YB-1 引發蕾莎瓦抗藥性肝細胞癌產生表皮細胞間質化之機制探討

Abstract

肝細胞癌 (HCC) 是原發性肝癌中最常見的類型，在全球癌症統計中其致死率位居第四名。而Sorafenib 是美國FDA 第一個核可用於治療晚期肝細胞癌的標靶藥物，但患者在長期接受Sorafenib 治療後產生抗藥性的問題為治療上的主要困境。 為了探討HCC 細胞對於Sorafenib 產生抗藥性的機制，本實驗室建立了對Sorafenib 具有抗藥性的Huh7R 細胞，透過磷酸化蛋白質體學的分析與臨床TCGA資料庫內的數據進行交集，並利用生物資訊工具進行分析，進一步發現Y-Box binding protein 1 (YB-1) 是在Huh7R 細胞中高度表現的重要調控蛋白。YB-1 為轉錄因子且於許多癌症中有活化的情況。根據我們的研究發現，YB-1 在Huh7 細胞中是受MAPK 訊息傳導途徑所調控，而在Huh7R 細胞中則會受EGFR/PI3K/AKT路徑的調控而大量活化。因此，為了探討YB-1 在Huh7R 細胞中所扮演角色，我們利用shRNA 抑制YB-1 表現並進行後續研究。由體外功能分析實驗結果發現，knockdown YB-1 會抑制細胞的爬行及侵襲能力。由於表皮細胞間質化 (EMT) 被認為是癌症轉移中的關鍵機制，因此為探討轉錄因子YB-1 調控Huh7R 細胞轉移的機制，我們發現knockdown YB-1 會影響EMT 相關分子的表達，像是抑制Snail、Twist1、Zeb-1、MMP2、Vimentin 並增加ZO-1、E-cadherin 的RNA 及蛋白表現。此外，我們也發現，knockdown YB-1 會抑制藥物輸出通道蛋白MDR1/p-glycoprotein的表現，進而增加Huh7R 細胞對Sorafenib 的感受性。 由免疫螢光染色及核質分離的結果，我們發現PI3K 抑制劑LY294002 可抑制磷酸化YB-1S102 的核轉移。同時，我們也發現當細胞表現YB-1S102A 時可增加對Sorafenib 的感受性並抑制細胞爬行及侵襲的能力。顯示出YB-1 Serine 102 位點的磷酸化會使YB-1 進入細胞核並調控下游標的基因的轉錄活性，進而影響Huh7R 細胞的功能表現。最後，我們也利用其他對Sorafenib 具有抗藥性的HCC 細胞PLC-5R 進行功能分析的驗證。 綜合上述結果，我們發現Sorafenib 透過EGFR/PI3K/AKT 路徑活化YB-1 的磷酸化表現，並促使YB-1 進入細胞核進而調控抗藥性相關及EMT 相關基因的表達，造成Huh7R 細胞產生抗藥性及表皮細胞間質化的現象。因此，本篇研究說明了在對Sorafenib 產生抗藥性的HCC 細胞中，YB-1 是可用來作為預測腫瘤惡化發生轉移的重要預後生物標記。

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most common cause of cancer-related death worldwide. Sorafenib is the first FDA approval target therapy for advanced HCC. However, the major obstacle is that acquired resistance usually occurs in HCC patients after long term treatment of sorafenib. We established the sorafenib-resistant HCC cell line Huh7R to investigate the mechanism of sorafenib resistance. Based on bioinformatic integration of phosphoproteomics analysis and the clinical TCGA data, we found Y-Box binding protein 1 (YB-1) is a potential regulator and is highly expressed in sorafenib-resistant Huh7R cells. YB-1, a transcription factor, is activated in various cancers. According to our findings, YB-1 is regulated by MAPK signaling pathway in Huh7 cells and YB-1 is activated by EGFR/PI3K/AKT signaling pathway in Huh7R cells. Therefore, we knockdown YB-1 by shRNA to investigate its role in Huh7R cells. In vitro functional analysis showed that knockdown YB-1 impairs the ability of cell migration and invasion. Epithelial‐to‐mesenchymal transition (EMT) has been suggested to play crucial roles in tumor metastasis. To further elucidate the mechanisms, we found that knockdown YB-1 reduces the expression of EMT-related markers such as Snail, Twist1, Zeb-1, MMP2, Vimentin and increases the expression of ZO-1 and E-cadherin both in RNA and protein levels. Furthermore, we also found that knockdown YB-1 sensitizes cells to sorafenib through down-regulate the drug efflux pump MDR1/p-glycoprotein expression in Huh7R cells. Through immunofluorescence staining and cellular fractionation, the expression of p-YB-1S102 is suppressed in the nucleus level by PI3K inhibitor, LY294002. We also found that the mutant YB-1S102A increases the sensitivity to sorafenib and suppresses the migration and invasion ability. These data indicate that phosphorylation of YB-1 at serine-102 could enhance its nuclear translocation and mediate the transcriptional regulation of the downstream target genes, affecting the functional performance of Huh7R cells. Finally, we also validate the functional assays with another sorafenib-resistant HCC cell line PLC-5R. In conclusion, we reveal the mechanism of sorafenib activated the phosphorylation of YB-1 through EGFR/PI3K/AKT pathway in sorafenib-resistant Huh7R cells, which promotes the nuclear translocation of YB-1 to regulate the MDR1 and EMT-related genes expression and induces drug resistance and EMT in Huh7R cells. Therefore, our findings suggested that YB-1 is a prognostic biomarker for the metastatic potential in sorafenib-resistant HCC cells.