Gastrointestinal (GI) cancers are major health problems, being the most common cancers worldwide. Resistance to apoptosis is closely linked to carcinogenesis and enables malignant cells to evade therapy-induced cell death. In the recent past, the increasing understanding of molecular pathways of apoptosis has provided novel targets in cancer therapy. Several drugs, either inhibiting antiapoptotic signalling or actively inducing apoptosis in cancer cells, have already entered clinical trials. Until now, agents targeting apoptosis pathways are primarily being tested alone or in combination with chemotherapy. In the near future, personalised combination therapies will probaby be beneficial for patients with GI cancer. In this review, the current knowledge on defects in apoptosis signalling in GI cancer is summarised and the focus is on the potential clinical efficacy of apoptosis targeting agents.
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Key points 1
Gastrointestinal (GI) cancer is a major health burden worldwide.
Disruption of apoptosis signalling is frequently detected in GI cancer.
Various treatment approaches in oncology, such as chemotherapy and radiotherapy, act at least in part via induction of apoptosis.
The increasing understanding of impaired apoptosis signalling in GI cancer has identified promising therapeutic targets.
Apoptosis, also depicted as programmed cell death, is the most common form of cell death in the body. It is a fundamental event in the development and homeostasis of multicellular organisms. Virtually all cells contain the programmed machinery for the execution of apoptosis. In any multicellular organism, damaged and unwanted cells are removed by apoptosis in a programmed way every day. Apoptosis is actively executed by specific proteases, the caspases. Caspases cleave various proteins that are essential for cell integrity, resulting in DNA fragmentation, chromatin condensation, cell shrinkage, the formation of membrane blebs and, finally, the breakage of the cell into small vesicles. These apoptotic vesicles are removed by phagocytosis. In contrast to necrotic cell death, apoptosis does not cause inflammatory responses in physiological conditions.1
In human cancer, disruption of apoptosis signalling is frequently detected. Excessive cell survival due to defective apoptosis has emerged as a major mechanism by which neoplastic cells get a competitive advantage over normal cells. Since various treatment approaches in gastrointestinal (GI) oncology act at least in part via induction of apoptosis, resistance to apoptosis is a major obstacle for the treatment of advanced GI cancers. Many early-phase clinical trials are on the way which aim at reversing the apoptosis-resistant phenotype. Therapeutic strategies to inhibit antiapoptotic signals selectively in tumour cells have the potential to provide powerful tools to treat GI cancers.
INTRINSIC AND EXTRINSIC APOPTOTIC PATHWAYS
Apoptosis can be induced by various stimuli, including growth factor withdrawal, irradiation, cytotoxic drugs and death receptor ligands. There are two major signalling routes in mammalian cells leading to apoptosis, the extrinsic pathway (triggered by death receptors) and the intrinsic pathway (mediated by mitochondria) (fig 1).
In both the intrinsic and the extrinsic pathway, activation of caspases is a central event in the execution of apoptosis. Caspases are cysteine aspartyl proteases causing cell death by cleaving a variety of intracellular substrates. Caspases are constitutively expressed in cells as inactive precursors and are activated by proteolytic cleavage (for effector caspases) or by aggregation (for initiator caspases). Initiator caspases such as caspase-8, -9 and -10 are recruited to large protein complexes such as the so-called “death-inducing signalling complex” (DISC, see below). Initiator caspases cleave and activate effector caspases, such as caspase-3, -6 and -7, resulting in the characteristic biochemical and morphological changes of apoptosis.
The extrinsic pathway is initiated by binding of death receptor ligands to specific death receptors on the cell surface. The growing family of death receptors belongs to the tumour necrosis factor (TNF) receptor superfamily.2 Death receptors share an intracellular death domain (DD) which is essential for the transduction of the apoptotic signal.3 Among the members of the death receptor family known so far are TNFR1 (CD120a), TNFR2, CD95 (APO-1, Fas), TRAILR1 (TNF-related apoptosis-inducing ligand receptor 1) (APO-2, DR4) and TRAILR2 (DR5, KILLER, TRICK 2). The biological activity of TNFα depends upon ligation of TNFR1 and TNFR2.4 TNFR1 is not only a death receptor, but also promotes cell survival through activation of the transcriptional modulators nuclear facor-κB (NF-κB) and activator protein 1 (AP-1).5 6 CD95 is widely expressed on normal tissues, including the liver. The majority of GI cancers show one or more alterations in the CD95 pathway molecules (see below). TRAILRs attract special interest in oncology, since cancer cells are significantly more sensitive to TRAIL-induced apoptosis than normal epithelial cells. Herein, a panel of agonistic antibodies against TRAILRs have already been developed for the treatment of different GI cancers (table 1).
The crucial point of death receptor signalling is the formation of a multimeric complex of proteins triggered by receptor cross-linking with either agonistic antibodies or their natural ligands.7 The structure formed is called DISC.8 The DISCs of CD95 and TRAILR1/R2 consist of trimerised death receptors, the serine-phosphorylated adaptor Fas-associated death domain protein (FADD/Mort1), caspase-8 and caspase-10.9 The death signal is then propagated by caspase-8 and -10 via cleavage and activation of the effector caspases-3, -6 and -7.10 Both caspase-8 and -10 can also promote the activation of the intrinsic pathway, in a cell type-specific manner, by the cleavage of the protein Bid leading to direct activation of the proapoptotic Bcl-2 family members BAX and BAK.10 The intrinsic pathway is initiated at the mitochondrial level. Mitochondria are central players in apoptosis signalling and act as integrating sensors of various death stimuli. BH3-only members of the Bcl-2 family initiate the mitochondrial signalling cascade by sensing damage to the cells.11 After activation, BH3-only proteins are released to neutralise antiapoptotic Bcl-2 proteins. Subsequently, BAX and BAK trigger mitochondrial membrane leakage followed by the release of mitochondrial proteins into the cytosol, including cytochrome c, Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI) and apoptosis-inducing factor (AIF). Once cytochrome c is released into the cytosol, the “apoptosome” is assembled, a multiprotein complex in which apoptotic protease-activating factor-1 (Apaf-1) serves as an oligomerisation platform for assembly and autoproteolytic activation of caspase-9. Caspase-9 mediates activation of the effector caspases-3, -6 and -7, and produces a positive feedback loop in the extrinsic pathway through further activation of the initiator caspases-8 and -10.10
Smac/DIABLO proteins, which are released from activated mitochondria, inactivate the IAP (inhibitors of apoptosis proteins) protein family, which consists of XIAP, IAP1/2 and survivin. XIAP is a direct caspase inhibitor.12 Other IAPs including survivin, which is the one most differentially expressed between malignant and healthy tissue, have several functions apart from caspase inhibition—for example, triggering of ubiquitination processes.13
Bcl-2 proteins are a well-established family of proteins with important impact on mitochondrial integrity. These proteins exert their effects upstream of the mitochondria, integrating death and survival signals. The family includes at least 20 members of both proapoptopic and antiapoptotic effects, and share homology in the Bcl-2 homology regions (BH1–BH3). Antiapoptotic Bcl-2 family members possess all four BH homology regions (eg, Bcl-2, BCLX and MCL1). Proapoptotic members can either lack the BH4 domain (eg, BAX and BAK) or lack BH1, 2 and 4 domains (“BH3-only proteins”, eg, BAD and BIM). The antiapoptotic Bcl-2 family members interact with BAX and BAK to inhibit the activation of mitochondria.11
The prototypic apoptosis pathways require caspase activity. Opposing this established hypothesis, caspase-independent apoptosis models were established recently.14 In addition, it has become more and more clear that cancer cells also die by non-apoptotic mechanisms, such as autophagy, mitotic catastrophe and necrosis.15 However, a clear differentiation between death patterns is often difficult in individual cases due to morphological and molecular overlaps.16
PRINCIPAL APPROACHES TO MANIPULATE APOPTOSIS SIGNALLING IN CANCER CELLS
The application of antisense oligonucleotides (AS ODNs) is an option to downregulate antiapoptotic proteins efficiently in cancer cells. AS ODNs consist of nucleotide sequences that are complementary to a target RNA. Once delivered in the cell, AS ODNs bind to their RNA counterpart and suppress expression of the protein encoded by the target RNA. So far, phosphorothioate ODNs which are stable against cellular nuclease-mediated degradation, are the gold standard for AS therapy and have proved to be relatively non-toxic.17 AS ODNs usually need protracted intravenous or subcutaneous infusion for application. AS ODNs have already entered clinical trials for the treatment of GI cancer—for example, phase I/II in CRC with oblimersen, which targets Bcl-2 (www.clinicaltrials.gov).
Application of antibodies
The use of monoclonal antibodies (mAbs) for therapeutic purposes has become an important pillar in oncology in recent years. Despite the complex antigen structure of antibodies, severe treatment side effects remain the exception. In the coming years, numerous new antibodies will extend the therapeutic opportunities in cancer therapy. Antibodies may exert their antitumour effects in at least three different ways: (1) via induction of apoptosis (eg, agonistic mAbs specific for death receptors); (2) via competition with receptor ligands (eg, antibodies binding to growth factors); and (3) by preventing the expression of oncogenic or antiapoptotic genes. Many antibodies also stimulate the immune system mediating antibody-dependent cellular cytotoxicity (ADCC).18 One of the obstacles of antibody treatment is the immunogenicity of xenogeneic antibodies, and, if humanised immunoglobulins are used, the possible production of anti-idiotype antibodies. Structural modifications of antibodies—for example, coupling with radioisotopes—may further improve their antitumour efficacy.19 In addition, antibodies coupled to drug-loaded immunoliposomes may be used for site-specific delivery of anticancer agents.20
Tyrosine kinase inhibitors
Protein tyrosine kinases (TKs) are enzymes that provide a central switch mechanism in cellular signal transduction pathways including apoptosis signalling. Several TKs are known to be activated in cancer. Two classes of TK inhibitors (TKIs) have been developed. One acts by binding to the ATP-binding site and the other by binding to the substrate-binding site of the enzyme. TKIs are orally available, low molecular weight agents. Some TKIs specifically target one TK; others target multiple TKs in parallel (multi-TKIs). Together with mAbs, TKIs have already set a paradigm in cancer treatment, especially for the inhibition of growth factor receptors and other protein kinases.
Direct small molecule inhibitors/mimetics
This decade has witnessed a tremendous effort in the area of small molecule design. Small molecule inhibitors (SMIs) are organic molecules of low molecular weight. SMIs, currently developed—for example, for targeting Bcl-2 proteins, are orally available. There is substantial evidence that SMIs are in principal capable of triggering apoptosis in a variety of human cancers with minimal toxicity to normal cells. However, research is still at a starting point.21
Proapopototic (“suicide”) genes can be introduced into cancer cells via gene therapy approaches. Selected gene delivery systems—for example, adenoviral vectors—targeted to cancer cells are under investigation. The field of gene therapy and oncolytic virotherapy has now matured beyond the first high expectancy. Further development of second- and third-generation agents is necessary to ensure the therapeutic potential.22
Oncolytic viruses can selectively infect and replicate in cancer cells—for example in colorectal cancer (CRC).23 JX-594 is a first-in-class targeted oncolytic poxvirus designed selectively to replicate and destroy cancer cells. Intratumoural injection—for example, in metastasis of CRC—has been performed in phase I/II trials, showing reproducible efficacy.24 Parvoviruses preferentially replicate and induce apoptosis in tumour cells—for example, in HCC cells, and may thus be considered as therapeutic options for the treatment of GI cancer.25
RNA interference technology
A novel way for blocking antiapoptotic proteins in cancer cells is the application of RNA interference (RNAi) techniques. Here, double-stranded RNA (dsRNA) induces sequence-specific, post-transcriptional gene silencing.26 RNAi is now being extensively used to downmodulate gene expression in tumour cells in vitro. Several strategies, including chemical modification, nanoparticle formulation and application of viral vectors, are under development to improve the delivery of dsRNA into cancer tissues.27
MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that control gene expression post-transcriptionally via either the degradation of target mRNAs or the inhibition of protein translation. miRNAs play roles in almost all aspects of cancer biology. In the near future, more miRNAs will possibly emerge as players in the regulation of apoptosis signalling in cancer cells.28 The increasing knowledge of the role of miRNAs provides a rationale for the design of therapeutic agents that specifically target miRNAs (such as miRNA silencing or miRNA mimics). However, such agents have not entered large clinical trials so far.28
Dendrimer-based nanodevices, on the one hand, are able to improve cancer diagnosis as contrast agents, and, on the other hand, can be specifically targeted to cancer cells and thereby incorporate a proapoptotic function.29 The nanoparticle properties enable the agents to penetrate the tumour and to release therapeutic compounds. IT-101 is the first de novo designed experimental nanoparticle (linked to camptothecin).30
Transcription inhibitors can reduce the level of antiapoptotic proteins by binding to the promoter region of the corresponding genes. Terameprocol (currently in phase I/II trials) triggers apoptosis in cancer cells in part by the inhibition of survivin gene transcription.31
Triggering immune attack
Novel approaches to immune-based cancer treatment aim at augmenting antitumour immune responses by expanding tumour-reactive T cells, providing exogenous immune-activating stimuli, and antagonising immune tolerance.32 One approach is to target antiapoptotic proteins by peptide vaccines. Survivin peptide vaccines are already in phase I/II trials in solid cancers.33
DEFECTS IN APOPTOSIS SIGNALLING IN GI CANCER AS THERAPEUTIC TARGETS
Multiple sequential genetic changes need to occur to ensure GI cancer evolution. Cancer cells share at lest six features that distinguish them from normal cells: autocrine production of growth signals; inability to respond to antigrowth signals; sustained angiogenesis; unlimited replicative potential; tissue invasion/metastasis; and apoptosis avoidance.34 Tumour cells are subjected to pro-apoptotic stressful environmental factors, but nevertheless evade apoptosis induction. Different approaches in clinical development target these apoptosis defects.
Targeting defects in death receptor pathways
GI cancer cells are usually resistant to apoptosis triggered by death receptor activation. The majority of GI cancers show alterations in the CD95 pathway molecules—for example, in hepatocelluar carcinoma (HCC).35 36 Loss of response to CD95 triggering may be in part caused by downregulation of CD95 expression due to cleavage of the receptor by matrix metalloproteinases.37 Other alterations of CD95 signalling are enhanced expression of inhibitory downstream signalling mediators, such as cellular FLICE/caspase-8 inhibitory protein (c-FLIP or CFLAR), or antiapoptotic Bcl-2 proteins. c-FLIP, which is an inhibitor of caspase-8 activation, is constitutively expressed in GI cancer representing an important defect in apoptosis signalling at the death receptor level—for example, CRC.38 Although defects in CD95 signalling can be frequently found, GI cancer cells often show response to CD95 stimulation in vitro. Since many organs, especially the liver, constitutively express CD95, systemic application of agonistic antibodies or recombinant ligands would be too dangerous. However, for local therapies, triggering of CD95 might be a therapeutic option. APO010 is a recombinant CD95 ligand applied in phase I studies in solid tumours (www.clinicaltrials.gov). In addition, Fasaret is a recombinant adenovirus encoding CD95 ligand. Moreover, the application of bispecific antibodies directed to both target antigens on tumour cells and to CD95 may provide an attractive strategy for the selective stimulation of CD95 on the surface of tumour cells.39
Triggering TNFR1 by application of TNFα would cause severe toxic effects that are consistent with its role as a proinflammatory cytokine.40 In addition, activation of TNFR1 induces antiapoptotic proteins via NF-κB activation, limiting the antitumour effects. However, TNFerade which is a replication-deficient adenovector expressing TNFα under the control of a radiation-inducible promoter, might be applied intratumourally and afterwards activated by radiotherapy. Phase II trials in pancreatic, rectal and oesophageal cancer in combination with chemotherapy and radiotherapy are under way (www.clinicaltrials.gov).41
The death receptors which currently attract most attention in preclinical and clinical trials are TRAILR1 and 2. The natural ligand of these receptors, TRAIL, induces apoptosis in various transformed cell lines but lacks apoptosis induction in normal tissues.42 On the other hand, genetic lesions in various components of the TRAIL pathway have been described in human malignancies, suggesting that inactivation of the TRAIL pathway and escape from TRAIL-mediated immunosurveillance plays an important role in tumour progression.42 Two main approaches for triggering of TRAILRs on cancer cells have been developed: agonistic TRAILR antibodies, specific either for TRAILR1 (DR4) or TRAILR2 (DR5), and recombinant ligands (table 1).
Six agonistic mAbs against TRAILRs are currently in clinical trials and hold promise as novel selective tumour compounds. Mapatumumab (HGS-ETR1) is the only DR4 antibody currently in clinical development. Preclinically, mapatumumab is effective in killing various cancer cell lines and xenografts.42 In a phase I study in solid tumours including CRC, mapatumumab was well tolerated, but no objective responses were observed.44 Phase II studies exploring mapatumumab in combination with chemotherapy have been completed in non-small-cell lung carcinoma recently.42 Various DR5 agonistic antibodies are currently applied in clinical trials: apomab, AMG-655, CS-1008, LBY-135 and lexatumumab (HGS-ETR2) (table 1).42 Like mapatumumab, lexatumumab also showed liver toxicity at high dosis. In a phase Ib study, two patients with CRC received lexatumumab in combination with folinic acid, fluorouracil and irinotecan, and showed partial RECIST response.45 Apomab triggered minor responses in a patient with CRC.46 Moreover, early-phase studies of apomab in combination with chemotherapy have been started in CRC. AMG-655 was well tolerated and a PET response in a patient with CRC was detected.47 Recombinant human TRAIL (rhTRAIL) has been developed as a soluble zinc-coordinated homotrimer and was well tolerated in a phase I study.42
Stimulation of TRAILRs either by antibodies or by recombinant ligands may not be effective as a monotherapy in patients with GI cancer. Indeed, in phase I and II studies, effects of TRAIL stimulation were reported to be slow and to take several months to trigger responses. The reason is that cancer cells are mostly insensitive to TRAIL-mediated apoptosis, thus tempering the expectations of monotherapy in the clinic. Various mechanisms are being discussed with regard to mediating treatment resistance. Evidence is accumulating for the ability of TRAILR1 and 2 themselves to activate prosurvival signalling (fig 2).48 Moreover, decoy receptors, TRAILR3 and 4, which lack an intracytoplasmic death domain and act as non-functional binding factors for TRAIL, contribute to TRAIL resistance.10 O-Glycosylation of TRAIL receptors has recently been described as a mechanism of resistance at the receptor level.49 Importantly, increased expression of antiapoptotic proteins, such as c-FLIP, IAPs and BCL2, also contributes to TRAIL resistance.42 However, there are recent advances in the sensitisation of GI cancer to TRAlL-induced apoptosis. For example, proteasome inhibitors and histone deacetylase inhibitors (HDACIs) may sensitise GI cancer cells but not primary human hepatocytes for TRAIL-induced apoptosis.50 51 Given the potent inhibitory effect of c-FLIP in mediating resistance to TRAIL agonists, combination of TRAIL with c-FLIP inhibitors would be an attractive option for clinical development. Indeed, efforts to develop SMIs or peptide inhibitors for targeting c-FLIP are expanding.52 Another potent TRAIL sensitiser could be the multi-TKI sorafenib. A key mechanism for TRAIL sensitisation by sorafenib is downregulation of the antiapoptotic Bcl-2 protein MCL1, which is overexpressed, for example, in cholangiocellular carcinoma.53 Another approach is the combination with so-called Smac mimetics, which allow for a more potent caspase activation following TRAILR stimulation. In addition, Smac mimetics mediate antitumour activity by the induction of TNFα and TNFR1.42
In the near future, combination of TRAIL agonists with agents targeting TRAIL-antagonistic mechanisms will be potentially effective in cancer therapy. However, care should be taken when combining TRAIL with other therapeutic agents because of the possible toxicity to normal tissues, as illustrated, for example, by the finding that TRAIL/cisplatin combination was toxic towards primary hepatocytes.50 Moreover, it has been reported that hepatocytes have augmented TRAIL sensitivity during viral infections, alcohol intake and cholestasis.42
Targeting defects in mitochondrial activation
Many of the genetic alterations observed in GI cancer result in an imbalance of proapoptopic and antiapoptotic members of the Bcl-2 family. Bcl-2 proteins are critical regulators of the apoptotic machinery. Antiapoptotic Bcl-2 proteins are overexpressed in a great percentage of GI cancers conferring protective effects on malignant cells. In contrast, proapoptotic members such as BAX are downregulated. Thus, mitochondrial activation, which is a key event in a variety of apoptotic events—for example, in chemotherapy-induced cell death, is impaired. To target antiapoptotic Bcl-2 family proteins, AS ODNs, cell-permeable peptides as well as non-peptidic SMIs are under clinical investigation. Oblimersen, the furthest advanced AS ODN, targets Bcl-2 and is applied in phase II studies in HCC (in combination with doxorubicin), CRC (with FOLFOX or irinotecan), GI stroma tumours (with imatinib) and oesophageal cancer (with cisplatin and fluorouracil) (table 2). Upon continuous infusion in phase III trials, one of the main side effect was myelosuppression. Other Bcl-2 AS ODNs, designed by the use of so-called locked nucleic acid technology, which confers higher resistance to enzyme degradation (eg, SPC2996) are not applied in GI cancer yet. Another approach to target antiapoptotic Bcl-2 proteins is the use of cell-permeable peptides, which mimic the BH3-only class of proapoptotic Bcl-2 proteins and are thus also depicted as “BH3 mimetics”. They block the hydrophobic BH3-binding pocket of antiapoptotic Bcl-2 proteins that normally interacts with BH3-only proteins. Thereby, BH3-only proteins are released and activated. Attention is currently focused on another group of agents—that is, non-peptidic SMIs (table 2).
These agents also act as BH3 mimetics, since they bind to the hydrophobic BH3-binding pocket in antiapoptotic Bcl-2 proteins. Gossypol was the first such compound to reach the clinic, although its mechanism of action was not known at the time of development. It is a natural polyphenol binding to Bcl-2, BCLX and MCL1.55 An R-enantiomer (AT-101) is currently in phase I/II studies in oesophageal cancer in combination with chemotherapy and radiotherapy (www.clinicaltrials.gov). A number of other SMIs with diverse chemical structures including pan Bcl-2 SMIs are under study.54 Interestingly, SMIs show a large spectrum of activities apart from targeting Bcl-2 proteins (eg, activity against survivin or AKT). Among other combination therapies, a promising approach in GI cancer is the application of BH3 mimetics or SMIs in combination with TRAILR stimulation. In particular, targeting of MCL1 has been shown to be the gateway to TRAIL sensitisation in preclinical studies.53 In CRC cell lines specific downregulation of BCLX via RNAi strongly increases TRAIL sensitivity.56
Targeting of proteins downstream of activated mitochondria
Smac/DIABLO is released from mitochondria to the cytosol in response to diverse apoptotic stimuli and antagonises IAPs, thus allowing caspase activation and apoptosis induction (fig 1).12 Preclinical observations have shown that upregulation of Smac signalling can increase sensitivity of cancer cell lines and xenografts to chemotherapy while having few toxic effects on healthy tissues.57 The proapoptotic activity of Smac partly relies on an N-terminal motif that binds to a groove of IAPs, blocking its interaction with caspase-9. Smac mimetics bind to this groove on different IAP proteins.58 IAPs are frequently overexpressed in GI cancer.59 The IAP protein family consists of eight members, including survivin, XIAP and IAP1/2. They represent apoptosis-antagonising proteins which act independently of caspase activation by binding to other adaptor or cofactor molecules.59 Specific inhibitors of survivin are in development. LY2181308 is an AS ODN against survivin which is highly potent in preclinical studies and is currently applied in phase I/II studies in advanced HCC.33 In addition, immunotherapeutic approaches against survivin are under development, since survivin is differentially expressed in malignant cells compared with normal cells.60 Terameprocol (M4N, EM-1421) is a transcription inhibitor which reduces the levels of survivin by binding to the gene promoter.31 The discovery of a small molecule-binding groove distinct from the Smac-binding site opens up ways to develop SMIs of survivin.59 Another approach to target survivin is the inhibition of heat-shock proteins, such as HSP-90.61 Inhibition of the eukaryotic initiation factor-4E, which is upregulated in various cancers including CRC, also efficiently downregulates survivin and other antiapopototic proteins.62 Recently, it has become more and more clear that survivin is not only a caspase inhibitor, but also a nodal protein linking multiple pathways of cellular homeostasis and regulating cell division, non-apoptotic cell death and angiogenesis.59
XIAP has been extensively studied for its role in human neoplasia and is known to inhibit caspase-3, -7 and -9. Second-generation AS ODNs, such as AEG35156, have been developed against XIAP and are currently applied in phase I/II studies in pancreatic cancer (in combination with gemcitabine).63
Targeting of receptor tyrosine kinases
Receptor tyrosine kinases (RTKs) are often aberrantly activated in human malignancies and contribute to cancer development and progression. RTKs can protect cancer cells from apoptosis induced by stress, physiological factors or proapoptotic drugs. Specific RTK inhibition has been shown to be clinically effective in GI cancer patients.
The epidermal growth factor receptor (ErbB1 or EGFR) represents the main member of the TK type ErbB receptor family, together with ErbB2 (HER2/neu), ErbB3 and ErbB4. EGFR activation leads to the activation of downstream signalling cascades, such as Ras–Raf–mitogen-activated protein kinase (MAPK) and PI3K (phosphatidylinositol 3-kinase)/AKT. Consequently, the pathway exerts antiapoptotic functions, among other effects.64 In GI cancer studies, EGFR overexpression has frequently been detected, and correlates with an advanced stage of the disease and poor outcome.65 Several strategies targeting EGFR are in progress in GI cancer, but anti-EGFR mAbs (such as the chimeric antibody cetuximab) and TKIs (such as gefitinib and erlotininib) are the most developed. In preclinical studies in oesophageal cancer, gefitinib efficiently triggers apoptosis via PI3K/AKT pathway blockage.66 The same applies for application of the EGFR–ErbB2 dual blocker lapatinib, which is currently in phase III trials in oesophageal cancer in combination with chemotherapy (www.clinicaltrial.gov).
Vascular endothelial growth factor receptor (VEGFR)-dependent signalling is a key regulator of angiogenesis, but also exerts antiapopototic effects in GI cancer cells. Thus, blocking the VEGFR pathway (eg, by the VEGF antibody bevacizumab) also induces apoptosis in cancer cells. Other approaches resulting in VEGFR blockage are application of multi-TKIs such as sorafenib and sunitinib, or a selective TKI such as cediranib, which has recently been applied in phase II studies in CRC.67 In CRC, VEGFR expression correlates with tumour aggressiveness and can also be downregulated by cetuximab.68
c-Met is an RTK activated by hepatocyte growth factor/scatter factor, which is implicated in cancer cell survival. Overexpression of c-Met or Met gene amplification has been associated with poorer prognosis in GI cancer.69 Many inhibitor strategies are used clinically, and some inhibitors have entered phase I or II trials including antibodies to c-Met and c-Met TKI. ARQ 197, for example, is a non-ATP competitive SMI of c-Met applied in phase II trials in pancreatic cancer (www.clinicaltrials.gov).
The RTK IGF-IR (insulin-like growth factor-I receptor) activates various pathways involved in apoptosis signalling. More than 25 molecules are at different stages of development to inhibit IGF-IR in cancer cells, including TKIs and mAbs.70 IGF-IR is mainly activated by the growth hormones IGF-I and -II, which interact with their high-affinity binding proteins (IGF-binding proteins, IGFBPs). TK activity results in the activation of the Ras–Raf–MAPK and PI3K/AKT pathway. Various mAbs and TKIs (the latter with less experience) are currently tested in phase I and II studies (eg, CP-751,871, phase II in CRC as a single agent; IMC-A12, combination with cetuximab in CRC).70 The main side effects, reported so far, are hyperglycaemia and mild skin reactions. Other approaches to inhibit IGF-IR (peptides, proteins, AS ODNs) have not reached the clinic yet.
Transforming growth factor β inhibition
The intracellular signals of the transforming growth factor β (TGFβ) family of cytokines couple its intracellular signals to the apoptotic machinery by activation of the intrinsic pathway. However, TGFβ can induce cell death on the one side, but on another side it activates antiapoptotic signals, mainly via activation of EGFR. Dysregulation in TGFβ signalling contributes to carcinogenesis, for example in colorectal and pancreatic cancer.71 A way to target this pathway is, for example, application of AS ODNs. AP 12009 is specific for TGFβ2 mRNA and is currently tested in phase I/II trials in pancreatic cancer and CRC.72
Other RTKs contributing to apoptosis resistance are the PDGF (platetet-derived growth factor) and FGF (fibroblast growth factor) RTKs. CP-868 is a highly specific PDGFR inhibitor in phase I trials.73 Brivanib alaninate is a dual TKI of FGFR and VEGFR which has been tested in CRC in phase I studies.74
Targeting of intracellular survival signalling
Interference with intracellular protein kinase pathways, which mediate antiapoptotic prosurvival signals in tumour cells, represents an important approach to overcome apoptosis resistance in cancer. The PI3K pathway induces a diverse array of cancer-promoting events including the regulation of the protein kinases PDK1 and AKT, which directly bind to and are activated by PIP3 (fig 3). The AKT serine/threonine kinase phosphorylates a plethora of targets to activate the cell cycle, prevent apoptosis and trigger cellular growth.75 Over 20 direct AKT targets have been identified. Recently, the activation of the PI3K pathway has been shown to correlate significantly with the lack of efficacy of several targeted therapies, for example treatment with cetuximab.76 Interestingly, K-Ras mutations, which are known to activate PI3K, are also associated with poor outcome after cetuximab treatment.77 PI3K inhibitors are already in clinical trials. XL765, for example, is a well-tolerated, selective dual oral inhibitor of PI3K and mTOR (mammalian target of rapamycin; a downstream target of AKT), applied in phase I trials in solid tumours.33 Further potential targets in the PI3K/AKT pathway include the upstream RTKs (including growth factor receptors), Ras proteins, AKT and PDK1.
An important trigger for overactivation of PI3K in cancer cells is deletion or mutation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted from chromosome ten), an inhibitor of PI3K. PTEN is one of the most commonly mutated tumour suppressors in GI malignancies and affects diverse cellular processes such as cell proliferation, apoptosis and DNA damage responses (fig 3).78 However, interconnections of the PTEN network to many other tumour suppressor and oncogenic pathways are being constantly unravelled. Importantly, it has become clear that functional PTEN may be required for the effectiveness of targeted therapies, for example for the treatment of CRC with cetuximab.78
One of the downstream targets of the PI3K/AKT pathway is the kinase mTOR. mTOR is an important regulator of apoptosis signalling in cancer cells due to its key position on the crossroad of various signalling pathways including Ras and PI3K. There are two distinct mTOR complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). Several elements of these complexes have been identified in the recent past—for example, raptor (regulatory associated protein of mTOR) and rictor (rapamycin-insensitive companion of mTOR). The main functions of mTORC1 include protein synthesis and cell cycle progression, whereas mTORC2 plays an important role in actin cytoskeleton organisation and cell survival. In HCC, for example, mTOR signalling including aberrant expression of rictor plays an important pathophysiological role.79 These recent findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC. Examples of mTOR inhibitors already applied in phase III clinical trials are temsirolimus (for renal cell carcinoma) and everolimus (eg, for pancreatic neuroendocrine tumours).80
The MAPK pathway, which is often deregulated in GI cancer, includes the G-protein Ras and the kinases Raf, MEK1/2 and ERK1/2. Ras, which is frequently overexpressed in GI cancer, is activated by RTKs, including growth factor and cytokine receptors, and is the main trigger for the activation of Raf and MAPK. The MAPK pathway exerts antiapoptotic effects in cancer cells. For example, ERK1/2 phosphorylates and thereby inactivates the proapoptotic Bcl-2 protein BAD.81 A recent publication demonstrated that Ras activation results in DNA methylation of important components of the cell killing machinery, such as CD95 and its ligand.82
Various inhibitors of the Raf kinase have been developed for the inhibition of the MAPK pathway. LErafON is a chemically protected AS ODN with improved intracellular delivery by encapsulation in a cationic liposome, and is administered in phase I trials.83 Another way to block Raf kinase activity is the application of sorafenib, which blocks Raf, VEGFR and other kinases in parallel. Direct therapeutic intervention of Ras activation is being attempted by the use of farnesyl transferase inhibitors, which prevent membrane localisation and thereby activation of Ras.84 XL518 is an example for an oral MEK inhibitor applied in phase I trials in advanced cancer.85
Signal transducer and activator of transcription (STAT) proteins are a family of latent transcription factors that become activated by RTKs in response to cytokines and growth factors. STAT proteins are aberrantly activated in a diverse spectrum of GI tumours and exert antiapoptotic functions. STAT3 inhibition can reverse tumour growth in experimental systems while having few effects in normal cells. Molecules have been developed that block STAT3 function at a variety of steps including inhibitors of Janus kinases or inhibitors of STAT3 dimerisation.86
NF-κB is a key regulator of cellular survival. Its activation in response to cellular stress, originating from radiotherapy or chemotherapy or other proapopototic agents, leads to increased expression of antiapoptotic genes, for example BCLX or XIAP. Proteasome-mediated degradation of the NF-κB inhibitory protein, IκB-α, leads to NF-κB translocation to the nucleus and to the activation of target gene transcription.87 Thus, proteasome inhibitors are promising agents for interruption of NF-κB signalling (see below). Specific inhibitors of NF-κB signalling include the IKK inhibitor PS-1145 and the IκB-kinase 2 inhibitor AS6002868, and have been proven to sensitise pancreatic cancer cells, for example towards TRAIL-induced apoptosis.88 RTA 402 is a novel NF-κB/STAT3 inhibitor in phase I trials in solid tumours.33
The protein kinase C (PKC) family of proteins includes several cytoplasmic serine/threonine kinases with deregulated expression in various tumours. Enzastaurin is an oral kinase inhibitor suppressing PKC and PI3K activity entering phase I trials in advanced cancer.89
The protein kinase CK2 is one of the most pleiotropic serine/threonine kinase, with a plethora of endogenouse substrates implicated in apoptosis signalling. For example, CK2 is at the nexus of two signalling pathways that protect cancer cells from TRAIL-induced apoptosis. First, it inhibits TRAIL-mediated, caspase-8-dependent cleavage of Bid. Secondly, it promotes NF-κB-mediated expression of BCLX. A number of selective CK2 inhibitors are already available.90
Progression of GI cancer is often associated with an enhanced activity of the non-receptor Src family of TKs. Src is capable of hyperactivating EGFR by phosphorylating multiple intracellular receptor sites.91 Due to their antiapoptotic effects, Src kinases are potential targets in GI cancer. The TKI dasatinib targets Src kinases and other prosurvival kinases, and is currently being tested in phase III trials, for example in gastroeintestinal stroma tumours (www.clinicaltrials.gov).
Treatment options with a broad mechanism of action including downregulation of antiapoptotic signalling
Recently, the ubiquitinin–proteasome degradation pathway has become one of the most frequently investigated areas in oncology research, since it is responsible for the regulation of various proapoptopic and antiapoptotic proteins. The 26S proteasome degrades proteins targeted for destruction by ubiquitination. Inhibition of the proteasome might be directly proapoptotic (eg, via inhibition of the degradation of proapoptotic molecules) or indirectly apoptotic (via stress responses caused by an aberrant protein environment). The small molecule proteasome inhibitor bortezomib, which is already approved for the treatment of multiple myeloma and mantle cell lymphoma, reduces NF-κB levels in various cancer lines, thus resulting in transcriptional downregulation of antiapoptotic Bcl-2 proteins, among other effects.92 Since many chemotherapeutic agents trigger antiapoptotic responses via activation of NF-κB, combination of proteasome inhibitors with chemotherapy might result in an effective treatment response. Phase II studies in different solid tumours are on the way to analyse this approach. Since proteasome inhibitors can upregulate TRAILRs on cancer cells, they might be combined with TRAIL agonistic antibodies or ligands.92 However, concerns have been raised that this combination might exert hepatotoxic effects, since primary human hepatocytes show reduced viability after combined treatment.93
Targeting the p53 family
Chemotherapy and radiotherapy usually require function of the TP53 tumour-suppressor gene for antitumour activity. However, up to 50% of all cancers have aberrant p53 due to TP53 mutations (eg, gastric cancer: 60%), causing reduced apoptosis sensitivity.94 Therefore, the restoration of the p53 pathway is a therapeutic approach for tumours with defects in p53 signalling. One approach, which has already entered clinical testing, is reintroduction of functional p53 into tumour cells by gene transfer vectors of retroviral and adenoviral origin.94 95 In addition, small molecules are considered to influence p53 conformation and to restore p53 activity in tumour cells. Surprisingly, recent analysis of p53 activators found that they often act as repressors of NF-κB, and vice versa.96 Inhibition of the molecular chaperone hsp90—for example, by geldanamycin—contributes to depletion of mutant p53 in CRC.96 Another way to restore p53 activity is inhibition of negative p53 regulators, for example HDM2.97 The discovery of the p53-related genes p63 and p73 raised the possibility that p53 is not the only component in predicting prognosis and response to chemotherapy, but instead the status of a network that contains p53, p73 and p63 (fig 4). Deregulated dominant-negative p63 and p73 isoforms play an oncogenic role in human cancer and contribute to chemoresistance. Thus, therapeutic modulation of dominant-negative p63/p73 isoforms and mutant p53 levels might be used to target the large percentage of human tumours that harbour alterations in p53, p63 and p73 signalling.98
GI cancer cells often display perturbations in arachidonic acid metabolism. Cyclo-oxygenase (COX) catalyses the enzymatic conversion of arachidonic acid into prostaglandin H2, the common precursor of prostaglandins. The isoform COX-2 is frequently overactivated in GI cancer cells, contributing to apoptosis resistance, for example by upregulation of Bcl-2 or downregulation of BAX in oesophageal cancer.99 Targeting COX-2 by the inhibitor celecoxib has proved to be beneficial in GI cancer including HCC.100 Celecoxib has been well tolerated in phase II studies in oesophageal cancer in combination with radiotherapy or chemotherapy.99
Cell cycle inhibitors
Cyclin-dependent kinases (CDKs) promote the cell to progress through the cell cycle. Abnormal activity of CDKs has been described in GI cancer.101 CDK inhibitors have recently emerged as novel anticancer drugs due to their apoptosis-inducing effects.102 Flavoperidol, which is currently in clinical trials, inhibits different CDKs and enhances the antitumour activity of cytotoxic drugs by apoptosis induction in nanomolar concentrations. Co-administration with irinotecan and cisplatin for relapsed gastric and oesophageal cancer was evaluated in phase I trials with remarkable efficacy results.103 Aurora kinases (Aks) A, B and C also play a key role in orderly progression through mitosis and are often overactivated in GI cancer. SNS-314 is a pan-aurora kinase inhibitor applied in phase I studies in advanced GI cancer.104
Alterations in chromatin structure profoundly influence gene expression during malignant transformation. Methylation of cytosines by DNA methyltransferases (DNMTs) is an important epigenetic mechanism and facilitates recruitment of chromatin-remodelling proteins.105 Tumour suppressor genes involved in apoptosis signalling often become epigenetically silenced in cancer cells by DNA methylation or histone acetylation. DNMT and HDACIs represent novel chromatin-remodelling agents in cancer therapy. Combined treatment with HDAC and DNMT inhibitors has been shown markedly to induce re-expression of methylated proapoptotic genes in CRC.106 Inhibitors as well as AS ODNs of DNMT have entered clinical trials.107 HDACIs induce growth arrest, differentiation and apoptosis in cell lines derived from various GI malignancies.108 Among the proapoptototic mechanisms described are downregulation of antiapoptotic Bcl-2 proteins as well as c-FLIP, and upregulation of TRAILR2 as well as proapoptotic Bcl-2 proteins.51 109 Recently, it has been shown that HDACIs promote apoptosis in CRC cells at least in part by hyperinduction of canonical Wnt signalling. Wnt signalling, resulting from mutations in the adenomatous polyposis coli (APC) or β-catenin genes, has been implicated in the apoptosis resistance of CRC.110 Several HDACIs are presently in clinical trials for the treatment of GI cancer. For example, the HDACI SAHA, which is approved for the treatment of cutaneuous T cell lymphoma, is in clinical trials in CRC in combination with chemotherapy (www.clinicaltrials.gov). Interestingly, HDACIs are potent sensitisers for radiation therapy in CRC.108
Key points 2
Various compounds targeting defective apoptosis signalling in gastrointestinal (GI) cancer have already entered clinical trials.
Triggering of death receptors and activation of mitochondria are key approaches to induce apoptosis in GI cancer cells.
The selective triggering of tumour necrosis factor-related apoptosis-including ligand (TRAIL) death receptors is among the most promising approaches for apoptosis induction in cancer cells.
Novel microarray technologies help to select patients who would benefit from a specific proapoptotic treatment.
Cancer cells probably need to be attacked by a combination of proapoptotic and antisurvival stimuli to increase the effectiveness of antitumour therapy.
THERAPEUTIC CLUES FOR THE FUTURE
In cancer cells, dysregulation of the apoptotic programme is a prerequisite for cell survival and resistance to conventional therapy. Antitumour agents that restore apoptosis signalling in cancer cells might induce cancer regression and improve conventional treatment regimens including radiotherapy and chemotherapy. To achieve this therapeutic goal, cancer cells probably need to be attacked by more than one proapoptotic stimulus in combination with an antisurvival stimulus. A single hit will rarely result in tumour regression. The increasing knowledge about interacting pathways that regulate apoptosis will probably help to develop apoptosis-inducing therapies further. The development of microarray hybridisation technologies for parallel analysis of the whole genome. as well as mass spectrometry-based protein profiling, has yielded hundreds of candidate genes and proteins that are differentially expressed in GI cancer. In addition, profiling of miRNA signatures of cancer tissues will represent a new addition to the tools to be used by medical oncologists. This knowledge will improve prediction of therapy responses and survival rates, for example in HCC.111 Importantly, these novel technologies may help to select patients who would benefit from a specific proapoptotic treatment. Until now, in oncological practice, patients are preselected to therapeutics by a few single markers at maximum. This will probably change in the future. Oncologists may be enabled to evaluate and design specific and combinatorial treatment approaches.
Another key question will be if combined proapoptotic treatment strategies specifically target cancer cells. Keeping in mind that nearly all cells in the human body contain the entire programme for the apoptosis machinery, this problem remains a major challenge for years to come.
Moreover, it would be of interest to monitor the efficacy of treatment by the detection of apoptotic events in tumour tissues in vivo. This may help to cancel or change therapy as soon as possible if it is no longer benefical for the patient. However, diagnostic techniques are hampered by the fact that apoptotic cells are rapidly cleared by phagocytosis. There is still no routine method available to monitor apoptosis induction in vivo despite over a decade of intense research. The currently most promising approach for apoptosis detection in vivo is application of radiolabelled annexin V.112 Annexin V binds to phosphatidylserine, which is normally exclusively localised to the inner leaflet of the plasma membrane, but is exposed on the cell surface early in apoptosis.
In the near future, the concept of cancer as a stem cell disease has the potential to change our view of the problem of its treatment dramatically. A major cellular target of cancer therapy will be directed against neoplastic stem cells.
Apparently, the success of anticancer treatment does not depend solely on apoptosis induction. Tumour cells are intrinsically resistant to apoptosis, and other modes of cell death compensate for this apoptosis block upon radiotherapy or chemotherapy (eg, autophagy, mitotic catastrophe and necrosis). Exploring non-apoptotic types of cell death might therefore provide ways for a more effective cancer therapy.113
In summary, approaches for targeting apoptosis signalling in GI cancer are constantly developed and remain an exciting area of future research in oncology.
SUMMARY AND CLINICAL IMPLICATIONS
Apoptosis resistance is a hallmark of malignant tumours, including GI cancer. Cancer cells overcome the apoptotic machinery and, hence, the propensity to be naturally eliminated. Conventional treatment strategies in GI cancer such as chemotherapy and radiotherapy have limited effectiveness at least in part due to apoptosis resistance. Thus, within the past few years, therapeutic agents targeting apoptosis pathways have burst onto the scene. Many of these agents have shown early signs of activity in monotherapy studies and fairly few effects on healthy tissue. However, effectiveness is limited in monotherapeutic approaches. This is mainly due to the fact that tumours in a progressive stage are highly heterogenous despite their clonal origin. It is likely that different cells within a tumour have acquired different mechanisms of apoptosis resistance (fig 5). However, the ability to manipulate apoptosis pathways in cancer has significant therapeutic implications.
Among others, the following groups of compounds targeting defective apoptotic signalling in cancer cells are currently in clinical trials and hold promise as novel apoptosis-inducing agents in GI cancer (fig 6).
Application of ligands or agonistic mAbs selectively triggering the TRAIL death receptors. An increasing number of potential rationalised combination therapies are being tested to increase the antitumour properties of TRAILR stimulation.
Blockage of the IAP family of proteins including survivin. There are emerging data on the antiapoptotic functions of these proteins apart from caspase inhibition, all conferring resistance to radiotherapy or chemotherapy.
Targeting epigenetic modifications. Methylation and histone deacetylation in cancer cells often result in silencing of genes involved in apoptosis signalling.
Targeting antiapoptotic Bcl-2 proteins. Bcl-2 proteins are key regulators of mitochondrial integrity. SMIs or AS ODNs against antiapoptotic members of the Bcl-2 family represent a challenging strategy to augment antitumour activity of cancer therapeutics.
RTK inhibition. The TK activity of growth factor receptors such as EGFR exerts antiapoptotic functions via activation of multiple survival pathways. Blocking of RTKs via antibodies or TKIs has already become clinical routine.
The emerging data from the clinical development of apoptosis-inducing compounds have provided novel opportunities in the treatment of GI cancer that will probably translate into benefit in the treatment of these common malignancies.
▸ Competing interests: Declared (the declaration can be viewed on the Gut website at http://www.gut.bmj.com/supplemental).
Funding: HSB, JS and PRG are supported by grants from the German Research Foundation (DFG). MM is supported by the German Cancer Aid and Hector Foundation, Germany.
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