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Introduction: The novel tumor suppressor gene, HPP1 is downregulated in over 80% of colorectal cancers and mediates its effects by alterations in STAT (Signal Transducer and Activator of Transcription) signaling. HPP1 is a secreted transmembrane protein that contains a single Epidermal Growth Factor (EGF)-like domain which differs from EGF by having a His instead of an Arg at what is thought to be a critical amino acid (AA) site. We sought to investigate the impact of targeted site-directed mutagenesis at this AA site on HPP1’s biologic behavior.

Methods:
Site-directed mutagenesis technology was utilized to create a mutated HPP1 construct substituting an Arg moiety in the place of His at site 299 in its EGF-like domain. Computed 3-D protein folding models demonstrate no alterations in the overall configuration of HPP1 as a result of this mutation. Full-length wild-type HPP1, the mutated HPP1 (H299R) and empty vector control were transfected into the HPP1 non-expressing HCT116 colon cancer cell line. Biologic effects on STAT signaling were assessed by RT-PCR and Western Blot analyses. Effects on proliferation and anchorage-independent growth were evaluated by MTT and soft agar assays respectively.

Results:
We have previously demonstrated that HPP1 overexpression results in a substantial reduction in proliferation, growth in soft agar and tumorigenicity. These effects are associated with activation of suppressive STAT1 and -2 with down regulation of oncogenic STAT3, -5 and -6. Transfection of HPP1 H299R resulted in a reversal of this profile with a reduction in activated STAT1 and -2 and increased phosphorylation of STAT3, -4 and -5. Moreover, forced expression of mutated HPP1 abrogated tumor suppressive behavior with increased cell proliferation (Optical Density-OD: 0.78± 0.18 vs.; 0.33 ± 0.13 p≤0.001) and colony formation in soft agar (543±20 vs 2±1 colonies; p≤0.001) as compared to wild-type HPP1 transfectants. Cell growth parameters were similar between HPP1 H299R and EV control transfectants (OD 0.75±0.07; 591±176 colonies; P=NS).

Conclusion:
The EGF-like domain of HPP1 is essential for its tumor suppressive effects with the Histidine moiety at position 299 being critical for mediating its associated biologic and molecular signaling effects. Therapeutic targeting of the erbB family of receptors is of great interest and our findings may lead to a greater understanding of the complex and sometimes contradictory nature of their associated signaling pathways.