Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin PARK2
Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin PARK2
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E3 泛素 PARK2 对 Hippo/YAP 信号传导和食管鳞状细胞癌进展的调节
DOI：
10.7150/thno.46078
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Li, Xiumin
中科院分区：
文献类型：
--
作者：
Zhou, Xiaofeng;Li, Yajie;Li, Xiumin
目的：食管鳞状细胞癌是我国最常见的恶性肿瘤之一。最近的基因组测序分析表明Hippo/雅普信号通路的过度激活可能在ESCC患者的致癌过程和进展中起重要作用。然而，对ESCC中控制Hippo信号活性的分子机制知之甚少。我们前期的研究表明，PLCE 1-一个重要的ESCC危险因子-通过snail信号与ESCC进展相关，在此期间，我们发现PARK 2是PLCE 1-snail轴的重要下游靶点。PARK 2在ESCC人样本中减少，并且与ESCC患者的良好预后相关。进一步的研究表明，PARK 2可以抑制雅普，YAP是Hippo通路的关键下游效应子。本研究旨在揭示PARK 2调控Hippo通路在ESCC中的分子机制。方法：采用组织芯片技术（TMA）和免疫组织化学方法，分析PARK 2在223例ESCC患者中的表达与临床病理因素的关系，探讨PARK 2在ESCC中的作用。使用CRISPR/Cas9技术从ESCC细胞中消除内源性PARK 2和雅普。流式细胞术和EdU细胞增殖实验检测ESCC细胞增殖情况。裸鼠皮下注射和Ki-67染色用于评价体内肿瘤生长。进行迁移和侵袭测定。此外，使用小鼠肺转移模型来验证PARK 2在体内的功能。通过表达微阵列筛选、免疫荧光双染和免疫共沉淀法鉴定参与hippo通路的PARK 2。通过定量实时PCR（qRT-PCR）分析验证RNA-seq分析结果。结果：PARK 2低表达与食管鳞癌的临床分期（P < 0.001）、分化程度（P < 0.04）、淋巴结转移（P <0.001）和远处转移（P = 0.0087）有关。多因素考克斯比例回归分析进一步显示PARK 2表达（P = 0.032）是影响ESCC患者总生存期的独立预后因素。免疫组化结果显示PARK 2与雅普蛋白表达呈负相关（P < 0.001）。PARK 2缺失通过Hippo/雅普轴促进ESCC进展，而PARK 2过表达通过Hippo信号传导抑制ESCC肿瘤进展。Co-IP和泛素化分析表明PARK 2在细胞质中与雅普相互作用，促进雅普K48连接的K90位点的泛素化。多因素考克斯比例回归分析进一步显示PARK 2是影响食管鳞癌患者总生存率的独立预后因素。本研究的细胞和分子机制表明PARK 2与细胞质中的雅普蛋白结合，促进ESCC细胞中雅普的泛素化和蛋白酶体依赖性降解。因此，作为一种新的Hippo信号调节剂，PARK 2活性或基因表达水平的调节可能是一个有吸引力的策略来治疗食管癌。
Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC.Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo. Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo. Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays.Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites.Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal.