Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome that is often associated with squamous cell carcinomas. The syndrome appears to be mediated by the production by tumors of a novel parathyroid hormone-related peptide (PTHrP). This peptide is a naturally- occurring, local product of normal keratinocytes, but when produced by malignant tumors, PTHrP enters the systemic circulation and interacts with classical PTH receptors in bone and kidney, resulting in the prototypical biochemical features of HHM. We have characterized an experimental system of twelve human squamous tumor lines which will allow us to study in detail the molecular mechanisms regulating PTHrP gene expression, with the aim of identifying why some squamous tumors cause HHM and others do not. Using this system, we have observed that squamous cell tumor lines universally secrete PTHrP, but do so over a wide spectrum that is a direct function of the steady- state level of PTHrP mRNA. These observations suggested to us that a given squamous cell tumor's ability to cause HHM might be related to its quantitative ability to express the PTHrP gene, as opposed to a simple qualitative or """"""""on-off"""""""" defect in gene expression. This appears to be the case, for when these tumor lines were grown in athymic mice, the levels of calcium and PTHrP in the circulation correlated directly with the level of PTHrP mRNA expression in tumor tissue, confirming the quantitative relationship predicted. Furthermore, we present evidence that the mechanisms underlying the differential levels of PTHrP gene expression seen in these tumors are transcriptional in nature and operate in trans. We have thus far identified a 500 bp region of the PTHrP promoter that appears to contain regulator sequences that govern these differential rates of transcription. We propose to study further the mechanisms of PTHrP gene expression in this system: l) by mapping in some detail the regulator sequences responsible for the differential rates of PTHrP transcription in these different squamous tumor lines, 2) by defining the protein-DNA interactions important to these differential rates of PTHrP gene transcriptions and 3) by purifying and identifying transcription factors which appear able to differentially regulate PTHrP expression in these squamous tumor lines and, if they are novel, to clone their cDNAs. The principal investigator is an endocrinologist with an interest in studying mechanisms of gene regulation related to abnormal secretion of hormones by malignancies. The proposal offers an ideal training vehicle for a clinician-investigator with these interests, for it involves extensive use of a wide range of molecular techniques pertinent to the studs of gene expression directed to the study of a common clinical syndrome. This project combined with the richness and diversity of research at Yale, will maximize my opportunities for growth as an independent young investigator.
