A Chemical Screen Identifies Anisomycin as an Anoikis Sensitizer That Functions by Decreasing FLIP Protein Synthesis

Abstract

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 anoikisresistant 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. [Cancer Res 2007;67(17):8307-15]

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro

“…Our results showed SB203580 significantly inhibits activation of Hsp27 which is the downstream substrate of p38MAPK but does not inhibit the phosphorylation of p38MAPK, indicating that SB203580 inhibit p38MAPK activity but not its activation. For SP600125, we demonstrated it significantly inhibits the phosphorylation of JNK (Figure 2 Anisomycin induces U251 and U87 cell death independent of its ability to sensitize cells to anoikis Caspase-8 and the caspase-8 inhibitor FLIP have been reported to participate in anisomycin-induced apoptosis [6] . Anisomycin can induce apoptosis by decreasing levels of FLIP, resulting in a sensitization of cells to anoikis.…”

“…More recently, it was found that not only endothelial cells and nonmalignant epithelial cells but also malignant epithelial cells could undergo anoikis through self-initiated activation of the death receptor signaling pathway [17][18][19][20] . The results from these studies suggested that anisomycin initiates anoikis in a caspase-8-dependent manner, due to its inhibition of FLIP protein synthesis, and independent of its ability to activate JNK and p38 [6] . To investigate whether anisomycin initiates anoikis in U251 and U87 cells, we detected the expression of fulllength caspase-8, cleaved caspase-8 and FLIP protein by Western blotting.…”

“…These data led to the following important question: through which pathway does anisomycin induce U251 cell death? A recent study addressed a similar question [6] . Using a chemical screen, the authors identified a novel mechanism by which anisomycin induces apoptosis.…”

“…This decrease in FLIP expression sensitizes cells to anoikis, which is the initiation of apoptosis that is triggered by a loss of contact with the extracellular matrix [6] .…”

Molecular Mechanisms of Apoptosis and Roles in Cancer Development and Treatment

“…In addition, they showed that SAHA disrupts the FLIP/ Ku70 complex via increasing the acetylation of Ku70, which consequently triggers FLIP polyubiquitination and degradation by the proteasome (Kerr et al, 2012). C-FLIP has been demonstrated to be down-regulated by several compounds such as actinomycin D (Hernandez et al, 2001), cycloheximide (Kaminskyy et al, 2013), and anisomycin (Mawji et al, 2007a); the first is an RNA synthesis inhibitor, and the second and third are protein synthesis inhibitors. Moreover, protease inhibitors such as Bortezomib (PS-34) have been extensively studied and shown to have the ability to reduce c-FLIP in diverse cell lines (Koschny et al, 2007;Perez et al, 2010).…”