The Mechanism Of Ubiquitin-like Protein FAT10 In Tumor Cells Mediates The Bevacizumab Efficacy By Regulating Multiple Alternative Pro-angiogenesis Factors

Background and Objective:Neovascularization plays an important role in the growth and metastasis of solid tumors.The process of tumor blood vessel formation is complex.Studies have pointed out that under external environmental stimuli,endothelial cells are activated,basement membrane and extracellular matrix are degraded,endothelial cells proliferate and migrate to form blood vessels,and can invade and extend into solid tumors to fuse with existing blood vessels.Forms stable blood vessels.The new tumor blood vessels provide nutrition for the tumor body and provide a pathway for tumor cells to enter the circulatory system.It is a key step and necessary condition for tumor development and metastasis.Therefore,inhibiting tumor angiogenesis has become an important way to inhibit tumor growth and metastasis,and also an important strategy for clinical treatment of tumors.Studies have proved that a variety of pro-angiogenesis factors play a key role in promoting tumor angiogenesis.Vascular endothelial growth factor（VEGF）is currently recognized as the strongest pro-angiogenic factor.A large number of studies have confirmed that under hypoxia or inflammation,VEGF secreted by tumor cells and various tumor-re0lated stromal cells is significantly increased.VEGF can promote angiogenesis in tumor tissues by binding to VEGF receptors on vascular endothelial cells.It plays an important role in promoting tumor growth.Therefore,the application of drugs targeting the VEGF pathway to inhibit tumor angiogenesis has become an important strategy for clinical treatment of tumors.Bevacizumab（Avastin）is a recombinant human-derived anti-VEGF monoclonal antibody.Bevacizumab is the first anti-tumor angiogenesis drug approved by the FDA.It mainly neutralizes VEGF and blocks it.It binds to VEGF receptors on endothelial cells to produce effects.It is a targeted drug for anti-angiogenesis.However,the occurrence of drug resistance severely limits the efficacy and application of bevacizumab.Studies have shown that the key factors and mechanisms affecting bevacizumab resistance are complex.Research suggests that there are two main modes of resistance to VEGF-targeted drugs（including bevacizumab）:one is acquired resistance（also known as escape resistance）,and the other is inherent resistance.Researchers believe that one of the key mechanisms for the acquired resistance of vascular endothelial growth factor VEGF-targeted drugs is:when the activity of vascular endothelial growth factor VEGF signal is blocked,platelet derived growth factor（PDGF）,The expression of endothelial growth factor（Epidermal growth factor,EGF）,fibroblast growth factor（FGF）and transforming growth factor（Transforming growth factor,TGF）and other pro-angiogenesis signal factors increased.Studies have also confirmed that when stimulated by hypoxia or inflammation in tumor tissues,these non-VEGF pro-angiogenic signaling factors secreted by tumor cells can directly promote the proliferation and movement of endothelial cells,thereby promoting angiogenesis in tumor tissues.In addition,studies have shown that bevacizumab treatment can aggravate the inflammation and hypoxia of tumor tissues,and bevacizumab-resistant tumors can promote angiogenesis.Interestingly,the study found that after bevacizumab treatment,the expression levels of a variety of non-VEGF pro-angiogenic signaling factors,including b FGF,TGF-β,ANG2 and PDGF,increased significantly.Researchers believe that one of the key mechanisms for the acquired resistance of vascular endothelial growth factor VEGF-targeted drugs is:when the activity of vascular endothelial growth factor VEGF signal is blocked,platelet derived growth factor（PDGF）,The expression of endothelial growth factor（Epidermal growth factor,EGF）,fibroblast growth factor（FGF）and transforming growth factor（Transforming growth factor,TGF）and other pro-angiogenesis signal factors increased.Studies have also confirmed that when stimulated by hypoxia or inflammation in tumor tissues,these non-VEGF pro-angiogenic signaling factors secreted by tumor cells can directly promote the proliferation and movement of endothelial cells,thereby promoting angiogenesis in tumor tissues.In addition,studies have shown that bevacizumab treatment can aggravate the inflammation and hypoxia of tumor tissues,and bevacizumab-resistant tumors can promote angiogenesis.Interestingly,the study found that after bevacizumab treatment,the expression levels of a variety of non-VEGF pro-angiogenic signaling factors,including b FGF,TGF-β,ANG2 and PDGF,increased significantly.Methods:1.CRISPR-Cas9 technology was used to construct FAT10 knockout tumor cells and virus stable transformation technology was used to construct tumor cells that stable interfered with FAT10.Subcutaneous tumor-bearing model of nude mice was constructed.After BV treatment,the changes of tumor volume in nude mice were observed.QRT-PCR,ELISA,immunohistochemistry,immunofluorescence and other experiments were used to observe the expression changes of FAT10,VEGF and several non-VEGF pro-angiogenesis factors in tumor cells,as well as the angiogenesis in tumor tissues.2.QRT-PCR,immunohistochemistry,immunofluorescence and ELISA were used to analyze the expression changes of FAT10,VEGF and several non-VEGF pro-angiogenesis factors in the tumor tissue of the BV treatment group and the Aspirin+BV treatment group,as well as the tumor growth rate,tumor volume changes and angiogenesis in the tumor tissue of nude mice.Immunohistochemistry,RNA-seq,qRT-PCR and luciferase reporter gene were used to analyze the relationship between FAT10 andβ-catenin,STAT3,NF-κB and HIF1αsignaling pathways in tumor tissues of the Aspirin+BV treatment group and the BV treatment group alone.3.The molecular mechanisms of FAT10 regulatingβ-catenin,STAT3,NF-κB and HIF1αsignaling pathways were explored by immunoprecipitation,in vitro ubiquitination and immunofluorescence techniques.4.The expression of FAT10 and angiogenic factor in tumor cells after hypoxia and inflammation was detected by qRT-PCR.FAT10 overexpressed tumor cells were constructed,and supernatants of the control group and the overexpressed group were co-cultured with human umbilical vein endothelial cells（HUVECs）.The tubulation ability of HUVECs cells was detected by the tubulation experiment and the proliferation ability of HUVECs cells was detected by the multifunctional real-time cell function analyzer（RTCA）.Results:1.We found that the tumor volume of the FAT10^+/+mouse model was significantly increased after BV treatment,while no significant difference was observed in the tumor volume of the FAT10^-/-mouse model.The m RNA and protein levels of non-VEGF pro-angiogenic factors were significantly increased in the FAT10^+/+model,while no significant changes were found in the FAT10^-/-model.In addition,VEGF m RNA levels were increased and protein levels were decreased after BV treatment in both the FAT10^+/+model and the FAT10^-/-model.RNA-seq analysis showed that in the FAT10^+/+tumor model,the expression levels of FAT10,VEGF and non-VEGF pro-angiogenic factors were all upregulated after BV treatment,and hypoxia and inflammation-related genes were upregulated.However,in the FAT10^-/-model,the non-VEGF pro-angiogenic factors were not significantly changed after BV treatment,while the VEGF,hypoxia and inflammation-related genes were still upregulated.Hypoxia,macrophage infiltration and FAT10 expression levels of tumor cells were increased in the BV-treated group of the FAT10^+/+mouse model,but vascular density in the tumor tissue was significantly increased.However,hypoxia,macrophage infiltration and FAT10 expression levels in the tumor tissues of the FAT10^-/-mice treated with BV increased,and the vascular density in the tumor tissues decreased significantly.Further,we found that,in sh FAT10 group,m RNA expression levels of FAT10,VEGF and non-VEGF pro-angiogenic factors were significantly lower,while BV treatment significantly reduced the tumor growth speed and volume,compared with sh NC group,protein concentrations of non-VEGF pro-angiogenic factors within the tumor tissue of sh FAT10 model decreased significantly,and VEGF has no obvious change,in addition.Furthermore,within the tumor tissue of sh FAT10model,hypoxia and macrophage infiltration degree has no obvious change,and vascular density decreased obviously.2.In the FAT10^+/+model,BV treatment alone or Aspirin+BV treatment group,we found that compared with the BV treatment group,the tumor growth rate and volume in the Aspirin+BV treatment group decreased significantly,and the m RNA expression of FAT10,VEGF and non-VEGF pro-angiogenic factor decreased,the protein expression of non-VEGF pro-angiogenic factor decreased,and VEGF did not change significantly.The infiltration degree of macrophages,vascular density,β-catenin,STAT3,NF-κB and HIF1αin tumor tissues were significantly decreased,while the hypoxia level was not significantly changed.The above changes were not observed in the FAT10^-/-model.RNA-seq results showed that target genes,angiogenesis-related genes and FAT10 were all upregulated in FAT10^+/+tumor cells treated with TNF-α/IFN-γ.However,differences in expression levels of these genes were not observed in FAT10^-/-tumor cells.Furthermore,we found that TNF-α/IFN-γtreatment in FAT10^+/+tumor cells increased the expression of GLUT1,HOXB9,Bcl2and CCND1,enhanced the nuclear translocation and transcriptional activity of HIF1α,β-catenin,STAT3 and p65.However,these changes were not observed in FAT10^-/-tumor cells treated with TNF-α/IFN-γ.3.FAT10 was found to bind HIF1α,β-catenin,STAT3 and TAB3 in HCCLM3cells,respectively,and Ub and FAT10 competitively bind these substrate proteins.By adding proteasome inhibitor MG132,the expression of HA-FAT10-P62 was significantly increased,but the expression levels of HA-FAT10-HIF1α,HA-FAT10-β-catenin,HA-FAT10-STAT3 and HA-FAT10-TAB3 complexes remained unchanged.In addition,by adding CHX,a protein synthesis inhibitor with different dose gradient,we found that overexpression of RPN10 increased the degradation rate of HA-FAT10-P62,whereas interference with RPN10 increased the half-life of HA-FAT10-P62 degradation.However,changing the expression of RPN10 did not significantly affect the expression levels of HA-FAT10-HIF1α,HA-FAT10-β-catenin,HA-FAT10-STAT3 and HA-FAT10-TAB3 complexes.In addition,we also found that TNF-α/IFN-γcan increase the expression of FAT10^+/+in FAT10^+/+tumor cells and decrease the ubiquitination levels of HIF1α,β-catenin,STAT3 and TAB3,while these changes were not observed in FAT10^-/-tumor cells.4.The m RNA expression levels of FAT10,VEGF and non-VEGF pro-angiogenic factors were significantly increased after inflammation or hypoxia stimulation in FAT10^+/+tumor cells,however,these changes were not observed in FAT10-/-tumor cells.FAT10 was overexpressed in tumor cells with VEGF antibody,and no significant changes were found in VEGF protein levels,while m RNA expressions of VEGF,b FGF,TGF-β,Ang2 and PDGF were upregulated.Supernatant of FAT10 tumor cells overexpressed with VEGF antibody was collected,and co-culture with HUVEC cells showed that overexpression of FAT10 significantly enhanced the tubulation ability and proliferation ability of HUVEC cells.Conclusion:FAT10 exerted the function of simultaneous stabilization multiple substrates,resulting in an increase in FAT10 to simultaneously upregulate protein expression of multiple substrates,including HIF1α,β-catenin,STAT3,TAB3,in tumour cells.This simultaneously enhanced the transcriptional activity of HIF1α,WNT/β-catenin,JAK/STAT3,and NF-k B signaling pathways,thereby simultaneously upregulating the expression levels of VEGF,b FGF,TGF-β,ANG2,and PDGF in the tumour cells.Although BV blocked the activity of VEGF signal within tumour tissues,the simultaneous upregulation of multiple non-VEGF pro-angiogenic signaling factors,including b FGF,TGF-β,ANG2,and PDGF,mediated by FAT10 in tumour cells within tumour tissues compensated for the inhibition of the VEGF signal,resulting in accelerated VEGF-independent angiogenesis of tumour tissues to stimulated tumour growth.