Mo1161 Phase 2B Randomized Placebo-Controlled Spectral Markers As Biomarkers for Colonic Chemoprevention With Aspirin: Correlation With Rectal Apoptosis but Not Proliferation

Abstract

BACKGROUND AND AIM: In the early stages of tumorigenesis, transforming growth factor (TGF)-β1-driven signaling provides tumor-suppressive action. TGF-β1 activity is inhibited by SMAD7, a protein that binds to TGFβ receptor and prevents TGF-β1-induced signal transduction. Forced expression of SMAD7 in cancer cells differently regulates tumorigenesis depending on the cell context analyzed, and preliminary evidence suggests that amplification of SMAD7 associates with a significantly worse prognosis in patients with colorectal cancer (CRC). In this study we evaluated the role of SMAD7 in the process of colon carcinogenesis. METHODS: SMAD7 expression was evaluated in human CRC samples, CRC cell lines (i.e. HCT-116, DLD-1, HT-29 and HT-115) and normal colonic epithelial cells by immunohistochemistry and Western blotting. HCT-116 and DLD-1 cells were transfected with a specific oligonucleotide antisense (GED-0301) or control sense, and cell proliferation, cell cycle, caspase-3 activation and apoptosis were then evaluated by flow-cytometry. Expression of cell cycle-related proteins (e.g., CDK2, CDC25A, cyclin D1) was assessed byWestern blotting. To evaluate the effect of SMAD7 knock-down on CRC growth in vivo, xenografts were induced in Rag1-deficient mice by subcutaneous injection of HCT-116 cells and mice were treated either with SMAD7 sense or GED-0301. RESULTS: High SMAD7 was seen in CRC samples as compared to matched normal, adjacent, colonic samples. Consistently, SMAD7 was highly expressed in several CRC cell lines but not in normal colonic epithelial cells. Transfection of HCT-116 and DLD-1 cells with GED-0301 down-regulated SMAD7 and inhibited cell growth. Cell cycle analysis revealed that silencing of SMAD7 with GED-0301 induced cells to accumulate in S phase and this effect was associated with a sustained phosphorylation of CDK2 and decreased expression of CDC25A and cyclin D1. Moreover, time-course studies revealed that GED-0301-mediated cell cycle arrest was followed by caspase-dependent induction of CRC cell apoptosis. Finally, in vivo studies showed that daily intra-peritoneal administration of GED-0301 markedly inhibited the development of HCT-116-derived xenografts. CONCLUSIONS: SMAD7 knock-down causes accumulation of CRC cells in S phase thereby promoting cell growth arrest and apoptosis. Data suggest that SMAD7 could be a promising molecular target for pharmacological intervention in CRC patients.