植化素誘導之分泌性自噬促進RAB37媒介的TIMP1分泌與改善人類肝細胞癌的索拉非尼抗藥性

Abstract

Sorafenib常為肝細胞癌（原位肝癌主要類別）的標靶藥物，卻容易在用藥六個月後產生抗藥性。分泌性自噬不同於降解性自噬僅分解老廢胞器，此路徑協助癌細胞在飢餓狀態下活化RAB37媒介的TIMP1胞吐，使其與MMP9結合，抑制腫瘤形成。本研究透過活化分泌性自噬，改善抗藥性肝細胞癌的治療困境。G9A表現也與抗藥性相關。本研究發現肝癌患者G9A mRNA高表現與較低存活期有關，而Huh7是對Sorafenib最敏感的細胞株。以Huh7建立Sorafenib具抗藥性的Huh7R細胞株。與Huh7相比，Huh7R表現出較高的RAB蛋白轉譯後修飾及囊泡出芽與生合成。實驗顯示Huh7R具有顯著較高的細胞爬行能力。大量研究報導植化素具有治療腫瘤的能力，透過大數據推測薑黃素為具潛力的分泌性自噬誘導劑。結果發現，以薑黃素處理後可增加自噬標記LC3-II及TIMP1分泌，同時G9A表現與Huh7R細胞的爬行率皆降低。透過敲低ATG5證實，阻斷自噬路徑可降低LC3-II與薑黃素誘導的TIMP1分泌，並且細胞質中的LC3-II和TIMP1共定位減弱，雙層膜的類自噬體數量同時減少。這表示薑黃素誘導的TIMP1分泌依賴ATG5的路徑。合併給予Sorafenib和薑黃素使G9A表現量降低。實驗證實薑黃素是肝細胞癌細胞株的分泌性自噬誘導劑，並揭示G9A與分泌性自噬的新關聯性。Sorafenib is the targeted therapy for hepatocellular carcinoma (HCC), but often leading to drug resistance after six months of treatment. Unlike degradative autophagy, secretory autophagy activates RAB37-mediated TIMP1 exocytosis under starvation, enabling its binding to MMP9 to suppress tumor formation. This study aimed to improve drug sensitivity and reduce migration in resistant cells. The histone methyltransferase G9A is linked to drug resistance, and high expression of G9A mRNA in patients with liver cancer correlates with poor survival. Among HCC cell lines, Huh7 is most sensitive to Sorafenib. A resistant model (Huh7R) was developed to mimic acquired resistance. GSEA revealed increased RAB protein modifications and vesicle biogenesis in Huh7R. Huh7R also showed enhanced migration. Omics-based pathway prediction identified Curcumin as a potential secretory autophagy inducer. Curcumin treatment reduced G9A, increased LC3-II, promoted TIMP1 secretion, and decreased Huh7R migration. ATG5 knockdown suppressed autophagy, reduced Curcumin-induced TIMP1 secretion, and diminished the cytoplasmic co-localization of LC3-II and TIMP1, along with fewer double-membraned vesicles. These data indicate Curcumin-induced TIMP1 secretion is ATG5-dependent. Combined treatment with Sorafenib and Curcumin further lowered G9A in Huh7R. In summary, Curcumin is identified as a novel secretory autophagy inducer in HCC cells, uncovering a new link between G9A and secretory autophagy.