LOX-PP Inhibits FGF-2 Induced Ras Signaling in Oral Cancer

Objectives: Lysyl oxidase (LOX) is secreted as a 50-kDa inactive pro-enzyme (Pro-LOX) and cleaved to a functional 30-kDa enzyme and an 18-kDa pro-peptide (LOX-PP). We have previously shown that the LOX-PP domain, and not the 30-kDa enzyme, has the ras recission activity demonstrated by the inhibition of the ras-transformed phenotype in fibroblasts. It is reported that several members of the ras family are over-expressed in oral cancers. Furthermore, FGF-2 is reported to play an important role in the angiogenesis and growth of oral squamous cell carcinoma. The aim of the present study is to evaluate whether LOX-PP inhibits ras signaling in oral cancer cells by interfering with FGF-2 signaling.

Methods: LOX expression in pharyngeal squamous cell carcinoma (FADU) cells was evaluated compared to normal gingival epithelial cells using real time qPCR and Northern Blotting. Ras activation in FADU cells transfected with LOX-PP was evaluated using a Ras GTPase chemi ELISA kit (Active Motif). The effect of LOX-PP on phosphorylation of Erk, which is a down stream target of ras signaling, was determined by Western blotting using anti-phospho-Erk antibody and normalized using anti-total Erk antibody (Cell Signaling Technology).

Results: Real time qPCR and Northern blotting experiments revealed that LOX expression is significantly low in FADU cells. LOX-PP inhibited the ras activation in FADU cells by about 50%. In FADU cells, the Erk phosphorylation was induced by 20 ng/ml FGF-2 within 20 minutes determined by Western blotting. Addition of 10 ng/ml LOX-PP to FADU cells inhibited the Erk phosphorylation by about 50%.

Conclusions: FADU cells express low levels of lysyl oxidase compared to normal gingival epithelial cells. LOX-PP can inhibit the FGF-2 induced ras signaling in FADU cells determined by ras activation and Erk phosphorylation assays.

Supported by: NIH grants CA82742 and DE14066