Targeting Integrin αvβ3 With Indomethacin Inhibits Patient-derived Xenograft Tumor Growth And Recurrence In Esophageal Squamous Cell Carcinoma

Background and aimsEsophageal squamous cell carcinoma(ESCC)is a highly heterogeneous disease that accounts for 80%of esophageal cancers and produces variable clinical outcomes.Surgical resection,chemotherapy or chemoradiotherapy are often used to treat ESCC.However,curative second-or later-line chemotherapy drugs specific for ESCC have not been developed.Moreover,many chemotherapeutic agents exhibited deleterious effects in ESCC patients,producing adverse outcomes such as secondary leukemia,bone marrow suppression,and cardiac toxicity.The 5-year survival rate for ESCC ranges between 15%-25%due to inadequate therapeutic strategies and postoperative recurrence.Therefore,there is an urgent need to investigate novel strategies for preventing postoperative recurrence in ESCC patients.The membrane proteins,integrins,are transmembrane heterodimers including 18 integrin α subunits and 8 integrin β subunits.Integrin αv(ITGAV),the receptor for fibronectin,forms heterodimers with integrin β1,3,5,6,and 8.Ligand-integrin binding promotes the activation of many protein kinases(including FAK and SRC),and their respective downstream effectors.Several signaling cascades,including the ERK/JNK and PI3K/AKT pathways are essential for cell proliferation,migration,and invasion.Integrins are also known as activators of latent TGFβ.TGFβ has been shown to promote cancer cell growth upon binding with its receptor through regulating SMAD2/3 phosphorylation.Additionally,TGFβ prevents penetration of cancer cell by cytotoxic CD8+ T cells in the tumor immune microenvironment.Integrin heterodimer formation is mainly regulated by α subunit synthesis;therefore,regulation of α subunits is particularly important.Several studies have shown that ITGAV is frequently upregulated in various types of cancers.To date,clinical trials designed to assess the efficacy of ITGAV inhibitors have produced unsatisfactory results,suggesting a need to identify novel integrin inhibitors.Indomethacin has been shown to inhibit colon cancer cell growth and decrease esophageal cancer(EC)cell viability.Mechanistically,indomethacin is generally thought to reduce cancer risk by reducing prostaglandin synthesis through COX inhibition.However,indomethacin was also found to increase p53 expression in xenograft-derived acute lymphoblastic leukemia cells,induce apoptosis by upregulating BAX in esophageal adenocarcinoma cells,and blocking the translation of eIF2α kinase PKR in colon cancer cells.Methods(1)To evaluate the mRNA expression of integrin family members(ITGA1-11,ITGAV,ITGB1-8)in the context of ESCC,we searched the data in The Cancer Genome Atlas(TCGA)database(http://ualcan.path.uab.edu)and analyzed.We next verified the protein levels of these integrin α subunits suggested to be differentially represented between samples at the mRNA level,using 10 paired(tumor and adjacent)ESCC patient tissues by Western blot.We next performed IHC staining using a tissue array including tumor tissues(n=105)and adjacent tissues(n=72)to determine the protein levels of ITGAV.The protein levels of the ITGAV β subunits in tumor and adjacent tissues were determined by Western blot.(2)The ITGAV protein levels in SHEE and ESCC cells(KYSE30,KYSE70,KYSE140,KYSE410,and KYSE510)were next determined by Western blot.Furthermore,we detected the β subunits(ITGB1,ITGB3,ITGB5,ITGB6 and ITGB8)protein levels after knockdown of ITGAV by Western blot.Cell line-derived xenograft models were generated,through injecting athymic nude mice with shMock or shITGAV cells.To investigate the potential function of ITGAV in ESCC patient-derived tumors,a lentivirus-transduced PDX mouse model was generated.(3)In order to identify a new ITGAV inhibitor that can suppress ESCC growth and prevent recurrence,we performed a virtual screening of FDA-approved drugs.We next used a docking model to verify the results of virtual screening.Additionally,we performed SPR analysis and pulldown assays to explore the binding affinity of indomethacin to ITGAV.To further assess whether indomethacin can compete with the ITGAV for its interaction with fibronectin,we performed a competition IP assay using HEK-293T cells overexpressing ITGAV.To determine the effect of indomethacin on the downstream protein targets of ITGAV,we treated ESCC cell lines with indomethacin and quantified changes in protein levels using Western blot.(4)We examined ITGAV mRNAlevels by RT-PCR.The total RNA from indomethacintreated ESCC cells was extracted and subjected to RT-PCR.We performed the ubiquitination activity assay to verify indomethacin-mediated degradation of ITGAV We used Ubibrowser(http://ubibrowser.ncpsb.org.cn/ubibrowser/)to predict the E3 ubiquitin ligases that could potentially interact with ITGAV.we performed an IP assay with an anti-ITGAV antibody to detect if ITGAV interacts with the top 3(NEDD4,ITCH and SYVN1)predicted candidates.We overexpressed ITGAV and SYVN1 in HEK-293T cells and then detected the ubiquitination of ITGAV(5)To detect the inhibitory effect of indomethacin on ESCC cell proliferation,we treated ESCC cell lines with various concentrations of indomethacin at different time points and then measured cell viability using an MTT assay.To confirm the effect of indomethacin on cell cycle,cells were treated with indomethacin for 48 h prior to being analyzed using flow cytometry.We then examined the protein levels of G1-phaseassociated proteins by Western blot.Furthermore,to investigate whether indomethacin could induce apoptosis,annexin V staining was performed after indomethacin treatment for 72 h.In order to determine whether the effects of indomethacin are dependent upon ITGAV expression,we performed MTT and anchorage-independent cell growth assays in shMock or shITGAV cells after DMSO or indomethacin treatment(KYSE30 and KYSE510).We then compared the rates of cell growth inhibition with the respective ITGAV protein levels in the infected cells.(6)We next examined the effects of indomethacin in three different ESCC PDX models.IHC staining was performed to determine the Ki67,ITGAV,ITGB3,p-FAK(Tyr925),p-PI3K p85a(Tyr467),p-AKT(Ser473),and p-GSK3β(S9)protein levels in the indomethacin-treated group compared to vehicle-treated group.(7)We utilized a PDX model to investigate the potential antitumor effects of indomethacin,iRGD and cilengitide.We then performed IHC staining to detect ITGB3,p-FAK,ki67,ITGAV protein levels.We then stablished an in vivo model to determine if indomethacin,iRGD or cilengitide could affect ESCC recurrence.(8)We measured the concentrations of active TGFβ in the cell culturing medium after indomethacin treatment for 48 h by ELISA kit.Humanized PDX mouse model was generated.Human CD45 levels in mice blood were determined 13 and 23 days after hPBMCs injection.Next,we determined the concentration of active TGF β in the serum of mice from control and treatment groups.Flow cytometry analysis was performed to determine CD8+T cell population.We determined IFNγ protein levels in mice tumors by ELISA kit.Results(1)ITGAV is upregulated in ESCC and associated with poor prognosisITGA4,ITGA6,ITGA2,ITGA11,ITGA3,and ITGAV transcripts were increased in ESCC tumor tissues(n=95)compared with normal tissues(n=11).ITGB1,ITGB2,ITGB4-8 were highly expressed in tumor tissues compared with normal tissues.Among the integrin α subunits that were examined,ITGAV showed increased expression in all 10 paired ESCC tissues.The results of the IHC analysis showed that ITGAV was upregulated in ESCC tumor tissues compared to adjacent tissues.Increased ITGAV expression was associated with a poor survival outcome and clinical grade.We found that ITGB1(4/10),ITGB3(6/10),ITGB5(6/10),and ITGB6(5/10)protein levels were increased in tumor tissues compared to adjacent tissues.In contrast,ITGB8(10/10)protein levels were increased in adjacent tissues compared with tumor tissues.(2)ITGAV promotes ESCC cell proliferation and metastasisCompared to SHEE cells,KYSE30,KYSE140,KYSE410 and KYSE510 cells showed increased ITGAV protein levels.After knockdown or knockout of ITGAV,the cell growth was suppressed.The p-FAK,p-PI3K and p-AKT protein levels were also downregulated after knockdown of ITGAV.In contrast,overexpression of ITGAV promoted the colony formation ability in ESCC cells and activated the FAK/PI3K/AKT signaling pathway.ITGAV knockdown suppressed cell invasion and migration.ITGB3 expression was significantly decreased in the KYSE30 and KYSE510 knockdown groups,while ITGB1 expression was decreased in the KYSE30 knockdown group.The results showed that ITGAV knockdown suppressed tumor growth in the shITGAV KYSE30 or shITGAV KYSE510 groups and a significant reduction in the volume and weight of shITGAV-infected PDX tumors.(3)Indomethacin binds to ITGAV and modulates its downstream signaling effectorsThe top 20 drugs that showed significant docking scores(-7.2 to-4.7 kcal/mol)are listed in Table.It illustrates that,indomethacin(docking score:-5.30)and disulfiram(anti-alcoholic drug)(docking score:-4.93)are promising candidates.The docking results suggested that indomethacin can bind to ITGAV at PHE31 and PHE159 or LYS615,GLN614,and ASP613.SPR results indicated that indomethacin had a dosedependent affinity for ITGAV.Pulldown assay results illustrated that the indomethacinconjugated beads were able to bind to ITGAV protein.Indomethacin decreases the affinity between ITGAV and fibronectin in a concentration dependent manner.T KYSE30 and KYSE510 cells treated with 200 μM indomethacin exhibited decreased ITGAV,ITGB3,p-FAK(Tyr925),p-PI3K p85a(Tyr467),p-GSK3β(S9)and p-AKT(Ser473)protein levels.(4)Indomethacin promotes SYVN1-mediated degradation of ITGAVThe ITGAV mRNA levels were not changed after indomethacin treatment.The results showed that the half-life of ITGAV protein in the indomethacin plus CHX treatment group was shorter than that of the CHX treatment group.MG132 successfully attenuated the indomethacin-mediated degradation of ITGAV The ubiquitination activity assay showed that more ubiquitin bound to ITGAV in indomethacin treated cells than in DMSO treated cells.The results showed that the E3 ubiquitin ligase SYVN1 could bind to ITGAV in KYSE30 cells.We also observed that SYVN1 overexpression reduced integrin αvβ3 protein levels.Additionally,indomethacin treatment enhanced SYVN1-mediated ubiquitination of ITGAV(5)Indomethacin inhibits ESCC cell growth by targeting ITGAVWe found that indomethacin could significantly inhibit ESCC cell growth without inhibiting normal esophageal epithelial cell proliferation.Additionally,indomethacin treatment strongly decreased the colony numbers.The results showed that indomethacin treatment inhibited cell cycle at G1 phase and reduced CDK4/6,cyclin D1 protein levels.We found that indomethacin treatment increased the rate of apoptotic cells.The results showed that indomethacin treatment produced less of an inhibitory effect with respect to cell proliferation and anchorage-independent colony growth in ITGAV knockdown ESCC cells compared to shMock cells.Our results showed that cells with increased ITGAV protein levels were more sensitive to indomethacin treatment than cells with low ITGAV protein levels.(6)Indomethacin inhibits ESCC PDX tumor growthTumor volume and weight were decreased in indomethacin treatment groups in PDX mouse model.Indomethacin treatment did not reduce the mice body weight.IHC staining results of tumors excised from the mice showed reduced Ki67,ITGAV,ITGB3,p-FAK(Tyr925),p-PI3K p85a(Tyr467),p-AKT(Ser473),and p-GSK3β(S9)protein levels in the indomethacin-treated group compared to vehicle-treated group.Specifically,PDX tumors characterized by increased ITGAV protein levels(case LEG74)were more sensitive to indomethacin than those with low ITGAV protein levels(case LEG92 and LEG84).The enhanced inhibitory effect of indomethacin was not observed in tumors with increased COX protein levels.(7)Indomethacin exerts stronger antitumor activities than iRGD and cilengitide in vivoThe data indicated that each drug significantly inhibited ESCC tumor growth.Statistical analysis suggested that the inhibitory effect of indomethacin was more significant than that of iRGD and cilengitide.Indomethacin,iRGD,or cilengitide treatment did not decreased the mice body weight.The results showed that indomethacin and cilengitide treatment decreased ki67 protein levels.Both ITGAV and ITGB3 were decreased in the indomethacin treatment group,but not in the iRGD and cilengitide treatment groups.However,treatment with indomethacin,iRGD,and cilengitide suppressed p-FAK protein levels.The results showed that indomethacin treatment prevented ESCC recurrence.(8)Indomethacin suppresses TGFβ/SMAD2/3 signaling and enhances cancer immune responses.Results showed that the concentration of active TGFβ was decreased in the medium of the indomethacin treatment groups.SMAD2/3 phosphorylation levels were also decreased upon indomethacin treatment.We detected the antitumor activity of indomethacin in a humanized PDX mouse model.We observed that human CD45 protein levels were increased more than 30%in the hPBMC injection group 23 days after hPBMCs injection.Indomethacin treatment inhibited tumor growth in the nonhPBMC injection group and the PBMC injection group.However,the tumor volumes in the hPBMC+indomethacin group were decreased compared to the indomethacin group.The results suggested indomethacin treatment prevented the activation of TGFβ.Flow cytometry analysis demonstrated that indomethacin treatment increased the percentage of CD8+T cells in mice blood.We observed increased IFNγ protein levels in tumors after indomethacin treatment.SMAD2/3 phosphorylation levels were also decreased upon indomethacin treatment.ConclusionsIn summary,this study established that ITGAV is highly expressed in ESCC and promotes tumor progression.Indomethacin binds to ITGAV,induces ITGAV ubiquitin mediated degradation,and suppresses the integrin αvβ3/FAK/PI3K/AKT/GS3Kβsignaling axis.Moreover,indomethacin prevents activation of TGFβ/SMAD2/3 signaling and enhances antitumor immune responses.Importantly,indomethacin treatment suppressed ESCC tumor growth and recurrence.