The Study Of Molecular Mechanisms Of The Interaction Of Lysyl Oxidase-Like 2 (LOXL2)with GATA6 And MACC1 Induces Angiogenesis In Cholangiocarcinoma

Background:Cholangiocarcinoma(CCA),which has a dismal prognosis,is the second most common hepatobiliary cancer after hepatocellular carcinoma,and the overall incidence of CCA is increasing worldwide.Surgical resection is the preferred treatment for patients with early-stage CCA,but only some of these patients(approximately 35%)are candidates for surgical resection with a curative intent.Therefore,a better understanding of the molecular characteristics and biology of CCA will be helpful for treating this cancer and will be useful for identifying a more specific molecular-targeted therapy.Angiogenesis,which is defined as the formation of new blood vessels from pre-existing vasculature,has become a well-established hallmark of cancer.Angiogenesis maintains the aggressive growth and metastasis of the tumor by supplying nutrients and oxygen.Vascular endothelial growth factor A(VEGFA)plays a key role in tumor angiogenesis and correlates with the vessel count in many solid cancers.Patients with solid tumor with a high microvessel density(MVD)have a shorter survival than patients with a low MVD.Antiangiogenic therapy has been clinically administered to treat various solid tumors,including CCA.Nevertheless,the clinical benefits of antiangiogenesis therapy for cancer remain unsatisfactory,and high rates of clinical side effects,cytotoxicity,acquired resistance and patient relapse have been reported.Thus,treatments that address new mechanisms of angiogenesis are urgently needed.GATA-binding protein 6(GATA6)is a member of the GATA-binding protein family that shares two highly conserved zinc finger-containing transcription factors.GATA6 is essential for the proliferation,differentiation and development of the cardiovascular system,digestive system,and other tissues during embryonic development.Tumorigenesis is closely related to gene activation in the embryonic stage,and overexpression of GATA6 promotes the progression of gastric cancer,colorectal cancer,breast cancer and CCA.Some studies have linked GATA6 to cancer angiogenesis,however the relative molecular mechanism has not been investigated.Lysyl oxidase-like 2(LOXL2)is a member of the LOX family of extracellular matrix(ECM)-modifying enzymes,which includes the prototypic LOX and four different LOX-like proteins(LOXL1,LOXL2,LOXL3,and LOXL4).The C-terminal region of LOXL2 is responsible for its enzymatic activity,and the N-terminus contains four scavenger receptor cysteine-rich(SRCR)domains that are thought to be involved in protein-protein interactions.Intranuclear LOXL2 cooperates with certain transcription factors to regulate epithelial-mesenchymal transition(EMT)and promote tumor metastasis.Moreover,targeted inhibition of LOXL2 decreases angiogenesis in tumors and ophthalmic diseases.Nonetheless,the angiogenic mechanism of LOXL2 in CCA has not been investigated.Metastasis-associated in colon cancer-1(MACC1)was first identified in 2009 through a genome-wide search for differentially expressed genes in human colon cancer tissues and metastatic tissues.It has been reported that MACC1 mRNA expression might be an independent prognostic indicator of recurrence and disease-free survival in colorectal carcinoma,lung adenocarcinoma,pancreatic cancer,and hilar cholangiocarcinoma.MACC1promotes the proliferation,migration,and invasiveness of cancer cells via the hepatocyte growth factor(HGF)/c-Met/MAPK signaling pathway.Previous studies have also indicated that MACC1 participates in angiogenesis in gastric cancer and cervical cancer.However,the correlation between MACC1 and angiogenesis in CCA has not yet been investigated.In the first part of the study,we used an in silico analysis to reveal that GATA6 possibly binds to the VEGFA promoter.We verified that GATA6 transcriptionally regulates VEGFA expression,and we elucidated a new mechanism of angiogenesis in CCA in which LOXL2interacts with GATA6 to promote angiogenesis and tumor growth.In the second part of the study,we also found that MACC1 and VEGFA were significantly upregulated in CCA according to The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)datasets,as well as in human paraffin-embedded CCA samples.Moreover,MACC1 and VEGFA expression levels were positively correlated in CCA tissues.MACC1 was an independent predictor of overall survival.We further confirmed that MACC1 regulated the expression and secretion of VEGFA and promoted angiogenesis in CCA cells.Methods:PartⅠ1.We investigated the expression of GATA6 and LOXL2 and the correlation of GATA6and LOXL2 with VEGFA in The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)datasets.2.A total of 91 CCA patients undergoing surgical resection at Southwest Hospital between 2013 and 2016 were included;91 cancerous samples and 31 paracancerous samples were obtained.None of the patients received radiotherapy or chemotherapy before or after surgery.TMA(Tissue Microarray)from 91 cancerous and 31 paracancerous paraffin samples was constructed by Outdo Biotech Co.China;Expression levels of GATA6,LOXL2,VEGFA and MVD(CD34)on TMAs were investigated by immunohistochemistry(IHC).3.Correlations between GATA6/LOXL2 expression and overall survival and disease-free survival were investigated by Kaplan-Meier curves and Cox regression.Correlations between GATA6/LOXL2 expression with VEGFA expression and MVD were analyzed in TMAs.4.Real-time qPCR and Western blotting was performed to evaluate impact of intervening GATA6 and LOXL2 expression on the VEGFA.The localizations of GATA6 and LOXL2 expression were determined by confocal laser scanning microscope detection.Co-immunoprecipitation(Co-IP)was used to analyze the interaction between GATA6 and LOXL2.Dual-luciferase reporter assay and chromatin immunoprecipitation(ChIP)were performed to analyze transcriptional activation of VEGFA.Enzyme-linked immunosorbent assay(ELISA)was used to test the secretion level of VEGFA.Tube formation was analyzed by Matrigel-based angiogenesis assays.5.We constructed two LOXL2 deletion mutants lacking or retaining the SRCR domain(Flag-LOXL2-Δ1:deleted aa 548-774;Flag-LOXL2-Δ2:deleted aa 1-547)and full-length LOXL2(Flag-LOXL2-full)to further explore the interaction between LOXL2 and GATA6.The different mutants of LOXL2 interacting with GATA6 were determinated by Co-IP,and the different mutants of LOXL2 increasing VEGFA promoter activity were performed by dual-luciferase reporter assays.6.We determined the role of GATA6/LOXL2 complex in vivo using subcutaneous tumors of nude mice.Subcutaneous tumors were removed by surgery,and they were then measured.The tumors were sliced,and then stained for hematoxylin-eosin(HE)and IHC.PartⅡ1.Data from the TCGA(https://cancergenome.nih.gov/)and GEO(https://www.ncbi.nlm.nih.gov/geo/),accession numbers:GSE76297,GSE89749 databases are publicly available.The difference in MACC1 and VEGFA mRNA expression between CCA tissue and normal tissue was compared,and the relationship between the expressions of MACC1 and VEGFA was analyzed2.31 paracarcinoma specimens from 122 patients with CCA who underwent surgery between 2010 and 2016 at the Department of Hepatobiliary Surgery,Southwest Hospital.Seven paired CCA and matched paracarcinoma tissues were obtained during surgery in 2018and were immediately stored in liquid nitrogen.None of the patients either received(neo)adjuvant chemotherapy or underwent liver transplantation.Real-time qPCR and Western blotting was performed to expression levels of MACC1 and VEGFA in seven paired CCA.Immunohistochemistry(IHC)were used to investigate expression levels of MACC1,VEGFA and MVD(CD34)in paraffin-embedded CCA samples.3.Correlations between MACC1 and VEGFA expression with overall survival were investigated by Kaplan-Meier curves and Cox regression.Correlations between MACC1expression with VEGFA expression and MVD were analyzed.4.Real-time qPCR and Western blotting was performed to evaluate impact of intervening MACC1 expression on the VEGFA.The localizations of MACC1 and VEGFA expression were determined by confocal laser scanning microscope detection.Enzyme-linked immunosorbent assay(ELISA)was used to test the secretion level of VEGFA.Tube formation was analyzed by Matrigel-based angiogenesis assays.Results:PartⅠ1.GATA6 and LOXL2 were significantly upregulated in CCA tissues,and GATA6 and LOXL2 were significantly correlated with VEGFA.2.GATA6 was expressed only in the nucleus,but LOXL2 was expressed in both the nucleus and cytoplasm.The scores for the two protein-staining methods were calculated only for the nucleus.All paracancerous samples were negative for GATA6,LOXL2 and VEGFA staining in bile duct epithelium.Among these CCA TMA samples,38 GATA6 and LOXL2weak-positive or strong-positive samples were defined as double-positive,which was marked as GATA6/LOXL2-positive;28 were defined as double-negative,which was marked as GATA6/LOXL2-negative.The remaining 25 cases included 12 GATA6-negative/LOXL2-positive and 13 GATA6-positive/LOXL2-negative samples.VEGFA staining was observed in the cytoplasm.VEGFA positive expression(35/66)and negative expression(31/66)were observed.CD34 was expressed in vascular endothelial cells and marked MVD.The mean value of MVD,defined as the cut-off value,was 23.69/mm~2 in 38GATA6/LOXL2-positive and 28 GATA6/LOXL2-negative staining samples.High MVD(40/66)and low MVD(26/66)were observed.3.Kaplan-Meier analysis showed that CCA patients with GATA6/LOXL2-positive staining had worse overall survival(log rank P=0.01)and shorter disease-free survival(log rank P=0.02)than did CCA patients with GATA6/LOXL2-negative staining.Using Cox regression to assess hazard ratios(HRs)for age,gender,tumor location,TNM stage and GATA6/LOXL2 expression,we observed expression of GATA6/LOXL2 to be an independent predictor of poor overall survival and poor disease-free survival(HR=1.871,95%CI=1.070–3.271,P=0.028;HR=1.781,95%CI=1.013–3.129,P=0.045).Additionally,advanced TNM stage(Ⅲ–Ⅳ)was another independent predictor of overall survival and disease-free survival(HR=2.333,95%CI=1.311–4.150,P=0.004;HR=1.861,95%CI=1.053–3.291,P=0.033).In the same TMA section sample,we observed 38 GATA6/LOXL2-positive stained tissues,25(65.8%)VEGFA-positive stained tissues,and 27(71.1%)high MVD tissues.Notably,GATA6/LOXL2 was positively correlated with VEGFA(P=0.02)and MVD(P=0.04).4.Protein levels of VEGFA were significantly reduced in QBC939 cells by GATA6 and LOXL2 knockdown but were increased in RBE cells by GATA6 and LOXL2 overexpression.The same changes in mRNA levels were observed in CCA cells.Confocal laser scanning microscope detection showed that GATA6 was located in the nucleus and LOXL2 in the nucleus and cytoplasm.These results are consistent with the IHC results for CCA tissues.Co-immunoprecipitation confirmed that GATA6 and LOXL2 physically interacted with each other in CCA cell lines,whereby this interaction transcriptionally regulated VEGFA expression and secretion and promoted tube formation.5.Based on the results from the Co-IP experiment,GATA6 bound to the SRCR domain of LOXL2 independently of the catalytic domain in CCA cells.Moreover,Flag-LOXL2-Δ1and Flag-LOXL2-full increased VEGFA promoter activity;however,LOXL2-Δ2 did not increase VEGFA promoter activity.In addition,knockdown of GATA6 or LOXL2 in QBC939cells decreased the interaction of LOXL2 with GATA6.Based on the data,the SRCR domain of LOXL2 interacts with GATA6 to increase VEGFA promoter activity.6.In vivo analysis further demonstrated that GATA6/LOXL2 promoted VEGFA expression,angiogenesis and tumor growth.PartⅡ1.MACC1 and VEGFA were significantly upregulated in CCA tissues from the datasets and in paired CCA tissues compared with the levels in normal control tissues.MACC1 was significantly correlated with VEGFA in the TCGA,GSE76297 and GSE89749 datasets.2.The protein and mRNA levels of MACC1 and VEGFA were increased in 7 frozen CCA tissues compared with those in the matched paracarcinoma tissues.IHC showed that MACC1-high and VEGFA-high expression was observed in 53.3%(65/122 cases)and 54.9%(67/122 cases)of CCA cases,respectively.Combined MACC1-high/VEGFA-high expression was observed in 43 cases,and MACC1-low/VEGFA-low expression was observed in 33 cases.However,all 31 paracarcinoma samples showed low expression of MACC1 and VEGFA.122CCA patients,56(45.9%)had high MVD,while the remaining 66(54.1%)were considered to have low MVD.3.Kaplan–Meier analysis revealed that high expression of MACC1 and VEGFA was significantly associated with reduced overall survival(log-rank P<0.01 and P<0.05).MACC1-high/VEGFA-high expression also had a worse overall survival than those with MACC1-low/VEGFA-low expression(log-rank P<0.001).Cox regression analysis showed that MACC1 and lymph node metastasis were independent predictors of overall survival in CCA patients.66.2%(43/65)of CCA tissues with high MACC1 expression showed high VEGFA expression(P<0.01),and 60%(39/65)of CCA tissues with high MACC1 expression showed a high MVD.4.Confocal laser scanning microscopy showed that MACC1 and VEGFA were expressed in the cytoplasm and nucleus.Western blotting and real-time qPCR verified that knocking down MACC1 significantly downregulated the protein and mRNA expression of VEGFA in both CCA cell lines.ELISA showed that MACC1 knockdown significantly decreased the level of secreted VEGFA.Tube formation assay Matrigel using HUVECs showed that The number of tubes formed after exposure to medium from the MACC1 knockdown group was significantly lower than that after exposure to medium from the shControl group in both CCA cells(P<0.01 and P<0.01).Conclusion:In summary,our study identifies a new molecular mechanism for increased angiogenesis resulting from the interaction between LOXL2 and GATA6 that enhances VEGFA expression and promotes angiogenesis and tumor growth.The development of new candidate prognostic markers for the stratification of patients with CCA and of small-molecule,cell-permeable inhibitors that target both intracellular and extracellular LOXL2 are likely to have great therapeutic value.In addition,we also found that MACC1 and VEGFA are upregulated in CCA and that high expression of MACC1 and VEGFA predicts poor survival.Moreover,MACC1 is an independent predictor of overall survival and facilitates CCA angiogenesis by upregulating VEGFA.