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Glioblastoma (GBM) is the most malignant brain tumor of adults and is characterized by extensive cell dissemination within the brain parenchyma and enhanced angiogenesis. Effective preclinical modeling of these key features suffers from several shortcomings. Aim of this study was to determine whether modulating the expression of extracellular matrix (ECM) modifiers in proneural (PN) and mesenchymal (MES) cancer stem cells (CSCs) and in conventional glioma cell lines (GCLs) might improve tumor invasion and vascularization. To this end, we selected secreted, acidic and rich in cysteine-like 1 (SPARCL1) as a potential mediator of ECM remodeling in GBM. SPARCL1 transcript and protein expression was assessed in PN and MES CSCs as well as GCLs, in their xenografts and in patient-derived specimens by qPCR, WB and IHC. SPARCL1 expression was then enforced in both CSCs and GCLs by lentiviral-based transduction. The effect of SPARCL1 gain-of-function on microvascular proliferation, microglia activation and advanced imaging features was tested in intracranial xenografts by IHC and MRI and validated by chorioallantoic membrane (CAM) assays. SPARCL1 expression significantly enhanced the infiltrative and neoangiogenic features of PN and MES CSC/GCL-induced tumors, with the concomitant activation of inflammatory responses associated with the tumor microenvironment, thus resulting in experimental GBMs that reproduced both the parenchymal infiltration and the increased microvascular density, typical of GBM. Overall, these results indicate that SPARCL1 overexpression might be instrumental for the generation of CSC-derived preclinical models of GBM in which the main pathognomonic hallmarks of GBMs are retrievable, making them suitable for effective preclinical testing of therapeutics.
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Neoplasias Encefálicas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Xenoinjertos , Humanos , Ratones , Microglía/metabolismoRESUMEN
The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.
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Antineoplásicos/farmacología , Morfolinas/farmacología , Purinas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Benzotiazoles/farmacología , Western Blotting , Caspasas/metabolismo , Puntos de Control del Ciclo Celular , Muerte Celular , Desdiferenciación Celular , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular , Endorreduplicación , Activación Enzimática , Fibroblastos/efectos de los fármacos , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Citometría de Flujo , Silenciador del Gen , Células HeLa , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM displays poor prognosis and its resistance to common therapeutic approaches makes it a highly recurrent tumor. Several studies have identified a subpopulation of tumor cells, known as GBM cancer stem cells (CSCs) characterized by the ability of self-renewal, tumor initiation and propagation. GBM CSCs have been shown to survive GBM chemotherapy and radiotherapy. Thus, targeting CSCs represents a promising approach to treat GBM. Recent evidence has shown that GBM is characterized by a dysregulated expression of microRNA (miRNAs). In this study we have investigated the difference between human GBM CSCs and their paired autologous differentiated tumor cells. Array-based profiling and quantitative Real-Time PCR (qRT-PCR) were performed to identify miRNAs differentially expressed in CSCs. The Cancer Genome Atlas (TCGA) data were also interrogated, and functional interpretation analysis was performed. We have identified 14 miRNAs significantly differentially expressed in GBM CSCs (p < 0.005). MiR-21 and miR-95 were among the most significantly deregulated miRNAs, and their expression was also associated to patient survival. We believe that the data provided here carry important implications for future studies aiming at elucidating the molecular mechanisms underlying GBM.
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Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog-activated (SHH-activated) MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MB developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mTORC1 hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MB and CSC-derived MB resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patient samples, and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.
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Proteínas Hedgehog/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Meduloblastoma/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Humanos , Meduloblastoma/patología , RatonesRESUMEN
Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Glioblastoma/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Autorrenovación de las Células/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuronas/metabolismo , Neuronas/patología , Transducción de SeñalRESUMEN
The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted 'Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy'. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as 'Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico "P.Giaccone", Università di Palermo, Palermo, 90127, Italy'. The correct affiliation is 'Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy'. These errors have now been corrected in both the PDF and HTML versions of the Article.
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Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.
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Neoplasias de la Mama/metabolismo , Epigénesis Genética , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Carcinogénesis , Línea Celular Tumoral , Reprogramación Celular , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones SCID , Células Madre Neoplásicas/citologíaRESUMEN
Stem cells balance their self-renewal and differentiation potential by integrating environmental signals with the transcriptional regulatory network. The maintenance of cell identity and/or cell lineage commitment relies on the interplay of multiple factors including signaling pathways, transcription factors, and the epigenetic machinery. These regulatory modules are strongly interconnected and they influence the pattern of gene expression of stem cells, thus guiding their cellular fate. Embryonic stem cells (ESCs) represent an invaluable tool to study this interplay, being able to indefinitely self-renew and to differentiate towards all three embryonic germ layers in response to developmental cues. In this review, we highlight those mechanisms of signaling to chromatin, which regulate chromatin modifying enzymes, histone modifications, and nucleosome occupancy. In addition, we report the molecular mechanisms through which signaling pathways affect both the epigenetic and the transcriptional state of ESCs, thereby influencing their cell identity. We propose that the dynamic nature of oscillating signaling and the different regulatory network topologies through which those signals are encoded determine specific gene expression programs, leading to the fluctuation of ESCs among multiple pluripotent states or to the establishment of the necessary conditions to exit pluripotency.
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Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/ß-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/ß-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.
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Redes Reguladoras de Genes/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Autorrenovación de las Células/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Factor Inhibidor de Leucemia/farmacología , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Proteínas del Grupo Polycomb/metabolismo , Transcripción Genética/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem-like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor-dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5ß1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system.
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Células Madre Neoplásicas/inmunología , Neoplasias de la Próstata/inmunología , Linfocitos T/inmunología , Tenascina/fisiología , Escape del Tumor , Animales , Movimiento Celular , Proliferación Celular , Humanos , Integrina alfa5beta1/metabolismo , Metástasis Linfática , Activación de Linfocitos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias de la Próstata/patología , Fibras de Estrés/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment. METHODS: We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student's t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided. RESULTS: No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (P interaction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients. CONCLUSIONS: EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide-based therapies.
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Administración Metronómica , Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Receptores ErbB/genética , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Adulto , Análisis de Varianza , Antineoplásicos Alquilantes/administración & dosificación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirugía , Quimioterapia Adyuvante , Dacarbazina/administración & dosificación , Dacarbazina/uso terapéutico , Supervivencia sin Enfermedad , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/cirugía , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Modelos de Riesgos Proporcionales , Temozolomida , Regulación hacia ArribaRESUMEN
BACKGROUND: Issues regarding cancer stem cell (CSC) movement are important in neurosphere biology as cell-cell or cell-environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. AIMS: Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have been scarcely characterized from a morphological and ultrastructural point of view. RESULTS: Here we report a morphological and ultrastructural characterization performed by live imaging and scanning electron microscopy. Glioblastoma multiforme (GBM) CSC-derived neurospheres are heterogeneous and are constituted by cells, morphologically different, capable of forming highly dynamic structures. These dynamic structures are regulated by not serendipitous cell-cell interactions, and they synchronously pulsate following a cyclic course made of "fast" and "slow" alternate phases. Autocrine/paracrine non canonical Wnt signalling appears to be correlated with the association status of neurospheres. CONCLUSIONS: The results obtained suggest that GBM CSCs can behave both as independents cells and as "social" cells, highly interactive with other members of its species, giving rise to a sort of "multicellular organism".
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Comunicación Celular , Movimiento Celular , Células Madre Neoplásicas/fisiología , Células Cultivadas , Glioblastoma/patología , HumanosRESUMEN
According to the cancer stem cell (CSC) theory, therapies that do not target the CSC compartment have limited, if any, chances to eradicate established tumors. While cytotoxic T lymphocytes (CTLs) have the potential to recognize and kill single neoplastic cells within a tissue, whether CSCs can be targeted by the immune system during spontaneous or vaccination-elicited responses is poorly defined. Here, we provide experimental evidence showing that CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice expressed prostate cancer-associated antigens, MHC Class I and II molecules as well as ligands for natural killer (NK) cell receptors. Indeed, CSC were targets for both NK cell- and CTL-mediated cytotoxicity, both in vitro and in vivo. The administration of dendritic cells pulsed with irradiated CSCs induced a tumor-specific immune response that was more robust than that induced by dendritic cells pulsed with differentiated tumor cells, delayed tumor growth in mice challenged with prostate CSCs and caused tumor regression in TRAMP mice. Thus, CSC are targeted by both innate and adaptive immune responses and might be exploited for the design of novel immunotherapeutic approaches against cancer.
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The relevant social and economic impact of prostate adenocarcinoma, one of the leading causes of death in men, urges critical improvements in knowledge of the pathogenesis and cure of this disease. These can also be achieved by implementing in vitro and in vivo preclinical models by taking advantage of prostate cancer stem cells (PCSCs). The best-characterized mouse model of prostate cancer is the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice develop a progressive lesion called prostatic intraepithelial neoplasia that evolves into adenocarcinoma (AD) between 24 and 30 weeks of age. ADs often metastasize to lymph nodes, lung, bones, and kidneys. Eventually, approximately 5% of the mice develop an androgen-independent neuroendocrine adenocarcinoma. Here we report the establishment of long-term self-renewing PCSC lines from the different stages of TRAMP progression by application of the neurosphere assay. Stage-specific prostate cell lines were endowed with the critical features expected from malignant bona fide cancer stem cells, namely, self-renewal, multipotency, and tumorigenicity. Notably, transcriptome analysis of stage-specific PCSCs resulted in the generation of well-defined, meaningful gene signatures, which identify distinct stages of human tumor progression. As such, TRAMP-derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Células Madre Neoplásicas/metabolismo , Neoplasia Intraepitelial Prostática/genética , Neoplasias de la Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Efecto Fundador , Humanos , Masculino , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/patología , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
Glioblastoma multiforme (GBM) represents the most aggressive and deadliest brain tumor of adults. To date, cell heterogeneity within GBM has been explained by the "hierarchical" model of tumorigenesis, aka the "cancer stem cell" hypothesis. In agreement with this model, only rare tumor cells, namely the cancer stem cells (CSCs), are responsible for GBM initiation and, as such, are considered the favored target of therapy. However, multiple evidence has recently indicated that tumor-initiating cells (TICs) may not represent a restricted and infrequent GBM component; rather, they might constitute most of the cells within the tumor bulk. Here we review several studies that recently shed new light on the process of gliomagenesis. We critically analyze the methodological inconsistencies and drawbacks that are causing protracted controversy in the field. Finally, we discuss the clinical implications and the novel therapeutic scenarios that have been put forward by the presence of functionally and molecularly distinct subpopulations of GBM-initiating cells within the same tumor.
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Neoplasias Encefálicas/patología , Transformación Celular Neoplásica , Glioblastoma/patología , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Descubrimiento de Drogas , Glioblastoma/tratamiento farmacológico , Humanos , Células Madre Neoplásicas/patologíaRESUMEN
BACKGROUND: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. METHODS: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs) and mouse neural stem cells (NSCs) by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. RESULTS: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. CONCLUSIONS: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.
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Biomarcadores de Tumor/análisis , Centrosoma/enzimología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Anciano , Animales , Secuencia de Bases , Biopsia , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Genoma Humano/genética , Glioma/genética , Glioma/patología , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Células-Madre Neurales/enzimología , Células-Madre Neurales/patología , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment.
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Receptores ErbB/biosíntesis , Glioblastoma/enzimología , Células Madre Neoplásicas/enzimología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Silenciador del Gen , Técnicas de Transferencia de Gen , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/patología , Quinazolinas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tirfostinos/farmacologíaRESUMEN
PURPOSE: Cancer stem cells (CSC) have been isolated from human tumors, including glioblastoma multiforme (GBM). The aims of this study were the immunobiological characterization of GBM CSCs and the assessment of whether these cells represent suitable targets for immunotherapy. EXPERIMENTAL DESIGN: GBM CSC lines and their fetal bovine serum (FBS)-cultured non-CSC pair lines were generated and examined by flow cytometry for expression of known tumor antigens, MHC-I and MHC-II molecules, antigen-processing machinery components, and NKG2D ligands. In addition, immunogenicity and immunosuppression of such cell lines for autologous or allogeneic T lymphocytes were tested by cytokine secretion (ELISPOT) or proliferation (carboxyfluorescein diacetate succinimidyl ester) assays, respectively. RESULTS: Both GBM CSC and FBS lines were weakly positive and negative for MHC-I, MHC-II, and NKG2D ligand molecules, respectively. Antigen-processing machinery molecules were also defective in both cell types. Upregulation of most molecules was induced by IFNs or 5-Aza deoxycytidine, although more efficiently in FBS than in CSCs. Patient T-cell responses, mediated by both TH1 and the TH2 subsets, against autologous CSC could be induced in vitro. In addition, CSC but not their paired FBS tumor lines inhibited T-cell proliferation of healthy donors. Notably, a differential gene signature that was confirmed at the protein levels for some immunologic-related molecules was also found between CSC and FBS lines. CONCLUSIONS: These results indicate lower immunogenicity and higher suppressive activity of GBM CSC compared with FBS lines. The immunogenicity, however, could be rescued by immune modulation leading to anti-GBM T cell-mediated immune response.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Presentación de Antígeno , Línea Celular Tumoral , Perfilación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Linfocitos T/inmunologíaRESUMEN
The use of type I interferons (IFNs) in cancer therapy has been limited by ineffective dosing and significant toxicity. Here, we exploited the tumor-homing ability of proangiogenic Tie2-expressing monocytes (TEMs) to deliver IFN-alpha to tumors. By transplanting hematopoietic progenitors transduced with a Tie2 promoter/enhancer-driven Ifna1 gene, we turned TEMs into IFN-alpha cell vehicles that efficiently targeted the IFN response to orthotopic human gliomas and spontaneous mouse mammary carcinomas and obtained significant antitumor responses and near complete abrogation of metastasis. TEM-mediated IFN-alpha delivery inhibited tumor angiogenesis and activated innate and adaptive immune cells but did not impair myelopoiesis and wound healing detectably. These results illustrate the therapeutic potential of gene- and cell-based IFN-alpha delivery and should allow the development of IFN treatments that more effectively treat cancer.