The Functional Role Of Golgi Transmembrane Protein 73 (GP73) In The Invasion And Metastasis Of Hepatocellular Carcinoma And Its Sensitivity To Sorafenib

Hepatocellular carcinoma is the fifth most common cancer worldwide, which is predominant in developing countries. And its incidence is on the rise in the West.The pathogenesis of HCC is not completely understood, however a variety of risk factors have been found to be associated with HCC.Major factors include hepatitis viruses infection, alcoholic liver disease, nonalcoholic steatohepatitis, vinylchloride, foodstuffs contaminated with aflatoxin B1 (AFB1).In general, all these damages can induce hepatocellular carcinogenesis mainly through reactive oxygen species(ROS) production, cellular DNA damage, endoplasmic reticulum stress, and necrosis of damaged hepatocytes.HCC is a highly vascularized tumor, although neoplastic vessels are functionally abnormal and it will lead to HCC tissue hypoxia. HCC largely occurs within an established background of chronic liver disease, especially cirrhosis.(70%-90% of all detected HCC cases). This chronic disease causes fibrogenesis to demolish the normal liver blood system. Damage to the liver blood system leads to the shortage of blood circulation in the liver and consequently leads to hypoxia. Moreover the rapid proliferation of liver tumors will inevitably lead to an imbalance between energy supply and consumption, resulting in local tissue hypoxia. So hypoxia is a common phenomenon in HCC tissue. Reduced oxygen supply induces the upregulation of hypoxia-inducible factor 1 (HIF-1), a major transcription factor that regulates the expression of several genes with critical roles in angiogenesis, invasion, and metastasis. HIF1A induced by Hypoxia stimulates growth and blocks apoptosis of HCC, and is generally associated with chemoresistance and radioresistance.We used the technology of laser capture microdissection (LCM) and microarray previously to investigate the difference of gene expression profiling between metastasis tumor and primary tumor. And we found a 73 kD protein-cross Golgi membrane glycoprotein GP73-which may play an important role in HCC metastasis process and it may be an important target for intervention of HCC metastasis.GP73 is a 73-kD resident Golgi transmembrane glycoprotein. It was firstly isolated and identified in a patient with adult giant-cell hepatitis (GCH), an uncommon hepatitis presumed induced by adenovirus infection.Subsequent studies revealed the expression level of hepatocyte GP73 is obviously upregulated in HBV/HCV infection, alcohol-or autoimmune disease and during the progression of liver disease to cirrhosis. The expression pattern indicated that gp73 was increasing under various hepatocellular acute or chronic injury stress. Further studies demonstrated that the expression of gp73 was also increasing in the serum and resection specimens of patients with HCC. Although gp73 as a serum tumor marker for HCC is still controversial, its overexpression strongly correlates with poor prognosis, especially metastasis, in patient with HCC. Few articles related to the function of GP73. GP73 was shown to interact and co-localize with sCLU in Golgi. While sCLU is an important secretory protein correlates with stress response and carcinogenesis. In the research of Mice with C-Terminally Truncated GP73 model, the most important observations are microvesicular hepatic steatosis and subtle steatohepatitis in the liver of GP73tr/tr animals. Similar changes can be seen in human fatty liver disease. However these histological changes are associated with oxidative stress and mitochondrial stress. Above all, we may hypothesize that GP73 is upregulated by liver injury stress, in turn it may involve in the progression of stress.Given various liver injury stress induce hepatocellular carcinogenesis and oxidative stress can influence the progression and metastasis of hcc, we hypothesize that GP73 may participate in the malignant progressionof liver tumors. In this study, we demonstrate that GP73 is triggered by oxidative stress through the HIF1A dependent way. And the increasing of GP73 can induce HCC cells epithelial-mesenchymal transition (EMT) and HCC angiogenesis in hypoxia condition. Moreover, we validate that GP73 may localize to mitochondria and affect the production of ROS and HIF1A in hypoxia stress. At last our findings show that knock down GP73 may overcome hypoxia-Mediated Sorafenib resistance.PART ONEGP73 promotes the invasion and metastasis of HCC both in normixa and hypoxia conditions.Objective:To investigate the function of GP73 in promoting the invasion and metastasis of HCC.Lentivirus-mediatedRNAi or over-expression was used to obtain a stable HCC cell line with low or high expression of GOLPH2Methods:With the use of RNA interference/over-expression technology mediated by lentiviral, we established a stable HCC cell line with low or high expression of GP73(GP73 stable over-expression cell line PLC/PRF/5-LV-GP73,control cell line PLC/PRF/5-LV-EV; GP73 stable RNA interference cell line MHCC97H-sh-GP73,contol cell line MHCC97H-sh-NT).Then we investigated the function of GP73 in promoting the invasion and metastasis of HCC by using matrigel invasion assay, and We analysised the expression level of E-cadherin and Vimentin and other biomarkers related to EMT in liver tumor cells, in hypoxic and normoxic environment, by using western blotting technology. Furthermore, with the use of in situ xenograft model, we investigated the impact of GP73 on HCC invasion and metastasis in vivo. By using Western blotting,PCR, andimmunofluorescence and Matrigel invasion assay, we wanted to prove that HIF1A played a key role in GP73 promoting HCC invasion and metastasis and to make clear that how GP73 affects the expression of HIF1A.Results:We found that the ability of invasion and metastasis of PLC/PRF/5-LV-GP73 cells was significantly enhanced compared to the control cells (p<0.05) both in hypoxic and normoxic environmentOn the contrary,the ablity of invasion and metastasis of MHCC97H-sh-GP73 cells was significantly weakened compared with the control cells both in hypoxic and normoxic environment. MHCC97H-sh-GP73 cells exhibited the characteristics of epithelial transformation both under hypoxia and normoxia conditions, such as the strengthening of intercellular adhesion, cell growing in cluster and so on. Besides western blotting showed that the E-cadherin expression was significantly up-regulated and the Vimentin expression was significantly reduced in MHCC97H-sh-GP73 cells. However, the PLC/PRF/5-LV-GP73 cells exhibited EMT conversion both under hypoxic and normoxic environment, with the significantly reduction of E-cadherin and up-regulation of Vimentin. In vivo experiments showed that PLC/PRF/5-LV-GP73 cells exhibited high level of lung metastasis rate (83.3%vs 33.3%) and high grade of metastatic tumor (Ⅲ～Ⅳ vsⅠ， ～II) than the control group; MHCC97H-sh-GP73 cells exhibited low level of lung metastasis rate (16.7% vs 83.3%) and low grade of metastatic tumor (Ⅰ vs Ⅱ～Ⅲ) than the control group. Further studies showed that GP73 could affect the expression of HIF1A protein both under normoxic and hypoxic conditions, but the change of HIF1A mRNA expression was not obvious. PLC/PRF/5-LV-GP73 cells interfered with the expression of HIF1A exhibited low ability of invasion and metastasis than control cells (p<0.05) both under hypoxia and normoxia conditions, with significantly up-regulation of E-cadherin and reduction of Vimentin. All these results indicated that GP73 could promote tumor cells EMT transformation through stabilizing HIF1A expression. Then we used the confocal immunofluorescence technology to confirm the location of GP73, and we figured out that GP73 was partially located within the mitochondria. Furthermore, we found that the ROS generation declined in MHCC97H-sh-GP73 cells while increased significantly in PLC/PRF/5-LV-GP73 cells both under hypoxic and normoxic condition, (p<0.05). HIF1A protein half-life testing found that the the half-life of HIF1A in PLC/PRF/5-LV-GP73 was significantly longer than that in the control cells (57.3min vs 34.8min), at the same time, the PHD2/PHD3 expression level was also significantly lower than the control group.This evidence proved that GP73 promoted the stability of HIF1A protein by influence ROS production.Conclusion:GP73 promote invasion and metastasis of HCC by affecting HIF1A protein stability.PART TWO GP73 induce resistance to Sorafenib in HCCObjective:To investigate the effect of GP73 on Sorafenib sensitivity. And further clarify that how GP73 interact with HIF1 A to promote sorafenib resistant in HCC.Methods:We used in vitro cytotoxicity experiments to verify the effects of GP73 on sorafenib sensitivity in hypoxia and normoxiae nvironment. GP73 interference or overexpressing cell lines were injected into nude mice to form tumor. And the nude mice should take Sorafenib one week after injection. Dynamic change was observed in tumor size to validate the effect of GP73 on sorafenib sensitivity in HCC in vivo.Results:In vitro experiments showed that MHCC97H-sh-GP73 cell increased its sensitivity to sorafenib than that of the control cell (P<0.05).Under hypoxic conditions, MHCC97H cells gained resistence to Sorafenib (p<0.05). After interference with GP73, MHCC97H cells recovered its sensitivity to Sorafenib. PLC/PRF/5-LV-GP73 cells also gained resistence to Sorafenib than the control group under normoxic environment (p<0.05). Under hypoxic environment this cells get significantly resistence to Sorafenib compared with the control (p<0.01). PLC/PRF/5-LV-GP73 cells interfered with the expression of HIF1 A restored the sensitivity to Sorafenib (p<0.01) under both hypoxia and normoxia conditions. Under hypoxia conditions, PLC/PRF/5-LV-GP73 cells treated with Sorafenib, HIF1 A protein degradation rate become slower than that of control group.The different is more obvious when the drug concentration is 2.5 μm and 5μm. In vivo experiments demonstrated that the MHCC97H cells interfered with the expression of GP73, Sorafenib inhibition rate was 80.1% higher than the control group52.2%; PLC/PRF/5 cells overexpressing GP73 Sorafenib inhibition rate was 40.02%,lower than the control group 63.3%.Conclusion:GP73 induce resistance to Sorafenib in HCC by affecting HIF1 A protein stability.PART THREEGP73 expression is upregulated by hypoxia stress,which is mediated by HIF1A in HCC cells.Objective:This study aimed to investigate the mechanism of the upregulated expression of GP73 in liver cirrhosis, chronic liver inflammation and other kinds of damage. We also wanted to make clear that increased expression of HIF1A under hypoxic stress played a key role in the upregulation of the GP73 expression.Methods:We subjected human HCC cell lines to hypoxic conditions (cocl2 and hypoxia incubator) for up to 12 hours. Western-blot and real-time PCR technology were used to detect GP73 protein and transcription expression under hypoxic stress. Lentiviral RNA interference/overexpression techniques were used to verify the role of HIF1A in the upregulation of GP73. Finally we used the secreted Gaussia luciferase test system to verify the hypoxia response element activity in GP73 promoter.Results:Under COCL2 (200μM),1%O2condition, GP73 protein and transcription expression increased. However knockdown HIF1A under hypoxic environment, GP73 protein and transcription expression did not increas. Furthermore by using the secreted Gaussia luciferase test system,we proved that HIF1A can bind to the promoter of GP73 and upregulate the expression of GP73.Conclusion:GP73 expression is upregulated during hypoxia,which is mediated by HIF1A in HCC cells.