Lysyl oxidase is synthesized as a 50 kDa pro-enzyme, secreted into the extracellular environment where it is then processed by proteolytic cleavage to the functional 30 kDa enzyme and the 18 kDa propeptide. Expression of lysyl oxidase gene was found to inhibit the transforming activity of ras and was hence named as """"""""ras recision gene"""""""" (rrg). Over expression of k-ras and other members of ras family is found in oral cancer. Our preliminary data indicates that lysyl oxidase expression is low in several oral cancer cell lines. We recently reported that the 18 kDa pro-peptide domain, and not the 30 kDa active enzyme, inhibits the ras transformed phenotype of fibroblasts. We also reported that the lysyl oxidase pro-peptide inhibits epithelial to mesenchymal transition of breast cancer cells by affecting the ras dependent MEK/Erk and PI3K/Akt pathways. However, the mechanism by which the lysyl oxidase propeptide inhibits the ras signaling is not yet known. We have previously reported that lysyl oxidase interferes with a fibroblast growth factor loop in transformed fibroblasts. Our preliminary studies suggest that the lysyl oxidase pro-peptide inhibits ras activation and FGF-2 induced Erk phosphorylation in pharyngeal squamous cell carcinoma. Here we propose that the lysyl oxidase propeptide interferes with the FGF-2 signaling upstream of MEK/Erk, thereby affecting the ras signaling.
Two specific aims are proposed.
Aim 1 will identify the molecular targets of the lysyl oxidase propeptide. Effects of the lysyl oxidase pro-peptide on the FGF-2 signaling intermediates in two selected oral cancer cell lines will be evaluated. In particular, the effects on the phosphorylation of FGF receptors and other adaptor molecules will be determined. Furthermore, the effect on the formation of FGF receptor complexes will be determined. Indirect inhibition of ras signaling via SRC kinase and Sprouty 2 also will be evaluated.
Aim 2 will determine the structure function relationship of the lysyl oxidase pro-peptide. We hypothesize that the highly conserved regions within the lysyl oxidase propeptide sequence have a functional significance in inhibiting the transformed phenotype. We will use custom synthesized small peptides, which include those regions to treat oral cancer cells in order to evaluate the effect on ras dependent MEK/Erk pathway. Overall these studies will further our understanding of the signaling mechanisms involved in the inhibition of ras dependent pathways. Furthermore, the identification of functionally significant regions of the lysyl oxidase pro-peptide may help define novel therapeutic drugs for the treatment of ras dependent oral cancer. Project Narrative The lysyl oxidase pro-peptide inhibits the ras-dependent phenotype of cancer cells. Since most oral cancers are ras-dependent, we propose to investigate the mechanism by which the lysyl oxidase pro-peptide inhibits the ras signaling in oral cancer and to determine the shortest functional sequence of the lysyl oxidase pro-peptide. These studies will be of great importance to develop new therapeutic agents for oral cancer.
