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Abstract: Although it has been suggested that smad7 blocks downstream signaling of TGF-β, the role of smad7 in the EGF signaling pathway has not been fully elucidated. We determined the effect of smad7 on EGF-induced MMP-9 expression in SKBR3 breast cancer cells. The expression of smad7 and MMP-9 was increased by EGF or TGF-β1, respectively, and further increased by EGF and TGF-β1 co-treatment. EGF induced the phosphorylation of EGFR, smad3, ERK, and JNK, and MMP-9 expression was decreased by the EGFR inhibitor, AG1478. In addition, EGF-induced MMP-9 expression was inhibited by UO126 (a MEK1/2 inhibitor) or SIS3 (a smad3 inhibitor), but not by SP600125 (a JNK inhibitor). Interestingly, EGF-induced smad3 phosphorylation was completely blocked by smad7 over-expression, but not the phosphorylation of ERK and JNK. EGF- or TGF-β1-induced MMP-9 expression was completely decreased by adenoviral-smad7 (Ad-smad7) over-expression. We also investigated the role of smad3 on EGF-induced MMP-9 expression and showed that EGF-induced MMP-9 expression was decreased by smad3 siRNA transfection, whereas EGF-induced MMP-9 expression was further increased by smad3 over-expression, as expected. This study showed that EGF-induced smad3 phosphorylation mediates the induction of MMP-9, whereas smad7 inhibits TGF-β1 as well as the EGF signaling pathway in SKBR3 cells. [Copyright &y& Elsevier]

Abstract: The number of epidermal growth factor receptors (EGFRs) and their ligands are highly expressed in malignant tumor cells. The EGF signaling pathway is also activated in up to one-third of patients with breast cancer. In this study, we investigated the novel function of the JAK3 inhibitor, WHI-P131, on EGF-induced MMP-9 expression and the regulatory mechanism of EGF-induced MMP-9 expression in SKBR3 cells. We observed that EGF increased MMP-9 mRNA and protein expression in a dose-dependent manner. EGF also induced the phosphorylation of EGFR, ERK, and STAT-3, and these effects were inhibited by the EGFR inhibitor, AG1478. To investigate the involvement of the STAT-3 pathway on EGF-induced MMP-9 expression, we pretreated SKBR3 cells with JAK1, JAK2, and JAK3 inhibitors prior to EGF treatment. The results showed that the JAK3 inhibitor, WHI-P131, as well as JAK3 siRNA transfection, but not the JAK1 and JAK2 inhibitors, significantly decreased EGF-induced MMP-9 expression. In addition, EGF-induced STAT-3 phosphorylation was only inhibited by WHI-P131. We then transfected cells with adenoviral STAT-3 (Ad-STAT-3), followed by treatment with EGF. Interestingly, EGF-induced MMP-9 expression was decreased by Ad-STAT-3 overexpression in a dose-dependent manner, while it was significantly increased by STAT-3 siRNA transfection. Our results also showed that basal levels of MMP-9 expression were significantly increased by constitutive active-MEK (CA-MEK) overexpression. EGF-induced ERK phosphorylation was prevented by WHI-P131, but not by JAK1 and JAK2 inhibitors. On the other hand, EGF-induced MMP-9 expression was decreased by the MEK1/2 inhibitor, UO126. Therefore, for the first time, we suggest that the JAK3 inhibitor, WHI-P131, inhibits EGF-induced STAT-3 phosphorylation as well as ERK phosphorylation. The JAK3/ERK pathway may play an important role in EGF-induced MMP-9 expression in SKBR3 cells. [Copyright &y& Elsevier]