Quantitative Phosphoproteome Analysis to Reveal Alternative Signaling Pathways in Hepatocellular Carcinoma Cell under Sorafenib Treatment

• Main Authors: Chun-Yao Wang, 王俊堯
• Other Authors: Lu-Ping Chow
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/70486691544447851456

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 102 === Hepatocellular carcinoma(HCC) is now the third leading cause of cancer deaths worldwide, accounting for 90% of primary liver cancers. Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis). Advanced HCC is often a complex condition with highly aggressive tumor and poor prognosis to common therapies. The mainstay of therapy is surgical resection, but the majority of patients are not eligible because of tumor extent or underlying liver dysfunction. Recently, a therapeutic option for advanced HCC is the use of sorafenib, a muti-kinase inhibitor, which was showed to be able to increase survival in HCC patients. In this study, we applied quantitative proteomics approach using nanoflow LC MS/MS in combination with SILAC technology to compare HuH7 and sorafenib treated HuH7 cells. Through phosphopeptide enrichment by titanium dioxide affinity column, a total of 2180 phosphopeptides from 562 phosphorylated proteins were identified. The SILAC based quantification described 72 up-regulated and 116 down-regulated phosphorylation sites upon sorafenib treatment. By using Motif-x, STRING and IPA analyses, we found that most proteins showing significant fold change under sorafenib treatment are involved in MAPK-ERK signaling pathway. One of the down-regulated proteins we found is the Y-Box binding protein 1(YB-1), which was reported as a transcription factor regulated by MAPK-ERK and PI3K-AKT signaling pathway. YB-1 is overexpressed in many cancers and associated with tumor progression and drug resistance. In this study we found that YB-1 is a downstream target of MAPK-ERK signaling cascade, its phosphorylation is decreased under U0126(MEK inhibitor) treatment. Surprisingly, we found that phosphorylation of YB-1 is increased in sorafenib- resistant cell line HuH7R. Recent studies indicated YB-1 as a direct substrate of AKT. The functional significance of AKT-mediated phosphorylation remains largely unknown. Based on our findings, YB-1 is regulated by PI3K/AKT signaling pathway instead of MAPK signaling pathway in HuH7R cell. Invasion and migration ability are decreased in YB-1-knock-down HuH7R cell. After signaling pathway and functional analysis, results indicated that YB-1 regulates cell invasion and migration ability through activation of PI3K/AKT/GSK3βsignaling pathway. We further identified some YB-1-regulated downstream genes like PI3K, P53 and VEGFA that may contribute to cell viability. In conclusion, we defined different roles of YB-1 in HuH7 and HuH7R cell. YB-1 is regulated by MAPK signaling pathway in HuH7 cell but it is regulated by an alternative signaling pathway, PI3K/AKT signaling pathway, in HuH7R cell. Understanding different regulation mechanisms of the same signal molecule at different circumstances may contribute to further development of novel anti-cancer drug and alternative therapies.