Glioblastoma multiforme (GBM) is an aggressive central nervous system (CNS) cancer characterized by enhanced tumor cell motility, pernicious invasion into the normal brain, extensive tumor-induced angiogenesis, and adaptive resistance to current therapeutic paradigms. One of the difficulties associated with GBM is the ability of the tumor cells to infiltrate normal CNS tissue. Neurosurgeons can remove the primary tumor mass, but peripheral cells that are inaccessible will ultimately result in a secondary lesion that can lead to death. The matrix metalloproteinases (MMP) are well known for their abilities to facilitate processes of cellular motility and invasion through their clearance of extracellular matrix (ECM). A specific member of this family, MMP-1, is not observed in normal brain, yet its expression is a common characteristic of GBM. The various causes of MMP-1 expression, and its consequences in GBM cells are unknown. As such, functional studies were conducted related to the induction of MMP-1 expression via another molecule intrinsic to GBM, nitric oxide (NO). The exposure of GBM cell lines to nanomolar concentrations of NO produced significant inductions of MMP-1 expression and GBM cell motility. The specific removal of MMP-1 with siRNA elicited an abrogation of NO-stimulated motility, suggesting a pathological contribution by this enzyme. Furthermore, recent accumulating evidence suggests that MMP-1 contributes to tumor cell survival and related angiogenesis in other cancer settings. To investigate these capabilities in GBM, cell lines were stably engineered to have either MMP-1 over-expression or knock-down. Both tumor formation and size were significantly reduced with MMP-1 knock-down and significantly increased with over-expression. In a model of GBM cell-induced angiogenesis, the presence of MMP-1 contributed to an angiogenic phenotype. Further angiogenesis studies revealed a significant recruitment of host endothelium to the tumor interstitium in vivo. Proteomic studies suggest that one mechanism by which MMP-1 could influence angiogenesis is through the easement of the anti-angiogenic tissue inhibitor of metalloproteinases-4 (TIMP-4), since the removal of MMP-1 elicits a significant increase in TIMP-4 detection. Altogether, these functional data present MMP-1 as a promising target for future therapeutic investigation, because it is unique to the GBM environment and contributes to key overlapping GBM pathologies.

Introduction: Despite many advances in therapeutic and surgical techniques for glioblastoma multiforme (GBM), this form of brain cancer still remains incurable. A hallmark feature of GBM is the ability of the glioma cells to infiltrate surrounding brain tissue. The invasive nature of glioma cells is a key challenge in considering treatment for patients with GBM. Certain members of the matrix metalloproteinase (MMP) family play a role in tumor cell invasion and metastasis (Coussens, et al., 2002). A functional SNP resulting from an additional guanine at position -1607 in the MMP-1 promoter creates an erythroblastosis twenty six transcription factor protein (ETS) DNA consensus binding site, which results in significantly higher transcriptional activity of MMP-1 (Rutter et al., 1998). Several published studies show the incidence of this 2G allele is significantly higher in aggressive and metastatic tumors. Binding of an adjacent transcription factor DNA consensus site, activator protein -1 (AP1) site at -1607 has been shown to cooperate with ETS binding to activate transcription of the MMP-1 gene. We have reported a significant increase in the 2G/2G MMP-1 genotype in glioblastomas (pPurpose: To determine if a novel SNP decoy can inhibit the 2G genotype-dependent increase in MMP-1 transcriptional activity, three specific aims were tested: one, to verify specificity of binding of a transcription factor decoy designed to mimic the -1607 SNP site within the MMP-1 promoter; two, to determine the effect of transcription factor decoy ODN on transcriptional activity of an MMP-1 promoter containing the 2G SNP at -1607; and three, to assess the effect of the transcription factor decoy ODN on MMP-1 mRNA and protein expression in treated glioma cells. Methods: Modified and unmodified decoys were designed to mimic position -1607 to -1593 of the MMP-1 promoter. The SNP decoy contains both ETS and AP1 DNA consensus sites and MMP-1 flanking sequences. We first determined optimal binding conditions with electromobility shift assays (EMSAs). The EMSA assays were used to determine the presence of Ets-1 and AP1 DNA binding activity within the glioma cell lines, T98 and U87. EMSAs were also used to determine if these transcription factors could bind to the MMP-1 promoters with and without the SNP. Lastly, EMSAs were done to determine the binding characteristics of the two modified SNP decoys (LNA-locked nucleic acid, and a PS-phosphothioate modification). The effect of the decoy on MMP-1 transcriptional activity was assessed using a Dual-Luciferase Reporter Assay. The effect of the SNP decoys on mRNA was assessed using quantitative RT-PCR, and on protein expression using a sandwich enzyme-linked immunoassay (ELISAs). Statistical analysis was done using a two-way ANOVA to evaluate the effect of the decoy on MMP-1 transcriptional activity, and protein expression. Results: EMSA results indicate that Ets-1 and AP1 probes, and MMP-1 promoter probes effectively bind proteins from glioma cell nuclear extracts. Addition of excess decoy was able to inhibit protein interactions with the 2G MMP-1 promoter probe and to a lesser extent the 1G promoter probe. The scrambled decoy had no effect. Promoter studies showed a significant increase in transcriptional activity of the 2G promoter and addition of 5 mm PS-SNP decoy could effectively prevent the increase in activity (pConclusions: U87 and T98 cell lines contain DNA binding activity of the transcription factors of interest, namely ETS-1 and AP1. The candidate transcription factors can bind to the MMP-1 promoter in the presence or absence of the 2G. Both the LNA and PS-SNP modified decoys can inhibit nuclear proteins from binding to the MMP-1 2G promoter. The PS-SNP decoy was able to inhibit MMP-1 (2G) gene transcription in a dose dependent manner, whereas the control decoy showed a consistent non-specific effect. The PS-SNP decoy inhibited MMP-1 mRNA and protein expression in glioma cells containing the 2G genotype, and to lesser extent in glioma cells containing the 1G genotype. The results presented here support the conclusion that the chimeric SNP decoy can selectively inhibit the MMP-1 promoter containing the 2G genotype.

Glioblastoma multiforme (GBM) is an aggressive cancer that affects millions of patients per year. Conventional therapies combining chemotherapeutic agents with radiation can only extend survival by a few months; therefore, there is a dire need for an effective means of treating this deadly disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), currently in the early stages of FDA pre-IND drug trials, has proven to be an effective cancer specific cytokine, able to trigger the onset of mitochondrial dysfunction and/or autophagy. GBM’s have mutations that often result in the activation of cytoprotective cell signaling pathways, preventing cancer therapeutics and even MDA-7/IL-24 treatments from being effective. Since the discovery of MDA-7/IL-24 a number of groups have shown toxic effects in a variety of tumor cells. However, the lethality of MDA-7/IL-24 is not enough to eradicate the tumor. We hypothesized two xxiii rationales for this minimalistic effect. First, the MDA-7/IL-24 gene delivery mechanisms are not efficient or second, active pro-survival pathways are playing a role in protection. Here we have shown that the inhibition of cytoprotective cell-signaling pathways using small molecule inhibitors of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; mammalian target of rapamycin (mTOR) and MEK1/2; HSP90 inhibitor 17AAG; and the autophagy-inducing drug OSU-03012 (AR-12), enhances the toxicity of MDA-7/IL-24. In addition, the use of a modified recombinant adenovirus comprised of the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, proved to be a more effective, CAR-independent means of infecting and killing GBM cells in vitro and in vivo when compared to Ad.5-mda-7. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults, affecting thousands of people worldwide every year, with a life expectancy, post diagnosis of 12 months. Surgery, radiotherapy and chemotherapy together, result in an overall mean survival not exceeding 15 months. Targeted therapeutic agents sorafenib, an oral multi kinase inhibitor, and lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, used in combination have been shown to kill GBM cells be through inhibition of major growth mediating signaling pathways that are frequently over expressed in gliomas, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT). Sorafenib can restore lapatinib induced cytotoxicity by down regulation of myeloid cell leukaemia-1 (Mcl-1) expression. Prior studies have shown Mcl-1 to play an important role in resistance to lapatinib. Furthermore, data indicated that this drug combination is able to trigger activation of autophagic and apoptotic pathways and induce endoplasmic reticulum (ER) stress response in GBM cells, collectively resulting in cell death. In conclusion, data presented here demonstrates that the combination of sorafenib and lapatinib can kill GBM cells in a greater than additive fashion, through induction of autophagy, apoptotic events (extrinsic and intrinsic) and ER stress.

The Wilms' tumor 1 gene is overexpressed in many types of cancer and is associated with poor prognosis and resistance to anti-cancer therapies. In vitro studies in non-glioma cells types have demonstrated that WTl plays a role in increased proliferation, resistance to apoptosis, and increased cellular invasion. We aimed to thoroughly characterize the expression pattern of Wilms' tumor 1 in human malignant glioma and discern its function in this complex disease process. We screened a large sample of established human malignant glioma cell lines and glioma tissue specimens of all grades for WT1 expression. The majority of cell lines and 80% of all glioma tissue expressed WTI mRNA, all of which expressed WTl(+KTS) isoforms. Further screening of the glioblastoma specimens for p53 mutation followed by logistic regression analysis demonstrated a positive correlation between WTl expression and wild-type p53 (p = 0.04). To determine if WTl and p53 functionally interacted, we generated LN-229 glioblastoma cells that stably expressed WTl. As LN-229 cells harbor a p53 mutation, transient transfection with wild-type p53 induced apoptosis. However, stable WTI expression did not protect cells from p53-mediated cell death. We then generated U87MG cells (p53 wild-type) that stably expressed WT1 to model an endogenous p53 response. It is well known that after treatment with ionizing radiation, U87MG cells readily undergo p53-mediated apoptosis. Again, WTI expression did not protect against ionizing radiation induced p53-mediated cell death. We next examined the effect of transient WTI silencing on ionizing radiation induced cell death in T98G and LN-18 cells which express endogenous WTl. Combination treatment with ionizing radiation and silencing of WTI using short interfering RNA caused a decrease in viability and clonogenic survival relative to radiation alone in both cell lines. Lastly, we studied the effect of stable WTl silencing using short hairpin RNA on glioblastoma cell tumorigenicity. Stable transduction of U25 1MG and LN-18 cells with WTI short hairpin RNA resulted in a marked decrease in proliferation. WTI silencing in U251MG cells also caused a decrease in in vitro invasion. WTl silencing in U251MG cells caused an increase in tumor latency and a decrease in tumor growth rate when cells were used to subcutaneously inoculate nude mice. Not only do these studies support an oncogenic role for WTI in glioma biology, they provide encouraging evidence that WTl may be a therapeutic target for molecular treatment of glioblastoma.

DHEA has been determined to have medically significant activity and is the parent compound to the more active metabolites; 17α-AED, 17β-AED and 17β-AET, which exhibit strong biological activity that has been attributed to androgenic, estrogenic or anti-glucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17β-AED and 17β-AET for their ability to activate the human AR, ER and GR receptors and determine the relative androgenicity, estrogenicity and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERβ may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR receptor; only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors. 17β-AED and 17α-AED, androstene epimers that produce either survival or death, were utilized to treat T98G Glioblastoma cells. We identified 26 genes oppositely regulated by 17β-AED and 17α-AED to directly affect the cellular life or death decision. Network analysis demonstrated that these 26 genes are essential to regulating three critical Glioblastoma pathways. This report, for the first time, demonstrates that naturally occurring, chemically identical adrenal hormones (17β-AED or 17α-AED) direct a cellular life or death decision through contrasting modulation of identical signaling pathways and core regulators. Chronic pressure ulcers represent a significant health problem and are characterized by hypoxia, bacterial infection, repetitive ischemia/reperfusion and altered cellular and systemic stress responses. Whole genome microarray analysis was utilized in conjunction with IPA® premiere networking software to analyze chronic wound edge tissue. IPA® network analysis identified Ubiquitin C (UBC) as the most significant network. Sixteen (16) ubiquitin C associated genes were identified to be different in the chronic pressure ulcer and normal skin control. Targeted network analysis associated core regulators to 8 UBC associated genes that are unique to chronic pressure ulcers. The identification of these genes will allow the establishment of more effective treatments for Spinal Cord Injury (SCI) patients with chronic pressure ulcers.

YKL-40 is a secreted protein that is highly up-regulated in malignant glioblastoma (GBM). Its expression is correlated with the invasive nature of GBMs and poor diagnosis of patients (Nigro et al., 2005). Previous research has shown that in astrocytes and GBM cells, YKL-40 expression is regulated by two transcription factors, NFI-X3 and STAT3, which form a complex with each other (Singh et al., 2011). Here, we show that the N-terminal domain of NFI-X3 is sufficient and required for its interaction with STAT3. We also show that the DNA-binding domain of NFI-X3 is required to induce YKL-40 expression. Thus, the interaction of NFI-X3 with STAT3 may play a role in stabilizing the otherwise weak binding of NFI-X3 to the YKL-40 promoter. Collectively, the observations made in this study shed light on the mechanisms by which NFI-X3, in concert with STAT3 regulate YKL-40 expression.

The most common malignant glioma, glioblastoma multiforme (GBM), remains an intractable tumor despite advances in therapy. Its proclivity to infiltrate surrounding brain tissue contributes greatly to its treatment failure and the grim prognosis of patients. Radiation is a staple in modern therapeutic regimens, though cells surviving radiation become more aggressive and invasive. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/Syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in human melanoma models. We show that MDA-9/Syntenin is robustly expressed in GBM cell lines and patient samples, and expression increases by tumor grade. These findings are confirmed through database analysis, which revealed MDA-9/Syntenin expression correlates with shorter survival times and patient tumors high in MDA-9/Syntenin have a worse prognosis when undergoing radiotherapy. Modulating MDA-9/Syntenin levels produced changes in invasion, angiogenesis, and signaling, indicating MDA-9/Syntenin enhances glioma pathogenesis. Overexpression of MDA-9/Syntenin enhances invasion, while knockdown inhibits invasion, migration, and anchorage-independent growth in soft agar. MDA-9/Syntenin increases activation of c-Src, P38MAPK, and NF-kB, leading to elevated MMP2 expression and IL-8 secretion. Through an orthotopic tumor model, we show that shmda-9 tumor cells formed smaller tumors and had a less invasive phenotype in vivo. Knockdown of MDA-9/Syntenin radiosensitizes GBM cells and significantly reduces post-radiation invasion gains through abrogation of radiation-induced Src and EphA2 activity. In efforts to pharmacologically inhibit MDA-9/Syntenin, we describe the effects of a novel small molecule, PDZ1i, which targets the PDZ1 domain of MDA-9/Syntenin and successfully reduces invasion gains in GBM cells following radiation. While it does not effect astrocyte radiosensitivity, PDZ1i radiosensitizes GBM cells. PDZ1i inhibits crucial GBM signaling including FAK and mutant EGFR, EGFRvIII, and can negate gains in secreted proteases, such as MMP2 and MMP9, following radiation. In a model of glioma, PDZ1i treatment combined with radiation results in less invasive tumors and extends survival. Our findings indicate that MDA-9/Syntenin is a novel and important mediator of GBM pathogenesis, and further identify it as a targetable protein that enhances radiotherapy for treatment in glioma.

Rôle des kinésines mitotiques Eg5 et MKLP-2 dans l’angiogenèse physiologique etpathologique.L’angiogenèse est un phénomène biologique complexe qui correspond à la formation de nouveauxvaisseaux à partir de vaisseaux préexistants. Ce processus essentiel est régulé par des nombreuxfacteurs, dont le plus puissant est le facteur de croissance de l’endothélium vasculaire (VEGF).Des inhibiteurs du VEGF sont actuellement utilisés dans le traitement de nombreux cancerssolides. Leur efficacité est constatée dans plusieurs études mais des résistances contre cesmolécules sont fréquemment observées. Afin d’identifier de nouvelles cibles thérapeutiques dansla voie de signalisation de VEGF, nous avons utilisé le modèle de la membrane chorioallantoïdienne(CAM) de l’embryon de poulet. Les CAM traitées au VEGF pendant 24hdéveloppent de nombreux vaisseaux. Ces tissus ont été isolés pour effectuer une analysetranscriptomique. En dehors des gènes endothéliaux déjà connus pour être régulés par le VEGF,de nouveaux gènes ont été identifiés. Nous avons focalisé notre recherche sur des gènes codantpour les kinésines mitotiques KIF11/Eg5 et KIF20A/MKLP-2 qui ont été fortement induites.Nous avons démontré qu’Eg5 et MKLP-2 sont fortement exprimées au niveau de l’endothéliumdans des tissus sains et dans des cancers solides. Des inhibiteurs chimiques spécifiques d’Eg5(dimethylenastron et ispinesib mesylate) et MKLP-2 (paprotrain) bloquent les étapes clés de laformation des vaisseaux sanguins (prolifération, adhérence et migration des cellules endothéliales),la prolifération des cellules tumorales ainsi que la formation de néo-vaisseaux dans des culturesd’anneaux aortiques. De plus, sur la CAM et chez la souris, l’inhibition de cette même kinésinediminue significativement la croissance et la vascularisation des modèles tumoraux utilisés lors dece projet (le glioblastome et le carcinome rénal). En conclusion, Eg5 et MKLP-2 pourraient descibles potentielles dans les thérapies anti-angiogéniques.Mots clés : Eg5, MKLP-2, angiogenèse, kinésine, ispinesib, dimethylenastron, glioblastome,cancer rénal / Role of the mitotic kinesins Eg5 and MKLP-2 in physiologic and pathologic angiogenesis.Angiogenesis is a complex biological phenomenon which corresponds to the formation of newblood vessels from pre-existing vessels. This process is regulated by a plethora of differentmolecules with vascular endothelial growth factor (VEGF) being one of the most important ones.VEGF inhibitors are currently used in the treatment of numerous solid cancers. Even though theefficacy of such treatment is prouven by numerous studies, resistance to anti-angiogenic therapy isa common feature. To identify new therapeutic targets downstream of VEGF, we modelized itsaction on the chick chorioallantoic membrane (CAM). VEGF-treated CAMs develop a densevascular network 24h after application. We used chick microarrays to monitor global geneexpression changes in VEGF-induced CAMs. Beside a consistent number of genes alreadydescribed to be regulated by VEGF, numerous unknown genes have been identified. We havefocused our work on the characterization of Eg5/KIF11 and MKLP-2/KIF20A, members of thekinesin family, both strongly upregulated by VEGF.We demonstrated that Eg5 and MKLP-2 are strongly expressed by blood vessels in normal andcancer tissue sections. KIF20A is involved in the proliferation and migration of endothelial cellsin vitro. We showed that chemical inhibitors specific for KIF11/Eg5 (dimethylenastron andispinesib mesylate) affect key steps in the formation of blood vessels (proliferation, adhesion andmigration of endothelial cells) and proliferation of tumor cells (glioma and renal cancer).Furthermore, in experimental glioblastoma and renal cell carcinoma models (CAM and orthotopicimplantation in mice), anti-Eg5 treatment strongly reduces tumor angiogenesis and growth. Inconclusion, Eg5 and MKLP-2 could be potential targets in anti-angiogenic therapies.Keywords: Eg5, MKLP-2, angiogenesis, kinesin, ispinesib, dimethylenastron, glioblastoma, renalcell cancer

Les glioblastomes (GBMs) sont les tumeurs cérébrales primaires les plus courantes chez l’adulte avec un pronostic fatal dans les douze mois suivant le diagnostic. De nouvelles avancées dans la connaissance de la pathologie moléculaire des GBMs et de leurs régulateurs clés sont indispensables à l’émergence de nouvelles pistes thérapeutiques. Inositol Requiring Enzyme 1 α (IRE1α) est une protéine résidente du réticulum endoplasmique (RE) agissant en tant que détecteur proximal de l’Unfolded Protein Response (UPR) en conditions physiologiques ou pathologiques. IRE1α est une enzyme bivalente possédant une activité Ser/Thr kinase et endoribonucléasique (RNase). Récemment, des mutations ponctuelles dans le gène ERN1/IRE1α ont été détectées dans les cancers chez l’homme, (en particulier dans les GBMs), et IRE1α a été proposé comme un régulateur majeur de la progression tumorale parmi les protéines kinases. Dans ce travail, nous avons montré que le blocage des deux activités Set/thr kinase et RNase dans les cellules U87-MG réprime fortement l’angiogenèse tumorale, la perfusion des vaisseaux et l’expression de facteurs pro-angiogéniques dans des modèles tumoraux de xénogreffes. Ce changement phénotypique est accompagné d’une réponse dite« d’échappement » des cellules tumorales. Celles-ci envahissent le tissu cérébral sain par migration de long des vaisseaux (mécanismes appelé co-option vasculaire). De plus, ce phénotype a été montré comme étant fonctionnellement associé au processus de transition mésenchymateuse. Par mutagenèse dirigée, nous avons montré qu’IRE1α module les processus d’angiogenèse et d’invasion par ces deux domaines catalytiques : l’activité Ser/Thr kinase d’IRE1α est essentielle pour l’angiogenèse alors que le domaine RNase d’IRE1α contrôle le phénotype invasif et co-opté. IRE1α est ainsi identifié comme un régulateur clé de la croissance du glioblastome, agissant au carrefour de signalisations majeures dans le contrôle de l’adaptation de la cellule tumorale à son micro-environnement. / Glioblastomas (GBMs) are the most common primary brain tumors in humans and remain essentially incurable. New advances in the knowledge of GBM molecular pathology and their key regulators are crucial to identify new putative ways for GBM therapy. Inositol Requiring Enzyme 1 α (IRE1α) is a transmembrane Endoplasmic Reticulum (ER)-resident protein acting as proximal sensor of the Unfolded Protein Response (UPR) in both physiological and pathological situations. IRE1α is a bivalent enzyme, displaying Ser/Thr kinase and endoribonuclease (RNase) activities in its cytosolic side. Recently, single mutations in IRE1α gene were detected in human cancers, including GBM, and IRE1α was proposed as a major contributor to tumor progression among protein kinases. In this work, we have shown that blockade of both IRE1α Ser/Thr kinase and RNase activities in U87-MG cells highly repressed tumor angiogenesis, blood perfusion and the expression of pro-angiogenic factors in human xenograft tumor models. This phenotypic change is adversely associated to the so-called "evasive response" of tumors cells. The cells began to migrate along pre-existing brain capillaries and invade healthy tissue (a process named blood vessel co-option). Moreover, this phenotype was shown to be functionally linked to the mesenchymal differentiation process. By using site-directed mutagenesis, we demonstrated that IRE1α protein modulates both angiogenesis and invasive processes through its two catalytic domains: IRE1α Ser/Thr kinase domain was essential for IRE1-mediated angiogenesis, whereas IRE1's RNase domain drove the invasive, co-opted phenotype. IRE1α is therefore identified as a key regulator of glioma progression, acting at the crossroads of major signaling networks in the control of tumor cell adaptation to its microenvironment.