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1.
BMC Cancer ; 21(1): 1061, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34565342

RESUMO

BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.


Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
2.
Cancer Lett ; 499: 24-38, 2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33248210

RESUMO

The incidence of pediatric cancers is rising steadily across the world, along with the challenges in understanding the molecular mechanisms and devising effective therapeutic strategies. Pediatric cancers are presented with diverse molecular characteristics and more distinct subtypes when compared to adult cancers. Recent studies on the genomic landscape of pediatric cancers using next-generation sequencing (NGS) approaches have redefined this field by providing better subtype characterization and novel actionable targets. Since early identification and personalized treatment strategies influence therapeutic outcomes, survival, and quality of life in pediatric cancer patients, the quest for actionable biomarkers is of great value in this field. Fusion genes that are prevalent and recurrent in several pediatric cancers are ideally suited in this context due to their disease-specific occurrence. In this review, we explore the current status of fusion genes in pediatric cancer subtypes and their use as biomarkers for diagnosis and personalized therapy. We discuss the technological advancements made in recent years in NGS sequencing and their impact on fusion detection algorithms that have revolutionized this field. Finally, we also discuss the advantages of pairing liquid biopsy protocols for fusion detection and their eventual use in diagnosis and treatment monitoring.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer/métodos , Neoplasias/diagnóstico , Proteínas de Fusão Oncogênica/genética , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Criança , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Monitoramento de Medicamentos/métodos , Testes Genéticos/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/métodos , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/sangue , Medicina de Precisão/métodos , Resultado do Tratamento
3.
Oncotarget ; 11(40): 3633-3645, 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33088424

RESUMO

Intercellular communication between tumor cells within the hypoxic microenvironment promote aggressiveness and poor patient prognoses for reasons that remain unclear. Here we show that hypoxic Ewing's sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation. Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effects of EWS derived exosomal miRNAs on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS tumors.

4.
BMC Med Genomics ; 13(1): 87, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591022

RESUMO

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant cancers in children. MB is primarily classified into four subgroups based on molecular and clinical characteristics as (1) WNT (2) Sonic-hedgehog (SHH) (3) Group 3 (4) Group 4. Molecular characteristics used for MB classification are based on genomic and mRNAs profiles. MB subgroups share genomic and mRNA profiles and require multiple molecular markers for differentiation from each other. Long non-coding RNAs (lncRNAs) are more than 200 nucleotide long RNAs and primarily involve in gene regulation at epigenetic and post-transcriptional levels. LncRNAs have been recognized as diagnostic and prognostic markers in several cancers. However, the lncRNA expression profile of MB is unknown. METHODS: We used the publicly available gene expression datasets for the profiling of lncRNA expression across MB subgroups. Functional analysis of differentially expressed lncRNAs was accomplished by Ingenuity pathway analysis (IPA). RESULTS: In the current study, we have identified and validated the lncRNA expression profile across pediatric MB subgroups and associated molecular pathways. We have also identified the prognostic significance of lncRNAs and unique lncRNAs associated with each MB subgroup. CONCLUSIONS: Identified lncRNAs can be used as single biomarkers for molecular identification of MB subgroups that warrant further investigation and functional validation.


Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Meduloblastoma/genética , Meduloblastoma/patologia , RNA Longo não Codificante/genética , Adolescente , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Taxa de Sobrevida
5.
Mol Cancer Ther ; 19(6): 1351-1362, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32371591

RESUMO

The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.


Assuntos
Azepinas/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Meduloblastoma/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolinas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Feminino , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-myc/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mol Ther Nucleic Acids ; 19: 1379-1398, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32160708

RESUMO

Gene fusions that contribute to oncogenicity can be explored for identifying cancer biomarkers and potential drug targets. To investigate the nature and distribution of fusion transcripts in cancer, we examined the transcriptome data of about 9,000 primary tumors from 33 different cancers in TCGA (The Cancer Genome Atlas) along with cell line data from CCLE (Cancer Cell Line Encyclopedia) using ChimeRScope, a novel fusion detection algorithm. We identified several fusions with sense (canonical, 39%) or antisense (non-canonical, 61%) transcripts recurrent across cancers. The majority of the recurrent non-canonical fusions found in our study are novel, unexplored, and exhibited highly variable profiles across cancers, with breast cancer and glioblastoma having the highest and lowest rates, respectively. Overall, 4,344 recurrent fusions were identified from TCGA in this study, of which 70% were novel. Additional analysis of 802 tumor-derived cell line transcriptome data across 20 cancers revealed significant variability in recurrent fusion profiles between primary tumors and corresponding cell lines. A subset of canonical and non-canonical fusions was validated by examining the structural variation evidence in whole-genome sequencing (WGS) data or by Sanger sequencing of fusion junctions. Several recurrent fusion genes identified in our study show promise for drug repurposing in basket trials and present opportunities for mechanistic studies.

7.
BMC Cancer ; 19(1): 1056, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694585

RESUMO

BACKGROUND: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. METHODS: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. RESULTS: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. CONCLUSION: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.


Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Humanos , Isoquinolinas/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Pirimidinas/farmacologia , Interferência de RNA , Análise de Sobrevida
8.
Biochem Biophys Res Commun ; 520(2): 250-256, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31594641

RESUMO

Medulloblastoma (MB) is a highly aggressive, malignant brain tumor in children with poor prognosis. Cyclin-dependent kinase 9 (CDK9), a serine-threonine kinase, is widely implicated in the control of basal gene expression by phosphorylating Serine 2 (Ser2) of the heptad repeat in the RNA Polymerase II (RNA Pol II) C-terminal domain (CTD). Although CDK9 plays a pathogenic role in various cancers, its function in MB remains unknown. Here, we show that CDK9 is highly expressed in MB tumors and increased CDK9 expression is correlated with high risk MB patients. CDK9 expression along with phospho-Ser2 RNA Pol II (pRNA Pol II ser2) and bromodomain-binding protein 4 (BRD4), which recruits CDK9, were elevated in multiple MB cell lines and in MB tumors originated spontaneously from Ptch1+/-p53-/- mice. Inhibition of CDK9 with LDC067 suppressed MB cell growth, reduced pRNA Pol II ser2 level and expression of oncogenic markers, including MYC. Moreover, LDC067 treatment synergistically sensitizes MB cells to chemotherapeutic agent cisplatin. Further, LDC067 in combination with BRD4 inhibitor decreased MB cells growth, delayed cell migration and attenuated pRNA Pol II ser2 occupancy to CCND1 and BCL2 gene promoters as revealed by chromatin immunoprecipitation assay (ChIP). Together, these findings highlight the importance of CDK9 in MB pathogenesis and suggest that it may serve as a promising therapeutic target for the treatment of MB.


Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Quinase 9 Dependente de Ciclina/metabolismo , Meduloblastoma/tratamento farmacológico , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Cisplatino/administração & dosagem , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos Mutantes , Terapia de Alvo Molecular , Neoplasias Experimentais , Pirimidinas/administração & dosagem , RNA Polimerase II/metabolismo , Serina/metabolismo , Sulfonamidas/administração & dosagem , Fatores de Transcrição/metabolismo
9.
Leuk Lymphoma ; 60(5): 1214-1223, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30424705

RESUMO

Mantle cell lymphoma (MCL) represents an aggressive B-cell lymphoma with frequent relapse and poor survival. Recently, dysregulated histone-deacetylases (HDACs) and cell cycle CDK-Rb pathway have been shown to be commonly associated with MCL pathogenesis, and are considered promising targets for relapsed-lymphoma therapy. Therefore, we investigated the single agents and combination efficacy of HDACs inhibitor Vorinostat, CDK4/6 dual-inhibitor Palbociclib on MCL cell growth/survival and underlying molecular mechanism(s) using MCL cell lines including therapy-resistant MCL cell lines. Our results showed that both inhibitors as single agents or combined, significantly suppressed the cell growth and induced apoptosis in therapy-resistant and parental MCL lines. In addition, the combination of Vorinostat and Palbociclib significantly inhibited the activation of the key molecules of the CDK4/6-Rb pathway and HDAC activity and subsequently decreased the expression of Cyclin-D1 and Bcl-2. These studies demonstrated the potential for combining these two inhibitors as a novel therapeutic approach in refractory MCL therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Linfoma de Célula do Manto/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Linfoma de Célula do Manto/patologia , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Vorinostat/administração & dosagem
10.
Oncotarget ; 9(24): 16619-16633, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29682173

RESUMO

Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. In vivo results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients.

11.
Mol Carcinog ; 57(4): 536-548, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29280516

RESUMO

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Chaperonas Moleculares/genética , Peptídeos/farmacologia , Proteínas Inibidoras de STAT Ativados/genética , Fator de Transcrição STAT3/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Chaperonas Moleculares/metabolismo , Peptídeos/síntese química , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo
12.
Leuk Lymphoma ; 59(7): 1565-1573, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-28882083

RESUMO

Chronic lymphocytic leukemia (CLL) is a heterogeneous B cell malignancy that still remains incurable. Recent studies have highlighted cellular and non-cellular components of the tissue microenvironment in CLL that help nurture the growth of leukemic cells by providing the necessary stimuli for their proliferation and survival. The diverse stimuli in the specialized tissue microenvironment of CLL lead to constitutive activation of several signaling pathways that includes B cell receptor signaling and the associated mitogen-activated protein kinase (MAPK) signaling. Recent findings have described aberrant activation of MAPK signaling and its interactions with other cellular signaling pathways in the pathogenesis of CLL. These studies have shed light on the deregulated molecular mechanisms contributing to hyperactivation of MAPK signaling and provided avenues for therapeutic options for aggressive CLL. In this review, we describe and discuss the current status of our understanding into the role of MAPK signaling in the pathogenesis of CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/imunologia , Linfócitos B/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Antígenos de Linfócitos B/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
13.
BMC Cancer ; 16(1): 867, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27821095

RESUMO

BACKGROUND: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. METHODS: The activity of 12 ozonides, artemisinin, and two semisynthetic artemisinins were tested for activity against two neuroblastoma cell-lines (BE (2)-c and IMR-32) and the Ewing's Sarcoma cell line A673 in an MTT viability assay. Time course data indicated that peak effect was seen 18 h after the start of treatment thus 18 h pre-treatment was used for all subsequent experiments. The most active ozonide (OZ513) was assessed in a propidium iodide cell cycle flow cytometry analysis which measured cell cycle transit and apoptosis. Metabolic effects of OZ513 in BE (2)-c cells was evaluated. Western blots for the apoptotic proteins cleaved capase-3 and cleaved PARP, the highly amplified oncogene MYCN, and the cell cycle regulator CyclinD1, were performed. These in-vitro experiments were followed by an in-vivo experiment in which NOD-scid gamma immunodeficient mice were injected subcutaneously with 1 × 106 BE (2)-c cells followed by immediate treatment with 50-100 mg/kg/day doses of OZ513 administered IP three times per week out to 23 days after injection of tumor. Incidence of tumor development, time to tumor development, and rate of tumor growth were assessed in DMSO treated controls (N = 6), and OZ513 treated mice (N = 5). RESULTS: It was confirmed that five commonly used chemotherapy drugs had no cytotoxic activity in BE (2)-c cells. Six of 12 ozonides tested were active in-vitro at concentrations achievable in vivo with OZ513 being most active (IC50 = 0.5 mcg/ml). OZ513 activity was confirmed in IMR-32 and A673 cells. The Ao peak on cell-cycle analysis was increased after treatment with OZ513 in a concentration dependent fashion which when coupled with results from western blot analysis which showed an increase in cleaved capase-3 and cleaved PARP supported an increase in apoptosis. There was a concentration dependent decline in the MYCN and a cyclinD1 protein indicative of anti-proliferative activity and cell cycle disruption. OXPHOS metabolism was unaffected by OZ513 treatment while glycolysis was increased. There was a significant delay in time to tumor development in mice treated with OZ513 and a decline in the rate of tumor growth. CONCLUSIONS: The antimalarial ozonide OZ513 has effective in-vitro and in-vivo activity against a pleiotropic drug resistant neuroblastoma cell-line. Treatment with OZ513 increased apoptotic markers and glycolysis with a decline in the MYCN oncogene and the cell cycle regulator cyclinD1. These effects suggest adaptation to cellular stress by mechanism which remain unclear.


Assuntos
Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Biomarcadores , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Metaboloma , Metabolômica/métodos , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Oncotarget ; 7(27): 41081-41094, 2016 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-27232759

RESUMO

Molecular pathogenesis of Chronic Lymphocytic Leukemia (CLL) is not fully elucidated. Genome wide association studies have linked Interferon Regulatory Factor 4 (IRF4) to the development of CLL. We recently established a causal relationship between low levels of IRF4 and development of CLL. However, the molecular mechanism through which IRF4 suppresses CLL development remains unclear. Deregulation of Notch signaling pathway has been identified as one of the most recurrent molecular anomalies in the pathogenesis of CLL. Yet, the role of Notch signaling as well as its regulation during CLL development remains poorly understood. Previously, we demonstrated that IRF4 deficient mice expressing immunoglobulin heavy chain Vh11 (IRF4-/-Vh11) developed spontaneous CLL with complete penetrance. In this study, we show that elevated Notch2 expression and the resulting hyperactivation of Notch signaling are common features of IRF4-/-Vh11 CLL cells. Our studies further reveal that Notch signaling is indispensable for CLL development in the IRF4-/-Vh11 mice. Moreover, we identify E3 ubiquitin ligase Nedd4, which targets Notch for degradation, as a direct target of IRF4 in CLL cells and their precursors. Collectively, our studies provide the first in vivo evidence for an essential role of Notch signaling in the development of CLL and establish IRF4 as a critical regulator of Notch signaling during CLL development.


Assuntos
Apoptose/genética , Fatores Reguladores de Interferon/fisiologia , Leucemia Linfocítica Crônica de Células B/genética , Receptores Notch/metabolismo , Animais , Apoptose/imunologia , Transformação Celular Neoplásica/genética , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Genes Supressores de Tumor , Estudo de Associação Genômica Ampla , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores Notch/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
15.
Leuk Lymphoma ; 57(12): 2874-2889, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27074052

RESUMO

Mantle cell lymphoma (MCL) is characterized by a clinically aggressive course with frequent relapse and poor survival. The p53 pathway is frequently dysregulated and p53 status predicts clinical outcome. In this report, we investigated whether modulation of p73 isoforms by diclofenac inhibits cell growth, induces apoptosis and/or cell cycle arrest in MCL relative to p53 status. Wild-type p53 [Granta-519 and JVM-2], mutant p53 [Jeko-1 and Mino-1] expressing cells, therapy resistant cell lines, and primary human cells isolated from MCL patients were used. Overexpression of pro-apoptotic TAp73 enhanced MCL cell apoptosis. Diclofenac induced a concentration- and duration-dependent increase in TAp73, cell cycle arrest, cell death, and inhibited MCL cell growth independent of p53 status. Diclofenac treatment was associated with increased activity of caspases 3, 7, and 8 and induction of p53 transcriptional target genes. These studies demonstrate the potential for diclofenac as novel therapeutic agent in MCL independent of p53 status.


Assuntos
Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/genética , Diclofenaco/farmacologia , Humanos , Isoformas de Proteínas , Proteína Tumoral p73/metabolismo
16.
Oncotarget ; 7(12): 15215-29, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26934655

RESUMO

Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic.


Assuntos
Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Piridinas/farmacologia , Topotecan/farmacologia , Animais , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/patologia , Inibidores da Topoisomerase I/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Blood ; 127(19): 2310-21, 2016 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-26809508

RESUMO

Clinical heterogeneity is a major barrier to effective treatment of chronic lymphocytic leukemia (CLL). Emerging evidence suggests that constitutive activation of various signaling pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-Erk) signaling plays a role in the heterogeneous clinical outcome of CLL patients. In this study, we have investigated the role of Sprouty (SPRY)2 as a negative regulator of receptor and nonreceptor tyrosine kinase signaling in the pathogenesis of CLL. We show that SPRY2 expression is significantly decreased in CLL cells, particularly from poor-prognosis patients compared with those from good-prognosis patients. Overexpression of SPRY2 in CLL cells from poor-prognosis patients increased their apoptosis. Conversely, downregulation of SPRY2 in CLL cells from good-prognosis patients resulted in increased proliferation. Furthermore, CLL cells with low SPRY2 expression grew more rapidly in a xenograft model of CLL. Strikingly, B-cell-specific transgenic overexpression of spry2 in mice led to a decrease in the frequency of B1 cells, the precursors of CLL cells in rodents. Mechanistically, we show that SPRY2 attenuates the B-cell receptor (BCR) and MAPK-Erk signaling by binding to and antagonizing the activities of RAF1, BRAF, and spleen tyrosine kinase (SYK) in normal B cells and CLL cells. We also show that SPRY2 is targeted by microRNA-21, which in turn leads to increased activity of Syk and Erk in CLL cells. Taken together, these results establish SPRY2 as a critical negative regulator of BCR-mediated MAPK-Erk signaling in CLL, thereby providing one of the molecular mechanisms to explain the clinical heterogeneity of CLL.


Assuntos
Linfócitos B/metabolismo , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Apoptose/genética , Linfócitos B/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Quinase Syk/genética , Quinase Syk/metabolismo
18.
Exp Hematol ; 44(1): 30-7.e1, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26435347

RESUMO

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States. The tissue microenvironment, specifically the lymph nodes, influences the biological and clinical behavior of CLL cells. Gene expression profiling of CLL cells from peripheral blood, bone marrow, and lymph nodes revealed Cav-1 as one of the genes that might be involved in the pathogenesis of CLL. We have previously reported that the knockdown of Cav-1 in primary CLL cells exhibits a significant decrease in cell migration and immune synapse formation. However, the precise role of Cav-1 in CLL initiation and progression in vivo is not known. Therefore, we decreased the expression of Cav-1 in vivo by breeding Eµ-TCL1 with cav-1 knockout mice. We observed a significant decrease in the number of CLL cells and rate of proliferation of CLL cells in spleen, liver, and bone marrow from Eµ-TCL1-Cav1(-/+) and Eµ-TCL1-Cav1(-/-) mice as compared with Eµ-TCL1 mice. In addition, there was a significant increase in survival of Eµ-TCL1-Cav1(-/+) and Eµ-TCL1-Cav1(-/-) compared with Eµ-TCL1 mice. Mechanistically, we observed a decrease in MAPK-Erk signaling measured by p-Erk levels in Eµ-TCL1-Cav1(-/+) mice when compared with Eµ-TCL1-Cav(wt/wt). Together these results indicate that decreased Cav-1 in Eµ-TCL1 mice significantly delays the onset of CLL and decreases leukemic progression by inhibiting MAPK-Erk signaling, suggesting a role for Cav-1 in the proliferation and progression of CLL.


Assuntos
Caveolina 1/genética , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Camundongos
19.
BMC Res Notes ; 7: 912, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25510246

RESUMO

BACKGROUND: Computational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations. FINDINGS: We have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines. CONCLUSIONS: The experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.


Assuntos
Núcleo Celular/metabolismo , Biologia Computacional/métodos , Citoplasma/metabolismo , Organelas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Software , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes
20.
Mol Med ; 20: 290-301, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24800836

RESUMO

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eµ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.


Assuntos
Fator Ativador de Células B/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfonodos/metabolismo , NF-kappa B/genética , Microambiente Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Medula Óssea/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/genética , Baço/metabolismo
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