Abstract 929: CDKN2A/p16 knockout in LGR5+ progenitor cells augments mucous gland hyperplasia/metaplasia, inflammation, and dysplasia in mouse stomach squamocolumnar junction
Authors: Sun Jing , Komissarova Elena V. , Hills Caitlin , Seckar Tyler , Su Gloria H. , Sepulveda Jorge , Wang Timothy L. , Sepulveda Antonia R. 
Published: June 2022

Background: Esophageal adenocarcinoma (EAC) develops in pre-cancer columno-glandular hyperplasia, intestinal metaplasia and dysplasia that replaces squamous epithelium and characterizes Barrett’s esophagus (BE). We reported CDKN2A/p16 deletions in up to 69% of BE patients who progressed to dysplasia and EAC, suggesting that p16 loss may drive progression of BE to EAC. LGR5+ cardia like stem cells in the squamocolumnar junction (SCJ) of mouse stomach have been implicated in the cellular origins of BE, and glandular hyperplasia/metaplasia (MGHP) and dysplasia and enhanced inflammation in SCJ were reported in interleukin 1 beta (IL1b) transgenic mice. Our hypothesis is that p16 deletion in LGR5+ cells that give rise to glandular epithelium in SCJ of IL1b mice may accelerate glandular hyperplasia and metaplasia and augment progression to dysplasia and EAC.

Design: We tested the hypothesis using conditional knockout of CDKN2A/p16 in IL1b mouse targeting SCJ LGR5+ cells. Model A genotype is p16del: (Lgr5-CreERT2 +/-; IL1b wt/tg; p16 fl/fl) and control model C is p16 wild type (wt): (Lgr5-CreERT2 +/-; IL1b wt/tg; p16 wt/wt). Conditional knockout was induced with intraperitoneal tamoxifen injection. All mice were given bile acid in drinking water, daily. Mice were euthanized and gross and histologic alterations in SCJ, stomach, small intestine (SI) and colon were assessed over time, until 16 months. MGHP was scored as the number of glandular profiles in each section of SCJ, and inflammation and dysplasia were evaluated and scored (0 to 3) in H&E-stained sections.

Results: We examined 39 mice: 19 model A (p16del) and 20 model C (p16wt). Mice were grouped into 3 groups from 3 to 16 months. The SCJ of p16del mice exhibited larger areas of hyperplastic cardia-type mucous glands compared to p16wt mice, showing higher numbers of gland profiles (median 17 vs. 8 gland profiles, P=9-5). Higher inflammation scores were also seen in p16del mice ages 3-7 and 7-12 months (P=.036 and P=.013). P16del mice showed higher dysplasia scores overall (P=.013), particularly in mice ages 7-16 months (P=.038). There was a positive correlation between MGHP and inflammation (P=.004) and between inflammation and dysplasia (P=.021). No significant alterations were seen in the stomach, SI, or colon of p16del vs p16wt mice.

Conclusions: Loss of p16 alone is not sufficient to develop neoplastic alterations in stomach, SI and colon. Deletion of p16 in LGR5+ cardia-like stem cells in the squamocolumnar junction of mouse stomach, together with IL1b and BA-induced inflammation and cellular injury, is associated with increased MGHP, inflammation, and dysplasia. The model implicates p16 in the early stages of esophageal carcinogenesis, similar to human disease, providing a model to further dissect the contributing molecular mechanisms of progression to esophageal dysplasia and cancer.

Citation Format: Jing Sun, Elena V. Komissarova, Caitlin Hills, Tyler Seckar, Gloria H. Su, Jorge Sepulveda, Timothy L. Wang, Antonia R. Sepulveda. CDKN2A/p16 knockout in LGR5+ progenitor cells augments mucous gland hyperplasia/metaplasia, inflammation, and dysplasia in mouse stomach squamocolumnar junction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 929.