This project is directed at identifying biochemical mechanisms of tumor cells which may be targets for early detection, intervention and/or therapeutic strategies, with a focus on the enzymatic processes responsible for post-translational processing of inactive precursor prohormones into bioactive peptide hormones. Our studies of tumor cell enzymes required for processing of precursor prohormones to active peptide hormones are comprised of three parts: biochemistry of peptidyl amidating enzymes (PAM), biology of PAM enzymes in human tumors, and effect of inhibiting PAM on tumor growth. We have used a PCR approach to identify novel human-specific forms of human PAM mRNA. These forms, a major form PAM-C and a quantitatively minor form PAM-D, have the functional domains previously reported for PAM enzymes from several species, but contain a novel linker region. By analogy with the known linker region in rat PAM-A, the new linker C will have significant biochemical effects on the activity rate, the pH optimum, glycosylation and sulfation status, and post-translational endoproteolytic processing of the new proteins. We have cloned mRNA for each of the bifunctional PAM forms for expression in mammalian transfected cells to determine the functional significance of the new enzymes. We have previously reported that non-endocrine lung tumor cell lines express high levels of PAM enzymes. We have confirmed, using immunohistochemical and in situ mRNA/mRNA hybridization, that this finding reflects the biology of the tumors in lung epithelium and is not an artifact of cell culture. Using the techniques described above and a quantitative PCR approach, we have recently determined that non- endocrine lung tumor cell lines preferentially express a form of PAM with lower activity towards lipophilic substrates than the principal PAM form expressed by more-neuroendocrine cell types. This finding suggests that unrecognized peptide hormones may be important to the biology of non-endocrine lung tumors. We are attempting to extend these findings to individual cells, including non-neoplastic morphological abnormalities in cigarette-carcinogen-damaged lungs, using an in situ PCR approach on archived human tumor sections. Approval has been received for tumor xenograft experiments for pre- clinical evaluation of PAM inhibitors as potential intervention and therapeutic agents. These studies have commenced, and two candidate first-generation inhibitors have been tested by two routes of administration. Results so far have been mixed, but no severe toxicities have been identified.
