P-112 Oleic acid promotes the malignant transformation of Kras-mutant colonic organoids via the expansion of tumorigenic stem cells and abnormal Paneth cells through upregulation of NFATc family

Abstract

Although the five-year survival rate of colorectal cancer (CRC) is significantly high with localized stage (90%), only 38% of the patients are diagnosed at this stage. Therefore, finding high risk factors of CRC could prevent the disease at the early stage. Obesity and high-fat diet consumption are risk factors of CRC. In detail, oleic acids (OA) were found to accumulate in the adipose tissues of obese patients as well as being the most common long chain fatty acid in dietary lipid. The role of OA in colorectal cancer development is still controversary. On the other hand, organoids, which can be derived from adult tissue stem cells, are the miniatures of organs. Recent works suggest organoids are useful tools to dissect mechanisms involved in cancer development. The current work aims to utilize crypt stem cell-derived organoids to dissect how obesity can contribute to CRC development. Organoids were derived from Lgr5 + -EGFP mice (normal colon control), Lgr5 + -EGFP-creER; LSL-KrasG12D mice (colonic epithelium harbored KrasG12D mutation) and AOM/DSS-induced CRC mice (cancerous colon control). For in-vitro, the impact of OA on the malignant transformation of Kras-mutant colonic organoids was assessed by MTT proliferation assay, 2D transwell migration and invasion assay, irinotecan-resistant test and colony formation assay. The tumor formation in-vivo was induced by subcutaneous engrafting of OA-treated organoids on NOD/SCID mice. RNAseq was done to screen some differential expressed genes when treating OA. The existence of stem cell and abnormal Paneth cell population was demonstrated by immunostaining, flow cytometry and ELISA. OA enhanced the malignant of Kras-mutant colonic organoids through increasing proliferation, migration, invasion, drug resistance and colony formation. Besides, mice which were engrafted with OA-trained Lgr5-Kras organoids possessed larger tumors. The colonic stem cell population which were represented by positive Lgr5-GFP + cells were upregulated in OA-treated group. Interestingly, there is an existence of abnormal Lyz + /CD24 + Paneth cells in OA-treated Kras-mutant organoids. This suggested that OA may promote abnormal differentiation of colonic progenitor cells. Indeed, treatment of OA upregulated ATOH1 and GFI1, which contributed to select between goblet/Paneth and enteroendocrine cell fates. Besides, NFAT family, among which NFATc3 expressed the most, significantly increased in OA-treated organoids. The upregulation of NFATc was induced by inhibition of proteasomal degradation resulting from low expression of the complex RNF123, USP18 and TRIM30A. In addition, inhibition of the nuclear translocalization of NFATc family by VIVIT demonstrated the decline of stemness markers as well as Paneth cell markers. Finally, we showed the role of NFATc, especially NFATc3, in promoting the malignancy of OA-treated Kras-mutant organoids. Our findings suggested the impact of oleic acid on promoting the malignant transformation of Kras-mutant colonic organoids through NFATc-relating pathway. The expansion of tumorigenic stem cells via induction of abnormal Paneth cell, may contribute to the potential of cancer-toward metaplasia in Kras-mutant organoids when exposing to high level of oleic acid. Oleic acid is an unfavorable factor of colorectal cancer. The frequent usage of OA, which may benefit to cardiovascular diseases, may lead to the increased risk of CRC in latent sensitive Kras-mutant individuals.