Small Molecule T315 Promotes Casitas B-Lineage Lymphoma-Dependent Degradation of Epidermal Growth Factor Receptor via Y1045 Autophosphorylation

Despite the fact that tyrosine kinase inhibitors (TKIs) have been found effective in treating patients harboring activating mutations of epidermal growth factor receptor (EGFR), an acquired secondary mutation, T790M, which lowers the affinity to TKIs, can lead to EGFR TKI resistance after this stand...

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Veröffentlicht in:American journal of respiratory and critical care medicine 2016-04, Vol.193 (7), p.753-766
Hauptverfasser: Huang, Kuo-Yen, Kao, Shih-Han, Wang, Wen-Lung, Chen, Chi-Yuan, Hsiao, Tzu-Hung, Salunke, Santosh B, Chen, Jeremy J W, Su, Kang-Yi, Yang, Shuenn-Chen, Hong, Tse-Ming, Chen, Ching-Shih, Yang, Pan-Chyr
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container_issue 7
container_start_page 753
container_title American journal of respiratory and critical care medicine
container_volume 193
creator Huang, Kuo-Yen
Kao, Shih-Han
Wang, Wen-Lung
Chen, Chi-Yuan
Hsiao, Tzu-Hung
Salunke, Santosh B
Chen, Jeremy J W
Su, Kang-Yi
Yang, Shuenn-Chen
Hong, Tse-Ming
Chen, Ching-Shih
Yang, Pan-Chyr
description Despite the fact that tyrosine kinase inhibitors (TKIs) have been found effective in treating patients harboring activating mutations of epidermal growth factor receptor (EGFR), an acquired secondary mutation, T790M, which lowers the affinity to TKIs, can lead to EGFR TKI resistance after this standard treatment. To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Lung adenocarcinoma cells were treated with T315, and cell proliferation and apoptotic proportion were determined by the CellTiter 96 AQueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry. The effects of T315 on EGFR mRNA and protein levels, autophosphorylation, ubiquitination, and degradation were evaluated by real-time polymerase chain reaction and Western blot, respectively. Direct targeting of T315 to EGFR was confirmed by the in vitro kinase assay and mass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. We identified T315 as a novel, potent small molecule for suppressing cancer cell proliferation in vitro and in vivo. The therapeutic effect was verified after T315 was combined with a second-generation TKI, afatinib, compared with a single drug administration. We found a new mechanism of action, in that T315 appears to directly bind EGFR and triggers EGFR-Y1045 autophosphorylation, whereby its degradation is triggered through the ubiquitin-proteasome pathway. Our evidence suggests that T315 is a novel class of anticancer drug that is able to inhibit the growth of EGFR-TKI-resistant lung adenocarcinoma cells by inducing the degradation of EGFR.
doi_str_mv 10.1164/rccm.201502-0250OC
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To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Lung adenocarcinoma cells were treated with T315, and cell proliferation and apoptotic proportion were determined by the CellTiter 96 AQueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry. The effects of T315 on EGFR mRNA and protein levels, autophosphorylation, ubiquitination, and degradation were evaluated by real-time polymerase chain reaction and Western blot, respectively. Direct targeting of T315 to EGFR was confirmed by the in vitro kinase assay and mass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. We identified T315 as a novel, potent small molecule for suppressing cancer cell proliferation in vitro and in vivo. 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Kao, Shih-Han ; Wang, Wen-Lung ; Chen, Chi-Yuan ; Hsiao, Tzu-Hung ; Salunke, Santosh B ; Chen, Jeremy J W ; Su, Kang-Yi ; Yang, Shuenn-Chen ; Hong, Tse-Ming ; Chen, Ching-Shih ; Yang, Pan-Chyr</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c380t-e7d6e6cd0bb3c182d9014d126a9fe0b2f8d9cb2d2b16d93df489c24672d6ef4c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Adaptor Proteins, Signal Transducing - drug effects</topic><topic>Adaptor Proteins, Signal Transducing - genetics</topic><topic>Adenocarcinoma - drug therapy</topic><topic>Adenocarcinoma - genetics</topic><topic>Adenocarcinoma of Lung</topic><topic>Afatinib</topic><topic>Animals</topic><topic>Antineoplastic Agents - adverse effects</topic><topic>Antineoplastic Agents - therapeutic use</topic><topic>Blotting, Western</topic><topic>Cell Proliferation - drug effects</topic><topic>Drug Combinations</topic><topic>Drug Resistance, Neoplasm - drug effects</topic><topic>Enzyme Assays</topic><topic>ErbB Receptors - antagonists &amp; 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subjects Adaptor Proteins, Signal Transducing - drug effects
Adaptor Proteins, Signal Transducing - genetics
Adenocarcinoma - drug therapy
Adenocarcinoma - genetics
Adenocarcinoma of Lung
Afatinib
Animals
Antineoplastic Agents - adverse effects
Antineoplastic Agents - therapeutic use
Blotting, Western
Cell Proliferation - drug effects
Drug Combinations
Drug Resistance, Neoplasm - drug effects
Enzyme Assays
ErbB Receptors - antagonists & inhibitors
ErbB Receptors - drug effects
ErbB Receptors - genetics
Humans
Lung Neoplasms - drug therapy
Lung Neoplasms - genetics
Mass Spectrometry
Mice
Mutation - genetics
Protein Kinase Inhibitors - adverse effects
Protein Kinase Inhibitors - therapeutic use
Proto-Oncogene Proteins c-cbl - drug effects
Proto-Oncogene Proteins c-cbl - genetics
Quinazolines - adverse effects
Quinazolines - therapeutic use
Real-Time Polymerase Chain Reaction
Tumor Cells, Cultured
Xenograft Model Antitumor Assays
title Small Molecule T315 Promotes Casitas B-Lineage Lymphoma-Dependent Degradation of Epidermal Growth Factor Receptor via Y1045 Autophosphorylation
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