RESUMO
INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) and AXL tyrosine kinase receptor are known to be involved in the SARS-CoV-2 entry of the host cell. Therefore, targeting ACE2 and AXL should be an effective strategy to inhibit virus entry into cells. However, developing agents that can simultaneously target ACE2 and AXL remains a formidable task. The natural compound quercetin has been shown to inhibit AXL expression. MATERIALS AND METHODS: In this study, we employed PLGA nanoparticles to prepare nanoparticles encapsulated with quercetin, coated with ACE2-containing cell membranes, or encapsulated with quercetin and then coated with ACE-2-containing cell membranes. These nanoparticles were tested for their abilities to neutralize or inhibit viral infection. RESULTS: Our data showed that nanoparticles encapsulated with quercetin and then coated with ACE2-containing cell membrane inhibited the expression of AXL without causing cytotoxic activity. Nanoparticles incorporated with both quercetin and ACE2-containing cell membrane were found to be able to neutralize pseudo virus infection and were more effective than free quercetin and nanoparticles encapsulated with quercetin at inhibition of pseudo virus and SARS-CoV-2 infection. CONCLUSIONS: We have shown that the biomimetic nanoparticles incorporated with both ACE-2 membrane and quercetin showed the most antiviral activity and may be further explored for clinical application.
Assuntos
COVID-19 , Nanopartículas , Humanos , Enzima de Conversão de Angiotensina 2 , Quercetina/farmacologia , Quercetina/uso terapêutico , SARS-CoV-2RESUMO
The extracts from Psoralea corylifolia Linn. (P. corylifolia) seeds have been shown to display antitumor activity. To date, the prospects of this plant and its active compounds in the treatment of non-small-cell lung cancer (NSCLC) have not been thoroughly studied. In this study, we identified a novel psorachromene compound that displays selective cytotoxic effects on all NSCLC cells tested, including NSCLC cells harboring epidermal growth factor receptor (EGFR) activation mutants (H1975L858R/T790M and H1975-MS35L858R/T790M/C797S ). Psorachromene induces G1 arrest in NSCLC cells harboring wild-type EGFR but induces apoptosis in NSCLC cells harboring activating EGFR mutations. Psorachromene inhibits activated EGFR signaling and kinase activity and suppresses tumor growth of implanted H1975-MS35L858R/T790M/C797S cells in nude mice. Molecular docking analysis revealed that psorachromene could form stronger bonds with mutant EGFR than wild-type EGFR, which might account for the greater cytotoxic effects observed in NSCLC cells harboring activating EGFR mutations (H1975 and H1975-MS35) than wild-type EGFR (A549). In conclusion, it is suggested that psorachromene is an attractive agent to be further explored for its use in the treatment of NSCLC patients harboring EGFR L858R/T790M/C797S.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
RATIONALE: Despite the fact that tyrosine kinase inhibitors (TKIs) have been found effective in treating patients harboring activating mutations of epidermal growth factor receptor (EGFR), an acquired secondary mutation, T790M, which lowers the affinity to TKIs, can lead to EGFR TKI resistance after this standard treatment. OBJECTIVES: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. METHODS: Lung adenocarcinoma cells were treated with T315, and cell proliferation and apoptotic proportion were determined by the CellTiter 96 AQueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry. The effects of T315 on EGFR mRNA and protein levels, autophosphorylation, ubiquitination, and degradation were evaluated by real-time polymerase chain reaction and Western blot, respectively. Direct targeting of T315 to EGFR was confirmed by the in vitro kinase assay and mass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. MEASUREMENTS AND MAIN RESULTS: We identified T315 as a novel, potent small molecule for suppressing cancer cell proliferation in vitro and in vivo. The therapeutic effect was verified after T315 was combined with a second-generation TKI, afatinib, compared with a single drug administration. We found a new mechanism of action, in that T315 appears to directly bind EGFR and triggers EGFR-Y1045 autophosphorylation, whereby its degradation is triggered through the ubiquitin-proteasome pathway. CONCLUSIONS: Our evidence suggests that T315 is a novel class of anticancer drug that is able to inhibit the growth of EGFR-TKI-resistant lung adenocarcinoma cells by inducing the degradation of EGFR.
Assuntos
Adenocarcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Afatinib , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Western Blotting , Proliferação de Células/efeitos dos fármacos , Combinação de Medicamentos , Ensaios Enzimáticos , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Espectrometria de Massas , Camundongos , Mutação/genética , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Proto-Oncogênicas c-cbl/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-cbl/genética , Quinazolinas/efeitos adversos , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. OBJECTIVES: To screen drugs that target CSCs to improve the current treatment outcome and overcome drug resistance in patients with lung cancer. METHODS: We used publicly available embryonic stem cell and CSC-associated gene signatures to query the Connectivity Map for potential drugs that can, at least in part, reverse the gene expression profile of CSCs. High scores were noted for several phenothiazine-like antipsychotic drugs, including trifluoperazine. We then treated lung CSCs with different EGFR mutation status with trifluoperazine to examine its anti-CSC properties. Lung CSCs resistant to epidermal growth factor receptor-tyrosine kinase inhibitor or cisplatin were treated with trifluoperazine plus gefitinib or trifluoperazine plus cisplatin. Animal models were used for in vivo validation of the anti-CSC effect and synergistic effect of trifluoperazine with gefitinib. MEASUREMENTS AND MAIN RESULTS: We demonstrated that trifluoperazine inhibited CSC tumor spheroid formation and down-regulated the expression of CSC markers (CD44/CD133). Trifluoperazine inhibited Wnt/ß-catenin signaling in gefitinib-resistant lung cancer spheroids. The combination of trifluoperazine with either gefitinib or cisplatin overcame drug resistance in lung CSCs. Trifluoperazine inhibited the tumor growth and enhanced the inhibitory activity of gefitinib in lung cancer metastatic and orthotopic CSC animal models. CONCLUSIONS: Using in silico drug screening by Connectivity Map followed by empirical validations, we repurposed an existing phenothiazine-like antipsychotic drug, trifluoperazine, as a potential anti-CSC agent that could overcome epidermal growth factor receptor-tyrosine kinase inhibitor and chemotherapy resistance.
Assuntos
Antipsicóticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Quinazolinas/farmacologia , Trifluoperazina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Gefitinibe , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Sensibilidade e Especificidade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Immune checkpoint inhibitors (ICIs) targeting PD-L1 immunotherapy are state-of-the-art treatments for advanced non-small cell lung cancer (NSCLC). However, the treatment response of certain patients with NSCLC is unsatisfactory because of an unfavorable tumor microenvironment (TME) and poor permeability of antibody-based ICIs. In this study, we aimed to discover small-molecule drugs that can modulate the TME to enhance ICI treatment efficacy in NSCLC in vitro and in vivo. We identified a PD-L1 protein-modulating small molecule, PIK-93, using a cell-based global protein stability (GPS) screening system. PIK-93 mediated PD-L1 ubiquitination by enhancing the PD-L1-Cullin-4A interaction. PIK-93 reduced PD-L1 levels on M1 macrophages and enhanced M1 antitumor cytotoxicity. Combined PIK-93 and anti-PD-L1 antibody treatment enhanced T cell activation, inhibited tumor growth, and increased tumor-infiltrating lymphocyte (TIL) recruitment in syngeneic and human peripheral blood mononuclear cell (PBMC) line-derived xenograft mouse models. PIK-93 facilitates a treatment-favorable TME when combined with anti-PD-L1 antibodies, thereby enhancing PD-1/PD-L1 blockade cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Microambiente Tumoral , Linfócitos do Interstício TumoralRESUMO
Particulate matter 2.5 (PM2.5) is a risk factor for lung cancer. In this study, we investigated the molecular mechanisms of PM2.5 exposure on lung cancer progression. We found that short-term exposure to PM2.5 for 24 h activated the EGFR pathway in lung cancer cells (EGFR wild-type and mutant), while long-term exposure of lung cancer cells to PM2.5 for 90 days persistently promoted EGFR activation, cell proliferation, anchorage-independent growth, and tumor growth in a xenograft mouse model in EGFR-driven H1975 cancer cells. We showed that PM2.5 activated AhR to translocate into the nucleus and promoted EGFR activation. AhR further interacted with the promoter of TMPRSS2, thereby upregulating TMPRSS2 and IL18 expression to promote cancer progression. Depletion of TMPRSS2 in lung cancer cells suppressed anchorage-independent growth and xenograft tumor growth in mice. The expression levels of TMPRSS2 were found to correlate with nuclear AhR expression and with cancer stage in lung cancer patient tissue. Long-term exposure to PM2.5 could promote tumor progression in lung cancer through activation of EGFR and AhR to enhance the TMPRSS2-IL18 pathway.
Assuntos
Neoplasias Pulmonares , Material Particulado , Humanos , Camundongos , Animais , Material Particulado/toxicidade , Interleucina-18 , Transdução de Sinais , Neoplasias Pulmonares/patologia , Receptores ErbB/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
In our ongoing study of the desmosdumotin C (1) series, twelve new analogues, 21-32, mainly with structural modifications in ring-A, were prepared and evaluated for in vitro antiproliferative activity against several human tumor cell lines. Among them, the 4'-iodo-3,3,5-tripropyl-4-methoxy analogue (31) showed significant antiproliferative activity against multiple human tumor cell lines with ED(50) values of 1.1-2.8 µM. Elongation of the C-3 and C-5 carbon chains reduced activity relative to propyl substituted analogues; however, activity was still better than that of natural compound 1. Among analogues with various ether groups on C-4, compounds with methyl (2) and propyl (26) ethers inhibited cell growth of multiple tumor cells lines, while 28 with an isobutyl ether showed selective antiproliferative activity against lung cancer A549 cells (ED(50) 1.7 µM). The gene expression profiles showed that 3 may modulate the spindle assembly checkpoint (SAC) and chromosome separation, and thus, arrest cells at the G2/M-phase.
Assuntos
Alcenos/síntese química , Antineoplásicos/síntese química , Cetonas/síntese química , Modelos Biológicos , Neoplasias/tratamento farmacológico , Fuso Acromático/efeitos dos fármacos , Alcenos/química , Alcenos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cetonas/química , Cetonas/farmacologia , Estrutura MolecularRESUMO
EGFR is established as a driver of lung cancer, yet the regulatory machinery underlying its oncogenic activity is not fully understood. PTEN-induced kinase 1 (PINK1) kinase is a key player in mitochondrial quality control, although its role in lung cancer and EGFR regulation is unclear. In this study, we show that PINK1 physically directly interacts with EGFR via the PINK1 C-terminal domain (PINK1-CTD) and the EGFR tyrosine kinase domain. This interaction constituted an endogenous steric hindrance to receptor dimerization and inhibited EGFR-mediated lung carcinogenesis. Depletion of PINK1 from lung cancer cells promoted EGFR dimerization, receptor activation, EGFR downstream signaling, and tumor growth. In contrast, overexpression of PINK1 or PINK1-CTD suppressed EGFR dimerization, activation, downstream signaling, and tumor growth. These findings identify key elements in the EGFR regulatory cascade and illustrate a new direction for the development of anti-EGFR therapeutics, suggesting translational potential of the PINK1-CTD in lung cancer. SIGNIFICANCE: This study identifies PINK1 as a critical tumor suppressor that impedes EGFR dimerization and highlights PINK1-CTD as a potential therapeutic agent in EGFR-driven lung cancer.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Proteínas Quinases/fisiologia , Multimerização Proteica , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Estudos de Coortes , Regulação para Baixo , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Multimerização Proteica/genéticaRESUMO
The development of a new generation of tyrosine kinase inhibitors (TKIs) has improved the treatment response in lung adenocarcinomas. However, acquired resistance often occurs due to new epidermal growth factor receptor (EGFR) mutations. In particular, the C797S mutation confers drug resistance to T790M-targeting EGFR TKIs. To address C797S resistance, a promising therapeutic avenue is combination therapy that targets both total EGFR and acquired mutations to increase drug efficacy. We showed that combining vorinostat, a histone deacetylase inhibitor (HDACi), with brigatinib, a TKI, enhanced antitumor effects in primary culture and cell lines of lung adenocarcinomas harboring EGFR L858R/T790M/C797S mutations (EGFR-3M). While EGFR phosphorylation was decreased by brigatinib, vorinostat reduced total EGFR-3M (L858R/T790M/C797S) proteins through STUB1-mediated ubiquitination and degradation. STUB1 preferably ubiquitinated other EGFR mutants and facilitated protein turnover compared to EGFR-WT. The association between EGFR and STUB1 required the functional chaperone-binding domain of STUB1 and was further enhanced by vorinostat. Finally, STUB1 levels modulated EGFR downstream functions. Low STUB1 expression was associated with significantly poorer overall survival than high STUB1 expression in patients harboring mutant EGFR. Vorinostat combined with brigatinib significantly improved EGFR-TKI sensitivity to EGFR C797S by inducing EGFR-dependent cell death and may be a promising therapy in treating C797S-resistant lung adenocarcinomas.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organofosforados/farmacologia , Pirimidinas/farmacologia , Vorinostat/farmacologia , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Compostos Organofosforados/administração & dosagem , Pirimidinas/administração & dosagem , Distribuição Aleatória , Vorinostat/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: Claudin (CLDN)-1, a key component of tight junction complexes, was down-regulated in human lung adenocarcinomas. OBJECTIVES: To investigate the clinical significance of CLDN1 expression in patients with lung adenocarcinoma and its role in cancer invasion and metastasis. METHODS: We examined the CLDN1 mRNA expression in tumor specimens from 64 patients with lung adenocarcinoma and protein expression by immunohistochemistry in an independent cohort of 67 patients with lung adenocarcinoma. CLDN1 functions in cancer cell migration, invasion, and metastatic colonization were studied by overexpression and knockdown of CLDN1. Affymetrix microarrays were performed to identify gene expression changes associated with CLDN1 overexpression. MEASUREMENTS AND MAIN RESULTS: We found that low-CLDN1 mRNA expression had shorter overall survival (P = 0.027, log-rank test) in 64 patients with lung adenocarcinoma, and we confirmed by immunohistochemistry that low CLDN1 expression had shorter overall survival (P = 0.024, log-rank test) in an independent cohort of 67 patients with lung adenocarcinoma. Overexpression of CLDN1 inhibited cancer cell dissociation in time-lapse imaging of wound healing, and suppressed cancer cell migration, invasion, and metastasis. Knockdown CLDN1 expression increased cancer cell invasive and metastatic abilities. Affymetrix microarrays identified a panel of genes altered by CLDN1 overexpression. CLDN1 increased expressions of cancer invasion/metastasis suppressors (e.g., connective tissue growth factor [CTGF], thrombospondin 1 [THBS1], deleted in liver cancer 1 [DLC1], occludin [OCLN], zona occludens 1 [ZO-1]) and suppressed expressions of invasion/metastasis enhancers (e.g., secreted phosphoprotein 1 [SPP1], cut-like homeobox 1 [CUTL1], transforming growth factor alpha [TGF-alpha], solute carrier family 2 [faciliated glucose transporter] member 3 [SLC2A3], placental growth factor [PGF]), supporting a role for CLDN1 as an invasion and metastasis suppressor. CONCLUSIONS: CLDN1 is a cancer invasion/metastasis suppressor. CLDN1 is also a useful prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Idoso , Biomarcadores Tumorais/genética , Claudina-1 , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Prognóstico , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de SobrevidaRESUMO
Oncogenic mutations of epidermal growth factor receptor (EGFR) are responsive to targeted tyrosine kinase inhibitor (TKI) treatment in non-small-cell lung cancer (NSCLC). However, NSCLC patients harboring activating EGFR mutations inevitably develop resistance to TKIs. The acquired EGFR C797S mutation is a known mechanism that confers resistance to third-generation EGFR TKIs such as AZD9291. In this work, we employed CRISPR/Cas9 genome-editing technology to knock-in the EGFR C797S mutation into an NSCLC cell line harboring EGFR L858R/T790M. The established cell model was used to investigate the biology and treatment strategy of acquired EGFR C797S mutations. Transcriptome and proteome analyses revealed that the differentially expressed genes/proteins in the cells harboring the EGFR C797S mutation are associated with a mesenchymal-like cell state with elevated expression of AXL receptor tyrosine kinase. Furthermore, we presented evidence that inhibition of AXL is effective in slowing the growth of NSCLC cells harboring EGFR C797S. Our findings suggest that AXL inhibition could be a second-line or a potential adjuvant treatment for NSCLC harboring the EGFR C797S mutation.
RESUMO
Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype.
RESUMO
Midbody, a transient organelle-like structure, is known as central for abscission and is indispensable for termination of cytokinesis. Here, we used the midbody proteome inventories to construct the potential midbody protein-protein interaction (PPI) network. To delineate novel regulators participating in cytokinesis, the z-score, a standard statistic score, rather than hub degree was implemented to prioritize the novel hubs. Of these hubs, KIAA0133, SEPT1, KIAA1377, and CRMP-1 were localized to the midbody, whereas HTR3A and ICAM2 were associated with the cleavage furrow as examined by immunofluorescence. Knockdown of SEPT1 and KIAA1377 resulted in increasing numbers of cytokinesis defect cells, suggesting these newly identified hubs play critical roles in cytokinesis progression. Moreover, ectopic expression of CRMP-1 mutant in which Aurora-A phosphorylation sites have been replaced with Ala results in a cytokinesis defect. This subproteome network construction not only sheds light on the intimate interactions of the midbody proteomes, but also prioritizes novel hubs or protein complexes that may govern the process of cytokinesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese/fisiologia , Organelas/química , Mapeamento de Interação de Proteínas , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Aurora Quinases , Proteínas de Ciclo Celular/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos TestesRESUMO
Collapsin response mediator protein-1 (CRMP-1) controls neural development and axonal growth but also acts as a cancer invasion suppressor. In this study, we investigated the transcriptional regulation of CRMP-1 expression. Using a serial deletion strategy, we identified a basal promoter region between nucleotides -100 and -180 in the 5' flanking region of CRMP-1 (nucleotides -1,920 to +50) that contains multiple putative Sp1 and C/EBPalpha sites. Site-directed mutagenesis and deletion analysis revealed that the two C/EBPalpha sites, from nucleotides -122 to -133 and from nucleotides -101 to -113, are the most important regulatory elements. Gel-shift and antibody supershift assays showed that Sp1 protein was also present at this C/EBPalpha site, which overlaps with a Sp1 site. Overexpression of Sp1 decreased CRMP-1 promoter activity and protein expression, whereas overexpression of C/EBPalpha produced the opposite effect. Chromatin immunoprecipitation assays confirmed that Sp1 and C/EBPalpha compete for binding at the overlapping C/EBPalpha and Sp1 sites and reciprocally regulate CRMP-1 expression. Overexpression of cyclooxygenase-2 (COX-2) decreased CRMP-1 mRNA and protein expression. Conversely, the COX-2 inhibitor, celecoxib, induced a dose-dependent increase in CRMP-1 expression. COX-2 inhibition also decreased Sp1-DNA complex formation and inhibited cell invasion. We conclude that transcription of the invasion suppressor, CRMP-1, is reciprocally regulated at the promoter region by C/EBPalpha and Sp1. COX-2 inhibitors increase CRMP-1 expression by inhibiting Sp1-DNA complex formation and enhancing DNA binding of C/EBPalpha at the promoter.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ciclo-Oxigenase 2/metabolismo , Invasividade Neoplásica/patologia , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição Sp1/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Análise Mutacional de DNA , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Elementos de Resposta/genética , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
BACKGROUND: Lung cancer is the most common cause of cancer-related mortality worldwide despite diagnostic improvements and the development of targeted therapies, notably including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling has been shown to contribute to tumorigenesis, tumor progression, and resistance to therapy in most human cancer types, including lung cancer. Here, we explored the therapeutic effects of co-inhibition of PI3K and mTOR in non-small-cell lung cancer (NSCLC) cells with different EGFR status. METHODS: The antiproliferative activity of a dual PI3K/mTOR inhibitor BEZ235 was examined by the WST-1 assay and the soft agar colony-formation assay in 2 normal cell lines and 12 NSCLC cell lines: 6 expressing wild-type EGFR and 6 expressing EGFR with activating mutations, including exon 19 deletions, and L858R and T790 M point mutations. The combination indexes of BEZ235 with cisplatin or an EGFR-TKI, BIBW2992 (afatinib), were calculated. The mechanisms triggered by BEZ235 were explored by western blotting analysis. The anti-tumor effect of BEZ235 alone or combined with cisplatin or BIBW2992 were also studied in vivo. RESULTS: BEZ235 suppressed tumor growth in vitro and in vivo by inducing cell-cycle arrest at G1 phase, but without causing cell death. It also reduced the expression of cyclin D1/D3 by regulating both its transcription and protein stability. Moreover, BEZ235 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells by enhancing or prolonging DNA damage and BIBW2992-induced apoptosis in EGFR-TKI-resistant NSCLC cells containing a second TKI-resistant EGFR mutant. CONCLUSIONS: The dual PI3K/mTOR inhibition by BEZ235 is an effective antitumor strategy for enhancing the efficacy of chemotherapy or targeted therapy, even as a monotherapy, to restrict tumor growth in lung cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Imidazóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Quinolinas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células A549 , Afatinib/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina D3/genética , Ciclina D3/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Developing new therapeutic strategies to overcome drug resistance of cancer cells is an ongoing endeavor. From among 2 million chemicals, we identiï¬ed ethyl 4-oxo-2-phenyl-1,4-dihydroquinoline-6-carboxylate (AS1712) as a low-toxicity inhibitor of lung cancer cell proliferation and xenograft tumor growth. We show that AS1712 is active against broad cancer cell lines and is able to bind in the colchicine-binding pocket of ß-tubulin, thereby inhibiting microtubule assembly and, consequently, inducing mitotic arrest and apoptosis. Our cell-based structure-activity relationship study identified a new lead compound, RJ-LC-15-8, which had a greater anti-proliferative potency for H1975â¯cells than did AS1712, while maintaining a similar mechanism of action. Notably, AS1712 and RJ-LC-15-8 overcame P-glycoprotein efflux pump and ß-tubulin alterations that lead to acquired resistance against microtubule-targeting drugs of cancer cells. AS1712 and RJ-LC-15-8 may be lead compounds that overcome acquired resistance to microtubule-targeting agents of cancer cells.
Assuntos
Quinolonas/química , Quinolonas/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colchicina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tubulina (Proteína)/químicaRESUMO
PURPOSE: Neuropilin 1 (NRP1) is a mediator of lung branching and angiogenesis in embryonic development and angiogenesis in cancer. The role of NRP1 in cancer progression is not fully elucidated. We investigated the role of NRP1 in cancer invasion and tumor angiogenesis, its signaling pathways, prognostic significance, and therapeutic implications. EXPERIMENTAL DESIGN: Sixty patients with non-small cell lung cancer (NSCLC) were studied. NRP1 mRNA expression was measured using real-time quantitative reverse-transcription PCR. NRP1 and cancer cell invasion, angiogenesis, and signaling pathways were studied using NRP1 stimulation by vascular endothelial growth factor 165 (VEGF(165)) and NRP1 inhibition by small interfering RNAs (siRNA), soluble NRP1 (sNRP1), and NRP1-inhibition peptides. The NRP1-inhibition peptides were identified using a phage display peptide library. RESULTS: NSCLC patients with high expression of NRP1 had shorter disease-free (P = 0.0162) and overall survival (P = 0.0164; log-rank test). Multivariate analyses showed NRP1 is an independent prognostic factor in overall (HR, 2.37, 95% CI = 1.15 to 4.9, P = 0.0196) and disease-free survival [hazard ratio (HR), 2.38; 95% confidence interval (95% CI), 1.15-4.91; P = 0.0195] of NSCLC patients. Knockdown of NRP1 suppressed cancer cell migration, invasion, filopodia formation, tumorigenesis, angiogenesis, and in vivo metastasis. NRP1 signaling pathways involved VEGF receptor 2 and phosphoinositide-3-kinase (PI3K) and Akt activation. Two potent synthetic anti-NRP1 peptides, DG1 and DG2, which block NRP1 signaling pathways and suppress tumorigenesis, cancer invasion, and angiogenesis, were identified. CONCLUSIONS: NRP1 is a cancer invasion and angiogenesis enhancer. NRP1 expression is an independent predictor of cancer relapse and poor survival in NSCLC patients. NRP1 plays a critical role in tumorigenesis, cancer invasion, and angiogenesis through VEGF, PI3K, and Akt pathways. NRP1 may have potential as a new therapeutic target in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Neuropilina-1/fisiologia , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/terapia , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/etiologia , Neovascularização Patológica/prevenção & controle , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Mensageiro/análise , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Tolerância Imunológica , Leucemia Promielocítica Aguda/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Microambiente Tumoral , Ubiquitinação , Células A549 , Animais , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Metástase Neoplásica , Proteínas Nucleares/genética , Prognóstico , Interferência de RNA , Proteínas Supressoras de Tumor/genéticaRESUMO
PURPOSE: In a previous genome-wide gene expression profiling analysis using an invasion cancer cell lines model, we have identified Slug as selectively overexpressed in the highly invasive cancer cells. Here, we investigated the clinical significance of Slug in lung adenocarcinoma and the role of Slug in the process of cancer cell invasion and metastasis. EXPERIMENTAL DESIGN: Real-time quantitative reverse transcription-PCR was used to investigate Slug mRNA in surgically resected lung adenocarcinoma of 54 patients and its correlation with survival. We overexpressed Slug in a lung adenocarcinoma cell line with very low Slug levels and investigated the in vitro and in vivo effects of Slug expression. RESULTS: High expression of Slug mRNA in lung cancer tissue was significantly associated with postoperative relapse (P = 0.03) and shorter patient survival (P < 0.001). The overexpression of Slug enhanced xenograft tumor growth and increased microvessel counts in angiogenesis assay. Both inducible and constitutive overexpression of Slug suppressed the expression of E-cadherin and increased the in vitro invasive ability. Zymography revealed increased matrix metalloproteinase-2 activity in Slug overexpressed cells. ELISA, reverse transcription-PCR, and immunohistochemistry confirmed the increase of matrix metalloproteinase-2 proteins and mRNA in Slug overexpressed cells and xenograft tumors. CONCLUSIONS: Slug expression can predict the clinical outcome of lung adenocarcinoma patients. Slug is a novel invasion-promoting gene in lung adenocarcinoma.
Assuntos
Adenocarcinoma/patologia , Neoplasias Pulmonares/patologia , Fatores de Transcrição/genética , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Idoso , Animais , Northern Blotting , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Análise de Sobrevida , Fatores de Transcrição/fisiologia , Transfecção , Transplante Heterólogo , Resultado do TratamentoRESUMO
Clinical virotherapy has been successfully approved for use in cancer treatment by the U.S. Food and Drug Administration; however, a number of improvements are still sought to more broadly develop virotherapy. A particular challenge is to administer viral therapy systemically and overcome limitations in intratumoral injection, especially for complex tumors within sensitive organs. To achieve this, however, a technique is required that delivers the virus to the tumor before the body's natural self-defense eradicates the virus prematurely. Here we show that recombinant adeno-associated virus serotype 2 (AAV2) chemically conjugated with iron oxide nanoparticles (â¼5 nm) has a remarkable ability to be remotely guided under a magnetic field. Transduction is achieved with microscale precision. Furthermore, a gene for production of the photosensitive protein KillerRed was introduced into the AAV2 genome to enable photodynamic therapy (PDT), or light-triggered virotherapy. In vivo experiments revealed that magnetic guidance of "ironized" AAV2-KillerRed injected by tail vein in conjunction with PDT significantly decreases the tumor growth via apoptosis. This proof-of-principle demonstrates guided and highly localized microscale, light-triggered virotherapy.