Cancer Research Institute, Korea University College of Medicine, Seoul, Republic of Korea.
BK21 Plus program, Korea University College of Medicine, Seoul, Republic of Korea.
Cancer Med. 2021 Jan;10(1):372-385. doi: 10.1002/cam4.3635. Epub 2020 Dec 12.
Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently noted in non-small cell lung cancer (NSCLC). In recent decades, EGFR tyrosine kinase inhibitors (TKIs) have been developed, although the therapeutic efficacy of these inhibitor is restricted to EGFR-mutant patients. In this study, we investigated that clathrin-mediated EGFR endocytosis hampers the effects of gefitinib and sustains NSCLC cells with wild-type EGFR.
NSCLC cell lines (H358, Calu-3, SNU-1327, and H1703) were stimulated with the EGF and treated with gefitinib and endocytosis inhibitors (phenylarsine oxide (PAO) and Filipin III). Growth inhibition and apoptosis were evaluated. Immunofluorescence, immunoprecipitation, and western blot assay were performed to investigate EGFR endocytosis and determine the signaling pathway. Xenograft mouse models were used to verify the combination effect of gefitinib and PAO in vivo.
We confirmed the differences in EGFR endocytosis according to gefitinib response in wild-type EGFR NSCLC cell lines. EGFR in gefitinib-sensitive and -refractory cell lines tended to internalize through distinct routes, caveolin-mediated endocytosis (CVE), and clathrin-mediated endocytosis (CME). Interestingly, while suppressing CME and CVE did not affect cell survival in sensitive cell lines significantly, CME inhibition combined with gefitinib treatment decreased cell survival and induced apoptosis in gefitinib-refractory cell lines. In addition, blocking CME in the refractory cell lines led to downregulate of p-STAT3 and inhibit nuclear localization of STAT3 in vivo, combination treatment with gefitinib and a CME inhibitor resulted in tumor regression accompanying apoptosis in xenograft mouse models.
Clathrin-mediated EGFR endocytosis contribute primary resistance of gefitinib treatment and CME inhibition combined with gefitinib could be an option in treatment of wild-type EGFR NSCLC.
表皮生长因子受体（EGFR）信号的致癌改变在非小细胞肺癌（NSCLC）中经常被发现。在最近几十年中，已经开发出了 EGFR 酪氨酸激酶抑制剂（TKI），尽管这些抑制剂的治疗效果仅限于 EGFR 突变患者。在这项研究中，我们研究了网格蛋白介导的 EGFR 内吞作用会阻碍吉非替尼的作用，并维持具有野生型 EGFR 的 NSCLC 细胞。
用 EGF 刺激 NSCLC 细胞系（H358、Calu-3、SNU-1327 和 H1703），并用吉非替尼和内吞作用抑制剂（苯胂酸钠（PAO）和 Filipin III）处理。评估生长抑制和细胞凋亡。进行免疫荧光、免疫沉淀和 Western blot 分析以研究 EGFR 内吞作用并确定信号通路。使用异种移植小鼠模型来验证吉非替尼和 PAO 组合在体内的效果。
我们根据野生型 EGFR NSCLC 细胞系中对吉非替尼的反应证实了 EGFR 内吞作用的差异。吉非替尼敏感和耐药细胞系中的 EGFR 倾向于通过不同途径内化，即小窝蛋白介导的内吞作用（CVE）和网格蛋白介导的内吞作用（CME）。有趣的是，虽然抑制 CME 和 CVE 对敏感细胞系中的细胞存活没有明显影响，但 CME 抑制联合吉非替尼治疗可降低耐药细胞系中的细胞存活并诱导细胞凋亡。此外，在耐药细胞系中阻断 CME 可导致下调 p-STAT3 并抑制 STAT3 的核定位，在异种移植小鼠模型中联合使用吉非替尼和 CME 抑制剂可导致肿瘤消退和凋亡。
网格蛋白介导的 EGFR 内吞作用是吉非替尼治疗原发性耐药的主要原因，CME 抑制联合吉非替尼治疗可能是治疗野生型 EGFR NSCLC 的一种选择。
