Abstract C153: Both TRAIL receptor DR5 isoforms are required for the induction of cell death in human colon carcinoma

Colorectal cancer continues to be the third most common malignancy in the United States. This disease remains resistant to treatment, with only 25–30% of patients disease‐free long term; for the rest, median su rvival is only 18–22 months. We previously demonstrated synergistic interaction between FUra/LV combined with IFN‐ (which potentiates FUra/LV‐induced DNA damage and requires the death receptor Fas) in human colon carcinoma cell lines and xenografts. FUra/LV/IFN‐ also demonstrated responses in heavily pretreated and previously untreated patients in Phase I trial and is currently completing Phase II evaluation in Stage IV colorectal cancer. Further, lexatumumab, an agonistic TRAIL‐R2 (DR5) monoclonal antibody, is highly synergistic with FUra/LV/IFN‐ in xenograft models, dependent on TRAIL receptor activation. Lexatumumab demonstrated the broadest spectrum in vitro activity in a panel of 6 human colon carcinoma cell lines when compared to the activity of the recombinant ligand, TRAIL (targeting DR4 and DR5). To further understand the mechanism of lexatumumab‐induced cell death via DR5, DR5 expression, was determined in human colon carcinoma cell lines (Western, RT‐PCR) and primary colon tumors (RT‐PCR). The short isoform of the receptor (DR5S) was expressed at a higher level than the long isoform (DR5L). To determine the specific function of each isoform in regulating death signaling, a BJAB‐derived cell line lacking DR5 expression (DR5−/−) was employed. DR5 −/− cells were resistant to lexatumumab‐induced apoptosis, even when each DR5 isoform was expressed individually, despite the fact that the individual isoforms could still bind ligand (lexatumumab). Analysis of receptor complex formation indicated that DR5, FADD, and caspase‐8 were recruited; however, caspase‐8 was not processed. Apoptotic signaling was restored when both DR5 isoforms were re‐expressed in DR5−/− cells. Lexatumumab sensitivity of HCT8 cells was eliminated by stable shRNA knockdown of both DR5 isoforms, as was the recruitment of FADD and caspase‐8 to the receptor complex. In contrast, following 1 hr lexatumumab treatment in wild type HCT8 cells, both DR5 isoforms were recruited to the receptor complex; cell death correlated with recruitment of FADD, cleavage of caspase‐8, cleavage of RIP, and reduced expression of c‐FLIPS and c‐FLIPL. TRAF2 and c‐IAP2, but not TRAF1 or c‐IAP1, were also recruited; however, the significance of these signaling molecules in the cell death response is currently unknown. Overall, data indicate that both DR5S and DR5L are required for lexatumumab‐induced cell death via the DR5 receptor. Although the significance of this is currently unknown, factors that regulate lexatumumab‐induced cell death vs survival at the level of receptor complexes are currently being elucidated. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C153.