{"title":"磷蛋白组学分析发现FAK和Src相互磷酸化是表皮生长因子受体突变肺癌对奥希替尼产生耐药性的机制之一","authors":"Takehiro Tozuka MD , Rintaro Noro MD, PhD , Keisuke Yoshida PhD , Satoshi Takahashi MD, PhD , Mariko Hirao XX , Kuniko Matsuda XX , Yasuhiro Kato MD , Shinji Nakamichi MD, PhD , Susumu Takeuchi MD, PhD , Masaru Matsumoto MD, PhD , Akihiko Miyanaga MD, PhD , Shinobu Kunugi MD, PhD , Kazufumi Honda DDS, PhD , Jun Adachi PhD , Masahiro Seike MD, PhD","doi":"10.1016/j.jtocrr.2024.100668","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><p>Osimertinib is a standard treatment for patients with <em>EGFR</em>-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance.</p></div><div><h3>Methods</h3><p>We established two osimertinib-resistant cell lines from <em>EGFR</em> mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3.</p></div><div><h3>Results</h3><p>Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with <em>EGFR</em>-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib.</p></div><div><h3>Conclusions</h3><p>Phosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.</p></div>","PeriodicalId":17675,"journal":{"name":"JTO Clinical and Research Reports","volume":"5 4","pages":"Article 100668"},"PeriodicalIF":3.0000,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2666364324000389/pdfft?md5=7b6560c60e702d1983547e4ee3c5ef18&pid=1-s2.0-S2666364324000389-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer\",\"authors\":\"Takehiro Tozuka MD , Rintaro Noro MD, PhD , Keisuke Yoshida PhD , Satoshi Takahashi MD, PhD , Mariko Hirao XX , Kuniko Matsuda XX , Yasuhiro Kato MD , Shinji Nakamichi MD, PhD , Susumu Takeuchi MD, PhD , Masaru Matsumoto MD, PhD , Akihiko Miyanaga MD, PhD , Shinobu Kunugi MD, PhD , Kazufumi Honda DDS, PhD , Jun Adachi PhD , Masahiro Seike MD, PhD\",\"doi\":\"10.1016/j.jtocrr.2024.100668\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Introduction</h3><p>Osimertinib is a standard treatment for patients with <em>EGFR</em>-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance.</p></div><div><h3>Methods</h3><p>We established two osimertinib-resistant cell lines from <em>EGFR</em> mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3.</p></div><div><h3>Results</h3><p>Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with <em>EGFR</em>-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib.</p></div><div><h3>Conclusions</h3><p>Phosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.</p></div>\",\"PeriodicalId\":17675,\"journal\":{\"name\":\"JTO Clinical and Research Reports\",\"volume\":\"5 4\",\"pages\":\"Article 100668\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-04-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2666364324000389/pdfft?md5=7b6560c60e702d1983547e4ee3c5ef18&pid=1-s2.0-S2666364324000389-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"JTO Clinical and Research Reports\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2666364324000389\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"JTO Clinical and Research Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2666364324000389","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ONCOLOGY","Score":null,"Total":0}
Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer
Introduction
Osimertinib is a standard treatment for patients with EGFR-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance.
Methods
We established two osimertinib-resistant cell lines from EGFR mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3.
Results
Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib.
Conclusions
Phosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.
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