HPP1 Ectodomain Shedding is Mediated by ADAM17 and is Necessary for Tumor Suppression in Colon Cancer.

BACKGROUND: Hyperplastic polyposis protein 1 (HPP1) encodes a tumor-suppressive transmembrane cleavable epidermal growth factor-like ligand. It is unclear as to whether cleavage and shedding of HPP1 are essential steps in achieving its tumor suppressive properties. ADAM proteins are key players in cellular ectodomain shedding processes with ADAM17 being well characterized and representing the most likely sheddase for HPP1. In this study, we explore the mechanisms and importance of ectodomain shedding in contributing to HPP1-mediated tumor suppression. METHODS: Baseline characterization of HPP1 ectodomain shedding and ADAM family member expression was performed in HCT116 colon cancer cells with forced overexpression of HPP1 and controls. Subsequent impact of attenuation of ADAM expression by short interfering RNA on HPP1 shedding was evaluated. Furthermore, we examined the functional impact of an uncleavable HPP1 mutant construct (HPP1-Δstalk) generated by site-directed mutagenesis. Cellular growth potential functions were analyzed by MTT and soft agar assays. RESULTS: Select proinflammatory cytokines enhanced HPP1 ectodomain shedding, whereas short interfering RNA-mediated knockdown of ADAM17 resulted in abrogation of HPP1 ectodomain shedding. ADAM17 knockdown concomitantly resulted in increased cell proliferation and anchorage-independent growth. HPP1-Δstalk-transfected cells exhibited significantly higher proliferation and reduced STAT1 activation relative to full-length HPP1, further suggesting a critical role for ectodomain shedding in HPP1-mediated tumor suppression. CONCLUSION: The tumor-suppressive properties of HPP1 in colorectal cancer require cleavage and shedding of its ectodomain which in turn are mediated by ADAM17. Further investigations into the regulation of HPP1 may lead to a greater understanding of epidermal growth factor-like ligand family biology and potential novel therapeutic strategies.