| Background and ObjectivePancreatic ductal adenocarcinoma (PDAC) is one of the most aggressivemalignancies, with great invasive and metastatic ability. Most patients with PDAC haveextensive local invasion or remote metastasis when symptoms occur, making it impossiblefor curative resection, additionally, PDAC is largely resistant to chemoradiotherapy,leading to the5-year survival rate still less than5%. Thus, it is urgent to better understandthe mechanism of PDAC invasion and metastasis to identify new theraputic target forPDAC. Our previous study has found that eukaryotic elongation factor1alpha2(EEF1A2)is abberantly upregulated in PDAC, moreover overexpression of EEF1A2cansignificantly promote proliferation and migration of pancreatic cancer cells in vitro, thus,we speculate that EEF1A2may participate in PDAC invasion and metastasis. In this study,we first determine the expression of EEF1A2in PDAC specimen and analyze itscorrelation with clinicopathological characteristics and prognosis of PDAC patients, andfurther verify its biological role in PDAC invasion and metastasis by using in vitro and invivo experiments. Furthermore, we perform gene chip technique to investigate the effectof over expression of EEF1A2on metastasis-related genes and signal pathways, clarifying the molecular mechanism by which EEF1A2promotes PDAC invasion and metastasis.Methods1. Tissue microarray and immunohistochemistry were used to detect the EEF1A2protein expression in PDAC specimen, then analyze its correlation withclinicopathological characteristics and prognosis of PDAC patients. Real time-PCR,Western Blot and Matrigel invasion assay were then used for examining the EEF1A2mRNA, protein and invasive ability in SW1990, BxPC-3, Panc-1and Capan-1cells,respectively.2. SW1990cells were tranduced with lentivirus containing full length EEF1A2geneand empty lentivirus and then selected with puromycin to yeild the stably tranducedSW1990-EEF1A2and SW1990-GFP cells (control cells). Panc-1cells were transfectedwith siRNAs targetting EEF1A2specifically. Real time-PCR and Western Blot were usedto determine the EEF1A2expression in cells above, respectively. The alteration ofmigration and invasion ability were evaluated by wound healing assay and Matrigeltranswell assay, respectively.3.7-week-old BLAB/c nude mice were randomly assigned to two groups, and wereinjected with SW1990-EEF1A2and SW1990-GFP cells (1×106/mouse) into tail vein andperitoneal cavity, respectively, then after indicated time, mice were euthanized, the lungand peritoneal metastasis were examined.4. Gene chip microarray was used for detecting the differently expressedmetastasis-related genes between SW1990-EEF1A2and SW1990-GFP cells. Then thosegenes were validated by Real time-PCR. MMP-9protein and activity were determined bywestern Blot and gelatin zymography in pancreatic cancer cells treated with overexpression and downregulation of EEF1A2, respectively. Wound healing assay andMatrigel transwell assay were used to evaluate the effect of MMP-9specific inhibitor(MMP-9Inhibitor I) on SW1990-EEF1A2and SW1990-GFP cell migration and invasion.EEF1A2and MMP-9were detected by immunohistochemistry in PDAC specimen, andthe association between them was analyzed.5. Western Blot was used to detect the effetc of overexpression of EEF1A2onPI3K/AKT signal pathway in SW1990cells, then the migration、invasion、MMP-9proteinand activity were examined in SW1990-EEF1A2and SW1990-GFP cells exposed to AKTpathway specific inhibitor LY294002.Results1. EEF1A2expression was absent in normal pancreas, in contrast, EEF1A2showedpositive immunoreactivity in76of97(77.8%) PDA cases. There was significantcorelation between positive EEF1A2expression and the presence of lymph nodemetastasis (P=0.039), perineural invasion (P=0.043) and TNM stage (P=0.040).Kaplan-Meier survival analysis showed that the median survival time of EEF1A2-positivePDA patients (11.7months) was significantly shorter than that of EEF1A2-negative PDApatients (17.5months; P<0.001). Multivariate survival analysis showed positive EEF1A2expression is a relatively independent prognostic predictor for pancreatic cancer patients.By evaluating the invasive ability of a panel of pancreatic cancer cell lines with differentmetastatic potentials, EEF1A2expression in cells was positively associated with cellinvasive ability.2. The stably transducd SW1990-EEF1A2and SW1990-GFP cells were establishedsuccessfully. Real-time PCR and Western Blot results showed the expression of EEF1A2 was significantly upregulated in SW1990-EEF1A2cells compared to SW1990-GFP cells(P<0.05). Conversely, the expression of EEF1A2was significantly decreased in Panc-1cells transfected with specific siRNA targeting EEF1A2at48h compared to control cellswhich was transfected with scramble siRNA (P<0.05). Matrigel transwell assay revealedthat the number of invaded cells of SW1990-EEF1A2(60.13±4.31) was more than thatof control cells (33.43±3.57, P<0.05). Wound healing assay showed overexpression ofEEF1A2enhanced SW1990cell migration significantly (P<0.05). On the contrast,downregulation of EEF1A2expression reduced the cell migration and invasion in Panc-1cells compared with control cells (P<0.05).3. Peritoneal metastasis model results showed that the number of metastatic tumor inperitoneal cavity in SW1990-EEF1A2group (16.5±6.11) was significantly more than thatin control group (6.25±2.2, P<0.05). Consistently, the results of lung metastasis modeshowed that the metastatic rate in SW1990-EEF1A2group (100%,5/5) was significantlyhigher than control group (20%,1/5; P<0.05).4. Gene chip results showed that there were10differently expressedmetastasis-related genes (>1.5fold) between SW1990-EEF1A2and SW1990-GFP, amongwhich MMP-9was upregulated with the highest fold change (2.54fold). Real time-PCRand Western Blot results also confirmed that MMP-9was significantly increased inSW1990-EEF1A2compared with control cells (P<0.05). Gelatin zymography resultsindicated that MMP-9activity was markedly higher in SW1990-EEF1A2thanSW1990-GFP cells (P<0.05). Conversely, downregulation of EEF1A2reduced theMMP-9protein expression and activity in Panc-1cells (P <0.05). MMP-9inhibitor I couldsigificantly surpress both migration and invasion of SW1990-EEF1A2and SW1990-GFP cells. Immunohistochemistry results showed that MMP-9and EEF1A2expression waspositive in25/62(40.3%) and41/62(66.1%) of PDAC cases, and the EEF1A2expressionwas positively associated with MMP-9expression (P <0.05).5. Overexpression of EEF1A2activated PI3K/AKT signaling pathway in SW1990cells (P<0.05). AKT signaling pathway specific inhibitor (LY294002) inhibited AKTactivity, MMP-9protein expression and activity, as well as in vitro migration and invasionin a dose-dependent manner (P<0.05).Conclusion1. EEF1A2is pervasively expressed in human pancreatic cancer, but absent in normalpancreas and chronic pancreatitis tissue, positive EEF1A2expression is positivelycorelated with the presence of lymph node metastasis, perineural invasion and TNM stage.Median survival time of EEF1A2-positive PDA patients was significantly shorter than thatof EEF1A2-negative PDA patients. Positive EEF1A2expression is a relativelyindependent prognostic predictor for pancreatic cancer patients.2. EEF1A2expression level is positively associated with cell invasive capacity in apanel of pancreatic cancer cells. Overexpression of EEF1A2could significantly promotecell migration, invasion and metastasis, on the contrast, downregulation of EEF1A2couldinhibit cell migration and invasion.3. EEF1A2overexpression could upregulate MMP-9expression and activity whichmediated the promoting effect of EEF1A2on cell invasion and migration. EEF1A2protein expression is positively associated with MMP-9expression in pancreatic cancertissue. Overexpression of EEF1A2could activate PI3K/AKT signaling pathway inpancreatic cancer cells, subsquently promoting MMP-9expression and activity. |
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