Abstract
Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing.
Highlights
Human hepatocellular carcinoma (HCC) is one of the most lethal diseases around the world [1].One major reason for the high death ratio of HCC is due to the fast invasion and metastasis ability of tumor cells in primary liver cancers
We studied the phosphoproteome of a metastasis HCC cell line, MHCC97-H, by an off-line high-pH HPLC separation strategy combined with a multi-step IMAC method
In order to globally profile the phosphoproteome involved in liver cancer invasion and metastasis, we performed a systematic phosphorylation profiling with an off-line high-pH HPLC separation strategy combined with a multi-step IMAC (Immobilized Metal ion Affinity Chromatography) method for a human metastatic HCC cell line, MHCC97-H, from 2.5 mg cell lysates (Figure 1)
Summary
Human hepatocellular carcinoma (HCC) is one of the most lethal diseases around the world [1]. Phosphorylation or dephosphorylation usually activates and/or inactivates the transduction of many signal pathways, such as the MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinase) pathway [2], the cAMP (cyclic AMP)-dependent pathway [3] and the IP3/DAG (Inositol 1,4,5-triphosphate/1,2-Diacylglycerol) pathway [4] Dysfunction of these pathways and mutations of important kinases are always associated with various diseases, including cancers [5,6,7,8]. Li et al had established HCC cell lines with different metastatic potentials, MHCC97-L (low metastasis) and MHCC97-H (high metastasis), from the metastatic hepatocellular carcinoma cell line, MHCC97 [11] These cell lines have been widely used for studying the mechanism of metastasis in HCC [12,13,14]. The phosphopeptide enrichment strategy established in this study could provide novel phosphoproteins involved in liver cancer
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