Transplanted Melanoma Was Inhibited By JWA Deficiency Induced Changes In In Vivo Microenvironment

Recent studies have shown close relationships between microenvironmental components and tumorogenesis as well as tumor metastasis.On one hand,tumor cells could promote its growth and development by altering and maintaining the advantagerous condition via autocrine and paracrine secretion.On the other hand,the whole body and local tissue confine tumorogenesis and development through its homeostasis systems.It is well known that tumor and microenvironment are dependent of each other and can promote each other’s development while they have antagonistic and competitive effect to each other.IL-1α,a isoform of IL-1,is mainly produced by activated mononuclear-macrophage system.IL-1αnot only influences the inflammation process but also has the ability of promoting immunity and growth.It was reported that IL-1αcan advance the growth of melanoma by increased formation of blood vessels in xenograft mouse model.JWA,a gene known for its role in environmental response,had been found to be upregulated under different stress conditions.In recent years,we have investigated on the altering expression of JWA in tumor cells and their subsequent alterations of biological functions,JWA regulates microtubule reorganization and cell migration by modulating downstream molecules via MAPK signal pathway;JWA also regulates melanoma metastasis through intergrin signal pathway.Deficiency of JWA inhibits chemical induced skin papillomas via suppressing MAPK signaling in mice.Objective:to investigate the effects of JWA deficiency on tumor development in xenograft mouse model,and the involved molecular mechanism.Methods:Cre-Loxp mediated recombination was used to establish conditional conventional JWA deficiency mice.The melanoma growth was observed in two different genotypes mouse（knockout or wild type mice）in order to evaluate the influence of the altered microenvironment on melanoma induced by the deficiency of JWA.The potential differential cytokines in the serum of tumor-bearing mouse of two different genotypes were identified by two-dimensional electrophoresis and mass spectroscopy;Cell coculture model was applied to mimic in vivo interactions between tumor cells and macrophages in mice,and the tube formation assay was used to vandilate the role of identified cytokines on tumor growth.ELISA and Western blotting were used to evaluate expression of cytokines in serum,culture medium,cell extracts or tumor tissues.Results:1.JWA deficiency altered in vivo microenvironment inhibits melanoma growth in xenograft mouse.The body weights,tumor sizes in JWA^?2/?2 mice were less than those of JWA^+/+（P<0.05）mice;Moreover,the ratio of tumor size to body weight in JWA^?2/?2mouse was also reduced compared to wild type mice（P<0.05）.2.JWA deficiency suppressed clood vesselvessel formation in melanoma.Results of immunohistochemistry and Western blotting showed that the expression of vascular endothelial markers CD31 in JWA^?2/?2 mouse was significantly lower than that in JWA^+/+mouse（P<0.05）,and so did the inflammation-inducing factor COX-2 and the cell proliferation marker PCNA（P<0.05）.3.JWA deficiency blocked IL-1αproduction in xenograft mouse.Results of two-dimensional electrophoresis and mass spectroscopy showed that the production of IL-1αin the serum of JWA^?2/?2 mouse were much lesser than those of JWA^+/+mouse（P<0.05）,and these were confirmed by ELISA assay.4.JWA deficiency suppressed blood vessel formation in melanoma in mouse was partly due to the blockade of IL-1αproduction.The IL-1αin the supernatant of peritoneal primary macrophage of JWA^?2/?2 mice cocultured with B16F10 cells was less than that macrophages of the JWA^+/+mice cocultured with B16F10（P<0.05）.The cocultured supernatant of primary peritoneal macrophage of JWA^?2/?22/?2 mouse with B16F10 inhibited tube formation compared to the supernatant from JWA^+/+mice（P<0.05）.Transwell assays also showed that the cocultured supernatant of primary peritoneal macrophage of JWA^+/+mouse with B16F10 induced more HUVECs migration（P<0.05）.Moreover,after adding neutralizing antibody of IL-1αin coculture system of primary peritoneal macrophage of JWA^+/+mouse with B16F10,both the tube formation（P<0.05）and the migration ability（P<0.05）were reversed.5.IL-1αsuppressed the blood vessel formation in melanoma via reduce the secretion of VEGF and TNF-α.The IL-1α,as well as VEGF and TNF-αin the cocultured supernatant of primary peritoneal macrophage of JWA^?2/?2 mouse with B16F10 was lesser than that of the JWA^+/+with B16F10（P<0.05）.After adding neutralizing antibody of IL-1αin coculture system of primary peritoneal macrophage of JWA^+/+mouse with B16F10,the VEGF production was decreased as expected（P<0.05）,and the TNF-αproduction was also reduced to the level that equal to the cocultured supernatant of primary peritoneal macrophage of JWA^?2/?2 mouse with B16F10（P<0.05）.In conclusion,JWA played a vital role in B16F10 xenograft development.JWA can regulate the IL-1αproduction,effect on tumor blood vessel formation,thus influenced tumor progress.Our study provided new evidences that JWA defiency induced changes of in vivo microenvironment components in mice palys an important role on melanoma development,and the IL-1αlinked VEGF might be the downstream targets involved in this process.The detailed molecular mechanisms of how JWA deficiency induced changes of in vivo microenvironment are need to be further investigated.