Tozuka Takehiro, Noro Rintaro, Yoshida Keisuke, Takahashi Satoshi, Hirao Mariko, Matsuda Kuniko, Kato Yasuhiro, Nakamichi Shinji, Takeuchi Susumu, Matsumoto Masaru, Miyanaga Akihiko, Kunugi Shinobu, Honda Kazufumi, Adachi Jun, Seike Masahiro
Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Department of Bioregulation, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
JTO Clin Res Rep. 2024 Mar 21;5(4):100668. doi: 10.1016/j.jtocrr.2024.100668. eCollection 2024 Apr.
Osimertinib is a standard treatment for patients with -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.
We established two osimertinib-resistant cell lines from 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.
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 -mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib.
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.
奥希替尼是治疗表皮生长因子受体（EGFR）突变型非小细胞肺癌（NSCLC）患者的标准疗法。尽管已确定了一些奥希替尼耐药机制，但近50%的机制仍有待阐明。本研究旨在确定奥希替尼耐药的非遗传机制。
我们采用逐步培养法，从EGFR突变阳性的PC-9和HCC827 NSCLC细胞系（分别为PC-9OR和HCC827OR）中建立了两个奥希替尼耐药细胞系。我们使用质谱法比较了奥希替尼耐药细胞和亲本细胞的磷酸化蛋白质组图谱。使用激酶富集分析版本3鉴定上游激酶。
磷酸化蛋白质组分析揭示了PC-9OR和HCC827OR细胞中80个相互上调的磷酸化位点。激酶富集分析版本3分析确定粘着斑激酶（FAK）和原癌基因酪氨酸蛋白激酶Src（Src）为这些上调的磷酸化蛋白的上游激酶。在两个细胞系中，小干扰RNA介导的FAK敲低降低了Src磷酸化，而Src敲低降低了FAK磷酸化。此外，FAK或Src特异性小干扰RNA处理恢复了PC-9OR和HCC827OR细胞中的EGFR磷酸化。FAK和Src抑制剂的联合使用在体外抑制了PC-9OR和HCC827OR细胞的增殖，并在异种移植小鼠模型中抑制了肿瘤生长。对EGFR突变型NSCLC患者肿瘤的免疫组织化学分析表明，磷酸化的FAK和Src参与了对奥希替尼的初始和获得性耐药。
磷酸化蛋白质组分析可能有助于阐明肺癌分子靶向治疗的耐药机制。FAK和Src的相互磷酸化参与了奥希替尼耐药。因此，抑制FAK和Src可能是奥希替尼耐药NSCLC的新治疗策略。
