Barrett’s esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%–74% of patients with Barrett’s esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5 stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett’s esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett’s-like metaplasia and dysplasia development. We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRAS expression targeting SCJ LGR5 cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice ( = .0051). Combined p16KO+KRAS resulted in more frequent dysplasia and higher dysplasia scores ( = .0036), with 82% of p16KO+KRAS mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). p16 deletion in LGR5 cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRAS synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett’s high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett’s-like lesions to dysplasia in mice, representing an model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide modeling opportunities of esophageal precancer stages.

中文翻译：

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CDKN2A-p16缺失和激活的KRASG12D驱动巴雷特样腺体增生-化生并协同不典型增生癌前病变的发展

巴雷特食管是食管发育不良和食管腺癌的先兆。据报道，在进展为不典型增生和食管腺癌的 Barrett 食管患者中，有 34%–74% 存在 CDKN2A-p16 缺失，这表明 p16 缺失可能会导致肿瘤进展。 KRAS 激活经常发生在食管腺癌和癌前病变中。小鼠胃鳞柱状交界处 (SCJ) 中的 LGR5 干细胞充当巴雷特食管祖细胞。我们的目的是确定 p16 缺失和 KRAS 激活对 Barrett 样化生和发育不良发展的功能影响。我们在白细胞介素 1b 转基因小鼠中建立了条件性敲除 CDKN2A-p16 (p16KO) 和/或激活针对 SCJ LGR5 细胞的 KRAS 表达的小鼠模型，并表征了小鼠 SCJ 中的组织学改变（粘液腺增生/化生、炎症和发育不良）。通过 SCJ 组织和培养的 3 维类器官的微阵列、RNA 测序和免疫组织化学测定基因表达。与对照小鼠相比，p16KO 小鼠表现出粘液腺增生/化生增加 (= .0051)。 p16KO+KRAS 组合导致更频繁的不典型增生和更高的不典型增生评分 (= .0036)，82% 的 p16KO+KRAS 小鼠出现高度不典型增生。 SCJ 转录组分析显示，与对照小鼠相比，p16KO 小鼠有多个激活途径（细胞凋亡、肿瘤坏死因子-α/核因子-kB、蛋白酶体降解、p53 信号传导、MAPK、KRAS 和 G1 至 S 转变）。 LGR5 细胞前体中 p16 缺失会引发 SCJ 粘液腺增生/化生增加。 KRAS 与 p16 缺失协同作用，导致更高级别的 SCJ 腺体发育不良，类似于 Barrett 的高度发育不良。这些转基因小鼠模型建立了 p16 和激活的 KRAS 在小鼠 Barrett 样病变发展为不典型增生过程中的功能作用，代表了食管腺癌癌前病变的模型。衍生的 3 维类器官模型进一步提供了食管癌前期阶段的建模机会。