Saygideğer-Kont Yasemin, Minas Tsion Zewdu, Jones Hayden, Hour Sarah, Çelik Haydar, Temel Idil, Han Jenny, Atabey Nese, Erkizan Hayriye Verda, Toretsky Jeffrey A, Üren Aykut
Department of Oncology, Georgetown University Medical Center, Washington, DC, USA; Department of Molecular Medicine, Institute of Health Sciences, Dokuz Eylul University, Izmir, Turkey.
Department of Oncology, Georgetown University Medical Center, Washington, DC, USA.
Neoplasia. 2016 Feb;18(2):111-20. doi: 10.1016/j.neo.2016.01.002.
Ezrin is a scaffolding protein that is involved in oncogenesis by linking cytoskeletal and membrane proteins. Ezrin interacts with epidermal growth factor receptor (EGFR) in the cell membrane, but little is known about the effects of this interaction on EGFR signaling pathway. In this study, we established the biological and functional significance of ezrin-EGFR interaction in non-small cell lung cancer (NSCLC) cells. Endogenous ezrin and EGRF interaction was confirmed by co-immunoprecipitation and immunofluorescent staining. When expression of ezrin was inhibited, EGFR activity and phosphorylation levels of downstream signaling pathway proteins ERK and STAT3 were decreased. Cell fractionation experiments revealed that nuclear EGFR was significantly diminished in ezrin-knockdown cells. Consequently, mRNA levels of EGFR target genes AURKA, COX-2, cyclin D1, and iNOS were decreased in ezrin-depleted cells. A small molecule inhibitor of ezrin, NSC305787, reduced EGF-induced phosphorylation of EGFR and downstream target proteins, EGFR nuclear translocation, and mRNA levels of nuclear EGFR target genes similar to ezrin suppression. NSC305787 showed synergism with erlotinib in wild-type EGFR-expressing NSCLC cells, whereas no synergy was observed in EGFR-null cells. Phosphorylation of ezrin on Y146 was found as an enhancer of ezrin-EGFR interaction and required for increased proliferation, colony formation, and drug resistance to erlotinib. These findings suggest that ezrin-EGFR interaction augments oncogenic functions of EGFR and that targeting ezrin may provide a potential novel approach to overcome erlotinib resistance in NSCLC cells.
埃兹蛋白是一种支架蛋白，通过连接细胞骨架蛋白和膜蛋白参与肿瘤发生。埃兹蛋白在细胞膜中与表皮生长因子受体（EGFR）相互作用，但这种相互作用对EGFR信号通路的影响尚不清楚。在本研究中，我们确定了埃兹蛋白与EGFR相互作用在非小细胞肺癌（NSCLC）细胞中的生物学和功能意义。通过免疫共沉淀和免疫荧光染色证实了内源性埃兹蛋白与EGRF的相互作用。当埃兹蛋白的表达受到抑制时，EGFR活性以及下游信号通路蛋白ERK和STAT3的磷酸化水平降低。细胞分级实验表明，在埃兹蛋白敲低的细胞中，核EGFR显著减少。因此，在埃兹蛋白缺失的细胞中，EGFR靶基因AURKA、COX-2、细胞周期蛋白D1和诱导型一氧化氮合酶的mRNA水平降低。埃兹蛋白的小分子抑制剂NSC305787降低了EGF诱导的EGFR及其下游靶蛋白的磷酸化、EGFR核转位以及核EGFR靶基因的mRNA水平，其作用与抑制埃兹蛋白相似。NSC305787在表达野生型EGFR的NSCLC细胞中与厄洛替尼表现出协同作用，而在EGFR缺失的细胞中未观察到协同作用。发现埃兹蛋白Y146位点的磷酸化是埃兹蛋白与EGFR相互作用的增强剂，是增加增殖、集落形成以及对厄洛替尼耐药所必需的。这些发现表明，埃兹蛋白与EGFR的相互作用增强了EGFR的致癌功能，靶向埃兹蛋白可能为克服NSCLC细胞对厄洛替尼的耐药性提供一种潜在的新方法。
