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1.
BMC Cancer ; 18(1): 1025, 2018 Oct 22.
Article in English | MEDLINE | ID: mdl-30348136

ABSTRACT

BACKGROUND: The dismal prognosis of patients with glioblastoma (GBM) is attributed to a rare subset of cancer stem cells that display characteristics of tumor initiation, growth, and resistance to aggressive treatment involving chemotherapy and concomitant radiation. Recent research on the substantial role of epigenetic mechanisms in the pathogenesis of cancers has prompted the investigation of the enzymatic modifications of histone proteins for therapeutic drug targeting. In this work, we have examined the function of Krüppel-like factor 9 (KLF9), a transcription factor, in chemotherapy sensitization to histone deacetylase inhibitors (HDAC inhibitors). METHODS: Since GBM neurosphere cultures from patient-derived gliomas are enriched for GBM stem-like cells (GSCs) and form highly invasive and proliferative xenografts that recapitulate the features demonstrated in human patients diagnosed with GBM, we established inducible KLF9 expression systems in these GBM neurosphere cells and investigated cell death in the presence of epigenetic modulators such as histone deacetylase (HDAC) inhibitors. RESULTS: We demonstrated that KLF9 expression combined with HDAC inhibitor panobinostat (LBH589) dramatically induced glioma stem cell death via both apoptosis and necroptosis in a synergistic manner. The combination of KLF9 expression and LBH589 treatment affected cell cycle by substantially decreasing the percentage of cells at S-phase. This phenomenon is further corroborated by the upregulation of cell cycle inhibitors p21 and p27. Further, we determined that KLF9 and LBH589 regulated the expression of pro- and anti- apoptotic proteins, suggesting a mechanism that involves the caspase-dependent apoptotic pathway. In addition, we demonstrated that apoptosis and necrosis inhibitors conferred minimal protective effects against cell death, while inhibitors of the necroptosis pathway significantly blocked cell death. CONCLUSIONS: Our findings suggest a detailed understanding of how KLF9 expression in cancer cells with epigenetic modulators like HDAC inhibitors may promote synergistic cell death through a mechanism involving both apoptosis and necroptosis that will benefit novel combinatory antitumor strategies to treat malignant brain tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Glioblastoma/metabolism , Histone Deacetylase Inhibitors/pharmacology , Kruppel-Like Transcription Factors/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Flow Cytometry , Gene Expression , Glioblastoma/genetics , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Panobinostat/pharmacology
2.
Epigenetics ; 13(7): 751-768, 2018.
Article in English | MEDLINE | ID: mdl-30058478

ABSTRACT

Recent studies have revealed an unexpected role of DNA methylation at promoter regions in transcription activation. However, whether DNA methylation at enhancer regions activates gene expression and influences cellular functions remains to be determined. In this study, by employing the transcription factor krÜppel-like factor 4 (KLF4) that binds to methylated CpGs (mCpGs), we investigated the molecular outcomes of the recruitment of KLF4 to mCpGs at enhancer regions in human glioblastoma cells. First, by integrating KLF4 ChIP-seq, whole-genome bisulfite sequence, and H3K27ac ChIP-seq datasets, we found 1,299 highly methylated (ß >0.5) KLF4 binding sites, three-quarters of which were located at putative enhancer regions, including gene bodies and intergenic regions. In the meantime, by proteomics, we identified 16 proteins as putative targets upregulated by KLF4-mCpG binding at enhancer regions. By chromosome conformation capture (3C) analysis, we demonstrated that KLF4 bound to methylated CpGs at the enhancer regions of the B-cell lymphocyte kinase (BLK) and Lim domain only protein 7 (LMO7) genes, and activated their expression via 3D chromatin loop formation with their promoter regions. Expression of mutant KLF4, which lacks KLF4 ability to bind methylated DNA, or removal of DNA methylation in enhancer regions by a DNA methyltransferase inhibitor abolished chromatin loop formation and gene expression, suggesting the essential role of DNA methylation in enhancer-promoter interactions. Finally, we performed functional assays and showed that BLK was involved in glioblastoma cell migration. Together, our study established the concept that DNA methylation at enhancer regions interacts with transcription factors to activate gene expression and influence cellular functions.


Subject(s)
DNA Methylation , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Kruppel-Like Transcription Factors/metabolism , Cell Movement , CpG Islands , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , src-Family Kinases/genetics , src-Family Kinases/metabolism
3.
Oncogene ; 37(38): 5160-5174, 2018 09.
Article in English | MEDLINE | ID: mdl-29849122

ABSTRACT

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called "single base editing", and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix (ECM), and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin ß4 (ITGB4); and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by "single base editing", we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the "single base editing" strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Astrocytes/cytology , Down-Regulation/genetics , Heterozygote , Isocitrate Dehydrogenase/genetics , Mutation , Phosphoproteins/metabolism , Astrocytes/metabolism , Cell Movement/genetics , Cell Proliferation/genetics , Epigenesis, Genetic , Humans , Transcription Factors , YAP-Signaling Proteins
4.
Clin Neurol Neurosurg ; 171: 26-33, 2018 08.
Article in English | MEDLINE | ID: mdl-29803091

ABSTRACT

OBJECTIVES: Receptor tyrosine kinases (RTK), such as c-Met and epidermal growth factor receptor (EGFR), are implicated in the malignant progression of glioblastoma. Studies show that RTK systems can co-modulate distinct and overlapping oncogenic downstream signaling pathways. EGFRvIII, a constitutively activated EGFR deletion mutant variant, leads to increased tumor growth and diminishes the tumor growth response to HGF: c-Met pathway inhibitor therapy. Conversely, activation of the c-Met pathway diminishes the tumor growth response to EGFR pathway inhibitors. Previously we reported that EGFRvIII and c-Met pathway inhibitors synergize to inhibit tumor growth in isogenic GBM cell lines engineered to express EGFRvIII. More recently, studies suggest that despite targeting RTK signaling in glioblastoma multiforme, a subpopulation of stem-like tumor-propagating cells can persist to replenish the tumor cell population leading to tumor recurrence. PATIENTS AND METHODS: Mayo 39 and Mayo 59 xenograft lines were cultured and xenografts were maintained. Subcutaneous xenograft lines were serially passaged in nude mice to generate subcutaneous xenografts. Xenografts were implanted in 6-8 week old nude mice. Once tumors reached a substantial size (150 mm3), mice were randomly divided into 4 groups: 1) control vehicle, 2) Crizotinib (crizo), 3) Erlotinib (erlot), or 4) Crizotinib + Erlotinib, (n = 5 per group). RESULTS: Crizotinib (c-Met pathway inhibitor) and Erlotinib (EGFR pathway inhibitor) in combination significantly inhibited tumor growth, phospho-EGFRvIII, phospho-Met, phospho-AKT, phospho-MAPK, and neurosphere growth in Mayo 39 and Mayo 59 primary GBM subcutaneous xenografts. The expression of the stem cell markers Nestin, Musashi, Olig 2 and Sox2 were also significantly down-regulated by c-Met inhibition, but no additive down-regulation was seen by co-treatment with Erlotinib. CONCLUSIONS: These results are consistent with and corroborate our previous findings demonstrating that targeting these two parallel pathways with c-Met and EGFR inhibitor therapy provides substantial anti-tumor activity in glioblastoma models.


Subject(s)
Crizotinib/pharmacology , Erlotinib Hydrochloride/pharmacology , Glioblastoma/drug therapy , Proto-Oncogene Proteins c-met/drug effects , Animals , Antibodies, Monoclonal/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , ErbB Receptors/drug effects , ErbB Receptors/metabolism , Glioblastoma/pathology , Heterografts/drug effects , Humans , Mice, Nude , Neoplasm Recurrence, Local/drug therapy
5.
J Biol Chem ; 293(17): 6544-6555, 2018 04 27.
Article in English | MEDLINE | ID: mdl-29507094

ABSTRACT

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critical for the regulation of many cellular functions in both normal and neoplastic cells. Here, using human glioblastoma cells, we investigated KLF4's effects on cancer cell metabolism. We found that forced KLF4 expression promotes mitochondrial fusion and induces dramatic changes in mitochondrial morphology. To determine the impact of these changes on the cellular functions following, we analyzed how KLF4 alters glioblastoma cell metabolism, including glucose uptake, glycolysis, pentose phosphate pathway, and oxidative phosphorylation. We did not identify significant differences in baseline cellular metabolism between control and KLF4-expressing cells. However, when mitochondrial function was impaired, KLF4 significantly increased spare respiratory capacity and levels of reactive oxygen species in the cells. To identify the biological effects of these changes, we analyzed proliferation and survival of control and KLF4-expressing cells under stress conditions, including serum and nutrition deprivation. We found that following serum starvation, KLF4 altered cell cycle progression by arresting the cells at the G2/M phase and that KLF4 protected cells from nutrition deprivation-induced death. Finally, we demonstrated that methylation-dependent KLF4-binding activity mediates mitochondrial fusion. Specifically, the downstream targets of KLF4-mCpG binding, guanine nucleotide exchange factors, serve as the effector of KLF4-induced mitochondrial fusion, cell cycle arrest, and cell protection. Our experimental system provides a robust model for studying the interactions between mitochondrial morphology and function, mitochondrial dynamics and metabolism, and mitochondrial fusion and cell death during tumor initiation and progression.


Subject(s)
Cell Division , G2 Phase , Glioblastoma/metabolism , Kruppel-Like Transcription Factors/metabolism , Mitochondrial Dynamics , Neoplasm Proteins/metabolism , Oxygen Consumption , Cell Line, Tumor , Cell Survival , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Neoplasm Proteins/genetics
6.
Oncogene ; 37(20): 2615-2629, 2018 05.
Article in English | MEDLINE | ID: mdl-29479058

ABSTRACT

UDP-glucose 6-dehydrogenase (UGDH) produces UDP-α-D-glucuronic acid, the precursors for glycosaminoglycans (GAGs) and proteoglycans of the extracellular matrix. Elevated GAG formation has been implicated in a variety of human diseases, including glioblastoma (GBM). In our previous study, we found that Krüppel-like factor 4 (KLF4) promotes GBM cell migration by binding to methylated DNA, mainly methylated CpGs (mCpG) and transactivating gene expression. We identified UDGH as one of the downstream targets of KLF4-mCpG binding activity. In this study, we show that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4-induced cell migration in vitro. UGDH knockdown decreases GAG abundance in GBM cells, as well as cell proliferation and migration in vitro. In intracranial xenografts, reduced UGDH inhibits tumor growth and migration, accompanied by a decrease in the expression of extracellular matrix proteins such as tenascin C, brevican. Our studies demonstrate a novel DNA methylation-dependent UGDH upregulation by KLF4. Developing UGDH antagonists to decrease the synthesis of extracellular matrix components will be a useful strategy for GBM therapy.


Subject(s)
Brain Neoplasms/enzymology , Glioblastoma/enzymology , Kruppel-Like Transcription Factors/metabolism , Up-Regulation , Uridine Diphosphate Glucose Dehydrogenase/genetics , Animals , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , CpG Islands , DNA Methylation , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , Glioblastoma/genetics , Glycosaminoglycans/metabolism , Humans , Kruppel-Like Factor 4 , Mice , Neoplasm Transplantation , Proteoglycans/metabolism , Up-Regulation/drug effects , Uridine Diphosphate Glucose Dehydrogenase/antagonists & inhibitors
7.
Elife ; 62017 05 29.
Article in English | MEDLINE | ID: mdl-28553926

ABSTRACT

Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.


Subject(s)
Cell Movement , DNA Methylation , Gene Expression Regulation , Kruppel-Like Transcription Factors/metabolism , Regulatory Sequences, Nucleic Acid , Transcriptional Activation , Cell Line, Tumor , Chromatin Assembly and Disassembly , Humans , Kruppel-Like Factor 4 , Protein Binding
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