RESUMO
Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR-binding proteins are required to elucidate this network. We affinity-purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high-resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc-1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well-described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin-associated adaptor complex AP-2 showed 4.6-fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP-2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.
Assuntos
Receptores ErbB , Peptídeos e Proteínas de Sinalização Intracelular , Mapas de Interação de Proteínas/fisiologia , Proteômica/métodos , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Biologia de Sistemas/métodos , Espectrometria de Massas em TandemRESUMO
Exploring mechanisms of drug resistance to targeted small molecule drugs is critical for an extended clinical benefit in the treatment of non-small cell lung cancer (NSCLC) patients carrying activating epidermal growth factor receptor (EGFR) mutations. Here, we identified constitutive cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A) in the HCC4006rErlo0.5 NSCLC cell line adapted to erlotinib as a model of acquired drug resistance. Constitutive CIP2A resulted in a constitutive activation of Akt signaling. The proteasome inhibitor bortezomib was able to reduce CIP2A levels, which resulted in an activation of protein phosphatase 2A and deactivation of Akt. Combination experiments with erlotinib and bortezomib revealed a lack of interaction between the two drugs. However, the effect size of bortezomib was higher in HCC4006rErlo0.5, compared to the erlotinib-sensitive HCC4006 cells, as indicated by an increase in Emax (0.911 (95%CI 0.867-0.954) vs. 0.585 (95%CI 0.568-0.622), respectively) and decrease in EC50 (52.4 µM (95%CI 46.1-58.8 µM) vs. 73.0 µM (95%CI 60.4-111 µM), respectively) in the concentration-effect model, an earlier onset of cell death induction, and a reduced colony surviving fraction (0.38 ± 0.18 vs. 0.95 ± 0.25, respectively, n = 3, p < 0.05). Therefore, modulation of CIP2A with bortezomib could be an interesting approach to overcome drug resistance to erlotinib treatment in NSCLC.
Assuntos
Autoantígenos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Bortezomib/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Mitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células A549 , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006rERLO0.5 adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006rERLO0.5 cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006rERLO0.5 cells. Taken together, differential proteins in the EGFR interactome of HCC4006rERLO0.5 cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance.