Quantitative proteomics unveiled: Regulation of DNA double strand break repair by EGFR involves PARP1

Myllynen, Laura; Kwiatkowski, Marcel; Gleißner, Lisa; Riepen, Britta; Hoffer, Konstantin; Wurlitzer, M.; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai; Schlüter, Hartmut; Kriegs, Malte

doi:10.1016/j.radonc.2015.09.018

Cited by 14 publications

(11 citation statements)

References 31 publications

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“…We have recently published that EGFR targeting inhibits DNA DSB repair in HNSCC cells via MAPK signalling and PARP1 [26]. In this study we also observed elevated residual 53BP1 foci, indicative of an inhibition of DNA DSB repair (Figure 5B).…”

Section: Discussionsupporting

confidence: 75%

“…However, it does not correlate with cellular radiosensitization since an increased amount of residual 53BP1 was detected also in SAS cells, which do not become sensitized. Additionally, using delayed plating conditions, an increased number of foci was detected in UT-SCC 5 and SAS cells, too [26]. Whilst the quantification of residual DSB repair complexes using marker proteins such as 53PB1 is a very well established method, further endeavours have to be made to answer why residual repair foci do not correlate with cellular survival in the context of combined EGFR targeting and IR, a phenomenon which has been described already by other studies [10, 27].…”

Section: Discussionmentioning

confidence: 99%

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Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

et al. 2016

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The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

et al. 2016

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“…This indicates a biological response to the heavy amino acids, which can be detected only by including an adequate control. In future studies the number of quantified proteins might be increased by including also proteins smaller than 34 kDa, by an increased LC column length and by further reduction of the complexity of each sample (for example reduced size range of the SDS gel fractions [ 14 ] or by isoelectric focusing of the lysates [ 17 ]). Another way to increase the number of quantified proteins would be to decrease the cut-off of the SILACAnalyzer.…”

Section: Discussionmentioning

confidence: 99%

“…By analyzing the nuclear phospho–proteome using SILAC-based MS we have recently identified the molecular mechanism of EGFR-mediated DNA DSB repair inhibition in irradiated HNSCC cells [ 14 ]. In that study, erlotinib treatment led to multiple changes in the phospho-proteome but not in the composition of the chromatin fraction (unpublished data [ 18 ]).…”

Section: Discussionmentioning

confidence: 99%

“…To address this question we have recently established a quantitative mass spectrometry (MS) approach to analyze changes in nuclear protein phosphorylation. Using this approach we have already successfully unveiled the mechanism by which EGFR inhibition affects DNA DSB repair in HNSCC cells, demonstrating the effectiveness of this approach [ 14 ]. In the present study, we compare this phospho-proteomic approach with an approach analyzing proteins, which are altered in their interaction with the chromatin.…”

Section: Introductionmentioning

confidence: 99%

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Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins

et al. 2017

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The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising.

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Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets

Bußmann

Hoffer

Bargen

et al. 2021

Intl Journal of Cancer

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Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a testfunctional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

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Quantitative proteomics unveiled: Regulation of DNA double strand break repair by EGFR involves PARP1

Cited by 14 publications

References 31 publications

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets

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