Abstract 3380: The serine protease inhibitor PN-1 induces activation of FGFR-mediated signaling

We are studying the role of PN-1 (Protease Nexin-1) in breast cancer. PN-1 is a serine protease inhibitor (serpin) that blocks a broad spectrum of proteases including among others tPA, uPA and thrombin. Once bound to a target protease, the PN-1/protease complexes bind with high affinity to Low Density Lipoprotein Receptor related Protein (LRP-1). A bioinformatic analysis revealed that PN-1 is overexpressed in a significant proportion of human tumors from different origins. Moreover, PN-1 expression levels correlate with markers of poor prognosis in breast cancer. PN-1 is also expressed in breast tumor cell lines and we recently showed that PN-1 is required by the malignant 4T1 mammary cancer cell line to metastasize to the lungs. Indeed, when mammary fat pads of Balb/c mice were injected with PN-1-knock down (KD)-4T1 cells, primary mammary tumors developed and these grew similarly as control 4T1 cells. However, the PN-1 KD tumors displayed a significant decrease in their potential to form lung metastasis, in comparison to control tumors. The mechanism by which complexes of serpin/protease mediate their effects in cancer cells is still largely unknown. Thus, our current studies are aimed at deciphering how the protease/PN-1 complex impacts on intracellular signaling in order to promote metastasis. Using the PN-1 negative 168FARN mammary tumor cell line, we have found that PN-1/protease complexes bind LRP-1 receptor and induce MMP-9 expression via Erk1/2 pathway activation. Importantly, the PN-1-driven regulation of MMP-9 expression was also demonstrated to be central to PN-1's prometastatic effect in 4T1 cells. Here by the use of Western Blot and specific inhibitors, we show that PN-1/protease complex treatment of fibroblast growth factor receptor (FGFR)-expressing tumor cells induces Erk signaling. Furthermore, fibroblast growth factor receptor substrate 2 (FRS2), a lipid anchored docking protein that plays a crucial role in mediating FGFR and tropomyosin receptor kinase (Trk)-induced signaling, was also found to be phosphorylated upon PN-1/protease complex addition. Preincubation of the cells with the FGFR inhibitor, TKI 258, abrogated phosphorylation of FRS2, Erk and Shc upon PN-1/protease complex treatment. In contrast, inhibitors of EGFR or PDGR did not have any effect on PN-1-induced phosphorylation of these proteins. This suggests that the PN-1/protease complex via binding to LRP-1 may transactivate FGFR and potentially other receptors known to activ