GEP100在表皮生长因子诱导的肿瘤细胞迁移中作用机制的研究
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摘要
以往的研究表明,EGF是人类恶性肿瘤细胞迁移过程中起着关键趋化作用的诱导因子,EGF可通过GEP100起始下游信号。另一方面,GEP100参与调控与细胞迁移相关的细胞-基质黏附以及细胞骨架的重构,但目前GEP100在恶性肿瘤细胞迁移中的分子调节机制尚不清楚。本研究在建立体外EGF诱导MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移模型的基础上,研究了GEP100、Arf6和ERK1/2-uPAR-Rac1信号通路在EGF诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移中作用及作用的可能机制,以期为深入阐明恶性肿瘤浸润和转移机制提供理论依据。
本研究发现:
1、GEP100能调控EGF所诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移。
2、分别使用特异性化学阻断剂预处理细胞,或瞬时转染Arf6和Rac1的负显性突变Arf6-T27N和Rac1-T17N质粒,或siRNA干扰uPAR表达后,通过细胞划痕实验,证实GEP100、Arf6、ERK1/2、uPAR和Rac1参与EGF诱导的肿瘤细胞迁移。
3、因为EGF能诱导ERK1/2和Rac1的活化以及uPAR的表达,瞬时转染GEP100的PH区域缺失突变体(GEP100-ΔPH)能有效抑制EGF引起的ERK1/2和Rac1活化及uPAR的表达,ERK1/2特异性抑制剂U0126和siRNA干扰uPAR均可以显著地降低Rac1活化水平,结果证明EGF诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移中存在着GEP100→Arf6→ERK1/2→uPAR→Rac1信号调控途径。
4、在裸鼠模型中,GEP100的PH区域缺失突变体(GEP100-ΔPH)能显著地降低MDA-MB-231乳腺癌细胞在裸鼠体内的转移能力。
综上所述,我们第一次发现GEP100-Arf6调控的ERK1/2-uPAR-Rac1信号通路中介EGF诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞的迁移。
Epidermal growth factor (EGF)-induced cancer cell migration is key to tumor invasion and metastasis. Recent studies indicated that GEP100, a guanine nucleotide exchanging factor (GEF) that preferentially activates Arf6 in vitro and binds directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6 through its pleckstrin homology domain. In addition, formation of E-cadherin-mediated cell-cell adhesion is one of the major properties that maintain the non-invasive phenotypes of various carcinomas . Formation of E-cadherin-mediated cell-cell adhesion in MCF7 cells was substantially impaired in the presence of EGF by co-overexpression of GEP100 and Arf6. Likewise, over the past few years, GEP100, via its well-established roles in the regulation of Arf6 activation, has been shown in some tumor cells to regulate cell adhesion and organization of actin cytoskeleton , both of which are correlated with tumor cell migration.
Given these potential connections between GEP100 and different aspects of cancer progression, we sought to investigate whether GEP100 functioned in the EGF-signaling pathways operational in cancer cell migration and metastasis by using human breast cancer cell line MDA-MB-231 and human liver carcinoma cell line HepG2. We first found that GEP100 regulated EGF-induced cell migration of both MDA-MB-231 and HepG2 cells. We then found that not only GEP100 regulated tumor cell migration through the activation of Arf6 and extracellular signal-regulated kinase (ERK1/2), but that GEP100-dependent ERK1/2 activation was also required for Rac1 activation during EGF-induced tumor cell migration. We further showed that the urokinase-type plasminogen activator receptor (uPAR) signaling downstream of the GEP100-ERK1/2 pathway was critical for Rac1 activation. Using athymic nude mice as a model, we further found that the pleckstrin homology domain deletion mutant of GEP100 (GEP100-△P H) blocked tumor metastasis in vivo. The findings described here document for the first time an important role for GEP100 in regulating human breast and liver cancer cell migration in vitro, and delineate a signaling pathway downstream of EGF signaling that is essential to cell migration of different tumor types.
本研究发现:
1、GEP100能调控EGF所诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移。
2、分别使用特异性化学阻断剂预处理细胞,或瞬时转染Arf6和Rac1的负显性突变Arf6-T27N和Rac1-T17N质粒,或siRNA干扰uPAR表达后,通过细胞划痕实验,证实GEP100、Arf6、ERK1/2、uPAR和Rac1参与EGF诱导的肿瘤细胞迁移。
3、因为EGF能诱导ERK1/2和Rac1的活化以及uPAR的表达,瞬时转染GEP100的PH区域缺失突变体(GEP100-ΔPH)能有效抑制EGF引起的ERK1/2和Rac1活化及uPAR的表达,ERK1/2特异性抑制剂U0126和siRNA干扰uPAR均可以显著地降低Rac1活化水平,结果证明EGF诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞迁移中存在着GEP100→Arf6→ERK1/2→uPAR→Rac1信号调控途径。
4、在裸鼠模型中,GEP100的PH区域缺失突变体(GEP100-ΔPH)能显著地降低MDA-MB-231乳腺癌细胞在裸鼠体内的转移能力。
综上所述,我们第一次发现GEP100-Arf6调控的ERK1/2-uPAR-Rac1信号通路中介EGF诱导的MDA-MB-231乳腺癌细胞和HepG2肝癌细胞的迁移。
Epidermal growth factor (EGF)-induced cancer cell migration is key to tumor invasion and metastasis. Recent studies indicated that GEP100, a guanine nucleotide exchanging factor (GEF) that preferentially activates Arf6 in vitro and binds directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6 through its pleckstrin homology domain. In addition, formation of E-cadherin-mediated cell-cell adhesion is one of the major properties that maintain the non-invasive phenotypes of various carcinomas . Formation of E-cadherin-mediated cell-cell adhesion in MCF7 cells was substantially impaired in the presence of EGF by co-overexpression of GEP100 and Arf6. Likewise, over the past few years, GEP100, via its well-established roles in the regulation of Arf6 activation, has been shown in some tumor cells to regulate cell adhesion and organization of actin cytoskeleton , both of which are correlated with tumor cell migration.
Given these potential connections between GEP100 and different aspects of cancer progression, we sought to investigate whether GEP100 functioned in the EGF-signaling pathways operational in cancer cell migration and metastasis by using human breast cancer cell line MDA-MB-231 and human liver carcinoma cell line HepG2. We first found that GEP100 regulated EGF-induced cell migration of both MDA-MB-231 and HepG2 cells. We then found that not only GEP100 regulated tumor cell migration through the activation of Arf6 and extracellular signal-regulated kinase (ERK1/2), but that GEP100-dependent ERK1/2 activation was also required for Rac1 activation during EGF-induced tumor cell migration. We further showed that the urokinase-type plasminogen activator receptor (uPAR) signaling downstream of the GEP100-ERK1/2 pathway was critical for Rac1 activation. Using athymic nude mice as a model, we further found that the pleckstrin homology domain deletion mutant of GEP100 (GEP100-△P H) blocked tumor metastasis in vivo. The findings described here document for the first time an important role for GEP100 in regulating human breast and liver cancer cell migration in vitro, and delineate a signaling pathway downstream of EGF signaling that is essential to cell migration of different tumor types.
引文
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