TLR4 Ligand/H2O2 Enhances TGF-β1 Signaling to Induce Metastatic Potential of Non-Invasive Breast Cancer Cells by Activating Non-Smad Pathways

TGF-beta 1 has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-beta 1 was inefficient in inducing metastatic potential of many non-invasive human tumor cells. Here we report that the enhancement of TGF-beta 1 signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-beta 1 alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4 ligand (LPS) and H2O2 cooperated with TGF-beta 1 to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-kappa B. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-beta 1 signaling by down-regulating Nm23-H1 expression and up-regulating the expression of T beta RI and T beta RII, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-beta 1 signaling induced higher expression of SNAI2, which also promoted T beta RII expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-beta 1/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-beta 1/H2O2/LPS. The metastatic potential induced by TGF-beta 1/H2O2/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice. The findings in this study suggested that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, and that targeting one of stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.