Degree awarded (2004): PhDBmS, Neuroscience Program, George Washington University / Glutamate is critically important as an excitatory neurotransmitter in the central nervous system. Increasing evidence suggests additional signaling roles for glutamate in cell proliferation and migration in normal and oncogenic states. Recently, glutamate release from glioma cells has been shown to increase tumor growth in vivo. To investigate the mechanism of glutamate enhancement of tumor growth, we investigated the effect of glutamate on tumor cell proliferation, invasion, and glioma-induced cell death. Here we demonstrate that glutamate enhances tumor growth via increasing tumor cell proliferation and inducing excitotoxic death of cells surrounding the solid tumor mass, thereby facilitating tumor expansion. The evidence that glutamate enhances tumor growth suggests that regulating extracellular levels of glutamate may restrict tumor growth. In the normal brain, extracellular glutamate levels are maintained by a family of glutamate transporters. To investigate the therapeutic potential of regulating extracellular glutamate concentrations on tumor growth, we utilized a transgenic mouse model of EAAT2 glutamate transporter overexpression. In this report, we demonstrate that increased glutamate transport limits tumor growth in vivo and provides protection against glioma-associated neuronal cell death. In addition, seizure activity, often associated with the presence of a CNS tumor, is attenuated in transgenic mice overexpressing the glutamate transporter, EAAT2. These findings suggest that glutamate transporters may provide a new therapeutic target for limiting tumor expansion and secondary epileptogenesis. / Advisory Committee: Dr. Margaret Sutherland (Chair), Dr. Steven Patierno (Chair), Dr. Tim Hales, Dr. Vincent Chiappinelli, Dr. Linda Werling, Dr. Frances Noonan

<p>Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. </p><p>Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. <i>PDGFB</i> and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a <i>PDGFB</i> coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured <i>in vitro</i>. </p><p>In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the <i>PDGFB</i>-virus model was used with <i>p53</i> or <i>Ink4a-Arf</i> deficient mice, tumors arose with shorter latency and higher frequency. Loss of <i>p53</i> or <i>Ink4a-Arf</i> seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. </p><p>Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.</p>

La famille des protéines SIBLINGs comprend la sialoprotéine osseuse (BSP), lostéopontine (OPN), la sialophosphoprotéine de dentine (DSPP), la protéine de matrice de dentine 1 (DMP1), la phosphoglycoprotéine de matrice extracellulaire (MEPE) et lénameline (ENAM). Comme leur nom lindique, ces protéines ont dabord été identifiées au niveau de la matrice minéralisée de los et de la dent. Durant la dernière décennie, notre Laboratoire et dautres équipes ont démontré que cette famille de protéines est également exprimée par un certain nombre de tissus tumoraux. Nous avons entrepris ce doctorat dans la continuité des projets de recherche menés au Laboratoire sintéressant à létude des SIBLINGs au cours de la progression tumorale et métastatique. La première partie de notre projet a consisté à investiguer les mécanismes de régulation de lexpression du gène de la BSP humaine au niveau de cellules ostéoblastiques. Le facteur de transcription Runx2 est un facteur clé de la régulation des gènes osseux. Dès lors, nous avons émis lhypothèse selon laquelle linduction de lexpression du gène de la BSP observée au cours de la différenciation ostéoblastique pourrait être sous la dépendance de ce facteur. Nous avons ensuite étudié la régulation du gène de la BSP au niveau de cellules cancéreuses mammaires. En effet, nous avons voulu déterminer si lexpression de la BSP était sous la dépendance de mécanismes de régulation transcriptionnelle différents de ceux observés au niveau de cellules dorigine osseuse. Lobjectif final étant de bloquer spécifiquement lexpression de la BSP au niveau des tumeurs. Notre stratégie a dabord consisté à identifier les principaux facteurs transcriptionnels impliqués dans cette régulation puis à en étudier limpact sur lactivité du promoteur de la BSP au niveau des deux types cellulaires considérés. Dans la deuxième partie de ce projet, nous nous sommes consacrés à létude dun autre membre de la famille des SIBLINGs, lOPN, afin den identifier le rôle au niveau des gliomes humains. Précédemment, il a été démontré que lOPN est surexprimée dans les gliomes et ce, en corrélation avec le grade de la tumeur. Cependant, il ny a que peu détudes décrivant le rôle de lOPN dans les gliomes. Dès lors, nous avons voulu vérifier limportance de lOPN au cours du développement des gliomes en utilisant la technique dinterférence à lARN.

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. PDGFB and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a PDGFB coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured in vitro. In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the PDGFB-virus model was used with p53 or Ink4a-Arf deficient mice, tumors arose with shorter latency and higher frequency. Loss of p53 or Ink4a-Arf seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.

The leucine-rich repeats and immunoglobulin-like domains (LRIG) family consists of three integral membrane proteins that are important in human cancer. LRIG1 is a negative regulator of growth factor signaling. Its expression is associated with longer survival in several cancer types, and the gene has been shown to function as a tumor suppressor. The roles of LRIG2 and LRIG3 are less well known. The aim of this thesis was to improve our understanding of the expression and function of the LRIG protein family in psoriasis and cancer. To investigate their expression in psoriasis, the mRNA levels and subcellular localization of the LRIG proteins were analyzed and compared between normal and psoriatic human skin. There were no differences in the LRIG mRNA levels between psoriatic and normal skin samples. However, the subcellular localization of all three LRIG proteins differed between psoriatic and normal skin. To study the physiological and molecular functions of Lrig2, we generated Lrig2E12-/- mice. These mice were viable and born at a Mendelian rate, but Lrig2E12-/- mice had an increased rate of spontaneous mortality and a transient reduction in growth rate compared to Lrig2 wild-type (wt) mice. In an orthotopic platelet-derived growth factor (PDGF)B-driven brain tumor mouse model, we studied the effect of Lrig2 on gliomagenesis. All Lrig2 wt mice developed tumors; 82% developed grade II/III tumors, and 18% developed grade IV tumors. Only 77% of the Lrig2E12-/- mice developed tumors, and they were all grade II/III tumors. Thus, Lrig2 increased the incidence and malignancy rates of PDGFB-driven gliomas. We then analyzed the effect of Lrig2 on Pdgf receptor (Pdgfr) signaling. Lrig2 had no effect on Pdgfr steady-state levels, the starvation-induced up-regulation of Pdgfrs, the phosphorylation of Pdgfrs, primary cilium formation or the PDGFBB-induced phosphorylation of Akt or Erk1/2. However, the kinetics of induction of the immediate-early genes Fos and Egr2 were altered, resulting in a more rapid induction in Lrig2E12-/- cells. We then analyzed the clinical and biological importance of LRIG1 in lung cancer. In a human lung cancer tissue micro-array (TMA), LRIG1 expression was found to be an independent positive prognostic factor for adenocarcinoma. To study the importance of Lrig1 regarding lung cancer development in vivo, we used an inducible EGFRL858R-driven mouse lung cancer model. The mice developed diffuse lung adenocarcinoma, and the tumor burden was greater in Lrig1-/- mice than in Lrig1+/+ mice (p = 0.025) at 60 days. The human lung cancer cell line H1975, with either normal or Tet-induced expression of LRIG1, was injected into the flanks of Balb/cA nude mice. Tumors formed by LRIG1-overexpressing cells were smaller than those formed by parental cells, further indicating that LRIG1 is important during lung tumor formation or growth. In vitro, LRIG1 suppressed the proliferation of H1975 cells and down-regulated the phosphorylation of MET and RET. To investigate the molecular functions of LRIG proteins further, we performed a yeast two-hybrid (YTH) screen using a peptide from the cytosolic tail of LRIG3 as bait. This screen identified LMO7 and LIMCH1 as prominent interaction partners for LRIG3. Proximity ligation assays showed that LMO7 interacted with all of the LRIG proteins at endogenous expression levels. LMO7 and LIMCH1 were expressed in all human tissues analyzed. Their expression was dramatically decreased in lung cancer compared to normal lung tissue. The expression of LMO7 was analyzed in a human lung cancer TMA. LMO7 was expressed in respiratory epithelial cells in normal lungs. However, LMO7 was only expressed in a quarter of the lung tumors. LMO7 expression was found to be an independent negative prognostic factor for lung cancer. In summary, we found that the LRIG proteins were redistributed in psoriatic skin. In a mouse glioma model, Lrig2 promoted oligodendroglioma genesis. LRIG1 was an independent positive prognostic factor in human lung cancer. Lrig1 ablation increased the tumor size in an EGFRL858R-driven lung cancer mouse model. LRIG1 expression decreased the tumor growth of human lung cancer cells in a xenograft mouse model. LMO7 interacted with all three LRIG proteins and was an independent negative prognostic factor in human lung cancer. These data demonstrate the importance of LRIG proteins in human disease.

Hedgehog-signalling in malignant gliomas The Hedgehog signalling pathway is important for the development of the central nervous system. On the other hand, aberrant induction is observed in different tumors. Immunofluorescence and real-time qRT-PCR confirmed that in some gliomas, specifically in Glioblastoma multiforme (GBM), Gli1, a transcription factor activated by signalling, is present. In general, the hedgehog pathway is initiated by binding of extracellular ligands to the transmembrane receptor Patched and leads finally to the activation of the transcription factors Gli1, Gli2, Gli3 and Gli4. Whereas Gli1 acts as an activator, Gli2 appears to be an activator but retains some repressor activities and Gli3 and Gli4 are believed to act only as inhibitors. Therefore, the determination of hedgehog activity at the level of transcription requires additional experiments measuring gene activation. For that reason, cells isolated from 13 tumors of patients with glioblastoma (WHO Grade IV) and cells from two different glioma cell lines were transfected with reporter genes. These reporter genes carried the luciferase gene from Gaussia princeps under the control of two promoters (pT109 and pT81) conjugated to Gli binding sites. The activity of the reporter genes was compared to a control plasmid with mutant Gli-binding sites. In addition reporter gene activity was analysed in the absence and presence of the hedgehog signalling inhibitor cyclopamine and the effect of cyclopamine on cellular metabolism was studied. The analysis revealed that the two cell lines and cells from 6 glioblastomas exhibited enhanced reporter gene activity compared to the activity mutant control. This points towards an enhanced expression of Gli1. In three cultures a repression was detected suggesting that Gli3 may be active in these cells. Four cultures did neither show activation nor repression. This could provide evidence that Gli1 and Gli3 effects cancel each other out or that there is no effect at all. Enhanced luciferase activity in cells from the line T98G and in cells from four primary cultures was not influenced by the hedgehog inhibitor cyclopamine, whereas one cell line significantly responded to its presence with a decreased activity. Interestingly, ATP level was suppressed by cyclopamine in cells from the line T98G and also in cells from one primary culture that responded to the inhibitor. This may point towards an effect of cyclopamine independent of smo. Since cyclopamine is a potential new substance for the treatment of tumors, the observed effect of this inhibitor even in cells without an indication of hedgehog signalling activity should be investigated in further experiments in more detail. / Der Hedgehog (Hh) -Signalweg spielt während der Embryonalentwicklung eine wichtige Rolle, so auch bei der Entstehung des zentralen Nervensystems (Varjosalo & Taipale 2008). Andererseits führt seine unregulierte Aktivität zur Ausbildung verschiedenster Tumore (Bailey et al. 2009; Fiaschi et al. 2009; Shaw et al. 2009; Velcheti & Govindan 2007). Vorausgegangene Studien wiesen durch Immunfluoreszenz und real-time qRT-PCR nach, dass auch in Gliomen, speziell in Glioblastoma multiforme, dem agressivsten Hirntumor des Menschen, Effektoren des Signalweges (Gli1) überexprimiert werden (Wang et al. 2010). Die Aktivierung des Signalweges geschieht über Bindung des Hh-Liganden an den Rezeptor Ptch und endet mit der Aktiverung der Transkriptionsfaktoren der Gli Familie (Kinzler & Vogelstein 1990; Stone et al. 1996). Die aktuell bekannten Vertreter dieser Familie sind der Aktivator der Transkription Gli1, Gli2, der als Aktivator und Repressor agieren kann sowie Gli3 und Gli4, die die Transkription inhibieren (Marine et al. 1997; Ruppert et al. 1988). Ziel dieser Arbeit war es, herauszufinden, inwieweit die Transkriptionsfaktoren der Gli-Familie in Zellen von Glioblastoma multiforme aktiv sind. Dafür wurden Zellen aus Tumormaterial isoliert und daraus Primärkulturen hergestellt. In diese 13 Primärkulturen, wie auch in zwei Gliom-Zelllinien, wurden mittels transienter Transfektion Reporterplasmide eingebracht. Diese enthielten ein Gen der Gaussia-Luciferase, das unter der Kontrolle zweier verschiedener Promotoren (pT109 und pT81) mit Bindungsmotiven für die Transkriptionsfaktoren der Gli-Familie stand. Weiterhin wurde der Einfluss des Inhibitors des Hh-Signalweges Cyclopamin auf die Gli-Aktivität und die Metabolische Aktivität der Zellen untersucht. Die Beobachtungen ergaben, dass die zwei Zelllinien und sechs der primären Kulturen eine erhöhte Luciferaseaktivität und damit gesteigerte Aktivität von Gli1 zeigten. Weiterhin wiesen vier Kulturen eine verminderte Luciferaseaktivität auf. Dies ließ darauf schließen, dass in diesen Zellen Gli3 aktiv war. In den restlichen vier Kulturen zeigte sich keine Veränderung der Luciferaseaktiviät, was für einen Aufhebungseffekt von Gli1 und Gli3 oder gar keinen Effekt spricht. Weiterhin konnte gezeigt werden, dass die Luciferaseaktivität und damit die Aktivität von Gli1 in Zellen der Zelllinie T98G und von vier Primärkulturen nicht durch Cyclopamin beeinflusst wird. Lediglich eine Probe der Primärkulturen reagierte mit einer Abnahme der Luciferaseaktivität. Außerdem konnte Cyclopamin die ATP-Produktion sowohl in Zellen von T98G als auch in Zellen der Zelllinie, deren Gli-Aktivität durch Cyclopamin vermindert wurde, senken. Dies sprach für eine Smo unabhängige Wirkung des Cyclopamins. Da Cyclopamin ein potenzielles Pharmakon für die Antitumortherapie ist, bedarf dieser Umstand näherer Untersuchungen.

Giloma derive from glial cell, which is the most common malignant and deadly primary tumor that affects the brain and nervous system, and the possible causes are not fully understood. Glioma cells are highly invasive, and can spread to distant area of the brain, this invasive behavior makes complete tumor debulking virtually impossible. Glioma even resists to high dose of radiotherapy and chemotherapy, the prognosis of malignant glioma remains dismal and the estimated median survival time is 12¡Ð15 months. The previous studies showed that the interaction of hyaluronan (HA), the abundant component of the ECM in the adult central nervous system, with cell-surface receptors, CD44 is able to mediate motility, tumor formation and multidrug resistance of glioma. In addition, the interacted between HA and CD44, that could up-regulate glioma HA production. But the effect of hyaluronan synthases (HAS) expression in this regulation mechanism was not described clearly. In this study, the HAS expression was a target gene in the rat glioma cell line¡ÐC6 on the conditions of HA addition or cd44 gene silence, respectively. The results showed that HA addition increased the HAS expression, and cd44 gene silence caused the less expression of HAS, and which could restored by HA addition. Futher, the HA addition could prolong cell proliferation , decrease the expression of the CD44 and GFAP, the astrocyte differentiation marker, and increase brain tumor stem cell marker¡Ðnestin expression, and this result could reappear by the cd44 gene silence alone. However, instead the stemness of cell, the cell toward differentiation and proliferation by HA addition after the cd44 gene silence. From those results, the interaction between HA and CD44 could exist the positive feedback to trigger the HA production, and HA could regulate cells proliferation and differentiation by interaction with CD44 in the glioma cells.

No description available.

No description available.

<p>The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens. </p><p>Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes. </p><p>This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix. </p><p>In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.</p> / Dissertation