Introduction: The tumor suppressor gene; HPP1 is downregulated in over 80% of colorectal cancers (CRC) and has potential as a serum and stool-based biomarker. HPP1 is a secreted transmembrane protein that contains an Epidermal Growth Factor (EGF) - like domain, two follistatin modules, and a cytosolic tail with a potential G-protein activating motif. The soluble ectodomain of HPP1 gene may undergo cleavage and functions as an EGF-like ligand which may bind to erbB family receptors. We sought to investigate the importance of cleavage and the possiblity of a juxtacrine role of the flanking EGF-like domain in the context of HPP1's tumor suppressive function..

Methods: An HPP1 double deletion construct (DD) lacking the juxtamembrane stalk cleavage site and an additional 4 amino acid residues (299-320) that constitutes the critical portion of the EGF-like domain was developed by PCR-based overlap extension mutagenesis. HPP1 non-expressing HCT116 cell lines were transfected with either wild-type HPP1 (WT), empty vector (EV) or double deletion construct (DD). Effects on proliferation (MTT assay) and anchorage-indpendent growth (soft agar assay) were evaluated.

Results: We have previously shown that overexpression of HPP1 results in substantial reduction in proliferation, growth in soft agar, and tumorigenicity. We have also identified that transfection of HPP1 with a deleted stalk sequence cleavage site (amino acid residues 303-320) results in a near-complete attenuation of its tumor suppressive effects. In order to determine whether there is a contributory juxtacrine effect of non-cleaved HPP1, we transfected the double deletion construct with a non-functioning EGF-like domain. Successful transfections of EV, WT, and DD were confirmed by RT-PCR. DD cells had similar proliferative potential and growth in soft agar when compared to EV cells (p = 0.609 and p= 0.263, respectively). When compared to WT, DD cells demonstrated a loss of HPP1's growth suppressive impact on cellular proliferation (optical density; 0.16± 0.03 vs. 0.31±0.04, p= 0.0001) and colony formation in soft agar (14.7 ± 5.5 vs. 278 ± 95.0, p= 0.009).

Conclusion: HPP1's tumor suppressive effects in colorectal cancer require cleavage and ectodomain shedding. However, it is unlikely that HPP1's biologic functions are mediated via an additional juxtacrine mechanism in this setting.

Citation Format: Abul Elahi, Abidemi Ajidahun, Whalen Clark, Jonathan Hernandez, Leigh Ann Humphries, David Shibata. HPP1's tumor suppressive effects are mediated by ectodomain shedding but not by juxtacrine signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 579. doi:10.1158/1538-7445.AM2014-579