探討受AKT調控之下游表現蛋白於Sorafenib抗藥性之肝腫瘤細胞中所扮演之角色

To study AKT downstream effector proteins involved in acquired sorafenib resistance in Huh7 cells

何玟瑾(Wen-Ching Ho)

指導教授 ： 周綠蘋

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10710

摘要

肝細胞瘤是全球致死率極高的癌症之一， 現行肝癌治療臨床標靶藥物Sorafenib抑制細胞中酪胺酸激酶的活性消滅癌細胞，但從臨床上成效有限的病例顯示，Sorafenib對癌細胞Raf pathway的長期抑制誘發了其他替代訊息傳遞的活化，造成了Sorafenib的後天抗藥性。
為了探討Sorafenib抗藥性的問題，實驗室先前將肝癌細胞Huh7經過Sorafenib長期的藥物篩選，建立一株Sorafenib藥物敏感度低的細胞株Huh7R，發現其AKT相較於原生態Huh7高度活化。並且從先前差異蛋白質體學分析(Stable Isotope Labeling by Amino acids in Cell culture ，SILAC)，發現一小分子量14kDa凝集素在Huh7R中表現較多。由於Huh7R中AKT的高度活化，與14kDa凝集素的高表現量，我們推測AKT可能是14kDa凝集素的上游調控因子。透過PI3K 抑制劑LY294002的抑制，隨著AKT活化程度的降低可以看到14kDa凝集素的減少，進一步我們加入mTOR抑制劑rapamycin，同樣看到14kDa凝集素的表現下降，另外透過加入氯化亞鈷誘發轉錄因子HIF1α的上升，發現14kDa凝集素基因表現增加。以上結果顯示Huh7R細胞高度活化的AKT pathway 經由mTOR/HIF1α增加14kDa凝集素的基因表現。
為了更進一步了解14kDa凝集素在Huh7R細胞中扮演的角色，我們在Huh7R細胞抑制生成14kDa凝集素，以及在Huh7細胞促進表現14kDa凝集素，發現在Huh7R細胞中減少14kDa凝集素會使Sorafenib藥物感受性增加，細胞遷移與侵襲的能力降低。從以上實驗我們發現Sorafenib抗藥性細胞中誘發高度活化的PI3K/AKT pathway，經由mTOR與HIF1α調控14kDa凝集素的高表現，並影響癌細胞抗藥性與轉移能力。

關鍵字

肝細胞瘤 ； Sorafenib ； 抗藥性 ； 凝集素 ； AKT

並列摘要

Hepatocellular carcinoma (HCC) is one of the most serious health problems which results in cancer-related death worldwide. The targeted drug sorafenib has improved the survival of patients with advanced liver cancer. However, accumulatively clinical evidences have suggested that the blockade of Raf signaling might result in unexpected molecular events.
To understand the sorafenib unrevealed mechanism, our lab established a sorafenib-resistant HCC cell line , Huh7R, from Huh7. From SILAC-base (Stable Isotope Labeling by Amino acids in Cell culture) quantitative proteomic analysis, we found a 14kDa-lectin in Huh7R cells. In order to investigate the correlation between sorafenib resistance and the up-regulated lectin in Huh7R, we compared the protein expression levels between Huh7 and Huh7R cells, which established by long term exposure to sorafenib, and observed a dramatic increase of a 14kDa-lectin in Huh7R by Western blotting. We also found that the 14kDa-lectin is an AKT downstream effector which was up-regulated in Huh7R. After treatment with PI3K inhibitor LY294002, we observed the reduction of the protein expression along with the decrease of phosphorylated-AKT. Furthermore, to uncover the signaling pathway, we treated Huh7R cells with mTOR inhibitor rapamycin and hypoxia-mimetic agent cobalt chloride, and we detected the 14 kDa-lectin decreased as mTOR inhibited by rapamycin and the lectin increased after HIF1α was induced. We hypothesized that AKT/mTOR may regulate expression of the 14kDa-lectin through the induction of HIF1α.
To further examine the role of 14kDa-lectin in Huh7R, we knockdowned the endogenous 14kDa-lectin in Huh7R and overexpressed 14kDa-lectin in Huh7. Functional analysis indicated that the overexpression of the 14kDa-lectin in Huh7R contributes to sorafenib resistance, cell migration and invasion. In conclusion, 14kDa-lectin could be mediated by PI3K/AKT signaling pathway, and be involved in the acquired resistance to Sorafenib in HCC.

並列關鍵字

Hepatocellular carcinoma ； Sorafenib ； drug resistance ； lectin ； AKT

參考文獻

1.Vikram, R.P. and D.B. Alastair, Pathology of liver tumours. Surgery (Oxford), 2007. 25.

2.Farazi, P. and R. DePinho, Hepatocellular carcinoma pathogenesis: from genes to environment. Nature reviews. Cancer, 2006. 6(9): p. 674-687.

3.Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians, 2011. 61(2): p. 69-90.

4.Bruix, J., M. Sherman, and D. American Association for the Study of Liver, Management of hepatocellular carcinoma: an update. Hepatology (Baltimore, Md.), 2011. 53(3): p. 1020-1022.

5.Wu, X. and Y. Li, Signaling Pathways in Liver Cancer.