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1.  Identification of temozolomide resistance factors in glioblastoma via integrative miRNA/mRNA regulatory network analysis 
Scientific Reports  2014;4:5260.
Drug resistance is a major issue in the treatment of glioblastoma. Almost all glioblastomas are intrinsically resistant to chemotherapeutic temozolomide (TMZ) or develop resistance during treatment. The interaction networks of microRNAs (miRNAs) and mRNAs likely regulate most biological processes and can be employed to better understand complex processes including drug resistance in cancer. In this study, we examined if integrative miRNA/mRNA network analysis using the web-service tool mirConnX could be used to identify drug resistance factors in glioblastoma. We used TMZ-resistant glioblastoma cells and their integrated miRNA/mRNA networks to identify TMZ-sensitizing factors. TMZ resistance was previously induced in glioblastoma cell lines U87, Hs683, and LNZ308. miRNA/mRNA expression profiling of these cells and integration of the profiles using mirConnX resulted in the identification of plant homeodomain (PHD)-like finger 6 (PHF6) as a potential TMZ-sensitizing factor in resistant glioblastoma cells. Analysis of PHF6 expression showed significant upregulation in glioblastoma as compared to normal tissue. Interference with PHF6 expression in three TMZ-resistant subclones significantly enhanced TMZ-induced cell kill in two of these cell lines. Altogether, these results demonstrate that mirConnX is a feasible and useful tool to investigate miRNA/mRNA interactions in TMZ-resistant cells and has potential to identify drug resistance factors in glioblastoma.
doi:10.1038/srep05260
PMCID: PMC4052714  PMID: 24919120
2.  EFEMP1 induces γ-secretase/Notch-mediated temozolomide resistance in glioblastoma 
Oncotarget  2013;5(2):363-374.
Glioblastoma is the most common malignant primary brain tumor. Temozolomide (TMZ) is the standard chemotherapeutic agent for this disease. However, intrinsic and acquired TMZ-resistance represents a major obstacle for this therapy. In order to identify factors involved in TMZ-resistance, we engineered different TMZ-resistant glioblastoma cell lines. Gene expression analysis demonstrated that EFEMP1, an extracellular matrix protein, is associated with TMZ-resistant phenotype. Silencing of EFEMP1 in glioblastoma cells resulted in decreased cell survival following TMZ treatment, whereas overexpression caused TMZ-resistance. EFEMP1 acts via multiple signaling pathways, including γ-secretase-mediated activation of the Notch pathway. We show that inhibition of γ-secretase by RO4929097 causes at least partial sensitization of glioblastoma cells to temozolomide in vitro and in vivo. In addition, we show that EFEMP1 expression levels correlate with survival in TMZ-treated glioblastoma patients. Altogether our results suggest EFEMP1 as a potential therapeutic target to overcome TMZ-resistance in glioblastoma.
PMCID: PMC3964213  PMID: 24495907
Temozolomide resistance; glioblastoma; EFEMP1; γ-secretase; Notch; GSI
3.  Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway 
Neuro-Oncology  2011;13(11):1213-1224.
Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.
doi:10.1093/neuonc/nor067
PMCID: PMC3199161  PMID: 21757445
cardiac glycoside; glioblastoma; lanatoside C; non-apoptotic cell death; TRAIL
4.  Extracellular Vesicles and Their Convergence with Viral Pathways 
Advances in Virology  2012;2012:767694.
Extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.
doi:10.1155/2012/767694
PMCID: PMC3410301  PMID: 22888349
5.  Functional Drug Screening Assay Reveals Potential Glioma Therapeutics 
Abstract
Here we describe a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells. Using this assay, we identified small-molecule drugs that sensitized brain tumor cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. Human glioblastoma multiforme cells were engineered by gene transfer to express Gluc as a reporter for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay for screening and validated it using the National Institute of Neurological Disorders and Stroke (NINDS) custom collection II library consisting of 1,040 drugs and bioactive compounds, most of which are Food and Drug Administration-approved and are able to cross the blood–brain barrier. We found that the cardiac glycosides family sensitized glioblastoma multiforme cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. In conclusion, the Gluc secretion assay is a robust tool for functional drug screening and can be applied to many different fields including cancer.
doi:10.1089/adt.2010.0324
PMCID: PMC3102258  PMID: 21184646
6.  Human Glioma Growth is Controlled by MicroRNA-10b 
Cancer research  2011;71(10):3563-3572.
MicroRNA (miRNA) expression profiling studies revealed a number of miRNAs dysregulated in the malignant brain tumor, glioblastoma. Molecular functions of these miRNAs in gliomagenesis are mainly unknown. We show that inhibition of miR-10b, a miRNA not expressed in human brain and strongly up-regulated in both low-grade and high-grade gliomas, reduces glioma cell growth by cell cycle arrest and apoptosis. These cellular responses are mediated by augmented expression of the direct targets of miR-10b, including BCL2L11/Bim, TFAP2C/AP-2γ, CDKN1A/p21, and CDKN2A/p16, which normally protect cells from uncontrolled growth. Analysis of The Cancer Genome Atlas (TCGA) expression dataset reveals a strong positive correlation between numerous genes sustaining cellular growth and miR-10b levels in human glioblastomas, while pro-apoptotic genes anti-correlate with the expression of miR-10b. Furthermore, survival of glioblastoma patients expressing high levels of miR-10 family members is significantly reduced in comparison to patients with low miR-10 levels, indicating that miR-10 may contribute to glioma growth in vivo. Finally, inhibition of miR-10b in a mouse model of human glioma results in significant reduction of tumor growth. Altogether, our experiments validate an important role of miR-10b in gliomagenesis, reveal a novel mechanism of miR-10b-mediated regulation, and suggest the possibility of its future use as a therapeutic target in gliomas.
doi:10.1158/0008-5472.CAN-10-3568
PMCID: PMC3096675  PMID: 21471404
microRNA; glioma; apoptosis; cell cycle; cancer
7.  Regulation of monocyte functional heterogeneity by miR-146a and Relb 
Cell reports  2012;1(4):317-324.
SUMMARY
Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6Chi/Ly-6Clo) and human (CD14hi/CD14loCD16+) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6Chi monocyte response during inflammatory challenge whereas it did not affect Ly-6Clo cells. miR-146a–mediated regulation was cell-intrinsic and depended on Relb, a member of the non-canonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide novel mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify novel targets to manipulate Ly-6Chi monocyte responses while sparing Ly-6Clo monocyte activity.
doi:10.1016/j.celrep.2012.02.009
PMCID: PMC3334310  PMID: 22545247
8.  Microfluidic isolation and transcriptome analysis of serum microvesicles† 
Lab on a chip  2009;10(4):505-511.
Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.
doi:10.1039/b916199f
PMCID: PMC3136803  PMID: 20126692
9.  In vivo tomographic imaging of red-shifted fluorescent proteins 
Biomedical Optics Express  2011;2(4):887-900.
We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.
doi:10.1364/BOE.2.000887
PMCID: PMC3072128  PMID: 21483611
(170.6960) Tomography; (170.3880) Medical and biological imaging
10.  Mutant sodium channel for tumor therapy 
Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, non-infected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells should be susceptible and death is rapid with no time for the tumor cells to become resistant.
doi:10.1038/mt.2009.33
PMCID: PMC2751883  PMID: 19259066
11.  Real-time monitoring of NF-kappaB activity in cultured cells and in animal models 
Molecular imaging  2009;8(5):278-290.
Nuclear factor kappa B (NFkB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. Monitoring of expression of this transcription factor should facilitate a better understanding of NFkB activation in pathological processes. We designed a reporter system based on NFkB responsive promoter elements driving expression of the secreted Gaussia luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for non-invasive monitoring of the kinetics of NFkB activation and inhibition over time, both in conditioned medium of cultured cells, as well as in blood and urine of animals. NFkB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NFkB activation during tumor development as correlated to tumor formation using the NFkB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NFkB reporter system provides a powerful tool for monitoring NFkB activity in real time in vitro and in vivo.
PMCID: PMC2856067  PMID: 19796605
NF-kappaB; Gaussia luciferase; blood assay; bioluminescence imaging
13.  MicroRNA 21 Promotes Glioma Invasion by Targeting Matrix Metalloproteinase Regulators▿ † 
Molecular and Cellular Biology  2008;28(17):5369-5380.
Substantial data indicate that microRNA 21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This microRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense oligonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of gliomas in nude mice. Moreover, downregulation of miR-21 in glioma cells leads to decreases of their migratory and invasion abilities. Our data suggest that miR-21 contributes to glioma malignancy by downregulation of MMP inhibitors, which leads to activation of MMPs, thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir, like miR-21, with specific antisense molecules can provide a novel therapeutic approach for “physiological” modulation of multiple proteins whose expression is deregulated in cancer.
doi:10.1128/MCB.00479-08
PMCID: PMC2519720  PMID: 18591254
14.  The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions 
The Journal of Experimental Medicine  2007;204(12):3037-3047.
Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6Chi and -6Clo monocytes via CCR2 and CX3CR1, respectively. Ly-6Chi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6Clo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor. Consequently, Ly-6Chi monocytes digest damaged tissue, whereas Ly-6Clo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6Chi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.
doi:10.1084/jem.20070885
PMCID: PMC2118517  PMID: 18025128

Results 1-14 (14)