A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis.
Malignant epithelial cells with metastatic potential resist apoptosis that normally occurs upon loss of anchorage from the extracellular matrix, a process termed "anoikis." Resistance to anoikis enables malignant cells to survive in an anchorage-independent manner, which leads to the formation of distant metastases. To understand the regulation of anoikis, we designed, automated, and conducted a high-throughput chemical screen for anoikis sensitizers. PPC-1 anoikis-resistant prostate cancer cells were seeded in hydrogel-coated ultralow binding plates for suspension conditions and standard tissue culture plates to promote adhesion. After seeding, cells were treated with aliquots from a library of previously characterized small molecules, and viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, assay. From this chemical screen, we identified anisomycin that induced apoptosis in suspension conditions, but was not toxic to these cells grown under adherent conditions. Anisomycin sensitized cells to anoikis by decreasing levels of the caspase-8 inhibitor FLIP and subsequently activating the death receptor pathway of caspase activation. Although anisomycin activated c-Jun-NH(2)-kinase and p38, these kinases were not functionally important for the effect of anisomycin on anoikis and FLIP. Rather, anisomycin decreased FLIP and sensitized cells to anoikis by inhibiting its protein synthesis. Finally, we showed that anisomycin decreased distal tumor formation in a mouse model of prostate cancer metastases. Thus, a novel chemical screen identified anisomycin as an anoikis sensitizer that acts by decreasing FLIP protein synthesis. Our results suggest that FLIP is a suppressor of anoikis and inhibiting FLIP protein synthesis may be a useful antimetastatic strategy.
Mawji
Imtiaz A
IA
Ontario Cancer Institute, Princess Margaret Hospital, Mt Sinai Hospital, Toronto, Ontario, Canada.
Simpson
Craig D
CD
Gronda
Marcela
M
Williams
Moyo A
MA
Hurren
Rose
R
Henderson
Clare J
CJ
Datti
Alessandro
A
Wrana
Jeffrey L
JL
Schimmer
Aaron D
AD
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology* / Q000627:therapeutic use*; D023102:Anoikis / Q000187:drug effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000096:biosynthesis*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008297:Male; D051379:Mice; D008954:Models, Biological; D009362:Neoplasm Metastasis / Q000517:prevention & control*; D009360:Neoplastic Cells, Circulating / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000473:pathology; D014176:Protein Biosynthesis / Q000187:drug effects*; D053218:Receptors, Death Domain / Q000378:metabolism; D014407:Tumor Cells, Cultured