Role of non-receptor and receptor tyrosine kinases (TKs) in the antitumor action of alpha-difluoromethylornithine (DFMO) in breast cancer cells.

We have shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis, reduces the invasive and metastatic properties of MDA-MB-435 breast cancer cells while activating multiple signal transduction pathways, including MAPK, Stat3, Stat1, and JNK. Since the activity of these signaling mechanisms is frequently regulated by upstream tyrosine kinases (TKs), we tested whether non-receptor and receptor TKs may be involved in the signaling and biological effects of DFMO in MDA-MB-435 cells. Treatment with DFMO (1 mM for 48 h) did not affect Src phosphorylation (Tyr 416). Administration of the Src-family members inhibitor PP-1 (1 microM), blocked Src phosphorylation in the absence and in the presence of DFMO, but did not block the signaling effects of DFMO (increased phosphorylation of Stat3, Stat1, ERK and JNK). PP-1 treatment, on the other hand, inhibited the invasiveness of MDA-MB-435 cells in matrigel and potentiated the anti-invasive effect of DFMO. Next, we focused on the role of receptor TK. Western analysis of cell lysates from MDA-MB-435 cells failed to show the presence of EGF-R and HER-2neu but demonstrated the expression of c-Met, the receptor for hepatocyte growth factor (HGF). Therefore, we tested the effect of DFMO on the HGF/c-Met pathway which is strongly implicated in the progression of human breast cancer. We found that DFMO treatment blocked HGF-induced c-Met phosphorylation in MDA-MB-435 cells, suggesting that its anti-invasion action may be mediated, at least in part, by blocking c-Met signaling. Next, we showed that 1 mM DFMO suppressed HGF induced invasiveness of MDA-MB-435 cells in matrigel. Combination administration of DFMO with suboptimal doses of PHA-665752, a specific c-Met inhibitor, reduced invasiveness to an even greater extent than the individual treatment. These findings indicate that Src-family members, while not involved in DFMO action, promote invasiveness of breast cancer cells and their inhibition may enhance the antitumor effect of PA depletion. Our data also point to inhibition of HGF/c-Met pathway as a possible novel approach to enhancing the antitumor action of DFMO.