cell adhesion molecules that brain tumors tend to lack. Here, we describe a strategy to detect CTC based on telomerase activity, which is elevated in nearly all tumor cells but not normal cells. This strategy uses an adenoviral detection system that is shown to successfully detect CTC in patients with brain tumors. Clinical data suggest that this assay might assist interpretation of treatment response in patients receiving radiotherapy, for example, to differentiate pseudoprogression from true tumor progression. These results support further development of this assay as a generalized method to detect CTC in patients with cancer.