HDAC Inhibitors as Epigenetic Regulators

Indeed, while SAC genes are frequently mutated in various solid tumors including colon, breast, prostate and lung [66,67,68,69,70,71,72], a survey of four MB datasets included in the cBioPortal [4,73,74,75] revealed a lower-than-expected mutation rate in BUB1, BUB1B, BUB3, MAD1L1, MAD2L1, CDC20 (the maximal rate of inactivating mutations was 0.3%, as in the case on BUB1). could be an effective target for MB treatment. In MB cell lines, CENPE depletion induced defects in division and resulted in cell death. To consolidate CENPE as a target for MB treatment, we tested GSK923295, a specific inhibitor already in clinical trials for other cancer types. GSK923295 induced effects similar to CENPE depletion at low nM levels, supporting the idea that CENPEs inhibition could be a viable strategy for MB treatment. Abstract Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many individuals still die and those who survive suffer from neurological and endocrine disorders. Consequently, more effective therapies are needed. Main microcephaly (MCPH) is definitely a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for additional MCPH genes, CENPE is required BCL2 for VX-661 normal proliferation and survival of neural VX-661 progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, VX-661 CENPE knockdown induced mitotic problems and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE like a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in medical trial for additional tumor types. GSK923295, induced effects much like CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPEs inhibition could be a therapeutic strategy for MB treatment. < 0.05; **, < 0.01; n.s., not significant. In light of these results, to validate CENPE as encouraging target for MB, we selected DAOY and ONS-76 cells, two founded human being MB lines showing a transcriptional signature of SHH activation [1] and transporting mutated or crazy type TP53, respectively [55]. We induced transient CENPE knockdown in these cells (Number 2A), using a pool of specific siRNAs, and analyzed cellular phenotypes 48 h after transfection. As expected [40], CENPE knockdown did not alter mitotic spindle assembly (Number 2B). However, live cell imaging showed clear mitotic problems (Number 2CCE). In particular, as previously explained in HeLa, mitotic cells of both lines were strongly delayed in their metaphase-to-anaphase transition (Number 2C,D; observe also Video clips S1 and S2). After CENPE depletion, DAOY and ONS-76 showed long term metaphase in 50% and 40% of divisions, with and average period of 66 and 36 min, respectively (Number 2D,E). Interestingly, DAOY cells were dramatically affected, since 17% of divisions VX-661 ended up with mitotic catastrophe before completing cytokinesis. Consistent with the observed mitotic defects, CENPE knockdown strongly impaired the development of DAOY and ONS-76 cells, if compared to settings (Number 2F,G). Open in a separate window Open in a separate window Number 2 Medulloblastoma cells are sensitive to CENPE knockdown. (A) Western blot analysis of total lysate from DAOY and ONS-76 cell lines, 48 h after treatment with non-targeting (siCtrl) or CENPE-specific (siCENPE) siRNA. The level of CENPE was analyzed and the internal loading control was tubulin (TUB). The original images are demonstrated in the Numbers S1 and S2. (B) Representative image of DAOY cells processed for immunofluorescence 48 h after transfection with siCtrl or siCENPE and stained with anti-CENPE antibody, anti-Tubulin antibody and DAPI. (C) Representative images of live imaging performed on DAOY 30 h after transfection. Time lapses were recorded over night with an interval of 5 min. Magnification: 40 (D) Quantification of the time spent in metaphase by DAOY and ONS-76 cells analyzed as explained in.