RNF128 Regulation of TP53 in Barrett's Progression

摘要： 食管腺癌 (EAC) 是一种致命的癌症，发生在称为巴雷特氏癌的癌前区域 化生（BE）。 EAC 的发病率增长速度高于任何其他癌症，并且与 患有胃食管反流病（GERD）和肥胖症。了解巴雷特的哪些属性 化生上皮与 EAC 进展相关，可能会确定新的治疗方法 预防这种癌症的策略。我们利用从进展为高级的患者获得的 BE 发育异常和 EAC，并对基因表达以及亚型特异性 mRNA 进行 RNAseq 分析 非发育不良 Barrett 病、低度不典型增生 (LGD)、高度不典型增生 (HGD) 到 EAC 的分析。 重要的是，我们观察到从 Barrett’s/LGD 到 HGD/EAC 的转变与显着的亚型相关 个体基因内的变化。这种“异构体转换”与通路分析一致，表明 BE 进展到 EAC 时，剪接作为顶部通路的改变。我们观察到“异构体转换”同时发生 随着保护性粘蛋白的丧失、炎症增加、ATM/DNA 损伤反应激活 HGD 和 EAC 中的 H2AX 染色增加。最近有报道称 ATM 激活可以调节 拼接。我们的顶级基因之一是 RNF128 或 Grail，它是一种泛素 连接酶 (E3) 与野生型 (WT) TP53 的 N 末端功能性相互作用以靶向其降解 并调节其反式激活活性。 TP53 基因突变是 EAC 中最常见的事件 LGD 和 HGD 增加。有两种 Grail 同工型：RNF128-Iso1 和 RNF128-Iso2 单独的发起人。 Iso1 在 BE/LGD 中丰富，在 HGD 和 EAC 中显着减少，在 使用炎症剂 (γ-IFN) 或 DNA 损伤剂 (H2O2) 处理后的 EAC 细胞。另外， 我们发现 Iso1 的减少会导致 Iso2 糖基化形式的代偿性上调 蛋白，与 WT TP53 相比，它可以稳定突变 TP53* 蛋白以增加克隆存活率 永生化 BE 细胞对抗炎药物（他汀类药物）的耐药性。因此，了解角色 Grail 异构体在 BE/EAC 中调节野生型和 TP53* 功能/稳定性可能会导致有效的 调节这一重要基因并预防巴雷特氏症患者癌症发展的方法 化生。

Abstract: Esophageal adenocarcinoma (EAC) is a deadly cancer that arises within a premalignant field called Barrett’s metaplasia (BE). EAC is increasing in incidence at a rate greater than any other cancer and is associated with gastroesophageal reflux disease (GERD) and obesity. Understanding which properties of Barrett’s metaplastic epithelium are associated with progression to EAC may potentially identify novel therapeutic strategies to prevent this cancer. We utilized the BE obtained from patients who progressed to high-grade dysplasia and EAC and performed RNAseq analysis for gene expression as well as isoform-specific mRNA analysis in non-dysplastic Barrett’s, low-grade dysplasia (LGD), high-grade dysplasia (HGD) to EAC. Importantly, we observe that transition from Barrett’s/LGD to HGD/EAC is associated with significant isoform changes within individual genes. This “isoform switching” is consistent with pathway analysis indicating splicing as the top pathway altered in BE progression to EAC. We observe that “isoform switching” coincides with the loss of protective mucins, increased inflammation, activation of ATM/DNA damage response pathway and increased H2AX staining in HGD and EAC. ATM activation was recently reported to regulate splicing. One of our top genes that demonstrate dramatic isoform switching is RNF128 or Grail, an ubiquitin ligase (E3) that functionally interacts with the N-terminus of wild type (WT) TP53 to target its degradation and modulate its transactivation activity. Mutations in the TP53 gene are the most frequent event in EAC and increase in LGD and HGD. There are two Grail isoforms: RNF128-Iso1 and RNF128-Iso2 that have separate promoters. Iso1 is abundant in BE/LGD, dramatically reduced in HGD and EAC and decreased in EAC cells following treatment with either inflammatory (-IFN) or DNA damaging (H2O2) agents. Additionally, we found that the reduction in Iso1 causes a compensatory upregulation of the glycosylated form of Iso2 protein, which, in contrast to WT TP53, stabilizes mutant TP53* protein to increase clonogenic survival and resistance to anti-inflammatory drugs (statins) in immortalized BE cells. Thus, understanding the role of Grail isoforms in the regulation of wild type and TP53* function/stability in BE/EAC may lead to effective methods to modulate this important gene and prevent cancer development in patients with Barrett’s metaplasia.