A Novel Role Of Egf17 In Hepatocellular Carcinoma: Promoting Metastasis By Activating FAK Through EGFR

Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. Though the hepatic resection for HCC has evolved into a safe procedure with low operative mortality, the long-term survival remains unsatisfactory because of a high incidence of recurrence and metastasis after the hepatic resection. Thus, the inhibition of invasion and metastasis is of great importance in the HCC therapies.Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein and once believed to be specifically expressed in endothelial cells (ECs) and essential in the process of vascular development. Recent studies have demonstrated that Egfl7 can act as a chemoattractant for cell migration and regulate collective migration of ECs, indicating that Egfl7 may be important in cell motility. Interestingly, Egfl7 expression is high during embryonic development, down-regulated in almost all mature tissues, and increases again during tumorigenesis. Moreover, our previous results have shown the overexpression of Egfl7 in HCC tissues, implicating its potential role in HCC. However, its role in HCC remains unknown. Therefore, we carried out the present study to determine the expression of Egfl7 in human HCC tissues as well as cell lines and tried to elucidate the function of Egfl7 in the metastasis of HCC by characterizing its role in cell migration through both in vitro and in vivo models.1. Semi-quantitative PCR, real-time quantitative PCR and Western blot were employed to detect Egfl7 mRNA and protein in 31 cases of HCC tissues and corresponding adjacent nontumorous liver tissues (ANLTs). Our results showed that both of Egfl7 mRNA and protein expression levels increased significantly in HCC tissues. Meanwhile, Egfl7 mRNA and protein expression levels were significantly higher in nodular hepatocellular carcinoma than those in solitary large hepatocellular carcinoma. Immunohistochemistry was used to detect Egfl7 in 112 cases of HCC tissues and the results showed that Egfl7 expressed in 90.2% of HCC tissues (101/112) and its expression correlates significantly with multiple nodules, without capsular and vein invasion. ELISA determination also showed that serum Egfl7 level in HCC patients increased significantly compared with chronic liver disease patients, gastroenterological cancer patients, benigh liver tumor patients and normal people. Moreover, HCC patients within the high Egfl7 expression group had either poorer disease free survival or poorer overall survival than those within the low expression group. By univariable and multivariable Cox regression analysis, high Egfl7 expression was found to be independent prognostic factors for overall survival. 2. Double-labeled indirect histoimmunofluorescence was performed to detect the distribution of Egfl7 in HCC tissue. HCC cells in tissues, identified by anti-AFP antibody labeling, were positively stained by a special anti-Egfl7 antibody, suggesting that the Egfl7 protein in HCC tissues were mainly expressed in plasma of HCC carcinoma cells, but not ECs. We further confirm the Egfl7 expression in three HCC cell lines: HepG₂, MHCC97-L and HCCLM3, with a liver cell line CCL13 as a control. The results showed a significantly higher expression of Egfl7 in HCC cells than in normal live cells. Among the three cell lines analyzed, HCCLM3 cells have the highest Egfl7 expression, followed by MHCC97-L and HepG2. Of note is that the different expression levels of Egfl7 correlate with the metastatic potential of these HCC cell lines, suggesting a potential role for Egfl7 in HCC metastasis.3. To define the correlation of Egfl7 expression and HCC metastasis, we employed siRNA approach to inhibit expression of Egfl7 in HCCLM3 cells. We identified three putative candidate sequences and one control sequence and cloned them into the pLKO.1.puro lentiviral expression vector. After selection with puromycin, HCCLM3 cell lines stably expressed siRNA-expressing sequence and control sequences, were obtained and named as HCCLM3 ^Egfl7RNAi+ and HCCLM3 ^Egfl7RNAi- respectively. The expression of Egfl7 protein was decreased dramatically in HCCLM3 ^Egfl7RNAi+ compared with HCCLM3 ^Egfl7RNAi-, which showed a more than 70% inhibitory efficiency. Wound-healing assay and Transwell invasion assay were employed to determine the migration of HCCLM3 cells and the results showed the decreased migration of HCCLM3 ^Egfl7RNAi+ cells compared with HCCLM3 ^Egfl7RNAi- cells. Proliferation rates of HCCLM3 ^Egfl7RNAi+ and HCCLM3 ^Egfl7RNAi- cells were compared by MTT method and the results showed no significant deference between these two cells. At the same time, apoptosis in HCCLM3 ^Egfl7RNAi+ and HCCLM3 ^Egfl7RNAi- cells, as determined using FASC, showed no significant difference (P>0.05). Bcl-x_L and BAX were also detected by Western blot and their expression levels were not significantly changed between HCCLM3 ^Egfl7RNAi+ and HCCLM3 ^Egfl7RNAi- cells. These results suggest that Egfl7 contributes to metastasis without significantly effecting proliferation and apoptosis of HCCLM3 cell.4. In Egfl7 deficient mice, the focal adhesion kinase (FAK) phosphrylation of EC was significantly reduced, implicating the potential property of Egfl7 in regulation of cell motility. Based on this, we presumed that Egfl7 might enhance cell motility by activating FAK. Therefore, we documented the decreased phosphrylated FAK in HCCLM3 ^Egfl7RNAi+ cells without significant change of total FAK level by Western blot. Then we treated HCCLM3 ^Egfl7RNAi+ cells with recombinant Egfl7 protein and found the phosphorylated FAK level significantly elevated after this stimulation. Meantime, F-actin was reorganized in HCCLM3 ^Egfl7RNAi+ cells and the enhanced migration also was evidenced by wound healing assay and Transwell assay, implicating an important role of Egfl7 in controlling cell motility through mediating FAK phosphrylation.5. Based on two EGF-like domains included in the structure of Egfl7, we presumed Egfl7 may mediate FAK phosphrylation through EGFR just like EGF. Then we treated HCCLM3 ^Egfl7RNAi+cells with EGFR inhibitor (15μM) before recombinant Egfl7 protein (50ng/ml) stimulation and found that the FAK phosphrylation, F-actin reorganization level, and cell migration did not change siginificantly. The effects of Egfl7 protein on HCCLM3 ^Egfl7RNAi+cells were all blocked by EGFR inhibitor, which implicated that FAK activation by Egfl7 was EGFR-dependent and the Egfl7/EGFR/FAK pathway may be critical in controlling HCC cell motility.6. We examined the in vivo relevance of the potential role for Egfl7 in HCC tumorigenesis by using a mouse implantation model. The expression of Egfl7 and phosphorylated FAK in HCCLM3 tumor was determined by immunohistochemistry and the results showed that the expression of Egfl7 and phosphorylated FAK was all significantly decreased in HCCLM3 ^Egfl7RNAi+ tumor than that in HCCLM3 ^Egfl7RNAi- tumor. At the 35th day after implantation, we found that the average size of liver tumors in HCCLM3 ^Egfl7RNAi+ group was dramatically smaller than that of HCCLM3 ^Egfl7RNAi- group. Two of eight mice in the HCCLM3 ^Egfl7RNAi+ group showed intrahepatic metastasis (25%), which is significantly lower than that in the HCCLM3 ^Egfl7RNAi- group (seven of eight, 88%). The pulmonary metastasis was observed in the lung tissue sections of only one mouse in HCCLM3 ^Egfl7RNAi+ group (one of eight, 13%), much less than the ratio of pulmonary metastasis in HCCLM3 ^Egfl7RNAi- group (four of eight, 50%). Together, these data support an important role for Egfl7 in HCC metastasis.In conclusion, our study has shown for the first time that Egfl7 expresses in HCC carcinoma cells and its overexpression in HCC tissues and serum significantly correlates to poor prognosis of HCC. Furthermore, we have demonstrated the novel role of Egfl7 in metastasis of HCC by enhancing cell motility through EGFR- dependent FAK phosphorylation, implicating Egfl7 as a novel serum/ molecular marker for diagnosis, monitoring after hepatectomy and prognosis of HCC and a potential therapeutic target for metastasis of HCC.