Studies with animal models have revealed that c-FLIP plays an important role in T cell proliferation and heart development [88,89]. Moreover, abnormal c-FLIP expression has been found in various diseases such as cancer, multiple sclerosis, Alzheimer's disease, diabetes mellitus, and rheumatoid arthritis [24,44]. c-FLIP is also thought to be the main causal factor of “immune escape” [90]. c-FLIP is involved in TRAIL, Fas, TNF-α, and chemotherapeutic drug resistance in a wide range of human malignancies [24,44,72,91]. Moreover, studies using c-FLIP-deficient mice support a dual function for c-FLIP_L by confirming a role for c-FLIP in Fas L, TNF-α-induced and apoptosis and revealing that c-FLIP has a similar function to caspase-8 in heart development [89]. Nonetheless, a now extensive literature encompassing diverse types of human cancer cells indicates that the action of c-FLIP is generally anti-apoptotic in cancer cells. Furthermore, interference with c-FLIP expression sensitizes tumor cells to death ligands and chemotherapy in experimental models [24,45,91-94]. In addition to its function as an apoptosis modulator, c-FLIP exerts other cellular functions including increased cell proliferation and tumorigenesis [24,45,48,94-96] (Figure 1).

While the precise mechanism of c-FLIP regulation of apoptosis remains elusive, the profound structural differences between human c-FLIP variants clearly indicate distinct regulatory roles for c-FLIP_L and c-FLIP_S in apoptosis. In fact, c-FLIP_S inhibits TRAIL-induced DISC formation and apoptosis [24,45,48], while c-FLIP_L is responsible for the above described dual functions whereby it inhibits Fas-induced caspase-8 activation when expressed at high levels, but enhances caspase-8 activation when its expression level is low [24,45]. These opposing c-FLIP_L functions may reflect observations that c-FLIP_L activates caspases-8 and -10 in vitro by forming heterodimeric enzyme molecules with a substrate specificity and catalytic activity indistinguishable from caspase-8 homodimers, despite the fact that c-FLIP_L is protease dead [24,97,98]. Recent reports have clearly demonstrated that c-FLIP_S also plays a central role in preventing cancer cell apoptosis. c-FLIP_S has been shown to inhibit oxaliplatin-induced apoptosis through the sustained XIAP protein level and Akt activation [99]. c-FLIP_S also suppresses apoptosis by inhibiting caspase-8 activation [100-102], although at different levels of procaspase-8 process [103,104]. c-FLIP_L induces a conformation of procaspase-8 that triggers partial but incomplete proteolytic processing, while in contrast, c-FLIP_S even prevents partial procaspase-8 activation at the DISC [8]. Using an in vitro induced proximity assay, Boatright et al. [97] provide evidence that c-FLIP_L is an activator of caspase-8/-10 and demonstrate that the resulting heterodimer is enzymatically active with a substrate specificity identical to that of the caspase-8 homodimer.

We recently discovered that c-FLIP_L interacts with DR5, FADD, and caspase-8 forming an apoptotic inhibitory complex (AIC) in MCF-7 breast cancer cells [76]. Moreover, silencing the c-FLIP gene by a specific siRNA leads to death ligand-independent but DR5-, FADD-, and caspase-8- and -9-dependent apoptosis in these cells. Furthermore, we showed that the knockdown of c-FLIP expression inhibits breast cancer cell proliferation and triggers spontaneous apoptosis by activating both the death receptor and mitochondrial pathways [76]. Our data support the previous report by Jin et al. [105] demonstrating that the peptide corresponding to the DR5 binding domain of c-FLIP_L induces apoptosis in cancer cells. Therefore, inhibiting the interaction of DR5 and c-FLIP_L by peptides or small molecule inhibitors should provide a mechanism by which tumor selective apoptosis can be achieved. Interestingly, a recent report also identified a checkpoint of the autophagy pathway where cellular and viral FLIPs could limit the Atg3-mediated step of LC3 ubiquitin-like protein conjugation to regulate autophagosome biogenesis. Furthermore, the c-FLIP-derived short peptides hold promise as new cancer therapeutic agents since they induced growth inhibition by binding to and effectively suppressing Atg3-c-FLIP interactions [106].

As shown in Figure 1, c-FLIP activates several cytoprotective signaling pathways involved in regulating cell survival, proliferation, and carcinogenesis. Overexpression of c-FLIP_L 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 [48,107] (Figure 1). The caspase-8 processed N-terminal fragment of c-FLIP_L (p43cFLIP) is more efficient than c-FLIP_L at recruiting TRAF2 and RIP1, leading to more robust NF-κB activation [48,49,108,109]. Golks et al. [110] showed that in nonapoptotic cells, c-FLIP and the procaspase-8 heterodimer result in a novel NH₂-terminal fragment of c-FLIP (p22-FLIP) which is the key mediator of NF-κB activation by binding directly to the IKK complex. These results provide a new mechanism of c-FLIP-mediated NF-κB activation. Recently, Chang et al. [111] demonstrated that TNF-α-mediated JNK activation increases turnover of the NF-κB-induced c-FLIP. This is not the result of direct c-FLIP phosphorylation, but rather depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. Thus, JNK antagonizes NF-κB during TNF-α signaling by promoting the proteasomal elimination of c-FLIP_L.

Akt is a serine-threonine kinase that plays a major role in transducing cellular survival signals and also regulates a number of proteins involved in the apoptotic signaling pathways. Recent results showed that Akt interacts with c-FLIP_L protein and that c-FLIP_L enhances anti-apoptotic Akt functions by modulating Gsk3β activity. Moreover, through its effects on Gsk3β, c-FLIP_L overexpression in cancer cells induced resistance to TRAIL. This effect is mediated by regulation of p27(Kip1) and caspase-3 expression [112]. Downregulation of the DNA-PK/Akt pathway was also reported to correlate with high responsiveness to TRAIL-mediated growth inhibition and apoptosis [113]. siRNA-mediated suppression of DNA-PKcs or treatment with 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, led to decreased phosphorylation of Akt and Bad (a target molecule of Akt), increased expression of DR4/DR5, and down-regulation of c-FLIP [113]. Therefore, inhibition of the DNA-PK/Akt pathway may have clinical usefulness in treating TRAIL-resistant cancer cells. [97].

Panner et al. [114] initially reported that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates c-FLIP_S ubiquitination and stability in glioblastoma multiforme (GBM) cell lines and xenografts. However, how PTEN and Akt are linked to AIP4 activity was unclear. Recently, these authors described a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8) which is a downstream target of Akt, and it links Akt to AIP4 and the regulation of c-FLIP_S stability [115] (Figure 3). Overexpression of USP8 increased c-FLIP_S ubiquitination, decreased FLIP_S half-life, decreased FLIP_S steady-state levels, and decreased TRAIL resistance (Figure 3). Therefore, PTEN appears to use control of ubiquitination to regulate TRAIL sensitivity in GBM cells.

c-FLIP_L also interacts with Daxx (a death domain-associated protein that has been implicated in proapoptosis and transcriptional regulation) and prevents Fas-induced JNK activation [116]. Thus, c-FLIP_L acting on both the FADD- and Daxx-mediated signaling pathways may be involved in completely inhibiting Fas-induced cell death. Furthermore, Nakajima et al. [117] demonstrated that c-FLIP_L directly interacts with a JNK activator, MAP kinase kinase 7 (MKK7), in a TNF-α-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-FLIP_L with MKK7 might selectively suppress JNK activation (Figure 1).

Another regulator of the c-FLIP expression is the calcium/calmodulin-dependent protein kinase II (CaMK II) which mediates the upregulation of c-FLIP, thereby protecting cancer cells from TRAIL-induced apoptosis. Treating resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued Fas agonistic antibody (CH-11) sensitivity [118,119]. Targeting this pathway may provide novel therapeutic strategies in treating cancers with upregulated CaMK II. Interestingly, phosphorylation of c-FLIP variants by CaMK II appears to promote c-FLIP_L recruitment to the DISC and inhibit TRAIL-induced apoptosi [118,119], but phosphorylation of c-FLIP_L by protein kinase C or the bile acid glycochenodeoxycholate results in decreased c-FLIP_L recruitment to the DISC and increased the sensitivity of hepatocellular carcinoma cells to TRAIL-triggered apoptosis [120]. Thus, the particular site of phosphorylation on c-FLIP_L appears to influence the functional outcome of this protein on apoptosis.

Increased expression of c-FLIP can alter cell cycle progression and enhance cell proliferation and carcinogenesis [121,122] (Figure 1). Overexpression of c-FLIP_L inhibited the ubiquitination and proteasomal degradation of β-catenin, resulting in an increase in the target gene cyclin D1, colony formation, and invasive activity in prostate cancer cells. The c-FLIP/β-catenin/cyclin D1 signals contributing to colony formation and invasion were reversed by selective silencing of c-FLIP expression [123]. Similarly, c-FLIP_L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of β-catenin, thus suggesting a new mechanism involved with tumorigenesis [123]. Recent results also suggest a role for nuclear c-FLIP_L in the modulation of Wnt signaling [124]. Interestingly, a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of β- catenin can also lead to repression of c-FLIP expression through activation of c-Myc [125], c-FLIP upregulation may contribute to the carcinogenesis and aggressiveness of endometrial carcinomas and may serve as a useful prognostic factor for this tumor [24,126]. Wang et al. [127] demonstrated that c-FLIP overexpression is also significantly related to the presence of high-risk human papillomavirus (HR-HPV) infection during the progression of cervical squamous cell cancer and that c-FLIP is an early marker of cervical carcinogenesis. Moreover, HPV16 E2 protein interacts with and abrogates the apoptosis inhibitory function of c-FLIP and renders cervical cancer cell lines hypersensitive to Fas/FasL apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by human HPV16 E2 protein expression [112]. This observation is greatly significant for developing therapeutic strategies to silence c-FLIP for intervention with cervical carcinogenesis [128]. Furthermore, overexpression of c-FLIP_L also increases the hypoxia-inducible factor-1α (HIF1α) [129]. Overexpression of HIF1α can result in regulation of genes responsible for global changes in cell proliferation, metastasis, and invasion. Moreover, c-FLIP overexpression accelerated progression to androgen independence by inhibiting apoptosis in LNCaP prostate tumors implanted in nude mice [130].

Accumulating information clearly demonstrates that c-FLIP_S plays a major role in causing resistance to death ligands and chemotherapeutic agents. Park et al. [131] reported that MEK1/2 inhibitors synergistically interacted with the heat shock protein 90 (HSP90) inhibitor, geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin], to kill hepatoma and pancreatic carcinoma cells. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP_S that was connected to loss of MEK1/2 and AKT function. Moreover, overexpression of c-FLIP_S or Inhibition of caspase-8 abolished cell killing by MEK1/2 inhibitors and 17AAG. Interestingly, Panner et al. [132] reported that HSP90α recruits c-FLIP_S to the death-inducing signaling complex (DISC) and contributes to TRAIL resistance. Furthermore, combinations of low doses of sorafenib and vorinostat increased CD95 surface levels and CD95 association with caspase-8 and knockdown of CD95 or FADD expression reduced sorafenib/vorinostat cell death [133]. Signaling by CD95 caused protein kinase R (PKR)-like endoplasmatic reticulum kinase (PERK) activation that was responsible for both promoting caspase-8 association with CD95 and increased eIF2α phosphorylation. Suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK-and eIF2α-dependent lowering of c-FLIPs protein levels while overexpression of c-FLIP_S maintained cell viability [133]. Similarly, Zhang et al. [134] showed that expression of phosphorylation-insensitive eIF2α-S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP_S levels and overexpression of c-FLIP_S abolished lethality. Overexpression of c-FLIP_S function suppressed cell death by the multinuclear platinum chemotherapeutic BBR3610 [135].

Another important role of c-FLIP is its involvement in increasing cancer cell motility. The role of c-FLIP in cell motility has been investigated using a c-FLIP-specific siRNA. Shim et al. [136] showed that siRNA-mediated down-regulation of c-FLIP_L correlated with increased levels of reactive oxygen species (ROS), while over-expression of c-FLIP_L triggered the opposite effect. ROS generated by silencing c-FLIP induced phosphorylation of Akt and impaired cell motility [136]. The role of c-FLIP in the motility of HeLa cells was also shown using siRNA directed against c-FLIP. Silencing c-FLIP_L but not c-FLIP_S inhibited the adhesion and motility of the cells by activating FAK and extracellular regulated kinase (ERK), and increasing MMP-9 expression [137]. Additional evidence demonstrating the role of c-FLIP_L in triggering cell motility was recently provided in ovarian tumors [73]. In these tumors, c-FLIP_L played a role in chaperoning tumor cells from immunosurveillance and increasing their invasive potential by augmenting cell motility [73].

EMT is a process that induces morphological and genetic changes of cancer cells from an epithelial to a mesenchymal phenotype, which forms the basis for the metastatic potential of tumor cells [138]. Various tumor microenvironmental factors, including cytokines, growth factors, and chemotherapeutic agents trigger EMT [138], and this process is believed to be partly responsible for the chemotherapy-resistant phenotype [138,139]. A cancer-associated antigen gene (CAGE) which is widely expressed in various cancer tissues and cancer cell lines regulates expression of EMT-related proteins through ERK, Akt and NF-kB [140,141]. Snail, an EMT-related protein, mediates the effect of CAGE by inducing matrix metalloproteinase-2 (MMP-2) and cancer cell motility. Interestingly, c-FLIP mediates the effect of CAGE on the induction of MMP-2 and cell motility by the induction of Snail [141].

As shown in Table 1, DNA damaging agents are promising drugs with regard to downregulating levels c-FLIP variants. Pretreatment with chemotherapeutic drugs including cisplatin, doxorubicin, or topoisomerase I inhibitors (camptothecin, 9-NC, irinotecan) downregulated c-FLIP variants expression in various tumor cells by inhibiting its transcription and rendering cells sensitive to death receptor-triggered apoptosis (Table 1) [69,143-148]. Successful inhibition of malignant cell growth and apoptosis induction using histone deacetylase inhibitor (HDACi) compounds has highlighted the potential use of these compounds as anticancer agents. Several HDACi have been shown to downregulate c-FLIP expression in various cancer cells at the transcriptional and translational levels [149-152]. Among these, suberoylanilide hydroxamic acid (SAHA, vorinostat) is the most promising HDACi that causes robust inhibition of c-FLIP variants [149]. Recent results demonstrated that TRAIL-triggered apoptosis in breast cancer cells is blocked at the level of apical activation of caspase-8, and that SAHA enhances the TRAIL-induced processing and activation of procaspase-8. Interestingly, degradation of c-FLIPL and c-FLIPS by an ubiquitin/proteasome-dependent Itch/AIP4-independent mechanism is observed upon exposure to SAHA [149]. We recently showed that a new HDACi 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) or droxinostat [151,152], identified using a highthroughput chemical library screen [153,154], triggered apoptosis in the breast cancer cell line MCF-7 through c-FLIPL and c-FLIPS mRNA as well as protein downregulation [151]. Interestingly, this agent induced more robust apoptosis in a doxorubicin-resistant variant of MCF-7 cells [151]. As shown in Table 1, a number of agents with modulating effects on Akt, PI3K, NF-κB, and Ras pathways, as well as an inhibitor of STAT3 have also been shown to transcriptionally silence c-FLIP expression.

We have shown that CCRF-HSB-2 human lymphoblastic leukemia cells transfected with an antisense c-FLIP plasmid abrogated c-FLIP_S and c-FLIP_L expression and triggered a significant increase in Taxol-induced apoptosis [7]. Logan et al. [143] investigated whether using an antisense oligonucleotide to target c-FLIP was a clinically feasible approach (Table 2). These authors developed a novel c-FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO) and recently used it in vitro in transient transfection experiments and in vivo using xenograft models in Balb/c nude mice [143]. The AS PTO downregulated c-FLIP and resulted in caspase-8 activation and apoptosis induction in non-small cell lung cancer (NSCLC) cells, but not in normal lung cells. Similar results were observed in colorectal and prostate cancer cells. The AS PTO also sensitized cancer cells but not normal lung cells to apoptosis induced by TRAIL and increased chemotherapy-triggered apoptosis in NSCLC cells. Importantly, compared to a control non-targeted PTO, intraperitoneal delivery of c-FLIP AS PTO inhibited the growth of NSCLC xenografts and enhanced the in vivo antitumor effects of cisplatin. Therefore, this c-FLIP-targeted AS PTO may have potential for further pre-clinical development.

The development of RNA interference (RNAi)-based therapeutics to target the c-FLIP gene in vivo may change the way cancers are treated by inducing apoptosis [79] or by sensitizing cancers to chemotherapeutic agents. However, difficulties in siRNA design, delivery, and stability must be solved before RNAi-based therapeutics will be feasible for clinical use. We have used lipocomplexes of c-FLIP siRNA to successfully knock down the c-FLIP gene and induce spontaneous apoptosis in MCF-7 breast cancer cells in vitro [142] (Table 2), and in vivo by directly injecting the c-FLIP siRNA lipocomplexes into MCF-7 mouse xenografts [142]. Lipocomplexes of c-FLIP siRNA have also been used to successfully silence the c-FLIP gene and trigger spontaneous apoptosis in A549 lung cancer cells [165], HCT116 colorectal cancer cells [166], and LNCaP and PC3 prostate cancer cells [13]. Furthermore, c-FLIP siRNA lipocomplexes injected into HCT116 colorectal tumor xenografts decreased tumor growth [166]. These studies show that c-FLIP siRNA lipocomplex formulations can be used to successfully knock down the c-FLIP gene in various cancer cell types [76,95].

As discussed above, c-FLIP is predominately degraded by the ubiquitin-proteasome system. Downregulation of c-FLIP_L and c-FLIP_S due to degradation is observed in cells treated with various apoptosis-inducing agents (Table 2). Cycloheximide [167] and anisomycin [153], two protein synthesis inhibitors, as well as the RNA synthesis inhibitor actinomycin D [168] have been shown to downregulate c-FLIP_L and c-FLIP_S. Treating cancer cells with fluorouracil (5-FU) was also demonstrated to downregulate both isoforms in colon cancer cell lines [106,169] (Table 2).

Peroxisome proliferator-activated receptor γ (PPARγ) agonists sensitize cancer cells to TRAIL by ubiquitination and proteasome-dependent c-FLIP degradation [170-174]. Tiwary et al. [77] recently reported that α-tocopherol ether-linked acetic acid analogue (α-TEA) downregulation of c-FLIP is mediated by ER stress-dependent JNK/CHOP/DR5 signaling via JNK activation of Itch E3 ligase ubiquitination and involved in activation of the ER-stress-dependent events via reducing the inhibitory effect of c-FLIP on caspase-8.

Proteasome inhibitors are a new class of drugs that decrease proliferation and induce apoptosis in a variety of hematologic and solid malignancies [175-186]. Interestingly, several proteasome inhibitors lead to the downregulation of c-FLIP_L and c-FLIP_S [24,45,172]. The induction of apoptosis by the proteasome inhibitors MG-132 and PS-341 (bortezomib, Velcade^®) in primary chronic lymphocytic leukemia (CLL) cells and the Burkitt lymphoma cell line BJAB was associated with upregulation of TRAIL and its death receptors, DR4 and DR5, and decreased c-FLIP protein expression [179]. Similarly, bortezomib decreased c-FLIP expression in multiple myeloma and human esophageal squamous cell carcinoma cell lines [180,181]. However, the effect of PS-341 on the regulation of c-FLIP expression may be cancer cell-type specific. In contrast to what was observed human esophagil cancer cell lines, Liu et al. reported that PS-341 upregulates DR5 as well as c-FLIP and survivin in human non-small cell lung carcinomas (NSCLC) cells [183]. As discussed earlier, c-FLIP is degraded via a ubiquitin-proteosome system. Therefore, PS-341 should increase c-FLIP and prevent apoptosis. Interestingly, Zhao et al. have shown that PS-341 decreases c-FLIP at the gene level [184].

The Bcr-Abl kinase inhibitor imatinib mesylate (formerly known as CGP 57148B, STI571, or Gleevec) is currently the standard therapy for chronic myeloid leukemia (CML). Hamaï et al. [187] reported that Imatinib mesylate increases human melanoma cell sensitivity to TRAIL-induced cell death by directly downregulating protein levels of c-FLIP variants. Interestingly, Park et al. [188] showed that silencing the Bcr-Abl in K562 leukemia cells led to the downregulation of c-FLIP_L and subsequent increase to TRAIL sensitivity.

As shown in Table 2, a number of agents known to affect various targets and signaling pathways in cancer cells also cause degradation of c-FLIP variants (189-205). Moreover, several compounds have been shown to inhibit expression of c-FLIP variants, but whether these agents cause degradation of these proteins or silence their transcription remain to be found. Nutlin-3, a small molecule antagonist of MDM2 which inhibit the p53-MDM2 interaction and activates p53 signaling was recently shown to decrease expression of c-FLIP_S and c-FLIP_L and was synergistic with TRAIL in triggering cell death [206]. Moreover, Ozarelix, a gonadotropin-releasing hormone antagonist [207], celecoxib, a cyclooxygenase-2 inhibitor [208], the chemopreventive agent, all-trans-retinyl acetate (RAc) [125], smac mimetic compounds (SMC) [180], and sunitinib, an orally administered tyrosine kinase inhibitor (TKI) [210] decreased expression of c-FLIP. Furthermore, downregulation of c-FLIP by a specific microRNAs (i.e., miR-512-3p) increased taxol-induced apoptosis [211], supporting our previous report that silencing c-FLIP variants increases Taxol-triggered apoptosis [7]. Gemcitabine was also recently shown to inhibit expression of c-FLIP variant in pancreatic cancer cells [71], but whether it inhibits the transcription, enhances degradation, or prevents translation of c-FLIP remains to be found.

Recent data [122,212,213] clearly demonstrate that ataxia telangiectasia mutated (ATM) kinase activity modulates c-FLIP_L and c-FLIP_S protein levels in response to DNA damage (Figure 4). Moreover, the radiomimetic drug Neocarzinostatin (NCS) may trigger the down-regulation of c-FLIP isoforms by inducing the activation of the ATM kinase in response to DNA damage [122]. ATM kinase activity negatively modulates the stability of c-FLIP_L and c-FLIP_S at the protein level, thereby promoting the sensitivity to apoptosis induction by Fas (CD95/APO-1), a TRAIL-R1/R2-related death receptor [213]. NCS-triggered decrease in c-FLIP resulted in increased sensitivity to TRAIL which was inhibited by ATM kinase activity inhibition [122]. Upon NCS treatment, ATM promotes c-FLIP_L protein degradation through the ubiquitin-proteasome system but the mechanism of degradation of c-FLIP_S that ATM is linked to remains to be determined [122].