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Validation of EZH2 targets in GSCs treated with shNT (control), shMELK, shFOXM1, and EZH2 overexpression

ABSTRACT: We demonstrate that the catalytic subunit of Polycomb Repressive Complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are co-expressed in GBM and significantly induced in post-irradiation recurrent tumors whose expression inversely correlated with patient prognosis. Through gain-and loss-of-function study, our data show that MELK or FOXM1 contributes on GSC radioresistance by regulation of EZH2. We used microarrays to validate EZH2 target gene expression. GSCs were treated with shNT (control), shMELK, shFOXM1, and EZH2 overexpression. Total RNA was isolated using the Qiagen RNeasy kit (Qiagen).

ORGANISM(S): Homo sapiens

SUBMITTER: Ichiro Nakano

PROVIDER: E-GEOD-63963 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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EZH2 protects glioma stem cells from radiation-induced cell death in a MELK/FOXM1-dependent manner.

Kim Sung-Hak SH Joshi Kaushal K Ezhilarasan Ravesanker R Myers Toshia R TR Siu Jason J Gu Chunyu C Nakano-Okuno Mariko M Taylor David D Minata Mutsuko M Sulman Erik P EP Lee Jeongwu J Bhat Krishna P L KP Salcini Anna Elisabetta AE Nakano Ichiro I

Stem cell reports 20150115 2

Glioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postir ...[more]

PMID: 25601206

Similar Datasets

Validation of EZH2 targets in GSCs treated with shNT (control), shMELK, shFOXM1, and EZH2 overexpression

Project description:We demonstrate that the catalytic subunit of Polycomb Repressive Complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are co-expressed in GBM and significantly induced in post-irradiation recurrent tumors whose expression inversely correlated with patient prognosis. Through gain-and loss-of-function study, our data show that MELK or FOXM1 contributes on GSC radioresistance by regulation of EZH2. We used microarrays to validate EZH2 target gene expression.

2015-04-16 | GSE63963 | GEO

MELK mediates the stability of EZH2 throught site-specific phosphorylation in extranodal natural killer/T-cell lymphoma

Project description:Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry (MS) analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. Interestingly, FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme which deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to Bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor Bortezomib response.

2019-08-26 | PXD015008 | Pride

Affinify Purification of EZH2 and phosphodeficient EZH2 identifies phosphorylation dependent interactions of EZH2

Project description:Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. We have shown that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. Here, we explore the interactome of EZH2 and of a phosphodeficient mutant EZH2_T367A. We identified novel interactors of EZH2, and identified interactions that are dependent on the phosphorylation and cellular localization of EZH2 that may play a role in EZH2 dependent metastatic progression.

2018-07-03 | PXD010073 | Pride

Glioma stem cell treatment with MELK-signaling inhibitors

Project description:Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Recent studies including our own identified that the mitotic kinase, maternal embryonic leucine-zipper kinase (MELK), is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we provide evidence that the kinase activity of MELK is essential for the action of MELK in GSCs and vital for GBM growth. We utilized in silico structure-based analysis for protein-compound interaction to predict that a recently identified small molecule, Compound 1 (C1), binds to the kinase-active site of MELK protein and eliminates MELK kinase activity in nanomolar concentrations. When treated with C1, GSCs undergo mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed using MELK shRNA-mediated knockdown. C1 treatment strongly induces tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuates growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors. Microarray-based expression analysis of glioma stem cells treated with MELK-signaling inhibitors

2013-08-28 | E-GEOD-50227 | biostudies-arrayexpress

Glioma stem cell treatment with MELK-signaling inhibitors

2013-08-28 | GSE50227 | GEO

Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition [RNA-seq]

Project description:Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these features are associated with distinct transcriptional programs, with vascular regions showing a Proneural profile and hypoxic regions a Mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas Mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress. Using both genetic and pharmacologic inhibition, we found that Proneural GSCs are selectively sensitive to EZH2 disruption, whereas Mesenchymal GSCs are sensitive to BMI1 inhibition. Given that glioblastomas contain both Proneural and Mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be necessary to overcome resistance to therapies caused by intratumoral heterogeneity.

2017-10-01 | GSE86237 | GEO

Homo sapiens

Project description:Validation of EZH2 targets in GSCs treated with shNT (control), shMELK, shFOXM1, and EZH2 overexpression

| PRJNA269617 | ENA

Differential gene expression profile analysis of parental, Cas9-p15 control, and MELK knockout cells

Project description:Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. Our study showed that MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition (EMT), and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells. Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice. Our findings indicate that MELK supports metastasis by promoting EMT and the CSC phenotype in TNBC. In our microarray analysis, we identified potential downstream targets of MELK, including STAT5 and NF-kB target genes, as well as genes involved in tumor progression and metastasis (i.e., EMT, angiogenesis, hypoxia, and apical junction). EMT was the most strongly enriched hallmark among genes highly expressed in Cas9-p15 control cells, further confirming that EMT is a major factor contributing to MELK-induced metastasis in TNBC. We also identified a direct physical interaction partner (PRKAB2) of MELK and a set of intermediate proteins (CDC25B, EZH2, FOXM1, JUN, MAP3K5, PRKAB1, PRKAB2, and SMAD2), suggesting that these proteins are key components of MELK-induced signal transduction.

2023-05-01 | GSE227774 | GEO

Id1 knockdown in primary human glioma stem cells

Project description:Abnormal activation of stemness factors is a crucial signature of cancer stem cells (CSCs), a highly tumorigenic subpopulation in malignant tumors. However, it is unclear whether multi-signaling pathways are activated in CSCs, as like normal stem cells. I would like to report that an inhibitor of differentiation 1 (ID1) activates intracellular multi-signaling involved in proliferation, genesis, and maintenance of glioma stem cells (GSCs) by suppression of Cullin3, an E3 ubiquitin ligase that degrades Cyclin E and components of SHH and WNT signaling. ID1 inhibits BMP-dependent differentiation of GSCs by activation of BMPR2-targeting miR17/20a. ID1HIGH-Cullin3LOW signature correlates with a poor prognosis of GBM patients with a significant association to gene signatures enriched in EGF, WNT, SHH, and BMP signaling. Combinational inhibition of GSC intracellular multi-signaling network increases tumor-bearing mice survival. These results provide insights on molecular and cellular basis of GSC biology, and also suggest necessity of multi-signaling inhibition for GSCs therapy. Two human primary glioma stem cells (GSCs) such as GSC2 and GSC8 were isolated from two individual primary human glioma specimens. The GSCs were directly transfected with pSuper-GFP-ID1-shRNA and pSuper-GFP-Scrambled-shRNA using FuGENE 6 reagent (Roche). The RNA extraction in these cells was used to analyze gene expression.

2013-09-05 | E-GEOD-40614 | biostudies-arrayexpress

Gene expression profiles of H16N2 cells expressing EZH2 variants

Project description:A cytosolic role for EZH2 in regulating cell signaling via interaction with the VAV family of proteins has been reported by us previously. However, it is unclear whether EZH2 interactions with VAV are critical for tumorigenesis. Here, we show that cytosolic EZH2 exhibits greater transforming/metastatic capacity than wild-type EZH2 and that targeted disruption of the EZH2-VAV interaction abolishes EZH2-promoted tumorigenesis. We also found that interaction of cytosolic EZH2 with VAV is critical for EZH2-mediated talin methylation and subsequent STAT3 activation. Both cytosolic EZH2 and a methyl-mimicking talin mutant substantially promoted STAT3 activation and tumor growth. We therefore propose that the VAV-dependent cytosolic function of EZH2 may be a critical step in the initiation of cellular transformation, preceding the well-established function of EZH2 in epigenetic silencing of tumor suppressors. Thus, disrupting EZH2-VAV interaction could be an alternative intervention strategy for treatment of cancers associated with EZH2 overexpression.

2017-10-04 | E-MTAB-3599 | biostudies-arrayexpress

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