RNF128 Regulation of TP53 in Barrett's Progression

摘要： 食管腺癌(EAC)是一种发生在被称为巴雷特病的癌前区域的致命癌症。 化生(BE)。EAC的发病率增加的速度比任何其他癌症都要快，而且与 患有胃食道反流病(GERD)和肥胖。了解Barrett‘s的哪些特性 化生上皮与EAC进展相关可能寻找新的治疗方法 预防这种癌症的策略。我们利用从进展到高级别的患者获得的BE 并进行RNAseq分析基因表达和异构体特异性mRNA的表达 非发育不良Barrett‘s、低度异型增生(LGD)、高度异型增生(HGD)对EAC的影响 重要的是，我们观察到从Barrett‘s/LGD到HGD/EAC的转变与显著的亚型有关 个体基因内的变化。这种“异构体转换”与通路分析相一致，表明 作为顶端通路的剪接在BE进展到EAC的过程中发生了改变。我们观察到“同形转换”是一致的。 随着保护性粘蛋白的丧失，炎症加剧，ATM/DNA损伤反应被激活 在HGD和EAC中，-H_2AX染色增加。最近有报道称，自动取款机的激活可以调节 拼接。我们最重要的基因之一是RNF128或Grail，它是一种泛素 连接酶(E3)，功能上与野生型(WT)TP53的N末端相互作用，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、HGD升高。有两种Grail亚型：RNF128-Iso1和RNF128-Iso2 不同的发起人。Iso1在BE/LGD中含量丰富，在HGD和EAC中显著减少，在 炎症性(-干扰素)或DNA损伤(过氧化氢)试剂处理后的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 蛋白质，与WT TP53不同，它稳定突变的TP53*蛋白以增加克隆存活和 永生化BE细胞对抗炎药(他汀类药物)的耐药性。因此，理解 GRAIL异构体调控野生型和TP53*在BE/EAC中的功能/稳定性可能导致有效 调节这一重要基因和预防Barrett‘s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.