Critical Role for Fas-Associated Death Domain-Like Interleukin-1-Converting Enzyme-Like Inhibitory Protein in Anoikis Resistance and Distant Tumor Formation

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Abstract
Background Normal epithelial cells undergo anoikis, or apoptosis on loss of anchorage to the extracellular matrix, by initiating the death receptor pathway of caspase activation. However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. We hypothesized that c-Fas–associated death domain–like interleukin-1–converting enzyme–like inhibitory protein (FLIP), an endogenous inhibitor of death receptor signaling, may suppress anoikis. Methods We assessed viability and apoptosis of PPC-1 prostate cancer cells cultured in adherent and suspension conditions using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and Annexin V staining assays. Expression of the death receptor Fas and activation of caspase 8 were measured using flow cytometry. Expression of Fas ligand was measured by reverse transcription–polymerase chain reaction. FLIP protein expression was measured by immunoblotting. Small-molecule inhibitors of FLIP (including the death receptor sensitizer 5809354) and small-interfering (si) RNA directed against FLIP were used to assess the effects of FLIP inhibition on anoikis of prostate cancer cells in vitro and in vivo. All statistical tests were two-sided. Results PPC-1 cells cultured in suspension resisted anoikis, despite increased expression of Fas (0 versus 8 hours, mean relative percent expression = 100% versus 135%, difference = 35%, 95% confidence interval [CI] = 10% to 61%; P = .02) and Fas L (0 versus 24 hours, mean relative percent expression = 100% versus 208%, difference = 108%, 95% CI = 18% to 197%; P = .02). Knockdown of FLIP expression by siRNA or treatment with 5809354 sensitized prostate cancer cells to anoikis (control siRNA versus FLIP siRNA at 10 nM, mean relative percent viability = 95% versus 51%, difference = 44%, 95% CI = 34% to 54%; P<.001; control versus 5809354 at 20 μM, mean relative percent viability = 96% versus 52%, difference = 44%, 95% CI = 13% to 75%; P = .015). Inhibition of FLIP expression specifically activated caspase 8 in PPC-1 cells grown in suspension but not adherent conditions and decreased the metastatic potential of circulating PPC-1 cells in vivo. Conclusions FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.