Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid-induced genotoxicity

Background: Risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett’s esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. Methods: Esophageal biopsies from normal epithelium (NE), BE and EAC, were analysed using concurrent lipidomics and proteomics (n=30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n=22) and immunohistochemistry (IHC, n=80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for gH2AX. Results: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism and biosynthesis of specialized proresolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipids synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for immunohistochemistry validation: D4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates DHCer to Cer, and D5, and D6-Desaturases (fatty acid desaturases FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increase from BE through dysplasia to EAC, but transcript of DEGS1 was decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with 3 and 4 double bonds and reduced bile acid-induced DNA double strand breaks in FLO-1 cells in vitro. Conclusions: Integrated multiomics revealed sphingolipid and phosphopholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro in FLO-1 cells, potentially through reduced lipid peroxidation.

背景：食管腺癌（esophageal adenocarcinoma, EAC）的发病风险与胃食管反流病（gastro-esophageal reflux disease, GERD）及肥胖密切相关。以脂质代谢为靶点的治疗可降低巴雷特食管（Barrett’s esophagus, BE）进展为EAC的风险，但EAC发生过程中确切的脂质代谢改变及其在基因毒性中的作用仍有待阐明。
方法：本研究对正常上皮（normal epithelium, NE）、BE及EAC患者的食管活检组织进行同步脂质组学与蛋白质组学分析（n=30），随后采用独立样本通过RNA测序转录组学（RNAseq transcriptomics, n=22）与免疫组化（immunohistochemistry, IHC, n=80）进行正交验证。此外，用FADS2选择性抑制剂SC26196或/和胆汁酸混合物处理EAC细胞系FLO-1，随后对gH2AX进行免疫荧光染色。
结果：对蛋白质组学数据进行聚焦代谢的Reactome通路分析显示，EAC发病过程中富集了脂肪酸代谢、酮体代谢及特异性促消退介质（specialized proresolving mediators）的生物合成通路。脂质组学分析显示，从NE到BE再到EAC，甘油磷脂合成通路呈现进行性改变：甘油三酯水平下降，磷脂酰胆碱与磷脂酰乙醇胺水平升高；鞘脂合成通路同样呈现进行性改变：二氢神经酰胺水平下降，神经酰胺水平升高。此外，含C20脂肪酸的脂质及双键数≥4的多不饱和脂质水平也呈进行性升高。整合转录组数据后，筛选出可用于免疫组化验证的候选酶：Δ4-去饱和酶、鞘脂1（DEGS1，可将二氢神经酰胺DHCer转化为神经酰胺Cer），以及负责多不饱和修饰的Δ5、Δ6去饱和酶（脂肪酸去饱和酶FADS1与FADS2）。这三种酶的表达均从BE经异型增生阶段进展至EAC的过程中显著升高，但DEGS1的转录水平却出现下降，提示其存在翻译后调控。最后，FADS2选择性抑制剂SC26196可显著降低含3个与4个双键的多不饱和脂质水平，并在体外实验中减轻FLO-1细胞中胆汁酸诱导的DNA双链断裂。
结论：整合多组学分析揭示了EAC发生过程中鞘脂与磷脂代谢的重编程现象。在体外实验中，抑制FADS2并降低高水平多不饱和脂质可有效保护EAC细胞免受胆汁酸诱导的DNA损伤，其潜在机制可能与减轻脂质过氧化有关。