The Effect And Mechanism Of Immune Negative Regulator IL-37 On Tumor Growth And Metastasis

ObjectiveIt is well known that cytokines are important effectors and regulatory molecules in the immune response.In recent years,studies have found that cytokines also play an important role in tumorigenesis and development,and have become an important anti-tumor target.IL-37 is firstly discovered in 2000 years.It is a new negative immunoregulatory molecule and belongs to IL-1 family.Studies have shown that IL-37 can be expressed not only in a variety of immune cells,but also in cells of a variety of solid tissues and organs,including tumor cells.It has both intracellular and extracellular functions.In recent years,it has been reported that IL-37 exerts anti-tumor effect by affecting the immune response.IL-37 suppresses tumor growth by inducing an IL-12-dependent adaptive immune respons in mouse fibrosarcoma.IL-37 inhibits tumor growth via recruiting CD57 + NK cells in human hepatocellular carcinoma.However,the role of IL-37 in tumor metastasis is unclear.The direct functions of IL-37 in the malignant phenotype of tumor cells and its underling mechanisms remain to be further clarified.In the present study,we explore the direct effect of IL-37 on tumor,and search for the mechanism.MethodsI The direct effect of IL-37 on the malignant phenotype of tumor1.IHC was used to detect IL-37 expression in HCC and lung adenocarcinoma tissues.The relationship between IL-37 expression and clinical information was analyzed.2.Western blot,immunofluorescence and ELISA were used to detect IL-37 expression in tumor cell lines.3.Control siRNA and IL-37 siRNA or mock and IL-37b plasmids were transfected into cells.Cell proliferation was detected by CCK8.Cell migration ability was detected by transwell assay.4.Subcutaneous xenograft model in nude mice was established with A549 cells infected with NC or IL-37b lentivirus.Tumor size was measured every three days.At last,tumors were dissected,photographed and weighted.IL-37 expression in tumors was detected by IHC.5.A549 cells were infected with NC or IL-37b lentivirus and injected into nude mice via the tail vein to establish metastatic tumor model.We observed the status of the mice and recorded the survival period.At last,the mice were dissected and observed whether there is metastatic foci in face,limbs,spine,chest wall,lungs,etc,.The dissected lungs were weighted and photographed.The number of metastatic foci in lungs was counted under anatomical microscope.The metastatic foci were confirmed by HE staining.II The mechanism of IL-37 in inhibiting the malignant phenotype of tumor cells1.Tumor cells were transfected with control siRNA or IL-37 siRNA and mock or IL-37b plasmids,and then activity of Racl was detected by PAK-PBD pull down assay and western blot was applied to detect the phosphorylation of Racl downstream effector-PAK.2.Cells pretreated with NSC23766 or siRNA for Racl were transfected with IL-37 siRNA and applied for transwell assy.3.IF was used to detect the co-localization of IL-37 and Racl in tumor cells.Co-IP was used to detect the interaction between endogenous IL-37 and endogenous Racl in BEL-7402 and THP-1 cells.IL-37 precursor,two mature IL-37 and wild type Rac1 plasmids were transfected into tumor cells respectively and the interaction between exogenous IL-37 and exogenous Racl was detected by co-IP.4.Co-IP and GST-pull down assay were used to detect the interaction between recombinant IL-37 protein and recombinant Racl protein.5.Inactive Rac1,active Racl,various Rac1 truncated mutants and IL-37b?1-45 plasmids were co-transfected into 293T cells,and then the interaction was detected by co-IP.6,Wild Racl and mock or IL-37b?1-45 plasmids were co-transfected in 293T cells,or 293T cells were transfected with increasing amounts of IL-37b?1-45 plasmids,and the membrane proteins and cytoplasm proteins were extracted respectively.Western blot was used to detect the expression of Racl,Na,K-ATPase and actin.IF was used to observe the expression of Rac1 in the cell membrane and in the cytoplasm.7.293T cells were transfected with increasing amounts of IL-37b?1-45 plasmids and p-PAK and PAK expression were detected by western blot.Mock and IL-37b?1-45 plasmids were transfected into A549 cells respectively,and then p-PAK and PAK expression were detected by western blot;F-actin polymerization was observed by IF.ResultsI The effect of IL-37 on the malignant phenotype of tumor1.IL-37 expression in tumors was significantly associated with tumor metastasis and patients' prognosis(1)IL-37 expression was down-regulated in HCC tissuesIL-37 expression was investigated in 20 HCC tissues and their non-cancerous counterparts using immunohistochemical staining.The results were similar to the previous study.IL-37 was mainly expressed in the normal liver tissues.Although there was variation in the level of IL-37 expression in HCC tumor tissues,IL-37 expression was higher in adjacent non-tumor inflammatory tissues.Moreover,it has been reported that IL-37 expression is significant associated with microvascular invasion and poor IL-37 expression predicts poor prognosis.(2)Low IL-37 expression in lung adenocarcinoma tissues was significantly associated with tumor metastasis and predicted poor prognosisTo further elucidate the clinical significance of IL-37 in tumor,we analyzed the relationship between IL-37 expression and clinicopathological parameters in 84 patients with lung adenocarcinoma.We found that IL-37 was highly expressed in normal bronchial epithelial cells(mainly in the cytoplasm),while IL-37 expression in lung adenocarcinoma tissues was low.We next analyzed the relationship between IL-37 expression and the clinical information of the patients.IL-37 expression was significantly associated with lymph node metastasis,pathological grade,T stage and AJCC stage.Moreover,overall survival(OS)of patients with low IL-37 expression was much poorer than that of patients with high IL-37 expression.Univariate analysis indicated that IL-37 expression as well as lymph node metastasis and AJCC stage were prognostic factors for OS.2.IL-37 suppressed growth and metastasis of tumor cells in vitro(1)IL-37 was mainly expressed in the cytoplasm of tumor cells and could be secreted to the outside of the cellIn order to explore the biological functions of IL-37,we firstly detected the level of IL-37 expression in a variety of HCC cells by western blot and found all of them could express IL-37.Using IF,we found that IL-37 was mainly expressed in the cytoplasm.Using ELISA,we found that IL-37 also could be secreted to the outside of the cells.(2)Intracellular IL-37 inhibited growth of tumor cellsTo determine effect of IL-37 on growth of tumor cells,we used CCK8 assay and found that IL-37 knockdown increased cell growth.After added different concentrations of recombinant IL-37 protein,the proliferation of tumor cells did not change.The results indicate that intracellular IL-37 inhibits growth of tumor cells.(3)Intracellular IL-37 suppressed tumor cell migration abilityTo determine effect of IL-37 on tumor cell migration,we used transwell assay.Overexpression of IL-37 inhibited migration ability of tumor cells.In accordance with the overexpression experiments,IL-37 knockdown markedly promoted migration of tumor cells.Taken together,these data indicate that IL-37 inhibits migration of tumor cells.As both intracellular and extracellular forms of IL-37 are functional,we then investigated which form of IL-37 inhibits tumor cell migration.We found that administration of different concentrations of exogenous rhIL-37 did not affect the migration of A549 cells that did not express IL-37.Moreover,the blockade of IL-37 by neutralizing antibody had no effect on the migration of HepG2 cells that could release IL-37.Consistently,IL-37 overexpression significantly inhibited the migration of A549 cells,but this effect could not be blocked by IL-37 neutralizing antibody.Collectively,these results indicate that the intracellular form of IL-37 suppresses the migration of tumor cells.3.IL-37 suppressed tumor growth and metastasis in vivo(1)IL-37 inhibited tumor growthTo provide in vivo evidence that IL-37 suppresses tumor growth,we established subcutaneous xenograft tumor model.The nude mice were injected subcutaneously with A549 cells infected with NC or IL-37b lentivirus and monitored for tumor growth.Compared with LV-NC group,LV-IL-37 group showed a decreased tumor size,but the decrease of tumor weight had no statistical significance.Overexpression of IL-37 in tumor was confirmed by immunohistochemical staining.(2)IL-37 inhibited tumor metastasisTo provide in vivo evidence that IL-37 suppressed tumor metastasis,we established metastatic colonization model,we injected LV-NC or LV-IL-37 A549 cells into nude mice through the tail vein and found that the weight loss and paralysis appeared later in LV-IL-37 group than that in LV-NC group.IL-37 overexpression markedly increased the survival rate.More importantly,IL-37 overexpression not only significantly reduced the number of mice with distant metastasis,but also dramatically reduced the number and size of metastatic tumors in lungs.Additionally,LV-IL-37 group had lower lung weight compared with LV-NC group.These results demonstrate that IL-37 markedly suppresses tumor metastasis in vivo.? The mechanism of IL-37 in inhibiting the malignant phenotype of tumor cells1.Intracellular IL-37 suppressed Racl activationNext,we aimed to exploring the underlining mechanism by which intracellular IL-37 inhibits migration of tumor cells.Using a PAK-PBD pull-down assay,we found that IL-37 knockdown enhanced Racl GTPase activity in tumor cells,while IL-37 overexpression in tumor cells had the opposite effects.PAK is a key downstream effector of Racl,we further examined the effect of IL-37 on PAK activation and found that IL-37 knockdown increased PAK phosphorylation,while IL-37 overexpression markedly attenuated PAK phosphorylation.Taken together,these results indicate that IL-37 inhibits Racl activation and subsequent PAK phosphorylation.2.Intracellular IL-37 inhibited tumor cell migration in a Racl dependent mannerTo further investigate whether IL-37 could inhibit tumor cell migration via suppressing Rac1-induced signal pathway,a Rac1 specific inhibitor NSC23766 and siRNA that mediated Racl knockdown were used to manipulate Racl activity.NSC23766 treatment effectively attenuated migration of BEL-7402 cells.IL-37 knockdown dramatically enhanced migration of tumor cells.But,NSC23766 treatment blocked the enhancement of cell migration mediated by IL-37 knockdown.Consistently,Rac1 knockdown also reversed the effects of IL-37 knockdown on migration of tumor cells.These results indicate that intracellular IL-37 suppresses the migration of tumor cells via inhibiting Rac1 signal pathway.3.Intracellular IL-37(amino acids 46-218)directly bound to Racl(1)IL-37 co-localized with Racl in tumor cellsTo elucidate the mechanisms by which intracellular IL-37 inhibits Racl,we first examined the association between IL-37 and Racl.Co-localization of intracellular IL-37 and Racl was observed in BEL-7402 and HepG2 cells.In addition,similar result was obtained in THP-1 cells(a human monocyte cell line derived from acute leukemia),indicating that the co-localization of intracellular IL-37 and Racl is common in a variety kinds of cells.(2)Endogenous IL-37 interacted with endogenous RaclNext,we investigated the interaction between IL-37 and Racl by immunoprecipitation(IP).An association between IL-37 and Rac1 was detected in BEL-7402 cells.Additionally,the similar interaction was obtained in resting,LPS-stimulated and PDGF-stimulated THP-1 cells.(3)Exogenous mature IL-37 interacted with exogenous RaclTo investigate which form of IL-37 can interact with Racl,IL-37b precursor(Flag-tagged IL-37)and two mature IL-37b plasmids(IL-37b?1-20 and IL-37b?1-45)were constructed.We found that only IL-37b?1-45 could interact with Rac1,while IL-37b and IL-37b?1-20 could not.(4)Mature IL-37 directly bound to RaclWe next tested whether mature IL-37 could interact with Racl directly by in vitro binding assays.After mixing purified recombinant mature human IL-37(contains amino acids 46-218)and GST-Rac1 protein together in vitro,the interaction between IL-37 and Racl was detected by co-IP and GST-pu1l down assay.We found that IL-37 could directly interact with Rac1.Collectively,these results demonstrate that intracellular mature IL-37 directly binds to Rac1.4.Intracellular IL-37 bound to the CAAX motif of Racl(1)Mature IL-37 interacted with GDP-Racl and GTP-Rac1To further clarify the mechanism by which IL-37 inhibits Racl activity,we next determined whether the interaction between mature IL-37 and Racl depends on Racl-activating status.We found that mature IL-37 could bind to both inactive and active forms of Rac1,indicating that the interaction between mature IL-37 and Racl was not dependent on Racl-activating status.(2)Mature IL-37 interacted with C-terminal HVR of RaclTo search for the domains of Racl that is responsible for the interaction with IL-37,a series of Myc-tagged Rac1 truncated mutants were constructed.By co-IP,we found that the C terminal HVR but not switch I or the insert region was required for mature IL-37 interaction(3)Mature IL-37 bound to CAAX motif of HVRHVR contains a polybasic region(PBR,contains a stretch of adjacent lysine and arginine residues)and a CAAX motif.By co-IP,we found that the CAAX motif,but not the PBR,was required for the Racl-IL-37 interaction.Collectively,these data indicate that intracellular IL-37 binds to both active form and inactive form of Racl,and CAAX motif of Rac1 is responsible for the interaction.5.Mature IL-37 inhibited membrane translocation of RaclMembrane translocation of Racl is a crucial step for its activation.The hydrophobic C-terminal region of Racl is responsible for anchoring Racl to the plasma membrane.The fact that IL-37 binds to the C-terminus of Racl promotes us to investigate the effects of mature IL-37 on Racl membrane translocation.We found that IL-37b?1-45 reduced the membrane-bound Rac1.Consistently,membrane-bound GTP-Rac1 in 293T cells was dramatically decreased with IL-37b?1-45 overexpression by immunofluorescence staining.Collectively,these results indicate that IL-37bA 1-45 inhibits membrane translocation of Racl,and thus attenuates its activation.6.Intracellular mature form of IL-37 inhibited Racl signaling(1)Mature IL-37 suppressed the phosphorylation of Racl downstream effector-PAKWe next investigated whether intracellular mature form of IL-37(amino acids 46-218)possessed main inhibitory role in Racl signaling.Overexpression of Racl-61L,the constitutively active Rac1,dramatically induced PAK phosphorylation in 293T cells.Intracellular mature IL-37 inhibited PAK phosphorylation in a dose-dependent manner.Similarly,the inhibitory effects of intracellular mature IL-37 on PAK phosphorylation was observed in A549 cells.(2)Mature IL-37 inhibited F-actin polymerization mediated by RaclActivated Racl can induce the assembly of filamentous actin(F-actin)structure through PAK-dependent and PAK-independent signaling.We next examined the effect of mature IL-37 on F-actin polymerization and found that F-actin polymerization rate was significantly decreased in mature IL-37 overexpressed A549 cells,compared with that in control group.Taken together,these data indicate that the mature form of IL-37 effectively inhibits Rac1 signaling.Conclusions1.Low IL-37 expression in tumors was significantly associated with tumor metastasis and predicted poor prognosis.2.Intracellular IL-37 inhibited tumor growth and metastasis in vitro and in vivo.3.IL-37 inhibited Racl activation.4.Mature IL-37 directly bound to the CAAX motif of Racl' s C-terminus.5.Mature IL-37 inhibited membrane translocation of Racl and thus attenuated its activation,leading to the decrease of PAK phosphorylation and F-actin polymerization.Originality and Significance1.For the first time,we demonstrate that Intracellular IL-37 inhibits tumor metastasis by in vitro and in vivo experiments.The results are significant for new technology of tumor therapy.2.For the first time,we demonstrate that mature IL-37 directly binds to Racl and inhibits its activation.The finding provides new understanding of IL-37 function.Limitations1.The number of samples needs to be expanded to make the correlation analysis between IL-37 expression and clinicopathological parameters more accurate.2.We will try to find the detection methods that can distinguish different isoforms and further clarify their expression levels in tumor tissues.This is of great significance for studying the role of IL-37 in tumors.