磷酸化 ATF1 在 Thr184 促进胃癌转移并调节 MMP2 的表达。
Phosphorylated ATF1 at Thr184 promotes metastasis and regulates MMP2 expression in gastric cancer.
机构信息
Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China.
Department of Pathology, Chongqing Renji Hospital, University of Chinese Academy of Science, Chongqing, China.
出版信息
J Transl Med. 2022 Apr 9;20(1):169. doi: 10.1186/s12967-022-03361-3.
BACKGROUND
Studies have revealed an important role of activating transcription factor 1 (ATF1) and phosphorylated ATF1 at Ser63 in tumors. Our previous study identified Thr184 as a novel phosphorylation site of ATF1. However, the role of phosphorylated ATF1 at Thr184 (p-ATF1-T184) in tumor is unclear. This study figured out the role of p-ATF1-T184 in the metastasis of gastric cancer (GC) and in the regulation of Matrix metallopeptidase 2 (MMP2).
METHODS
Immunohistochemical analysis (IHC) was performed to analyze the level of p-ATF1-T184 and its relationship with clinicopathological characteristics. Wound scratch test, Transwell assay were used to observe the role of p-ATF1-T184 in the invasion and metastasis of GC. The regulation of MMP2 by p-ATF1-T184 was investigated by a series of experiments including quantitative RT-PCR, western blot, gelatin zymography assay, Chromatin immunoprecipitation (ChIP), luciferase reporter assay and cycloheximide experiment. The Cancer Genome Atlas (TCGA) data were used to analyze the expression and prognostic role of ATF1 and MMP2 in GC. Mass spectrometry (MS) following co-immunoprecipitation (co-IP) assay was performed to identify potential upstream kinases that would phosphorylate ATF1 at Thr184.
RESULTS
High expression level of p-ATF1-T184 was found and significantly associated with lymph node metastasis and poor survival in a GC cohort of 126 patients. P-ATF1-T184 promoted migration and invasion of gastric cancer cells. Phosphorylation of ATF1-T184 could regulate the mRNA, protein expression and extracellular activity of MMP2. P-ATF1-T184 further increased the DNA binding ability, transcription activity, and stabilized the protein expression of ATF1. Moreover, TCGA data and IHC results suggested that the mRNA level of ATF1 and MMP2, and protein level of p-ATF1-T184 and MMP2 could be prognosis markers of GC. Two protein kinase related genes, LRBA and S100A8, were identified to be correlated with the expression ATF1 in GC.
CONCLUSION
Our results indicated that p-ATF1-T184 promoted metastasis of GC by regulating MMP2.
背景
研究表明激活转录因子 1（ATF1）和磷酸化 ATF1 在 Ser63 在肿瘤中具有重要作用。我们之前的研究确定 Thr184 是 ATF1 的一个新磷酸化位点。然而，磷酸化 ATF1 在 Thr184 上的作用（p-ATF1-T184）在肿瘤中尚不清楚。本研究探讨了 p-ATF1-T184 在胃癌（GC）转移和基质金属蛋白酶 2（MMP2）调节中的作用。
方法
采用免疫组织化学分析（IHC）分析 p-ATF1-T184 的水平及其与临床病理特征的关系。划痕试验和 Transwell 试验用于观察 p-ATF1-T184 在 GC 侵袭和转移中的作用。通过一系列实验，包括定量 RT-PCR、western blot、明胶酶谱分析、染色质免疫沉淀（ChIP）、荧光素酶报告基因检测和环己酰亚胺实验，研究了 p-ATF1-T184 对 MMP2 的调节作用。使用癌症基因组图谱（TCGA）数据分析了 ATF1 和 MMP2 在 GC 中的表达和预后作用。采用免疫共沉淀（co-IP）后质谱（MS）分析鉴定潜在的上游激酶，这些激酶可将 ATF1 磷酸化至 Thr184。
结果
在 126 例 GC 患者队列中发现 p-ATF1-T184 高表达水平，并与淋巴结转移和不良预后显著相关。p-ATF1-T184 促进了胃癌细胞的迁移和侵袭。ATF1-T184 的磷酸化可调节 MMP2 的 mRNA、蛋白表达和细胞外活性。p-ATF1-T184 进一步增加了 ATF1 的 DNA 结合能力、转录活性和蛋白表达稳定性。此外，TCGA 数据和 IHC 结果表明，ATF1 和 MMP2 的 mRNA 水平以及 p-ATF1-T184 和 MMP2 的蛋白水平可作为 GC 的预后标志物。两个与 GC 中 ATF1 表达相关的蛋白激酶相关基因 LRBA 和 S100A8 被鉴定。
结论
我们的结果表明，p-ATF1-T184 通过调节 MMP2 促进 GC 的转移。
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