表皮生長因子受體L858R突變調控粒線體代謝之研究

Abstract

腫瘤生成（tumor initiation）是一個由基因突變驅動的早期過程，其中驅動性突變（driver mutations）賦予細胞異常的生長優勢，使其跳脫正常細胞對增殖與分化的調控，進而持續擴增並朝向惡性轉化。為因應快速增殖所需的能量與物質，腫瘤細胞會重新編排其代謝模式，此一過程稱為代謝重編程（metabolic reprogramming），粒線體作為細胞能量核心器官，在其中扮演不可或缺的角色，特別是在能量產生與代謝訊號傳導方面。 在東亞地區，表皮生長因子受體（epidermal growth factor receptor, EGFR）突變是非小細胞肺癌中最常見的驅動事件，尤其以 exon 19 缺失與 exon 21 的 L858R 點突變最為常見。儘管 EGFR 在腫瘤形成中的角色已有諸多研究，但其是否與粒線體功能調控直接相關仍未被充分闡明。因此，本研究旨在探討 EGFR L858R突變是否會影響粒線體功能與細胞代謝的變化。 本研究建立了穩定過表現 EGFR L858R 的 NIH3T3 細胞株，並發現其增殖速率與集落形成能力皆高於對照組。此外，EGFR L858R 細胞培養液呈現加速變黃，並有顯著乳酸累積，搭配 Seahorse 分析也觀察到其氧化磷酸化與糖解能力均上升，顯示代謝活性顯著改變。透過萃取細胞中的粒線體蛋白，發現EGFR L858R有粒線體轉位的現象，進一步探索可能參與代謝重編程的粒線體蛋白，我們細胞的粒線體蛋白質體，交叉比對 MitoCarta3.0 資料庫後篩選出五個具有粒線體功能的蛋白，其中包含STING1與MAVS。我們發現 EGFR L858R 細胞中STING1與MAVS蛋白表現均明顯上升，MAVS也呈現高分子量寡聚體化（oligomerization）的現象。這類 MAVS 聚集型態類似病毒感染時所誘發的 prion-like 聚集模式，但有趣的是，第一型干擾素（如 Ifn-α, Ifn-β）並未被顯著上調，顯示此聚集可能並未活化典型免疫路徑，反而誘導 Cxcl10 等與發炎及代謝壓力相關的基因上升。此外，cleaved caspase-3 表現下降與PARP上升的現象，也暗示 EGFR L858R 細胞可能透過重新調控 MAVS/STING1 訊號，壓抑細胞凋亡並促進代謝適應，進一步支持先前觀察到的細胞型態與代謝功能變化。

Tumor initiation is a complex, multistep process in which cells acquire oncogenic mutations that provide clonal growth advantages. To sustain unchecked proliferation and evade nutrient limitations, tumor cells undergo profound metabolic reprogramming. Mitochondria, as the central hub of energy production and metabolic integration, are believed to play critical roles in this transformation. Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide. In East Asian populations, mutations in the epidermal growth factor receptor (EGFR), particularly the L858R point mutation in exon 21, are highly prevalent. Although oncogenic signaling of EGFR has been extensively studied, its direct involvement in regulating mitochondrial metabolism during early tumorigenesis is not well understood. In this study, we aimed to elucidate the mitochondrial mechanisms underlying EGFR L858R-driven metabolic remodeling during tumor initiation. We established a stable NIH3T3 cell line overexpressing EGFR L858R to model early oncogenic transformation. These cells exhibited enhanced proliferation, altered morphology, and increased colony-forming ability compared to control cells. Notably, the culture medium of EGFR L858R cells turned yellow more rapidly, corresponding with a significant increase in extracellular lactate levels, indicating a shift toward glycolytic metabolism. Seahorse analysis confirmed enhanced glycolysis and oxidative phosphorylation in EGFR L858R cells. We further isolated the mitochondrial fraction and confirmed the translocation of EGFR L858R protein to mitochondria. To explore EGFR's influence on mitochondrial function, we performed proteomic profiling of mitochondrial extracts. Bioinformatic filtering using the MitoCarta3.0 database revealed five EGFR-regulated mitochondrial proteins, among which STING1 and MAVS were of particular interest due to their dual roles in immune signaling and metabolism. We observed elevated STING1 expression and increased MAVS oligomerization in EGFR L858R cells, but without corresponding activation of canonical type-I interferon responses. Interestingly, only Cxcl10 expression was significantly upregulated, suggesting a non-canonical signaling route. Moreover, the apoptosis landscape appeared dysregulated, with reduced cleaved CAPSASE-3 and BCL-2 expression but increased PARP levels, pointing toward survival-promoting mitochondrial adaptation rather than cell death. In summary, our findings suggest that EGFR L858R not only rewires mitochondrial metabolism but may also redirect immune-related adaptors such as STING1 and MAVS toward supporting tumor-like metabolic states. Further investigation into their mechanistic interactions with mitochondrial EGFR may uncover novel regulatory axes in early tumorigenesis.