內質網(endoplasmic reticulum, ER)是真核細胞內最主要負責新合成蛋白質與蛋白質修飾的胞器。當細胞遭受到環境影響，使它無法正確的折疊蛋白質，就會累積許多未摺疊的蛋白質(unfolded proteins)，造成內質網壓力(ER stress)。許多研究指出很多癌細胞都會誘導內質網壓力的上升，例如：乳癌與肝癌。此外也有文獻報導指出不正常活化脂質代謝會造成乳癌細胞的發生率上升。但較少研究顯示到底內質網壓力是如何去調控脂質代謝。在我們實驗室學長與學姊的研究發現內質網壓力可調控長鏈型醯基輔酵素A合成酵素3(acyl-CoA synthetase long-chain family member 3, ACSL3)，這個酵素為脂質進入代謝的第一個步驟，最主要是將長鏈脂肪酸接上CoA。產生的acyl-CoA可參與生合成新的脂質(anabolic)與將它當作能量消耗掉(catabolic)兩個途徑。ACSL家族共含有5個成員，分別為ACSL1, ACSL3, ACSL4, ACSL5,和ACSL6。我的研究目標則是去觀察乳癌細胞中是否ACSL也同樣扮演重要的角色？首先我們將內質網壓力誘導物處理兩種乳癌細胞，可造成ACSL1的上升與脂質的累積。我們也使用Triacsin C (ACSL1, 3, and 4的抑制劑) 和C75 (脂質合成的抑制劑，inhibitor of Fatty acid synthase)處理細胞，發現Triacsin C可有效的去抑制脂質累積。接著也去利用Triacsin C處理MDA-MB-231細胞，可觀察到Triacsin C會抑制生長與爬行。為了確認ACSL1扮演重要的角色，我們在乳癌細胞中建立了專一降低ACSL1的stable clones。這些細胞株中我們發現細胞生長速度沒有明顯的差異，但細胞爬行則是有明顯的變慢。接著也進一步去分析到底哪些爬行因子受到改變，可觀察到磷酸化的paxillin, 磷酸化的FAK與上皮生長因子接受體(EGFR)皆有下降的情形。由結果告訴我們ACSL1未來可做為目標去治療乳癌細胞。

The endoplasmic reticulum (ER) is a major organelle for folding and maturation of newly synthesized proteins. Excess of unfolded proteins induce ER stress, when cells encounter adverse physiological conditions that impact on protein folding in ER. Several studies have showed that ER stress was induced in various tumors, such as breast tumors and hepatocellular carcinomas. Besides, many evidences indicated that dysregulation of lipid metabolism increases risk of developing cancer. However, relatively little information links ER stress to dysregulation of lipid metabolism. Our previous studies have showed that ER stress can induce lipid accumulation via acyl-CoA synthetase long-chain family member 3 (ACSL3) which catalyzes the first step in fatty acid metabolism by converting long-chain fatty acid into acyl-CoA thioesters and enters both anabolic and catabolic pathways. The family of mammalian ACSLs include five members, ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. Aim of my research is to investigate which ACSL member plays an essential role in breast cancer. Our results showed that ER stress could induce lipid accumulation and expression of ACSL1 in several breast cancer cell lines. Triacsin C (an inhibitor of ACSL1, 3, and 4) repressed lipid accumulation under ER stress. However, C75 (an inhibitor Fatty acid synthase, the rate-limiting step for the biosynthesis) could not repress it. Then, we investigated whether downregulation of ACSLs could impair cell growth and migration in MDA-MB-231 cells. Therefore we established ACSL1 knockdown stable clones in breast cancer cell line. We further studied whether ACSL1 is an essential role in tumorigenesis. We determined cell growth and migration with MTT assay and boyden chamber respectively. The migration rate decreased but growth rate had no significant change in ACSL1 knockdown stable clones. We also analyzed the expression of migration-associated molecules such as phospho-paxillin, phospho-FAK and EGFR. Those molecules were downregulated in stable clones, but lipid accumulation was not attenuated in those cells. Our results implied that ACSL1 will become a therapeutic agent in breast cancer.

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