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

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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. The retinoblastoma protein (pRB) was the first human tumor suppressor to be described, and it works by limiting the activity of the E2F transcription factor. The pRB pathway is inactivated in most forms of cancer, and, accordingly, most tumor cells have deregulated E2F. Uncontrolled E2F drives cell proliferation, but it also sensitizes cells to die (apoptosis). E2F-induced apoptosis is not well understood, but it affects the development of cancer and, potentially, could be exploited for cancer treatment. To date, however, there have been very few studies of E2F-induced apoptosis in animal models. The authors describe a series of genetic tools that allow systematic studies of E2F-induced apoptosis in Drosophila. As validation, this approach identified some known regulators of E2F-dependent apoptosis and also identified Api5, a little-studied gene that had not previously been linked to E2F, as a potent suppressor of E2F-induced cell death. The effects of Api5 on E2F occur in several different tissues and are conserved from flies to humans. This last point is significant since Api5 is upregulated in cancer cells. The discovery of the E2F–Api5 interaction demonstrates that important modulators of E2F-induced apoptosis are waiting to be discovered and that they can be found using Drosophila.