TGF-beta-p38MAPK signaling contributes to tumor invasion and pulmonary metastases by increasing MMP9 activity and cell motility

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Transforming growth factor beta (TGF-b) is a potent tumor suppressor but at the late stages of cancer it can promote formation of highly metastatic tumors by stimulating epithelial to mesenchymal transition (EMT), cell migration, and changes in tumor microenviroment. We have shown that the p38 mitogen activated protein kinase (p38MAPK) pathway contributes in TGF-b-mediated EMT and tumor cell migration (Bakin et al., JCS, 2002). In this study we investigated the contribution of p38MAPK signaling in TGF-beta-mediated metastasis of breast cancer MDA-MB-231 in spontaneous and experimental metastasis mouse models. Expression of constitutively active TGF-b type I receptor Alk5-204D in MDA-MB-231 (MB-231) cells enhanced cell migration, whereas kinase-inactive receptor Alk5-232R reduced cell migration. Alk5-204D increased by 4-fold tumor cell invasion in the matrigel invasion assay. Examination of matrix metalloproteinase activity in zymography assays showed an 8-fold increase in MMP9 activity but no effect on MMP2 and MMP1. Application of p38MAPK inhibitors reduced MMP9 activity and blocked cell invasion. RT-PCR showed a 4-fold increase in MMP9 mRNA in Alk5-204D cells compared to control cells. Alk5-204D-expressing cells formed 4-times more pulmonary macrometastatic lesions compared to control cells when injected in tail vein of SCID mice (P<0.01). To study TGF-b effects on the spontaneous metastasis, tumor cells were placed into mammary fat pad of SCID female mice. Alk5-204D significantly reduced tumor latency and enhanced primary tumor growth compared to control and Alk5-232R cells. Analysis of metastasis showed that Alk5-204D cells formed significantly more pulmonary metastases compared to control and Alk5-232R cells. To investigate the role of p38MAPK in metastasis, we generated cell lines expressing dominant negative (dn) MKK6AL and p38AGF. MMP9 in MDA-MB-231 and Alk5-204D cells was suppressed using shRNA to human MMP9. DN-mutants reduced cell motility, while shMMP9 reduced MMP9 activity examined in zymography assays. The effect of the dn-mutants and shMMP9 on metastasis is under investigation. These studies suggest that TGF-b signaling enhances tumor cell invasion and metastasis by increasing MMP9 activity and cell migration via p38MAPK dependent mechanisms.