RESUMO
Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here.
Assuntos
Citosina Desaminase/genética , Genes Transgênicos Suicidas/genética , Terapia Genética , Neoplasias/terapia , Timidina Quinase/genética , Animais , Citosina Desaminase/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Vetores Genéticos/genética , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Simplexvirus/enzimologia , Timidina Quinase/farmacologia , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
PURPOSE: Due to the recent rise in immunotherapy research to treat glioblastoma (GBM), immunocompetent mouse models have become increasingly crucial. However, the character and kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor has the impact of commonly-used marker proteins and foreign antigens. METHODS: In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry as well as investigated several factors that influence the immune response, including kinetics, tumor size, and expression of commonly-used marker proteins and foreign antigens. We hypothesize that these factors influence the immune response enough to warrant consideration when studying new immunotherapeutic approaches for GBM. RESULTS: CT2A-Luc, but not GL261-Luc2, drastically increased the number of T cells in the brain compared with wild-type controls, and significantly altered CT2A's responsiveness to anti-PD-1 antibody therapy. Additionally, a larger cell inoculum size in the GL261 model increased the T cell response's magnitude at day 28 post-injection. CT2A and GL261 models both stimulate a peak T cell immune response at day 21 post-injection. CONCLUSIONS: Our results suggest that the impact of foreign proteins like luciferase on the intracranial immune response is dependent upon the model, with CT2A being more sensitive to added markers. In particular, luciferase expression in CT2A could lead to meaningful misinterpretations of results from immune checkpoint inhibitor (ICI) studies.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Imunidade Adaptativa , Animais , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Glioblastoma/terapia , Glioma/terapia , Luciferases , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.
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Neoplasias Encefálicas , Modelos Animais de Doenças , Glioblastoma , Metaloproteinase 9 da Matriz/metabolismo , Transplante Heterólogo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismoRESUMO
SPRY2 is a purported tumor suppressor in certain cancers that promotes tumor growth and resistance to receptor tyrosine kinase inhibitors in glioblastoma. Here, we identify a SPRY2-dependent bypass signaling mechanism in glioblastoma that drives resistance to EGFR and MET inhibition. In glioblastoma cells treated with EGFR and MET inhibitors, SPRY2 expression is initially suppressed but eventually rebounds due to NF-κB pathway activation, resultant autocrine FGFR activation, and reactivation of ERK, which controls SPRY2 transcription. In cells where FGFR autocrine signaling does not occur and ERK does not reactivate, or in which ERK reactivates but SPRY2 cannot be expressed, EGFR and MET inhibitors are more effective at promoting death. The same mechanism also drives acquired resistance to EGFR and MET inhibition. Furthermore, tumor xenografts expressing an ERK-dependent bioluminescent reporter engineered for these studies reveal that this bypass resistance mechanism plays out in vivo but can be overcome through simultaneous FGFR inhibition.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Ligantes , Camundongos Nus , Modelos Biológicos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The cholesterol-lowering statins have known anti-cancer effects, but the mechanisms and how to utilize statins in oncology have been unclear. We noted in the CellMiner database that statin activity against cancer lines correlated with higher expression of TGF-ß target genes such as SERPINE1 and ZYX. This prompted us to assess whether statins affected TGF-ß activity in glioblastoma (GBM), a cancer strongly influenced by TGF-ß and in dire need of new therapeutic approaches. We noted that statins reduced TGF-ß activity, cell viability and invasiveness, Rho/ROCK activity, phosphorylation and activity of the TGF-ß mediator Smad3, and expression of TGF-ß targets ZYX and SERPINE1 in GBM and GBM-initiating cell (GIC) lines. Statins were most potent against GBM, GIC, and other cancer cells with high TGF-ß activity, and exogenous TGF-ß further sensitized mesenchymal GICs to statins. Statin toxicity was rescued by addition of exogenous mevalonolactone or geranylgeranyl pyrophosphate, indicating that the observed effects reflected inhibition of HMG CoA-reductase by the statins. Simvastatin significantly inhibited the growth of subcutaneous GIC grafts and prolonged survival in GIC intracranially grafted mice. These results indicate where the statins might best be applied as adjunct therapies in oncology, against GBM and other cancers with high TGF-ß activity, and have implications for other statin roles outside of oncology.
RESUMO
Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Metaloproteinase 9 da Matriz/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas de Fase Aguda/metabolismo , Neoplasias Encefálicas/enzimologia , Ativação Enzimática , Fibronectinas/química , Gelatina/química , Glioblastoma/enzimologia , Humanos , Lipocalina-2 , Lipocalinas/metabolismo , Metaloproteinase 9 da Matriz/química , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
OBJECT: We analyzed MMP-9 expression using mRNA and protein level determinations and explored the possibility that matrix metalloproteinase-9 (MMP-9) is a potential biological marker of pituitary adenoma invasiveness and whether MMP-9 could be used to discriminate the extent of invasiveness among different hormonal subtypes, tumor sizes, growth characteristics, and primary versus recurrent tumors. MATERIALS AND METHODS: 73 pituitary tumor specimens were snap frozen in liquid nitrogen immediately after surgical resection. RNA and protein were extracted. MMP-9 mRNA transcripts were analyzed by quantitative RT-PCR. MMP-9 protein activity was analyzed by gelatin zymography and validated by western blot analysis. Immunohistochemistry was performed to identify the presence and localization of MMP-9 in pituitary adenomas. Statistical differences between results were determined using Student's t-test or one way ANOVA. RESULTS: Comparing different hormonal subtypes of noninvasive and invasive pituitary tumors, MMP-9 mRNA expression was significantly increased in the majority of invasive adenomas. Considering the protein levels, our data also showed a significant increase in MMP-9 activity in the majority of invasive adenomas and these differences were confirmed by western blot analysis and immunohistochemistry. In addition, consistent differences in MMP-9 expression levels were found according to tumor subtype, tumor size, tumor extension and primary versus redo-surgery. CONCLUSIONS: MMP-9 expression can consistently distinguish invasive pituitary tumors from noninvasive pituitary tumors and would reflect the extent of invasiveness in pituitary tumors according to tumor subtype, size, tumor extension, primary and redo surgery, even at early stages of invasiveness. MMP-9 may be considered a potential biomarker to determine and predict the invasive nature of pituitary tumors.
Assuntos
Adenoma/enzimologia , Biomarcadores Tumorais/análise , Metaloproteinase 9 da Matriz/análise , Neoplasias Hipofisárias/enzimologia , Adenoma/genética , Adenoma/patologia , Adenoma/cirurgia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Western Blotting , Criança , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/cirurgia , Valor Preditivo dos Testes , RNA Mensageiro/análise , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Adulto JovemRESUMO
Background: The mesenchymal phenotype in glioblastoma (GBM) and other cancers drives aggressiveness and treatment resistance, leading to therapeutic failure and recurrence of disease. Currently, there is no successful treatment option available against the mesenchymal phenotype. Methods: We classified patient-derived GBM stem cell lines into 3 subtypes: proneural, mesenchymal, and other/classical. Each subtype's response to the inhibition of diacylglycerol kinase alpha (DGKα) was compared both in vitro and in vivo. RhoA activation, liposome binding, immunoblot, and kinase assays were utilized to elucidate the novel link between DGKα and geranylgeranyltransferase I (GGTase I). Results: Here we show that inhibition of DGKα with a small-molecule inhibitor, ritanserin, or RNA interference preferentially targets the mesenchymal subtype of GBM. We show that the mesenchymal phenotype creates the sensitivity to DGKα inhibition; shifting GBM cells from the proneural to the mesenchymal subtype increases ritanserin activity, with similar effects in epithelial-mesenchymal transition models of lung and pancreatic carcinoma. This enhanced sensitivity of mesenchymal cancer cells to ritanserin is through inhibition of GGTase I and downstream mediators previously associated with the mesenchymal cancer phenotype, including RhoA and nuclear factor-kappaB. DGKα inhibition is synergistic with both radiation and imatinib, a drug preferentially affecting proneural GBM. Conclusions: Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Diacilglicerol Quinase/antagonistas & inibidores , Glioblastoma/patologia , Ritanserina/farmacologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Diacilglicerol Quinase/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Glioblastoma (GBM) is the most common and lethal brain tumor. Gene expression profiling has classified GBM into distinct subtypes, including proneural, mesenchymal, and classical, and identifying therapeutic vulnerabilities of these subtypes is an extremely high priority. We leveraged The Cancer Genome Atlas (TCGA) data, in particular for microRNA expression, to seek druggable core pathways in GBM. The E2F1-regulated miR-17Ë92 cluster and its analogs are shown to be highly expressed in proneural GBM and in GSC lines, suggesting the E2F cell cycle pathway might be a key driver in proneural GBM. Consistently, CDK4/6 inhibition with palbociclib preferentially inhibited cell proliferation in vitro in a majority of proneural GSCs versus those of other subtypes. Palbociclib treatment significantly prolonged survival of mice with established intracranial xenografts of a proneural GSC line. We show that most of these sensitive PN GSCs expressed higher levels of CDK6 and had intact Rb1, while two GSC lines with CDK4 overexpression and null Rb1 were highly resistant to palbociclib. Importantly, palbociclib treatment of proneural GSCs upregulated mesenchymal-associated markers and downregulated proneural-associated markers, suggesting that CDK4/6 inhibition induced proneural-mesenchymal transition and underscoring the enhanced role of the E2F cell cycle pathway in the proneural subtype. Lastly, the combination of palbociclib and N,N-diethylaminobenzaldehyde, an inhibitor of the mesenchymal driver ALDH1A3, showed strong synergistic inhibitory effects against proneural GSC proliferation. Taken together, our results reveal that proneural GBM has increased vulnerability to CDK4/6 inhibition, and the proneural subtype undergoes dynamic reprogramming upon palbociclib treatment-suggesting the need for a combination therapeutic strategy.
RESUMO
Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed.Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib.Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958-68. ©2017 AACR.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Glioblastoma/metabolismo , Glioblastoma/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Sinergismo Farmacológico , Everolimo/farmacologia , Feminino , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Modelos Biológicos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Local disruption of the integrity of both the myoepithelial cell layer and the basement membrane is an indispensable prerequisite for the initiation of invasion and the conversion of human breast ductal carcinoma in situ (DCIS) to infiltrating ductal carcinoma (IDC). We previously reported that human endometase/matrilysin-2/matrix metalloproteinase (MMP) 26-mediated pro-gelatinase B (MMP-9) activation promoted invasion of human prostate carcinoma cells by dissolving basement membrane proteins (Y. G. Zhao et al., J. Biol. Chem., 278: 15056-15064, 2003). Here we report that tissue inhibitor of metalloproteinases (TIMP)-2 and TIMP-4 are potent inhibitors of MMP-26, with apparent K(i) values of 1.6 and 0.62 nM, respectively. TIMP-2 and TIMP-4 also inhibited the activation of pro-MMP-9 by MMP-26 in vitro. The expression levels of MMP-26, MMP-9, TIMP-2, and TIMP-4 proteins in DCIS were significantly higher than those in IDC, atypical intraductal hyperplasia, and normal breast epithelia adjacent to DCIS and IDC by immunohistochemistry and integrated morphometry analysis. Double immunofluorescence labeling and confocal laser scanning microscopy revealed that MMP-26 was colocalized with MMP-9, TIMP-2, and TIMP-4 in DCIS cells. Higher levels of MMP-26 mRNA were also detected in DCIS cells by in situ hybridization.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Carcinoma Ductal/enzimologia , Metaloproteinases da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Inibidores Teciduais de Metaloproteinases/farmacologia , Carcinoma Ductal/patologia , Ativação Enzimática , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cinética , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz Secretadas , Invasividade Neoplásica , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
Breast cancer metastasis suppressor 1 (BRMS1) is downregulated in non-small cell lung cancer (NSCLC), and its reduction correlates with disease progression. Herein, we investigate the mechanisms through which loss of the BRMS1 gene contributes to epithelial-to-mesenchymal transition (EMT). Using a short hairpin RNA (shRNA) system, we show that loss of BRMS1 promotes basal and transforming growth factor beta-induced EMT in NSCLC cells. NSCLC cells expressing BRMS1 shRNAs (BRMS1 knockdown [BRMS1(KD)]) display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers. Mesenchymal phenotypes observed in BRMS1(KD) cells are dependent on RelA/p65, the transcriptionally active subunit of nuclear factor kappa B (NF-κB). In addition, chromatin immunoprecipitation analysis demonstrates that loss of BRMS1 increases Twist1 promoter occupancy of RelA/p65 K310-a key histone modification associated with increased transcription. Knockdown of Twist1 results in reversal of BRMS1(KD)-mediated EMT phenotypic changes. Moreover, in our animal model, BRMS1(KD)/Twist1(KD) double knockdown cells were less efficient in establishing lung tumors than BRMS1(KD) cells. Collectively, this study demonstrates that loss of BRMS1 promotes malignant phenotypes that are dependent on NF-κB-dependent regulation of Twist1. These observations offer fresh insight into the mechanisms through which BRMS1 regulates the development of metastases in NSCLC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Transição Epitelial-Mesenquimal , Genes Supressores de Tumor , Histonas/metabolismo , Humanos , Metástase Linfática , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Nucleares/genética , Fenótipo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína 1 Relacionada a Twist/genética , CicatrizaçãoRESUMO
Expression of the breast cancer metastasis suppressor 1 (BRMS1) protein is dramatically reduced in non-small cell lung cancer (NSCLC) cells and in primary human tumors. Although BRMS1 is a known suppressor of metastasis, the mechanisms through which BRMS1 functions to regulate cell migration and invasion in response to specific NSCLC driver mutations are poorly understood. To experimentally address this, we utilized immortalized human bronchial epithelial cells in which p53 was knocked down in the presence of oncogenic K-RasV12 (HBEC3-p53KD-K-RasV12). These genetic alterations are commonly found in NSCLC and are associated with a poor prognosis. To determine the importance of BRMS1 for cytoskeletal function, cell migration and invasion in our model system we stably knocked down BRMS1. Here, we report that loss of BRMS1 in HBEC3-p53KD-K-RasV12 cells results in a dramatic increase in cell migration and invasion compared to controls that expressed BRMS1. Moreover, the loss of BRMS1 resulted in additional morphological changes including F-actin re-distribution, paxillin accumulation at the leading edge of the lamellapodium, and cellular shape changes resembling mesenchymal phenotypes. Importantly, re-expression of BRMS1 restores, in part, cell migration and invasion; however it does not fully reestablish the epithelial phenotype. These finding suggests that loss of BRMS1 results in a permanent, largely irreversible, mesenchymal phenotype associated with increased cell migration and invasion. Collectively, in NSCLC cells without p53 and expression of oncogenic K-Ras our study identifies BRMS1 as a key regulator required to maintain a cellular morphology and cytoskeletal architecture consistent with an epithelial phenotype.
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Células Epiteliais Alveolares/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Citoesqueleto de Actina/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Humanos , Camundongos Nus , Mutação , Paxilina/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Repressoras , Proteínas ras/metabolismoRESUMO
The mechanisms through which the metastasis suppressor gene BRMS1 functions are poorly understood. Herein, we report the identification of a previously undescribed E3 ligase function of BRMS1 on the histone acetyltransferase p300. BRMS1 induces polyubiquitination of p300, resulting in its proteasome-mediated degradation. We identify BRMS1 as the first eukaryote structural mimic of the bacterial IpaH E3 ligase family and establish that the evolutionarily conserved CXD motif located in BRMS1 is responsible for its E3 ligase function. Mutation of this E3 ligase motif not only abolishes BRMS1-induced p300 polyubiquitination and degradation, but importantly, dramatically reduces the metastasis suppressor function of BRMS1 in both in vitro and in vivo models of lung cancer metastasis.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Mutação , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Interferência de RNA , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transplante Heterólogo , Carga Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.
Assuntos
Neoplasias Encefálicas/genética , Diacilglicerol Quinase/genética , Glioblastoma/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Diacilglicerol Quinase/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Terapia de Alvo Molecular , Piperidinas/administração & dosagem , Pirimidinonas/administração & dosagem , Quinazolinonas/administração & dosagem , RNA Interferente Pequeno , Tiazóis/administração & dosagemRESUMO
BACKGROUND AND OBJECTIVE: Elevated expression of matrix metalloproteinases (MMPs) has been found in multiple carcinoma tissues. MMP-26 is highly expressed in prostate and breast cancer tissues, and promotes the invasion of human prostate cancer cells not only through the cleavage of fibronectin and type IV collagen but also by the activation of pro-MMP-9, a powerful gelatinase. This study was to present a comprehensive protein expression profile of MMP-26 in multiple human cancer tissues. METHODS: The protein expression pattern of MMP-26 was examined using immunohistochemistry and multiple-tissue microarray. MMP-26 mRNA expression in coronary artery smooth muscle cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of MMP-26 in breast, colon, lung, brain, head and neck, prostate cancer, and melanoma tissues was significantly elevated when compared with parallel normal tissues (P<0.05), while not significantly elevated in kidney cancer, ovarian cancer, and non-Hodgkin's lymphoma (P>0.05). MMP-26 was also detected to express in gastric, rectal, thyroid, esophageal, and pancreatic cancers. MMP-26 protein was expressed in smooth muscle cells of the prostate and associated blood vessels. MMP-26 mRNA was also detected to express in human coronary artery smooth muscle cells. CONCLUSIONS: MMP-26 expression may be associated with multiple human carcinomas, and it may serve as a molecular marker for the early diagnosis of these carcinomas. MMP-26 may also contribute to smooth muscle function in the human prostate and cardiovascular system.
Assuntos
Metaloproteinases da Matriz Secretadas/metabolismo , Miócitos de Músculo Liso/enzimologia , Neoplasias/enzimologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Vasos Coronários/citologia , Vasos Coronários/enzimologia , Detecção Precoce de Câncer , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Metaloproteinases da Matriz Secretadas/genética , Análise em Microsséries/métodos , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/patologia , Próstata/citologia , Próstata/enzimologia , RNA Mensageiro/metabolismoRESUMO
Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas.
Assuntos
Astrócitos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Análise de Variância , Animais , Neoplasias Encefálicas/patologia , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transfecção/métodos , Proteínas ras/metabolismoRESUMO
Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.
Assuntos
Astrocitoma/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Proteína Quinase C-alfa/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Astrocitoma/terapia , Linhagem Celular Tumoral , Movimento Celular , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos SCID , Invasividade Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C-alfa/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The complete resection of pituitary adenomas (PAs) is unlikely when there is an extensive local dural invasion and given that the molecular mechanisms remain primarily unknown. DNA microarray analysis was performed to identify differentially expressed genes between nonfunctioning invasive and noninvasive PAs. Gene clustering revealed a robust eightfold increase in matrix metalloproteinase (MMP)-9 expression in surgically resected human invasive PAs and in the (nonfunctioning) HP75 human pituitary tumor-derived cell line treated with phorbol-12-myristate-13-acetate; these results were confirmed by real-time polymerase chain reaction, gelatin zymography, reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and Northern blot analyses. The activation of protein kinase C (PKC) increased both MMP-9 activity and expression, which were blocked by some PKC inhibitors (Gö6976, bisindolylmaleimide, and Rottlerin), PKC-alpha, and PKC-delta small interfering (si)RNAs but not by hispidin (PKC-beta inhibitor). In a transmembrane invasion assay, phorbol-12-myristate-13-acetate (100 nmol/L) increased the number of invaded HP75 cells, a process that was attenuated by PKC inhibitors, MMP-9 antibody, PKC-alpha siRNA, or PKC-delta siRNA. These results demonstrate that MMP-9 and PKC-alpha or PKC-delta may provide putative therapeutic targets for the control of PA dural invasion.
Assuntos
Adenoma , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica/genética , Neoplasias Hipofisárias , Adenoma/enzimologia , Adenoma/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Neoplasias Hipofisárias/enzimologia , Neoplasias Hipofisárias/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pironas/farmacologia , RNA Interferente Pequeno , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.