Identification of changes in posttranslational modifications of EGFR and in EGFR-binding partners in head and neck cancer cells upon treatment with EGF or the EGFR antibody mixture Sym004
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell, Cell Culture
DISEASE(S): Tongue Squamous Cell Carcinoma
SUBMITTER: 
Silvia Surinova
LAB HEAD: Silvia Surinova
PROVIDER: PXD016208 | Pride | 2020-05-27
REPOSITORIES: Pride
Dataset's files
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| EGFRinteractome.xlsx | Xlsx | |||
| GelImage.PNG | Other | |||
| MQtxtfilesforPRIDE.7z | Other | |||
| andromeda.7z | Other | |||
| experimentalDesignTemplate.txt | Txt |
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Publications
Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation.
Jones Sylwia S King Peter J PJ Antonescu Costin N CN Sugiyama Michael G MG Bhamra Amandeep A Surinova Silvia S Angelopoulos Nicos N Kragh Michael M Pedersen Mikkel W MW Hartley John A JA Futter Clare E CE Hochhauser Daniel D
Scientific reports 20200120 1
Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR cl ...[more]
Similar Datasets
Project description:Sym004 represents a novel EGF receptor (EGFR)-targeting approach comprising a mixture of two anti-EGFR antibodies directed against distinct epitopes of EGFR. In contrast with single anti-EGFR antibodies, Sym004 induces rapid and highly efficient degradation of EGFR. In the current study, we examine the capacity of Sym004 to augment radiation response in lung cancer and head and neck cancer model systems. We first examined the antiproliferative effect of Sym004 and confirmed 40% to 60% growth inhibition by Sym004. Using clonogenic survival analysis, we identified that Sym004 potently increased cell kill by up to 10-fold following radiation exposure. A significant increase of γH2AX foci resulting from DNA double-strand breaks was observed in Sym004-treated cells following exposure to radiation. Mechanistic studies further showed that Sym004 enhanced radiation response via induction of cell-cycle arrest followed by induction of apoptosis and cell death, reflecting inhibitory effects on DNA damage repair. The expression of several critical molecules involved in radiation-induced DNA damage repair was significantly inhibited by Sym004, including DNAPK, NBS1, RAD50, and BRCA1. Using single and fractionated radiation in human tumor xenograft models, we confirmed that the combination of Sym004 and radiation resulted in significant tumor regrowth delay and superior antitumor effects compared with treatment with Sym004 or radiation alone. Taken together, these data reveal the strong capacity of Sym004 to augment radiation response in lung and head and neck cancers. The unique action mechanism of Sym004 warrants further investigation as a promising EGFR targeting agent combined with radiotherapy in cancer therapy.
| S-EPMC3960925 | biostudies-literature
Project description:As a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array. To accomplish this, mRNA expression levels of these genes were measured in the glioblastoma cell lines, U87 and U178, engineered with EGFR by retrovirus transduction (termed U87-EGFR and U178-EGFR respectively), with or without 20 ng/mL EGF treatment for 3 h. U87 and U178 cells engineered to express EGFR were stimulated with or without EGF. The experiment was replicated twice for each U87 and U178 cells.
2011-11-05 | E-GEOD-33442 | biostudies-arrayexpress
Project description:Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a poor prognosis with a 5-year survival rate of ~45%. PRDM2/RIZ, a member of Positive Regulatory Domain (PRDM) gene family, expresses two main molecular variants, the PR-plus isoform (RIZ1) and the PR-minus (RIZ2). The imbalance in their expression levels in favour of RIZ2 is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumour suppressor, whereas few studies have explored the RIZ2 oncogenic properties. PRDM2 is often target of frameshift mutations and aberrant DNA methylation in CRC. Accordingly, our analysis of Exome- and transcriptome public datasets available at The Cancer Genome Atlas (TCGA) portal revealed that PRDM2 gene is frequently mutated and transcriptionally deregulated in CRC. However, little is known about its role in CRC. In this study, we first evaluated the expression of the different PRDM2 transcripts by in silico analysis on TCGA CRC datasets. Our in-silico analysis on TCGA datasets revealed a highly significant downregulation of RIZ1 in CRC samples whereas a RIZ2 increase was observed in the same samples. Then, we assayed several CRC cell lines by qRT-PCR analysis for the main PRDM2 transcripts and selected DLD-1 cell line, which showed the lowest RIZ2 levels to mimic TCGA datasets analysis results and consequently to assess the PRDM2/RIZ2 role in CRC cells. RNAseq analysis revealed that RIZ2 overexpression induced an EGF overexpression suggesting that RIZ2 could be involved in the EGF autocrine regulation of DLD-1 cell behaviour. Noteworthy, the forced RIZ2 expression increased cell viability, growth, colony formation, migration and organoid formation thus confirming our previous findings on HEK-293 cells. These effects could be mediated by the release of high EGF levels by RIZ2 overexpressing DLD-1 cells. Our findings add novel insights on the putative RIZ2 tumor-promoting functions in CRC, although additional attempts are warranted to depict the underlying molecular mechanism of action
2023-10-20 | E-MTAB-13103 | biostudies-arrayexpress
Project description:UnlabelledEpidermal growth factor receptor (EGFR) is a well-characterized protooncogene that has been shown to promote tumor progression in solid cancers. Clinical results for EGFR targeting with specific monoclonal antibodies (mAbs) such as cetuximab and panitumumab are promising; however, most studies indicate that only a subgroup of patients receiving the mAbs benefit from the immunotherapy, independent of EGFR expression level. To understand the in vivo kinetics of antibody delivery and localization, we performed small-animal PET studies with (64)Cu-labeled panitumumab in xenografts derived from 3 cell lines of human head and neck squamous cell carcinoma (HNSCC).MethodsNude mice bearing HNSCC tumors with different levels of EGFR expression were imaged with small-animal PET using (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-panitumumab. Antibody distribution in the tumors was confirmed by ex vivo immunostaining using panitumumab and fluorescein 5(6)-isothiocyanate (FITC) panitumumab. CD31 immunostaining and Evans blue assay were also performed to assess the tumor vascular density and permeability.ResultsAmong these 3 tumor models, UM-SCC-22B tumors with the lowest EGFR protein expression showed the highest (64)Cu-DOTA-panitumumab accumulation, whereas SQB20 tumors with the highest EGFR expression showed the lowest (64)Cu-DOTA-panitumumab accumulation. Ex vivo staining demonstrated that SQB20 cells still had extremely high EGFR expression after forming tumors in nude mice, indicating that the low uptake of (64)Cu-DOTA-panitumumab in SQB20 tumors was not due to the loss of EGFR expression. The results from CD31 immunostaining and Evans blue permeability assay suggest that the low vessel density, poor vascular permeability, and binding site barrier are likely responsible for the overall low tumor uptake of the highly EGFR-expressing SQB20 tumors.ConclusionThe results from this study provide a possible explanation for the lack of an observed correlation between therapeutic efficacy of cetuximab and panitumumab and EGFR expression level as determined by immunohistochemistry or fluorescent in situ hybridization and may shed new light on the complications of anti-EGFR mAb therapy for HNSCC and other malignancies.
| S-EPMC6435376 | biostudies-literature
Project description:The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence.
| S-EPMC3674118 | biostudies-literature
Project description:Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase is overexpressed in 90% of Head and neck squamous cell carcinoma (HNSCC) patients. Clinical trials with EGFR-targeted tyrosine kinase inhibitors such as erlotinib have shown a modest activity in HNSCC alternate mechanisms of resistance are acquired. To investigate these acquired mechanisms of resistance and identify novel therapeutic targets we employed whole exome sequencing of an isogenic pair of erlotinib sensitive (SCC-S) and resistant (SCC-R) HNSCC cell line. We observed single nucleotide variations and copy number alterations in genes related to RAS-RAF-MEK-ERK pathway. To assess the effects of these variations on cellular kinome and we employed SILAC-based phosphoproteomic analysis of SCC-S and SCC-R cell lines. Quantitative phosphoprotein profiling led to identification of 5558 unique phosphopeptides and 5025 unique phosphosites corresponding to 2344 proteins. We observed, 903 phosphopeptides belonging to 579 proteins and 518 phosphopeptides belonging to 368 proteins to be hyper and hypophosphorylated (≥2 fold) in SCC-R cells, respectively. Bioinformatics analysis of differentially phosphorylated proteins showed enrichment of proteins involved in MAPK pathway downstream of EGFR. We identified and validated activation of proteins related to MAPK pathway and its downstream targets in SCC-R cells. We further demonstrated that MAP2K1 inhibitor can be used as an alternative to erlotinib in HNSCC.
2019-12-18 | PXD007785 | Pride
Project description:Although Epidermal growth factor receptor (EGFR) is overexpressed in 90% of Head and neck squamous cell carcinoma (HNSCC) patients. Clinical trials with EGFR-targeted small molecule inhibitors such as erlotinib have shown a modest activity in recurrent or advanced HNSCC. To investigate the acquired mechanisms of erlotinib resistance we employed SILAC-based total proteomic analysis of an isogenic pair of erlotinib sensitive (SCC-S) and resistant (SCC-R) HNSCC cell line. This resulted in the identification of 5,427 proteins of which 532 proteins were overexpressed and 527 proteins were downregulated in SCC-R cells as compared to SCC-S cells (≥2 fold). Several proteins known to mediate erlotinib resistance in HNSCC and lung cancer were found to be dysregulated. Bioinformatics analysis of differentially expressed proteins showed enrichment of proteins involved in focal adhesion kinase (FAK) pathway downstream of EGFR. We identified CUB-domain containing protein 1 (CDCP1) and integrin β1 as upstream regulators of FAK signalling pathway to be overexpressed. We further demonstrated that CDCP1 and FAK can be targeted in combination as an alternative to erlotinib in HNSCC.
2019-12-18 | PXD007768 | Pride
Project description:Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase is overexpressed in 90% of Head and neck squamous cell carcinoma (HNSCC) patients. Clinical trials with EGFR-targeted tyrosine kinase inhibitors such as erlotinib have shown a modest activity in HNSCC alternate mechanisms of resistance are acquired. To investigate these acquired mechanisms of resistance and identify novel therapeutic targets we employed SILAC-based tyrosine phosphoteomic analysis of an isogenic pair of erlotinib sensitive (SCC-S) and resistant (SCC-R) HNSCC cell line. Quantitative phosphotyrosine profiling revealed 98 phosphopeptides belonging to 64 proteins and 66 phosphopeptides belonging to 52 proteins to be hyper and hypophosphorylated (≥2 fold) in SCC-R cells, respectively. Several proteins such MET proto-oncogene, receptor tyrosine kinase (MET) and CRK like proto-oncogene, adaptor protein (CRKL) known to mediate erlotinib resistance in HNSCC and lung cancer were found to be dysregulated. Bioinformatics analysis of differentially phosphorylated proteins showed enrichment of proteins involved in focal adhesion kinase (FAK) pathway downstream of EGFR. We identified and validated activation of several phosphorylation sites of protein tyrosine kinase 2 (PTK2) in SCC-R cells and its downstream targets. We further demonstrated that CUB-domain containing protein 1 (CDCP1), an upstream regulator of PTK2 activity and PTK2 can be targeted in combination as an alternative to erlotinib in HNSCC.
2022-10-13 | PXD007769 | Pride
Project description:PurposeEGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity.Experimental designWe measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab.ResultsBoth monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype-dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8+ T cells in patients treated with cetuximab compared with those treated with panitumumab.ConclusionsAlthough panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229-37. ©2016 AACR.
| S-EPMC5093040 | biostudies-literature
Project description:Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.
2023-03-11 | PXD031988 | Pride