JOURNAL OF CELLULAR PHYSIOLOGY 196:9–18 (2003)

REVIEW ARTICLES

Overcoming Resistance of Cancer Cells to Apoptosis

PETER HERSEY* AND XU DONG ZHANG

Oncology and Immunology Unit, Newcastle Mater Misericordiae Hospital,David Maddison Clinical Sciences Building, Newcastle, New South Wales, Australia

Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to the concept thatdevelopment of cancers required the simultaneous acquisition, not only ofderegulated cell division, but also of resistance to programmed cell death orapoptosis. Apoptosis is arguably the commonpathway to cell death resulting fromarange of therapeutic initiatives, so that understanding the basis for the resistance ofcancer cells to apoptosis may hold the key to development of new treatmentinitiatives. Much has already been learnt about the apoptotic pathways in cancercells and proteins regulating these pathways. In most cells, apoptosis is dependenton the mitochondrial dependent pathway. This pathway is regulated by pro- andanti-apoptotic members of the Bcl-2 family, and manipulation of these proteinsoffers scope for a number of treatment initiatives. Effector caspases activated by themitochondrial pathway or fromdeath receptor signaling are under the control of theinhibitor of apoptosis protein (IAP) family. Certain proteins frommitochondrial can,however, competitively inhibit their binding to effector caspases. Informationabout the structure of these proteins has led to initiatives to develop therapeuticagents to block the IAP family. In addition to development of selective agents basedon these two (Bcl-2 and IAP) protein families, much has been learnt about signalpathways that may regulate their activity. These in turn might provide additionalapproaches based on selective regulators of the signal pathways. J. Cell. Physiol.196: 9–18, 2003. � 2003 Wiley-Liss, Inc.

Cancer cells acquire a number of characteristics thatallow them to establish in the host. Two key properties,however, that appear essential for survival of the cancercells are unregulated cell division and increased sur-vival and resistance to cell death (Cory and Adams,2002). In particular, there is evidence that cancercells are resistant to a process of cell death calledapoptosis. This is a form of cell death associated withcell shrinkage, chromatin condensation, and extensivenuclear and cellular fragmentation. The molecularmachinery that induces apoptosis is believed to bepresent in all cells and may be activated in manydisparate situations such as remodeling of tissuesduring development, tissue homeostasis, removal ofinfected cells, or cells that are damaged, particularly intheir DNA. Although chemotherapy may induce manydifferent biochemical lesions, induction of apoptosis isbelieved to be a final common pathway to cell death(Martin et al., 1997). The same may apply for immu-notherapy (Zhang et al., 2002) and irradiation althoughat high concentrations of chemotherapy or irradiationthe cell is damaged so much that cell division is notpossible (mitotic cell death) and the cell dies by necrosis(Coultas and Strasser, 2000). Some authors have alsodescribed a form of non-apoptotic cell death followingirradiation of solid tumors but the mode of cell deathinvolved is uncertain (Brown and Wouters, 1999;Roninson et al., 2001).

Traditionally, two principal pathways to apoptosishave been recognized, the transmembrane ‘‘extrinsic’’pathway and the mitochondrial ‘‘intrinsic’’ pathway.

Both depend on activation of cysteine proteases thatcleave at aspartate residues (caspases). These enzymesare synthesized as proenzymes that become activated byadaptor proteins. Initiator caspases once activated canactivate so-called effector caspases, which act on a widerange of substrates to cause apoptosis. The initiatorcaspases for the extrinsic pathway are caspase 8 and 10,whereas caspase 9 and 2 are initiator caspases for theintrinsic pathway. The effector caspases are believed tobe similar for both pathways, i.e., 3, 6, and 7.

Apoptosis induced by TNF family death receptorsand proteins regulating the pathway

Several of the TNF family receptors contain cytoplas-mic death domains (DDs) that interact with DD contain-ing proteins such as Fas associated death domain(FADD), which contain death effector domains (DED)that can interact with similar domains on procaspases 8and 10. Once activated these initiator caspases can

� 2003 WILEY-LISS, INC.

*Correspondence to: Peter Hersey, Oncology and ImmunologyUnit, Room 443, David Maddison Clinical Sciences Building, Cnr.King & Watt Streets, Newcastle, NSW 2300, Australia.E-mail: Peter.Hersey@newcastle.edu.au

Received 31 October 2002; Accepted 11 December 2002

DOI: 10.1002/jcp.10256

undergo an autocatalytic reaction and activate the ef-fector caspases 3, 6, and 7 (Ashkenazi, 2002).

A number of proteins and protein families are believedto regulate this pathway. Flice-like inhibitory proteins(FLIP) contain two DEDs that can bind to DEDs ofFADD and inhibit recruitment of procaspase 8 to thedeath inducing signal complex (DISC) (Irmler et al.,1997). Several studies in animal models (French andTschopp, 1999) suggested that FLIP expression intumors was important in escape from the immunesystem. However, studies on a large number of humanmelanoma did not show any evidence that FLIP wasinvolved in regulation of TNF related apoptosis inducingligand (TRAIL) induced apoptosis of melanoma (Zhanget al., 1999). Several other proteins, Flice-associatedhugh protein (FLASH) (Imai et al., 1999) and smallaccelerator of death signaling (SADS) (Suzuki et al.,2001) also contain DED and appear to increase recruit-ment of procaspase 8 to the DISC and increase apoptosis.

Inhibitor of apoptosis proteins (IAPs) consist of atleast six family members (Deveraux and Reed, 1999)—IAP1 and 2, XIAP, ML-IAP (Vucic et al., 2000), Livin(Kasof and Gomes, 2001), Bruce, and Survivin (Verha-gen et al., 2001). IAPs are characterized by the presenceof one to three baculoviral IAP repeat (BIR) domainsthat bind to caspases. Most also have really interestingnew gene (RING) domains which are involved inubiquitin ligation of proteins (Joazeiro and Weissman,2000). XIAP, IAP1, and IAP2 have three BIR domainsand a RING domain, whereas ML-IAP (Livin) has onlyone BIR domain similar to the BIR3 domain. Survivinhas a single BIR domain and may be involved physio-logically in cell division during mitosis (Uren et al.,2000).

XIAP is located in the cytosol and is widely distrib-uted. It can efficiently inhibit caspase 3, 6, and 7. cIAP1and two interact with TNF-receptor associated factors(TRAFs) and are localized in the membrane and in aperinuclear location. They are regulated by NF-kBactivation and can inhibit caspase 3 and 7 (Verhagenet al., 2001). ML-IAP has a more restricted distributionin adult tissues and cancer cell lines, particularlymelanoma. It may be expressed in the nucleus andfilamentous structures in the cytoplasm. It binds to andinhibits caspase 3, 7, and 9 (Vucic et al., 2000).

The BIR3 domain of XIAP binds to the p10 subunitof processed caspase 9, whereas the linker region up-stream of BIR2 interacts with the catalytic site ofcaspase 3 and 7, blocking their activity (Huang et al.,2001; Riedle et al., 2001). Binding of IAPs to caspases

can be inhibited by several proteins released frommitochondria. Second mitochondria derived activatorof caspases, direct IAP binding protein with low p1(Smac/DIABLO) is located in the intermembrane spaceof mitochondria and is released into the cytosol duringchanges in mitochondrial membrane permeability(MMP) (Du et al., 2000; Verhagen et al., 2000). Smac/DIABLO is synthesized as a precursor molecule of 239amino acids. The N terminal 55 residues serve as amitochondrial targeting sequence that is removed afterimport (Wu et al., 2000). It has a similar structure to thep10 subunit of caspase 9 and competes for binding to theBIR3 domain of XIAP, thus freeing caspase 9. It may alsocompete with binding of caspase 3 and 7 to BIR2domains of XIAP and dislodge the caspases from XIAP.

Omi is also a mitochondrial protein released duringchanges in MMP, which binds to the IAPs (Hegde et al.,2002). It is, therefore, similar in action to Smac/DIABLOand can facilitate apoptosis by freeing caspase 3 and 7from IAP inhibition. In addition, it can induce apoptosisby a caspase independent pathway due to intrinsicserine protease activity (Suzuki et al., 2001; Verhagenet al., 2002).

Another protein that can compete for binding to theIAPs is an interferon inducible protein, referred to as X-linked inhibitor of apoptosis associated factor-1 (XAF-1)(Leaman et al., 2002). It was postulated the IFN-b up-regulates this protein in human melanoma cells andthereby sensitizes the cells to TRAIL induced apoptosis.

Bcl-2 protein family and mitochondrialdependent pathways to cell death

The second pathway to apoptosis is dependent onrelease of pro-apoptotic molecules from the mitochon-dria, which directly or indirectly (via activation ofcaspases) leads to apoptosis. Release of these pro-apoptotic factors is regulated by the B cell lymphomagene 2 (Bcl-2) family of proteins. The initiating event isbelieved to be the production in the cells of pro-apoptoticmembers of the Bcl-2 family referred to as Bcl-2homology region 3 (BH3) only proteins (Table 1). Thelatter refers to a 9 amino acid homology region, which iscommon to the Bcl-2 family. The anti-apoptotic mem-bers have up to four homology domains and pro-apoptotic Bax, Bak, and Bok have three homologydomains (Cory and Adams, 2002). At least ten BH3only members have been described (Puthalakath andStrasser, 2002). They appear to act as sensors of celldamage and different apoptotic stimuli release differ-ent BH3 only proteins, for example, agents acting on

TABLE 1. BH3 only pro-apoptotic proteins*

Name Activation Regulation

Bid Caspase 8, granzyme B Post-translationalNoxa DNA damage Up-regulation by p53Puma DNA damage Up-regulation by P53Bim/Bod Microtubule damage (taxol, vincristine) Forkhead t.f., AP-1 transcription, post-translational MAP/AktBmf Action on myosin V actin motor complex (e.g., cytochalasin) Post-translational anoikis, calmodulin kinaseBad Released from endoplasmic reticulum Post-translational phosphorylationhrk/dp5 Neurones/hematopoietic cells Transcription SAPK/JNK kinase

*See Puthalakath and Strasser (2002).

10 HERSEY AND ZHANG

microtubules (e.g., taxol and vincristine) release Bimand agents acting on the actin complex release Bmf. Bidis activated by caspase 8 and hence is the main mediatorof death receptor initiated apoptosis, such as TRAILinduced apoptosis of melanoma (Thomas et al., 2000;Zhang et al., 2001). Granzyme B released by cytotoxicT cells also activates Bid and initiates apoptosis by thispathway (Sutton et al., 2000).

Damage to DNA results in p53 up-regulation of Noxaand Puma. It is not clear whether apoptosis due to up-regulation of p53 is mediated solely by production ofNoxa and Puma or whether other proteins may beinvolved, such as p63 and p73 (Flores et al., 2002).Caspase-2 has also been implicated as a mediator ofapoptosis following DNA damage (Lassus et al., 2002).

There is general agreement that the pro-apoptoticBH3 only proteins bind to the anti-apoptotic Bcl-2, Bcl-XL, Mcl-1, Bcl-W, and A1 proteins and thereby neu-tralize their binding capacity (Cheng et al., 2001). Thereis also agreement that Bax and Bak are needed to induceapoptosis (Wei et al., 2001), most probably by formationof oligomer complexes in the outer mitochondrialmembrane. There is a difference of opinion, however,as to whether the anti-apoptotic Bcl-2 members bindBax and Bak. Although earlier studies suggested thisoccurred (Korsmeyer, 1999) more recent studies suggestthat the BH3 only proteins may activate Bax and Bak,leading to a conformational change that allows oligo-merization and relocation of Bax and Bak to themitochondria. Bcl-2 or Bcl-XL in the mitochondrialmembrane may also have a role in preventing oligomer-ization (Mikhailov et al., 2001).

Bax is located in most cells in the cytosol and relocatesto the mitochondria during apoptosis. The basis for thisrelocation is not clear. Some authors have suggestedthat changes in MMP are responsible for relocation ofBax to mitochondria (De Giorgi et al., 2002). Others havesuggested that changes in pH in the cell (e.g., inducedby ceramide) result in a conformational change in Baxand thereby relocation to the mitochondria (von Haefenet al., 2002). Inhibitors of phosphatases preventedrelocation to mitochondria, suggesting that the phos-phorylation state of certain proteins is needed to retainBax in the cytosol (Ganju and Eastman, 2002). Cellswith mitochondria lacking DNA were resistant toTRAIL and Bax did not relocate to mitochondria whenBid was activated by TRAIL (Kim et al., 2002). Thisimplied that mitochondria played an active role inrelocation of Bax. The role of the protein bifunctionalapoptosis regulator (BAR) in activation of Bax isuncertain. BAR is a protein in the outer mitochondrialmembrane that binds to both Bcl family proteins andcaspases, and suppresses Bax induced apoptosis (Zhanget al., 2000).

Irrespective of the mechanism involved, it is clear thatBax and Bak are essential for induction of apoptosis andmice lacking both genes have a number of develop-mental abnormalities (Lindsten et al., 2000). Apoptosisof cancer cells induced by several chemotherapy agentsis dependent on Bax (Zhang et al., 2000; Bellosillo et al.,2002; Deng et al., 2002; LeBlanc et al., 2002; Ravi andBedi, 2002). Similarly, epithelial cancer cells lackingBax were resistant to apoptosis (Theodorakis et al.,2002).

Mitochondrial release of proteinsleading to apoptosis

Mitochondria release several factors that can triggerapoptosis. These include cytochrome-c, Smac/DIABLO,Omi, apoptosis inducing factor (AIF), and endonucleaseG (Ravagnan et al., 2002). These factors are located inthe membrane or intermembranous space between theouter and inner mitochondrial membranes. Two modelshave been proposed to explain the release of these pro-teins during apoptosis. In one model, an autonomouschannel formed by Bax or Bak is formed and this allowsthe release of the factors from the intermembrane space(Martinou and Green, 2001). Another model depends onspecific interaction of Bax or Bcl-2 with components ofthe permeability transition pore (PTP), which exists atsites of contact between outer and inner mitochondrialmembranes. This results in opening of the PTP, swellingof the mitochondrial matrix, and rupture of the outermitochondrial membrane (Zamzami and Kroemer,2001). Evidence for both models exist with possiblymore evidence in favor of the PTP model (Zamzami andKroemer, 2001).

In the classical description of apoptosis, cytochrome-creleased from mitochondria binds to a cytoplasmicprotein called apoptosis activating factor-1 (APAF-1),which binds to caspase 9 to form the apoptosome.Procaspase 9 becomes activated by undergoing a con-formational change and thereby activates the effectorcaspases 3, 6, and 7, resulting in apoptosis. This path-way often does not operate in melanoma cells, mostprobably due to low or absent levels of APAF-1, due tomethylation of the promoter for APAF-1 (Soengas et al.,2001). Instead, apoptosis appears dependent on releaseof Smac/DIABLO and/or Omi, which binds to andinhibits members of the IAP family (IAP1 and 2, XIAP,ML-IAP, and others) (Zhang et al., 2001). Smac/DIABLO and Omi, therefore, facilitate apoptosis byblocking the inhibitory effect of IAP proteins againstactivated caspases. In the case of Smac/DIABLO, thecaspases need to be activated either by death receptorinteractions or by the apoptosome. Omi also has serineprotease activity and can activate caspase 9 indepen-dent of the apoptosome (Verhagen et al., 2002).

AIF is another protein released from mitochondriathat induces apoptosis by caspase independent activa-tion of an unknown nuclease (Hunot and Flavell, 2001;Ye et al., 2002). AIF was shown to be essential fordevelopment but its role in induction of apoptosis incancer remains to be established.

Endonuclease G is responsible for the major nucleaseactivity of mitochondria and has specificity for singlestranded DNA. It is believed to participate in DNAfragmentation during apoptosis independent of caspaseactivated DNAse (CAD) (van Loo etal., 2001; Ikeda etal.,2002).

Regulation of Bcl-2 family proteins byMAP kinases

From the discussion above it is evident that the Bcl-2and IAP protein families are key determinants of apop-tosis. Bcl-2 family member activities can be regulatedat different levels; such as transcriptional activity,post-transitional modifications, and protein–protein

RESISTANCE OF CANCER CELLS TO APOPTOSIS 11

interactions. Information about the signal pathwaysregulating these proteins is as yet incomplete. Table 2summarizes some of the pathways involved in regula-tion of the Bcl-2 anti-apoptotic family. The microphthal-mia transcription factor (Mitf ) is one of the factorsregulating Bcl-2 in melanocytes and melanoma, mostprobably by c-kit activation of MAP-kinases (McGillet al., 2002). The MEK/ERK pathway was also shown toregulate Bcl-2, Bcl-X(L), and Mcl-1 in pancreaticcarcinoma cells (Boucher et al., 2000).

We have shown that TRAIL can activate Erk1/2 inmelanoma cells and inhibition of this pathway with theMEK inhibitor U0126 or a dominant negative MKK1was associated (after 16 h) with a decrease in Bcl-2, Bcl-XL, and Mcl-1 levels. However, treatment with U0126induced a rapid increase in sensitivity of melanoma cellsto TRAIL induced apoptosis well before detection ofchanges in the Bcl-2 family. Changes in mitochondrialmembrane potential were detected as early as 30 minafter exposure to TRAIL and were associated with moreconformational change and localization of Bax tomitochondria (Zhang et al., 2003). These results mayindicate that phosphorylation of proteins in the cell bythe Erk kinases may be necessary to localize Bax to thecytosol and would be consistent with studies referred toabove showing that inhibitors of phosphatases pre-vented relocalization of Bax to mitochondria (Ganjuand Eastman, 2002). The mechanism by which TRAILactivates MAPK kinases is not clear. The kinases NIKand MEKK1 were reported to be involved in activation ofNF-kB and this pathway may also be involved inactivation of Erk1/2 (Lee et al., 1997; Dhawan andRichmond, 2002). Akt/protein kinase B (PkB) wasshown to up-regulate Mcl-1 but not other members ofthe Bcl-2 family in macrophages and neutrophils(Epling-Burnette et al., 2001; Liu et al., 2001).

Regulation of apoptosis by theAkt/PkB signal pathway

A number of studies have shown the importance of theAkt pathway in promoting the survival of cells, forexample, a dominant negative allele of Akt blocked

survival of neurons mediated by insulin-like growthfactors (Dudek et al., 1997). Akt is constitutivelyactivated in a number of malignant tumors, such ascarcinomas from prostate, breast, ovary, lung, and liver(Vivanco and Sawyers, 2002). This was also the case instudies on melanoma lines (Zhang et al., submitted).

The mechanism(s) underlying the constitutive activa-tion of this pathway in cancer cells is complex and likelyto vary between cancers. In some autocrine or paracrineextracellular factors, such as platelet-derived growthfactors and insulin-like growth factors, may play a role.In other instances, genetic alterations of proteins in thepathway may be involved, such as activating mutationsin the Ras oncogene or downstream components. Ofparticular interest in melanoma are activating muta-tions in BRAF, reported to be present in approximatelytwo-thirds of melanoma (Davies et al., 2002). The geneencoding the P110 catalytic unit of P13K was amplifiedin some cases of ovarian cancer and the Akt2 form of Aktwas amplified in breast, ovarian, and pancreatic cancers(Cheng et al., 1996; Ruggeri et al., 1998; Shayesteh et al.,1999). Other activating mutations in the P13K pathwaywere reported in colon and ovarian cancers (Philp et al.,2001).

Inactivation or deletion of the gene for PTEN (phos-phatase and tension homologue) is present in a widerange of cancer cells (Ali et al., 1999; Vivanco andSawyers, 2002). This phosphatase is needed for conver-sion of PIP3 back to PIP2 to facilitate continued activa-tion. We have confirmed these findings in a number ofmelanoma lines used in our studies but whether this isthe sole cause of the constitutive activation of Akt inmelanoma cells is not known.

Events downstream of Akt/PkB activation that mightinhibit apoptosis are complex and in many systems,poorly defined (Table 3). Events upstream of mitochon-dria appear to be involved in some cancers, for example,studies in non-small cell lung cancer suggested thatactivation of Akt inhibited TRAIL induced apoptosis byblocking Bid cleavage (Kandasamy and Srivastava,2002). Fas mediated apoptosis of lymphocytes wasinhibited by activation of Akt and this was associated

TABLE 2. Regulation of Bcl-2 anti-apoptotic proteins

Mechanism Protein involved References

Microphthalmia transcription factor (Mitf) Bcl-2 McGill et al., 2002Map kinases Erk1/2 Bcl-2, Bcl-XL, Mcl-1 Boucher et al., 2000NF-kB transcription factor Bcl-XL, A1 Romashkova and Makarov, 1999Akt/PkB (via NF-kB or via inhibition of forkhead t.f. Mcl-1, Bcl-XL Brunet et al., 1999; Shin et al., 2001

TABLE 3. Akt mediated inhibition of apoptosis

Possible mechanisms Consequence Reference

Up-regulation of FLIP Inhibition of DISC formation Panka et al., 2001; Jones et al., 2002Inhibition of caspase 8 and Bid

cleavageDecreased MMP Chen et al., 2001; Nesterov et al., 2001; Kandasamy and

Srivastava, 2002Phosphorylation of Bad Retention of Bad in cytosol Hayakawa et al., 2000Phosphorylation of caspase 9 Inhibition of apoptosome formation Cardone et al., 1998NF-kB activation Increased Bcl-XL, IAP1 and 2, XIAP Romashkova and Makarov, 1999; Hofer-Warbinek et al.,

2000Inhibition of forkhead transcription

factor FKHRDecreased Bim, Fas ligand, increased

Bcl-XL, Mcl-1Brunet et al., 1999; Lin et al., 2001; Shin et al., 2001;

Tang et al., 2002Degradation of p53 by

phosphorylation of mdm-2Decreased Noxa, Puma, PTEN Mayo and Donner, 2001; Stambolic et al., 2001;

Zhou et al., 2001

12 HERSEY AND ZHANG

with reduced recruitment of caspase 8 to the DISC,reduced activation of caspase 8, and Bid (Jones et al.,2002). Akt activation was reported to up-regulatec-FLIP in a number of different cancer cell lines (Pankaet al., 2001) but was not involved in the Fas inducedapoptosis of lymphocytes. Similarly, inhibition of apop-tosis proteins (IAP1 and 2, XIAP) were not involved inthe study on Fas mediated apoptosis of lymphocytes.Studies on prostate cancer cell lines also suggested thatAkt protected against TRAIL induced apoptosis byinhibition of the cleavage of Bid (Chen et al., 2001;Nesterov et al., 2001).

Akt/PkB mediated down-regulation of pro-apoptoticBH3 only proteins or up-regulation of anti-apoptotic IAPand Bcl-2 family proteins has been implicated in somestudies, for example, cisplatin induced apoptosis ofovarian cancer cell lines was inhibited by Akt mediatedphosphorylation of the BH3 only pro-apoptotic protein,Bad, on serine at position 136. This was believed toresult in the sequestration of Bad on the cytoskeletalprotein 14-3-3 and thereby inhibited its binding to theanti-apoptotic Bcl-2 proteins (Hayakawa et al., 2000).

Up-regulation of the anti-apoptotic IAP and Bcl-2family proteins may be mediated by activation of thetranscription factor NF-kB by phosphorylation of IkBkinase that induces degradation of the NF-kB inhibitor,IkB (Ozes et al., 1999; Romashkova and Makarov, 1999).NF-kB induces a number of anti-apoptotic proteins,such as IAP1 and 2, XIAP, Bcl-XL, and A1 (Wang et al.,1998; Grumont et al., 1999; Mayo and Baldwin, 2000;Lee and Collins, 2001). Akt also phosphorylates amember of the forkhead family (FKHR) of transcriptionfactors. This prevents relocation to the nucleus, therebyinhibiting transcription of several pro-apoptotic genes,such as Bim and Fas ligand, and possible Bcl-XL (Brunetet al., 1999). Inhibition of the Akt pathway also resultedin decreased levels of Mcl-1 in macrophages (Liu et al.,2001). Akt indirectly down-regulates p53 by phosphor-ylation of MDM2. This facilitates entry of MDM2 intothe nucleus, binding and degradation of p53 (Mayo andDonner, 2001; Zhou et al., 2001) (Table 3).

We have shown that inhibitors of the Akt pathwaysensitize human melanoma cells to TRAIL inducedapoptosis but the mechanisms involved are not clear.Events upstream of the mitochondria did not appear tobe involved in that we were unable to show any increasein activation of caspase 8 or Bid in melanoma cellsexposed to TRAIL and the specific inhibitor of phosphoi-nositol 3 kinase (LY294002). We could also not demon-strate any change in the phosphorylation status of Bad(Zhang et al., submitted). Nevertheless, inhibition ofthis pathway was associated with rapid conformationalchange in Bax and marked increase in changes in MMP.The NF-kB pathway was constitutively activated inmany of the melanoma lines under study but again therapidity of increased sensitivity to TRAIL arguedagainst involvement of NF-kB dependent transcriptionof anti-apoptotic proteins.

Cancer cells exhibit a number of differentabnormalities in apoptotic pathways

We have studied TRAIL induced apoptosis of mela-noma as a model system and have found that melanomalines from different patients exhibit a range of abnorm-

alities that inhibit apoptosis induced by TRAIL. Theseinclude complete loss of death receptors for TRAIL, lossof caspase 8, and failure of signaling from the DISC. Thecommonest of these defects (upstream of mitochondria)was loss of death receptors seen in approximately 14% ofthe lines tested (Zhang et al., 1999). The signalingabnormality from the DISC was not due to FLIP or tomutations in the TRAIL-R2 receptor but the abnorm-ality has not been further defined. Loss of caspase 8 isreported to be a frequent finding in neuroblastoma cells(Hopkins-Donaldson et al., 2000) and in some tumorsthis could be reversed by treatment with a demethylat-ing agent (Grotzer et al., 2000).

In approximately 20% of cell lines but in a muchhigher percentage of fresh isolates of melanoma (Nguyenet al., 2001), TRAIL death receptors were expressed atlow levels and most probably below the levels needed toinduce apoptosis (Zhang et al., 1999). This appearedrelated to post-transcriptional control in that mRNAlevels were similar to those in melanoma cells with‘‘normal’’ TRAIL death receptor expression. The basisfor this translational control remains under study.

In approximately 40% of the melanoma lines, TRAILdeath receptor expression was ‘‘normal’’ (or equivalentto that on TRAIL sensitive lines) but the cell lines wererelatively resistant to TRAIL induced apoptosis. Thiswas shown to be due to inhibition of effector caspase 3 bythe IAP family and could be reversed by transfection ofSmac/DIABLO into the cells (Zhang et al., 2001). Theresistant lines had relatively less Smac/DIABLO releasefrom mitochondria compared to the TRAIL sensitivelines, even though Bid appeared to be activated tosimilar levels in both sensitive and resistant melanomalines. Inhibitors of Erk1/2 activated by TRAIL resultedin increased release of Smac/DIABLO and cytochrome-cin response to TRAIL. Akt/PkB was constitutivelyactivated in both TRAIL resistant and sensitive lines.Inhibition of Akt resulted in an increase in release ofSmac/DIABLO and cytochrome-c. Hence, inhibitors oftwo signal pathways could reverse the relatively lowrelease of pro-apoptotic proteins from mitochondria andresistance to apoptosis. The basis for these findings isthe subject of ongoing studies.

We consider these studies on cell lines provide a‘‘snapshot’’ of defects in the apoptotic signaling pathwaythat may be seen in patients with melanoma andperhaps other cancers. It is possible that many of thedefects may be seen in different tumors from the onepatient. If this is correct, treatment approaches mayneed to target a number of defects to prevent clonalevolution of apoptosis resistant tumor cells.

THERAPEUTIC OPPORTUNITIESBcl-2 proteins as therapeutic targets

From the above discussion, it is evident that severalapproaches may increase apoptosis of cancer cells. Oneis to increase the concentration or activity of pro-apoptotic BH3 only proteins by utilizing agents thatrelease different BH3 only proteins, for example, DNAdamaging agents to release Noxa and Puma, and agentsthat damage the cytoskeleton like Vincristine andTaxol to release Bim or Bmf. Biological agents thattarget death receptors, may increase activation of Bid.Together, the different pro-apoptotic BH3 proteins may

RESISTANCE OF CANCER CELLS TO APOPTOSIS 13

overwhelm the capacity of the anti-apoptotic Bcl-2proteins and trigger apoptosis.

Several approaches have been made to use mimics ofthe BH3 proteins in therapy to inhibit the anti-apoptoticBcl-2 family (Huang, 2000; Wang et al., 2000; Degterevet al., 2001; Tzung et al., 2001) and such agents may actin concert with BH3 only proteins induced with conven-tional cytotoxic agents. The mimics might be partic-ularly effective in tumors with mutations of p53 thatmay not respond to DNA damaging agents by up-regulating Noxa and Puma.

Reduction in the Bcl-2 anti-apoptotic family of pro-teins could also be expected to facilitate apoptosis.Clinical trials are already in progress using antisenseto Bcl-2 sponsored by Genta/Aventis in patients withmelanoma (Jansen et al., 2000), chronic lymphaticleukaemia (CLL) (O’Brien et al., 2001), small cellcarcinoma of the lung, acute myeloid leukaemia (Mar-cucci et al., 2001), and multiple myeloma (Gazitt et al.,2001). Responses in patients with CLL have often beenquite dramatic.

The randomized study in patients with melanoma isplanned to accrue 750 patients. Responses appear lessdramatic than in CLL and may indicate that othermembers of the Bcl-2 family are more important or athigher concentrations than Bcl-2. Bcl-2 was found to beabsent in sections from approximately 39% of metastaticmelanoma (Ramsay et al., 1995; Vlaykova et al., 2002)and was found at lower concentrations than Bcl-XL andMcl-1 (Tang et al., 1998). This may indicate that use ofantisense molecules targeted at both Bcl-2 and Bcl-XLmay be more effective (Olie et al., 2002) or even that useof antisense to all three proteins, Bcl-2, Bcl-XL, and Mcl-1 may be needed. Antisense to Mcl-1 was found to triggerapoptosis in U937 cells and Bcl-2 could not compensatefor the loss of Mcl-1 (Moulding et al., 2000). Antisense toBcl-XL was also shown to sensitize pancreatic carci-noma cells to TRAIL mediated apoptosis (Hinz et al.,2000). Bcl-2, Bcl-XL, and Mcl-1 are believed to havesimilar functions so that reducing any one might beeffective, depending on the concentration of BH3 pro-teins. There are, however, differences in the intracel-lular location of the proteins, with Bcl-2 being locatedentirely in membranes and Bcl-XL located predomi-nantly in the cytosol.

From the discussion above it is also possible thatinhibitors of particular signal pathways may also reduceBcl-2 anti-apoptotic proteins. Inhibitors of the Erk1/2pathway were shown to reduce Mcl-1 and Bcl-XL levelsin melanoma cells (Zhang et al., 2003) and Boucheret al. (2000) reported that inhibitors of Erk1/2 down-regulated Bcl-2, Bcl-XL, and Mcl-1 in pancreatic cells.The Erk1/2 inhibitor, CI-1040, produced by Pfizer isalready in clinical trials as a sole agent (LoRusso et al.,2002) but it could be expected to be more effective ifcombined with agents, which increase the pro-apoptoticBH3 proteins.

Inhibition of the Akt pathway can be carried outin vitro with inhibitors of P13 kinase, such as wortmaninand LY294002. They do not appear to have been usedclinically, perhaps because of a narrow therapeuticindex. Genistein is an isoflavonoid found in soy productswhich inhibits Akt (Li and Sarkar, 2002) and poten-tiated TRAIL induced apoptosis of A549 breast cancer

cells (Park and Seol, 2002). Rapamycin targets mTORkinase that is activated by Akt and which causes growthof cells. It was shown to have activity against cancerswith mutations in PTEN, such as glioblastoma, pros-tate, and breast carcinomas (Neshat et al., 2001; Gubaet al., 2002).

Inhibition of the transcription factor NF-kB mayreduce certain Bcl-2 anti-apoptotic proteins, such asBcl-XL and AI. Proteasome inhibitors, such as PS341,are already in clinical trials (Aghajanian et al., 2002;Tan and Waldmann, 2002) and might have such effectsbut proteasomes are involved in degradation of manyproteins, such as p53, which may be involved in regu-lation of apoptosis.

IAP proteins as therapeutic targets

The IAP proteins are believed to provide the lastlevel of regulation of apoptosis by inhibition of effectorcaspases 3, (6), 7, and 9. As discussed above, they bindto the caspases via BIR domains. One domain (BIR3)has selectivity for caspase 9, whereas the BIR2 domainhas selectivity to caspase 3 and 7. Binding to the caspa-ses can be competitively inhibited by the two proteinsSmac/DIABLO and Omi released from mitochondria.Binding of caspase 9 to XIAP can be competitivelyinhibited by the N terminal 4 amino acids (AVP1) ofSmac/DIABLO. On release from the mitochondria,Smac/DIABLO exists as a dimer in solution. The N ter-minal 4 amino acids of the monomeric form can fit into asurface groove of XIAP-BIR3 and inhibit its binding tocaspase 9. Dimeric forms of Smac/DIABLO are requiredfor binding to XIAP-BIR2 and appear of lower affinity(Wu et al., 2000). XIAP binds to the catalytic site ofcaspase 3 and 7 upstream of the BIR2 domain althoughan additional interaction of the processed p10 fragmentof caspase 3 may occur with the BIR2 domain (Huanget al., 2001; Riedle et al., 2001). Competition by Smac/DIABLO for this binding site presumably disruptsbinding of XIAP to the catalytic site of caspase 3(Verhagen et al., 2001).

The peptide sequence of Smac/DIABLO, which inhi-bits binding of caspase 3 to XIAP, is not well defined,perhaps because of the need for dimeric interactionsbetween Smac/DIABLO and BIR2 (Wu et al., 2000). Itwas reported (Srinivasula et al., 2000) that binding ofthe N terminal 30 amino acids to BIR2 was superior tothat shown by the terminal N7 residues. The N terminal10 amino acids of Smac also efficiently bound to the BIRdomain of ML-IAP (Vucic et al., 2000). Omi interactswith XIAP via its N terminal amino acid, AVPA, but hashigher affinity for the BIR2 domain (Verhagen et al.,2002). It is, therefore, more important in disrupting theIAP mediated inhibition of caspase 3 and 7.

The potential therapeutic implications of these find-ings have been investigated in a number of studies.Vucic et al. found that the N terminal 10 amino acidpeptide of Smac/DIABLO could potentiate Adriamycininduced apoptosis of MCF-7 breast carcinoma cells. Itwas reported (Guo et al., 2002) that the N terminal sevenor 4 amino acids of Smac/DIABLO potentiated TRAIL oran anti-microtubule agent (epothilone) induced apopto-sis of Jurkat T cells. Neuroblastoma cells and melanomacells were sensitized to TRAIL induced apoptosis bySmac/DIABLO peptides. Glioma xenografts could be

14 HERSEY AND ZHANG

completely eliminated by combining TRAIL with theSmac/DIABLO peptides (Fulda et al., 2002). N terminal4 or 8 amino acid peptides from Smac/DIABLO wereshown to enhance apoptosis of MCF-7 and other breastcarcinoma lines to a range of chemotherapeutic agents,including paclitaxel, etoposide, camptothecin, and dox-orubicin (Arnt et al., 2002). The use of peptide agonistsin vivo may present difficulties and for this reason thereis much interest in the production of small molecularweightmimicsof thepeptides, asdescribed (Gloveretal.,2002).

The regulation of the IAP proteins has receivedrelatively little attention. Activation of NF-kB appearsto play a key role in up-regulation of IAP1 and 2, andXIAP. NF-kB p65 knockout mice die at an early age fromapoptosis of their liver unless the gene for TNF-a is alsoknocked out (Doi et al., 1999). XIAP may also have apositive feedback effect on NF-kB activation by activa-tion of a MAP kinase TAK-1 (Hofer-Warbinek et al.,2000). Focal adhesion kinase (FAK) was also shown toactivate the Akt pathway and thereby NF-kB, resultingin up-regulation of IAP1 and 2, and XIAP (Sonoda et al.,2000). Given that the majority of melanoma cells haveconstitutive activation of Akt, it is possible this may bethe basis of the high IAP levels in melanoma.

Specific inhibitors of NF-kB are not yet in clinical use.Certain derivatives of a compound called Rocaglamidewere reported to specifically inhibit activation of NF-kBin T cells (Baumann et al., 2002). Nevertheless, the mostpractical agent in use clinically is the proteasome in-hibitor, PS341 (Millenium Pharmaceuticals, Cambridge,MA), which was shown to induce partial inhibition ofproteasome activity in phase I clinical studies (Aghaja-nian et al., 2002; Tan and Waldmann, 2002). The mainlimitation is the lack of specificity for inhibition of NF-kBas the proteasome degrades a large number of otherproteins involved in apoptosis. Certain histone deace-tylase inhibitors may have more selective effects thanPS341 but further work on this exciting class of drugs isneeded.

In conclusion, insights from studies on apoptosis aregradually providing a framework for new treatmentinitiatives. Selective up-regulation of pro-apoptotic BH3only proteins combined with agents which down-regulate the anti-apoptotic Bcl-2 family are feasibleapproaches that are at the first stages of clinical testing.Down-regulation or inhibition of the IAP family mayalso be a powerful addition to these new approaches.Given the range of defects in the apoptotic pathways incancer cells that are generated under selective pres-sures in the host, it is highly likely that judicious use ofseveral concurrent approaches will be needed to controlprogression of the cancer.

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