阻斷DHODH透過甲羥戊酸途徑重編程、SQLE表現下調與核內轉移，誘發神經母細胞瘤細胞死亡

Abstract

近期研究指出，二氫乳清酸脫氫酶（dihydroorotate dehydrogenase, DHODH）表現與癌症進程密切相關，尤其在具 MYCN 擴增的高風險神經母細胞瘤中呈現最高表達，暗示 DHODH 可能在腫瘤致病機制中扮演關鍵角色。透過分子對接分析、熱轉移試驗與酵素螢光活性分析，我們系統性地鑑定出 FDA 已核准的多重激酶抑制劑 Regorafenib 具有潛在 DHODH 抑制作用。功能驗證顯示，DHODH 基因敲降與 Regorafenib 處理均顯著誘導細胞凋亡並降低細胞遷移能力，但不影響細胞週期進程。為闡明其分子機制，我們實驗室之前利用 tandem mass tag （TMT）定量蛋白體分析，比較 Regorafenib 處理組與 DHODH 敲降組之蛋白質表現譜。液相層析–串聯質譜（LC-MS/MS）共鑑定 31,518 條胜肽與 4,472 種蛋白質。差異表現蛋白的基因本體論富集分析顯示，DHODH 抑制顯著干擾脂質代謝並下調甲羥戊酸途徑，最終指向鐵致凋亡（ferroptosis）為下游結果。為驗證此機制鏈結，我們檢測膽固醇生合成，發現總膽固醇與游離膽固醇皆顯著下降。鑑於膽固醇代謝與脂滴形成及鐵致凋亡調控之關聯，我們進一步以共軛焦顯微鏡與流式細胞儀檢視脂滴動態，證實 DHODH 阻斷顯著抑制脂滴形成。重新分析我們的蛋白體資料並對照加州大學柏克萊分校 Olzmann 實驗室發表之脂滴蛋白體，鑑定出在膽固醇生合成中具限速作用的角鯊烯環氧化酶（squalene epoxidase, SQLE）為關鍵下游分子。值得注意的是，SQLE 主要定位於脂滴周邊，且在 DHODH 抑制後呈現顯著的核內累積，此現象為文獻中未曾報導。為評估臨床相關性，我們建立了病人衍生的神經母細胞瘤類器官。Regorafenib 處理導致類器官明顯形態解離，伴隨顯著的細胞凋亡與鐵致凋亡增加，以及脂滴含量的顯著減少，與體外細胞實驗結果一致。綜合而言，本研究證實透過 Regorafenib 或基因沉默抑制 DHODH，可藉由調控甲羥戊酸途徑、擾動脂質代謝並抑制 SQLE 活性與定位，同時誘發神經母細胞瘤細胞的凋亡與鐵致凋亡。此發現凸顯 DHODH 作為高風險神經母細胞瘤新穎代謝脆弱點的治療潛力。

Recent studies have underscored a strong association between dihydroorotate dehydrogenase (DHODH) expression and cancer progression, particularly in high-risk neuroblastoma characterized by MYCN amplification. This correlation suggests a potential role for DHODH in neuroblastoma pathogenesis. Through molecular docking, thermal shift, and enzymatic fluorescence-based activity assays, we systematically identified the FDA-approved multi-kinase inhibitor Regorafenib as a potential DHODH inhibitor. Functional validation revealed that both DHODH knockdown and Regorafenib treatment significantly induced apoptosis and reduced cell migratory capacity, without affecting cell cycle progression. To elucidate the underlying molecular mechanisms, we previously performed tandem mass tag (TMT)-based quantitative proteomics, comparing protein expression profiles between Regorafenib-treated cells and those with DHODH knockdown. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis yielded 31,518 peptides corresponding to 4,472 proteins. Gene ontology enrichment analysis of differentially expressed proteins revealed marked disruption of lipid metabolic processes and suppression of the mevalonate pathway, ultimately implicating ferroptosis as a downstream consequence of DHODH inhibition. To validate this mechanistic link, we examined cholesterol biosynthesis and observed a significant reduction in both total and free cholesterol levels following DHODH inhibition. Given the known role of cholesterol metabolism in lipid droplet formation and ferroptosis regulation, we further evaluated lipid droplet dynamics using confocal microscopy and flow cytometry, which confirmed that DHODH blockade markedly suppressed lipid droplet formation. Reanalysis of our proteomic data, in comparison with the lipid droplet proteome curated by Dr. Olzmann’s laboratory (UC Berkeley), identified squalene epoxidase (SQLE)—a rate-limiting enzyme in cholesterol biosynthesis—as a key downstream effector. SQLE was found to localize near lipid droplets and showed significantly increased nuclear accumulation upon DHODH inhibition, a novel observation not previously reported in the literature. Finally, to assess the clinical relevance of our findings, we established patient-derived neuroblastoma organoids. Regorafenib treatment induced substantial morphological disintegration of the organoids, accompanied by increased apoptosis, ferroptosis, and a marked reduction in lipid droplet content, in line with our in vitro findings. In summary, this study demonstrates that DHODH inhibition, via Regorafenib or genetic silencing, induces both apoptosis and ferroptosis in neuroblastoma through modulation of the mevalonate pathway, disruption of lipid metabolism, and suppression of SQLE activity and localization. These findings highlight the therapeutic potential of targeting DHODH as a novel metabolic vulnerability in high-risk neuroblastoma.