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Advanced breast cancer, as well as ineffective treatments leading to surviving cancer cells, can result in the dissemination of these malignant cells from the primary tumor to distant organs. Recent research has shown that microRNA 200c (miR‐200c) can hamper certain steps of the invasion–metastasis cascade. However, it is still unclear whether miR‐200c expression alone is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR‐200c expression in MDA‐MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs, including lung, liver, brain, and spleen, revealed a dramatically reduced metastatic burden in mice with miR‐200c‐expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR‐200c on collective‐ and single‐cell migration in vitro, utilizing MDA‐MB 231 and MCF7 cell systems with genetically modified miR‐200c expression. Analysis of collective‐cell migration revealed confluence‐dependent motility of cells with altered miR‐200c expression. Additionally, scratch assays showed an enhanced predisposition of miR‐200c‐negative cells to leave cell clusters. The in‐between stage of collective‐ and single‐cell migration was validated using transwell assays, which showed reduced migration of miR‐200c‐positive cells. Finally, to measure migration at the single‐cell level, a novel assay on dumbbell‐shaped micropatterns was performed, which revealed that miR‐200c critically determines confined cell motility. All of these results demonstrate that sole expression of miR‐200c impedes metastasis formation in vivo and migration in vitro and highlights miR‐200c as a metastasis suppressor in breast cancer. [ABSTRACT FROM AUTHOR]

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high‐throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho‐AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR‐200c was overexpressed in lung metastases, implicating an inhibitory feed‐back loop to PI3K‐AKT. Moreover, transfection of miR200c‐mimic in U2‐OS cells reduced phospho‐AKT levels but increased cellular migration and proliferation. Notably, miR‐200c expression strongly correlated with miR‐141 and with the osteogenic inhibitor miR‐375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR‐200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR‐200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho‐AKT in the primary tumor and miR‐200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas.

Our most recent studies demonstrate that Rho GDIβ is able to promote human bladder cancer ( BC) invasion and metastasis in an X-link inhibitor of apoptosis protein-dependent fashion accompanied by increased levels of matrix metalloproteinase ( MMP)-2 protein expression. We also found that Rho GDIβ and MMP-2 protein expressions are consistently upregulated in both invasive BC tissues and cell lines. In the present study, we show that knockdown of Rho GDIβ inhibited MMP-2 protein expression accompanied by a reduction of invasion in human BC cells, whereas ectopic expression of Rho GDIβ upregulated MMP-2 protein expression and promoted invasion as well. The mechanistic studies indicated that MMP-2 was upregulated by Rho GDIβ at the transcriptional level by increased specific binding of the transcription factor Sp1 to the mmp-2 promoter region. Further investigation revealed that Rho GDIβ overexpression led to downregulation of miR-200c, whereas miR-200c was able directly to target 3′- UTR of jnk2 mRNA and attenuated JNK2 protein translation, which resulted in attenuation of Sp1 mRNA and protein expression in turn, inhibiting Sp1-dependent mmp-2 transcription. Collectively, our studies demonstrate that Rho GDIβ overexpression inhibits miR-200c abundance, which consequently results in increases of JNK2 protein translation, Sp1 expression, mmp-2 transcription, and BC invasion. These findings, together with our previous results showing X-link inhibitor of apoptosis protein mediating mRNA stabilization of both Rho GDIβ and mmp-2, reveal the nature of the MMP-2 regulatory network, which leads to MMP-2 overexpression and BC invasion. [ABSTRACT FROM AUTHOR]

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high-throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho-AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR-200c was overexpressed in lung metastases, implicating an inhibitory feed-back loop to PI3K-AKT. Moreover, transfection of miR200c-mimic in U2-OS cells reduced phospho-AKT levels but increased cellular migration and proliferation. Notably, miR-200c expression strongly correlated with miR-141 and with the osteogenic inhibitor miR-375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR-200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR-200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho-AKT in the primary tumor and miR-200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas. [ABSTRACT FROM AUTHOR]

Our most recent studies demonstrate that RhoGDIβ is able to promote human bladder cancer (BC) invasion and metastasis in an X-link inhibitor of apoptosis protein-dependent fashion accompanied by increased levels of matrix metalloproteinase (MMP)-2 protein expression. We also found that RhoGDIβ and MMP-2 protein expressions are consistently upregulated in both invasive BC tissues and cell lines. In the present study, we show that knockdown of RhoGDIβ inhibited MMP-2 protein expression accompanied by a reduction of invasion in human BC cells, whereas ectopic expression of RhoGDIβ upregulated MMP-2 protein expression and promoted invasion as well. The mechanistic studies indicated that MMP-2 was upregulated by RhoGDIβ at the transcriptional level by increased specific binding of the transcription factor Sp1 to the mmp-2 promoter region. Further investigation revealed that RhoGDIβ overexpression led to downregulation of miR-200c, whereas miR-200c was able directly to target 3'-UTR of jnk2mRNA and attenuated JNK2 protein translation, which resulted in attenuation of Sp1mRNA and protein expression in turn, inhibiting Sp1-dependent mmp-2 transcription. Collectively, our studies demonstrate that RhoGDIβ overexpression inhibits miR-200c abundance, which consequently results in increases of JNK2 protein translation, Sp1 expression, mmp-2 transcription, and BC invasion. These findings, together with our previous results showing X-link inhibitor of apoptosis protein mediating mRNA stabilization of both RhoGDIβ and mmp-2, reveal the nature of the MMP-2 regulatory network, which leads to MMP-2 overexpression and BC invasion., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)