The Mechanism Of FNDC4 Regulating Tiam1/Rac1 To Promote The Invasion And Metastasis Of Hepatocellular Carcinoma Through PI3K/AKt Pathway

Background:Primary liver cancer is one of the most common malignant tumors in China,its incidence ranks fourth among all malignant tumors,and its mortality ranks second.Primary liver cancer is divided into three pathological types:hepatocellular carcinoma（HCC）,intrahepatic cholangiocarcinoma and mixed hepatocellular carcinoma-cholangiocarcinoma,of which 75%to 85%are HCC.For medical workers,there is an urgent clinical need to study the pathological mechanism and prevention strategies of hepatocellular carcinoma.At present,there are various treatment methods for liver cancer,but the therapeutic effect has not been significantly improved.Up to 70%of patients with hepatocellular carcinoma have recurrence and metastasis 5 years after surgery.Although targeted immunotherapy has brought hope to some patients with liver cancer,the objective response rate（ORR）is still low,and the total ORR is only 12.6%～31.8%,and some patients showed treatment resistance.Like an intelligent organism,liver cancer cells can constantly adapt and transform themselves through changes in the extracellular environment,while maintaining a high degree of invasion and metastasis.So,how does a hepatocellular carcinoma cell sense the extracellular environment it faces（including medical treatment measures）and continuously maintain its high invasive and metastatic properties?Just like the"seed soil theory",hepatocellular carcinoma is composed of liver cancer cells,related stromal cells and extracellular matrix.The high invasion and metastasis characteristics of liver cancer are closely related to the tumor microenvironment of hepatocellular carcinoma,and their invasion and metastasis almost always receive abnormal signals from the extracellular matrix.It is triggered by the activation of multiple intracellular invasion and metastasis pathways.It can be said that the invasion and metastasis of HCC cells is a process of cell response to its surrounding environment,and is a process of action of extracellular molecules through intracellular effector molecules,however,the specific molecular mechanism has not been fully explained.Fibronectin（FN）is one of the important components of the extracellular matrix and is an important extracellular macromolecular glycoprotein that binds to certain integrin receptors on the cell surface through some short peptide sequences（such as RGD sequences）,and then transmit extracellular information to intracellular effector molecules to play a biological role.Fibronectin type III domain-containing protein 4（FNDC4）has this molecular function.It belongs to the fibronectin type III domain-containing protein family,and the extracellular domain can be cleaved and secreted into the extracellular matrix,and transmit the extracellular signal to the intracellular effector molecules to play a biological role.Bosma M et al.reported in Nature Communications that FNDC4,as an anti-inflammatory factor,acts on macrophages to reduce the expression of pro-inflammatory chemokines and can reduce the inflammatory response of mouse colon.Wuensch T et al found that FNDC4 expression was significantly elevated in the analysis of mucosal samples from colorectal cancer（CRC）patients.Studies have reported that the RGD sequence of FNDC4 interacts with integrinβ1 to activate focal adhesion kinase to promote cell migration,and interact with the membrane LRP6 receptor of the Wnt/β-catenin signaling pathway to affect the differentiation of C2C12cells.FNDC4 can also act as an extracellular factor.,can act on human visceral adipocytes to reduce adipogenesis and fat browning.FNDC4 is highly expressed in liver tissue,and whether it acts as an extracellular molecule to induce or promote the invasion and metastasis of hepatocellular carcinoma and its possible mechanism have not been reported yet,so we intend to conduct related experimental studies.As an extracellular inflammatory factor,FNDC4 needs intracellular effector molecules to play its role in the invasion and metastasis of hepatocellular carcinoma.At present,there have been considerable studies on the intracellular molecular mechanisms involved in tumor cell invasion and metastasis,and most of them have finally focused on the remodeling of the cytoskeleton by the Rho GTPases family.Among them,Rac1 activation mediates the formation of lamellipodia,which is an important mechanism of tumor cell invasion and metastasis.T-lymphoma invasion and metastasis 1（Tiam1）was identified as a Rac1-specific Guanine nucleotide exchange factor（GEF）in 1995,so Tiam1 exerts a wide range of biological functions through the composition-specific Tiam1/Rac1 pathway,including overexpression that causes extensive membrane wrinkling and pseudopodia formation,promoting cellular migration,etc.Studies have reported that Tiam1 is highly expressed in various solid tumors,including hepatocellular carcinoma,and is closely related to metastasis.However,Tiam1 can not only promote tumor migration,but also promote adhesion and antagonize tumor migration.This seemingly contradictory effect mainly depends on tumor cell type,intracellular localization of Tiam1 and induction of extracellular molecules,among which Tiam1 membrane localization is a key link in determining the activity of Tiam1/Rac1,regulated by signaling pathways including PI3K/Akt.As an extracellular inflammatory molecule,whether FNDC4 is involved in the regulation of the invasion and migration of hepatocellular carcinoma,and whether its molecular mechanism is related to the action of the intracellular molecule Tiam1/Rac1,has not been reported yet.In order to further study the role and molecular mechanism of FNDC4 in the invasion and metastasis of hepatocellular carcinoma,we propose the following research assumptions:We speculate that FNDC4 may be involved in the regulation of hepatocellular carcinoma invasion and metastasis as an extracellular inflammatory molecule.The mechanism may be that FNDC4 acts on the cell surface receptors,activate PI3K/Akt signaling pathway,induce Tiam1 to enrich in HCC cells membrane,Tiam1 enrichment at the cell membrane is more conducive to the activation of Rac1,thereby promoting Rac1-mediated invadopodia formation,and ultimately enhancing hepatocellular carcinoma invasion and metastasis.Objectives:1.The expression of FNDC4 in hepatocellular carcinoma was explored through database analysis,and the relationship between FNDC4 and clinical characteristics of hepatocellular carcinoma was verified by detecting hepatocellular carcinoma tissue samples,which will confirm that the follow-up molecular mechanism research has important research value.2.To clarify the role of FNDC4 in the invasion and metastasis of hepatocellular carcinoma,explore the mechanism of FNDC4 in promoting the invasion and metastasis of hepatocellular carcinoma,and verify that the extracellular domain of FNDC4 affects the activity of the PI3K/Akt pathway,which will clarify the extracellular mechanism of FNDC4in promoting the invasion and metastasis of hepatocellular carcinoma.3.Elucidated the intracellular effector mechanism of FNDC4 promoting the invasion and metastasis of hepatocellular carcinoma through PI3K/Akt pathway;confirmed that PI3K/Akt pathway affected Tiam1 membrane enrichment,thereby promoting the intracellular mechanism of Rac1-mediated invadopodia formation.4.Through the analysis of clinical cases and animal experiments,it was verified that FNDC4 can up-regulate the activity of Tiam1/Rac1 and enhance the ability of HCC cells to invade and metastasize.Methods:1.Key words such as Fibronectin and FN were searched through Human Protein Atlas database,multi-gene comparison analysis was performed using GEPIA database,and survival analysis was performed in combination with TCGA database to explore the relationship between the expression of FNDC4 protein and m RNA and hepatocellular carcinoma.Immunohistochemistry was used to detect the expression of FNDC4 in HCC tissue chips.According to the staining intensity,they were divided into high-expression FNDC4 group and low-expression FNDC4 group.The relationship between FNDC4 and tumor pathological and clinical characteristics of the two groups was statistically analyzed.2.Through exogenous overexpression or interference of FNDC4,and TGF-β1stimulation of endogenous high expression of FNDC4,Transwell and scratch assays were used to observe the effect on the invasion and migration of Hep G2（low invasive HCC cells）and Hu H-7（highly invasive HCC cells）.After the role of FNDC4 was identified,the protein lysates of FNDC4-overexpressing Hu H-7 cells and control cells were subjected to label free quantitative analysis of human protein solution,and the mechanism of FNDC4 was explored through GO and KEGG database analysis.The exogenous stimulation of FNDC4 increased,and the phosphorylation level of Akt was detected by western blot to verify the results of bioinformatics analysis.Finally,Hu H-7 cells were transfected with FNDC4Δ1-188extracellular truncated,Δ168-234 intracellular truncated plasmids and transferred to the medium of overexpressed FNDC4 cells to simulate the effect of extracellular molecules,and the phosphorylation of Akt was detected by Western Blot.The extracellular domain of FNDC4 affects the phosphorylation level of Akt and enhances the invasion and metastasis of hepatoma cells.3.First,suitable experimental cells are screened.Western blot and q PCR methods were used to detect the expression level of Tiam1 in liver cancer cell line.It was confirmed that the plasmid overexpressing Tiam1 was transfected into Hep G2 and PLC/PRF/5 cells,and Hu H-7and MHCCLM3 cells were subjected to si RNA interference experiments.Next,we treated Hep G2 Tiam1FLAG cells with PI3K inhibitor LY294002,extracted membrane proteins at0h/6h/12h/24h,and detected Tiam1 expression in membrane proteins by Western blot.After treatment with Akt-specific activator SC79,overexpression of PIP5K1A up-regulated PIP2and Neomycin inhibited PIP2 activity,Rac1 pull-down,Dot blot and Western blot were used to detect the expression levels of PIP2,membrane Tiam1,Rac1-GTP and cortactin.Finally,it was verified again by IF,transwell and scratch experiments,etc.that FNDC4 may promote Rac1-mediated invadopodia formation through the intracellular effector molecule Tiam1 to enhance the invasion and migration of hepatocellular carcinoma.4.The hepatocellular carcinoma clinical cases in two independent time periods were grouped as two independent cohorts for statistical analysis,and the correlation between the extracellular molecule FNDC4 and the intracellular effector molecule Tiam1 was confirmed by IHC analysis based on FNDC4,Tiam1,and cortactin tissue chips.Finally,Hu H-7 cells overexpressing FNDC4 and Tiam1 were used to conduct in situ liver tumorigenesis experiments in nude mice.After 4 weeks,the number and size of tumors on the liver surface of nude mice were counted.Immunohistochemistry analysis of liver cancer tissue in nude mice confirmed that FNDC4 can up-regulate the activity of intracellular effector Tiam1 and promote the invasion and migration of HCC cells at the level of animals.Results:1.Analysis of Human Protein Atlas and GEPIA database indicated that FNDC4 was correlated with hepatocellular carcinoma at both protein and m RNA levels.TCGA database analysis showed that the survival rate of FNDC4 high expression group was significantly lower than that of low expression group（p=0.0059）,suggesting that FNDC4 may play an important role in liver cancer.FNDC4 was differentially expressed in HCC tissues,mainly expressed in the extracellular matrix,with only a small amount of distribution in the cytoplasm and membrane,confirming that FNDC4 belongs to the extracellular matrix molecule.In 205 HCC patients,high expression of FNDC4 was associated with tissue differentiation（8.8%vs.69.3%vs.p=0.018）,microvascular invasion（36.6%vs.63.4%,p=0.035）was significantly associated,and FNDC4 high expression group was significantly associated with shorter overall survival（p=0.002）,while patients were more likely to have tumor recurrence（p=0.0277）,the clinical case survival analysis results are basically consistent with the results of the TCGA database.2.Overexpression of FNDC4 lentivirus transfected Hep G2 and Hu H-7 cells,the m RNA level was up-regulated by about 140%and 170%,and the esi RNA interference rate reached about 40%and 50%,respectively.FNDC4 interference had a considerable inhibitory effect on the migration and invasion ability of Hep G2 and Hu H-7 cells in vitro,while the migration and invasion ability of cells were significantly increased after overexpression of FNDC4.The expression of endogenous FNDC4 was significantly increased in Hu H-7 cells stimulated by TGF-β1,and the migration and invasion ability of Hu H-7 cells was also significantly enhanced.The expression of 1124 proteins was up-regulated and the expression of 1351proteins was down-regulated in Hu H-7 cells in the FNDC4 overexpression group detected by protein mass spectrometry.Through database analysis,it mainly affected membrane-related proteins and was closely related to the PI3K/Akt pathway.Then,it was verified by experiments that if the expression of FNDC4 was interfered,the phosphorylation level of Akt was significantly reduced by Western blot,while the expression of FNDC4 increased by exogenous or endogenous sources significantly increased the phosphorylation level of Akt.In the truncation experiment,Western blot detected a significant increase in Akt phosphorylation in the full-length FNDC4 and extracellular truncation groups,while the intracellular truncation did not change significantly.In the medium transfer experiment,the Akt phosphorylation level of the experimental group was significantly increased.These results thus confirm that the cellular extracellular domain of FNDC4 affects cellular PI3K/Akt pathway activity.3.The intracellular effector molecule Tiam1 is lowly expressed in Hep G2,PLC/PRF/5cells,and highly expressed in Hu H-7 and MHCCLM3 cells.It was confirmed that the overexpression of Tiam1 plasmid was transfected into Hep G2,PLC/PRF/5 cells,Hu H-7 and MHCCLM3 Cells were subjected to si RNA interference experiments.After LY294002treatment of Hep G2 Tiam1FLAG cells,the localization and expression of Tiam1 in the membrane of Hep G2 Tiam1FLAG cells gradually weakened and reached the lowest value at24h.The content of Tiam1 in the control group remained basically stable.After overexpression of PIP5K1A,the concentration of PIP2 in cells increased,while the content of Tiam1 membrane increased,and the content of Tiam1 membrane decreased after Neomycin inhibited PIP2.After SC79 treatment of cells,the expression levels of p Akt and Rac1-GTP were increased.When SC79 was used to treat Hep G2 Tiam1^FLAG cells that had been treated with LY294002,the expression levels of p Akt,cortactin and Rac1-GTP returned to almost normal levels.Up-regulation of PIP2 or overexpression of Tiam1FLAG,Rac1-GTP/Rac1ratio and cortactin level were significantly increased（p<0.01）,while inhibition of PIP2 or sh Tiam1 interference,Rac1-GTP/Rac1 ratio and cortactin level were significantly decreased（p<0.01）.After overexpression of FNDC4 and Tiam1^FLAG,the number of pseudopodia in Hu H-7 cells increased significantly.Overexpression of Tiam1 induced a significant increase in the migration and invasion of PLC/PRF/5 cells,while sh Tiam1 interference inhibited migration and invasion.4.Statistical analysis of clinical cases showed that patients with high expression of FNDC4 and Tiam1,Tiam1 and Cortactin had a low 5-year survival rate after surgery.There was a significant positive correlation between the expressions of FNDC4 and Tiam1,and Tiam1 and Cortactin.In animal experiments,the relative invasiveness of tumors in nude mice overexpressing FNDC4 and Tiam1 was higher than that in the control group（n=8,p<0.05）.In nude mouse liver cancer sections overexpressing FNDC4,it was observed that the cancer cells in the tumor foci had obviously invaded the surrounding tissues,and the expression level of Tiam1 was also significantly enhanced.Conclusions:1.Both the database and the clinical case analysis confirmed that the high expression of FNDC4 was positively correlated with the microvascular invasion of liver cancer tissue,and was significantly correlated with the poor prognosis of hepatocellular carcinoma patients.2.It was confirmed that FNDC4 can promote the invasion and migration of liver cancer cells in vitro,and then it was confirmed that the extracellular domain of FNDC4 affects the PI3K/Akt pathway activity of liver cancer cells by mass spectrometry analysis,truncated transfection and medium transfer experiments.However,the mechanism of interaction between FNDC4 and PI3K/Akt is not fully explained.We speculate that FNDC4 may bind to membrane integrin receptors（such as ITGβ1）through the extracellular domain,thereby transducing extracellular signals into cells to affect the activity of PI3K/Akt.Related research work still needs to be further advanced.3.PI3K/Akt pathway affects the intracellular accumulation of Tiam1 molecules under the synergistic effect of PIP2,which is conducive to the activation of Rac1-mediated invadopodia formation,thereby enhancing the invasion and metastasis of hepatocellular carcinoma.The conclusions of this part mainly explain the intracellular molecular mechanism of FNDC4 enhancing the invasion and metastasis of hepatocellular carcinoma by activating the PI3K/Akt pathway.4.Analysis of clinical cases and animal experiments in vivo confirmed that the extracellular molecule FNDC4 can promote the invasion and metastasis of liver cancer cells through the intracellular effector molecule Tiam1.In this study,we explored the molecular mechanism of FNDC4 through the PI3K/Akt pathway regulating Tiam1/Rac1 to promote the invasion and metastasis of hepatocellular carcinoma by asking questions and solving them in depth.For the first time,it was proposed and confirmed that FNDC4 affects PI3K/Akt activity through its extracellular domain,and promotes the intracellular effector molecule Tiam1 to achieve membrane enrichment under the coordination of PIP2.Tiam1 membrane localization is more conducive to the activation of Rac1 activity,resulting in increased invadopodia formation in HCC cells,thereby enhancing the invasion and metastasis of hepatocellular carcinoma.The findings of this study will further enrich the theory that the extracellular matrix affects the invasion and metastasis of tumor cells by regulating intracellular effector molecules,and also provide a direction for reversing the invasion and metastasis of hepatocellular carcinoma by changing the extracellular matrix in the future.