Biological Significance of Apoptosis in Ovarian Cancer: TRAIL Therapeutic Targeting

An apoptotic program is present in almost all cell types. Functional characterization of the apoptotic cascade has revealed how the apoptotic program is activated in response to di‐ verse stresses such as DNA damage, signaling imbalance provoked by oncogene activation, survival factors insufficiency or hypoxia. One of the hallmarks of tumor cells is their ability to resist apoptosis. The concept that apoptosis serves as a barrier to cancer development has been well established (Evan and Littlewood, 1998; Hengartner, 2000; Lowe et al., 2004; Adams and Cory, 2007). This is especially relevant for ovarian cancer (OC) where most pa‐ tients presenting with advanced OC (most commonly high grade serous OC) will respond to the initial chemotherapy treatment suggesting that most tumor cells present are sensitive to chemotherapy. However, only 10-15% of these patients maintain a complete response to the initial therapy implying that a fraction of the tumor cells escaped apoptosis induced by che‐ motherapeutic drugs. Thus, one of the main obstacles to an effective treatment in OC is the failure of the initial chemotherapy to eradicate a sufficient number of tumor cells to prevent disease recurrence. Attenuation of apoptosis in those tumor cells contributes to the resist‐ ance to subsequent therapy and likely plays an important role in OC progression.


Introduction
An apoptotic program is present in almost all cell types. Functional characterization of the apoptotic cascade has revealed how the apoptotic program is activated in response to diverse stresses such as DNA damage, signaling imbalance provoked by oncogene activation, survival factors insufficiency or hypoxia. One of the hallmarks of tumor cells is their ability to resist apoptosis. The concept that apoptosis serves as a barrier to cancer development has been well established (Evan and Littlewood, 1998;Hengartner, 2000;Lowe et al., 2004;Adams and Cory, 2007). This is especially relevant for ovarian cancer (OC) where most patients presenting with advanced OC (most commonly high grade serous OC) will respond to the initial chemotherapy treatment suggesting that most tumor cells present are sensitive to chemotherapy. However, only 10-15% of these patients maintain a complete response to the initial therapy implying that a fraction of the tumor cells escaped apoptosis induced by chemotherapeutic drugs. Thus, one of the main obstacles to an effective treatment in OC is the failure of the initial chemotherapy to eradicate a sufficient number of tumor cells to prevent disease recurrence. Attenuation of apoptosis in those tumor cells contributes to the resistance to subsequent therapy and likely plays an important role in OC progression.
This chapter focuses on the molecular pathways that lead to apoptotic resistance and the need to move towards new targeted treatment in OC. Particular attention will be given to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling cascade. TRAIL is a cytokine that triggers apoptosis in a wide variety of tumor cells with apparent little effect on normal cells. We will discuss the various mechanisms that OC cells may develop to suppress TRAIL cytotoxicity. Furthermore, we will review the emerging TRAIL-tar-geting strategies for treating OC and provide information about the latest clinical studies of TRAIL agonists that are being conducted for the treatment of OC.

Treatment for ovarian cancer
Because of the limited efficacy of current treatments for advanced OC, novel and more effective therapies are being investigated. An emerging option for the treatment of OC is the targeting of the TRAIL signaling cascade. Because of its unique ability to trigger apoptosis in cancer cells and spare normal cells, in contrast to other cytokines such as FasL and TNFα, TRAIL is an attractive and promising treatment for cancer therapy. Preclinical studies in mice provided the first evidence that the soluble recombinant TRAIL suppresses the growth of human tumor xenografts with no apparent systemic toxicity (Walczak et al., 1999;Ashkenazi et al., 1999). More recently, recombinant TRAIL has entered clinical trials for the treatment of various malignancies (Ashkenazi, 2008;Ashkenazi et al., 2008; Abdulghani and El-Deiry, 2010; Hellwig and Rehm, 2012). In addition to soluble TRAIL, several agonistic antibodies targeting TRAIL R1 or TRAIL R2 death receptors have been developed and entered into clinical trials that included OC patients Hellwig and Rehm, 2012). As for standard chemotherapy, tumor cells have developed various mechanisms to escape the apoptosis induced by TRAIL. This underscores the need to understand the mechanisms of TRAIL resistance, and based on this knowledge, identify and validate novel combinations that could be used with TRAIL to potentiate its therapeutic efficacy. For example, TRAIL resistance has been often associated with overexpression of anti-apoptotic proteins. Therefore, the identification of combination treatments that abrogate anti-apoptotic protein function is promising.

Apoptosis overview
Deregulation of the apoptotic cascade not only plays a key role in the pathogenesis and progression of cancer, but also leads to resistance to chemotherapy. There are two major cellular death pathways that transduce the effects of various death inducers: the extrinsic and the intrinsic pathway ( Figure 1). The extrinsic pathway is triggered when TRAIL binds to TRAIL R1 or TRAIL R2. Receptor trimerization, along with the subsequent oligomerization and clustering of the receptors, leads to the recruitment of the adaptor protein Fas-associated protein with death domain (FADD). FADD allows the recruitment of the inactive procaspase-8 or -caspase-10 via a shared death effector domain (DED) leading to the formation of the death-inducing signaling complex (DISC). Depending on the cell type, apoptosis activation through the extrinsic pathways may or may not depend on the intrinsic pathway. For example, in type I cells, upon DISC activation, sufficient caspase-8 is activated and, in turn, directly activates the effector caspases (caspase-3, -6, -7) leading to the execution of apoptosis (Abdulghani and El-Deiry, 2010). FLICE-inhibitory protein (c-FLIP) shares structural homology with pro-caspase-8 and possesses a death effector domain that lacks protease activity. In specific conditions, its structure allows c-FLIP to be recruited to the DISC where it inhibits the processing and activation of pro-caspase-8. Although many isoforms of c-FLIP have been identified, only three are expressed in human cells (Djerbi et al., 2001). They consist of two short variants, c-FLIP S and c-FLIP R , and a long splice variant, c-FLIP L . Both c-FLIP L and c-FLIP S contain two DEDs and compete with pro-caspase-8 for association with FADD (Bagnoli et al., 2010). Depending on the level of c-FLIP L expression, its function at the DISC will vary. When present in high amounts, c-FLIP L will exert an anti-apoptotic effect at the DISC (Krueger et al., 2001). When present in low amounts, it may heterodimerize with caspase-8 at the DISC and promotes apoptosis (Chang et al., 2002). c-FLIP is thus seen as a major inhibitor of the extrinsic pathway of apoptosis. In so-called type II cells, less caspase-8 is activated at the DISC and efficient apoptosis requires further signal amplification via the intrinsic or mitochondrial pathway. This is achieved by caspase-8-mediated Bid cleavage to generate a truncated form of Bid (tBid) which subsequently engages Bax/Bak to activate the mitochondria.
The intrinsic pathway is usually triggered in response to DNA damage, hypoxia or oncogene overexpression. As a sensor of cellular stress, p53 is a critical initiator of the intrinsic pathway. In response to cellular damage, p53 translocates from the cytoplasm to the nucleus where it promotes the transcription of pro-apoptotic members of the Bcl-2 family. Pro-apoptotic Bcl-2 family members Bax and Bak form pores in the outer mitochondrial membrane causing the release of cytochrome c and other apoptogenic factors such as apoptosis inducing factor (AIF) and SMAC/DIABLO into the cytoplasm. The released of cytochrome c, along with apoptosis protease activating factor-1 (APAF-1) and pro-caspase-9 form the apoptosome. Within the apoptosome, clustered pro-caspase-9 gets activated and cleaves downstream effector caspases, leading to the hallmark of apoptosis (Youle and Strasser, 2008;Brunelle and Letai, 2009). The release of SMAC/DIABLO from the mitochondria promotes apoptosis by binding to and neutralizing members of the family of inhibitor of apoptosis proteins (IAPs), which can block caspase-3 activity through its baculovirus IAP repeat domains. Although the extrinsic and intrinsic pathways are activated by different mechanisms, these two pathways are interconnected ( Figure 1). In type II cells, activated caspase-8 cleaves pro-apoptotic Bcl-2 family member Bid to form truncated Bid (tBid), which can then interact with Bax/Bak. This interaction increases the release of cytochrome c from the mitochondria. Thus, Bid provides a connection between extrinsic and intrinsic pathways (so called mitochondrial amplification loop). The reasons that determine whether tumor cells rely on type I or II signaling are not well understood but resistance has been attributed to dysfunction of different steps in the TRAIL-induced apoptosis pathway and/or elevation of survival signals (Zhang and Fang, 2005). In particular, it has been proposed that the levels of c-FLIP and XIAP relative to caspase-8 and SMAC/DIABLO might be important determinants (Kim et al., 2000).
Bcl-2 family proteins are involved in the regulation of apoptosis by controlling mitochondrial membrane permeability. Several studies have demonstrated that these proteins can interact with each other and these interactions can neutralize their pro-or anti-apoptotic functions. The balance between anti-and pro-apoptotic members dictates the fate of cell sur-vival or death. Pro-apoptotic Bcl-2 members can be divided into 2 groups according to their function and the number of BH domains that they possess. Proteins containing BH domains 1-3 are known as multidomain pro-apoptotic proteins such as Bax, Bak and Bok (Youle and Strasser, 2008). BH-3-only pro-apoptotic proteins such as Bik, Bid, Bad, Bim, Bmf, Noxa, Puma and others can form heterodimers with the multidomain proteins Bax and Bak leading to the activation of the mitochondria. Anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 can also form hetero-dimeric interactions with Bax and Bak, thereby neutralizing their proapoptotic activity. Anti-apoptotic proteins can form hetero-dimers with BH-3-only proteins and this interaction neutralizes the pro-survival function of anti-apoptotic proteins.

TRAIL and its receptors
TRAIL is a member of the TNF ligand superfamily of cytokines and is a type II transmembrane protein, which is anchored to the plasma membrane and presented to the cell surface. The extracellular domain of TRAIL can be shed from the cell surface by cysteine proteases to produce soluble TRAIL. Both the soluble and the membrane-bounded TRAIL can trigger apoptosis by interacting with its cognate death receptors expressed by target cells. Of the five human TRAIL receptors that have been identified, both TRAIL R1 (DR4) and TRAIL R2 (DR5) contain a functional death domain in their intracellular portion, unlike decoy receptors TRAIL R3 (DcR1) and TRAIL R4 (DcR2), which lack a functional death domain and are thus incapable of transmitting an apoptotic signal (Pan et al., 1997a;Pan et al., 1997b; Sheridan et al., 1997; Marsters et al., 1997). Soluble TRAIL also binds with low affinity to soluble osteoprotegerin (OPG), which is a decoy receptor for RANKL that blocks the RANK-RANKL interaction (Hofbauer et al., 2000). OPG negatively regulates osteoclastogenesis and soluble OPG can act as a scavenger for soluble TRAIL and therefore inhibits TRAIL-induced apoptosis (Vitovski et al., 2007).

Expression of apoptosis-related proteins in ovarian cancer
Because the susceptibility of tumor cells to apoptosis appears to be determined, at least in part, by the ratio between pro-and anti-apoptotic proteins, the expression pattern of antiapoptotic proteins, Bcl-2, Bcl-X L and Mcl-1 has been assessed in OC tissues. For example, higher Bcl-2 expression has been generally associated with a favorable outcome in OC (Henriksen et al., 1995; Herod et al., 1996;Marx et al., 1997;Marone et al., 1998). This apparent paradox may be explained by the observation that high Bcl-2 expression delays cell cycle progression by promoting accumulation of cells in S phases without affecting the rate of apoptosis in OC cells (Bélanger et al., 2005). Bcl-X L expression is generally higher in OC tissues when compared to normal tissues (Marone et al., 1998) but has not been consistently associated with worse outcome (Shigemasa et al., 2002;Williams et al., 2005). This could be related to the observation that the ability of Bcl-X L to attenuate apoptosis appears to be cell context-dependent in OC (Dodier and Piché, 2006). In at least one study, increased Mcl-1 ex-pression has been correlated with poor prognostic for patients with OC (Shigemasa et al., 2002). Elevated expression of c-FLIP L has been reported in a substantial percentage of OC tissues from patients with advanced diseases (Mezzanzanica et al., 2004;Horak et al., 2005a) and has been associated with adverse outcome in some studies (Ouellet et  In patients with OC, high TRAIL expression in either tumor or stromal cells is a predictor of overall survival (Lancaster et al., 2003;Horak et al., 2005a). Interestingly in Horak's study, almost 50% of the tumor analyzed expressed elevated level of c-FLIP L and about 80% of tumors displayed low expression of TRAIL R1 and/or TRAIL R2, which could contribute to protect OC cells from TRAIL-induced apoptosis. Loss of TRAIL expression has been associated with worse outcome (Duiker et al., 2010). Furthermore, this group reported that epigenetic silencing of TRAIL R1 occurred in 8% to 27% OC tumor samples (Horak et al., 2005b). Higher expression of TRAIL receptors in OC cells has been associated with a worse outcome

Resistance in OC cells
The mechanisms of resistance to TRAIL can be divided into three categories based on their mode of acquisition: intrinsic resistance, acquired resistance and environment-mediated resistance (Goncharenko-Khaider et al., 2012). Each of them will be discussed separately.

Intrinsic resistance
Intrinsic resistance is observed when tumor cells are resistant to a specific drug without previous exposure to this drug. The incidence of intrinsic resistance to TRAIL among patients presenting with OC is not known but intrinsic TRAIL resistance among OC cell lines and primary OC cells is roughly 50% ( In summary, intrinsic TRAIL resistance appears to be multi-factorial and can be influenced by the activation of survival pathways such as Akt. In this context, the identification of informative and validated biomarkers of TRAIL resistance will be important for selecting patients and predicting the clinical outcome.

Acquired resistance
Acquired resistance is a mechanism by which tumor cells that were initially sensitive to a drug adapted to survive to prolonged exposure to this drug. Acquired drug resistance constitutes a major problem in the management of OC. This type of resistance is believed to be caused by sequential genetic alterations in tumor cells often associated with sub-lethal exposure to apoptosis-inducing drugs that eventually result in a therapy-resistant phenotype.
For example, in an OC cell line model, resistance to the anti-TRAIL-R2 antibody TRA-8 was induced by repeated exposure to non-apoptosis-inducing doses of the antibody (Li et al., 2006). Interestingly, the apoptotic responses induced by TRAIL, a TRAIL-R1 agonist antibody (2E12), and other apoptotic stimuli were not impaired. Lane et al. demonstrated that TRAIL acquired resistance was due to a rapid degradation of active caspase-3 subunits by the proteasome in the TRAIL resistant variant OC cells OVCAR3 (Lane et al., 2006). Interestingly, TRAIL resistant OVCAR3 cells remained sensitive to chemotherapeutic drugs.
One reassuring finding of these studies in OC and other in different tumor types is the fact that acquired TRAIL resistance does not confer cross-resistance to chemotherapeutic drugs such as cisplatin. In fact, combining standard chemotherapy with TRAIL treatment appears to be beneficial because treatment with platinum compounds upregulates the expression of TRAIL death receptors regardless of the p53 status which leads to increase apoptosis in OC cells (El-Gazzar et al., 2010b).  1997). Interestingly, it was recently shown that elevated ascites levels of IL-6, but not IL-8, were an independent predictor of shorter progression-free survival (Lane et al., 2011). Whether IL-6 is a critical soluble factor in ascites-mediated TRAIL resistance is unclear but a recent study suggests that IL-6 may indeed be an important component of the tumor environment that support tumor growth (Kulbe et al., 2012). Recently, high levels of IL-10, OPG and leptin in ascites were found to correlate with shorter PFS (Matte et al., 2012). Furthermore, in this study, Il-10 neutralizing antibodies attenuated the protective effect of ascites against TRAIL-induced apoptosis suggesting that IL-10 is one of the factors in ascites that promote ascites-induced TRAIL resistance. Collectively, these data support the role of ascites to promote resistance to TRAIL-induced apoptosis, at least in vitro. Whether this is relevant in vivo remains unclear for the moment. However, the prosurvival activity of ascites against TRAIL-induced apoptosis has been associated with shorter PFS in women with OC suggesting that ascites-mediated resistance might be clinically relevant (Lane et al., 2010b).

TRAIL targeting agents
Different strategies have been used to activate the TRAIL signaling pathway in cancer therapy. A variety of recombinant forms of soluble TRAIL have been developed and fused with different tags (Pitti et al., 1996;Schneider et al., 2000;Ganten et al., 2006). Major limitations however of recombinant soluble TRAIL (rsTRAIL) include the short half-life in vivo and relative lack of specificity as rsTRAIL can also bind decoy receptors TRAIL R3 and TRAIL R4. Despite these potential limitations, rsTRAIL (dulanermin) has entered phase I and phase II clinical trials. Alternatively, various humanized TRAIL receptor agonist antibodies have been developed which target TRAIL R1 (Mapatumumab) or TRAIL R2 (Apomab, Conatumumab, Lexatumumab, Tigatuzumab and LBY-135), and are currently being evaluated clin-ically (Table 1). These antibodies have a significantly increased half-life and consequently their bioavailability is increased at the tumor site.

Combination therapy
Several studies demonstrated that the combination of TRAIL with cisplatin was more efficient than either molecule alone in various OC cell lines in vitro (Cuello et  The enhanced efficacy of TRAIL in combination with other agents in preclinical models is encouraging and suggests that combination therapies with TRAIL probably represent the best clinical option at this point. Because TRAIL resistance in OC can be induced by various pathways, a combination of molecules that targets critical steps in the TRAIL signaling cascade is likely to be the most efficient approach.

Clinical trials with TRAIL targeting agents in OC patients
A large number of phase I/II clinical trials have been undertaken with TRAIL targeting agents either as monotherapy or in combination with chemotherapeutic drugs in a wide range of solid and haematological malignancies (

Conclusions and future directions
The inherent properties of TRAIL or its agonists offer a new targeted therapy for OC. Preclinical studies using TRAIL or its agonists have demonstrated the therapeutic potential of these molecules and formed the basis of ongoing phase I/II clinical trials. Although these treatments appear to be clinically well tolerated so far, intrinsic, acquired and environmentmediated resistance may limit the effectiveness of these approaches. However, the development of combination treatments appears to be capable of overcoming, at least in part, some of these limitations. As the search for more effective treatment for OC continues, the morbidity and mortality will hopefully improve. TRAIL treatment strategies have been used so far in the context of salvage treatment and the optimal patient population that will mostly benefit from these treatments remains to be defined. Although significant progress has been made in our understanding of the molecular basis of TRAIL resistance in OC, efforts should continue to further improve this knowledge as this will likely lead to the development of specific biomarkers of resistance and more efficient targeted therapies. Pro-caspase-8 binds to FADD leading to DISC formation and resulting in its activation. Activated caspase-8 directly activates executioner caspases (caspase-3, -6, and -7) (type I cells) or cleaves Bid (type II cells). Translocation of the truncated Bid (tBid) to the mitochondria promotes the assembly of Bax-Bak oligomers and mitochondria outer membrane permeability changes. Cytochrome c is released into cytosol resulting in apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of effector caspases activation that leads to morphological hallmarks of apoptosis. Further cleavage of pro-caspase-8 by effector caspases generates a mitochondrial amplification loop that further enhances apoptosis. When FLIP levels are elevated in cells, caspase-8 preferentially recruits FLIP to form a caspase-8-FLIP heterodimer which does not trigger apoptosis. Chemotherapeutic drugs such as cisplatin cause DNA damage which is sensed by the ataxia telangiectasia mutated homolog (ATM) leading to the activation of p53 dependent activation of genes such as PUMA and Noxa which can bind to anti-apoptotic proteins Bcl-2/ Bcl-XL thereby opposing their effect. This leads to mitochondrial permeabilization and activation.

Author details
Nadzeya