EGFR Signal-induced PKM2/β-catenin Nuclear Translocation Promoting Early Recurrence After Hepatocellular Carcinoma Radical Resection

Radical resection is the most ideal means for the treatment of early hepatocellular carcinoma (HCC). However, there are still30-50%of patients with poor prognosis. Among the various factors affecting prognosis, early recurrence is one of the most important determinants. The early recurrence of HCC is defined as the occurrence of visible intrahepatic or extrahepatic metastases within one year after radical excision for HCC, and this metastasis is commonly of high malignant degree. Therefore, prevention of early recurrence is the most key strategy to improve the prognosis of patients with HCC. Unfortunately, there are no clinical markers for accurately predicting early recurrence of HCC radical resection, and the underlying molecular mechanisms are largely unknown. Therefore, it is urgent to discover accurate markers for HCC early recurrence and to elucidate the molecule mechanisms.HCC early recurrence after radical resection is mainly attributed to intrahepatic or extrahepatic metastasis and diffusion of liver cancer cells. Our group has extensively studied the mechanisms for liver cancer metastasis and found that activation of signaling pathways downstream of certain growth factors is a key characteristic of hepatoma carcinogenesis. Recent clinical evidence indicates that activation of EGFR signaling can be found in40-70%patients with HCC. However, little is known about its biological relevance with HCC early recurrence after radical resection. Given the key role of EGFR signaling in occurrence and development of HCC, we put forward the hypothesis that EGFR signaling activation could mechanistically account for HCC early recurrence after radical resection.We initially explored the relationship between EGFR signal and HCC early recurrence after radical resection. We performed immunohistochemical assays to determine the p-EGFR levels in fresh liver cancer tissue specimens from62patients with primary HCC, and used Kaplan-Meier analysis to evaluate the relevance of p-EGFR level with HCC early recurrence. Results showed that58%HCC patients had positive expression of p-EGFR, and statistical analysis showed significant association between p-EGFR level and HCC early recurrence after radical resection. Experiments in vitro demonstrated that EGFR was highly expressed in HepG2and SMMC-7721cells among the five tested human HCC cell lines. Similarly, these two cells were also mostly sensitive to EGF (100ng/ml) stimulation for12h manifested by apparent mesenchymal morphology and enhanced migration capability, suggesting that activation of EGFR induced epithelial-mesenchymal transition (EMT) in HCC cells. It has been realized that EMT is a common route for intrahepatic metastasis in HCC. Therefore we postulated that EGFR-induced EMT could be a pivotal mechanism underlying HCC early recurrence after radical resection. We subsequently investigated the following two aspects:1) the molecular mechanisms for EGFR signaling-induced HCC early recurrence after radical resection;2) reliable markers for predicting EGFR signaling upregulation associated with HCC early recurrence.It is known that many growth factors such as TGF-p and HGF can promote EMT in HCC. Interestingly, β-catenin plays a key role in the process. We herein demonstrated that EGF-induced EMT in HCC and migration in HCC cells also required β-catenin activity. Further mechanistic evidence indicated that β-catenin mediated EGF-induced EMT in HCC and migration in HCC cells via upregulating β-catenin-TCF/LEF transcriptional activity and its target gene Snail. We further demonstrated that EGF-induced β-catenin-TCF/LEF transcriptional activity was not dependent on Wnt pathway but on PI3K/AKT and ERK pathways. Further experiments showed that PI3K/AKT signaling regulated β-catenin nuclear translocation and ERK pathway regulated PKM2nuclear translocation. These two pathways co-regulated TCF/LEF transcriptional activity.In order to further verify the mechanisms, we detected the expression of E-cadherin, Snail, P-catenin and PKM2in human HCC specimens from62patients and performed statistical analysis. Correlation analysis showed that p-EGFR expression was positively correlates with Snail and nuclear levels of PKM2and β-catenin but negatively with E-cadherin. We also found that Snail expression was positively correlated with nuclear co-expression of PKM2and β-catenin, and that E-cadherin expression was negatively correlated with nuclear co-expression of PKM2and β-catenin. Additionally, we performed Kaplan-Meier analysis on the association of expression of E-cadherin and Snail and nuclear PKM2/β-catenin co-expression with HCC early recurrence. Results showed that expression of E-cadherin and Snail had no significant relevance to HCC early recurrence, but nuclear PKM2/β-catenin co-expression was significantly related to HCC early recurrence. Patients with positive expression of p-EGFR and PKM2/β-catenin had the highest recurrence rate of68.64%. COX multivariate analysis confirmed that nuclear PKM2/p-catenin co-expression is an independent factor affecting HCC early recurrence after radical resection.A good relevance of PKM2/β-catenin co-expression with HCC early recurrence had been established, but its downstream Snail and E-cadherin expression had no such relevance. Then we investigated the mechanisms underlying these seemingly paradox findings. Our data demonstrated that EGFR signal not only induced HCC cell invasion, but also on the proliferation of HCC cells. Interestingly, the influence of EGFR signaling on HCC proliferation correlated with EGFR activation time. Short time (24h) activation of EGFR could inhibit proliferation in SMMC-7721and HepG2cells associated with cell cycle arrest. Long time (72h) activation of EGFR could significantly promote proliferation in SMMC-7721and HepG2cells associated with upregulation of C-myc and CyclinDl. EGFR signaling is continuously activated in vivo and clinical specimen analysis also confirmed that Ki-67expression was not only positively related to the expression of p-EGFR, but also positively correlated with nuclear PKM2/β-catenin co-expression. Reoccurrence analysis found that there was very high reoccurrence rate in E-cadherin (-)/Ki-67(+) patients with HCC after radical resection. Continuous activation of EGFR signaling promoted cell invasion and proliferation in HCC cells leading to HCC early recurrence after radical resection in patients.The current study studied the key role of EGFR signaling in HCC early recurrence after radical resection in vivo and in vitro and elucidated the underlying molecular mechanisms. We provided reliable markers for predicting HCC early recurrence and prevention strategies by targeting EGFR signaling in patients with HCC after radical resection.