Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

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
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Abstract

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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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
导言奥西替尼是治疗表皮生长因子受体突变型 NSCLC 患者的标准疗法。尽管已经发现了一些奥希替尼的耐药机制,但仍有近50%的机制有待阐明。本研究旨在确定奥希替尼耐药的非遗传机制。方法我们采用逐步法从表皮生长因子受体突变阳性的PC-9和HCC827 NSCLC细胞系中建立了两种奥希替尼耐药细胞系(分别为PC-9OR和HCC827OR)。我们使用质谱法比较了奥希替尼耐药细胞和亲代细胞的磷酸化蛋白质组谱。结果磷蛋白组学分析发现,PC-9OR和HCC827OR细胞中有80个磷酸化位点相互上调。Kinase Enrichment Analysis version 3分析确定了病灶粘附激酶(FAK)和原癌基因酪氨酸蛋白激酶Src(Src)是这些上调磷酸化蛋白的上游激酶。在这两种细胞系中,小干扰 RNA 介导的 FAK 敲除可减少 Src 磷酸化,而 Src 敲除可减少 FAK 磷酸化。此外,FAK或Src特异性小干扰RNA处理可恢复PC-9OR和HCC827OR细胞的表皮生长因子受体磷酸化。FAK和Src抑制剂的组合抑制了体外PC-9OR和HCC827OR细胞的增殖,并抑制了异种移植小鼠模型中肿瘤的生长。对表皮生长因子受体突变型 NSCLC 患者肿瘤的免疫组化结果表明,磷酸化的 FAK 和 Src 参与了奥希替尼的初始耐药性和获得性耐药性的产生。FAK和Src的相互磷酸化参与了奥希替尼的耐药性。因此,抑制FAK和Src可能是治疗奥希替尼耐药NSCLC的新策略。
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CiteScore
4.20
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0.00%
发文量
145
审稿时长
19 weeks
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