Introduction: We have previously demonstrated that HPP1 is an epigenetically-silenced tumor suppressor gene that exerts its effects via an erbB4-JAK-STAT signaling pathway. Although identified as a secreted multi-moiety transmembrane protein, the specific component responsible for its growth suppressive function has not been elucidated. Notably, HPP1 contains a single Epidermal Growth Factor (EGF)-like domain that differs from EGF by the substitution of arginine (R) for histidine (H) at the critical 299 position. We sought to investigate the isolated biologic effects of HPP1’s EGF-like domain.

Methods: Synthetic wild type (WT; H299) and mutant (MUT; R299) peptides were prepared at our crystallographic core facility. The change in amino acid did not alter the predicted 3D structure of the peptide. A total of 50ng/ml of each peptide per well (6-well plate) was used to treat the HPP1 non-expressing HCT116 colon cancer cell line. Cells were starved for 24 hours with a peptide exposure time of 7 minutes. We subsequently examined alterations in relevant erbB-, JAK- and STAT-family proteins as well as associated changes in cell growth in soft agar.

Results: Treatment of HCT116 with WT peptide resulted in increased phosphorylation of erbB4, JAK1, JAK2, STAT1 and STAT2 with a concomitant downregulation of STAT3 activation as compared to untreated controls. Mutant peptide delivery did not result in any alterations in erbB4-JAK-STAT signaling components. WT peptide treatment resulted in a dramatic reduction in colony formation in soft agar as compared to mutant peptide-treated (p=0.0002) and control cells (p=0.0019). Significant increase in colony formation was observed in the mutant vs. control cells (p=0.0047).

Conclusion: The isolated effects of HPP1’s EGF-like domain mimic the observed signaling and growth suppressive alterations observed with full-length HPP1. This EGF-like domain along with its histidine residue at position 299 appear critical for HPP1 downstream signaling and its tumor suppressive effects. Further elaboration of these findings may have therapeutic implications.

Citation Format: Abul Elahi, Abidemi Ajidahun, Mazher S. Hussain, Leah Hendrick, Irina Getun, Andreas Becker, Yan Yang, Evan S. Glazer, David Shibata. The tumor suppressive effects of HPP1 in colorectal cancer are mediated by its EGF-like domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4283.