BACKGROUND & AIMS Barrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53. METHODS We obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53. RESULTS Progression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3β-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice. CONCLUSIONS We found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

中文翻译：

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RNF128的同工型调节Barrett食管细胞中突变体P53的稳定性。

背景与目的Barrett食管（BE）可能发展为不典型增生和食道腺癌（EAC），并伴有TP53突变，从而增加了其产品p53的稳定性。我们分析了BE组织中与BE进程相关的信使RNA（mRNA），并确定了一种影响p53稳定的信使RNA。方法从1992年至2015年，我们从高度不典型增生（HGD）或食道腺癌（EAC）的患者中收集了54份BE样品，并进行了RNA序列分析，包括同工型特异性分析。我们对166个样品进行了逆转录聚合酶链反应分析，并对组织微阵列进行了免疫组织化学分析，该组织微阵列包含来自100例HGD或EAC和正常食管鳞状黏膜（对照）的BE组织。从转染的质粒表达蛋白质，或在BE细胞中用小的干扰RNA敲除蛋白质，然后通过免疫印迹以及免疫沉淀和泛素连接酶分析进行分析。对携带EAC异种移植肿瘤（由OE-33细胞生长）的无胸腺裸鼠进行辛伐他汀腹膜内注射；监测肿瘤生长并收集肿瘤并通过免疫印迹分析RNF128，p53和乙酰化p53的水平。结果BE向HGD或EAC的进展与编码粘蛋白并促进ATM的炎症和活化以及DNA损伤反应的mRNA表达变化有关。随着组织从BE到HGD到EAC的发展，它们增加了编码RNF128（Iso1）的同工型1的mRNA的表达，并降低了RNF128的Iso2的表达。RNF128是E3泛素连接酶，靶向p53降解。BE细胞与干扰素γ一起孵育会导致它们增加Iso1的表达并降低Iso2的表达。Iso1的糖基化程度很高，对p53的泛素连接酶活性有限，导致p53稳定。敲除BE和EAC细胞中的Iso1会导致p53突变形式的降解并降低克隆形成的存活率。相反，Iso2是有效的连接酶，可降低BE细胞中p53突变形式的水平。在BE细胞中，Iso2通过ATM和GSK3β介导的磷酸化以及含β-TrCP1的SCF泛素连接酶复合物的激活而被低糖基化和降解。辛伐他汀可降解p53的突变形式，也可降解BE细胞中的RNF128 Iso1蛋白，并减缓小鼠EAC异种移植肿瘤的生长。结论我们发现BE细胞中RNF128的同工型2减少，