Project description:Cancer stem-like cells (CSCs) is a cell population in glioma with capacity of self-renewal and is critical in glioma tumorigenesis. Parallels between CSCs and normal stem cells suggest that CSCs give rise to tumors. Oncogenic roles of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) have been reported to play a crucial role in glioma tumorigenesis. Herein, we focus on mechanistic contributions of downstream molecules to maintaining stemness of glioma stem-like cells (GSCs). Transcriptional factor, NF-?B, co-locates with MELK/EZH2 complex. Clinically, we observe that the proportion of MELK/EZH2/NF-?B complex is elevated in high-grade gliomas, which is associated with poor prognosis in patients and correlates negatively with survival. We describe the interaction between these three proteins. Specifically, MELK induces EZH2 phosphorylation, which subsequently binds to and methylates NF-?B, leading to tumor proliferation and persistence of stemness. Furthermore, the interaction between MELK/EZH2 complex and NF-?B preferentially occurs in GSCs compared with non-stem-like tumor cells. Conversely, loss of this signaling dramatically suppresses the self-renewal capability of GSCs. In conclusion, our findings suggest that the GSCs depend on EZH2 phosphorylation to maintain the immature status and promote self-proliferation through NF-?B methylation, and represent a novel therapeutic target in this difficult to treat malignancy.
Project description:Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.
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
Project description:Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner.
Project description:Glioblastoma is dependent on a specific signaling pathway to maintain its tumor phenotype. The receptor tyrosine kinase (RTK) family mediates the multiple oncogenic growth factor receptor signaling and contributes to the pathogenesis of glioblastoma. Recently, many studies have shown that the expression of stem cell marker in glioblastoma tissue has prognostic significance, which indicates that the quantification of stem cell markers and RTK genes yields biological information about glioblastoma. In this study, we quantified RNA expression levels of stem cell markers [CD133, Nestin, BMI-1, maternal embryonic leucine zipper kinase (MELK), and Notch1-4] as well as RTKs (EGFR, ErbB4, VEGFR1-3, FGFR1, -2, PDGFR?, and PDGFR?) in 42 clinical samples of glioblastoma by the real-time RT-PCR method. We demonstrated that the expression of MELK is exclusively upregulated in glioblastoma tissue. Notch receptor expression is moderately upregulated and is correlated with that of VEGFR2, VEGFR3, and PDGFR?. Unsupervised clustering identified one unique sample group that showed high expression of most of the genes analyzed. Our results suggest that quantification of these stem cell markers and RTK genes can stratify patients based on the expression profile, which might provide insight into the glioma biology in each cluster.
Project description:Maternal embryonic leucine zipper kinase (MELK) is a highly conserved serine/threonine kinase initially found to be expressed in a wide range of early embryonic cellular stages, and as a result has been implicated in embryogenesis and cell cycle control. Recent evidence has identified a broader spectrum of tissue expression pattern for this kinase than previously appreciated. MELK is expressed in several human cancers and stem cell populations. Unique spatial and temporal patterns of expression within these tissues suggest that MELK plays a prominent role in cell cycle control, cell proliferation, apoptosis, cell migration, cell renewal, embryogenesis, oncogenesis, and cancer treatment resistance and recurrence. These findings have important implications for our understanding of development, disease, and cancer therapeutics. Furthermore understanding MELK signaling may elucidate an added dimension of stem cell control.
Project description:Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy.CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model.CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment.Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas.
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 a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we 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 demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated 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.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:Triple-negative breast cancer (TNBC) is an aggressive, highly recurrent breast cancer subtype, affecting approximately one-fifth of all breast cancer patients. Subpopulations of treatment-resistant cancer stem cells within the tumors are considered to contribute to disease recurrence. A potential druggable target for such cells is the maternal embryonic leucine-zipper kinase (MELK). MELK expression is upregulated in mammary stem cells and in undifferentiated cancers, where it correlates with poor prognosis and potentially mediates treatment resistance. Several MELK inhibitors have been developed, of which one, OTSSP167, is currently in clinical trials. In order to better understand how MELK and its inhibition influence TNBC, we verified its anti-proliferative and apoptotic effects in claudin-low TNBC cell lines MDA-MB-231 and SUM-159 using MTS assays and/or trypan blue viability assays together with analysis of PARP cleavage. Then, using microarrays, we explored which genes were affected by OTSSP167. We demonstrate that different sets of genes are regulated in MDA-MB-231 and SUM-159, but in both cell lines genes involved in cell cycle, mitosis and protein metabolism and folding were regulated. We identified p53 (TP53) as a potential upstream regulator of the regulated genes. Using western blot we found that OTSSP167 downregulates mutant p53 in all tested TNBC cell lines (MDA-MB-231, SUM-159, and BT-549), but upregulates wild-type p53 in the luminal A subtype MCF-7 cell line. We propose that OTSSP167 might have context-dependent or off-target effects, but that one consistent mechanism of action could involve the destabilization of mutant p53.