Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years. In particular recent new avenues regarding the role of stem cells and microRNAs along with further understanding of the importance of angiogenesis, immunotherapy and explanations for the resistance of the tumors to chemotherapeutic agents and radiation therapy has been developed. It is hopeful that this new information will lead to efficacious treatment strategies for these tumors which remain a challenge. In this book a review of the latest information on these topics along with a variety of new therapeutic treatment strategies with an emphasis on molecular targeted therapies is provided

Brain tumor and brain endothelial cells' response to ionizing radiation and phytochemical treatments

Le glioblastome multiforme (GBM) représente la tumeur cérébrale primaire la plus agressive et la plus vascularisée chez l’adulte. La survie médiane après le diagnostic est de moins d’un an en l’absence de traitement. Malheureusement, 90% des patients traités avec de la radiothérapie après la résection chirurgicale d’un GBM développent une récidive tumorale. Récemment, le traitement des GBM avec radiothérapie et témozolomide, un agent reconnu pour ses propriétés antiangiogéniques, a permis de prolonger la survie médiane à 14,6 mois. Des efforts sont déployés pour identifier des substances naturelles capables d’inhiber, de retarder ou de renverser le processus de carcinogenèse. Epigallocatechin-3-gallate (EGCG), un polyphénol retrouvé dans le thé vert, est reconnu pour ses propriétés anticancéreuses et antiangiogéniques. L’EGCG pourrait sensibiliser les cellules tumorales cérébrales et les cellules endothéliales dérivées des tumeurs aux traitements conventionnels. Le chapitre II décrit la première partie de ce projet de doctorat. Nous avons tenté de déterminer si l’EGCG pourrait sensibiliser la réponse des GBM à l’irradiation (IR) et si des marqueurs moléculaires spécifiques sont impliqués. Nous avons documenté que les cellules U-87 étaient relativement radiorésistantes et que Survivin, une protéine inhibitrice de l’apoptose, pourrait être impliquée dans la radiorésistance des GBM. Aussi, nous avons démontré que le pré-traitement des cellules U-87 avec de l’EGCG pourrait annuler l’effet cytoprotecteur d’une surexpression de Survivin et potentialiser l’effet cytoréducteur de l’IR. Au chapitre III, nous avons caractérisé l’impact de l’IR sur la survie de cellules endothéliales microvasculaires cérébrales humaines (HBMEC) et nous avons déterminé si l’EGCG pouvait optimiser cet effet. Bien que les traitements individuels avec l’EGCG et l’IR diminuaient la survie des HBMEC, le traitement combiné diminuait de façon synergique la survie cellulaire. Nous avons documenté que le traitement combiné augmentait la mort cellulaire, plus spécifiquement la nécrose. Au chapitre IV, nous avons investigué l’impact de l’IR sur les fonctions angiogéniques des HBMEC résistantes à l’IR, notamment la prolifération cellulaire, la migration cellulaire en présence de facteurs de croissance dérivés des tumeurs cérébrales, et la capacité de tubulogenèse. La voie de signalisation des Rho a aussi été étudiée en relation avec les propriétés angiogéniques des HBMEC radiorésistantes. Nos données suggèrent que l’IR altère significativement les propriétés angiogéniques des HBMEC. La réponse aux facteurs importants pour la croissance tumorale et l’angiogenèse ainsi que la tubulogenèse sont atténuées dans ces cellules. En conclusion, ce projet de doctorat confirme les propriétés cytoréductrices de l’IR sur les gliomes malins et propose un nouveau mécanisme pour expliquer la radiorésistance des GBM. Ce projet documente pour la première fois l’effet cytotoxique de l’IR sur les HBMEC. Aussi, ce projet reconnaît l’existence de HBMEC radiorésistantes et caractérise leurs fonctions angiogéniques altérées. La combinaison de molécules naturelles anticancéreuses et antiangiogéniques telles que l’EGCG avec de la radiothérapie pourrait améliorer l’effet de l’IR sur les cellules tumorales et sur les cellules endothéliales associées, possiblement en augmentant la mort cellulaire. Cette thèse supporte l’intégration de nutriments avec propriétés anticancéreuses et antiangiogéniques dans le traitement des gliomes malins pour sensibiliser les cellules tumorales et endothéliales aux traitements conventionnels.Glioblastoma multiform (GBM) represents the most aggressive and vascularised primary cerebral neoplasm in adults. Median length of survival without further therapy is usually less than one year from the time of diagnosis. Unfortunately, 90% of patients receiving radiotherapy following GBM resection develop a tumor recurrence. More recently, treatment of GBM with combined radiotherapy and temozolomide, an agent recognized for its antiangiogenic activity, increased the median survival to 14,6 months. Efforts have been oriented towards identifying naturally occurring substances capable of inhibiting, delaying or reversing the multi-stage carcinogenesis process. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has been recognized for its anticancerous and antiangiogenic property. EGCG may represent a potential agent capable of sensitizing brain tumor cells and their derived endothelial cells (ECs) to conventional treatments. In chapter II, the first part of this doctorate project aimed at determining if EGCG, in synergy with radiotherapy, can sensitize GBM’s response to radiation and whether specific molecular markers are involved. We documented that U-87 cells were relatively radioresistant and that Survivin, an inhibitor of apoptosis protein, may be involved in GBM’s radioresistance. We also found that pre-treatment of U-87 cells with EGCG could overcome the cytoprotective effect of Survivin overexpression and potentiate the cytoreductive effect of irradiation (IR). In chapter III, we characterized the impact of IR on human brain microvascular endothelial cell (HBMEC) survival and determined whether EGCG, could optimize this effect. We found that although EGCG treatment and IR individually decreased HBMEC survival, the combined treatment synergistically reduced survival. We documented that the combined treatment increased cell death, more specifically necrosis. In chapter IV, we investigated the impact of IR exposure on the angiogenic functions i.e. cell proliferation, cell migration in response to brain tumor-derived growth factors, and capacity for tubulogenesis of surviving human brain tumor-derived ECs. The Rho signalling pathway was also investigated in relation to the functional properties of radioresistant HBMEC. Our data suggests that IR significantly alters radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis. Response to growth factors important for tumor expansion and angiogenesis is significantly attenuated in these cells. In conclusion, this doctorate project confirmed IR’s cytoreductive properties on malignant gliomas. We proposed a novel mechanism to explain GBMs’ radioresistance. This project documented for the first time IR’s cytotoxic effect in HBMEC. It also described the existence of radioresistant HBMEC and characterized their altered angiogenic functions. The combination of natural anticancerous and antiangiogenic molecules such as EGCG with radiotherapy could improve IR’s effect on human malignant glioma cells and microvascular ECs, especially through increased necrosis of HBMEC. The thesis supports integrating nutrients bearing anticancerous and antiangiogenic properties, such as EGCG, in the management of gliomas to sensitize tumor and tumor-associated ECs to conventional therapies

Antioxidants stimulate BACH1-dependent tumor angiogenesis

Reactive oxygen species (ROS) can inactivate tumor suppressors, activate transcription factors, and stimulate tumor progression, which partly explains the belief that antioxidant supplements should fight cancer. Yet, despite decades of studies, the cancer-fighting potential of antioxidants has never been clearly established. Large clinical trials show that antioxidant supplementation either has no effect or increases cancer risk. Moreover, studies from my host group showed that antioxidants promote malignant melanoma and lung cancer metastasis. Indeed, endogenous and exogenous antioxidants stabilize the transcription factor BTB and CNC homology 1 (BACH1) which increases GAPDH and HK2 gene expression, stimulates aerobic glycolysis, and thus fuels metastasis. In this thesis I have addressed two questions that arose from the earlier studies. First, as glycolysis is often linked with angiogenesis, I wanted to test the hypothesis that antioxidants through BACH1 can stimulate angiogenesis. Second, our team wanted to study the impact of antioxidant administration on the progression of hematological malignancies, such as B cell lymphoma, where cancer cells can be professional ROS-producing cells, and determine if they respond similarly as solid tumors. In paper I, we demonstrate that antioxidant-stabilized BACH1 directly controls angiogenesis and glycolysis gene expression in lung cancer spheroids, tumor organoids, and xenograft tumors. Moreover, while HIF1α can control BACH1 expression under both normoxia and hypoxia, BACH1’s ability to stimulate angiogenesis gene expression is HIF1α-independent. In vivo, we found that antioxidant administration increased BACH1-dependent tumor angiogenesis and renders tumors more sensitive to anti-angiogenesis therapy. Notably, BACH1 expression in lung cancer patients’ tumor sections correlates with angiogenesis markers. We conclude that BACH1 is a novel redox and oxygen-sensitive transcription factor. In paper II, we found that high MYC expression appears to promote proliferation and shield B lymphoma cells from apoptosis at steady-state ROS levels and that compounds such as VitC and NAC in doses that reduce ROS levels induce apoptosis. Injections of VitC and NAC effectively curbed tumor growth from B lymphoma cells with high but not low MYC expression. Consequently, MYC knockdown imparts resistance to VitC and NAC whereas MYC activation makes B cells responsive to these compounds. Mechanistically, VitC and NAC stimulate MYC’s binding to EGR1 via Cys117 of MYC, shifting its transcriptional focus from cell cycle to apoptosis gene expression. Our findings establish a redox-regulated mechanism through which MYC sustains proliferation and averts apoptosis, suggesting a potential use of VitC or NAC as therapeutic agents for MYC-driven B cell lymphoma. Overall, the studies put forward in this thesis provide a refined understanding of the complex role of ROS, oxidative stress, and antioxidants in cancer

The regulatory mechanisms of enhancing IGF-1R expression and activity and the impact on therapy resistance of melanoma cells

Der IGF-1R spielt unter den RTKs mit die wichtigste Rolle in der Krebs-Entwicklung. Der IGF-1R und seine Liganden regulieren verschiedenste Zellfunktionen, die wesentlich für die Entstehung des malignen Phänotyps sind; einschließlich Zellproliferation, Überleben, Wachstum unabhängig von Zellkontakthemmung, Tumorneovaskularisierung sowie Migration, Invasion und Metastasierung der Tumorzellen. In der Klinik konnte jedoch bisher kein klarer Mechanismus von dysreguliertem IGF-1R gefunden werden: Der Rezeptor weist keine intrinsische fehlerhafte Veränderung auf und auch eine erhöhte Expression des Rezeptors durch Genamplifikation ist äußerst selten. Deshalb ist das Verständnis der molekularen Mechanismen, die die Expression und Aktivität des IGF-1Rs in Melanom-Zellen regulieren, unentbehrlich. Unsere Studie zeigt, dass die Expression von IGF-1R in Melanomzellen durch einen intrinsischen Mechanismus reguliert wird; außerdem spielen die erhöhte Expression des IGF-1R durch Hyperaktivierung von BRAF sowie der Phosphatase-Aktivität von PTEN eine Rolle. Darüber hinaus stabilisiert der Kontakt von Melanomzellen mit dermalen Fibroblasten die Expression von IGF-1R auf deren Zellmembran. Erhöhte Expression und Aktivität von IGF-1R können Resistenz gegenüber der Behandlung mit z.B. BRAF-Inhibitoren wie PLX4032 vermitteln. Interessanterweise können PTEN-negative Melanomzellen der negativen Wirkung der IGF-1R Inhibition durch eine verminderte Expression des IGF-1R entgehen. Das bedeutet, dass nur in Melanompatienten mit einem PTEN-positiven Tumor die Behandlung mit Hemmstoffen gegen IGF-1R eine passende Strategie darstellt, um die Therapieresistenz zu bekämpfen. Unsere Studie hebt hervor, dass das Mikromilieu des Tumors und Tumorzell-intrinsische Mechanismen die Expression des IGF-1R regulieren und dadurch das Therapieansprechen auf zielgerichtete Hemmstoffe beeinflussen können

Integrins in Tumorigenesis and Cancer Cell Invasion

The integrin family of transmembrane receptors are important for cell-matrix adhesion and signal transmission to the interior of the cell. Integrins are essential for many physiological processes and defective integrin function can consequently result in a multitude of diseases, including cancer. Integrin traffic is needed for completion of cytokinesis and cell division failure has been proposed to be an early event in the formation of chromosomally aberrant and transformed cells. Impaired integrin traffic and changes in integrin expression are known to promote invasion of malignant cells. However, the direct roles of impaired integrin traffic in tumorigenesis and increased integrin expression in oncogene driven invasion have not been examined. In this study we have investigated both of these aspects. We found that cells with reduced integrin endocytosis become binucleate and subsequently aneuploid. These aneuploid cells display characteristics of transformed cells; they are anchorage-independent, resistant to apoptosis and invasive in vitro. Importantly, subcutaneous injection of the aneuploid cells into athymic nude mice produced highly malignant tumors. Through gene expression profiling and analysis of integrin-triggered signaling pathways we have identified several molecules involved in the malignancy of these cells, including Src kinase and the transcription factor Twist2. Thus, even though chromosomal aberrations are associated with reduced cell fitness, we show that aneuploidy can facilitate tumor evolution and selection of transformed cells. Invasion and metastasis are the primary reason for deaths caused by cancer and the molecular pathways responsible for invasion are therefore attractive targets in cancer therapy. In addition to integrins, another major family of adhesion receptors are the proteoglycans syndecans. Integrins and syndecans are known to signal in a synergistic manner in controlling cell adhesion on 2D matrixes. Here we explored the role of syndecans as α2β1 integrin co-receptors in 3D collagen. We show that in breast cancer cells harbouring mutant K-Ras, increased levels of integrins, their co-receptors syndecans and matrix cleaving proteases are necessary for the invasive phenotype of these cells. Together, these findings increase our knowledge of the complicated changes that occur during tumorigenesis and the pathways that control the ability of cancer cells to invade and metastasize.Siirretty Doriast

Uncovering p53 Mutations and Abnormal Gene Expression in Pediatric Adrenocortical Cancer

Pediatric adrenocortical cancer is extremely rare and often fatal (approximately 0.3-0.4 cases per million worldwide; 50% 5-year survival). The incidence of pediatric adrenocortical cancer in southern Brazil is 10-15 times higher than the worldwide incidence. Due to the rarity of adrenocortical cancer, especially in children, underlying gene dysregulation and mechanisms of tumorigenesis of the adrenal gland are very poorly described in the literature. However, it is well-known that the tumor suppressor p53, which is mutated in over 50% of all human cancers, is commonly mutated in pediatric adrenocortical cancer. In addition, evidence strongly suggests that if a child has adrenocortical cancer, it indicates a germline p53 mutation exists. In order to provide an understanding of the etiology and the biology of this disease, blood and tumor samples from 35 pediatric adrenocortical tumor patients, including 24 from southern Brazil, were screened for p53 mutations. Matched blood and tumor samples were obtained as available. Of the 35 patient samples screened, 24 samples were entered into a novel gene expression study that exclusively investigated gene dysregulation in pediatric adrenocortical cancer; the first study of its kind. Overall findings from this study reveal the importance of screening for germline p53 mutations and provide fundamental insight into pediatric adrenocortical cancer

Investigation of apoptosis by conditional gene expression

Apoptosis is a form of cell death that occurs in individual cells in normal and diseased tissues. It occurs as a consequence of activation of a program of molecular events that results in the dismantlement and clearance of cells, often without induction of an inflammatory response. Many of the molecular components that regulate and execute apoptosis have been identified. Some of these regulators are, or are related to, genes that are altered in oplasia, such as c- myc,p53 and Bcl -2. The effector molecules belong to a class of cysteine proteases, known the ICE -like proteases (e.g. Nedd2). Whilst the known components can have identifiable activity in apoptosis id (or) cell cycle control, it has been suggested that they interact to control apoptosis. Further, it is not known nv the regulatory molecules interact upon downstream effectors to control apoptosis induction. Until recently, has not been possible to examine the role of apoptosis genes in combination other than by combinatorial mouse lockouts - a very time- consuming and expensive exercise. It was therefore decided to direct the expression of , veral apoptosis -related genes (namely c -myc, p53, p21 WAF /CIP, and Nedd2) in tissue culture in order to .ovide useful tools to answer questions about the role of such genes in the control of apoptosis.Owing to the limitations of existing conventional expression technology, where test genes are constitutively ,overexpressed from a strong viral promoter often in transient expression assays, use was made of conditional inigenes: 1) A temperature- sensitive (ts) murine p53 (p53va1135) was employed to investigate the sensitivity of :tivated c- Ha -ras- transformed rat embryo fibroblasts (Clone 6) to DNA -damage induced by the genotoxic iemotherapeutic drugs etoposide and bleomycin; 2) An oestrogen -regulable c- myc -oestrogen receptor hormone nding domain fusion protein (myc -ER) was used in conjunction with p53va1135 in order to investigate whether ,rced expression of phenotypically wild -type p53 was sufficient to trigger apoptosis by c -myc in Clone 6 and at -1 fibroblasts; and 3) Vectors were constructed that contain apoptosis genes under the control of semi - rnthetic promoters based upon the inducible E. coli lac operator- repressor system or a promoter containing east Gal-4 binding sites inducible by a tamoxifen -sensitive VP16GaElen chimaeric trans- activator protein.Results showed that: 1) Expression of ts p53 at the permissive temperature protected Clone 6 cells from rtotoxic drug- induced apoptosis, probably by enforcing a cell cycle arrest in G1. 2) Co- expression of myc -ER id ts p53 yielded no stable cell lines probably due to biologically significant basal activation of both p53val 135 id myc -ER under the culture conditions used. This is consistent with a co- operative role for c -myc and p53 in apoptosis triggering. 3) Control of expression in Rat -1 cells of the ICE -like protease Nedd2 (the mouse 3mologue of human ICH- I) by the VP I6GalER`m system was tight enough to allow development of stably - ansfected cells, which upon induction with 4- hydroxytamoxifen, rapidly underwent apoptosis. In contrast, ansfections with a LacI- repressible Nedd2 expression vector could not produce repressed stable expression vets that were low enough to be compatible with colony survival following selection in tissue culture.In this work, conditional expression technology was applied to the problem of control of apoptosis and shows tat gene expression experiments that increase the susceptibility of cells to apoptosis can be carried out in a :gulated fashion. Using this approach, a cytoprotective role of wild type p53 -mediated growth arrest was discovered that was abrogated by c -myc. In addition, cell lines were developed that are suitable for the biochemical characterisation of the action of Nedd2 protease

Mechanism Based Anticancer Drugs that Degrade Sp Transcription Factors

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. We demonstrated that curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of survivin, VEGF and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on downregulation of Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. Curcumin decreases expression of Sp1, Sp3 and Sp4 in blader cancer cells indicating that the cancer chemotherapeutic activity of curcumin is due, in part, to decreased expression of Sp transcription factors and Sp-dependent genes. Betulinic acid (BA) and curcumin are phytochemical anticancer agents, and we hypothesized that both compounds decrease EGFR expression in bladder cancer through downregulation of specificity protein (Sp) transcription factors. BA and curcumin decreased expression of EGFR, Sp1, Sp3, Sp4 and Sp-dependent proteins in 253JB-V and KU7 cells; EGFR was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3 and Sp4 showing that EGFR is an Sp-regulated gene. Methyl 2-cyano-3,11-dioxo-18?-olean-1,12- dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid which inhibits proliferation of KU7 and 253JB-V bladder cancer cells. CDODA-Me also decreased expression of specificity protein-1 (Sp1), Sp3 and Sp4 transcription factors. Similar results were observed for a structurally-related triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which is currently in clinical trials for treatment of leukemia. Celastrol, a naturally occurring triterpenoid acid from an ivy-like vine exhibits anticancer activity against bladder cancer cells. Celastrol decreased cell proliferation, induced apoptosis and decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-dependent genes like Fibroblast growth factor receptor 3 (FGFR3). In vivo studies using KU7 cells as xenografts showed that celastrol represents novel class of anticancer drugs that acts, in part, through targeting downregulation of Sp transcription factors