Proteomics in kanker onderzoek: methoden en toepassingen van ProteinChip arrays

IntroductionThe completion of the human genome project officiallyintroduced the era of the proteome. The new discipline of proteomicswas born, which refers to the analysis of the proteome or thecollection of all proteins that are expressed in a tissue, cell ororganism at a given point in time and under certain physiologicalconditions.Proteins are directly involved in the regulation ofbiochemical processes and aberrant or deregulated protein expression isrelated with many diseases, including cancer. Knowledge of cancerrelated proteins not only offers diagnostic perspectives, but alsoallows development of new molecules which specifically interact withthe deregulated proteins and are thereby able to inhibit cancerprogression.Two unanswered proteomic questions existed at theLaboratory of Experimental Oncology. The first question was whethercancer specific variants of the Vascular Endothelial Growth Factor A(VEGF-A) exist. VEGF-A plays an important role in tumor growth,metastasis and resistance, but is also crucial for the maintenance ahealthy vascular system. Knowledge of tumor specific VEGF-A variantsmight enable us to increase the therapeutic index of anti-VEGF-Atherapies. A second proteomic question was whether GastrointestinalStromal Tumors (GISTs) that easily develop resistance to treatment withImatinib express different proteins than GISTs that rarely develop suchresistance. MethodsThemost important challenges for proteomics are the high complexity of theproteome, the relatively low protein expression levels and the enormousinter and intra individual variability. The most important requirementfor proteomic analysis is therefore a sensitive and reproducible methodthat enables a relatively high sample throughput. We decided toinvestigate the usefulness of ProteinChip technology for the study ofthe proteomic questions mentioned above. ProteinChip technology isbased on the principle of retention chromatography for thefractionation of proteins from complex samples such as body fluids ortissue extracts. These proteins are subsequently analysed with SurfaceEnhanced Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry(SELDI TOF MS). ResultsThe high molecular weightvarieties of VEGF-A were associated with different types ofmalignancies, while the low molecular weight VEGF-A varieties ratherseemed common and were also observed in normal physiological conditions.Acomparative proteomic analysis of GISTs pointed out to 21 SELDI peaksthat were statistically significantly differentially expressed betweena group of GISTs with a high risk for development of Imatinibresistance and a group with a low risk for development of suchresistance. Multivariate analysis further pointed out to 14 additionalinteresting SELDI peaks. In total, forty proteins were identified andthe differential expression pattern of a selection of three of theseproteins could be confirmed with western blot analysis: the 40 kDaATP-ase domain of Heat Shock Protein 70 (HSP70), Cu/Zn SuperoxideDismuatase (SOD1) and Protein 14-3-3 Zeta. Conclusions and discussionThetherapeutic index of anti-VEGF-A therapies might be improved byspecific inhibition of tumor associated high molecular weight VEGF-Avarieties. Our data indicate that the low molecular weight varieties ofVEGF-A play an important role in normal physiology and should thereforebe spared by anti-VEGF-A therapies to minimize the risk of adverseeffects.Stress related proteins might play an important role in thedevelopment of Imatinib resistance of GISTs. The fact that theseproteins are regulated by one and the same factor, the Heat ShockFactor 1 (HSF1), offers interesting perspectives for the treatment ofImatinib resistant GISTs, especially because inhibitors of HSF1 alreadyexist. In addition, the inhibition of signal transduction pathwayscontrolled by 14-3-3 zeta and Calmodulin also seems promising.ProteinChiptechnology has showed to be suitable for both the targeted analysis ofproteins as for the analysis of differential protein expression betweendifferent groups of patients.status: publishe