YB-1 促進 PI3K 及 EGFR 激酶表現並引發蕾莎瓦抗藥性
肝癌細胞之絲狀偽足生成及表皮細胞間質化

肝癌是全世界最致命的惡性腫瘤之一,其中肝細胞癌 (hepatocellular carcinoma , HCC)佔了 70 %-85 %的發生率,為最常見的肝癌。針對晚期肝癌的患者,標靶藥物蕾莎瓦 (sorafenib)為第一線的治療策略,可以有效地延長晚期肝癌病人的存活期。蕾莎瓦是一種多重激酶抑制劑,透過阻擋 VEGFR, PDGFR, RAF 及 MAPK 訊息傳遞路徑以抑制腫瘤生長及血管新生。但是近來發現接受蕾莎瓦治療的病患有逐漸出現後天抗藥性 (acquired resistance)的隱憂。為了要了解蕾莎瓦抗藥性的機制,本實驗室建立了對蕾莎瓦有抗藥性的肝細胞癌細胞株-Huh7R。透過磷酸化蛋白質體學的分析,發現 YB-1 (Y-Box binding protein 1)在 Huh7R 細胞中會經由 AKT 活化而大量表現。YB-1 是一種轉錄因子, 在許多癌症中會大量活化。它會透過調控某些基因表現進而促進細胞增生及表皮細胞間質化 (epithelial-mesenchymal transition, EMT)。接著我們利用小髮夾 RNA (short hairpin RNA, shRNA)抑制 Huh7R 細胞中的 YB-1 表現。發現 knockdown YB-1 會抑制細胞移動及侵襲能力,除此之外細胞型態也會從拉長的紡錘狀變回表皮細胞的外型。經螢光染色後透過共軛焦螢光顯微鏡觀察肌動蛋白纖維的排列變化,發現 knockdown YB-1 會抑制絲狀偽足的表現。細胞型態的改變及絲狀偽足 (filopodia)的形成對於細胞的移動能力及 EMT 是很重要的,因此進一步想探討 YB-1 會透過調控什麼基因促進絲狀偽足的生成。我們發現在 Huh7R 細胞中 YB-1 會進核促進 PI3K 及 EGFR 表現。先前的研究已知PI3K會透過活化GTPase Rho家族蛋白促進肌動蛋白纖維 (actin filament)的重新排列。在本篇研究中阻斷 Huh7R 細胞的 PI3K 活性會抑制絲狀偽足的生成,而 knockdown YB-1 會減少活化態 Cdc42 的表現。我們推測在對蕾莎瓦有抗藥性的肝癌細胞中,YB-1 會透過促進 PI3K 表現以活化 Cdc42,進而增進絲狀偽足的生成。總結以上結果,我們探討了 Huh7R 細胞中 YB-1 促進絲狀偽足的生成及 EMT 的機制,顯示出 YB-1 對於癌細胞侵襲力的重要性。因此 YB-1 可作為一個治療標的,以解決蕾莎瓦抗藥性肝癌細胞轉移能力增強的隱憂。

關鍵字

肝細胞癌；蕾莎瓦抗藥性； Y-Box binding protein 1 ； PI3K ；絲狀偽足；表皮細胞間質化

並列摘要

Liver cancer is one of the most lethal malignancy in worldwide and hepatocellular carcinoma (HCC) accounts for 70%-85% of the total liver cancer incidences. For advanced HCC, targeted therapy sorafenib is a first-line treatment that can effectively prolong overall survival of late stage HCC patients. Sorafenib is a multi-kinase inhibitor that suppresses tumor progression and angiogenesis through blocking VEGFR, PDGFR, RAF and MAPK signaling pathways. However, acquired resistance has been concerned as a challenge to sorafenib treatment. We developed the sorafenib resistant hepatoma cell line Huh7R to understand the mechanism of sorafenib resistance. Based on phosphoproteomics analysis, we found Y-Box binding protein 1(YB-1) is highly expressed and activated by AKT in Huh7R cells. YB-1 is a transcription factor, which is highly activated in cancers. It can regulate gene expression resulted cell proliferation and epithelial-mesenchymal transition (EMT). To understand the role of YB-1 in Huh7R cells, we used shRNA to knockdown YB-1 in Huh7R cells and found that knockdown YB-1 suppressed the ability of cell migration and cell invasion. In addition, the morphology was changed from spindle type to epithelial-like cell in YB-1 knockdown cells. Through confocal microscopy, we observed that the expression of filopodia was repressed in YB-1 knockdown cells. Morphology changes and the formation of filopodia are essential for cell to increase the ability of cell migration and EMT. We also found that YB-1 can translocate into nucleus inducing PI3K and EGFR expression. It is reported that PI3K can induce actin rearrangement through activating GTPase Rho family protein. In our study, inhibiting PI3K activity reduced filopodia formation in Huh7R cells and the activity of Cdc42 was lower in YB-1 knockdown cells. We presumed that YB-1 would induce PI3K-mediated Cdc42 activation and promote filopodia formation and EMT in Huh7R cells. In conclusion, we investigated the mechanism of YB-1 promoting filopodia formation and EMT in Huh7R cells. It reveals the importance of YB-1 in cell invasion. Therefore YB-1 can be a therapeutic target to overcome the problem of metastatic potential in sorafenib resistant HCC cells.

並列關鍵字

Hepatocellular carcinoma ； Sorafenib resistance ； Y-Box binding protein 1 ； PI3K ； Filopodia ； Epithelial-mesenchymal transition

參考文獻

1. Lindsey A. Torre, M., et al., Global Cancer Statistics, 2012. CA CANCER J CLIN, 2015. 65: p. 87-108.

2. Laursen, L., A preventable cancer. Nature Reviews Cancer, 2014. 516: p. S2-S3.

3. Whittaker, S., R. Marais, and A.X. Zhu, The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene, 2010. 29(36): p. 4989-5005.

4. Villanueva, A., V. Hernandez-Gea, and J.M. Llovet, Medical therapies for hepatocellular carcinoma: a critical view of the evidence. Nat Rev Gastroenterol Hepatol, 2013. 10(1): p. 34-42.

5. Llovet, J.M., A. Burroughs, and J. Bruix, Hepatocellular carcinoma. Lancet, 2003. 362(9399): p. 1907-17.

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YB-1 促進 PI3K 及 EGFR 激酶表現並引發蕾莎瓦抗藥性肝癌細胞之絲狀偽足生成及表皮細胞間質化

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