RESUMO
PURPOSE: Glioblastoma (GBM) is the most aggressive adult brain cancer, with a 15 month median survivorship attributed to the existence of treatment-refractory brain tumor initiating cells (BTICs). In order to better understand the mechanisms regulating the tumorigenic properties of this population, we studied the role of the polycomb group member BMI1 in our patient-derived GBM BTICs and its relationship with CD133, a well-established marker of BTICs. METHODS: Using gain and loss-of-function studies for Bmi1 in neural stem cells (NSCs) and patient-derived GBM BTICs respectively, we assessed in vitro self-renewal and in vivo tumor formation in these two cell populations. We further explored the BMI1 transcriptional regulatory network through RNA sequencing of different GBM BTIC populations that were knocked down for Bmi1. RESULTS: There is a differential role of BMI1 in CD133-positive cells, notably involving cell metabolism. In addition, we identified pivotal targets downstream of BMI1 in CD133+ cells such as integrin alpha 2 (ITGA2), that may contribute to regulating GBM stem cell properties. CONCLUSIONS: Our work sheds light on the association of three genes with CD133-BMI1 circuitry, their importance as downstream effectors of the BMI1 signalling pathway, and their potential as future targets for tackling GBM treatment-resistant cell populations.
Assuntos
Antígeno AC133/metabolismo , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Complexo Repressor Polycomb 1/metabolismo , Antígeno AC133/genética , Animais , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-ß ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica/genética , Metástase Neoplásica/fisiopatologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias Encefálicas/fisiopatologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Estudos Prospectivos , Proteoglicanas/genética , Proteoglicanas/metabolismo , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Oligodendrocyte progenitor cell (OPC) migration is critical for effective myelination of the central nervous system. Not only during normal myelination but also during remyelination, the growth factors (GFs) and extracellular matrix (ECM) protein affect the OPC migration. Studies showed the altered levels of GFs and ECM in the demyelinating lesions. In our earlier studies, we have shown that the effect of platelet-derived growth factor alpha (PDGF-A) on OPC migration is dose- and time-dependent. In that we have shown that the physiological concentration (1 ng/ml) of PDGF-A was unable to induce OPC migration at transient exposure (30 min). However, the involvement of ECM in the regulation of PDGF-A mediated OPC migration was not clear. In the present study, we have used fibronectin (FN) as ECM. PDGF-A and FN have similar and overlapping intracellular signaling pathways including the extracellular regulated kinases 1 and 2 (ERK1/2). Here we demonstrate how physiological concentration of PDGF-A combines with FN to augment OPC migration in vitro. The present study is first of its kind to show the importance of the synergistic effects of PDGF-A and FN on peripheral recruitment of phosphorylated/activated ERK1/2 (pERK1/2), actin-pERK1/2 co-localization, and filopodia formation, which are essential for the enhanced OPC migration. These findings were further confirmed by ERK1/2 inhibition studies, using the pharmacological inhibitor U0126. An understanding of these complex interactions may lead to additional strategies for transplanting genetically modified OPCs to repair widespread demyelinated lesions.
Assuntos
Fibronectinas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Oligodendroglia/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pseudópodes/metabolismo , Células-Tronco/metabolismo , Animais , Butadienos/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Fibronectinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ligação Proteica/fisiologia , Pseudópodes/efeitos dos fármacos , Ratos , Células-Tronco/efeitos dos fármacosRESUMO
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with average disease relapse at 9 months and median survival rarely extending beyond 15 months. Brain tumor stem cells (BTSCs) have been implicated in not only initiating GBM but also conferring resistance to therapy. However, it is not clear whether the BTSC population that initiates tumor growth is also responsible for GBM recurrence. In this study, we have developed a novel in vitro treatment model to profile the evolution of primary treatment-naïve GBM BTSCs through chemoradiotherapy. We report that our in vitro model enriched for a CD15+/CD133- BTSC population, mirroring the phenotype of BTSCs in recurrent GBM. We also show that in vitro treatment increased stem cell gene expression as well as self-renewal capacity of primary GBMs. In addition, the chemoradiotherapy-refractory gene signature obtained from gene expression profiling identified a hyper-aggressive subtype of glioma. The delivery of in vitro chemoradiotherapy to primary GBM BTSCs models several aspects of recurrent GBM biology, and could be used as a discovery and drug-screening platform to uncover new biological drivers and therapeutic targets in GBM.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antígenos CD/metabolismo , Antinematódeos/farmacologia , Antineoplásicos/farmacologia , Autorrenovação Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: Brain metastases (BM) are mainly treated palliatively with an expected survival of less than 12 months after diagnosis. In many solid tumors, the human neural stem cell marker glycoprotein CD133 is a marker of a tumor-initiating cell population that contributes to therapy resistance, relapse, and metastasis. EXPERIMENTAL DESIGN: Here, we use a variant of our previously described CD133 binder to generate second-generation CD133-specific chimeric antigen receptor T cells (CAR-T) to demonstrate its specificity and efficacy against multiple patient-derived BM cell lines with variable CD133 antigen expression. RESULTS: Using both lung- and colon-BM patient-derived xenograft models, we show that a CD133-targeting CAR-T cell therapy can evoke significant tumor reduction and survival advantage after a single dose, with complete remission observed in the colon-BM model. CONCLUSIONS: In summary, these data suggest that CD133 plays a critical role in fueling the growth of BM, and immunotherapeutic targeting of this cell population is a feasible strategy to control the outgrowth of BM tumors that are otherwise limited to palliative care. See related commentary by Sloan et al., p. 477.
Assuntos
Neoplasias Encefálicas , Receptores de Antígenos Quiméricos , Humanos , Ensaios Antitumorais Modelo de Xenoenxerto , Recidiva Local de Neoplasia/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/metabolismo , Linfócitos T , Linhagem Celular Tumoral , Antígeno AC133/metabolismoRESUMO
Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Gânglios Espinais/metabolismo , Esclerose Múltipla/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Avaliação da Deficiência , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Imuno-Histoquímica , Vértebras Lombares/metabolismo , Vértebras Lombares/patologia , Esclerose Múltipla/patologia , Ratos , Ratos Endogâmicos LewRESUMO
PURPOSE: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.
Assuntos
Neoplasias Encefálicas/terapia , Ligante CD27/metabolismo , Glioblastoma/terapia , Imunoterapia/métodos , Proteômica/métodos , Transcriptoma/genética , Microambiente Tumoral/imunologia , Animais , Neoplasias Encefálicas/imunologia , Proliferação de Células , Glioblastoma/imunologia , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia , PrognósticoRESUMO
Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/patologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/genética , Células-Tronco Neoplásicas/patologia , ProteômicaRESUMO
Medulloblastoma (MB) remains a leading cause of cancer-related mortality among children. The paucity of MB samples collected at relapse has hindered the functional understanding of molecular mechanisms driving therapy failure. New models capable of accurately recapitulating tumor progression in response to conventional therapeutic interventions are urgently needed. In this study, we developed a therapy-adapted PDX MB model that has a distinct advantage of generating human MB recurrence. The comparative gene expression analysis of MB cells collected throughout therapy led to identification of genes specifically up-regulated after therapy, including one previously undescribed in the setting of brain tumors, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4). Subsequent functional validation resulted in a markedly diminished in vitro proliferation, self-renewal, and longevity of MB cells, translating into extended survival and reduced tumor burden in vivo. Targeting endothelial nitric oxide synthase, a downstream substrate of BPIFB4, impeded growth of several patient-derived MB lines at low nanomolar concentrations.
RESUMO
Mortality rates in patients diagnosed with central nervous system (CNS) tumors, originating in the brain or spinal cord, continue to remain high despite the advances in multimodal treatment regimens, including surgery, radiation, and chemotherapy. Recent success of adoptive cell transfer immunotherapy treatments using chimeric antigen receptor (CAR) engineered T cells against in chemotherapy resistant CD19 expressing B-cell lymphomas, has provided the foundation for investigating efficacy of CAR T immunotherapies in the context of brain tumor. Although significant efforts have been made in developing and translating the novel CAR T therapies for CNS tumors, including glioblastoma (GBM), researchers are yet to achieve a similar level of success as with liquid malignancies. In this review, we discuss strategies and considerations essential for developing robust preclinical models for the translation of T cell-based therapies for CNS tumors. Some of the key considerations include route of delivery, increasing persistence of T cells in tumor environment, remodeling of myeloid environment, establishing the window of treatment opportunity, harnessing endogenous immune system, designing multiple antigen targeting T cells, and rational combination of immunotherapy with the current standard of care. Although this review focuses primarily on CAR T therapies for GBM, similar strategies, and considerations are applicable to all CNS tumors in general.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Imunoterapia Adotiva , Neoplasias Experimentais , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapiaRESUMO
Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Vírus Oncolíticos , Animais , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , PeptídeosRESUMO
CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Antígeno AC133 , Animais , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Humanos , Imunoterapia , Camundongos , Células-Tronco NeoplásicasRESUMO
The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.
Assuntos
Neoplasias Encefálicas/patologia , Efrinas/metabolismo , Citometria de Fluxo/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Anticorpos/metabolismo , Sobrevivência Celular , Humanos , Microesferas , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.
Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Complexo Repressor Polycomb 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Criança , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor beta domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRalpha1 (GDNF family receptor alpha1), and heparin-bound GDNF is able to bind GFRalpha1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRalpha1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF-GFRalpha1 interaction, and the extracellular domain of GFRalpha1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF-GFRalpha1 engagement.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Heparina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Heparitina Sulfato , Humanos , Mutagênese Sítio-Dirigida , Plasmídeos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2 Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124-34. ©2018 AACR.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular , Metástase Neoplásica/tratamento farmacológico , Apomorfina/farmacologia , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas Serina-Treonina Quinases/genética , Selenoproteína W/genéticaRESUMO
Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023-37. ©2018 AACR.
Assuntos
Efrina-A2/genética , Glioblastoma/genética , Recidiva Local de Neoplasia/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Efrina-A2/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Células-Tronco Neoplásicas/patologia , Prognóstico , Radiação , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptor EphA3 , Receptores da Família Eph/antagonistas & inibidores , Receptores da Família Eph/genética , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain tumors are typically composed of heterogeneous cells that exhibit distinct phenotypic characteristics and proliferative potentials. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. This unit describes protocols for the culture and isolation BTICs. We applied culture conditions and assays originally used for normal neural stem cells (NSCs) in vitro to a variety of brain tumors. Using fluorescence-activated cell sorting for the neural precursor cell surface marker CD133/CD15, BTICs can be isolated and studied prospectively. Isolation of BTICs from GBM bulk tumor will enable examination of dissimilar morphologies, self-renewal capacities, tumorigenicity, and therapeutic sensitivities. As cancer is also considered a disease of unregulated self-renewal and differentiation, an understanding of BTICs is fundamental to understanding tumor growth. Ultimately, it will lead to novel drug discovery approaches that strategically target the functionally relevant BTIC population.
Assuntos
Neoplasias Encefálicas , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Separação Celular/métodos , Células-Tronco Neoplásicas , Células-Tronco Neurais , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Fucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Humanos , Antígenos CD15/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Peptídeos/metabolismoRESUMO
PURPOSE: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population. EXPERIMENTAL DESIGN: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. RESULTS: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/ß-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality. CONCLUSIONS: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicoproteínas/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Peptídeos/antagonistas & inibidores , Compostos de Pirvínio/farmacologia , Antígeno AC133 , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Proliferação de Células , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/mortalidade , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.