Amplification, Expression Alteration And Function Analysis Of SKP2 In Esophageal Squamous Cell Carcinoma

Ubiquitin-dependent degradation plays an essential role in many important cellular processes including cell-cycle,differenciation,apoptosis,gene transcription and signal transduction.The gene of SKP2,located on chromosome 5p13,is an F-box protein constituting the substrate recognitionsubunit of the SCF^SKP2 ubiquitin ligase complex and is implicated in ubiquitin-mediated degradation of several cyclin-dependent kinase inhibitors including p27^KIP1,p21^cip1,p57^kip2.Previous studies in our laboratory revealed that gain of chromosome 5p was often observed in esophageal squamous cell carcinoma(ESCC).The present study is to investigate the amplification status and expression level of SKP2 in ESCC and the underlying mechanisms through which SKP2 takes part in the carcinogenesis and development of the disease.Amplification of SKP2 by analysis of fluorescence in situ hybridization(FISH) was observed in 23 out of 50(46%) ESCC.Amplification of SKP2 was confirmed by real-time PCR expremient.Positive correlation was found between SKP2 amplification and tumor stage,lymph node metastase(P＜0.05).RT-PCR and Western-blotting results revealed that expression of SKP2 in the cases with amplification was increased as compared with that in non-amplification cases. Immunohistochemistry in 140 ESCC and adjacent normal tissues showed that the level of SKP2 protein was higher in tumors than that in normal epithelia.SKP2 overexpression was significantly associated with tumor stage and lymph nude metastase(P＜0.05),but no correlation was found between SKP2 expression and prognosis. In vivo assay showed that inhibition of SKP2 expression decreased tumor growth and lung metastasis of esophageal cancer cells.At the molecular level,knockdown of SKP2 by RNA interference had no effect on cell cycle and proliferation of EC9706 cells,but inhibited cell migration and invasion.Decreased SKP2 expression sensitized cancer cells to anoikis,as demonstrated by activation of caspase-3 and increase of cleaved-PARP level.Treatment with phosphoinositidyl-3 kinase(PI3K) inhibitor (LY294002) also rendered cells sensitive to anoikis.These results provide the first evidence for SKP2 amplification and overexpression in ESCC.Elevated expression of SKP2 protected cancer cells from anoikis,which was mediated at least in part by the PI3K-Akt pathway.The data suggest that SKP2 is an oncogene candidate in the 5p13 amplicon and exerts functions on tumor metastasis in ESCC.