Human type 1 Epidermal Growth Factor

Epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation drive glioblastoma (GBM) pathogenesis, but their regulation remains elusive. Here we characterized the EGFR/EGFRvIII "interactome" in GBM and identified thyroid receptor-interacting protein 13 (TRIP13), an AAA + ATPase, as an EGFR/EGFRvIII-associated protein independent of its ATPase activity. Functionally, TRIP13 augmented EGFR pathway activation and contributed to EGFR/EGFRvIII-driven GBM growth in GBM spheroids and orthotopic GBM xenograft models. Mechanistically, TRIP13 enhanced EGFR protein abundance in part by preventing Cbl-mediated ubiquitination and proteasomal degradation. Reciprocally, TRIP13 was phosphorylated at tyrosine(Y) 56 by EGFRvIII and EGF-activated EGFR. Abrogating TRIP13 Y56 phosphorylation dramatically attenuated TRIP13 expression-enhanced EGFR signaling and GBM cell growth. Clinically, TRIP13 expression was upregulated in GBM specimens and associated with poor patient outcome. In GBM, TRIP13 localized to cell membrane and cytoplasma and exhibited oncogenic effects in vitro and in vivo, depending on EGFR signaling but not the TRIP13 ATPase activity. Collectively, our findings uncover that TRIP13 and EGFR form a feedforward loop to potentiate EGFR signaling in GBM growth and identify a previously unrecognized ATPase activity-independent mode of action of TRIP13 in GBM biology.

Background: Hyperplastic polyposis protein 1 (HPP1) encodes a tumor-suppressive trans membrane 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 over expression 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-Dstalk) 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-Dstalk-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. Published by Elsevier Inc.