Functional Identification of Api5 as a Suppressor of E2F-Dependent Apoptosis In Vivo

Abstract

Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

Helical Repeat Structure of Apoptosis Inhibitor 5 Reveals Protein-Protein Interaction Modules

“…Non-small cell lung cancer patients whose cancers express API5 have a poorer prognosis than patients with non-API5-expressing cancers, and overexpression of API5 promotes the invasion and inhibits apoptosis in cervical cancer cells (15,17,20). It has been suggested that API5 suppresses E2F1 transcription factor-induced apoptosis (21). A recent study also suggested that API5 is phosphorylated by PIM2 kinase and inhibits apoptosis in hepatocellular carcinoma cells through NF-B (nuclear factor-B) (22).…”

Life and death decisions by the E2F transcription factors

“…Dyson and colleagues identified the anti-apoptotic protein Api5 in a screen for suppressors of E2F-mediated apoptosis in Drosophila [63]. Api5 specifically affects E2F-driven apoptosis both in flies and human cells, having no effect on other inducers of cell death.…”

Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo

“…First, global Drosophila genetic screening identified API-5 as a suppressor of apoptosis mediated by E2 promoter-binding factors (E2F) in multiple cell types and developmental contexts (5). This genetic interaction was confirmed in human cancer cells in vitro and appears to be mediated by API-5 binding and inactivation of acinus, a protein necessary for apoptotic DNA fragmentation (5,6). Second, API-5 protected tumor cells against the stress generated by suboptimal growth conditions or death-triggering chemicals, as demonstrated by Rigou et al, who found a marked increase of etoposide-and camptotecininduced apoptosis in various types of cancer cells caused by a depletion in the level of API-5 (6).…”

“…API-5 was originally identified based upon its marked anti-apoptotic action, demonstrated in mouse BALB/c3T3 fibroblasts, human cervical carcinoma CUMC-6 cells and immortalized primary liver THLE-3 cells, where ectopic API-5 expression significantly enhanced the cell survival following serum withdrawal or UV irradiation (1)(2)(3)(4). Similarly, a decrease in API-5 levels sensitized human squamous cell carcinoma (JHU-029), colon cancer (HCT116), cervical cancer (HeLa), lung adenocarcinoma (A549), acute lymphoblastic leukemia (Molt-4), endothelial (HUVEC) and osteosarcoma (U20S) cells to a number of forms of apoptosis, demonstrating the pro-survival signaling of API-5 in several experimental tumor cell systems in vitro (4)(5)(6).…”