[1]余腾骅,王智亮,彭美茜,等.肿瘤相关成纤维细胞中雌激素/GPER/PKA/FGF2信号轴促进乳腺癌细胞侵袭[J].第三军医大学学报,2019,41(18):1730-1737.
 YU Tenghua,WANG Zhiliang,PENG Meiqian,et al.Estrogen/GPER/PKA/FGF2 signal axis in cancer-associated fibroblasts enhances the invasion of breast cancer cells[J].J Third Mil Med Univ,2019,41(18):1730-1737.
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肿瘤相关成纤维细胞中雌激素/GPER/PKA/FGF2信号轴促进乳腺癌细胞侵袭(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第18期
页码:
1730-1737
栏目:
基础医学
出版日期:
2019-09-30

文章信息/Info

Title:
Estrogen/GPER/PKA/FGF2 signal axis in cancer-associated fibroblasts enhances the invasion of breast cancer cells
作者:
余腾骅王智亮彭美茜邱宇安
江西省肿瘤医院:乳腺肿瘤外科,重症医学科;重庆医科大学附属第二医院妇产科;重庆医科大学临床检验诊断学教育部重点实验室
Author(s):
YU Tenghua WANG Zhiliang PENG Meiqian QIU Yuan

Department of Breast Surgery, 4Department of Critical Care Medicine, Jiangxi Cancer Hospital, Nanchang, Jiangxi Province, 330029; 2Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010; 3Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing, 400016, China

关键词:
肿瘤相关成纤维细胞G蛋白偶联雌激素受体成纤维细胞生长因子2细胞侵袭乳腺癌
Keywords:
cancer-associated fibroblasts G protein-coupled estrogen receptor fibroblast growth factors 2 cell invasion breast cancer
分类号:
R73-37; R730.23; R737.9
文献标志码:
A
摘要:

目的 探讨共培养下乳腺癌肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)中细胞质G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)活化对成纤维细胞生长因子2(fibroblast growth factors 2,FGF2)的表达调控机制及其对肿瘤细胞侵袭能力的影响。 方法 细胞免疫荧光法检测单独及与雌激素受体阳性(estrogen receptor positive, ER+)乳腺癌MCF-7或ER阴性(ER-)乳腺癌MDA-MB-468细胞共培养下CAFs中GPER的细胞内定位;荧光定量PCR法与ELISA法分别检测不同处理后CAFs细胞内FGF2的mRNA表达及细胞上清液中的分泌水平;Western blot法检测CAFs细胞中GPER下游ERK、AKT及PKA信号通路活化情况;Transwell小室实验检测不同药物处理下乳腺癌细胞侵袭能力的改变。 结果 两种亚型的乳腺癌细胞均可诱导CAFs中GPER由细胞核转位入细胞质。共培养条件下,17-β雌二醇(E2)可显著促进CAFs中FGF2的mRNA表达及细胞上清液中的分泌量,突变GPER氨基酸序列中的核输出信号(nucleus export signal, NES)可显著抑制以上变化(P<0.05)。CAFs单独培养下则无以上现象。同时,E2处理共培养下CAFs细胞后显著活化细胞内GPER/AKT与GPER/PKA信号通路,促进下游p-AKT与p-PKA的蛋白表达(P<0.05)。突变GPER中NES序列后可显著抑制下游PKA信号的活化,反而促进p-ERK的表达(P<0.05)。用PKA特异性抑制剂(H-89)可明显阻断CAFs中E2诱导的FGF2表达(P<0.05),AKT特异性抑制剂(WM)则不能。CAFs中E2刺激下的FGF2旁分泌明显提高乳腺癌细胞侵袭能力,该效应被FGF2中和抗体所阻断(P<0.05)。 结论 雌激素活化CAFs中细胞质GPER依赖性的下游PKA信号旁分泌FGF2进而促进乳腺癌细胞侵袭,上述通路或在肿瘤进展中发挥重要作用。

Abstract:

Objective To investigate the effect of activated cytoplasmic G protein-coupled estrogen receptor (GPER) from co-cultured cancer-associated fibroblasts (CAFs) in the regulation of fibroblast growth factor 2 (FGF2) and the invasion of breast cancer cells. MethodsThe intracellular GPER localization in CAFs cultured alone or co-cultured with ER- positive (MCF-7) and ER- negative (MDA-MB-468) breast cancer cells was examined using immunofluorescence assays. Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were utilized to detect the expression of FGF2 mRNA in the CAFs and FGF2 secretion in the supernatant, respectively. The activation of GPER downstream ERK, AKT and PKA signal pathways was determined by Western blotting. The changes in the invasiveness of breast cancer cells were examined by Transwell assays. ResultsCo-culture with the 2 breast cancer cells both induced translocation of GPER from the cell nucleus to the cytoplasm in the CAFs. In the cell co-cultures, 17-β estradiol (E2) significantly increased FGF2 levels in the CAFs and the supernatant, and this effect was obviously blocked by mutation of the nucleus export signal (NES) in GPER amino acid sequence (P<0.05); these results could not be observed in the CAFs cultured alone. E2 treatment of the co-cultured CAFs resulted in obvious activation of GPER/AKT and GPER/PKA signal pathways (P<0.05) and increased the protein expression of p-AKT and p-PKA; Mutation of the NES in GPER sequence significantly inhibited PKA signaling and up-regulated the expression of p-ERK (P<0.05). The E2-induced increase of FGF2 expression in CAFs was significantly blocked by the application of the PKA inhibitor H-89 but not by the AKT inhibitor WM (P<0.05). More importantly, the secreted FGF2 from E2-treated CAFs significantly promoted the invasion of the breast cancer cells, which was abolished by the neutralizing antibody of FGF2. ConclusionEstrogen enhances the activation of the downstream PKA signaling in a cytoplasmic GPER-dependent manner in the CAFs, and the enhanced FGF2 paracrine promotes the invasion of breast cancer cells. These signal pathways may play important roles in the progression of breast cancer.

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更新日期/Last Update: 2019-09-21