Small molecule T315 promotes casitas b-lineage lymphoma dependent degradation of epidermal growth factor receptor via Y1045 autophosphorylation

Kuo Yen Huang, Shih Han Kao, Wen Lung Wang, Chi Yuan Chen, Tzu Hung Hsiao, Santosh B. Salunke, Jeremy J.W. Chen, Kang Yi Su, Shuenn Chen Yang, Tse-Ming Hong, Ching Shih Chen, Pan Chyr Yang

Research output: Contribution to journalArticle

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Abstract

Rationale: 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. Objectives: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Methods: 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 bythe in vitro kinase assayandmass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. Measurements and Main Results: 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. Conclusions: Our evidence suggests that T315 is a novel class of anticancerdrugthatisabletoinhibitthegrowthofEGFR-TKI resistant lung adenocarcinoma cells by inducing the degradation of EGFR.

Original languageEnglish
Pages (from-to)753-766
Number of pages14
JournalAmerican Journal of Respiratory and Critical Care Medicine
Volume193
Issue number7
DOIs
Publication statusPublished - 2016 Apr 1

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Epidermal Growth Factor Receptor
Lymphoma
Protein-Tyrosine Kinases
Cell Proliferation
Tetrazolium Salts
Mutation
Ubiquitination
Therapeutic Uses
Proteasome Endopeptidase Complex
Ubiquitin
Heterografts
Real-Time Polymerase Chain Reaction
Spectrum Analysis
Flow Cytometry
Phosphotransferases
Western Blotting
Messenger RNA
Therapeutics
Pharmaceutical Preparations
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Pulmonary and Respiratory Medicine
  • Critical Care and Intensive Care Medicine

Cite this

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. / Small molecule T315 promotes casitas b-lineage lymphoma dependent degradation of epidermal growth factor receptor via Y1045 autophosphorylation. In: American Journal of Respiratory and Critical Care Medicine. 2016 ; Vol. 193, No. 7. pp. 753-766.
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abstract = "Rationale: 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. Objectives: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Methods: 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 bythe in vitro kinase assayandmass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. Measurements and Main Results: 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. Conclusions: Our evidence suggests that T315 is a novel class of anticancerdrugthatisabletoinhibitthegrowthofEGFR-TKI resistant lung adenocarcinoma cells by inducing the degradation of EGFR.",
author = "Huang, {Kuo Yen} and Kao, {Shih Han} and Wang, {Wen Lung} and Chen, {Chi Yuan} and Hsiao, {Tzu Hung} and Salunke, {Santosh B.} and Chen, {Jeremy J.W.} and Su, {Kang Yi} and Yang, {Shuenn Chen} and Tse-Ming Hong and Chen, {Ching Shih} and Yang, {Pan Chyr}",
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Small molecule T315 promotes casitas b-lineage lymphoma dependent degradation of epidermal growth factor receptor via Y1045 autophosphorylation. / 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.

In: American Journal of Respiratory and Critical Care Medicine, Vol. 193, No. 7, 01.04.2016, p. 753-766.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Small molecule T315 promotes casitas b-lineage lymphoma dependent degradation of epidermal growth factor receptor via Y1045 autophosphorylation

AU - Huang, Kuo Yen

AU - Kao, Shih Han

AU - Wang, Wen Lung

AU - Chen, Chi Yuan

AU - Hsiao, Tzu Hung

AU - Salunke, Santosh B.

AU - Chen, Jeremy J.W.

AU - Su, Kang Yi

AU - Yang, Shuenn Chen

AU - Hong, Tse-Ming

AU - Chen, Ching Shih

AU - Yang, Pan Chyr

PY - 2016/4/1

Y1 - 2016/4/1

N2 - Rationale: 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. Objectives: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Methods: 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 bythe in vitro kinase assayandmass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. Measurements and Main Results: 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. Conclusions: Our evidence suggests that T315 is a novel class of anticancerdrugthatisabletoinhibitthegrowthofEGFR-TKI resistant lung adenocarcinoma cells by inducing the degradation of EGFR.

AB - Rationale: 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. Objectives: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. Methods: 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 bythe in vitro kinase assayandmass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. Measurements and Main Results: 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. Conclusions: Our evidence suggests that T315 is a novel class of anticancerdrugthatisabletoinhibitthegrowthofEGFR-TKI resistant lung adenocarcinoma cells by inducing the degradation of EGFR.

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