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Silencing of KDM2B leads to deregulation of apoptosis related genes in GBM

ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer protein that can specifically kill tumor cells while sparing healthy ones. Emerging evidences suggest that TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we aimed to identify novel epigenetic mechanisms regulating TRAIL response in Glioblastoma Multiforme (GBM) by a short-hairpin RNA (shRNA) screen. We employed an shRNA-mediated loss of function approach to interrogate the role of 48 genes in DNA and histone modification pathways. From this we identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of Caspase-8, Caspase-3, Caspase-7, and cleavage of PARP. KDM2B knockdown also accelerated the apoptosis process, as revealed by live cell imaging experiments. Moreover, simultaneous knockdown of the methyltransferases responsible for generating the histone marks removed by KDM2B significantly recovered the cell death phenotype observed with KDM2B inhibition. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing and quantitative PCR upon KDM2B loss, which revealed de-repression of pro-apoptotic genes HRK, caspase-7, and DR4 and repression of anti-apoptotic gene Mcl-1. The apoptosis phenotype was dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. In vivo, KDM2B-silenced tumors exhibited slower growth and reduced angiogenic capacity compared to controls. Taken together, our findings suggest a novel mechanism regulating apoptotic response, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE81043 | GEO | 2017/05/03

SECONDARY ACCESSION(S): PRJNA320464

REPOSITORIES: GEO

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Project description:Glioblastoma (GBM) carries a dismal prognosis largely due to acquired resistance to the standard treatment, which incorporates the chemotherapy temozolomide (TMZ). Inhibiting the proteasomal pathway is an emerging strategy, where combination treatments are under clinical investigation. We hypothesized that pre-treatment of GBM with bortezomib (BTZ) might sensitize glioblastoma to TMZ by abolishing autophagy survival signals to augment DNA damage and apoptosis. P3 patient-derived GBM cells as well as the tumor cell lines U87, HF66, A172 and T98G were investigated for clonogenic survival after single or combined treatment with TMZ and BTZ in vitro. Change in autophagic flux was examined after experimental treatments in conjunction with inhibitors of autophagy or downregulation of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in TMZ-resistant P3 and T98G cells as indicated by diminished levels of the autophagy markers LC3A/B-II and increased STX17, higher protein degradation and no formation of p62 bodies nor induction of apoptosis. In contrast, BTZ treatment attenuated ULK1 mRNA, total and phosphorylated protein, and accumulated LC3A/B-II, p62 and autophagosomes analogously to Baf1 and chloroquine autophagy inhibitors. These autophagosomes did not fuse with lysosomes, indicated by attenuated STX17 expression and reduced degradation of long-lived proteins, which culminated in enhanced caspase-3/8 dependent apoptosis. BTZ synergistically enhanced TMZ efficacy, attenuated tumor cell proliferation, triggered ATM/Chk2 DNA damage signalling to further augment caspase-3/8 mediated apoptosis in the TMZ resistant P3 and T98G GBM cells. Genetic or chemical inhibition of autophagy (with CRISPR-CAs9 ATG5, ATG7 shRNA, MRT68921 or VPS34-IN1) abrogated BTZ efficacy and rescued BTZ+ TMZ treated GBM cells from death. We conclude that Bortezomib ameliorates temozolomide resistance through ATG5/7-dependent abrogated autophagic flux and may be amenable in combination treatment regimens for TMZ refractory GBM patients.

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Silencing of KDM2B leads to deregulation of apoptosis related genes in GBM

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