RESUMO
p27 shifts from CDK inhibitor to oncogene when phosphorylated by PI3K effector kinases. Here, we show that p27 is a cJun coregulator, whose assembly and chromatin association is governed by p27 phosphorylation. In breast and bladder cancer cells with high p27pT157pT198 or expressing a CDK-binding defective p27pT157pT198 phosphomimetic (p27CK-DD), cJun is activated and interacts with p27, and p27/cJun complexes localize to the nucleus. p27/cJun up-regulates TGFB2 to drive metastasis in vivo. Global analysis of p27 and cJun chromatin binding and gene expression shows that cJun recruitment to many target genes is p27 dependent, increased by p27 phosphorylation, and activates programs of epithelial-mesenchymal transformation and metastasis. Finally, human breast cancers with high p27pT157 differentially express p27/cJun-regulated genes of prognostic relevance, supporting the biological significance of the work.
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Movimento Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/genéticaRESUMO
The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.
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Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/toxicidade , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/química , Pirimidinas/uso terapêutico , Pirimidinas/toxicidade , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 in 95% of patients. MCL patients experience frequent relapses resulting in median survival of 3 to 5 years, requiring more efficient therapeutic regimens. Interleukin (IL)-21, a member of the IL-2 cytokine family, possesses potent antitumor activity against a variety of cancers not expressing the IL-21 receptor (IL-21R) through immune activation. Previously, we established that IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B-cell lymphoma cells. Herein, we demonstrate that IL-21 possesses potent cytotoxicity against MCL cell lines and primary tumors. We identify that IL-21-induced direct cytotoxicity is mediated through signal transducer and activator of transcription 3-dependent cMyc upregulation, resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. IL-21-mediated cMyc upregulation is only observed in IL-21-sensitive cells. Further, we demonstrate that IL-21 leads to natural killer (NK)-cell-dependent lysis of MCL cell lines that were resistant to direct cytotoxicity. In vivo treatment with IL-21 results in complete FC-muMCL1 tumor regression in syngeneic mice via NK- and T-cell-dependent mechanisms. Together, these data indicate that IL-21 has potent antitumor activity against MCL cells via direct cytotoxic and indirect, immune-mediated effects.
Assuntos
Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Interleucinas/imunologia , Interleucinas/uso terapêutico , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/terapia , Animais , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais/imunologia , Linfoma de Célula do Manto/patologia , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/imunologia , Receptores de Interleucina-21/imunologia , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/imunologiaRESUMO
Erratum to: Breast Cancer Res Treat (2013),138:369381,DOI 10.1007/s10549-012-2389-6. In the original publication of the article, the Fig. 4c and d were published erroneously. The revised Fig. 4 is given in this erratum.
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In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.
Assuntos
Neoplasias da Mama , Inibidor de Quinase Dependente de Ciclina p27 , Hiperplasia , Células-Tronco Neoplásicas , Fator de Transcrição STAT3 , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Humanos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Animais , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Feminino , Fosforilação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hiperplasia/metabolismo , Camundongos , Regulação Neoplásica da Expressão Gênica , Autorrenovação Celular/genética , Linhagem Celular Tumoral , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Animais/citologia , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genéticaRESUMO
Oncogenic PI3K/mTOR activation is frequently observed in human cancers and activates cell motility via p27 phosphorylations at T157 and T198. Here we explored the potential for a novel PI3K/mTOR inhibitor to inhibit tumor invasion and metastasis. An MDA-MB-231 breast cancer line variant, MDA-MB-231-1833, with high metastatic bone tropism, was treated with a novel catalytic PI3K/mTOR inhibitor, PF-04691502, at nM doses that did not impair proliferation. Effects on tumor cell motility, invasion, p27 phosphorylation, localization, and bone metastatic outgrowth were assayed. MDA-MB-231-1833 showed increased PI3K/mTOR activation, high levels of cytoplasmic p27pT157pT198 and increased cell motility and invasion in vitro versus parental. PF-04691502 treatment, at a dose that did not affect proliferation, reduced total and cytoplasmic p27, decreased p27pT157pT198 and restored cell motility and invasion to levels seen in MDA-MB-231. p27 knockdown in MDA-MB-231-1833 phenocopied PI3K/mTOR inhibition, whilst overexpression of the phosphomimetic mutant p27T157DT198D caused resistance to the anti-invasive effects of PF-04691502. Pre-treatment of MDA-MB-231-1833 with PF-04691502 significantly impaired metastatic tumor formation in vivo, despite lack of antiproliferative effects in culture and little effect on primary orthotopic tumor growth. A further link between cytoplasmic p27 and metastasis was provided by a study of primary human breast cancers which showed cytoplasmic p27 is associated with increased lymph nodal metastasis and reduced survival. Novel PI3K/mTOR inhibitors may oppose tumor metastasis independent of their growth inhibitory effects, providing a rationale for clinical investigation of PI3K/mTOR inhibitors in settings to prevent micrometastasis. In primary human breast cancers, cytoplasmic p27 is associated with worse outcomes and increased nodal metastasis, and may prove useful as a marker of both PI3K/mTOR activation and PI3K/mTOR inhibitor efficacy.
Assuntos
Neoplasias Ósseas/prevenção & controle , Neoplasias da Mama/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Piridonas/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/secundário , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citoplasma/metabolismo , Intervalo Livre de Doença , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human germinal center-associated lymphoma (HGAL) is an adaptor protein specifically expressed in germinal center lymphocytes. High expression of HGAL is a predictor of prolonged survival of diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma. Furthermore, HGAL expression is associated with early-stage DLBCL, thus potentially limiting lymphoma dissemination. In our previous studies, we demonstrated that HGAL regulates B-cell receptor signaling and cell motility in vitro and deciphered some molecular mechanisms underlying these effects. By using novel animal models for in vivo DLBCL dispersion, we demonstrate here that HGAL decreases lymphoma dissemination and prolongs survival. Furthermore, by using an unbiased proteomic approach, we demonstrate that HGAL may interact with multiple cytoskeletal proteins thereby implicating a multiplicity of effects in regulating lymphoma motility and spread. Specifically, we show that HGAL interacts with tubulin, and this interaction may also contribute to HGAL effects on cell motility. These findings recapitulate previous observations in humans, establish the role of HGAL in dissemination of lymphoma in vivo, and explain improved survival of patients with HGAL-expressing lymphomas.
Assuntos
Proteínas do Citoesqueleto , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma Difuso de Grandes Células B , Proteínas dos Microfilamentos/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias , ProteômicaRESUMO
Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.
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Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial-mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. IMPLICATIONS: Our studies identified the SOX9 protein as a "master regulator" of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.
Assuntos
Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Fatores de Transcrição SOX9/genética , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima , Animais , Apoptose , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Células MCF-7 , Camundongos , Transplante de Neoplasias , Regiões Promotoras Genéticas , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has been identified as an estrogen-responsive gene and was recently identified as a tumor-promoting and prognostic factor for renal cell carcinoma. We investigated whether EBAG9 expression was correlated with primary tumor growth and distant tumor metastasis in a murine breast carcinoma model. Knockdown expression of EBAG9 by small interfering RNA significantly suppressed tumor growth and metastasis in vivo in a highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model. 4T1 cells stably overexpressing EBAG9 developed larger and faster tumor growth and lung metastasis compared with parental 4T1 or 4T1 expressing vector alone. Strong specific cytotoxic T lymphocyte activity and enhanced gamma-interferon and interleukin-2 productions were induced in mice that received EBAG9 small interfering RNA therapy. Gene silencing of EBAG9 prolonged the survival of tumor-bearing mice and induced more intensive infiltration of CD8+ T cells in tumor mass. EBAG9 induced apoptosis of T cells, enhanced glycogen synthase kinase 3beta phosphorylation and inhibited gamma-interferon production of T cells when T lymphocytes were cocultured with 4T1 cells overexpressing EBAG9. Furthermore, overexpression of EBAG9 in 4T1 cells was accompanied with enhanced expression of chemokine (C-X-C motif) receptor 4, which might be involved in tumor metastasis. Taken together, our results suggested that EBAG9 promoted primary 4T1 mammary carcinoma growth and distant metastasis, and EBAG9 small interfering RNA exerted overt regression of tumor growth and metastasis. These findings might provide insights into the mechanism through which tumors evade immunosurveillance and provide a strategy for therapeutic intervention of cancer metastases.
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Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Fosfosserina/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Vitiligo is an acquired pigmentary disorder and its pathogenesis remains unclear. Oxidative stress is considered to be the initial pathogenic event in the melanocyte destruction. NF-E2-related factor2 (Nrf2) is a transcription factor regulating the expression of detoxifying and antioxidant genes. To investigate the association of the Nrf2 gene promoter polymorphisms with vitiligo in Chinese Han population, the genotypes of -686A/G, -684G/A and -650C/A and the genotyping of variable number of tandem repeat were detected. The data were analysed by the chi-square test and the risk was evaluated by calculating OR and 95% CI. There was statistically significant difference in genotypic and allelic frequencies of -650C/A between the two groups (P < 0.05). A(-650) allele was significantly associated with the risk for vitiligo (OR = 1.724, chi(2) = 18.096). Polymorphism of the Nrf2 gene promoter at -650C/A was associated with the development of vitiligo and A(-650) allele may be one of the risk factors.
Assuntos
Fator 2 Relacionado a NF-E2/genética , Polimorfismo Genético , Vitiligo/genética , Adolescente , Adulto , Fatores Etários , Povo Asiático , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Fatores Sexuais , Sequências de Repetição em Tandem/genética , Vitiligo/patologia , Adulto JovemRESUMO
Melagenine, extracted from human placenta, has been shown to be effective in treating patients with vitiligo, yet the mechanisms of melagenine in inducing the repigmentation of vitiligo patients have not been fully investigated. Recent studies have suggested that melagenine stimulates melanocyte proliferation and melanogenesis. In this study, we utilized the NCCmelb4M5 melanoblast cell line to investigate the effects of melagenine on proliferation and differentiation of immature melanocytes or melanoblasts. NCCmelb4M5 cells were treated with different concentrations of melagenine (50-400 microg/ml), and MTT assay was performed to evaluate the effects of melagenine on proliferation of melanoblasts. RT-PCR and Western blotting were used to determine the expression of c-KIT and tyrosinase (TYR). Our results show that melagenine stimulates proliferation of NCCmelb4M5 cells in a dose-dependent manner with an optimal concentration of 100 microg/ml. Multipolar and highly branched dendritic network, as well as cluster-like growing cell assembly were visible in melagenine-treated NCCmelb4M5 cells. Melagenine induced expression of c-KIT, TYR and MITF. Our results provide insights into the molecular mechanism of the beneficial effect of melagenine in the treatment of vitiligo.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lipoproteínas/farmacologia , Melanócitos/efeitos dos fármacos , Melanócitos/fisiologia , Extratos Placentários/farmacologia , Linhagem Celular , Forma Celular , Humanos , Lipoproteínas/uso terapêutico , Melanócitos/citologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Extratos Placentários/uso terapêutico , Vitiligo/tratamento farmacológicoRESUMO
OBJECTIVE: To investigate the association of the single nucleotide polymorphisms (SNPs) in Nrf2 promoter region with the susceptibility to risk of vitiligo. METHODS: Samples of peripheral blood were collected from 300 vitiligo patients and 300 healthy persons. The genotypes of -686A/G, -684G/A, and -650C/A were detected by direct-sequencing. Genotyping of variable number of tandem repeat (VNTR) was performed by gene scan analysis with an ABI 310 Sequencer. Genetic and allelic frequencies were analyzed by Chi-square test and the risk was evaluated by calculating OR and 95% CI. RESULTS: There was statistical significant difference in genotypic and allelic frequencies of -650C/A between the vitiligo group and healthy control group (P < 0.05), and A -650 allele was associated with risk for vitiligo statistically significantly (OR = 1.724, 95% CI: 1.345-2.211, chi2 = 18.096, P < 0.01). Homozygote of A allele increased the risk for vitiligo obviously (OR = 2.902, 95% CI: 1.624-5.188, P < 0.01). No significant difference was found in other three polymorphisms between the two groups. CONCLUSION: polymorphism of Nrf2 promoter region -650C/A was associated with the development of vitiligo and A -650 allele may be one of risk factors for vitiligo.
Assuntos
Fator 2 Relacionado a NF-E2/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Vitiligo/genética , Adulto , Alelos , Sequência de Bases , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Fatores de RiscoRESUMO
Purpose: Rational targeted therapies are needed for treatment of ovarian cancers. Signaling kinases Src and MAPK are activated in high-grade serous ovarian cancer (HGSOC). Here, we tested the frequency of activation of both kinases in HGSOC and the therapeutic potential of dual kinase inhibition.Experimental Design: MEK and Src activation was assayed in primary HGSOC from The Cancer Genome Atlas (TGGA). Effects of dual kinase inhibition were assayed on cell-cycle, apoptosis, gene, and proteomic analysis; cancer stem cells; and xenografts.Results: Both Src and MAPK are coactivated in 31% of HGSOC, and this associates with worse overall survival on multivariate analysis. Frequent dual kinase activation in HGSOC led us to assay the efficacy of combined Src and MEK inhibition. Treatment of established lines and primary ovarian cancer cultures with Src and MEK inhibitors saracatinib and selumetinib, respectively, showed target kinase inhibition and synergistic induction of apoptosis and cell-cycle arrest in vitro, and tumor inhibition in xenografts. Gene expression and proteomic analysis confirmed cell-cycle inhibition and autophagy. Dual therapy also potently inhibited tumor-initiating cells. Src and MAPK were both activated in tumor-initiating populations. Combination treatment followed by drug washout decreased sphere formation and ALDH1+ cells. In vivo, tumors dissociated after dual therapy showed a marked decrease in ALDH1 staining, sphere formation, and loss of tumor-initiating cells upon serial xenografting.Conclusions: Selumetinib added to saracatinib overcomes EGFR/HER2/ERBB2-mediated bypass activation of MEK/MAPK observed with saracatinib alone and targets tumor-initiating ovarian cancer populations, supporting further evaluation of combined Src-MEK inhibition in clinical trials. Clin Cancer Res; 24(19); 4874-86. ©2018 AACR.
Assuntos
MAP Quinase Quinase 1/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Proteômica , Quinases da Família src/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Benzodioxóis/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Feminino , Humanos , MAP Quinase Quinase 1/genética , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/genéticaRESUMO
Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.
Assuntos
Adipócitos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Citocinas/metabolismo , Obesidade/complicações , RNA Mensageiro/metabolismo , Quinases da Família src/metabolismo , Adipócitos/citologia , Animais , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Humanos , Camundongos , RNA Mensageiro/genética , Fatores de Transcrição SOXB1 , Transdução de Sinais , Transfecção , Quinases da Família src/genéticaRESUMO
Estrogen receptor-negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor-negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Proteínas Quinases Associadas com Morte Celular/fisiologia , Genes p53 , Mutação , Animais , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/genética , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44(+) CD24(neg/low) cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44(+) CD24(low+) subpopulation generates CD44(+) CD24(neg) progeny with reduced sphere formation and tumourigenicity. CD44(+) CD24(low+) populations contain subsets of ALDH1(+) and ESA(+) cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44(+) CD24(low+) but not CD44(+) CD24(neg) express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44(+) CD24(low+) cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44(+) CD24(low+) cells, but CD44(+) CD24(neg) were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Família Aldeído Desidrogenase 1 , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Antígeno CD24/metabolismo , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/toxicidade , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Isoenzimas/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Receptores Notch/metabolismo , Retinal Desidrogenase/metabolismo , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
Phosphorylation of the cyclin-dependent kinase inhibitor p27 by upstream mitogenic signaling pathways regulates its stability, localization, and biological function. In human cancers, loss of the antiproliferative action of p27 can arise through reduced protein levels and/or cytoplasmic mislocalization, leading to increased cell proliferation and/or cell migration, respectively. Reduced p27 expression levels and p27 mislocalization have potential prognostic and therapeutic implications in various types of human cancers. This review highlights mechanisms of functional deregulation of p27 by oncogenic signaling that provide an important molecular rationale for pathway targeting in cancer treatment.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Neoplasias/terapia , Transdução de Sinais , Movimento Celular , Proliferação de Células , Humanos , Neoplasias/diagnóstico , Neoplasias/patologia , Fosforilação , Prognóstico , Transdução de Sinais/efeitos dos fármacosRESUMO
Here, we investigated the antitumor effect of adenovirus-mediated gene transfer of LIGHT, the tumor-necrosis factor (TNF) superfamily member also known as TNFSF14, in the murine A20 B-cell lymphoma. LIGHT gene modification resulted in upregulated expression of Fas and the accessory molecule--intercellular adhesion molecule-1 (ICAM-1) on A20 cells and led to enhanced A20 cell apoptosis. LIGHT-modified A20 cells effectively stimulated the proliferation of T lymphocytes and interferon (IFN)-gamma production in vitro. Immunization of BALB/c mice with a LIGHT-modified A20 cell vaccine efficiently elicited protective immunity against challenge with the parental tumor cell line. Adenovirus-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against pre-existing A20 cell lymphoma in BALB/c mice. This adenovirus-mediated LIGHT therapy induced substantial splenic natural killer (NK) and cytotoxic T lymphocyte (CTL) activity, enhanced tumor infiltration by inflammatory cells and increased chemokine expression of CC chemokine ligand 21 (CCL21), IFN-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) from tumor tissues. Thus, adenovirus-mediated LIGHT therapy might have potential utility for the prevention and treatment of B-cell lymphoma.