The Molecular Mechanism Of JWA Regulates Melanoma Metastasis Via Integrin Signaling

Tumor metastasis is the most common cause of death in cancer patients. The metastatic process of cancer is consist of a number of distinct steps including detachment of tumor cells from the primary site, penetration of tumor cells through the endothelium of blood or lymphatic vessels to enter the circulation systems, and finally, in the new host environment, stick and reinvasion, proliferation into a secondary tumor. There is increasing amount of data show that in the process of cancer metastasis, many molecules are activated and interact with each other,which consist a complex signal network. Determining the signal network and key mechanism in the cancer metastasis pathways may aid in inhibiting metastasis and curing cancer.JWA, a newly identified novel microtubule-associated protein （MAP）, was recently demonstrated to be indispensable for the rearrangement of actin cytoskeleton and activation of MAPK cascades induced by arsenic trioxide （As2O3） and phorbol ester （PMA）. JWA depletion blocked the inhibitory effect of As2O3 on HeLa cell migration, but enhanced cell migration after PMA treatment. As cancer cell migration is a hallmark of tumor metastasis and the functional role of JWA in cancer metastasis is not understood, here we show that JWA plays an important role in melanoma metastasis.Objectives: To investigate the role and the behind molecular mechanisms of JWA in the metastasis of melanoma.Methods:?Two metastatic melanoma cell lines B16-F10 and A375 were selected as our in vivo and in vitro models. Stable clones were generated by G418 screening that with knock-down JWA expression （B16-As and A375-As） and vector control （B16-Vec and A375-Vec）. Mouse in vivo metastasis assay was used to test the metastatic ability of melanoma cells by tail vein injecting the screened melanoma cells. The wound healing assay, transwell assay and immunofluorescence analysis were further used to examine whether JWA regulates cell migration; the adhesion assay, invasion assay and zymography were used to test if JWA regulates cell adhesion and invasion abilities. Finally, we examined those molecules identified in cell culture and animal models in clinic samples. Results:For murine B16 melanoma cell line in vivo metastasis animal model, extensive tumor formation was found in B16-As group. In contrast, the lungs from the mice injected with B16-Vec cells showed much fewer and smaller tumor nodules compared to B16-As group. For A375 human melanoma cell line in vivo metastasis animal model, the metastatic tumors were found in the lymph nodes of the neck of about 40% mice in A375-As group, however, no metastatic tumor was found in the lymph nodes of the neck in A375-Vec group. Cell proliferation assay indicated very similar results between control cells and the JWA knockdown cells. Therefore the increased number of tumor nodules in JWA knockdown group is due to the promotion of metastasis, not the promotion of cell proliferation by pEGFP-C1-As JWA plasmid. We previously reported that JWA knockdown could promote B16 melanoma cell migration. Here we confirmed this conclusion by using wound healing assay, transwell assay and immunofluorescence analysis. Our data showed that knockdown of JWA significantly promoted cell migration by regulating actin cytoskeleton rearrangements when compared with the cells carrying control vector. There were more stress fibers running across the cells in the JWA knockdown cells while the fibers in the control mainly localized at the periphery of the cells. JWA knockdown cells also showed higher degree of cell spreading when compared to the ILK knockdown cells.Since JWA is involved in actin organization in cancer cells, we tested if JWA regulated cell attachment on fibronectin and vitronectin coated plates. Data showed that in B16 cell line, JWA knockdown increased cell attachment ability of melanoma cell in fibronectin- and vitronectin-coated plates by 103% and 69%, respectively. To further confirm the result, JWA shRNA was used to knockdown the JWA protein expression in human melanoma cell line A375. The silencing of JWA in A375 cells also increased cell attachment ability in fibronectin- and vitronectin-coated plates by 75% and 58%, respectively.We next investigated the role of JWA in invasion of melanoma cells. We found that cells from B16-As cells and A375-shRNA cells had increased the ability to invade through Matrigel-coated Boyden chamber by 173% and 79%, respectively, compared to the control cells. Invasive ability of cancer cells can be regulated by matrix metalloproteinases. We performed zymography to measure the MMPs activities in melanoma cells. Our results showed that there was no significant difference in MMP-9 activity between B16-As cells and B16-vector cells, but MMP-2 activity was dramatically increased in B16-As cells by 4.5-fold.When JWA is knocked down, integrinα_Vβ₃ signaling is intensified. Furthermore, inhibitions of integrinα_Vβ₃ function in B16-As and A375-sh cells by siRNA or LM609 strongly reduced the JWA-mediated adhesion and invasion abilities in melanoma cells. Analysis of the integrinα_Vβ₃ promoter revealed several Sp1 binding sites that may be important for integrinα_Vβ₃ transcription in B16 and A375 cells. To confirm this proposal, we knock down Sp1 in B16-As and A375-sh cells by using siRNA, data indicated that expression of integrinα_Vβ₃ was highly reduced, as well as the adhesion and invasion abilities of melanoma cells.We further examined JWA,αV andβ3 protein expression in human melanoma tissues. The high level of immunoreactivity for JWA was detected in normal nevi. In contrast, very weak immunoreactivity of JWA was observed in dysplastic nevi and malignant melanoma. JWA staining had a significant inverse correlation with integrinαV andβ3 expression in these biopsies. Western blot results also showed that JWA is reduced in melanoma cell lines compared to normal melanocytes. Conclusion: Our data demonstrated that JWA knockdown increased the adhesion and invasion abilities of melanoma cells. Furthermore, JWA knockdown in B16-F10 and A375 melanoma cells significantly promoted the formation and growth of metastatic colonies in vivo. Moreover, in the tumor biopsies from human melanoma, JWA expression was significantly decreased compared with normal nevi. In addition, we found that JWA knockdown could intensify tumor integrinα_Vβ₃ signaling by regulating nuclear factor Sp1. These findings suggest that JWA suppresses melanoma metastasis and may serve a potential therapeutic target for human melanoma.