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1.
Neuro Oncol ; 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232320

RESUMO

BACKGROUND: Glioblastoma is the most common primary malignancy of the central nervous system with dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, has failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. METHODS: We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. RESULTS: Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. CONCLUSION: This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

2.
Front Oncol ; 11: 681130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34150645

RESUMO

Glioblastoma (GBM) is the most common and aggressive type of tumour arising from the central nervous system. GBM remains an incurable disease despite advancement in therapies, with overall survival of approximately 15 months. Recent literature has highlighted that GBM releases tumoural content which crosses the blood-brain barrier (BBB) and is detected in patients' blood, such as circulating tumour cells (CTCs). CTCs carry tumour information and have shown promise as prognostic and predictive biomarkers in different cancer types. Currently, there is limited data for the clinical utility of CTCs in GBM. Here, we report the use of spiral microfluidic technology to isolate CTCs from whole blood of newly diagnosed GBM patients before and after surgery, followed by characterization for GFAP, cell-surface vimentin protein expression and EGFR amplification. CTCs were found in 13 out of 20 patients (9/20 before surgery and 11/19 after surgery). Patients with CTC counts equal to 0 after surgery had a significantly longer recurrence-free survival (p=0.0370). This is the first investigation using the spiral microfluidics technology for the enrichment of CTCs from GBM patients and these results support the use of this technology to better understand the clinical value of CTCs in the management of GBM in future studies.

3.
Cell Death Discov ; 7(1): 81, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863878

RESUMO

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

4.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924599

RESUMO

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.


Assuntos
Autorrenovação Celular , Quinase 5 Dependente de Ciclina/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Transdução de Sinais/efeitos dos fármacos
5.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919246

RESUMO

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6-9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Glioblastoma/tratamento farmacológico , Organoides , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/fisiopatologia , Células Cultivadas , Glioblastoma/fisiopatologia , Humanos , Medicina de Precisão , Microambiente Tumoral
6.
Biochem Pharmacol ; 186: 114437, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33571503

RESUMO

MerTK has been identified as a promising target for therapeutic intervention in glioblastoma. Genetic studies documented a range of oncogenic processes that MerTK targeting could influence, however robust pharmacological validation has been missing. The aim of this study was to assess therapeutic potential of MerTK inhibitors in glioblastoma therapy. Unlike previous studies, our work provides several lines of evidence that MerTK activity is dispensable for glioblastoma growth. We observed heterogeneous responses to MerTK inhibitors that could not be correlated to MerTK inhibition or MerTK expression in cells. The more selective MerTK inhibitors UNC2250 and UNC2580A lack the anti-proliferative potency of less-selective inhibitors exemplified by UNC2025. Functional assays in MerTK-high and MerTK-deficient cells further demonstrate that the anti-cancer efficacy of UNC2025 is MerTK-independent. However, despite its efficacy in vitro, UNC2025 failed to attenuate glioblastoma growth in vivo. Gene expression analysis from cohorts of glioblastoma patients identified that MerTK expression correlates negatively with proliferation and positively with quiescence genes, suggesting that MerTK regulates dormancy rather than proliferation in glioblastoma. In summary, this study demonstrates the importance of orthogonal inhibitors and disease-relevant models in target validation studies and raises a possibility that MerTK inhibitors could be used to target dormant glioblastoma cells.


Assuntos
Proliferação de Células/fisiologia , Glioblastoma/enzimologia , Células-Tronco Neoplásicas/enzimologia , c-Mer Tirosina Quinase/antagonistas & inibidores , c-Mer Tirosina Quinase/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cicloexanóis/farmacologia , Relação Dose-Resposta a Droga , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
J Nanobiotechnology ; 19(1): 60, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637089

RESUMO

BACKGROUND: Approximately 80% of brain tumours are gliomas. Despite treatment, patient mortality remains high due to local metastasis and relapse. It has been shown that transferrin-functionalised porous silicon nanoparticles (Tf@pSiNPs) can inhibit the migration of U87 glioma cells. However, the underlying mechanisms and the effect of glioma cell heterogeneity, which is a hallmark of the disease, on the efficacy of Tf@pSiNPs remains to be addressed. RESULTS: Here, we observed that Tf@pSiNPs inhibited heterogeneous patient-derived glioma cells' (WK1) migration across small perforations (3 µm) by approximately 30%. A phenotypical characterisation of the migrated subpopulations revealed that the majority of them were nestin and fibroblast growth factor receptor 1 positive, an indication of their cancer stem cell origin. The treatment did not inhibit cell migration across large perforations (8 µm), nor cytoskeleton formation. This is in agreement with our previous observations that cellular-volume regulation is a mediator of Tf@pSiNPs' cell migration inhibition. Since aquaporin 9 (AQP9) is closely linked to cellular-volume regulation, and is highly expressed in glioma, the effect of AQP9 expression on WK1 migration was investigated. We showed that WK1 migration is correlated to the differential expression patterns of AQP9. However, AQP9-silencing did not affect WK1 cell migration across perforations, nor the efficacy of cell migration inhibition mediated by Tf@pSiNPs, suggesting that AQP9 is not a mediator of the inhibition. CONCLUSION: This in vitro investigation highlights the unique therapeutic potentials of Tf@pSiNPs against glioma cell migration and indicates further optimisations that are required to maximise its therapeutic efficacies.


Assuntos
Glioma/tratamento farmacológico , Nanopartículas/uso terapêutico , Porosidade , Silício/farmacologia , Aquaporinas/genética , Neoplasias Encefálicas , Linhagem Celular Tumoral , Movimento Celular , Glioblastoma/tratamento farmacológico , Humanos , Células-Tronco Neoplásicas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos
8.
Ther Adv Med Oncol ; 12: 1758835920946259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33014146

RESUMO

Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP's transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) in vitro, but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer. Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up (n = 371), and patient-matched breast and metastatic brain tumours (n = 91; 30 pairs). Results: Membrane localisation of phospho-HER4 [pHER4(Y1162)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% versus 75% positive), where it was usually co-expressed with pHER3(Y1289) (p < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T218/Y210) (p = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S127) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53; p = 9.6E-03), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78; p = 1.7E-02), particularly when co-expressed with nuclear HER4-ICD (p = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin; p < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S127) in ER-negative cases (p < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases. Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S127) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S127) associations with breast cancer clinical outcomes were dependent on ER status. Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.

9.
Clin Transl Immunology ; 9(10): e1191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33082953

RESUMO

Objectives: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples. Methods: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short-term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high-parameter flow cytometry and single-cell transcriptomics (scRNAseq). Results: Compared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single-cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel-localised FAP is because of expression on both ECs and pericytes. Conclusion: Fibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.

10.
Neurooncol Adv ; 2(1): vdaa030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32642689

RESUMO

Background: High-grade glioma (HGG) remains a recalcitrant clinical problem despite many decades of research. A major challenge in improving prognosis is the inability of current therapeutic strategies to address a clinically significant burden of infiltrating tumor cells that extend beyond the margins of the primary tumor mass. Such cells cannot be surgically excised nor efficiently targeted by radiation therapy. Therapeutic targeting of this tumor cell population is significantly hampered by the presence of an intact blood-brain barrier (BBB). In this study, we performed a preclinical investigation of the efficiency of MR-guided Focused Ultrasound (FUS) to temporarily disrupt the BBB to allow selective delivery of a tumor-targeting antibody to infiltrating tumor. Methods: Structural MRI, dynamic-contrast enhancement MRI, and histology were used to fully characterize the MR-enhancing properties of a patient-derived xenograft (PDX) orthotopic mouse model of HGG and to develop a reproducible, robust model of nonenhancing HGG. PET-CT imaging techniques were then used to evaluate the efficacy of FUS to increase 89Zr-radiolabeled antibody concentration in nonenhancing HGG regions and adjacent non-targeted tumor tissue. Results: The PDX mouse model of HGG has a significant tumor burden lying behind an intact BBB. Increased antibody uptake in nonenhancing tumor regions is directly proportional to the FUS-targeted volume. FUS locally increased antibody uptake in FUS-targeted regions of the tumor with an intact BBB, while leaving untargeted regions unaffected. Conclusions: FUS exposure successfully allowed temporary BBB disruption, localized to specifically targeted, nonenhancing, infiltrating tumor regions and delivery of a systemically administered antibody was significantly increased.

11.
Theranostics ; 10(14): 6361-6371, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32483457

RESUMO

The clinical translation of new nanoparticle-based therapies for high-grade glioma (HGG) remains extremely poor. This has partly been due to the lack of suitable preclinical mouse models capable of replicating the complex characteristics of recurrent HGG (rHGG), namely the heterogeneous structural and functional characteristics of the blood-brain barrier (BBB). The goal of this study is to compare the characteristics of the tumor BBB of rHGG with two different mouse models of HGG, the ubiquitously used U87 cell line xenograft model and a patient-derived cell line WK1 xenograft model, in order to assess their suitability for nanomedicine research. Method: Structural MRI was used to assess the extent of BBB opening in mouse models with a fully developed tumor, and dynamic contrast enhanced MRI was used to obtain values of BBB permeability in contrast enhancing tumor. H&E and immunofluorescence staining were used to validate results obtained from the in vivo imaging studies. Results: The extent of BBB disruption and permeability in the contrast enhancing tumor was significantly higher in the U87 model than in rHGG. These values in the WK1 model are similar to those of rHGG. The U87 model is not infiltrative, has an entirely abnormal and leaky vasculature and it is not of glial origin. The WK1 model infiltrates into the non-neoplastic brain parenchyma, it has both regions with intact BBB and regions with leaky BBB and remains of glial origin. Conclusion: The WK1 mouse model more accurately reproduces the extent of BBB disruption, the level of BBB permeability and the histopathological characteristics found in rHGG patients than the U87 mouse model, and is therefore a more clinically relevant model for preclinical evaluations of emerging nanoparticle-based therapies for HGG.


Assuntos
Barreira Hematoencefálica/fisiopatologia , Glioma/patologia , Nanomedicina/métodos , Nanopartículas/administração & dosagem , Animais , Barreira Hematoencefálica/química , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Permeabilidade Capilar , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Nanopartículas/química , Ensaios Antitumorais Modelo de Xenoenxerto
12.
BMC Biol ; 18(1): 45, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32354330

RESUMO

BACKGROUND: The traditional concept that heritability occurs exclusively from the transfer of germline-restricted genetics is being challenged by the increasing accumulation of evidence confirming the existence of experience-dependent transgenerational inheritance. However, questions remain unanswered as to how heritable information can be passed from somatic cells. Previous studies have implicated the critical involvement of RNA in heritable transgenerational effects, and the high degree of mobility and genomic impact of RNAs in all organisms is an attractive model for the efficient transfer of genetic information. RESULTS: We hypothesized that RNA may be transported from a somatic tissue, in this case the brain, of an adult male mouse to the germline, and subsequently to embryos. To investigate this, we injected one hemisphere of the male mouse striatum with an AAV1/9 virus expressing human pre-MIR941 (MIR941). After 2, 8 and 16 weeks following injection, we used an LNA-based qPCR system to detect the presence of virus and human MIR941 in brain, peripheral tissues and embryos, from injected male mice mated with uninjected females. Virus was never detected outside of the brain. Verification of single bands of the correct size for MIR941 was performed using Sanger sequencing while quantitation demonstrated that a small percentage (~ 1-8%) of MIR941 is transported to the germline and to embryos in about a third of the cases. CONCLUSIONS: We show that somatic RNA can be transported to the germline and passed on to embryos, thereby providing additional evidence of a role for RNA in somatic cell-derived intergenerational effects.


Assuntos
Encéfalo/fisiologia , Epigênese Genética , Células Germinativas/metabolismo , Padrões de Herança , MicroRNAs/metabolismo , Transporte de RNA , Animais , Hereditariedade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem
13.
Neurooncol Pract ; 7(1): 31-37, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32257282

RESUMO

Background: A key component of cancer research is the availability of clinical samples with appropriately annotated clinical data. Biobanks facilitate research by collecting/storing various types of clinical samples for research. Brain Cancer Biobanking Australia (BCBA) was established to facilitate the networking of brain cancer biobanking operations Australia-wide. Maximizing biospecimen utility in a networked biobanking environment requires the standardization of procedures and data across different sites. The aim of this research was to scope and develop a recommended clinical annotation dataset both for pediatric and adult brain cancer biobanks. Methods: A multidisciplinary working group consisting of members from the BCBA Consortium was established to develop clinical dataset recommendations for brain cancer biobanks. A literature search was undertaken to identify any published clinical dataset recommendations for brain cancer biobanks. An audit of data items collected and stored by BCBA member biobanks was also conducted to survey current clinical data collection practices across the BCBA network. Results: BCBA has developed a clinical annotation dataset recommendation for pediatric and adult brain cancer biobanks. The clinical dataset recommendation has 5 clinical data categories: demographic, clinical and radiological diagnosis and surgery, neuropathological diagnosis, patient treatment, and patient follow-up. The data fields have been categorized into 1 of 3 tiers; essential, preferred, and comprehensive. This enables biobanks and researchers to prioritize appropriately where resources are limited. Conclusion: This dataset can be used to guide the integration of data from multiple existing biobanks for research studies and for planning prospective brain cancer biobanking activities.

14.
Nat Commun ; 11(1): 1343, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165640

RESUMO

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.


Assuntos
Efrina-A2/genética , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Transportadores de Sulfato/genética , Sequência de Aminoácidos , Animais , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A2/química , Efrina-A2/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Bócio Nodular/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação Puntual , Ligação Proteica , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismo
15.
Cancers (Basel) ; 12(3)2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32168910

RESUMO

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While TP53 deletion is rare in glioblastomas, these tumors often carry TP53 mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53wt stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact TP53 gene. However, targeting MK2 in tumors with TP53 mutations may accelerate disease progression. These findings are highly relevant since TP53 mutations occur in over 50% of all cancers.

16.
Mol Cancer Res ; 18(5): 709-722, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32079743

RESUMO

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A. IMPLICATIONS: High expression of CIP2A in gliomas is maintained by a CHK1-dependent pSTAT3-CIP2A recursive loop; interrupting CIP2A induces cell senescence and slows GBM growth adding impetus to the development of CIP2A as an anticancer drug target.


Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Autoantígenos/genética , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Quinase 1 do Ponto de Checagem/genética , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Fosforilação , Prognóstico , Fator de Transcrição STAT3/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cells ; 9(2)2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31973233

RESUMO

Glioblastoma (GBM) is a treatment-refractory central nervous system (CNS) tumour, and better therapies to treat this aggressive disease are urgently needed. Primary GBM models that represent the true disease state are essential to better understand disease biology and for accurate preclinical therapy assessment. We have previously presented a comprehensive transcriptome characterisation of a panel (n = 12) of primary GBM models (Q-Cell). We have now generated a systematic, quantitative, and deep proteome abundance atlas of the Q-Cell models grown in 3D culture, representing 6167 human proteins. A recent study has highlighted the degree of functional heterogeneity that coexists within individual GBM tumours, describing four cellular states (MES-like, NPC-like, OPC-like and AC-like). We performed comparative proteomic analysis, confirming a good representation of each of the four cell-states across the 13 models examined. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified upregulation of a number of GBM-associated cancer pathway proteins. Bioinformatics analysis, using the OncoKB database, identified a number of functional actionable targets that were either uniquely or ubiquitously expressed across the panel. This study provides an in-depth proteomic analysis of the GBM Q-Cell resource, which should prove a valuable functional dataset for future biological and preclinical investigations.


Assuntos
Técnicas de Cultura de Células/métodos , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteômica , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Glioblastoma/genética , Humanos , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo
19.
J Neurooncol ; 146(1): 41-53, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31760595

RESUMO

INTRODUCTION: Malignant astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation to alter the behaviour of high-grade astrocytomas may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation. Here, we investigate whether family members NFIA and NFIB act as effectors of cellular differentiation in glioblastoma. METHODS: We analysed expression of NFIA and NFIB in mRNA expression data of high-grade astrocytoma and with immunofluorescence co-staining. Furthermore, we induced NFI expression in patient-derived subcutaneous glioblastoma xenografts via in vivo electroporation. RESULTS: The expression of NFIA and NFIB is reduced in glioblastoma as compared to lower grade astrocytomas. At a cellular level, their expression is associated with differentiated and mature astrocyte-like tumour cells. In vivo analyses consistently demonstrate that expression of either NFIA or NFIB is sufficient to promote tumour cell differentiation in glioblastoma xenografts. CONCLUSION: Our findings indicate that both NFIA and NFIB may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal astrocyte differentiation. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Glioblastoma/patologia , Fatores de Transcrição NFI/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fatores de Transcrição NFI/genética , Gradação de Tumores , Neurogênese , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Neuro Oncol ; 22(2): 216-228, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31504812

RESUMO

BACKGROUND: Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. METHODS: In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. RESULTS: Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell-like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. CONCLUSION: Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.


Assuntos
Neoplasias Encefálicas/patologia , Replicação do DNA/efeitos dos fármacos , Glioblastoma/patologia , Piranos/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Descoberta de Drogas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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