Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line.
Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line.
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DOI：
10.1186/1476-4598-7-42
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发表时间：
2008-05-21
期刊：
影响因子：
37.3
通讯作者：
Itoh K
中科院分区：
文献类型：
--
作者：
Nishimura Y;Yoshioka K;Bereczky B;Itoh K
吉非替尼（易瑞沙）-表皮生长因子受体（EGFR）酪氨酸激酶的特异性抑制剂-已被证明可以抑制非小细胞肺癌（NSCLC）细胞系中细胞存活和增殖所需的EGFR信号转导的激活。我们最近提供了新的证据，吉非替尼敏感的PC 9细胞显示正常的EGFR内吞作用：EGF刺激后15分钟，内化的EGF-EGFR复合物被转运到晚期内体/溶酶体，然后在溶酶体内降解。然而，吉非替尼耐药QG 56细胞在内化60分钟后显示内化的EGFR在早期内体中积聚，而不是其运输至溶酶体，表明EGF-EGFR从早期内体运输至晚期内体/溶酶体的某些步骤中存在异常。因此，我们推测EGF-EGFR从早期内体向晚期内体/溶酶体运输的某些步骤中的损伤可能赋予NSCLC细胞系吉非替尼耐药性。为了进一步证实吉非替尼敏感细胞和吉非替尼耐药细胞的详细内化机制，我们使用共聚焦免疫荧光显微镜检查了在吉非替尼存在或不存在的情况下磷酸化EGFR（pEGFR）的内吞转运。在没有EGF刺激的PC 9和QG 56细胞中，大量的pEGFR阳性的小泡状结构不与晚期内体/溶酶体共定位分布在整个细胞质中，并且一些pEGFR染色分布在细胞核中。这意味着pEGFR从胞质囊泡到细胞核的新的细胞内运输途径。此外，在QG 56细胞的核周区域中观察到早期内体的聚集囊泡结构;它被揭示与SNX 1相关，SNX 1最初被鉴定为与EGFR相互作用的蛋白质。因此，我们证实了我们先前的数据，即在QG 56细胞中EGF-EGFR从早期内体到晚期内体/溶酶体的运输的某些步骤中发生畸变。此外，在PC 9细胞中，在EGF刺激后3 min观察到EGFR的有效磷酸化和pEGFR的快速内化;这些内化的pEGFR阳性囊泡在15 min时被运输到晚期内体，表明PC 9细胞中EGF-pEGFR复合物从早期内体快速运输到晚期内体。吉非替尼处理强烈降低EGFR的磷酸化水平，并且随后在PC 9细胞中EGFR的内吞作用被显著抑制。相反，在QG 56细胞中，EGFR通过早期内吞途径的运输基本受损;因此，吉非替尼似乎轻微抑制pEGFR的内化。总的来说，我们的数据提供了新的证据，广泛的损害pEGFR内吞作用通过早期内吞途径可能会赋予吉非替尼耐药QG 56细胞。
Gefitinib (Iressa)–a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase–has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.
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