Nuclear MYH9-induced CTNNB1 transcription, targeted by staurosporin, promotes gastric cancer cell anoikis resistance and metastasis
Nuclear MYH9-induced CTNNB1 transcription, targeted by staurosporin, promotes gastric cancer cell anoikis resistance and metastasis
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星形孢菌素靶向核MYH9诱导的CTNNB1转录促进胃癌细胞失巢凋亡抵抗和转移
DOI：
10.7150/thno.46001
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Li, Guoxin
中科院分区：
文献类型：
--
作者：
Ye, Gengtai;Yang, Qingbin;Li, Guoxin
理由：腹膜转移预示胃癌患者预后不良，其潜在机制尚不清楚。研究方法：采用2-DIGE、MALDI-TOF/TOF MS和单细胞转录组技术检测正常胃黏膜、原发性胃癌和腹膜转移癌组织中差异表达的蛋白质。利用慢病毒载体介导的shRNA和转录激活因子样效应子核酸酶技术，敲低胃癌细胞株中肌球蛋白重链9（MYH 9）的表达。进行免疫荧光、免疫透射电子显微镜、染色质分级分离、免疫共沉淀以及染色质免疫沉淀、双荧光素酶报告基因、琼脂糖-寡核苷酸下拉、流式细胞术和细胞失巢凋亡测定，以揭示细胞核MYH 9诱导的β-连环蛋白（CTNNB 1）体外转录。裸小鼠和条件转基因小鼠被用于研究体内的发现。结果：我们观察到MYH 9在转移性GC组织中上调，并且与GC患者的不良预后相关。从机制上讲，我们证实MYH 9主要通过四种潜在的核定位信号定位在GC细胞核中。核MYH 9通过其DNA结合域与CTNNB 1启动子结合，并与肌球蛋白轻链9、β-肌动蛋白和RNA聚合酶II相互作用，促进CTNNB 1的转录，从而在体内外赋予GC细胞抗失巢凋亡的能力。星形孢菌素减少核MYH 9 S1943磷酸化以抑制CTNNB 1转录、Wnt/β-连环蛋白信号传导激活和正交异性异种移植GC裸鼠和转基因GC小鼠模型中的GC进展。结论：该研究确定了核MYH 9诱导的CTNNB 1表达促进GC转移，这可以被星形孢菌素抑制，这表明GC腹膜转移的新疗法。
Rationale: Peritoneal metastasis predicts poor prognosis of gastric cancer (GC) patients, and the underlying mechanisms are poorly understood. Methods: The 2-DIGE, MALDI-TOF/TOF MS and single-cell transcriptome were used to detect differentially expressed proteins among normal gastric mucosa, primary GC and peritoneal metastatic tissues. Lentiviruses carrying shRNA and transcription activator-like effector nuclease technology were used to knock down myosin heavy chain 9 (MYH9) expression in GC cell lines. Immunofluorescence, immune transmission electron microscopy, chromatin fractionation, co-immunoprecipitation, and assays for chromatin immunoprecipitation, dual luciferase reporter, agarose-oligonucleotide pull-down, flow cytometry and cell anoikis were performed to uncover nuclear MYH9-induced β-catenin (CTNNB1) transcription in vitro. Nude mice and conditional transgenic mice were used to investigate the findings in vivo. Results: We observed that MYH9 was upregulated in metastatic GC tissues and was associated with a poor prognosis of GC patients. Mechanistically, we confirmed that MYH9 was mainly localized in the GC cell nuclei by four potential nuclear localization signals. Nuclear MYH9 bound to the CTNNB1 promoter through its DNA-binding domain, and interacted with myosin light chain 9, β-actin and RNA polymerase II to promote CTNNB1 transcription, which conferred resistance to anoikis in GC cells in vitro and in vivo. Staurosporine reduced nuclear MYH9 S1943 phosphorylation to inhibit CTNNB1 transcription, Wnt/β-catenin signaling activation and GC progression in both orthotropic xenograft GC nude mouse and transgenic GC mouse models. Conclusion: This study identified that nuclear MYH9-induced CTNNB1 expression promotes GC metastasis, which could be inhibited by staurosporine, indicating a novel therapy for GC peritoneal metastasis.