TLR4Ligand/H₂O₂Enhances TGF-β1Signaling To Induce Metastatic Potential Of Non-invasive Breast Cancer Cells By Activating Non-Smad Pathways

Aim:The efficient activation of multiple signaling pathways is the important driving force for tumor cell metastasis. TGF-β1has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-β1was inefficient in inducing metastatic potential of many non-invasive human tumor cells. It has been found that TLR4ligand and H2O2also have the potential to activate non-Smad pathways which could be activated by TGF-β1. Therefore, in this study we investigated whether TGF-β1could induce the metastatic potential of non-invasive tumor cells in presence of TLR4ligand and/or H2O2, and whether TLR4ligand and/or H2O2could enhance TGF-β1signaling in non-invasive tumor cells.Methods:Invasive capacity was evaluated using matrigel invasion assay and actin polymerization analysis. Anoikis-resistance was determined by flow cytometry and colony formation in soft-agar. The expression of genes was detected using real-time RT-PCR and Western blot. The activation of signaling pathways was detected by Western blot. The metastasis and proliferation of tumor cell in experimental mouse metastasis model were analyzed by immunohistochemistry.Results:Here we report that the enhancement of TGF-β1signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-β1alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4ligand (LPS) and H2O2cooperated with TGF-β1to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-κB. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-β1signaling by down-regulating Nm23-H1expression and up-regulating the expression of TβRⅠ and TβRⅡ, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-β1signaling induced higher expression of SNAI2, which also promoted TβRⅡ expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-β1/H2O2/LPS. Consistent with the activation pattern of signaling pathways, the invasive capacity and anoikis-resistance of non-invasive breast cancer cells were gradually increased after prolonged stimulation with TGF-β1/H2O2/LPS. The metastatic potential induced by TGF-β1/H2O2/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice.Conclusion:The findings in this study demonstrated that H2O2/LPS could enhance TGF-β1signaling to induce the sustained activation of both Smad and non-Smad pathways in non-invasive breast cancer cells, and thus promoting the metastatic capability of non-invasive breast cancer cells. Metastases are responsible for most cancer deaths. Given that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, targeting one of these stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.