c-FLIP, A MASTER ANTI-APOPTOTIC REGULATOR

INTRODUCTION

The FADD-like interleukin-1β–converting enzyme (FLICE)-inhibitory (c-FLIP) proteins negatively regulate the signaling complex downstream of death receptors. In this review, I discuss (1) apoptosis signaling pathways and the role of c-FLIP isoforms as critical anti-apoptotic and drug resistance factors, (2) assess the potential for improving the outcome of cancer therapy by targeting c-FLIP and exploring the possibility of increasing its degradation and/or decreasing its expression to provide a safer approach to treat cancer, and (3) discuss novel c-FLIP targeted agents for cancer therapy that improve the efficacy of TRAIL and cytotoxic drugs.

APOPTOSIS SIGNALING PATHWAYS

Apoptosis is the orderly and tightly regulated cellular programmed cell death involving signal transduction pathways that induce cells to self-destruct during embryonic development, or in response to environmental hazards (e.g., radiation-induced DNA damage) or anticancer therapeutics. Apoptosis pathways provide control against cancer development, but specific mutations enable malignant cells to escape apoptosis and lead to tumor formation. Two major well-studied pathways, the intrinsic or mitochondrion-initiated pathway and the extrinsic or cell surface deathreceptors pathway, are involved in apoptosis (Fig. 1) [1–3]. In the mitochondrial pathway, cytochromec, certain caspases, apoptosis-inducing factor, Smac/DIABLO,andother apoptosis-inducing factors are released from the mitochondrial intramembranespace to the cytosol [4]. Once released, cytochrome c and dATPbind to apoptotic proteinase-activating factor-1 (Apaf-1), andthis complex along with adenine nucleotides promote procaspase-9autoactivation [5], which in turn activates caspases-2, -3,-6, -7, -8, and -10. Apoptosis triggered by various stimuli requires direct activation of Bax and BAK at the mitochondria by a member of the Bcl-2 homology domain-3 (BH3)-only family of proteins including Bid, Bim, or PUMA [6]. The various anti- and pro-apoptotic members of the Bcl-2 family form an interactive network that finally regulates the release of apoptosis triggering factors such as cytochrome c to the cytoplasm [7]. Apoptosis initiated by the endoplasmic reticulum (ER) stress signaling pathway is also mainly dependent on therelease of cytochrome c into the cytosol [8]. This release is associated with openingthe permeability transition pore (PTP) and a collapse of mitochondrial transmembrane potential (∆Ψ_m) due to the intake of Ca²⁺ following its release into the cytosolfrom the ER. Certain members of the Bcl-2 family on the ER appear to have a comprehensivefunction in the maintenance of ER homeostasis, participation in ER stress signal transduction pathways, and apoptosis [8].

In the extrinsic or death receptor-mediated apoptosis pathway (e.g., Fas/Fas ligand interaction, tumor necrosis factor-α [(TNF-α)/TNF receptor 1 (TNFR1), or TRAIL/DR5 interaction and cell death], the initiator caspa­ses-8 and -10 activate the downstream caspases including caspase-3 [9–11]. Active caspases-8 and -10 are known to cleave a pro-apoptotic Bcl-2 family member, Bid, and the truncated Bid induces mitochondrial cytochrome c release [26–29], 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 [2, 12–14]. Another pathway related to the intrinsic apoptotic pathway has also been identified [15]. In this pathway, BID is cleaved in response to several death-inducing stimuli (staurosporine, UV radiation, cycloheximide, etoposide) and this BID cleavage is blocked by Bcl-2, suggesting that degradation of BID occurred distal to cytochrome c release. Moreover, addition of cytochrome c to Jurkat post-nuclear extracts triggered cleavage of BID at Asp-59 which was catalyzed by caspase-3 rather than caspase-8. These results provide evidence that caspase-3 mediated cleavage of BID represents a feedback loop for the amplification of mitochondrial cytochrome c release during cytotoxic drug- and UV radiation-induced apoptosis [15].

CELLULAR FLICE-LIKE INHIBITORY PROTEIN (c-FLIP)

Structure of c-FLIP. The anti-apoptotic protein c-FLIP is a death effector domain (DED)-containing protein that is recruited to the DISC and regulates activation of caspases-8 and -10 in the death receptor signaling pathways (Fig. 1). c-FLIP has 13 distinct spliced variants, three of which are expressed as proteins: the 26 kDa short form (c-FLIP_S), the 24 kDa form of c-FLIP (c-FLIP_R), and the 55 kDa long form (c-FLIP_L) [16–18] (Fig. 2). The structures of c-FLIP_S and the viral v-FLIP proteins are similar, except that the two DEDs of c-FLIP_S are followed by 20 amino acids that appear to be crucial for its ubiquitaation and targeting for proteasomal degradation [11, 16–18]. c-FLIP_R also contains two DEDs but lacks the additional carboxy (C)-terminal amino acids that are present in c-FLIP_S. The C-terminus of c-FLIP_L is longer than that of c-FLIP_S and closely resembles the structure of caspases-8 and -10 [11, 16–18], but this region of c-FLIP_L does not contain a functional caspase domain. This lack of caspase activity is the result of several amino acid substitutions, particularly the crucial cysteine residue in the catalytic domain which is necessary for the catalytic activity of caspases [11, 16]. Additionally, c-FLIP_L has a caspase-8 cleavage site at position Asp-376 (LEVD); c-FLIP_L cleavage at this site produces the proteolytic fragment variant p43c-FLIP [11, 16–18]. The C-terminal region of c-FLIP_S and c-FLIP_R play a crucial role in ubiquitnation and degradation as well as the anti-apoptotic function of these isoforms [11, 16–18]. In humans, the decision to make c-FLIP_S or c-FLIP_R is defined by a single nucleotide polymorphism in a 3´ splice site of the c-FLIP gene. An intact splice site directs production of c-FLIP_S, but the splice-dead variant causes production of c-FLIP_R [19]. Because of differences in protein translation rates, higher levels of c-FLIP_S protein are produced compared with c-FLIP_R [19]. The three c-FLIP variants can interact with the adaptor protein FADD.

Fig. 1. Apoptosis pathways and roles of c-FLIP in preventing apoptosis. TRAIL interaction with DR4 and DR5 transduces the death receptor (extrinsic) and mithochondrial apoptosis signaling pathways through activation of caspases-8 and -10 (see the text for detailed information). c-FLIP isoforms are major anti-apoptotic proteins that suppress caspase-8 and -10 activation, and therefore prevent the downstream apoptosis cascade

Fig. 2. Structures of c-FLIP variants. Three c-FLIP variants, c-FLIP_L, c-FLIP_S, and c-FLIP_R, contain two death effector domains (DEDs) at their N termini. In addition to two DEDs, c-FLIP_L contains a large (p20) and a small (p12) caspase-like domain without catalytic activity. c-FLIP_S and c-FLIP_R consist of two DEDs and a small C terminus. Depending on its cellular level at the DISC, c-FLIP_L may act as an anti-apoptotic or pro-apoptotic factor [9, 16, 18]. c-FLIP_S, c-FLIP_S, c-FLIP_R, and two cleavage products of c-FLIP (p43-FLIP and p22-FLIP) act as anti-apoptotic proteins [9, 16, 18]. p43-FLIP and p22-FLIP are generated from c-FLIP_L by procaspase-8 cleavage at D376 [9] (adapted from [9, 16, 18])

c-FLIP transcription and translation. c-FLIP can be transcriptionally activated by various stimuli. These include TNF ligands, growth factors, interleukins, chemokines, and chemotherapeutic agents [11]. Several transcription factors are known to transcriptionally regulate the c-FLIP gene [11, 18]. These include NF-κB, p53 tumor suppressor protein, p63, E2F1, c-myc, IRF5, c-Fos, nuclear factor of activated T cells (NFAT), heterogeneous nuclear ribonucleoprotein K (hnRNP K), the forkhead transcription factor FOXO3a, early growth response-1 transcription factor (EGR1), androgen receptor (AR), E2F1, AP-1, and SP1. While NF-κB, p63, NFATc2, EGR1, hnRNP K, AR and SP1 induce c-FLIP, c-myc, FOXO3a, c-Fos, IRF5, and SP3 suppress c-FLIP transcription [11, 18]. p53 may transcriptionally upregulate the c-FLIP gene and also promote the degradation of c-FLIP protein [16]. Moreover, Gli2 transcription factor upregulates c-FLIP_S and c-FLIP_L [20]. c-FLIP_S is highly induced upon activation of T cells, primarily via the calcineurin-NFAT pathway [16]. The human T-cell leukemia virus type 1 (HTLV-1) Tax protein upregulates c-FLIP in HTLV-1-infected cells through activation of NF-κB [6]. c-FLIP_S is also upregulated at the translational level and causes TRAIL resistance in glioblastoma multiforme (GBM) cells due to activation of the Akt mammaliantarget of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway [21, 22]. Conversely,inhibition of mTOR or its targetS6K1 suppressed polyribosomal accumulation of c-FLIP_S mRNA, c-FLIP_Sprotein expression, and promoted TRAIL resistance in GBM cells. Moreover, it has been shown that Rocaglamide (Roc) sensitizes resistant adult T-cell leukemia/lymphoma (ATL) cells to DR4- and DR5-mediated apoptosis by translational suppression of c-FLIP_S through inactivation of the translation initiation factor 4E (eIF4E) [16, 23].

c-FLIP degradation. Both c-FLIP_L and c-FLIP_S isoforms are short-lived proteins that are predominately degraded by the ubiquitin-proteasome degradation system [11, 16, 18]. Both c-FLIP isoforms can be degraded by the proteasome. However, c-FLIP_S is particularly sensitive to ubiquitination and proteasomal degradation, partly due to two crucial lysine residues in the C-terminal 20 amino acids that are unique to c-FLIP_S [24]. The sensitivity of c-FLIP_S to ubiquitin-mediated degradation adds a novel concept to DISC regulation and its control of apoptosis [24]. c-FLIP_L and c-FLIP_S levels are also regulated by JNK activation via the E3 ubiquitin ligase Itch [11, 18, 25]. Phosphorylation events also play important roles in the regulation of c-FLIP protein levels. For instance, protein kinase C (PKC) phosphorylation at the serine 193 (S193) residue of c-FLIP_S inhibits its polyubiquitination, stabilizes c-FLIP_S levels, and increases cell survival [66]. S193 phosphorylation is markedly increased by treatment with the PKC activator 12-O-tetradecanoylphorbol-13-acetate and decreased by inhibition of PKCα and PKCβ. Phosphorylation of the S193 residue also decreased the ubiquitination of c-FLIP_L but did not affect its stability, indicating that S193 phosphorylation has a different function in c-FLIP_L than in c-FLIP_S. Moreover, pretreatment with the PKCδ-selective inhibitor rottlerin or transfection with PKCδ siRNA inhibited phorbol myristate acetate (PMA)-induced c-FLIP expression, which identifies a role for PKCδ in c-FLIP induction [26].

Upregulation of c-FLIP in human cancers. Increased expression of c-FLIP isoforms has been shown in cell lines from various types of cancer including colorectal [11, 16, 18]. Elevated levels of c-FLIP in tumor tissue from patients with colorectal cancer [28, 29], bladder urothelial cancer [30], cervical cancer [31], Burkitt’s lymphoma [32], non-Hodgkin’s lymphoma [33], head and neck squamous cell carcinoma (HNSCC) [34], and hepatocellular cancers [35] have been correlated with a poor clinical outcome and could be a reliable prognostic factor in these types of cancer. c-FLIP upregulation is also seen in gastric cancer and plays an important role in lymph node metastasis, which ultimately contributes to tumor progression [36]. c-FLIP isoforms are also expressed in pancreatic intraepithelial neoplasms as well as pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP expression [37]. McCourt et al. [38] reported that c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer tissue relative to normal prostate epithelium. Significantly, these authors found that maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC).

c-FLIP function

c-FLIP prevents apoptosis. Initial studies with animal models have revealed that c-FLIP plays an important role in T-cell proliferation and heart development [39, 40]. Moreover, abnormal c-FLIP expression has been found in various diseases such as cancer, multiple sclerosis, Alzheimer’s disease, diabetes mellitus, and rheumatoid arthritis [11, 17]. c-FLIP is also thought to be the main causal factor of “immune escape” [41]. c-FLIP is involved in TRAIL, Fas, TNF-α, and chemotherapeutic drug resistance in a wide range of human malignancies [11, 16–18]. 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 apoptosis and revealing that c-FLIP has a similar function to caspase-8 in heart development [40]. Nevertheless, extensive literature encompassing diverse types of human cancer cells now 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 [11, 18, 42–45]. In addition to its function as an apoptosis modulator, c-FLIP exerts other cellular functions including increased cell proliferation and tumorigenesis [11, 17–19].

The structural differences between human c-FLIP variants indicate distinct regulatory roles for c-FLIP_L and c-FLIP_S in apoptosis. c-FLIP_S inhibits TRAIL-induced DISC formation and apoptosis [24, 45], while c-FLIP_L is responsible for the dual functions described above whereby it inhibits Fas-induced caspase-8 activation when expressed at high levels, but enhances caspase-8 activation when its expression level is low [11, 18]. c-FLIP_S also suppresses apoptosis by inhibiting caspase-8 activation [46, 47]. Moreover, c-FLIP_S inhibits oxaliplatin-induced apoptosis through the sustained XIAP protein level and Akt activation [48].

We have demonstrated that c-FLIP_L interacts with DR5, FADD, and caspase-8 forming an apoptotic inhibitory complex (AIC) in MCF-7 breast cancer cells [49]. Moreover, silencing the c-FLIP gene by a specific siRNA leads to (1) death ligand-independent but DR5-, FADD-, and caspase-8- and -9-dependent apoptosis in these cells, and (2) the knockdown of c-FLIP expression inhibits breast cancer cell proliferation and triggers spontaneous apoptosis by activating both the death receptor and mitochondrial pathways [49]. Our data support the previous report by Jin et al. [50] demonstrating that the peptide corresponding to the DR5 binding domain of c-FLIP_L induces apoptosis in cancer cells. It is possible that inhibiting the interaction of DR5 and c-FLIP_L by peptides or small molecule inhibitors could provide a mechanism by which tumor-selective apoptosis can be achieved.

c-FLIP activates cytoprotective pathways. As shown in Fig. 3, 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 [19, 51] (see Fig. 3). 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 [19, 20, 52, 53]. Golks et al. [54] found that in nonapoptotic cells, c-FLIP and the procaspase-8 heterodimer result in a novel NH₂-terminal fragmentof c-FLIP (p22-FLIP) which is the key mediatorof NF-κB activation by binding to the IKK complex. Moreover, TNF-α-mediated JNK activation increases turnover of NF-κB-induced c-FLIP [25]. 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 [25].

Akt, a serine-threonine kinase, interacts with c-FLIP_L protein and c-FLIP_L enhances the anti-apoptotic functions of Akt [55, 56] by modulating Gsk3β activity. Moreover, through its effects on Gsk3β, c-FLIP_L overexpression in cancer cells induces resistance to TRAIL. This effect is mediated by regulation of p27 (Kip1) and caspase-3 expression [56]. Downregulation of the DNA-PK/Akt pathway is also reported to correlate with high responsiveness to TRAIL-mediated growth inhibition and apoptosis [57]. siRNA-mediated suppression of DNA-PK or treatment with 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), an inhibitor of DNA-PK, led to decreased phosphorylation of Akt and Bad (a target molecule of Akt), increased expression of DR4/DR5, and downregulation of c-FLIP [57]. Therefore, inhibition of the DNA-PK/Akt pathway may be clinically useful in treating TRAIL-resistant cancer cells [58].

Fig. 3. Multifunctional roles of c-FLIP on various signaling pathways. As discussed in the text, in addition to its functional role in inhibiting apoptosis by binding to procaspases-8 and -10 and inhibiting their activation, c-FLIP activates various anti-apoptotic and cell survival signaling pathways, leading to proliferation and cell survival

Panner et al. [59] demonstrated that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates c-FLIP_S ubiquitination and stability in GBM cell lines and xenografts. However, how PTEN and Akt are linked to AIP4 activity was unclear. These authors described a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), which is a downstream target of Akt, and how it links Akt to AIP4 and the regulation of c-FLIP_S stability [60]. Overexpression of USP8 increased c-FLIP_S ubiquitination, decreased c-FLIP_S half-life, decreased c-FLIP_S steady-state levels, and decreased TRAIL resistance. Therefore, PTEN appears to use control of ubiquitination to regulate TRAIL sensitivity in GBM cells.

c-FLIP_L also interactswiththe death domain-associated protein Daxx and prevents Fas-induced JNK activation [61]. Thus, c-FLIP_L acting on both the FADD- and Daxx-mediated signaling pathways may be involved in inhibiting Fas-induced cell death. Furthermore, 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 [62] (see Fig. 3).

Another regulator of c-FLIP upregulation is calcium/calmodulin-dependent protein kinase II (CaMK II), which protects 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 [63, 64]. Phosphorylation of c-FLIP variants by CaMK II appears to promote c-FLIP_L recruitment to the DISC and inhibit TRAIL-induced apoptosis [63, 64], but phosphorylation of c-FLIP_L by PKC or the bile acid glycochenodeoxycholate results in decreased c-FLIP_L recruitment to the DISC [65]. Thus, the particular site of phosphorylation on c-FLIP_L appears to influence the functional effect of this protein on apoptosis. It has been shown that calmodulin (CaM) binds directly to c-FLIP_L in a calcium-dependent manner and prevents Fas-triggered apoptosis [66]. Interestingly, a point mutation at H204 of c-FLIP_L markedly decreases CaM binding [67]. Therefore, inhibition of CaM/c-FLIP interaction may provide a new therapeutic strategy for cancer treatment.

Overexpression of c-FLIP can alter cell cycle progression and enhance cell proliferation and carcinogenesis [68, 69]. Increased expression 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 [70]. Similarly, c-FLIP_L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of β-catenin, thus suggesting an additional mechanism of tumorigenesis [70]. Moreover, a role for nuclear c-FLIP_L in modulating Wnt signaling has been established [71]. Interestingly, a deficiency of 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 [72], c-FLIP upregulation may contribute to the carcinogenesis and aggressiveness of endometrial carcinomas and serve as a useful prognostic factor for this tumor [71, 73]. 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 c-FLIP is an early marker of cervical carcinogenesis [74]. 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. This observation may be useful for developing therapeutic strategies to silence c-FLIP for intervention with cervical carcinogenesis [75]. Overexpression of c-FLIP_L also increases hypoxia-inducible factor-1α (HIF1α) [76]. In turn, overexpression of HIF1α can result in regulation of genes responsible 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 [77].

Much evidence has clearly demonstrated that c-FLIP_S plays a major role in causing resistance to death ligands and chemotherapeutic agents. Park et al. [78] reported that MEK1/2 inhibitors synergistically interact with the heat shock protein 90 (HSP90) inhibitor and geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin] to kill hepatoma and pancreatic carcinoma cells. Treating 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. [79] reported that HSP90α recruits c-FLIP_S to the DISC and contributes to TRAIL resistance. Furthermore, combinations of sorafenib and vorinostat increased CD95 surface levels and CD95 association with caspase-8, and knockdown of CD95 or FADD expression reduced sorafenib/vorinostat-induced cell death [80]. 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 sorafenib/vorinostat lethality. Cell death was paralleled by PERK- and eIF2α-dependent reduction of c-FLIPs protein levels, while overexpression of c-FLIP_S maintained cell viability [80]. Similarly, expression of phosphorylation-insensitive eIF2α-S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP_S levels and overexpression of c-FLIP_S [81]. Overexpression of c-FLIP_S also suppressed cell death by the multinuclear platinum chemotherapeutic BBR3610 [82].

c-FLIP as a therapeutic target for cancer treatment. c-FLIP can serve as a critical target for therapeutic intervention aimed at inhibiting its transcription and posttranscriptional changes [11, 16]. Ectopic expression of c-FLIP variants decreases apoptosis caused by death ligands and anticancer agents [24], indicating that overexpression of these proteins may cause resistance to multiple anticancer drugs. It does not appear possible to inhibit c-FLIP function with small molecule ligands since c-FLIP has significant structural similarity to caspase-8 (Fig. 2). This resemblance to caspase-8 makes c-FLIP protein a very difficult target for drugs to inhibit its function, since small molecules capable of blocking its recruitment to the DISC would also likely inhibit recruitment of caspase-8, thereby inhibiting apoptosis. Therefore, to reduce or inhibit c-FLIP expression, small molecules which target c-FLIP without inhibiting caspases-8 and -10 are needed. Several review articles have discussed classes of agents that decrease c-FLIP expression and sensitize cancer cells to TRAIL or anticancer drugs [11, 16, 18, 83]. These agents affect c-FLIP transcription, trigger c-FLIP degradation through the ubiquitin-proteasome system, or decrease c-FLIP translation. Moreover, DNA damaging agents are promising drugs with regard to downregulating levels of c-FLIP variants. Pretreatment with chemotherapeutic drugs including cisplatin, doxorubicin, or topoisomerase I inhibitors (camptothecin, 9-NC, irinotecan) downregulates c-FLIP variants expression in various tumor cells by inhibiting its transcription and rendering cells sensitive to death receptor-triggered apoptosis [84–90].

Several histone deacetylase inhibitors (HDACi) have been shown to significantly downregulate c-FLIP expression in various cancer cells at the transcriptional and translational levels [11, 91–94]. Among these, suberoylanilide hydroxamic acid (SAHA, vorinostat) is the most promising HDACi that causes robust inhibition of c-FLIP variants [91]. TRAIL-triggered apoptosis in breast cancer cells was blocked at the level of apical activation of caspase-8, and SAHA enhanced the TRAIL-induced processing and activation of procaspase-8. Interestingly, degradation of c-FLIP_L and c-FLIP_S by a ubiquitin/proteasome-dependent Itch/AIP4-independent mechanism is observed upon exposure to SAHA [91]. We recently showed that a new HDACi, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) [93] or droxinostat [94], identified using a high-throughput chemical library screen [95, 96], triggered apoptosis in the breast cancer cell line MCF-7 through c-FLIP_L and c-FLIP_S mRNA as well as protein downregulation [93]. Interestingly, this agent induced more robust apoptosis in a doxorubicin-resistant variant of MCF-7 cells [93]. Among c-FLIP inhibitors, histone deacetylase inhibitors have been very effective agents. Particularly significant is the recent discovery by Kerr et al. [97] reporting a novel interaction between c-FLIP and Ku70, a key component of non-homologous end joining machinery in the DNA damage pathway in the HCT-116 human colon cancer cell line. Interestingly, Ku70 regulates c-FLIP protein stability by inhibiting its polyubiquitination [97]. Moreover, these authors showed that vorinostat (SAHA) increased the acetylation of Ku70, thereby disrupting the c-FLIP/Ku70 complex and initiating c-FLIP polyubiquitination and degradation by the proteasome. Furthermore, the HDAC6-specific inhibitor Tubacin mimicked the effects of SAHA, suggesting that HDAC6 is a critical regulator of Ku70 acetylation and c-FLIP protein stability [97].

Small molecule therapeutics that selectively downregulate c-FLIP_S or c-FLIP_L and gene therapy strategies that knock down a specific c-FLIP variant have been used to downregulate these variants. Developing these innovative therapeutic strategies in conjunction with TRAIL and chemotherapeutic agents could potentially overcome the barrier of dose-limiting toxicity in cancer chemotherapy. TRAIL or chemotherapy resistance in diverse cancer cell types can be reversed by parallel treatment with agents known to downregulate c-FLIP variants.

Conclusions

Accumulating evidence shows that c-FLIP variants induce resistance to death receptor ligands and chemotherapeutic agents in various cancer cells. Moreover, c-FLIP upregulation correlates with a poor clinical outcome and could be a reliable prognostic factor in several types of cancer. Therefore, c-FLIP isoforms may be a relevant clinical target for counteracting therapy-resistant human malignancies. Various classes of agents can downregulate c-FLIP expression. Since c-FLIP has significant structural similarity to caspase-8, it is very difficult to target c-FLIP directly since small molecules capable of blocking the recruitment of c-FLIP to the DISC could simultaneously inhibit the recruitment of caspase-8 and thereby inhibit apoptosis. Therefore, to reduce or inhibit c-FLIP expression, small molecules which target c-FLIP without inhibiting caspases-8 and -10 are needed. Compounds that inhibit or downregulate c-FLIP mRNA expression or cause degradation of c-FLIP at the protein level through proteasome degradation will be of particular interest.

ACKNOWLEDGMENT

I would like to thank 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.

REFERENCES

1. Cereghetti GM, Scorrano L. The many shapes of mitochondrial death. Oncogene 2006; 25: 4717–24.
2. Gogvadze V, Orrenius S. Mitochondrial regulation of apoptotic cell death. Chem Biol Interact 2006; 163: 4–14.
3. Meng XW, Lee S, Kaufmann SH. Apoptosis in the treatment of cancer: A promise kept? Curr Opin Cell Biol 2006; 18: 668–76.
4. Ferri KF, Jacotot E, Blanco J, et al. Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex: Role of mitochondria and caspases. J Exp Med 2000; 192: 1081–92.
5. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 1996; 86: 147–57.
6. Ren D, Tu HC, Kim H, et al. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program.Science 2010; 330: 1390–3.
7. Danial NN, Gimenez-Cassina A, Tondera D. Homeostatic functions of BCL-2 proteins beyond apoptosis. Adv Exp Med Biol 2010; 687: 1–32.
8. Szegezdi E, MacDonald DC, Ni Chonghaile T, et al. Bcl-2 family on guard at the ER. Am J Physiol Cell Physiol 2009; 296: C941–53.
9. Lavrik IN, Krammer PH. Regulation of CD95/Fas signaling at the DISC. Cell Death Differ 2012; 19: 36–41.
10. Yang A, Wilson NS, Ashkenazi A. Proapoptotic DR4 and DR5 signaling in cancer cells: Toward clinical translation. Curr Opi Cell Biol 2010; 22: 837–44.
11. Safa AR, Day TW, Wu CH. Cellular FLICE-like inhibitory protein (c-FLIP): A novel target for cancer therapy. Curr Cancer Drug Targets 2008; 8: 37–46.
12. Li H, Zhu H, Xu CJ, et al. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the fas pathway of apoptosis. Cell 1998; 94: 491–501.
13. He B, Lu N, Zhou Z. Cellular and nuclear degradation during apoptosis. Curr Opin Cell Biol 2009; 21: 900–12.
14. Kurokawa M, Kornbluth S. Caspases and kinases in a death grip. Cell 2009; 138: 838–54.
15. Slee, EA, Keogh SA, Martin SJ. Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: A potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ 2000; 7: 556–65.
16. Safa AR, Pollok KE. Targeting the anti-apoptotic protein c-FLIP for cancer therapy. Cancers (Basel) 2011; 3: 1639–71.
17. Micheau O. Cellular FLICE-inhibitory protein: An attractive therapeutic target? Expert Opin Ther Targets 2003; 7: 559–73.
18. Bagnoli M, Canevari S, Mezzanzanica D. Cellular FLICE-inhibitory protein (c-FLIP) signalling: a key regulator of receptor-mediated apoptosis in physiologic context and in cancer. Int J Biochem Cell Biol 2010; 42: 210–3.
19. Ueffing N, Singh KK, Christians A, et al. A single nucleotide polymorphism determines protein isoform production of the human c-FLIP protein. Blood 2009; 114: 572–9.
20. Kump E, Ji J, Wernli M, et al. Gli2 upregulates cFlip and renders basal cell carcinoma cells resistant to death ligand-mediated apoptosis. Oncogene 2008; 27: 3856–64.
21. Panner A, Nakamura JL, Parsa AT, et al. mTOR-independent translational control of the extrinsic cell death pathway by RalA. Mol Cell Biol 2006; 26: 7345–57.
22. Panner A, Parsa AT, Pieper RO. Translational regulation of TRAIL sensitivity. Cell Cycle 2006, 5: 147–50.
23. Bleumink M, Köhler R, Giaisi M, et al. Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-Cell leukemia/lymphoma by translational suppression of c-FLIP expression. Cell Death Differ 2011; 18: 362–70.
24. Poukkula M, Kaunisto A, Hietakangas V, et al. Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail. J Biol Chem 2005; 280: 27345–55.
25. Chang L, Kamata H, Solinas G, et al. The E3 ubiquitin ligase itch couples JNK activation to TNFα-induced cell death by inducing c-FLIP_L turnover. Cell 2006; 124: 601–13.
26. Kaunisto A, Kochin V, Asaok T, et al. PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability. Cell Death Differ 2009; 16: 1215–26.
27. Jung SN, Park IJ, Kim MJ, et al. Down-regulation of AMP-activated protein kinase sensitizes DU145 carcinoma to Fas-induced apoptosis via c-FLIP degradation. Exp Cell Res 2009; 315: 2433–41.
28. McLornan DP, Barrett HL, Cummins R, et al. Prognostic significance of TRAIL signaling molecules in stage II and III colorectal cancer. Clin Cancer Res 2010; 16: 3442–51.
29. Ullenhag GJ, Mukherjee A, Watson NF, et al. Overexpression of FLIP_L is an independent marker of poor prognosis in colorectal cancer patients. Clin Cancer Res 2007; 13: 5070–5.
30. Korkolopoulou P, Goudopoulou A, Voutsinas G, et al. c-FLIP expression in bladder urothelial carcinomas: Its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations. Urology 2004; 63: 1198–204.
31. Wang W, Wang S, Song X, et al. The relationship between c-FLIP expression and human papillomavirus E2 gene disruption in cervical carcinogenesis. Gynecol Oncol 2007; 105: 571–7.
32. Valnet-Rabier MB, Challier B, Thiebault S, et al. c-FLIP protein expression in Burkitt’s lymphomas is associated with a poor clinical outcome. Br J Haematol 2005; 128: 767–73.
33. Valente G, Manfroi, F, Peracchio C, et al. cFLIP expression correlates with tumour progression and patient outcome in non-Hodgkin lymphomas of low grade of malignancy. Br J Haematol 2006; 132: 560–70.
34. Li X, Pan X, Zhang H, et al. Overexpression of cFLIP in head and neck squamous cell carcinoma and its clinicopathologic correlations. J Cancer Res Clin Oncol 2008; 134: 609–15.
35. Du X, Bao G, He X, et al. Expression and biological significance of c-FLIP in human hepatocellular carcinomas. J Exp Clin Cancer Res 2009; 28: 24.
36. Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (Lond.) 2004; 106: 397–405.
37. Haag C, Stadel D, Zhou S, et al. Identification of c-FLIP_L and c-FLIP_S as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells. Gut 2011; 60: 225–37.
38. McCourt C, Maxwell P, Mazzucchelli R, et al. Elevation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy. Clin Cancer Res 2012; 18: 3822–33.
39. Zhang N, Hopkins K, He YW. The long isoform of cellular FLIP is essential for T lymphocyte Proliferation through an NF-кB-independent pathway. J Immunol 2008; 180: 5506–11.
40. Yeh WC, Itie A, Elia AJ, et al. Requirement for casper (c-FLIP) in regulation of death receptor-Induced apoptosis and embryonic development. Immunity 2000; 12: 633–42.
41. Melki MT, Saпdi H, Dufour A, et al. Escape of HIV-1-infected dendritic cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk — A pivotal role of HMGB1. PLoS Pathog 2010; 6: e1000862.
42. Bagnol M, Canevari S, Mezzanzanica D. Cellular FLICE-inhibitory protein (c-FLIP) signaling: A key regulator of receptor-mediated apoptosis in physiologic context and cancer. Int J Biochem Cell Biol 2010; 42: 210–3.
43. Testa U. TRAIL/TRAIL-R in hematologic malignancies. J Cell Biochem 2010; 110: 21–34.
44. Yang JK. FLIP as an anti-cancer therapeutic target. Yonsei Med J 2008: 49: 19–27.
45. Yu JW, Shi Y. FLIP and the death effector domain family. Oncogene 2008; 27: 6216–27.
46. Li FY, Jeffrey PD, Yu JW, et al. Crystal structure of a viral FLIP: Insights into FLIP-mediated inhibition of death receptor signaling. J Biol Chem 2006; 281: 2960–8.
47. Budd RC, Yeh WC, Tschopp J. cFLIP regulation of lymphocyte activation and development. Nat Rev Immunol 2006; 6: 196–204.
48. Kim S, Lee TJ, Park JW, et al. Overexpression of cFLIPs inhibits oxaliplatin-mediated apoptosis through enhanced XIAP stability and Akt activation in human renal cancer cells. J Cell Biochem 2008; 105: 971–9.
49. Day TW, Huang S, Safa AR. c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8, and caspase-9-dependent apoptosis in breast cancer cells. Biochem Pharmacol 2008; 76: 1694–704.
50. Jin TG, Kurakin A, Benhaga N, et al. Fas-associated protein with death domain (FADD)-independent recruitment of c-FLIP_L to death receptor 5. J Biol Chem 2004; 279: 55594–601.
51. Chaudhary PM, Eby MT, Jasmin A, et al. Activation of the NF-кB pathway by caspase 8 and its homologs. Oncogene 2000; 19: 4451–60.
52. Fang LW, Tai TS, Yu WN, et al. Phosphatidylinositide 3-kinase priming couples c-FLIP to T cell activation. J Biol Chem 2004; 279: 13–8.
53. Dohrman A, Kataoka T, Cuenin S, et al. Cellular FLIP (long form) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation. J Immunol 2005; 174: 5270–8.
54. Golks A, Brenner D, Krammer PH, et al. The c-FLIP-NH2 terminus (p22-FLIP) induces NF-кB activation. J Exp Med 2006; 203: 1295–305.
55. Iyer AK, Azad N, Talbot S, et al. Antioxidant c-FLIP inhibits Fas ligand-induced NF-кB activation in a phosphatidylinositol 3-kinase/Akt-dependent manner. J Immunol 2011; 187: 3256–66.
56. Quintavalle C, Incoronato M, Puca L, et al. c-FLIPL enhances anti-apoptotic Akt functions by modulation of Gsk3β activity. Cell Death Differ 2010; 17: 1908–16.
57. Kim MJ, Kim HB, Bae JH, et al. Sensitization of human K562 leukemic cells to TRAIL-induced apoptosis by inhibiting the DNA-PKcs/Akt-mediated cell survival pathway. Biochem Pharmacol 2009; 78: 573–82.
58. Boatright KM, Deis C, Denault J-B, et al. Activation of caspases-8 and -10 by FLIP_L. Biochem J 2004; 382: 651–7.
59. Panner A, Crane CA, Weng C, et al. Ubiquitin-specific protease 8 links the PTEN-Akt-AIP4 pathway to the control of FLIP_S stability and TRAIL sensitivity in glioblastoma multiforme. Cancer Res 2010; 70: 5046–53.
60. Panner A, Crane CA, Weng C, et al. A novel PTEN-dependent link to ubiquitination controls FLIP_S stability and TRAIL sensitivity in glioblastoma multiforme. Cancer Res 2009; 69: 7911–6.
61. Kim YY, Park BJ, Seo GJ, et al. Long form of cellular FLICE-inhibitory protein interacts with daxx and prevents Fas-induced JNK activation. Biochem Biophys Res Commun 2003; 312: 426–33.
62. Nakajima A, Komazawa-Sakon S, Takekawa M, et al. An antiapoptotic protein, c-FLIP_L, directly binds to MKK7 and inhibits the JNK pathway. EMBO J 2006; 25: 5549–59.
63. Yang BF, Xiao C, Roa WH, et al. Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells. J Biol Chem 2003; 278: 7043–50.
64. Xiao C, Yang BF, Song JH, et al. Inhibition of CaMKII-mediate c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis. Exp Cell Res 2005; 304: 244–55.
65. Higuchi H, Yoon JH, Grambihler A, et al. Bile acids stimulate cFLIP phosphorylation enhancing TRAIL-mediated apoptosis. J Biol Chem 2003; 278: 454–61.
66. Pawar PS, Micoli KJ, Ding H, et al. Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling. Biochem J 2008; 412: 459–68.
67. Jing G, Yuan K, Liang Q, et al. Reduced CaM/FLIP binding by a single point mutation in c-FLIP(L) modulates Fas-mediated apoptosis and decreases tumorigenesis. Lab Invest 2012; 92: 82–90.
68. Gilot D, Serandour AL, Ilyin GP, et al. A role for caspase-8 and c-FLIP_L in proliferation and cell-cycle progression of primary hepatocytes. Carcinogenesis 2005; 26: 2086–94.
69. Stagni V, Mingardi M, Santini S, et al. ATM kinase activity modulates cFLIP protein levels: Potential interplay between DNA damage signalling and TRAIL-induced apoptosis. Carcinogenesis 2010; 31: 1956–63.
70. Naito M, Katayama R, Ishioka T, et al. Cellular FLIP inhibits β-catenin ubiquitylation and enhances Wnt signaling. Mol Cell Biol 2004; 24: 8418–27.
71. Katayama R, Ishioka T, Takada S, et al. Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L. J Cell Sci 2010; 123: 23–8.
72. Zhang L, Ren X, Alt E, et al. Chemoprevention of colorectal cancer by targeting APC-deficient cells for apo­ptosis. Nature 2010; 464: 1058–61.
73. Chen HX, Liu YJ, Zhou XD, et al. Expression of cellular FLICE/caspase-8 inhibitory protein is associated with malignant potential in endometrial carcinoma. Int J Gynecol Cancer 2005; 15: 663–70.
74. Wang W, Wang S, Song X, et al. The relationship between c-FLIP expression and human papillomavirus E2 gene disruption in cervical carcinogenesis. Gynecol Oncol 2007; 105: 571–7.
75. Wang W, Fang Y, Sima N, et al. Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell linesis mediated by interaction with c-FLIP. Apoptosis 2011; 16: 55–66.
76. Ishioka T, Katayama R, Kikuchi R, et al. Impairment of the ubiquitin-proteasome system by cellular FLIP. Genes Cells 2007; 12: 735–44.
77. Gao S, Wang H, Lee P, et al. Androgen receptor and prostate apoptosis response factor-4 target the c-FLIP gene to determine survival and apoptosis in the prostate gland. J Mol Endocrinol 2006; 36: 463–83.
78. Park MA, Zhang G, Mitchell C, et al. Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95. Mol Cancer Ther 2008; 7: 2633–48.
79. Panner A, Murray JC, Berger MS, et al. Heat shock protein 90α recruits FLIP_S to the death-inducing signaling complex and contributes to TRAIL resistance in human glioma. Cancer Res 2007; 67: 9482–89.
80. Walker T, Mitchell C, Park MA, et al. Sorafenib and vorinostat kill colon cancer cells by CD95-dependent and -independent mechanisms. Mol Pharmacol 2009; 76: 342–55.
81. Zhang G, Park MA, Mitchell C, et al. Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation. Clin Cancer Res 2008; 14: 5385–99.
82. Mitchell C, Kabolizadeh P, Ryan J, et al. Low-dose BBR3610 toxicity in colon cancer cells is p53-independent and enhanced by inhibition of epidermal growth factor receptor (ERBB1)-phosphatidyl inositol 3 kinase signaling. Mol Pharmacol 2007; 72: 704–14.
83. Plati J, Bucur O, Khosravi-Far R. Apoptotic cell signaling in cancer progression and therapy. Integr Biol (Camb) 2011; 3: 279–96.
84. Longley DB, Wilson TR, McEwan M, et al. c-FLIP inhibits chemotherapy-induced colorectal cancer cell death. Oncogene 2006; 25: 838–48.
85. Logan AE, Wilson TR, Fenning C, et al. In vitro and in vivo characterisation of a novel c-FLIP-targeted antisense phosphorothioate oligonucleotide. Apoptosis 2010; 15: 1435–43.
86. Kinoshita H, Yoshikawa H, Shiiki K, et al. Cisplatin (CDDP) sensitizes human osteosarcoma cell to Fas/CD95-mediated apoptosis by down-regulating FLIP-L expression. Int J Cancer 2000; 88: 986–91.
87. Abedini MR, Muller EJ, Brun J, et al. Cisplatin induces p53-dependent FLICE-like inhibitory protein ubiquitination in ovarian cancer cells. Cancer Res 2008; 68: 4511–7.
88. Song JH, Song DK, Herlyn M, et al. Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells. Clin Cancer Res 2003; 9: 4255–66.
89. El-Zawahry A, McKillop J, Voelkel-Johnson C. Doxorubicin increases the effectiveness of Apo2L/TRAIL for tumor growth inhibition of prostate cancer xenografts. BMC Cancer 2005; 5: 2.
90. Chatterjee D, Schmitz I, Krueger A, et al. Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIPshort. Cancer Res 2001; 61: 7148–54.
91. Yerbes R, Lуpez-Rivas A. Itch/AIP4-independent proteasomal degradation of cFLIP induced by the histone deacetylase inhibitor SAHA sensitizes breast tumour cells to TRAIL. Invest New Drugs 2012; 30: 541–7.
92. Lucas DM, Alinari L, West DA, et al. The novel deacetylase inhibitor AR-42 demonstrates pre-clinical activity in B-cell malignancies in vitro and in vivo. PLoS One 2010; 5: e10941.
93. Bijangi-Vishehsaraei K, Saadatzadeh MR, Huang S, et al. 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) Targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells. Mol Cell Biochem 2010; 342: 133–42.
94. Wood TE, Dalili S, Simpson CD, et al. Selective inhibition of histone deacetylases sensitizes malignant cells to death receptor ligands. Mol Cancer Ther 2010; 9: 246–56.
95. Schimmer AD, Thomas MP, Hurren R, et al. Identification of small molecules that sensitize resistant tumor cells to tumor necrosis factor-family death receptors. Cancer Res 2006; 66: 2367–75.
96. Mawji IA, Simpson CD, Gronda M, et al. A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis. Cancer Res 2007; 67: 8307–15.
97. Kerr E, Holohan C, McLaughlin KM, et al. Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis. Cell Death Differ 2012; 19: 1317–27.

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