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1.
Oncogene ; 43(19): 1431-1444, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38485737

RESUMO

MET amplification/mutations are important targetable oncogenic drivers in NSCLC, however, acquired resistance is inevitable and the majority of patients with targetable MET alterations fail to respond to MET tyrosine kinase inhibitors (TKIs). Furthermore, MET amplification is among the most common mediators of TKI resistance. As such, novel therapies to target MET pathway and overcome MET TKI resistance are clearly needed. Here we show that the epithelial-mesenchymal transition (EMT) transcription factor, TWIST1 is a key downstream mediator of HGF/MET induced resistance through suppression of p27 and targeting TWIST1 can overcome resistance. We found that TWIST1 is overexpressed at the time of TKI resistance in multiple MET-dependent TKI acquired resistance PDX models. We have shown for the first time that MET directly stabilized the TWIST protein leading to TKI resistance and that TWIST1 was required for MET-driven lung tumorigenesis as well as could induce MET TKI resistance when overexpressed. TWIST1 mediated MET TKI resistance through suppression of p27 expression and genetic or pharmacologic inhibition of TWIST1 overcame TKI resistance in vitro and in vivo. Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome resistance in MET-driven NSCLC as well as in other oncogene driven subtypes in which MET amplification is the resistance mechanism.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento de Hepatócito , Neoplasias Pulmonares , Proteínas Nucleares , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-met , Proteína 1 Relacionada a Twist , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Oncogenes/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
BMC Cancer ; 24(1): 233, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38373988

RESUMO

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.


Assuntos
Antineoplásicos , Neoplasias de Cabeça e Pescoço , Proteínas Quinases S6 Ribossômicas 90-kDa , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Anoctamina-1/genética , Anoctamina-1/metabolismo
3.
Oncogene ; 38(5): 656-670, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30171258

RESUMO

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Acrilamidas , Substituição de Aminoácidos , Compostos de Anilina , 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/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação de Sentido Incorreto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína 1 Relacionada a Twist/genética
4.
Mol Cancer Res ; 15(12): 1764-1776, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28851812

RESUMO

TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Harmina/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Células A549 , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética
5.
Mol Cancer Ther ; 16(8): 1658-1668, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28566436

RESUMO

A subset of non-small cell lung cancers (NSCLC) are dependent upon oncogenic driver mutations, including the most frequently observed driver mutant KRAS, which is associated with a poor prognosis. As direct RAS targeting in the clinic has been unsuccessful to date, use of Hsp90 inhibitors appeared to be a promising therapy for KRAS-mutant NSCLC; however, limited clinical efficacy was observed due to rapid resistance. Furthermore, the combination of the Hsp90 inhibitor (Hsp90i), ganetespib, and docetaxel was tested in a phase III clinical trial and failed to demonstrate benefit. Here, we investigated the mechanism(s) of resistance to ganetespib and explored why the combination with docetaxel failed in the clinic. We have not only identified a critical role for the bypass of the G2-M cell-cycle checkpoint as a mechanism of ganetespib resistance (GR) but have also found that GR leads to cross-resistance to docetaxel. Reactivation of p90RSK and its downstream target, CDC25C, was critical for GR and mediated the bypass of a G2-M arrest. Overexpression of either p90RSK or CDC25C lead to bypass of G2-M arrest and induced ganetespib resistance in vitro and in vivo Moreover, resistance was dependent on p90RSK/CDC25C signaling, as synthetic lethality to ERK1/2, p90RSK, or CDC25C inhibitors was observed. Importantly, the combination of ganetespib and p90RSK or CDC25C inhibitors was highly efficacious in parental cells. These studies provide a way forward for Hsp90 inhibitors through the development of novel rationally designed Hsp90 inhibitor combinations that may prevent or overcome resistance to Hsp90i. Mol Cancer Ther; 16(8); 1658-68. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Triazóis/uso terapêutico , Fosfatases cdc25/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Docetaxel , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Mitose/efeitos dos fármacos , Transdução de Sinais , Taxoides/farmacologia , Taxoides/uso terapêutico , Triazóis/farmacologia , Regulação para Cima/efeitos dos fármacos
6.
Mol Cancer Ther ; 16(5): 793-804, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28167505

RESUMO

Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations, and no effective therapeutic strategy exists for these patients. The use of Hsp90 inhibitors in KRAS-mutant NSCLC appeared to be a promising approach, as these inhibitors target many KRAS downstream effectors; however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations, which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS-mutant NSCLC ganetespib-resistant cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that ganetespib-resistant cells are "addicted" to these pathways, as ganetespib resistance leads to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK), were also increased in the ganetespib-resistant cells. Genetic or pharmacologic inhibition of p90RSK in ganetespib-resistant cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors, providing an effective therapeutic strategy for KRAS-mutant NSCLC. Mol Cancer Ther; 16(5); 793-804. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Serina-Treonina Quinases TOR/genética , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Int J Cancer ; 139(4): 841-53, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27037842

RESUMO

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.


Assuntos
Antígenos de Neoplasias/imunologia , Mastócitos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Xenoenxertos , Humanos , Interleucina-9/biossíntese , Mastócitos/metabolismo , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Receptor 3 Toll-Like/metabolismo
8.
Mol Ther Nucleic Acids ; 4: e237, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25919090

RESUMO

Blocking the immunoinhibitory PD-1:PD-L1 pathway using monoclonal antibodies has led to dramatic clinical responses by reversing tumor immune evasion and provoking robust and durable antitumor responses. Anti-PD-1 antibodies have now been approved for the treatment of melanoma, and are being clinically tested in a number of other tumor types as both a monotherapy and as part of combination regimens. Here, we report the development of DNA aptamers as synthetic, nonimmunogenic antibody mimics, which bind specifically to the murine extracellular domain of PD-1 and block the PD-1:PD-L1 interaction. One such aptamer, MP7, functionally inhibits the PD-L1-mediated suppression of IL-2 secretion in primary T-cells. A PEGylated form of MP7 retains the ability to block the PD-1:PD-L1 interaction, and significantly suppresses the growth of PD-L1+ colon carcinoma cells in vivo with a potency equivalent to an antagonistic anti-PD-1 antibody. Importantly, the anti-PD-1 DNA aptamer treatment was not associated with off-target TLR-9-related immune responses. Due to the inherent advantages of aptamers including their lack of immunogenicity, low cost, long shelf life, and ease of synthesis, PD-1 antagonistic aptamers may represent an attractive alternative over antibody-based anti PD-1 therapeutics.

9.
Mol Oncol ; 8(2): 337-50, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24388361

RESUMO

The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV-like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (K(D) 16 ± 3 nM) and interacts with itself (K(D) 100 ± 17 nM) and more tightly to the IgC-like A(3) domain (K(D) 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A(3)B(3) domains, or by adding soluble recombinant forms of the CEA N, A(3) or A(3)B(3) domains or a peptide corresponding to residues 108-115 of CEA resulted in the inhibition of CEA-mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA-expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A(3)B(3) modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV-like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Animais , Antígeno Carcinoembrionário/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Estrutura Terciária de Proteína
10.
Mol Oncol ; 7(4): 799-811, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23656757

RESUMO

The formation of metastatic foci occurs through a series of cellular events, initiated by the attachment and aggregation of cancer cells leading to the establishment of micrometastases. We report the derivation of synthetic DNA aptamers bearing anti-adhesive properties directed at cancer cells expressing the carcinoembryonic antigen (CEA). Two DNA aptamers targeting the homotypic and heterotypic IgV-like binding domain of CEA were shown to block the cell adhesion properties of CEA, while not recognizing other IgV-like domains of CEACAM family members that share strong sequence and structural homologies. More importantly, the pre-treatment of CEA-expressing tumour cells with these aptamers prior to their intraperitoneal implantation resulted in the prevention of peritoneal tumour foci formation. Taken together, these results highlight the effectiveness of targeting the cell adhesion properties of cancer cells with aptamers in preventing tumour implantation.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Antígeno Carcinoembrionário/metabolismo , Animais , Antígenos CD/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Imunidade Inata/efeitos dos fármacos , Camundongos , Modelos Biológicos , Estrutura Secundária de Proteína
11.
J Control Release ; 164(1): 58-64, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23075769

RESUMO

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Citosol/metabolismo , Portadores de Fármacos/administração & dosagem , Exotoxinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Técnicas de Cultura de Células , Rastreamento de Células , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacocinética , Portadores de Fármacos/farmacocinética , Exotoxinas/genética , Exotoxinas/farmacocinética , Citometria de Fluxo , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética , Exotoxina A de Pseudomonas aeruginosa
12.
Int J Cancer ; 131(12): 2839-51, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22495743

RESUMO

Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.


Assuntos
Antígeno Carcinoembrionário/imunologia , Metástase Neoplásica/imunologia , Neoplasias Experimentais/imunologia , Transferência Adotiva , Animais , Vacinas Anticâncer/imunologia , Camundongos , Neoplasias Experimentais/patologia
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