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1.
Proc Natl Acad Sci U S A ; 112(30): E4055-64, 2015 Jul 28.
Article in English | MEDLINE | ID: mdl-26159421

ABSTRACT

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.


Subject(s)
Brain Neoplasms/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Histone Demethylases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Gene Expression Profiling , Gene Silencing , Humans , Male , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms/metabolism , Stochastic Processes
2.
Cancer Biol Ther ; 16(3): 392-401, 2015.
Article in English | MEDLINE | ID: mdl-25692226

ABSTRACT

BACKGROUND AND PURPOSE: Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT. MATERIALS AND METHODS: Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday-Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting. RESULTS: USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition. CONCLUSIONS: These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.


Subject(s)
Biomarkers, Tumor/biosynthesis , Carcinoma, Non-Small-Cell Lung/genetics , Myeloid Cell Leukemia Sequence 1 Protein/biosynthesis , Ubiquitin Thiolesterase/biosynthesis , Apoptosis/genetics , Apoptosis/radiation effects , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/radiotherapy , Cell Line, Tumor , Cyanoacrylates/administration & dosage , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Pyridines/administration & dosage , Radiation , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/genetics
3.
Oncotarget ; 5(12): 4026-39, 2014 Jun 30.
Article in English | MEDLINE | ID: mdl-24994119

ABSTRACT

MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs and characterized the top candidate, miR-603. Transfection of miR-603 suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. Moreover, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents.


Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Glioblastoma/genetics , Glioblastoma/metabolism , Tumor Suppressor Proteins/genetics , Brain Neoplasms/drug therapy , Brain Neoplasms/mortality , Cell Proliferation , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioblastoma/mortality , Humans , MicroRNAs/genetics , Prognosis , Transfection , Tumor Suppressor Proteins/metabolism
4.
Oncotarget ; 5(4): 882-93, 2014 Feb 28.
Article in English | MEDLINE | ID: mdl-24658464

ABSTRACT

Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within two years of diagnosis. The available data suggest that simultaneous inactivation of critical nodes within the glioblastoma molecular circuitry will be required for meaningful clinical efficacy. We conducted parallel genome-wide shRNA screens to identify such nodes and uncovered a number of G-Protein Coupled Receptor (GPCR) neurotransmitter pathways, including the Dopamine Receptor D2 (DRD2) signaling pathway. Supporting the importance of DRD2 in glioblastoma, DRD2 mRNA and protein expression were elevated in clinical glioblastoma specimens relative to matched non-neoplastic cerebrum. Treatment with independent si-/shRNAs against DRD2 or with DRD2 antagonists suppressed the growth of patient-derived glioblastoma lines both in vitro and in vivo. Importantly, glioblastoma lines derived from independent genetically engineered mouse models (GEMMs) were more sensitive to haloperidol, an FDA approved DRD2 antagonist, than the premalignant astrocyte lines by approximately an order of magnitude. The pro-proliferative effect of DRD2 was, in part, mediated through a GNAI2/Rap1/Ras/ERK signaling axis. Combined inhibition of DRD2 and Epidermal Growth Factor Receptor (EGFR) led to synergistic tumoricidal activity as well as ERK suppression in independent in vivo and in vitro glioblastoma models. Our results suggest combined EGFR and DRD2 inhibition as a promising strategy for glioblastoma treatment.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , ErbB Receptors/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , RNA, Small Interfering/genetics , Receptors, Dopamine D2/metabolism , Animals , Brain Neoplasms/pathology , Cell Line, Tumor , Glioblastoma/pathology , Heterografts , Humans , Mice , Mice, Nude , Phosphorylation , RNA, Small Interfering/metabolism , Signal Transduction , Transfection
5.
PLoS One ; 8(9): e73710, 2013.
Article in English | MEDLINE | ID: mdl-24040035

ABSTRACT

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80-90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.


Subject(s)
Carcinoma, Non-Small-Cell Lung/therapy , DNA Repair/genetics , Lung Neoplasms/therapy , Proteasome Endopeptidase Complex/genetics , RNA Interference , Animals , BRCA1 Protein/genetics , Boronic Acids/pharmacology , Bortezomib , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/radiation effects , Combined Modality Therapy , Fanconi Anemia Complementation Group D2 Protein/genetics , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , NF-kappa B/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Protein Subunits/genetics , Protein Subunits/metabolism , Pyrazines/pharmacology , Rad51 Recombinase/genetics , Radiation, Ionizing , Radiation-Sensitizing Agents/pharmacology , Radiotherapy/methods , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays
6.
Neuro Oncol ; 14(6): 712-9, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22570426

ABSTRACT

Genome-wide microRNA (miRNA) profiling of 82 glioblastomas demonstrated that miR-181d was inversely associated with patient overall survival after correcting for age, Karnofsky performance status, extent of resection, and temozolomide (TMZ) treatment. This association was validated using the Cancer Genome Atlas (TCGA) dataset (n= 424) and an independent cohort (n= 35). In these independent cohorts, an association of miR-181d with survival was evident in patients who underwent TMZ treatment but was not observed in patients without TMZ therapy. Bioinformatic analysis of potential genes regulated by miR-181d revealed methyl-guanine-methyl-transferase (MGMT) as a downstream target. Indeed, transfection of miR-181d downregulated MGMT mRNA and protein expression. Furthermore, luciferase reporter assays and coprecipitation studies showed a direct interaction between miR-181d and MGMT 3'UTR. The suppressive effect of miR-181d on MGMT expression was rescued by the introduction of an MGMT cDNA. Finally, MGMT expression inversely correlated with miR-181d expression in independent glioblastoma cohorts. Together, these results suggest that miR-181d is a predictive biomarker for TMZ response and that its role is mediated, in part, by posttranscriptional regulation of MGMT.


Subject(s)
Antineoplastic Agents, Alkylating/therapeutic use , Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Dacarbazine/analogs & derivatives , Glioblastoma/genetics , MicroRNAs/genetics , Tumor Suppressor Proteins/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/mortality , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/genetics , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/genetics , Dacarbazine/therapeutic use , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioblastoma/mortality , Humans , Immunoblotting , Luciferases/metabolism , Oligonucleotide Array Sequence Analysis , Prognosis , Survival Rate , Temozolomide , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics
8.
Cancer Biomark ; 10(3-4): 185-93, 2011.
Article in English | MEDLINE | ID: mdl-22674304

ABSTRACT

BACKGROUND: The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) confers therapeutic resistance to DNA alkylating agents, including temozolomide. It is largely believed that MGMT promoter methylation is associated with down regulation of MGMT transcription and corresponding protein expression, thereby predisposing tumor cells to the toxic effect of temozolomide. Here we rigorously examined this underlying assumption. METHODS: We examined the correlation between MGMT promoter methylation, transcription, and protein expression using The Cancer Genome Atlas (TCGA) glioblastoma database as well as an independent collection of glioblastoma specimens. RESULTS: In both analyses, we found that MGMT promoter methylation status correlates well with low MGMT mRNA levels (p = 0.04). On the other hand, glioblastomas with unmethylated MGMT promoters exhibited a wide range of MGMT mRNA expression. Intriguingly, the MGMT mRNA levels correlated poorly with MGMT protein levels by Western blotting (R(2) = 0.04, p = 0.34) or by ImmunoHistoChemical (IHC) stain quantitation (R(2) = 0.02, p = 0.50). To exclude the possibility that the poor correlation was due to substandard specimens, we determined the mRNA and protein levels of Colony Stimulating Factor 1 (CSF1), a gene previously shown to exhibit excellent mRNA/protein correlation. In contrast to MGMT, the mRNA level of CSF1 correlated well with the protein level (R(2) = 0.47, p = 0.001). Importantly, long-term passaged glioblastoma cell lines with comparable MGMT transcript levels differed in MGMT protein levels, suggesting mechanisms of post-transcriptional regulation. Accordingly, the correlation between MGMT promoter methylation and MGMT protein expression was poor (p = 0.27). In silico analysis predicted potential binding sites for several miRNA within the 3'UTR of MGMT, suggesting a mechanism for the post-transcriptional of MGMT. CONCLUSION: Our results suggest mechanisms such as miRNA mediated regulation for post-transcriptional regulation of MGMT. Identification of these mechanisms should enhance the value of MGMT based prognostic or predictive biomarker strategies.


Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , O(6)-Methylguanine-DNA Methyltransferase/genetics , RNA Processing, Post-Transcriptional , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , DNA Methylation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Immunohistochemistry , Macrophage Colony-Stimulating Factor/genetics , Macrophage Colony-Stimulating Factor/metabolism , MicroRNAs/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction
9.
PLoS One ; 5(5): e10767, 2010 May 24.
Article in English | MEDLINE | ID: mdl-20532243

ABSTRACT

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.


Subject(s)
ErbB Receptors/metabolism , Glioblastoma/enzymology , Glioblastoma/therapy , Oxidative Stress , Poly(ADP-ribose) Polymerase Inhibitors , Cell Line, Tumor , DNA Damage , DNA Repair/genetics , Enzyme Activation , Gene Expression Regulation, Neoplastic , Genomic Instability , Glioblastoma/genetics , Humans , Radiation Tolerance , Reactive Oxygen Species/metabolism , Survival Analysis , Treatment Outcome
10.
J Biol Chem ; 285(25): 19246-58, 2010 Jun 18.
Article in English | MEDLINE | ID: mdl-20404327

ABSTRACT

Aging and DNA polymerase beta deficiency (beta-pol(+/-)) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in beta-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in beta-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in beta-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD beta-pol haploinsufficient mice.


Subject(s)
Colonic Neoplasms/drug therapy , Colonic Neoplasms/prevention & control , DNA Polymerase beta/genetics , Folic Acid Deficiency/metabolism , 1,2-Dimethylhydrazine/pharmacology , Animal Feed , Animals , Apoptosis , DNA Damage , DNA Repair , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Vitamins/metabolism
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