Neuropeptide receptors and cell signals as targets in the development of novel therapies for small cell lung cancer

Neuropeptides including bombesin and its mammalian homologue gastrin releasing peptide, stimulate proliferation and are implicated as growth factors in a variety of biological processes, including autocrine and paracrine growth of small cell lung cancer (SCLC). These peptides bind to seven transmembrane domain receptors which lack intrinsic tyrosine kinase activity and activate second messenger pathways via heterotrimeric G proteins. This thesis covers two main areas of investigation: (1) NEUROPEPTIDES AND TYROSINE PHOSPHORYLATION Recently, neuropeptides including bombesin have been shown to induce tyrosine phosphorylation in Swiss 3T3 cells, a useful model system for elucidating signal transduction pathways. The mechanism by which tyrosine phosphorylation is induced by G protein coupled receptors is unknown. This thesis shows that the non-hydrolyzable GTP analogue, GTP?S, induced tyrosine phosphorylation of multiple proteins including the focal adhesion associated proteins p125 focal adhesion kinase and paxillin in permeabilized Swiss 3T3 cells. The pattern of tyrosine phosphorylation closely resembled that induced by neuropeptides in intact cells. Our results suggest that the small GTP binding protein rhop21 rather than heterotrimeric GTP binding proteins mediates GTP?S induced tyrosine phosphorylation. The role of tyrosine phosphorylation in bombesin mediated mitogenesis is unknown. Here, it is shown that addition of the protein tyrosine kinase inhibitor [(3,4,5,-trihydroxyphenyl)-methylene]-propanedinitrile (tyrphostin) to intact Swiss 3T3 cells reduces bombesin induced tyrosine phosphorylation and inhibits bombesin mediated c-fos expression and DNA synthesis. The results demonstrate that inhibitors of tyrosine kinase activity prevent neuropeptide mediated mitogenesis and may be of therapeutic value for SCLC. (2) NOVEL SCLC THERAPIES Substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP and [Arg6, DTrp7,9, MePhe8] SP (6-11) inhibit the action of many different calcium mobilizing neuropeptides including vasopressin and bombesin in Swiss 3T3 cells and SCLC cell lines and block the growth of SCLC xenografts in nude mice. However, their mechanism of action is not understood. This thesis shows that these SP analogues reversibly inhibit neuropeptide- but not GTP?S-stimulated inositol phosphate production in permeabilized Swiss 3T3 cells and competitively inhibit ligand binding in intact cells or membrane preparations. The results suggest that these SP analogues block the action of vasopressin and bombesin at the receptor level. Identification of factors which stimulate SCLC cell growth is relevant to the development of novel SCLC therapies. Results presented here demonstrate that although serum mobilizes calcium and stimulates growth of SCLC cells, Ca2+ mobilization is not required for serum induced growth of these cells. The polypeptide growth factors hepatocyte growth factor and stem cell growth factor were found to stimulate colony growth of SCLC cells through a Ca2+ independent pathway. Since tyrphostin is known to inhibit polypeptide receptor tyrosine kinases and inhibits neuropeptide stimulated mitogenesis, the effect of tyrphostin with or without SP analogues was tested on SCLC growth in vitro. It is shown that tyrphostin and SP analogues synergistically inhibit SCLC cell growth