Study Of The Role Of ADAMTS18 In The Metastasis Of Melanoma

ADAMTS18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs（ADAMTS）family of proteases,which plays an important role in the physiological and pathological processes such as organ development,reproduction,angiogenesis,blood clotting,inflammation,and tumor.Mutation and knockout of Adamts18 are especially associated with tumorigenesis.This study focuses on the effects of Adamts18 on the metastasis of melanoma,which provides an important theoretical basis for the treatment of melanoma in the later stage.This study is divided into four parts:1.Generation of Adamts18 knockout mice with C57BL/6 genetic backgroundFirst,Adamts18 heterozygote mice(Adamts18^+/-)with C57BL6/129SV background were backcrossed to wild-type（WT）C57BL/6 mice for 10 generations to obtain Adamts18^+/-mice with C57BL/6 background.They were intercrossed to get pure C57BL/6 genetic background WT control(Adamts18^+/+)and Adamts18 KO mice(Adamts18^-/-)for further experiments.2.The effect of ADAMTS18 on experimental melanoma metastasisExperimental metastasis refers to the later steps of the metastatic migration process（extravasation from the bloodstream and then growth into pulmonary tumor）.We next evaluated the effect of Adamts18 deficiency on experimental lung metastases of B16melanoma cells inoculated via the tail vein in mice（2×10⁵ viable cells per mouse）.B16 melanoma cells disseminated to multiple organs including lung,spleen,enterocoelia,heart and liver.Among these organs,lung metastasis demonstrated the significant difference between Adamts18 KO mice and WT controls.The surface nodules and mean volumes of nodules of metastatic tumors were significantly increased in Adamts18 KO mice compared with WT controls.Thus,ADAMTS18 deficiency leads to increased lung metastatic focuses in experimental metastasis model.3.The effect of ADAMTS18 on ECM remodelingBecause the extracellular matrix（ECM）microenvironment plays important roles in tumor growth and expansion,we thus to clarify whether enhanced spontaneous and experimental lung metastasis in Adamts18 KO mice is related to ECM remodeling.The key tumor metastasis-associated ECM proteins were examined.The results showed that the expressions of laminin（LN）was significantly increased in lung tissues of Adamts18KO mice compared to those in WT controls.Because LN is the critical regulator of the PI3K-Akt-mTOR-SK6 signaling pathways,which plays important roles in tumor metastasis and expansion,we further examined the expression levels of PI3K and SK6by western blot.The results showed that PI3K and SK6 were significantly activated in the lung tissues of Adamts18 KO mice compared to those in WT controls.4.LN promotes the adhesion,spreading and chemotaxis of B16 cellsBy transwell chemotaxis assays,we found that the numbers of cells migrated to the lower chamber were increased as the concentration of LN increased.When detached and replated,B16 cells attached and spreaded faster on LN with the increase of LN concentration.Therefore,ADAMTS18 deficiency promotes melanoma growth in lung linked to remodeling of lung LN.