Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036, Rende, Italy.
MSD K.K, Tokyo, 102-8667, Japan.
J Exp Clin Cancer Res. 2019 Feb 6;38(1):58. doi: 10.1186/s13046-019-1056-8.
Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells.
Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA.
We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation.
The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.
黏着斑激酶（FAK）是一种细胞质酪氨酸激酶，它与整合素和生长因子受体结合，促进癌细胞的黏附、迁移和侵袭。G 蛋白偶联雌激素受体（GPER）参与了雌激素对乳腺癌的刺激作用。在这项研究中，我们研究了 GPER 信号在三阴性乳腺癌（TNBC）细胞中对 FAK 的作用。
利用公共大型数据库和来自“癌症基因组图谱”（TCGA；www.cbioportal.org）的患者数据集来评估 TNBC、非 TNBC 肿瘤和正常乳腺组织中的 FAK 表达。使用 MDA-MB 231 和 SUM159 TNBC 细胞作为模型系统。通过 Western blot 分析检测磷酸化 FAK、其他转导介质和靶基因的水平。进行黏附斑分析以确定黏附斑点和黏附斑（FA）的形成。进行荧光素酶测定以评估 GPER 激活后 c-FOS、EGR1 和 CTGF 启动子的活性。通过实时 PCR 测量上述基因的 mRNA 表达。使用 Boyden 室和划痕愈合试验来评估细胞迁移。通过方差分析进行统计分析。
我们首先通过生物信息学分析确定，编码 FAK 的基因即 PTK2 的 mRNA 表达水平在 TNBC 中高于非 TNBC 和正常乳腺组织。接下来，我们发现雌激素 GPER 信号触发 Y397 FAK 磷酸化以及 TNBC 细胞中黏附斑点（FAs）的增加。此外，我们确定 GPER 和 FAK 的激活参与了 STAT3 核积累和基因表达变化。作为生物学对照，我们表明 FAK 抑制可防止 TNBC 细胞在 GPER 激活后迁移。
本研究数据提供了关于 FAK 在 TNBC 中作用的新见解。此外，根据我们的发现，雌激素 GPER 信号可能被认为是参与 FAK 向乳腺癌进展的转导机制之一。
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