Human colorectal cancer (CRC) cells must detach from a substratum to metastasize to distant sites. During this process CRC are susceptible to detachment-induced cell death or anoikis - a form of programmed cell death (apoptosis) that occurs when anchorage-dependent cells go into suspension. Our purpose was to identify the mechanism that mediates anoikis in human CRC. Since we have previously shown that metastatic potential of CRC is inversely associated to anoikis, we used weakly metastatic (MIP-101 and Clone A), and highly metastatic (CX-1 and MIP-101.8) CRC cell lines. CRC were cultured on PolyHEMA-coated surfaces for 1 - 4 days to induce apoptosis which was measured by TUNEL or Annexin V/Propidium Iodide flow cytometry. CRC cultured for at least 24 hr had death rates >5% compared to <1% for CRC in attached monolayers. We then assessed whether caspases of the extrinsic (Caspase 8) or intrinsic (Caspase 9) death pathway were active and found that Caspase 8 and Caspase 3 were cleaved during exposure to suspension culture in four CRC lines and cell death was inhibited by caspase 3 and 8 inhibitors but not by a caspase 9 inhibitor. Gene expression analysis of death receptors in CRC 24 hr in suspension was confirmed at the protein and gene transcript level and revealed increased expression of DR5 and osteoprotegerin but not DR4, Fas, CD27, CD30 or CD40. Treatment with 5 μg/ml of antagonistic antibody to DR5 inhibited anoikis in 3 of 4 human CRC lines but treatment with a similar DR-4 antibody reduced anoikis only in CX-1 and MIP-101. Anti-DR5 antibody decreased caspase 8 activation in MIP-101 cells in suspension. siRNA to DR5 specifically reduced expression of DR5 and inhibited anoikis to levels significantly below transfection controls that included siRNA to DR4 for up to 4 days. In summary, DR5 receptor mediates death signals for anoikis in human CRC cells through the extrinsic apoptotic pathway.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]