Innovative assays for oncogenic hedgehog signaling Basal Cell Carcinoma (BCC) along with pancreatic cancer (PC) and medulloblastoma (Med) have been associated with mutations in Smoothened (Smo), or with dysregulation of the Smo modulators Patched (Ptc) and Hedgehog (Hg). Intracellular signals generated from the interplay among these three proteins are fundamental to normal organ development and tissue patterning, and suggest that abnormalities in their regulation may augment or underlie the metastatic potential observed in many cancerous tumors. In contrast to the classical G protein-coupled receptors (GPCRs), Smo signaling has not been conclusively demonstrated to occur through conventional G protein mediated pathways. However, Smo activation does result in arrestin recruitment, a feature also exhibited by GPCRs when they desensitize after agonist activation. Thus, changes in arrestin recruitment to Smo can serve as a measure of its activation or deactivation by ligands, or as a consequence of the pharmacological modulation of its upstream regulatory partners, Ptc and Hg. We propose to construct cell-based assay systems composed of Smo receptors and arrestin-fluorescent proteins or Smo-fluorescent protein chimeras that can be used in the high-throughput screening of large compound libraries.
The specific aims of this proposal are (I) Identification and pharmacological targeting of Smo/Ptc/Hg signaling components underlying malignancies including BCC, PC, and Med. A. To establish an assay protocol using cell lines with each permanently expressing components of the Hg pathway including Smo and a fluorescent arrestin, or a chimeric fluorescent Smo in which the efficacy of compounds for affecting Smo activity can be evaluated. B. To screen known upstream signaling partners such as Ptc for their effects on Smo activity. (II) Identification of a putative endogenous ligand for Smo by the screening of a Natural Compound Library of Small Molecules. (III) To study the effects of the screened compounds antagonizing Smo activity on the growth inhibition of BCC, PC, and Med cell lines. Relevance to Public Health: The availability of a cell-based model in which large numbers of compounds can be quickly screened for their efficacy in modulating Smo/Ptc/Hg signaling would make an important contribution in both developmental research and cancer therapeutics. In particular it should be possible to identify critical proteins modulating altered signaling pathways in pancreatic adenocarcinoma and design pharmacologic strategies to target them for therapeutic benefit. ? ? ?
