| Research BackgroundLiver cancer is one of the cancers with the highest mortality rate and ranks 6th incidence and mortality in global cancer.Hepatocellular carcinoma is the most common type of liver cancer in clinical practice,with the characteristics of high recurrence rate and high mortality rate.Current treatments for hepatocellular carcinoma include surgical resection,radiofrequency ablation,targeted therapy and more.Among them,the drugs such as Sorafenib and Lenvatinib,which have been approved by FDA,can respectively inhibit PDGFR/VEGFR and FGFR/RET family of RTKs of hepatocellular carcinoma cells and corresponding vascular endothelial cells.In this way,it can effectively inhibit angiogenesis and kill tumor cells,thereby delaying the progress of liver cancer.At present,the two are used as first-line clinical treatment drugs for liver cancer.However,higher rate of liver cancer recurrence and metastasis,especially extrahepatic metastasis,are important factors affecting the treatment effect and survival time of patients with liver cancer.Studies have confirmed that sorafenib prolonged the survival time of patients by 2.8 or 2.3 months in two clinical trials of whites and Asians.At the end point of clinical trials,most patients died because of liver cancer metastasis,which proved evidences that the treatment effect was not satisfactory.Therefore,searching for more effective therapeutic targets is of great significance to clinical treatment for preventing liver cancer metastasis,increasing the therapeutic effect,and prolonging the survival time of patients.Circulating Tumor Cells(CTCs)refer to tumor cells shed from cancer tissues into the blood.Existing forms of CTCs include single circulating tumor cell,circulating tumor microemboli,and circulating tumor cell clusters.Circulating tumor cells are considered to be sources which cause cancer recurrence,also,they play important roles in cancer metastasis.It has also been clinically confirmed that the existence of circulating tumor cells,especially circulating tumor emboli,is closely related to shortening progression-free survival and overall survival,as well as the increasement in probability of recurrence and distant metastasis.Anoikis resistance is one characteristic of circulating tumor cells,which help them to survive in the circulatory system after detaching from the extracellular matrix.A variety of signaling pathways,such as over-activated of Ras/MAPK pathway,are involved in anoikis resistance.The mechanism of anoikis resistance is still unclear.Therefore,finding the mechanism of circulating tumor cells to survival in circulating system and anoikis resistance are of great significance to finding methods for preventing liver cancer metastasis.Receptor Tyrosine Kinases(RTKs)are currently considered to play key roles in tumorigenesis and cancer metastasis.The human genome can encode 90 proteins with activity of tyrosine kinases,of which 58 proteins are located on the cell membrane.These 58 proteins can be further divided into 20 subfamilies.They belong to the same membrane protein family,which can catalyze the transferring of ATP y phosphate to the hydroxyl group of tyrosinase on the target protein.RTKs play important roles in cell cycle,migration,metabolism,proliferation and differentiation.Some RTKs,such as VEGFR/PDGFR family,can be used for clinical targeted therapy of primary liver cancer.The effect of targeting VEGFR/PDGFR family includes direct killing of liver cancer cells,interrupting tumor-induced angiogenesis and blocking tumor blood supplement,which exert anti-cancer effects.Also,RTKs such as EGFR and MET,can participate in tumor metastasis through mutation,amplification,formation of coreceptors with integrins and other molecules to induce resistance to targeted drugs.RTKs may be essential for tumor cell survival and distant metastasis in the circulatory system.Research confirmed that hyper-phosphorylation of EGFR was observed in suspension tumor cells.EGFR and ErbB2 have also been used as markers to identify circulating tumor cells in some clinical studies.Clinical reports showed that MET amplification was observed in circulating tumor cell in patients with non-small cell lung cancer.These all prove that RTKs are involved in survival and metastasis of circulating tumor cells.However,due to the scarcity of circulating tumor cells,the mechanism of RTKs in the survival and metastasis of circulating tumor cells is not completely clear.The expression and phosphorylation levels of RTKs are very different in various tumors.Therefore,choosing the most expressive and functional RTKs is the key for treatment of liver cancer.In summary,this experiment is mainly divided into three parts.In the first part,we performed protein quantitative analysis on clinical liver cancer tissues and liver cancer cells to screen out EGFR and MET,two molecules with high expression and strong functions in RTKs family.Also,we used the mouse model with liver cancer to prove evidence that EGFR and MET are up-regulated in circulating tumor emboli and downregulated in lung metastases of liver cancer.In the second part,through establishment of vitro model of anoikis,we proved states of EGFR and MET in suspended hepatocellular carcinoma cells,relationship between EGFR/MET and the stability of HCC cells in anoikis and the synergy between EGFR and MET in regulation of downstream pathways.We also proved EGFR/MET protect HCC cells avoid killing effect of leukocytes.In the third part,we verified influence of EGFR and MET on phosphorylation of other RTKs and possible mechanisms by inhibitor,siRNA,and more.This study reveals mechanism of EGFR and MET in promoting liver cancer metastasis and provided a new perspective for prevention,detection and treatment of lung metastasis in patients with liver cancer.Part 1 EGFR and MET are elevated in circulating tumor emboli.AimEGFR and MET play important roles in metastasis of liver cancer,but expression of EGFR and MET during metastasis and in circulating tumor microemboli are still unclear.This study aims to find out expression and phosphorylation level of EGFR and MET in primary mass and lung metastasis.Also,expression of EGFR and MET in circulating tumor microemboli need to be investigated.Methods1.Detection the expression levels of multiply RTKs family in liver cancer.Enzyme-linked immunosorbent assay and Luminex were used to quantitatively detect the expression levels of multiply RTKs in liver cancer tissues and cells to screen out EGFR and MET,the two RTKs molecules with the highest expression we detected.Western blot was used to compare the expression and phosphorylation levels of EGFR and MET in different HCC cell lines.At the same time,enzyme-linked immunosorbent assay and Luminex were used to quantitatively detect RTKs ligands EGF and HGF in HCC tissues and cells.2.Researches on the expression and phosphorylation of EGFR and MET in primary mass and lung metastasis.We established C57 mouse model with liver cancer metastasis.Mice models with only hepatic primary tumor mass and lung metastasis were selected for further experiments.Hepatic primary carcinoma and lung metastasis tissues were collected and separated into single cells for RNA sequence.According to result of RNA sequence,cells were dividing into different clusters through UMAP method.Tumor cell clusters were picked out by specific marker.Also,mouse RTKs phosphorylation detection kit assay was used for comparing phosphorylation level of EGFR and MET in hepatic primary tumor mass and lung metastasis of mice.3.Researches on the expression of EGFR and MET in circulating tumor emboli.We collected hepatic tumor tissues and lung metastases of mouse models,and then looking for circulating tumor emboli in tumor tissues by immunohistochemistry and immunofluorescence.ImageJ was used to quantitatively analyze the staining intensity and compare the expression differences of EGFR and MET between of tumor tissue and circulating tumor emboli in the same immunohistochemistry slice.Results1.Compared with other RTKs molecules,EGFR and MET are highly expressed in liver cancer tissues and cells.Through Luminex and enzyme-linked immunosorbent assay methods,we proved higher expression of EGFR and MET in liver cancer tissues and cells compared to most of other RTKs we detected.Especially in cells,the expression levels of EGFR and MET are much higher than those of other RTKs in cells.Moreover,expression levels of EGFR in different cell lines are almost the same,and phosphorylation levels of EGFR and MET generally increase in cell lines with MET amplification.We also found that EGF expression was very low in hepatic tumor tissues and HCC cells,while HGF was highly expressed in hepatic tumor tissues but low in HCC cells.2.Expression of EGFR and MET is significantly down-regulated after lung metastasisThrough analysis of single-cell sequencing and presented by heat map,we found that EGFR and MET are concentratedly expressed in HCC cells of hepatic tumor tissues.After lung metastasis,the overall expression level of RTKs family including EGFR and MET were rapidly decreased in tumor tissues.However,mice phosphorylation kit assay confirmed that the phosphorylation levels of EGFR and MET kept stable before and after tumor metastasis.3.EGFR and MET have different distributions in hepatic primary tumor mass and lung metastasis,and their expression in circulating tumor microemboli increased significantly.After immunohistochemistry and fluorescence analysis on the hepatic primary tumor mass and lung metastases,we found that expression of EGFR and MET showed great differences in different tissues.Among them,in hepatic primary tumor mass,the expression level of EGFR in HCC cells is lower than that in hepatic peritumor tissue,but MET is higher than that in hepatic peritumor tissue;in lung metastases,the expression level of EGFR in HCC cells is higher than that of most lung peritumor tissues,while the expression level of MET is similar to that in lung peritumor tissues.Also,compared with tumor tissues in the same organ,the expression of EGFR and MET in circulating tumor microemboli were significantly up-regulated,and especially EGFR rises more obviously in hepatic primary tumor mass.Part 2 Anoikis resistance induced by EGFR and MET promotes the survival of circulating tumor microemboli and resistance to the killing effect of leukocytes.AimIn previous part,we have proved expression advantages of EGFR and MET in RTKs family in hepatic tumor tissues and up-regulation of EGFR and MET in circulating tumor microemboli.However,effect of up-regulation of EGFR and MET expression on circulating tumor microemboli is still unclear.An important characteristic of circulating tumor microemboli is anoikis resistance.An anoikis model in vitro was established to simulate circulating tumor cells.The purpose of this study was to study relationship between EGFR/MET and anoikis resistance and following research would further investigate roles and mechanisms of EGFR and MET in the stabilizing HCC cells in anoikis and resistance to killing effect of leukocytes.Methods1.Research on the expression and phosphorylation levels of EGFR and MET in suspended HCC cells.Western blot and RTKs phosphorylation kit assay were performed to detect expression and phosphorylation of EGFR and MET in different HCC cell lines at suspension time of 0,24,48,72 hours.Low concentration of cells was used for detection of expression and phosphorylation of EGFR and MET to reduce cell cluster formation.2.Detection of relationship between EGFR/MET and secretion of cytokines and resistance of leukocytes killing effect.Enzyme-linked immunosorbent assay was used to determine the expression of intracellular and extracellular interleukin 8 in suspended HCC cells after EGFR and MET inhibition.Also,HCC cell lines and leukocytes were co-cultured,the survival rates of HCC cells and leukocytes was observed to analyze the relationship between EGFR/MET and resistance of leukocyte killing effect.3.Determining the regulatory effects of EGFR and MET on Ras/MAPK pathway.We used RTKs ligand and inhibitor to activate or inhibit phosphorylation of RTKs to evaluate the regulatory effects of RTKs on Ras/MAPK pathway,and the relationship between basic phosphorylation level of EGFR/MET in HCC cells and effect of activation/inhibition on Ras/MAPK pathway.We also use RTKs ligand to activate Ras/MAPK pathway in HCC cells with EGFR/MET knockdown to find out if activation of other RTKs could reverse the effect of EGFR/MET down-regulation.Moreover,we observed EGFR/MET activation or inhibition in HCC cells with MET/EGFR knockdown to prove that synergistic regulation was existed in EGFR and MET on regulating Ras/MAPK pathway.4.Determine the effect of Ras/MAPK pathway on stabilization suspended HCC cells in cell proliferation,cell cycle,apoptosis,and resistance of leukocytes killing effect.Firstly,we determine the activation of Ras/MAPK pathway in suspended HCC cells,then,proliferation,cell cycle,DNA synthesis,apoptosis and expression of cyclin were observed in HCC cells after the addition of Ras/MAPK inhibitors or cultured in suspension.Also,Ras/MAPK inhibitors was used for observation that the role of Ras/MAPK pathway plays in resistance of suspended HCC cells to leukocyte killing effect.Results1.Expression and phosphorylation levels of EGFR and MET in suspended HCC cells were up-regulated.We first established anoikis HCC cell model in vitro to simulate circulating tumor cells.We found that expression and phosphorylation level of EGFR and MET were upregulated in suspended HCC cells and reached at peak levels within about 24-48 hours,and then fell back to lower level.2.EGFR and MET were involved in the secretion of interleukin 8 and resistance of suspended HCC cells in leukocyte killing effect.We use enzyme-linked immunosorbent assay to find out the correlation between interleukin 8 and EGFR/MET.In HCC cells with MET amplification,EGFR and MET phosphorylation levels are extremely high.Inhibiting phosphorylation of EGFR and MET in HCC cell line with MET amplification leaded to significant down-regulation in interleukin 8 synthesis and secretion.However,synthesis and secretion of interleukin 8 were less affected by EGFR and MET inhibitor in HCC cells with low phosphorylation level.Also,HCC cells with EGFR and MET inhibited showed a decline in resisting leukocyte killing effect,while leukocytes showed a higher survival rate.3.EGFR and MET were powerful in regulation of Ras/MAPK pathway.3.1 The ability of EGFR and MET to regulate the Ras/MAPK pathway is related to the phosphorylation level of HCC cells.We found that the ability of EGFR and MET in regulation of Ras/MAPK pathway is various in different HCC cells.In other words,the ability of EGFR and MET in regulation of Ras/MAPK pathway is related to basic phosphorylation of EGFR/MET in HCC cells.In detail,Ras/MAPK pathway in HCC cells with higher phosphorylation levels of EGFR and MET is not sensitive to EGF and HGF,but sensitive to EGFR/MET inhibitors.HCC cells with lower phosphorylation levels of EGFR and MET are sensitive to EGF and HGF,but not sensitive to EGFR/MET inhibitors.3.2 EGFR and MET are more effective in regulation of Ras/MAPK pathway other RTKs.We stimulated RTKs which have been clinically used for liver cancer target therapy by related ligands,and compared their ability in activation of Ras/MAPK pathway.We found that EGF has the strongest ability in activation of Ras/MAPK pathway,and followed by HGF.FGF2,Insulin,VEGF and PDGF-BB have hardly effects on activating Ras/MAPK pathway in HCC cells.Also,EGF and HGF could reverse activation of Ras/MAPK pathway reduced by MET/EGFR knockdown,but FGF2,VEGF and PDGF-BB cannot completely restore the activation of Ras/MAPK pathway in HCC cells with EGFR/MET knockdown,even VEGF and PDGF-BB could hardly activate Ras/MAPK pathway in HCC cells.3.3 EGFR and MET have synergistic effect on the regulation of Ras/MAPK pathway.We use EGFR inhibitors to inhibit HCC cells with MET activation or knockdown,then observe changes in Ras/MAPK pathway activation.We found if HCC cells are sensitive to EGFR inhibitors,EGFR inhibitors also have a significant effect on MET and effectively interrupt phosphorylation of MET induced by exogenous or endogenous HGF.While HCC cells are not sensitive to EGFR inhibitors,EGFR inhibitors have no significant effect on MET.Also,the sensitivity of HCC cells to EGFR inhibitors decreased while MET was down-regulated.4.Ras/MAPK pathway reduces the damage of anoikis to HCC cells and help resist the killing effect of leukocytes.Like EGFR and MET,we found that Ras/MAPK pathway in suspended HCC cells was over-activated.Moreover,the ratio of S phase of suspended HCC cells was increased.Relatively,Cyclin E was also up-regulated in suspended HCC cells,while Cyclin A and Cyclin B was down-regulated.After inhibiting Ras/MAPK pathway,expression of all Cylin proteins/CDK4 proteins and HCC cells in S phase was decreased significantly.DNA synthesis were significantly inhibited.Also,we found that the apoptotic rate of suspended HCC cells increased and the survival rate decreased.Deactivation of Ras/MAPK pathway caused less resistance to apoptosis of HCC cells,and Ras/MAPK pathway inhibitor leaded to more apoptotic rate and less survival rate in supended HCC cells.Moreover,while inhibiting Ras/MAPK pathway,the resistance of suspended HCC cells to the killing effect of leukocytes was also significantly reduced.Part 3 EGFR and MET are involved in the activation of multiple RTKs.AimPrevious studies have confirmed the relationship between EGFR/MET and survival of circulating tumor microemboli.In clinical,high metastasis rate of liver cancer become an important factor leading to shortened progress-free time and median survival time of patients.Therefore,this part is aimed to clarify roles of EGFR and MET in drug resistance and related mechanisms.Methods1.Research on phosphorylation level of RTKs after inhibiting EGFR and MET.We used EGFR and MET inhibitors and siRNA to down-regulate phosphorylation level of EGFR and MET,then observed phosphorylation level of VEGFR,FGFR,RET,PDGFR and other RTKs receptors in different HCC cell lines.2.Determine inhibitory effects of Sorafenib and Lenvatinib on RTKs in HCC cells.Sorafenib and Lenvatinib,two RTKs inhibitors which were for treatment of liver cancer,were used to observe the inhibition of different RTKs phosphorylation in HCC cells.Western blot was used to analyze relationship between EGFR/MET phosphorylation and targeted RTKs phosphorylation in HCC cells with RTKs inhibitor.3.Research on related mechanisms of EGFR and MET in promoting phosphorylation of RTKs.We used immunoprecipitation to find out if there existed hetero-dimer or homodimer between RTKs.Also,Co-incubation of cell lysis and ATP/GTP solution was performed and mixture was used for RTKs phosphorylation detection by Western blot and Human phosphorylation kit assay.Results1.EGFR and MET can effectively promote the phosphorylation level of multiply RTKs.HCC cells with EGFR/MET inhibitors or siRNA were cultured in suspension and Western blot was used for detecting phosphorylation level of RTKs.We found that EGFR and MET inhibitors can directly inhibit the phosphorylation level of most RTKs in HCC cell lines with MET amplification.Knockout of MET has the same effect on HCC cells.Moreover,the stimulation of the ligand barely increases the level of phosphorylation related RTKs.But inhibition effects in HCC cells with low phosphorylation levels of EGFR and MET is relatively poor.2.EGFR and MET are involved in the drug resistance in HCC cells.We found that sorafenib was barely effective on EGFR and MET,also,PDGFR,one of RTKs sorafenib aimed to,was no sensitive to sorafenib in all HCC cells.Meanwhile EGFR and MET were effectively inhibited by Lenvatinib,and phosphorylation level of most of RTKs Lenvatinib targeted on were down-regulated in HCC cells with MET amplification.It suggested drugs without inhibition of EGFR and MET have a very limited effect on liver cancer,especially HCC cell with hyperphosphorylation of EGFR and MET.3.EGFR and MET cannot participate in the activation of RTKs by heterodimers.As previous part mentioned,EGFR and MET are two highly expressed molecules in RTKs family and EGFR and MET in HCC cell lines with MET amplification were hyper-phosphorylation.Therefore,we firstly considered the possibility of dimer formation of RTKs.We found that EGFR and MET were easily to form homo-dimer.However,even in HCC cell lines with MET amplification,dimer of EGFR-MET was barely existed and it is difficult for EGFR or MET to form heterodimers with other RTKs.4.High-energy phosphoric acid compounds are keys in activation of RTKs.As previous part mentioned,EGF was rarely in both HCC tissues and cells.So,there may exists ligand-independent pathway for RTKs stimulation.By Human phosphorylation kit assay,we found that sufficient ATP could activate most of RTKs in the cell lysate,even HCC cells with MET amplification.but the phosphorylation level of MET is hardly affected by ATP,which means ATP cannot directly phosphorylate MET.Western blot also confirmed that ATP could directly phosphorylate EGFR,but cannot phosphorylate MET,while GTP could directly phosphorylate both EGFR and MET.Phosphorylation of EGFR activated by ATP was not affected by MET but factor that cause ATP degradation,such as creatine kinase,leads to insufficient activation of EGFR.Conclusions1.EGFR and MET are high expressed molecules in RTKs family,and play important roles in metastasis of liver cancer.2.Expression levels of EGFR and MET was down-regulated in lung metastasis,but upregulated in circulating tumor microemboli,proving the therapy value in targeting circulating tumor microemboli.3.Anoikis resistance is an important reason which leads to increase expression and phosphorylation level of EGFR and MET in circulating tumor microemboli.4.Compared with other RTKs,EGFR and MET are more effective in regulation of Ras/MAPK pathway,which means EGFR and MET play important roles in stabilizing circulating tumor emboli and resisting leukocyte killing effect.5.EGFR and MET are involved in drug resistance of liver cancer therapy,and the mechanism may be related to high-energy phosphate compounds. |
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