Integration of the Mitochondrial and Death Receptors (DRs) Pathways and Role of c-FLIP
In TNF-α-triggered apoptosis, the internalization of the TNFR1 complex induces formation of Complex II that contains RIP, TRADD, FADD, and caspase-8. Caspase-8 is autoactivated to trigger activation of the executor caspases-3 and -7, resulting in apoptosis, and c-FLIP inhibits capsase-8 activation and apoptosis (Safa et al. 2008; Wang and Lin 2008). Active caspases-8 and -10 are known to cleave a pro-apoptotic Bcl-2 family member, Bid, and the truncated Bid (tBid) induces mitochondrial cytochrome c release, thereby linking the two pathways. After activation, both caspases-8 and -9 activate caspase-3, which in turn cleaves other caspases and many cellular proteins leading to apoptosis.
Mitochondria play a critical role in maintaining cellular respiration and homeostasis in the cells and transfer various signals to the cytosol necessary for survival and cell death. Recent data (Ranjan and Pathak 2016a) demonstrated that FADD and c-FLIP expression participates in balancing redox potential by regulating antioxidant levels. Further, we noticed that knockdown of c-FLIPL and induced expression of FADD results in rapid accumulation of intracellular ROS accompanied by JNK1 activation to substantiate apoptosis. Therefore, apart from their death receptor signaling, c-FLIPL and FADD play important roles in preventing mitochondrial-mediated apoptosis.
The TRAIL receptors, DR4 or DR5, can also promote alternative signaling pathways such as JNK, MAPK, or NF-κB by recruiting RIP1 and TRAF2 or TRAF5 to form secondary signaling complex (Yang et al. 2010; Trivedi and Mishra 2015). Activation of NF-κB in this pathway also results in increased expression of c-FLIP (Fig. 2). Studies with TRADD-deficient mouse embryo fibroblasts (MEFs) have documented that RIP1 was also recruited to the TRAIL receptor by interacting with TRADD and both RIP1 and TRADD protect against TRAIL-induced apoptosis (Cao et al. 2011). In these TRADD-deficient MEFs, MAPK and NF-κB pathway activation was impaired, confirming the role of TRADD as the key adaptor protein mediating nonapoptotic signaling by DRs (Cao et al. 2011). Indeed, Human T-Cell Leukemia Virus Type 1 Tax induces c-FLIP expression through DR4/DR5-dependent activation of the IKK-I-κBα-NF-κB pathway (Wang et al. 2014). It is also known that TNF-α and Fas trigger the cleavage of mitogen-activated protein kinase/ERK kinase kinase (MEKK)1, resulting in production of a constitutive active form of MEKK1 and leading to JNK activation in c-FLIP knockdown cells (Nakajima et al. 2008).
In the absence of caspase-8 activity, the death receptors can promote death by programmed necrosis (necroptosis) which requires the kinases receptor-interacting kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase-like protein (MLKL) (Silke and Strasser 2013).
c-FLIP Spliced Variants
Thome et al. (1997) initially identified viral FLICE-inhibitory proteins (v-FLIPs) by a bioinformatic search for novel death effector domains (DED-containing virus-encoded apoptotic regulatory proteins). These authors described six v-FLIPs and showed that cells expressing v-FLIPs were resistance to Fas (CD95/APO-1)- ,TRAILR1-, and TNFR1-induced apoptosis (Thome et al. 1997). v-FLIPs contain two DEDs, bind to the DED of FADD and interfere with the FADD-procaspase-8 interaction, leading to suppressed recruitment of procaspase-8 to the DISC and its activation (Thome et al. 1997). Shortly after the characterization of v-FLIPs, the mammalian cellular homologue was identified and termed c-FLIP (Irmler et al. 1997). c-FLIP consists of 13 distinct spliced variants, three of which are expressed as proteins: the 26 kDa short form (c-FLIPS), the 24 kDa form of c-FLIP (c-FLIPR), and the 55 kDa long form (c-FLIPL) (Lavrik and Krammer 2012; Safa 2013) (Fig. 3). The three c-FLIP variants can interact with the adaptor protein FADD. c-FLIPR is smaller than c-FLIPS and has a similar pattern of expression as c-FLIPS during activation of primary human T cells and is strongly induced in T cells upon CD3/CD28 costimulation (Golks et al. 2005).
Roles of c-FLIP in Necroptosis and Autophagy
In addition to its role in apoptosis, c-FLIPL also plays an important role in necroptotic cell death and autophagy (He and He 2013; Safa 2013). Recent results have demonstrated that necroptosis is a programed receptor-interacting protein-1/receptor-interacting protein-3 (RIP-1/RIP-3)-dependent necrotic cell death induced when caspase-8 activation is inhibited (Walsh and Edinger 2010). c-FLIPL is known to regulate necroptosis through the formation of the signaling platform Ripoptosome, a RIP1/caspase-8-containing intracellular cell death complex (Tenev et al. 2011; Feoktistova et al. 2011). While c-FLIPL prevents Ripoptosome formation, interestingly, c-FLIPS promotes its assembly (Feoktistova et al. 2011; Safa 2013). Therefore, c-FLIP isoforms in the Ripoptosome determine whether cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis (Feoktistova et al. 2011). c-FLIPL mediates the regulation of autophagic signaling and suppresses autophagic stress as well as regulating the activation of p53 and JNK1 to maintain the interaction of Bcl-2 and Beclin-1, a primary protein involved in the autophagic process (Ranjan and Pathak 2016b). To maintain the integrity of the autophagic process, c-FLIPL also simultaneously regulates oxidative stress and proautophagic accumulation of high-mobility group box 1 (HMGB1), a protein that can promote the pathogenesis of inflammatory reaction (Ranjan and Pathak 2016b).
Normal Function of c-FLIP
Animal models provide evidence that c-FLIP plays a critical role in T-cell proliferation and heart development (Micheau 2003). Abnormal c-FLIP expression has been identified in various diseases such as multiple sclerosis, Alzheimer’s disease, diabetes mellitus, rheumatoid arthritis, and various cancers (Micheau 2003). c-FLIP overexpression is known to evade the natural immunity mediated by TRAIL-induced cell death as well as by augmenting cell motility and invasion in vivo (El-Gazzar et al. 2010). Elevated expression of c-FLIP is also seen in Alzheimer disease (Mezache et al. 2015), systemic lupus erythematosus (SLE) (Tao et al. 2009), inflammation diseases (Kang et al. 2009), and lung miofibrosis (Golan-Gerstl et al. 2012). c-FLIPL plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes (He and He 2013). Moreover, recent data suggest that c-FLIPR is an important modulator of apoptosis and enforced expression leads to autoimmunity (Ewald et al. 2014). Experiments with c-FLIP-deficient mice demonstrated that c-FLIP has two distinct roles: (1) cooperates with FADD and caspase-8 during embryonic development and (2) cytoprotects against death-factor triggered apoptosis (Yeh et al. 2000). c-FLIP also has a role in cardiac remodeling following myocardial infarction (MI). c-FLIP also protects against the development of postinfarction cardiac remodeling (Xiao et al. 2012). Furthermore, c-FLIP is essential for myofibroblast accumulation and may serve as a potential target to manipulate tissue fibrosis (Golan-Gerstl et al. 2012). Recent results demonstrated that expression of c-FLIPR in hematopoietic cells supports an efficient immune response against bacterial infections (Telieps et al. 2013).
Upregulation of c-FLIP in Human Cancers
Overexpression of c-FLIP has been found in various types of malignancies and could be associated with cancer progression due to its ability to inhibit cancer cell death. Elevated expression levels of c-FLIP have been reported in several tumor types (Du et al. 2009; Pallares et al. 2009; Safa and Pollok 2011; Tian et al. 2012) including melanoma, hepatocellular carcinoma (HCC), nonsmall cell lung carcinoma, endometrial, colon, breast cancer, bladder cancer (BC), and prostate cancer. Significantly, increased c-FLIP expression was an independent negative prognostic indicator for disease-free survival (DFS) in cervical squamous cell carcinoma (CSCC) (Yao et al. 2016). c-FLIP overexpression has been found to be associated with disease progression and/or poor prognosis in BL, HCC, ovarian, endometrial, colon, prostate, and head and neck squamous cell carcinoma (HNSCC) (Safa et al. 2008; Korkolopoulou et al. 2007; Du et al. 2009). c-FLIPL is highly expressed in invasive breast carcinomas, and its expression level is closely related to the molecular subtypes and clinical prognosis of breast cancer patients (Zang et al. 2014). In addition to blocking apoptotic signaling pathways, c-FLIP has been implicated in contributing to tumor promotion through its influence on several critical cellular survival signaling mechanisms (Safa et al. 2008; Safa 2012).
Structure of c-FLIP Isoforms
In humans, three isoforms of c-FLIP, FLIPL (55 kDa), c-FLIPS (27 kDa) and c-FLIPR (25 kDa) have been identified (Oztürk et al. 2012; Safa 2013). All c-FLIP isoforms contain two DEDs that are structurally similar to the N-terminal part of procaspase-8 (Fig. 1). c-FLIPL contains additional caspase-like domains (p20 and p12) that are catalytically inactive. Moreover, c-FLIPS has an additional isoform-specific C-terminal tail of 19 amino acids. c-FLIPR also contains two DEDs but lacks the additional carboxy (C)-terminal amino acids that are present in c-FLIPS. Moreover, c-FLIPL has a caspase-8 cleavage site at position Asp-376 (LEVD); c-FLIPL cleavage at this site produces the proteolytic fragment variant p43c-FLIP, p43-FLIP, and p22-FLIP, respectively (Oztürk et al. 2012; Safa 2013). The C-terminal region of c-FLIPS and c-FLIPR plays a crucial role in ubiquitination and degradation as well as the antiapoptotic function of these isoforms (Oztürk et al. 2012; Safa 2013).
c-FLIP Activates Cytoprotective and Proliferation Pathways
Several cytoprotective and survival signaling pathways with crucial roles in regulating cell survival, proliferation, and carcinogenesis are activated by c-FLIP (Oztürk et al. 2012; Safa et al. 2008; Safa and Pollak 2011; Safa 2012; Seidelin et al. 2013). Increased expression of c-FLIPL activates NF-κB and ERK signaling by binding to adaptor proteins in each pathway, such as TNFR-associated factors 1 (TRAF1) and 2 (TRAF2), receptor-interacting protein 1 (RIP), and Raf-1 (Fig. 2). The caspase-8 cleaved N-terminal fragment of c-FLIPL (p43-cFLIP) is more efficient than c-FLIPL at recruiting TRAF2 and RIP1, leading to more robust NF-κB activation (Kataoka and Tschopp 2004; Yu and Shi 2008). Interestingly, c-FLIP may also provide the molecular switch from Fas-triggered apoptosis to Fas-promoting cell proliferation (Gilot et al. 2005).
It is known that interaction between Akt and c-FLIPL enhances the antiapoptotic functions of Akt by modulating Gsk3β activity (Quintavalle et al. 2010) and that c-FLIPL overexpression interferes with Gsk3-β phosphorylation levels and induces resistance to TRAIL in cancer cells. DNA-PK/Akt pathway is also reported to play a role in expression of c-FLIP (Kim et al. 2012). Interestingly, siRNA-mediated silencing of DNA-PK or treatment with its inhibitor 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB) led to decreased phosphorylation of Akt and Bad (a target molecule of Akt), elevated expression of DR4/DR5, and decreased expression of c-FLIP (Mizushima and Levine 2010). Furthermore, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase 7 (MKK7), in a TNFα-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1 (MEKK1), apoptosis signal-regulating kinase 1 (ASK1), and TGF-β-activated kinase 1. This interaction of c-FLIPL with MKK7 might selectively suppress JNK activation (Nakajima et al. 2006).
In addition to its role as a critical regulator of apotosis, necroptosis, and autophagy, c-FLIP may also regulate potentially harmful signaling pathways regulating the production of inflammatory cytokines, tumor cell migration and metastasis, and the activation of transcription factors critical during tumorigenesis (Naito et al. 2004; Gilot et al. 2005; Leverkus et al. 2008). Furthermore, overexpression of c-FLIP can alter cell cycle progression and enhance cell proliferation and carcinogenesis (Safa 2013).
c-FLIP is a master antiapoptotic protein and an important protein in causing resistance to cytokines and chemotherapeutic agents. It regulates apoptosis, necroptosis, autophagy, and cytoprotective signaling pathways. c-FLIP is expressed as c-FLIPL, c-FLIPS, and c-FLIPR isoforms in human cells. c-FLIP isoforms bind to FADD and caspase-8 or -10 and TRAIL receptor 5 (DR5), which prevents autocatalytic activation of these caspases and DISC formation. c-FLIPL and c-FLIPS are also known to have multifunctional activities in various cellular signaling pathways, as well as activating and/or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, β-catenin, and NF-κB. Upregulation of c-FLIP has been found in various tumor types, and its silencing has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Moreover, since elevated expression of c-FLIP has been documented in Alzheimer disease, systemic lupus erythematosus (SLE), inflammation diseases, and lung fibrosis, it is a potentially important target for identifying therapeutic agents for treating these diseases.
I would like to thank Dr. Mary D. Kraeszig for her editorial assistance. The work in the author’s laboratory was supported by research grants from the National Cancer Institute (CA 080734, CA 90878, and CA 101743), Department of Defense (DOD) (OC 06095), and the Indiana University Cancer Center Translational Research Acceleration Collaboration (ITRAC) initiative.
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