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1.
Neuro Oncol ; 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38831719

RESUMO

Brain metastases (BM) constitute an increasing challenge in oncology due to their impact on neurological function, limited treatment options, and poor prognosis. BM occur through extravasation of circulating tumor cells across the blood-brain barrier. However, the extravasation processes are still poorly understood. We here propose a brain colonization process which mimics infarction-like microenvironmental reactions, that is dependent on Angiopoietin (Ang-2) and vascular endothelial growth factor (VEGF). In this study, intracardiac BM models were used, and cerebral blood microcirculation was monitored by 2-photon microscopy through a cranial window. BM formation was observed using cranial magnetic resonance, bioluminescent imaging, and post-mortem autopsy. Ang-2/VEGF targeting strategies and Ang-2 gain-of-function (GOF) mice were employed to interfere with BM formation. In addition, vascular and stromal factors as well as clinical outcome were analyzed in BM patients. Blood vessel occlusions by cancer cells were detected, accompanied by significant disturbances of cerebral blood microcirculation, and focal stroke-like histological signs. Cerebral endothelial cells showed an elevated Ang-2 expression both in mouse and human BM. Ang-2 GOF resulted in an increased BM burden. Combined anti-Ang-2/anti-VEGF therapy led to a decrease in brain metastasis size and number. Ang-2 expression in tumor vessels of established human brain metastases negatively correlated with survival. Our observations revealed a relationship between disturbance of cerebral blood microcirculation and brain metastasis formation. This suggests that vessel occlusion by tumor cells facilitates brain metastatic extravasation and seeding, while combined inhibition of microenvironmental effects of Ang-2 and VEGF prevent the outgrowth of macrometastases.

2.
J Cell Physiol ; 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38775127

RESUMO

Primary, glioblastoma, and secondary brain tumors, from metastases outside the brain, are among the most aggressive and therapeutically resistant cancers. A physiological barrier protecting the brain, the blood-brain barrier (BBB), functions as a deterrent to effective therapies. To enhance cancer therapy, we developed a cancer terminator virus (CTV), a unique tropism-modified adenovirus consisting of serotype 3 fiber knob on an otherwise Ad5 capsid that replicates in a cancer-selective manner and simultaneously produces a potent therapeutic cytokine, melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24). A limitation of the CTV and most other viruses, including adenoviruses, is an inability to deliver systemically to treat brain tumors because of the BBB, nonspecific virus trapping, and immune clearance. These obstacles to effective viral therapy of brain cancer have now been overcome using focused ultrasound with a dual microbubble treatment, the focused ultrasound-double microbubble (FUS-DMB) approach. Proof-of-principle is now provided indicating that the BBB can be safely and transiently opened, and the CTV can then be administered in a second set of complement-treated microbubbles and released in the brain using focused ultrasound. Moreover, the FUS-DMB can be used to deliver the CTV multiple times in animals with glioblastoma  growing in their brain thereby resulting in a further enhancement in survival. This strategy permits efficient therapy of primary and secondary brain tumors enhancing animal survival without promoting harmful toxic or behavioral side effects. Additionally, when combined with a standard of care therapy, Temozolomide, a further increase in survival is achieved. The FUS-DMB approach with the CTV highlights a noninvasive strategy to treat brain cancers without surgery. This innovative delivery scheme combined with the therapeutic efficacy of the CTV provides a novel potential translational therapeutic approach for brain cancers.

3.
J Extracell Vesicles ; 12(10): e12363, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37759347

RESUMO

Melanoma has the highest propensity of all cancers to metastasize to the brain with a large percentage of late-stage patients developing metastases in the central nervous system (CNS). It is well known that metastasis establishment, cell survival, and progression are affected by tumour-host cell interactions where changes in the host cellular compartments likely play an important role. In this context, miRNAs transferred by tumour derived extracellular vesicles (EVs) have previously been shown to create a favourable tumour microenvironment. Here, we show that miR-146a-5p is highly expressed in human melanoma brain metastasis (MBM) EVs, both in MBM cell lines as well as in biopsies, thereby modulating the brain metastatic niche. Mechanistically, miR-146a-5p was transferred to astrocytes via EV delivery and inhibited NUMB in the Notch signalling pathway. This resulted in activation of tumour-promoting cytokines (IL-6, IL-8, MCP-1 and CXCL1). Brain metastases were significantly reduced following miR-146a-5p knockdown. Corroborating these findings, miR-146a-5p inhibition led to a reduction of IL-6, IL-8, MCP-1 and CXCL1 in astrocytes. Following molecular docking analysis, deserpidine was identified as a functional miR-146a-5p inhibitor, both in vitro and in vivo. Our results highlight the pro-metastatic function of miR-146a-5p in EVs and identifies deserpidine for targeted adjuvant treatment.


Assuntos
Neoplasias Encefálicas , Vesículas Extracelulares , Melanoma , MicroRNAs , Humanos , Astrócitos , Interleucina-6 , Interleucina-8 , Simulação de Acoplamento Molecular , MicroRNAs/genética , Microambiente Tumoral
4.
Nat Protoc ; 18(5): 1510-1542, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36859615

RESUMO

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.


Assuntos
Barreira Hematoencefálica , Células Endoteliais , Humanos , Camundongos , Animais , Citometria de Fluxo , Células Endoteliais/fisiologia , Barreira Hematoencefálica/metabolismo , Astrócitos , Neurônios
5.
Tidsskr Nor Laegeforen ; 142(14)2022 10 11.
Artigo em Norueguês | MEDLINE | ID: mdl-36226420

RESUMO

An ageing population as well as improved diagnostics, monitoring and treatment mean that an increasing incidence of brain metastases can be expected. Patients with brain metastases were previously regarded as a homogenous group with a very poor prognosis. However, the current picture is more complex. The development of new treatment methods, better molecular understanding and personalised medicine require a focus on multidisciplinary collaboration to provide optimal treatment for individual patients. This clinical review article provides an overview of important factors related to the diagnosis and treatment of patients with brain metastases.


Assuntos
Neoplasias Encefálicas , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/terapia , Humanos , Prognóstico
6.
PLoS One ; 17(9): e0273711, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36084109

RESUMO

Melanomas frequently metastasize to the brain. Despite recent progress in the treatment of melanoma brain metastasis, therapy resistance and relapse of disease remain unsolved challenges. CCT196969 is a SRC family kinase (SFK) and Raf proto-oncogene, serine/threonine kinase (RAF) inhibitor with documented effects in primary melanoma cell lines in vitro and in vivo. Using in vitro cell line assays, we studied the effects of CCT196969 in multiple melanoma brain metastasis cell lines. The drug effectively inhibited proliferation, migration, and survival in all examined cell lines, with viability IC50 doses in the range of 0.18-2.6 µM. Western blot analysis showed decreased expression of p-ERK, p-MEK, p-STAT3 and STAT3 upon CCT196969 treatment. Furthermore, CCT196969 inhibited viability in two B-Raf Proto-Oncogene (BRAF) inhibitor resistant metastatic melanoma cell lines. Further in vivo studies should be performed to determine the treatment potential of CCT196969 in patients with treatment-naïve and resistant melanoma brain metastasis.


Assuntos
Neoplasias Encefálicas , Melanoma , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Melanoma/patologia , Mutação , Recidiva Local de Neoplasia , Compostos de Fenilureia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazinas
8.
Int J Mol Sci ; 22(22)2021 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-34830178

RESUMO

Melanomas have a high potential to metastasize to the brain. Recent advances in targeted therapies and immunotherapies have changed the therapeutical landscape of extracranial melanomas. However, few patients with melanoma brain metastasis (MBM) respond effectively to these treatments and new therapeutic strategies are needed. Cabozantinib is a receptor tyrosine kinase (RTK) inhibitor, already approved for the treatment of non-skin-related cancers. The drug targets several of the proteins that are known to be dysregulated in melanomas. The anti-tumor activity of cabozantinib was investigated using three human MBM cell lines. Cabozantinib treatment decreased the viability of all cell lines both when grown in monolayer cultures and as tumor spheroids. The in vitro cell migration was also inhibited and apoptosis was induced by cabozantinib. The phosphorylated RTKs p-PDGF-Rα, p-IGF-1R, p-MERTK and p-DDR1 were found to be downregulated in the p-RTK array of the MBM cells after cabozantinib treatment. Western blot validated these results and showed that cabozantinib treatment inhibited p-Akt and p-MEK 1/2. Further investigations are warranted to elucidate the therapeutic potential of cabozantinib for patients with MBM.


Assuntos
Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética
9.
Oncogene ; 40(8): 1425-1439, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33420375

RESUMO

Dysregulated iron metabolism is a hallmark of many cancers, including glioblastoma (GBM). However, its role in tumor progression remains unclear. Herein, we identified coatomer protein complex subunit zeta 1 (COPZ1) as a therapeutic target candidate which significantly dysregulated iron metabolism in GBM cells. Overexpression of COPZ1 was associated with increasing tumor grade and poor prognosis in glioma patients based on analysis of expression data from the publicly available database The Cancer Genome Atlas (P < 0.001). Protein levels of COPZ1 were significantly increased in GBM compared to non-neoplastic brain tissue samples in immunohistochemistry and western blot analysis. SiRNA knockdown of COPZ1 suppressed proliferation of U87MG, U251 and P3#GBM in vitro. Stable expression of a COPZ1 shRNA construct in U87MG inhibited tumor growth in vivo by ~60% relative to controls at day 21 after implantation (P < 0.001). Kaplan-Meier analysis of the survival data demonstrated that the overall survival of tumor bearing animals increased from 20.8 days (control) to 27.8 days (knockdown, P < 0.05). COPZ1 knockdown also led to the increase in nuclear receptor coactivator 4 (NCOA4), resulting in the degradation of ferritin, and a subsequent increase in the intracellular levels of ferrous iron and ultimately ferroptosis. These data demonstrate that COPZ1 is a critical mediator in iron metabolism. The COPZ1/NCOA4/FTH1 axis is therefore a novel therapeutic target for the treatment of human GBM.


Assuntos
Proteína Coatomer/genética , Ferritinas/genética , Glioblastoma/genética , Coativadores de Receptor Nuclear/genética , Oxirredutases/genética , Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Ferroptose/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/genética
10.
Neurooncol Adv ; 3(1): vdab151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34988446

RESUMO

Brain metastasis (BM) is a major cause of cancer patient morbidity. Clinical magnetic resonance imaging (MRI) and positron emission tomography (PET) represent important resources to assess tumor progression and treatment responses. In preclinical research, anatomical MRI and to some extent functional MRI have frequently been used to assess tumor progression. In contrast, PET has only to a limited extent been used in animal BM research. A considerable culprit is that results from most preclinical studies have shown little impact on the implementation of new treatment strategies in the clinic. This emphasizes the need for the development of robust, high-quality preclinical imaging strategies with potential for clinical translation. This review focuses on advanced preclinical MRI and PET imaging methods for BM, describing their applications in the context of what has been done in the clinic. The strengths and shortcomings of each technology are presented, and recommendations for future directions in the development of the individual imaging modalities are suggested. Finally, we highlight recent developments in quantitative MRI and PET, the use of radiomics and multimodal imaging, and the need for a standardization of imaging technologies and protocols between preclinical centers.

11.
Cell Death Differ ; 28(1): 367-381, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32814880

RESUMO

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/ß and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.


Assuntos
Glioblastoma/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , NF-kappa B/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/patologia , Humanos , Quinase I-kappa B/metabolismo , Masculino , Camundongos , Camundongos Nus , Antígenos de Histocompatibilidade Menor/genética , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação , Proteínas Repressoras/genética , Transdução de Sinais , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
12.
ACS Appl Mater Interfaces ; 12(39): 43408-43421, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32885649

RESUMO

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. Currently, interventions are lacking, the median overall survival of patients with GBM is less than 15 months, and the postoperative recurrence rate is greater than 60%. We proposed an innovative local chemotherapy involving the construction of gene therapy-based iron oxide nanoparticles (IONPs) as a treatment for patients with glioblastoma after surgery that targeted ferroptosis and apoptosis to address these problems. The porous structure of IONPs with attached carboxyl groups was modified for the codelivery of small interfering RNA (siRNA) targeting glutathione peroxidase 4 (si-GPX4) and cisplatin (Pt) with high drug loading efficiencies. The synthesized folate (FA)/Pt-si-GPX4@IONPs exerted substantial effects on glioblastoma in U87MG and P3#GBM cells, but limited effects on normal human astrocytes (NHAs). During intracellular degradation, IONPs significantly increased iron (Fe2+ and Fe3+) levels, while Pt destroyed nuclear DNA and mitochondrial DNA, leading to apoptosis. Furthermore, IONPs increased H2O2 levels by activating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). The Fenton reaction between Fe2+, Fe3+, and intracellular H2O2 generated potent reactive oxygen species (ROS) to initiate ferroptosis, while the co-released si-GPX4 inhibited GPX4 expression and synergistically improved the therapeutic efficacy through a mechanism related to ferroptosis. As a result, superior therapeutic effects with low systemic toxicity were achieved both in vitro and in vivo, indicating that our nanoformulations might represent safe and efficient ferroptosis and apoptosis inducers for use in combinatorial glioblastoma therapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Cisplatino/farmacologia , Glioblastoma/tratamento farmacológico , Nanopartículas/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Compostos Férricos/química , Glioblastoma/metabolismo , Glioblastoma/cirurgia , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Tamanho da Partícula , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Porosidade , RNA Interferente Pequeno/química , Propriedades de Superfície
13.
Front Immunol ; 11: 1281, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32765489

RESUMO

Background: The tumor microenvironment (TME) of human glioblastoma (GBM) exhibits considerable immune cell infiltration, and such cell types have been shown to be widely involved in the development of GBM. Here, weighted correlation network analysis (WGCNA) was performed on publicly available datasets to identify immune-related molecules that may contribute to the progression of GBM and thus be exploited as potential therapeutic targets. Methods: WGCNA was used to identify highly correlated gene clusters in Chinese Glioma Genome Atlas glioma dataset. Immune-related genes in significant modules were subsequently validated in the Cancer Genome Atlas (TCGA) and Rembrandt databases, and impact on GBM development was examined in migration and vascular mimicry assays in vitro and in an orthotopic xenograft model (GL261 luciferase-GFP cells) in mice. Results: WGCNA yielded 14 significant modules, one of which (black) contained genes involved in immune response and extracellular matrix formation. The intersection of these genes with a GO immune-related gene set yielded 47 immune-related genes, five of which exhibited increased expression and association with worse prognosis in GBM. One of these genes, TREM1, was highly expressed in areas of pseudopalisading cells around necrosis and associated with other proteins induced in angiogenesis/hypoxia. In macrophages induced from THP1 cells, TREM1 expression levels were increased under hypoxic conditions and associated with markers of macrophage M2 polarization. TREM1 siRNA knockdown in induced macrophages reduced their ability to promote migration and vascular mimicry in GBM cells in vitro, and treatment of mice with LP-17 peptide, which blocks TREM1, inhibited growth of GL261 orthotopic xenografts. Finally, blocking the cytokine receptor for CSF1 in induced macrophages also impeded their potential to promote tumor migration and vascular mimicry in GBM cells. Conclusions: Our results demonstrated that TREM1 could be used as a novel immunotherapy target for glioma patients.


Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Glioblastoma/genética , Glioblastoma/imunologia , Imunidade/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Biologia Computacional , Bases de Dados Genéticas , Modelos Animais de Doenças , Progressão da Doença , Imunofluorescência , Perfilação da Expressão Gênica , Inativação Gênica , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Prognóstico , Transcriptoma , Receptor Gatilho 1 Expresso em Células Mieloides/genética
14.
Cancer Res ; 80(20): 4314-4323, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32641416

RESUMO

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood-brain barrier, blood-tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.


Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias Experimentais/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Humanos , Camundongos , Ratos , Tropismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Am J Cancer Res ; 10(2): 545-563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32195026

RESUMO

Brain metastasis is a major cause of mortality in melanoma patients. The blood-brain barrier (BBB) prevents most anti-tumor compounds from entering the brain, which significantly limits their use in the treatment of brain metastasis. One strategy in the development of new treatments is to assess the anti-tumor potential of drugs currently used in the clinic. Here, we tested the anti-tumor effect of the BBB-penetrating antipsychotic trifluoperazine (TFP) on metastatic melanoma. H1 and Melmet1 human metastatic melanoma cell lines were used in vitro and in vivo. TFP effects on viability and toxicity were evaluated in proliferation and colony formation assays. Preclinical, therapeutic efficacy was evaluated in NOD/SCID mice, after intracardial injection of tumor cells. Molecular studies using immunohistochemistry, western blots, immunofluorescence and transmission electron microscopy were used to gain mechanistic insight into the biological activity of TFP. Our results showed that TFP decreased cell viability and proliferation, colony formation and spheroid growth in vitro. The drug also decreased tumor burden in mouse brains and prolonged animal survival after injection of tumor cells (53.0 days vs 44.5 days), TFP treated vs untreated animals, respectively (P < 0.01). At the molecular level, TFP treatment led to increased levels of LC3B and p62 in vitro and in vivo, suggesting an inhibition of autophagic flux. A decrease in LysoTracker Red uptake after treatment indicated impaired acidification of lysosomes. TFP caused accumulation of electron dense vesicles, an indication of damaged lysosomes, and reduced the expression of cathepsin B, a main lysosomal protease. Acridine orange and galectin-3 immunofluorescence staining were evidence of TFP induction of lysosomal membrane permeabilization. Finally, TFP was cytotoxic to melanoma brain metastases based on the increased release of lactate dehydrogenase into media. Through knockdown experiments, the processes of TFP-induced lysosomal membrane permeabilization and cell death appeared to be STAT3 dependent. In conclusion, our work provides a strong rationale for further clinical investigation of TFP as an adjuvant therapy for melanoma patients with metastases to the brain.

16.
Brain ; 143(2): 512-530, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891366

RESUMO

Long non-coding RNAs play critical roles in tumour progression. Through analysis of publicly available genomic datasets, we found that MIR22HG, the host gene of microRNAs miR-22-3p and miR-22-5p, is ranked among the most dysregulated long non-coding RNAs in glioblastoma. The main purpose of this work was to determine the impact of MIR22HG on glioblastoma growth and invasion and to elucidate its mechanistic function. The MIR22HG/miR-22 axis was highly expressed in glioblastoma as well as in glioma stem-like cells compared to normal neural stem cells. In glioblastoma, increased expression of MIR22HG is associated with poor prognosis. Through a number of functional studies, we show that MIR22HG silencing inhibits the Wnt/ß-catenin signalling pathway through loss of miR-22-3p and -5p. This leads to attenuated cell proliferation, invasion and in vivo tumour growth. We further show that two genes, SFRP2 and PCDH15, are direct targets of miR-22-3p and -5p and inhibit Wnt signalling in glioblastoma. Finally, based on the 3D structure of the pre-miR-22, we identified a specific small-molecule inhibitor, AC1L6JTK, that inhibits the enzyme Dicer to block processing of pre-miR-22 into mature miR-22. AC1L6JTK treatment caused an inhibition of tumour growth in vivo. Our findings show that MIR22HG is a critical inducer of the Wnt/ß-catenin signalling pathway, and that its targeting may represent a novel therapeutic strategy in glioblastoma patients.


Assuntos
Glioblastoma/genética , MicroRNAs/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Masculino , Camundongos Nus , RNA Longo não Codificante/genética
17.
Oncogene ; 39(5): 1125-1139, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31605013

RESUMO

The Hippo signaling pathway controls organ development and is also known, in cancer, to have a tumor suppressing role. Within the Hippo pathway, we here demonstrate, in human gliomas, a functional interaction of a transmembrane protein, prostate transmembrane protein, androgen induced 1 (PMEPA1) with large tumor suppressor kinase 1 (LATS1). We show that PMEPA1 is upregulated in primary human gliomas. The PMEPA1 isoform PMEPA1a was predominantly expressed in glioma specimens and cell lines, and ectopic expression of the protein promoted glioma growth and invasion in vitro and in an orthotopic xenograft model in nude mice. In co-immunoprecipitation experiments, PMEPA1a associated with the Hippo tumor suppressor kinase LATS1. This interaction led to a proteasomal degradation of LATS1 through recruitment of the ubiquitin ligase, neural precursor cell expressed, developmentally downregulated 4 (NEDD4), which led to silencing of Hippo signaling. Alanine substitution in PMEPA1a at PY motifs resulted in failed LATS1 degradation. Targeting of a downstream component in the Hippo signaling pathway, YAP, with shRNA, interfered with the growth promoting activities of PMEPA1a in vitro and in vivo. In conclusion, the presented work shows that PMEPA1a contributes to glioma progression by a dysregulation of the Hippo signaling pathway and thus represents a promising target for the treatment of gliomas.


Assuntos
Progressão da Doença , Glioblastoma/patologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Transdução de Sinais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Invasividade Neoplásica , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Estabilidade Proteica
18.
EMBO Mol Med ; 12(1): e10924, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31777202

RESUMO

Dysregulated cholesterol metabolism is a hallmark of many cancers, including glioblastoma (GBM), but its role in disease progression is not well understood. Here, we identified cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for the elimination of cholesterol through the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), as one of the most dramatically dysregulated cholesterol metabolism genes in GBM. CYP46A1 was significantly decreased in GBM samples compared with normal brain tissue. A reduction in CYP46A1 expression was associated with increasing tumour grade and poor prognosis in human gliomas. Ectopic expression of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. RNA-seq revealed that treatment of GBM cells with 24OHC suppressed tumour growth through regulation of LXR and SREBP signalling. Efavirenz, an activator of CYP46A1 that is known to penetrate the blood-brain barrier, inhibited GBM growth in vivo. Our findings demonstrate that CYP46A1 is a critical regulator of cellular cholesterol in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target.


Assuntos
Colesterol 24-Hidroxilase , Colesterol/metabolismo , Glioblastoma , Encéfalo/metabolismo , Colesterol 24-Hidroxilase/genética , Colesterol 24-Hidroxilase/metabolismo , Glioblastoma/metabolismo , Homeostase , Humanos
19.
J Cell Mol Med ; 24(5): 2847-2856, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31778016

RESUMO

Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.


Assuntos
Apoptose/genética , Autofagia/genética , Neoplasias Encefálicas/genética , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Proteínas com Domínio MARVEL/genética , Proteolipídeos/genética , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Glioblastoma/ultraestrutura , Humanos , Proteínas com Domínio MARVEL/metabolismo , Masculino , Camundongos , Prognóstico , Proteolipídeos/metabolismo , Fator de Transcrição CHOP/metabolismo
20.
J Exp Clin Cancer Res ; 38(1): 434, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31665089

RESUMO

BACKGROUND: Breast cancer (BC) is the most frequent malignant tumor in females and the 2nd most common cause of brain metastasis (BM), that are associated with a fatal prognosis. The increasing incidence from 10% up to 40% is due to more effective treatments of extracerebral sites with improved prognosis and increasing use of MRI in diagnostics. A frequently administered, potent chemotherapeutic group of drugs for BC treatment are taxanes usually used in the adjuvant and metastatic setting, which, however, have been suspected to be associated with a higher incidence of BM. The aim of our study was to experimentally analyze the impact of the taxane docetaxel (DTX) on brain metastasis formation, and to elucidate the underlying molecular mechanism. METHODS: A monocentric patient cohort was analyzed to determine the association of taxane treatment and BM formation. To identify the specific impact of DTX, a murine brain metastatic model upon intracardial injection of breast cancer cells was conducted. To approach the functional mechanism, dynamic contrast-enhanced MRI and electron microscopy of mice as well as in-vitro transendothelial electrical resistance (TEER) and tracer permeability assays using brain endothelial cells (EC) were carried out. PCR-based, immunohistochemical and immunoblotting analyses with additional RNA sequencing of murine and human ECs were performed to explore the molecular mechanisms by DTX treatment. RESULTS: Taxane treatment was associated with an increased rate of BM formation in the patient cohort and the murine metastatic model. Functional studies did not show unequivocal alterations of blood-brain barrier properties upon DTX treatment in-vivo, but in-vitro assays revealed a temporary DTX-related barrier disruption. We found disturbance of tubulin structure and upregulation of tight junction marker claudin-5 in ECs. Furthermore, upregulation of several members of the tubulin family and downregulation of tetraspanin-2 in both, murine and human ECs, was induced. CONCLUSION: In summary, a higher incidence of BM was associated with prior taxane treatment in both a patient cohort and a murine mouse model. We could identify tubulin family members and tetraspanin-2 as potential contributors for the destabilization of the blood-brain barrier. Further analyses are needed to decipher the exact role of those alterations on tumor metastatic processes in the brain.


Assuntos
Antineoplásicos/administração & dosagem , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/secundário , Neoplasias da Mama/tratamento farmacológico , Docetaxel/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Neoplasias Encefálicas/genética , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Claudina-5/genética , Docetaxel/farmacocinética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Camundongos , Microscopia Eletrônica , Análise de Sequência de RNA , Tubulina (Proteína)/genética , Ensaios Antitumorais Modelo de Xenoenxerto
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