Mathematical Modeling Highlights the Complex Role of AKT in TRAIL-Induced Apoptosis of Colorectal Carcinoma Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionApoptosis is executed by caspases that are activated via intrinsic and extrinsic signaling pathways (). The intrinsic pathway is initiated by DNA damage, substrate detachment, or growth factor withdrawal and involves mitochondrial outer membrane permeabilization (MOMP), and the release of cytochrome c (). The extrinsic pathway is induced by ligand binding to plasma membrane receptors of the tumor necrosis factor superfamily, and the downstream molecular cascade that is triggered is believed to be genetically determined. This pathway can trigger two types of cell death signaling. First, Type I cells such as lymphocytes undergo mitochondria-independent cell death, relying solely on a receptor or ligand-instigated caspase cascade (, ). In Type II cells, however, amplification through MOMP and cytochrome c release is necessary (). Understanding how specific cells coordinate apoptotic responses contributes to our appreciation of cell death dynamics in disease.AKT (protein kinase B) is a promiscuous serine/threonine-specific protein kinase that influences protein synthesis (), proliferation (), glucose metabolism (), synaptic signaling (), autophagy (, ), and nuclear factor-κB signaling (). Several studies have also revealed a pivotal role for AKT in apoptosis. AKT inhibits apoptosis via inhibitory phosphorylation of the pro-apoptotic BCL-2 homology domain 3 (BH3-only) protein BAD (), triggering a cascade of inhibitory reactions impinging on pro-apoptotic BAX (AKT ┤ BAD ┤ BCL-2 ┤ BAX; ┤ denoting inhibition). The BCL-2-BAX and BAD-BCL-2 interactions are direct binding associations dependent on their respective BCL-2 homology (BH) domains, whereas AKT inactivates BAD through phosphorylation at Ser¹³⁶ leading to AKT sequestration by 14-3-3 proteins (). AKT also phosphorylates BAX at Ser¹⁸⁴, preventing the conformational changes in BAX needed for oligomerization and pore-forming capabilities during MOMP (). Downstream of MOMP, AKT phosphorylates procaspase-9 at Ser¹⁹⁶, preventing its processing and activation (href="#bib11" rid="bib11" class=" bibr popnode">Cardone et al., 1998). It also phosphorylates the X-linked inhibitor of apoptosis protein (XIAP) (href="#bib19" rid="bib19" class=" bibr popnode">Deveraux and Reed, 1999), an E3 enzyme that ubiquitylates caspases 9, 3, and 7, targeting them for proteasomal degradation. XIAP also regulates its own stability through autoubiquitylation (href="#bib41" rid="bib41" class=" bibr popnode">Nakatani et al., 2013), a process that is blocked by AKT-mediated Ser⁸⁷ phosphorylation (href="#bib16" rid="bib16" class=" bibr popnode">Dan et al., 2004). Robust cell death initiation requires XIAP inhibition via SMAC (second mitochondria-derived activator of caspases) that is released during MOMP and binds to the tetrapeptide IAP-binding motif of XIAP (href="#bib49" rid="bib49" class=" bibr popnode">Scott et al., 2005). AKT phosphorylates SMAC at Ser⁶⁷ to increase its binding to XIAP, conferring resistance to apoptosis (href="#bib31" rid="bib31" class=" bibr popnode">Jeong et al., 2015).Any systems-level study of the role of AKT during apoptosis must consider PTEN (phosphatase and tensin homolog). PTEN acts as a positive regulator of apoptosis by antagonizing AKT activation (href="#bib4" rid="bib4" class=" bibr popnode">Baehrecke, 2005); however, it is also downregulated via XIAP-mediated ubiquitylation and degradation (href="#bib52" rid="bib52" class=" bibr popnode">Van Themsche et al., 2009). In this study, we have constructed a deterministic model of apoptosis incorporating the interactions between AKT, PTEN, and the apoptotic machinery. System dynamics predictions generated using this model describe how individual protein species as well as the apoptotic system as a whole are affected in different genetic backgrounds. This model accurately predicts protein dynamics for three of four HCT116 cell lines (wild-type; BAX^−/−; XIAP^−/−), and further investigations uncover the possible mechanisms behind the dynamics of the fourth (PTEN^−/−). Subsequent laboratory studies show that AKT regulation of apoptosis is significantly stronger during TRAIL-mediated extrinsic apoptosis than in TRAIL-independent apoptosis in these cells, and that its effects are more prominent at early points in the apoptotic response.