Department of Pathology, George Washington University School of Medicine and Health Sciences, Washington, DC.
Department of Pharmacology & Physiology, George Washington University, School of Medicine and Health Sciences, Washington, DC.
Cell Mol Gastroenterol Hepatol. 2024;17(5):769-784. doi: 10.1016/j.jcmgh.2024.01.014. Epub 2024 Jan 29.
BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRAS resulted in more frequent dysplasia and higher dysplasia scores (P = .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). CONCLUSIONS: 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 in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.
背景与目的：巴雷特食管是食管发育不良和食管腺癌的前体。据报道，34%-74%进展为发育不良和食管腺癌的巴雷特食管患者存在 CDKN2A-p16 缺失，这表明 p16 缺失可能推动肿瘤进展。KRAS 激活在食管腺癌和癌前病变中经常发生。小鼠胃的鳞柱状交界处（SCJ）中的 LGR5 干细胞作为巴雷特食管的前体。我们旨在确定 p16 缺失和 KRAS 激活在巴雷特样化生和发育不良发展中的功能影响。
方法：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/或靶向 SCJ LGR5 细胞中激活的 KRAS 表达的小鼠模型，并对小鼠 SCJ 的组织学改变（粘液腺增生/化生、炎症和发育不良）进行了特征描述。通过微阵列、RNA 测序和 SCJ 组织及培养的 3 维类器官的免疫组织化学确定基因表达。
结果：p16KO 小鼠的粘液腺增生/化生明显多于对照组（P =.0051）。p16KO+KRAS 联合导致发育不良更频繁，发育不良评分更高（P =.0036），82%的 p16KO+KRAS 小鼠发生高级别发育不良。SCJ 转录组分析显示，p16KO 与对照组相比，有多个通路被激活（细胞凋亡、肿瘤坏死因子-α/核因子-kB、蛋白酶体降解、p53 信号转导、MAPK、KRAS 和 G1 至 S 期过渡）。
结论：LGR5 细胞前体中的 p16 缺失触发了 SCJ 粘液腺增生/化生的增加。KRAS 与 p16 缺失协同作用，导致 SCJ 腺发育不良程度更高，类似于巴雷特高级别发育不良。这些基因修饰的小鼠模型在小鼠中建立了 p16 和激活的 KRAS 在巴雷特样病变进展为发育不良中的功能作用，代表了食管腺癌前癌的体内模型。衍生的 3 维类器官模型进一步提供了食管前癌阶段的体外建模机会。
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