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De Kreukel, Oct 2008
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Neoplasia, 2013
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Journal of Cellular Physiology, Jan 20, 2020
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OncoImmunology, Jul 19, 2019
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Experimental and Molecular Medicine, Dec 16, 2016
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Current Pharmaceutical Design, Feb 1, 2013
Combination of drugs with different targets is a logical approach to overcome multilevel cross-st... more Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.
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Introduction Tyrosine kinases play an important role in tumor biology. Their activity can be meas... more Introduction Tyrosine kinases play an important role in tumor biology. Their activity can be measured using a kinase peptide substrate array consisting of 144 Tyr residue-containing peptides (PamChip®, PamGene, Den Bosch, The Netherlands). We evaluated this platform for the measurement of kinase activity in tumor tissue and cancer cell lines under various experimental conditions. Methods Lysates of colorectal and renal cancer cell lines, HCT116 and 786-0 respectively, were made using both Mammalian and Tissue Protein Extraction Reagent (M-PER and T-PER, Thermo Scientific) and Radio-Immunoprecipitation Assay (RIPA, home made) buffer. Lysates from patient-derived tumor tissues were prepared by adding T-PER to several 10 μm cryoslides containing >50% tumor. After lysate incubation with reaction buffer containing a fluorescent labeled antibody against phospo-tyrosine and ATP, kinase activity profiles were determined on kinetics of recorded peptide substrate phosphorylation intensities. The effect of protein and ATP concentration, different lysis buffers and number of freeze-thaw cycles on basal kinase activity was studied. Sunitinib, sorafenib and dasatinib, clinically available tyrosine kinase inhibitors (TKIs), were used to differentially inhibit kinase activity in the lysates. Results Application of 2.5-15 µg protein in 40 µl sample mix per array revealed linearly increasing phosphorylation signal intensities and initial velocities (Vini) of the kinetic curve (R2 = 0.98). Increasing ATP concentrations induced phosphorylation signal intensities, but above 400 µM the curve deviated from linearity. Basal kinase activity profiles of cell lines and tumor tissues were reproducible with CV's below 15%, with good signal-to-background ratios and low aspecific binding. Different lysis buffers resulted in a maximum variation of phosphorylation signal intensity of 47±5.7% in both cell lines without affecting the actual profile. Quadruple freeze-thawing of lysates did not affect signal intensities by more than 10%. Inhibition profiles of treated vs. control lysates were reproducible within and between experiments, showing a higher and differential number of inhibited peptides at increasing TKI concentrations. In contrast to the ATP-independent inhibition of dasatinib, ATP-dependent inhibition for sunitinib and sorafenib was demonstrated by combining a fixed drug concentration with increasing concentrations of ATP up to 800 µM. Conclusion Kinase activity in lysates from cancer cell lines and patient-derived tumor tissue can be reproducibly profiled with a tyrosine kinase peptide substrate array. In addition, TKIs show differential ATP-dependent inhibition profiles on this array. Taken together, we expect that array-based tumor kinase activity profiling may lead to specific TKI-phosphorylation fingerprints for personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3608. doi:1538-7445.AM2012-3608
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Background Sunitinib is a multi-targeted tyrosine kinase inhibitor (TKI), that is used for treatm... more Background Sunitinib is a multi-targeted tyrosine kinase inhibitor (TKI), that is used for treatment of patients with advanced renal cell cancer (RCC) Drug resistance to sunitinib is a major clinical problem. We recently reported that continuous exposure to sunitinib causes acquired resistance of tumor cells. The receptor tyrosine kinase Axl has been implicated in acquired resistance to multiple TKIs, including erlotinib and lapatinib. In addition, sunitinib was previously reported to be a potential inhibitor of Axl catalytic activity. We therefore investigated the potential role of Axl in sunitinib resistance. Methods 786-O cells resistant to sunitinib were obtained by continuous exposure to the drug as previously published (Gotink et al. Clin Cancer Res 2011). AXL expression was studied in 786-O cell lines and EC-RF24 endothelial cells by RT-PCR and Western blot. Sunitinib and AXL inhibitor R428 sensitivity of cells transfected with siRNAs against AXL or scrambled siRNAs was determined by MTT cell viability assays. For kinase activity experiments recombinant AXL and tyrosine peptide arrays (PamGene, ‘s Hertogenbosch, the Netherlands) were used. Phospho-AXL and total AXL in cell lysates were measured by ELISA. Results Axl phosphorylation in vitro in both 786-O and EC-RF24 cells was dose-dependently inhibited by sunitinib, with a 50% inhibitory concentration of 5 μM. Activity of recombinant Axl kinase was measured on tyrosine containing peptide arrays in presence of 0, 1 or 4 μM sunitinib or 2 μM R428. Sunitinib induced dose-dependent inhibition of Axl activity, where 2 μM R428 blocked all peptide phosphorylations. In addition, 786-O sunitinib resistant cells (786-O SUN) showed a 4-fold overexpression of Axl protein compared to 786-O parental cells. However, gene silencing of AXL in 786-O SUN cells by RNA interference did not sensitize the cells to sunitinib. Sunitinib resistant cells were shown to be cross-resistant to the Axl inhibitor R428 with respective IC50 values of 0.9 μM (±0.1 μM) and 1.8 μM (±0.1 μM). Conclusion Phosphorylation of Axl is inhibited by sunitinib in both 786-O RCC and EC-RF24 endothelial cells. Despite its overexpression in sunitinib resistant cells, gene silencing of AXL did not sensitize 786-O resistant cells to sunitinib, indicating that sunitinib resistance is not mediated by AXL. Citation Format: Johannes C. Van der Mijn, Kristy J. Gotink, Richard R. de Haas, Henk Dekker, Connie R. Jimenez, Henk J. Broxterman, Henk M.w. Verheul. Sunitinib inhibits AXL phosphorylation in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5641. doi:10.1158/1538-7445.AM2013-5641
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Introduction Exosomes are nanometer-sized vesicles secreted by tumor cells after fusion of multiv... more Introduction Exosomes are nanometer-sized vesicles secreted by tumor cells after fusion of multivesicular bodies with the plasma membrane that reflect tumor cell biology. Exosomes contain distinct classes of molecules including mRNA, miRNA and proteins. Because they can be isolated from blood they are promising candidate biomarkers. Diagnostic biomarkers for selection of patients for treatment with tyrosine kinase inhibitors are urgently warranted. We hypothesize that exosomes contain tumor derived kinases that may serve as biomarkers for treatment with kinase inhibitors. Methods The U87 glioma cell line with or without EGFRvIII (U87ΔEGFR) was used as an in vitro model in this study. Cell viability was studied with the MTT-assay. Exosomes were isolated from 32h conditioned media either serum-free or containing 10% exosome-deprived serum and from 5-9mL of fresh human serum from 3 cancer patients. The isolation procedure consisted of subsequent centrifugation at 500G, 2000G, 10.000G and 100.000G. The exosome pellet was treated with proteases for 1h at 37°C to digest soluble proteins, washed in PBS and lysed. Western blots for phospho-EGFR (y1068), EGFR, phospho-AKT, AKT, phospho-ERK, ERK, Alpha-Tubulin and exosome markers CD63, ALIX and CD81 were performed. Results The sensitivity of U87ΔEGFR to erlotinib (ERL) was significantly higher compared to parental U87 cells with IC50s of 1µM and 5.4µM (p<0.001), respectively. Treatment with 1 μM ERL resulted in reduced phosphorylation of EGFR, AKT and ERK in U87ΔEGFR, but not in U87 cells. The overexpression of constitutively active EGFRvIII mutant increased the phosphorylation of EGFR and downstream AKT and ERK in cells as well as in exosomes. Trypsin treatment only affected membrane proteins, where AKT and ERK levels were not altered. Exosomes from U87ΔEGFR cells treated with 0, 1 and 10µM ERL reflected the phosphorylation patterns of the cells with dose-dependent effects on AKT and ERK. Phosphorylated AKT and ERK could also be detected in exosomes from the cancer patients. Conclusion We here show that kinase activity and treatment response are reflected by exosomes derived from tumor cells in vitro. In addition we found that phosphorylated proteins can be detected in exosomes derived from blood of patients with cancer. Our aim is to further develop exosomal kinase activity analysis as an early biomarker for treatment response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 176. doi:1538-7445.AM2012-176
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Background: EGFR is an established target in advanced NSCLC, and the EGFR tyrosine kinase inhibit... more Background: EGFR is an established target in advanced NSCLC, and the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have been approved for the treatment of patients harbouring activating-EGFR mutations. Unfortunately, their efficacy is limited by acquired resistance, caused in ≈50% of the cases by the T790M secondary point-mutation. Several EGFR inhibitors have been developed with the aim to overcome such resistance. However, emergence of in vitro resistance due to T790M amplification has been reported for second-generation EGFR-TKIs. Therefore we evaluated the efficacy of CNX-2006, a prototype of the novel mutant-selective EGFR-TKI CO-1686, which is currently in a phase I clinical trial in previously treated mutant EGFR NSCLCs. Methods: CNX-2006 was provided by Celgene Avilomics Research. Its antiproliferative activity was tested by sulforhodamine B assay in 12 NSCLC cell lines, previously characterized for EGFR and K-Ras mutational status and gefitinib sensitivity, including PC9GR4 and PC9DR1 (kindly provided by Dr. Jänne, Harvard University, Boston, USA). Novel CNX-2006 resistant clones have been established starting from the EGFR T790M cells H1975 and PC9GR4, and several markers have been characterized by RT-PCR, kinase array and Western blot. Results: CNX-2006 inhibited cell proliferation independently from K-Ras mutations while it was as effective as gefitinib in activating-EGFR mutation positive cells. In the cell lines expressing wild-type EGFR CNX-2006 and gefitinib had limited anti-proliferative activity. CNX-2006 inhibited EGFR-T790M cells growth up to 1000-fold more compared to wild-type EGFR cells. EGFR inhibition was observed in cells harbouring the T790M mutation at IC50 values below 20 nM after 1 hour exposure to the drug. In contrast to gefitinib, CNX-2006 also significantly reduced the volume of tumor spheres derived from H1975 cells. Multiple CNX-2006 resistant clones were generated by exposing H1975 or PC9GR4 cells to increasing drug concentrations, leading to 30-fold resistant clones, which grow in CNX-2006 concentrations 16-20 times the initial IC50s. This resistance was retained for at least 3 months after drug removal. CNX-2006 resistant clones showed differences in expression of several biomarkers associated with EMT, such as a 3-fold reduction of E-cadherin mRNA and a 60-fold increase in MMP9 compared to the parental cells. Conclusions: CNX-2006 is a potent, mutant-selective EGFR inhibitor with excellent in vitro activity in cells with activating EGFR mutations, as well as in cells harbouring the T790M mutation. Future studies in mechanisms underlying EMT are warranted and might be used to prevent CNX-2006 resistance. Citation Format: Elena Galvani, Elisa Giovannetti, Annette O. Walter, Robert Tjin, Henk Dekker, Pier Giorgio Petronini, Egbert F. Smit, Godefridus J. Peters. Role of epithelial-mesenchymal transition (EMT) in sensitivity to CNX-2006, a novel mutant-selective EGFR inhibitor which overcomes in vitro T790M-mediated resistance in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3244. doi:10.1158/1538-7445.AM2013-3244
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Clinical Cancer Research, Feb 11, 2022
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Cancers, Feb 1, 2020
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European journal of medicinal chemistry, Mar 1, 2020
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Journal of Clinical Oncology, May 20, 2016
11609Background: Mass spectrometry-based phosphoproteomics of tumor tissue lysates provides a pot... more 11609Background: Mass spectrometry-based phosphoproteomics of tumor tissue lysates provides a potential personalized medicine approach based on its global information on aberrantly activated signal...
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Oncologist, Jul 17, 2018
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Biochemical Pharmacology, Aug 1, 2019
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Journal of Proteomics, Jun 1, 2017
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