Tumor necrosis factor-related apoptosis-inducing ligand

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1 (2005) 12, r 2005 Nature Publishing Group All rights reserved /05 $ Review Mechanisms of resistance to Lidong Zhang, 1 and Bingliang Fang 1 1 Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, considerable numbers of cancer cells, especially some highly malignant tumors, are resistant to apoptosis induction by TRAIL, and some cancer cells that were originally sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance). Understanding the mechanisms underlying such resistance and developing strategies to overcome it are important for the successful use of TRAIL for cancer therapy. Resistance to TRAIL can occur at different points in the signaling pathways of TRAILinduced apoptosis. Dysfunctions of the death receptors DR4 and DR5 due to mutations can lead to resistance. The adaptor protein Fas-associated death domain (FADD) and caspase-8 are essential for assembly of the death-inducing signaling complex, and defects in either of these molecules can lead to TRAIL resistance. Overexpression of cellular FADD-like interleukin-1b-converting enzymeinhibitory protein (cflip) correlates with TRAIL resistance in several types of cancer. Overexpression of Bcl-2 or Bcl-X L, loss of Bax or Bak function, high expression of inhibitor of apoptosis proteins, and reduced release of second mitochondria-derived activator of caspases (Smac/Diablo) from the mitochondria to the cytosol have all been reported to result in TRAIL resistance in mitochondriadependent type II cancer cells. Finally, activation of different subunits of mitogen-activated protein kinases or nuclear factor-kappa B can lead to development of either TRAIL resistance or apoptosis in certain types of cancer cells. (2005) 12, doi: /sj.cgt Published online 19 November 2004 Keywords: TRAIL; resistance; apoptosis; cancer Received October 14, Address correspondence and reprint requests to: Dr Bingliang Fang, Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. Bfang@mdanderson.org Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has attracted attention not only for its strong antitumor activity in a wide range of cancer cell types but also for its minimal cytotoxity to most normal cells and tissues. 1 Although we and others have demonstrated that introduction of TRAIL to normal human hepatocytes induced massive cell death, 2,3 we have also demonstrated that the human telomerase reverse transcriptase (htert) promoter, the gene that is highly active in more than 85% of human cancers but inactive in most normal cells, can be used to target TRAIL-mediated apoptosis to cancer cells, thus reducing TRAIL s toxicity to normal hepatocytes. 2 In mice, targeted expression of the TRAIL or Baxgene by the htert promoter also elicited strong antitumor activity without obvious systemic toxicity, even after systemic administration. 2,4 Nevertheless, resistance to TRAIL-mediated apoptosis induction in cancer cells remains a challenging issue for the successful application of TRAIL in gene therapy. Although many types of cancers are sensitive to TRAILinduced apoptosis, substantial numbers of cancer cells are resistant to TRAIL, especially some highly malignant tumors such as pancreatic cancer, 5 melanoma, 6 and neuroblastoma. 7 Moreover, we found that repeated application of TRAIL protein or TRAIL-expressing adenovectors to TRAIL-susceptible cancer cells results in selection and expansion of TRAIL-resistant cells, leading to acquired resistance. 8 Thus, characterizing the mechanisms of resistance to TRAIL-mediated apoptosis will not only provide insight regarding transduction of the death signal from membrane to nucleus, but will also be essential for designing strategies to overcome resistance to TRAIL for future clinical applications. Although the detailed mechanisms underlying TRAIL-mediated apoptosis remain to be characterized, some important components and steps in the signaling pathways of this process have been elucidated. A growing body of evidence shows that resistance to TRAIL-mediated apoptosis can occur at different levels in these pathways. Here we summarize some possible mechanisms related to cellular resistance to TRAIL-induced apoptosis. The signaling pathway of TRAIL-induced apoptosis The interaction of TRAIL with its two death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) is the initial

2 step in TRAIL-induced apoptosis. 9,10 The binding of TRAIL leads to trimerization of the death receptors and activation of receptor-mediated death pathway (Fig 1). The activated death receptors recruit and activate an adaptor protein called Fas-associated death domain (FADD) through interactions between the death domain (DD) on the death receptors and FADD. The death effector domain (DED) of FADD recruits and activates caspase-8, leading to the formation of the death-inducing signaling complex(disc). In type I cells, the presence of activated caspase-8, a so-called initiator caspase, is sufficient to induce activation of one or more effector caspases (e.g., caspase-3 or -7), which then act on final death substrates in apoptosis. 7,11 However, in type II cells, even a small amount of activated caspase-8, although not enough to activate the effector caspases, is sufficient to trigger a mitochondria-dependent apoptotic amplification loop by activating Bid, which induces the accumulation of Baxin mitochondria, the release of cytochrome c from mitochondria, the activation of caspase-9, caspase-3, and caspase-7, and finally, programmed cell death In addition to inducing apoptosis by caspase-8 recruitment through FADD, TRAIL binding to its receptors also leads to activation of the transcription factor nuclear factor-kappa B (NF-kB) (Fig 1). TRAIL death receptors, like the TNF receptor 1 (TNFR1), 14 activate NF-kB through the TNFR1-associated death domain protein Figure 1 The signaling pathway of TRAIL-induced apoptosis. Death signal from TRAIL receptors may transduce via activated caspase-8 directly by activating effector caspase-3, or indirectly by activating Bid, which in turn translocates into mitochondria and causes cytochrome c and Smac release to cytosol. The released cytochrome c interacts with Apaf-1 and causes caspase-9 activation, which then activates effector caspase-3. The released Smac inhibits IAPs by preventing their binding to capspase-9 and caspase-3. Death receptor apoptosis pathways induced by TRAIL can also activate NF-kB. Different subunit activation of NF-kB determines whether NF-kB favors apoptosis or survival. indicates activation. B indicates inactivation. (TRADD). 15 Activated TRADD recruits the DD-containing protein RIP and TNF receptor-associated factor-2 (TRAF2), leading to activation of the NF-kB pathway; in contrast, dominant-negative TRADD can block the NFkB activation induced by TRAIL receptors. 15 In TNFR1 signaling, TRADD is believed to be upstream of FADD. However, in TRAIL signaling, TRADD may be mediated by FADD, because TRADD recruitment to the DISC is observed only in the presence of FADD. 16 TRAIL death receptors and TRAIL resistance So far, five TRAIL receptors have been discovered: the two agonistic receptors DR4 9 and DR5 10 and the three antagonistic decoy receptors DcR1 (TRAIL-R3), 17 DcR2 (TRAIL-R4), 18 and osteoprotegerin. 19 Apart from their extracellular domains, which are capable of binding TRAIL, DR4 and DR5 contain intracellular DD that can trigger the apoptotic signal upon binding TRAIL. 1,11 On the other hand, DcR1 and DcR2 cannot transmit the apoptotic signal, because they either lack or have a functionally truncated DD. 9,10,11,17,18 The third decoy receptor, osteoprotegerin, is a secreted TNF receptor family member that can be detected in circulation, suggesting that osteoprotegerin may be a soluble antagonist receptor for TRAIL. 19 More data are needed before the function of osteoprotegerin in the signal pathway of TRAIL-induced apoptosis can be elucidated. Overall, it seems that all of the decoy receptors can inhibit TRAILinduced apoptosis by competing with DR4 or DR5 for TRAIL binding In humans, the decoy receptors are widely expressed in various tissues. DcR1 mrna has been detected in peripheral blood leukocytes, ovary, testis, prostate, thymus, spleen, liver, lung, placenta, heart, kidney, and bone marrow, 20,21 whereas DcR2 mrna expression has been found in all tissues tested, including peripheral blood leukocytes, ovary, prostate, thymus, spleen, colon, and small intestine. 18 Most of the human tissues tested also expressed the agonistic death receptors DR4 or DR5. 18,20,21 As all of the normal tissues that expressed decoy receptors and real death receptors at the same time were resistant to TRAIL-induced apoptosis, and because ectopic expression of DcR1 or DcR2 in some cancer cell lines reduced sensitivity to TRAIL-induced apoptosis, it is reasonable to postulate that DcR1 or DcR2 expression may protect human normal cells and tissues from TRAILinduced apoptosis. 18,20,21 However, no correlation has been found between decoy receptor expression and TRAIL resistance in any form of cancer studied thus far. In one study, the TRAIL-resistant melanoma cell line WM3211 expressed mrna for DR5, but not for DcR1 and DcR2. 22 In contrast, three TRAIL-sensitive melanoma cell lines (WM9, WM793, and WM1205) expressed DcR1, DcR2 or both. 22 In another study, 7 only five of 18 neuroblastoma cell lines tested were sensitive to TRAIL, but all five of those cell lines expressed DcR1, DcR2, or both, at moderate to strong levels. These findings indicate 229

3 230 that, in cancer, some mechanisms other than protection by decoy receptors exist, which confer resistance to TRAIL. DR4 and DR5, the two agonistic death receptors for TRAIL, are able to transmit apoptotic signals upon TRAIL binding. Theoretically, dysfunctions in either receptor could cause TRAIL resistance. The genes for both DR4 and DR5 have been mapped to chromosome 8p21 22, a segment noted in genome-wide searches to be one of the most common sites of loss of heterozygosity (LOH) in several types of cancers. Thus, it is rational to hypothesize that one or more tumor suppressor genes are located in this region, and to explore the possibility of mutational inactivation of DR4 and DR5 in those forms of cancer A polymorphism in DR4 has been described in the human ovarian cancer cell line SKOV3 and the human bladder cancer cell line J An A-to-G alteration at nucleotide 1322 of DR4 in both cell types results in substitution of an arginine for lysine at codon 441 (K441R) in the DD of DR4. About 20% of normal individuals also had the same base change. 24 Transfection of SW480 colon cancer cells with a vector expressing polymorphic DR4 showed that the polymorphic form was less effective in cell killing than its wild-type counterpart. 24 Another study by the same investigators 23 showed two missense nucleotide substitutions in DR4 in lung cancer, head and neck squamous cell cancer, and gastric adenocarcinoma cells. The first of these substitutions occurred at nucleotide 626 of DR4, where a C-to-G change results in substitution of an arginine for threonine at codon 209 (T209R). The other missense alteration occurred at nucleotide 422 of DR4, where a G-to-A change results in substitution of a histidine for arginine at codon 141 (R141H). Only 13% of a normal control group was homozygous for both the T209R and R141H changes, but 35% of non-small-cell lung cancer specimens, 47% of primary head and neck squamous cell cancer specimens, and 44% of gastric adenocarcinomas specimens were homozygous for both changes. These two amino-acid changes occurred in or near the ligandbinding domain of DR4, suggesting that these changes may cause abnormal death receptor trimerization or TRAIL binding. 23 In another study, 26 mutations in DR4 were identified in three of 34 specimens from breast cancers that had metastasized, but none were found in any of the 23 specimens of breast cancer that had not metastasized. All the three mutations were single-nucleotide substitutions, which resulted in missense mutations within the DD regions of DR4. Breast cancer that had metastasized also showed higher frequency of LOH than breast cancer that had not metastasized, suggesting that DR4 may act like a tumor suppressor gene in some breast cancer and but may lose this function during the progression into metastatic stages. Indeed, expression of mutated DR4 in 293 cells led to the suppression of apoptosis. 26 In one other report, 27 mutation screening in the DD region of DR4 revealed only two missense mutations among 117 samples of human non-hodgkin s lymphoma (1.7%). Mutations in the DR5 gene have been identified in head and neck cancer, non-small-cell lung cancer, breast cancer, non-hodgkin s lymphoma, and hepatocellular carcinoma. A screening of 40 primary head and neck cancer specimens for mutations in DR5 revealed two such mutations, one of which was a 2-bp insertion in the DD of DR5 that resulted in a premature stop codon and a truncated DR5. 28 This insertion was also present in the germ line of the affected patient. Transfection of the truncated DR5 mutant into head and neck squamous cell carcinoma and colon and ovarian carcinoma cell lines led to a loss of growth-suppressive function. 28 In another study, 11 of 104 non-small-cell lung cancer specimens (10.6%) were found to have mutations in the DD of DR5, which could not be detected in the corresponding normal tissue samples. Of these 11 samples, seven were hemizygous without LOH. The authors postulated that hemizygously mutated DR5 may bind with other, normal DR5 proteins to form a structurally abnormal DR5 trimer, which could affect binding to adaptor proteins, such as FADD. 29 In the study of DR4 in breast cancer discussed in the previous paragraph, 26 four mutations in DR5 were identified among 34 breast cancer that had metastasized (11.8%), but no mutations in DR5 were found in any of 23 breast cancers that had not metastasized. In contrast to the DR4 mutations in that study, the two DR5 mutations were identified within the DD, and another two mutations were detected in the flanking region of the DD. Still, expression of these mutated DR5 in 293 cells led to suppression of apoptosis. 26 In other studies, one missense mutation was detected in the DD of DR5 among 100 samples of hepatocellular carcinoma (1%), 30 and sixpoint mutations were detected in the coding regions of the DR5 gene among 117 samples of non-hodgkin s lymphoma (5.1%). 27 DISC assembly and TRAIL resistance Assembly of the DISC is an early molecular event in the signaling pathway of TRAIL-induced apoptosis. Several molecules, including TRAIL, TRAIL death receptors, FADD, caspase-8 or caspase-10, and cflip, participate in the formation of the DISC. Most components of the DISC are essential to TRAIL-induced apoptosis, and dysfunction in any of these DISC components can lead to TRAIL resistance. 7,11 Evidence regarding the roles of FADD, caspase-8, and cflip in TRAIL-mediated apoptosis is reviewed below. FADD FADD is an adaptor molecule containing both DD and DED. Its essential role in death signal transduction was first identified in FasL-induced apoptosis. Through its interactions with activated Fas at the DD and caspase-8 at the DED, FADD transmits a death signal from Fas to caspase The involvement and importance of FADD in TRAIL signaling are highly controversial. Different

4 groups have delivered a FADD dominant negative (FADD-DN) construct into TRAIL-sensitive cell lines and obtained conflicting conclusions, with some showing that TRAIL-activated apoptosis depends on FADD and others showing that activation is independent of FADD. 9,16,20,21,32 34 As the use of FADD-DN mutants may inhibit other proteins in addition to FADD, a better model for the study of FADD function is FADDdeficient cell lines. 35 In one study using such cells, 36 the overexpression of DR4 by transient transfection into homozygously FADD-deficient (FADD / ) mouse embryonic fibroblasts induced apoptosis of those cells. The authors of that study concluded that FADD was not essential, as it was not required for DR4- and possibly DR5-mediated apoptosis. The use of transient transfection for the ectopic expression of death receptors, however, has raised concerns about nonspecific aggregation of the DD in death receptors. 35 In fact, evidence has already shown that overexpression of DR4 or DR5 by transient transfection into TRAIL-sensitive cell lines can lead to apoptosis independent of the TRAIL ligand. 20,21,35 However, in stably transfected cells or in physiological situations, expression of these receptors does not lead to cell death, and TRAIL binding to the death receptors is necessary to initiate apoptosis. 35 Specifically, FADD / embryonic fibroblasts stably transfected with mouse or human TRAIL death receptors were all resistant to TRAIL-induced cell death, but FADD þ / fibroblasts stably transfected with mouse TRAIL death receptors, and FADD / fibroblasts in which FADD had been reconstituted with a retroviral construct, were all sensitive to TRAIL-induced apoptosis. 35 The conclusions from this study, that FADD is essential in TRAIL-induced apoptosis and that dysfunction of FADD can cause TRAIL resistance, are also supported by another study that used Jurkat cells. 37 In that study, FADD-deficient Jurkat cells were resistant to TRAIL even at very high concentrations (1 mg/ml), but wild-type Jurkat cells underwent extensive apoptosis at TRAIL concentrations as low as 10 ng/ml. Caspase-8 Convincing evidence is accumulating, which shows caspase-8 to be a key and irreplaceable molecule in TRAIL-induced as well as Fas L- and TNF-a-induced apoptosis Downregulation or loss of caspase-8 expression can lead to TRAIL resistance. 6,7 In the first line of evidence, caspase-8-deficient Jurkat cells were shown to be completely resistant to TRAIL, whereas the corresponding wild-type cells remained sensitive to TRAIL. Second, the specific caspase-8 inhibitor Z- IETD-FMK could inhibit TRAIL-induced apoptosis in most TRAIL-sensitive cell lines. 38,41 However, because Z- IETD-FMK may also inhibit other caspases, especially at high doses, use of a more specific approach such as small inhibitory RNA should be considered to confirm the specificity of caspase-8 inhibition. 42 Third, caspase-10, although structurally similar to caspase-8 and having a DED, is not a functional substitute for caspase Whether caspase-10 acts as an initiator caspase is controversial. Two groups that used the same mutant caspase-8-deficient Jurkat cells both showed that caspase- 10 does act as an initiator caspase in TRAIL deathreceptor signaling and that caspase-10 could replace caspase-8 function in the absence of caspase-8. 43,44 However, a subsequent study by a third group using the same caspase-8-deficient cell line specifically indicated that caspase-10 does not initiate apoptosis. 39 Although TRAIL could induce cell death in the caspase-8-deficient Jurkat cells, much higher concentrations of TRAIL ligand were needed to achieve the same degree of death induction when compared with caspase-8-expressing wild-type Jurkat cells. 39 Also, the two earlier studies used a transient caspase-10 expression system, which may have led to caspase-10 levels that were too high to be tolerated by the host cells. 43,44 Moreover, some of the cells in the third study that expressed little or no caspase-10 showed no apparent defects in TRAIL-induced apoptosis. 39 The conclusion from the current data, therefore, is that caspase-8 has one or more unique functions that caspase-10 cannot replace for apoptosis initiation. A fourth line of evidence implicating caspase-8 is the observation that several types of cancer cells, including Ewing s tumor, neuroblastoma, malignant brain tumors, melanoma, and small-cell lung cancer, show resistance to TRAIL-induced apoptosis that correlates with downregulation or absence of caspase-8 expression. Importantly, restoration of caspase-8 expression in these resistant cells rendered them sensitive to TRAIL. 6,7,45 47 In a very few cases, loss of caspase-8 expression resulted from a gene deletion, as demonstrated by Southern blot analysis. In most cases, however, absence of caspase-8 expression resulted from gene silencing by DNA methylation in its promoter region. 6,7,45 47 Treatment of such cells with the demethylation agent 5-aza-2 0 -deoxycytidine reversed the hypermethylation of the caspase-8 promoter, thereby restoring expression of caspase-8 and sensitivity to TRAIL-induced apoptosis. 6,7 cflip The apoptosis inhibitor cflip is structurally similar to caspase So far, only two forms of cflip have been detected, although multiple splicing variants may exist. cflip L contains two DEDs and a caspase-like domain, but it cannot activate caspase cascades because that domain lacks a cysteine residue essential for catalytic activity. cflip S also contains two DEDs, but it lacks almost the entire caspase-like domain. Both cflip L and cflip S can be recruited into the DISC, where they bind to either FADD or caspase-8 through DED DED interactions, resulting in inhibition of caspase-8 activation and inhibition of subsequent apoptosis. 48 Initially, cflip was reported to have both proapoptotic and antiapoptotic effects; however, the proapoptotic effect was soon recognized to occur only in transient transfection systems, where excessive cflip expression could result in nonspecific cell death. 57 On the other hand, use of stable expression systems and cflip-deficient models 231

5 232 resulted in only the antiapoptotic effect being observed. 58,59 Expression of cflip was recently reported to correlate strongly with malignant potential in colonic adenocarcinomas, melanoma, and hepatocellular carcinoma Changes in the cflip/caspase-8 ratio have also been reported to correlate with TRAIL resistance in several different tumors, including melanoma, hepatocellular carcinoma, Burkitt s lymphoma, and B-cell chronic lymphocytic leukemia. 62,63 Downregulation of cflip expression by using antisense RNA or sirna may be a worthwhile strategy to explore for overcoming TRAIL resistance in types of cancer in which cflip overexpression is a key determinant of TRAIL resistance. Despite findings that several types of malignant tumor express high levels of cflip and that overexpression of cflip can confer resistance to TRAIL and other death ligands, the physiological function of cflip remains unclear. However, it was recently postulated that cancer cells may acquire a certain degree of immune privilege by becoming resistant to death ligands such as TRAIL or FasL, and cflip may help in this process. 64 One group assessed the effect of cflip in tumorigenesis and immune escape by using stable cflip transfectants. In that system, inoculation of transfectants expressing little or no cflip into immunocompetent mice resulted in rejection of the transfectants in most mice, but inoculation of transfectants with high cflip expression into the same types of mice led to tumor development. In contrast, inoculation of either type of transfected cells into nude mice led to the formation of tumors that grew at the same rate regardless of cflip expression level. Thus, it seems that tumor cells that express little or no cflip can be eliminated to some extent through selective pressure by the immune system, but tumor cells that express high levels of cflip can escape immune surveillance. These findings suggest that immune therapy may be another useful approach for cancer therapy. 65 Bcl-2 family and TRAIL resistance During the process of TRAIL-induced apoptosis, activation of the initiator caspase-8 can transmit death signals either through direct activation of the effector caspase-3 or 7, or by means of the proapoptotic Bcl-2 family member Bid, through a mitochondrial pathway. 11 In this mitochondrial death pathway, the ratio of expression of the proapoptotic Baxprotein and the antiapoptotic Bcl-2 or Bcl-X L proteins ultimately determines cell death or survival. 12,13 The importance of the mitochondrial pathway in TRAIL-induced apoptosis depends on the cell type. Evidence implicating Bcl-2, Bcl-X L, Bax, and Bak in the mitochondrial pathway of TRAIL-induced apoptosis is reviewed in the following paragraphs. Bcl-2 and Bcl-X L Overexpression of Bcl-X L or Bcl-2 can protect some types of cells against TRAIL-mediated apoptosis, suggesting that the mitochondrial pathway predominates in these types of cells. Bcl-X L expression correlated highly with sensitivity to TRAIL-induced apoptosis in three pancreatic adenocarcinoma cell lines. 5 The cell line Colo357, originally sensitive to TRAIL and expressing low levels of Bcl- X L, became resistant to TRAIL after Bcl-X L expression was restored by means of a retrovirus. Interestingly, Bcl- X L -overexpressing Colo357 cells, although resistant to TRAIL, had the same degree of caspase-8 cleavage as did the parental Colo357 cells, suggesting that caspase-8 activation was independent and upstream of the mitochondrial pathway. In another study, 66 overexpression of Bcl-2 conferred protection against TRAIL in neuroblastoma, glioblastoma, and breast cancer cell lines, but reduced TRAIL-induced caspase-8 cleavage, suggesting that caspase-8 was activated both upstream and downstream of the mitochondria in these cells upon treatment with TRAIL. As apoptosis induced by chemotherapy acts mainly through the mitochondrial pathway, 11 downregulation of Bcl-2 or Bcl-X L might restore sensitivity not only to chemotherapy but also to TRAIL in some types of cancer. Bax and Bak Mutational inactivation of the proapoptotic genes of Bax or Bak can render cancer cells resistant to apoptosis induced by TRAIL or chemotherapy. The importance of both molecules in apoptosis was demonstrated by the discovery that TRAIL could induce cytochrome c release and apoptosis in wild-type, Bax / or Bak / mouse embryonic fibroblasts, but not in double-knockout Bax / /Bak / cells. 67 These findings suggest that, functionally, Baxand Bak can substitute for each other in these cells, because knocking out only one of the two genes was not sufficient to confer resistance to TRAILinduced apoptosis. This does not seem to be the case for other types of cells, however. For example, the human colon cancer cell line HCT116, which expresses Bak, required Baxfor TRAIL-induced apoptosis. 68 In that study, TRAIL produced rapid apoptosis in a single-alleleinactivated clone (Bax þ / ) but not in a double-alleleinactivated clone (Bax / ). In another study, Bakdeficient Jurkat cells were more resistant than wild-type Jurkat cells to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. Restoring the Bak gene restored cytochrome c release and the sensitivity of the Bak-deficient cells to VP-16. Recombinant Bak could also induce cytochrome c release from mitochondria purified from Bax / mice, suggesting that Bak plays an essential role, independent of Bax, in cytochrome c release and overcoming chemoresistance in Jurkat cells. 69 In conclusion, the importance of Bax, Bak or both in TRAIL- or chemotherapy drug-induced apoptosis seems to depend on the cell type. Inhibitors of apoptosis (IAP) proteins, Smac/Diablo, and TRAIL resistance IAP proteins can block apoptotic events by inhibiting the catalytic activity of effector caspases (e.g., -3 and -7) or by

6 blocking the activation of the apopsomal caspase-9 by directly interacting with the active sites of these caspases. Each IAP contains one or three tandem repeats of an evolutionarily conserved domain termed baculovirus inhibitory repeat (BIR). Different BIRs are thought to have different preferences for distinct caspases. So far, six mammalian IAPs have been identified: ciap1, ciap2, X- linked inhibitor of apoptosis (XIAP), neuronal apoptosis inhibitory protein (NAIP), survivin, and BIR repeatcontaining ubiquitin-conjugating enzyme (BRUCE). XIAP is the most potent inhibitor of caspase activity. 70,71 High expression of IAPs in cancer cells can confer resistance to TRAIL-induced apoptosis. 72,73 The activity of IAPs can be blocked by Smac/Diablo, a mitochondrial protein that is released into the cytosol at some point during the apoptotic cascade, where it promotes cell death by eliminating IAP inhibition of caspases. 74,75 Smac/Diablo is thought to interact with the BIR regions on IAPs, thereby releasing the caspases and promoting apoptosis. Blocking the release of Smac/ Diablo from the mitochondria has been associated with resistance to TRAIL in some, but not all, melanoma cell lines. 76 In mitochondria-dependent type II cells, regulation of apoptosis by IAPs and Smac/Diablo is a major determinant of TRAIL sensitivity. TRAIL-resistant cancer cell lines, compared with TRAIL-sensitive cancer cell lines, show a reduced release of Smac/Diablo from the mitochondria to the cytosol. 72,73,76 However, overexpression of Smac/Diablo by transfection restored sensitivity to TRAIL in those cells, in addition to downregulating or cleaving XIAP, ciap1, and ciap2. 72,73,76 The release of Smac/Diablo from mitochondria, like cytochrome c, can be blocked by Bcl-2 overexpression. 76 NF-jB and TRAIL resistance NF-kB is a transcription factor that participates in the control of immune regulation, inflammatory responses, cell growth, and apoptosis. The five component subunits of NF-kB, crel, crela/p65, crelb, NF-kB1/p50, and NF-kB2/p52 form homodimeric or heterodimeric complexes and control transcription by binding NF-kB consensus sequences in the promoter regions of target genes. 77,78 The effects of NF-kB on TRAIL signaling are controversial, with some reports showing that NF-kB activation protects cells from TRAIL-induced apoptosis 79,80 and others showing the opposite effect that NF-kB promotes apoptosis. 81 These discrepancies, however, may reflect differences in the function and relative amounts of the NF-kB subunits. 82 Overexpression of the crela subunit, for example, inhibits caspase-8, DR4, and DR5 expression, and enhances IAP1 and IAP2 expression after TRAIL treatment. On the other hand, overexpression of crel enhances DR4, DR5, and Bcl-X S expression and inhibits ciap1 and IAP2 expression after TRAIL treatment. The relative amounts of crel and crela in activated NF-kB molecules seem to determine whether the NF-kB favors apoptosis or survival. Thus, regulation of the expression of the different NF-kB subunits may be another new strategy for cancer therapy. Mitogen-activated protein (MAP) kinases and TRAIL resistance The MAP kinases are a superfamily of proteins that transmit signaling cascades from extracellular stimuli into cells; examples of MAP kinases include extracellular signal-regulated kinases (ERKs), c-jun N-terminal protein kinases (JNKs), and p38 MAP kinases. Like NF-kB, MAP kinases participate in a wide variety of cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. 83 Usually, activation of ERKs in response to death stimuli is believed to have an antiapoptotic effect. In support of this conclusion were findings that TRAIL induced rapid ERK1/2 activation in a group of melanoma cell lines, and the inhibition of that activation sensitized TRAIL-resistant melanoma cells to TRAIL-induced apoptosis, suggesting that ERK1/2 activation can itself protect against TRAIL-induced cell death in these TRAIL-resistant cell lines. 84 However, TRAIL also induced rapid ERK1/2 activation in TRAIL-sensitive melanoma cell lines, indicating that ERK1/2 activation by itself is not sufficient to protect against TRAIL-induced cell death in these TRAIL-sensitive cell lines. We hypothesize here that one or more anti-erk cofactors exist in the TRAIL-sensitive melanoma cell lines but not in the TRAIL-resistant cell lines. On the other hand, ERK activation has also been reported to have proapoptotic effects. 85 In that study, sensitization of lung cancer cells to TRAIL-induced apoptosis by PG490, a candidate chemotherapeutic agent, seemed to require ERK2 activation in that the sensitization could be blocked by inhibiting ERK2 activation. 85 Studies of p38 MAP kinase and JNK activation in TRAIL-induced apoptosis have also produced inconsistent results. In one such report, 86 use of doxorubicin and cisplatin enhanced the apoptosis-inducing activity of antibodies to TRAIL receptors; this effect was assumed to result from activation of p38 MAP kinase and JNK, as inhibition of these kinases suppressed the apoptotic effect. In another study, 87 inhibition of JNK activation was shown to block apoptosis induced by a combination of the translation inhibitor anisomycin and TRAIL. JNK activation seems to be required for TRAIL-plus-anisomycin-induced apoptosis, but it is not sufficient because activation of JNK by its upstream activator MEKK2 did not sensitize cells to TRAIL-induced apoptosis. Finally, we have noted p38 MAP kinase activation in colon cancer DLD1 cell line upon TRAIL treatment; however, inhibition of that activation was not related to development of TRAIL resistance in that cell line. 88 The functions of MAP kinases in TRAIL-induced apoptosis are further complicated by their involvement in cytokine secretion, particularly TNF-a, IL-1b, and IL

7 234 We hypothesize that TRAIL treatment involves different MAP kinases, different cell environments, and different cytokines, all of which interact to tip the balance in favor of cell survival or cell death. Resistance of normal human cells or tissues to TRAILinduced apoptosis Many approaches have been employed to overcome TRAIL resistance in cancer cells, notably by combination therapy of TRAIL with chemotherapy, or radiotherapy. However, a concern has been raised about the toxicity of such combination therapy on normal cells or tissues. 90 It is possible that methods used to sensitize TRAIL-resistant cancer cells can sensitize normal cells as well, leading to side effects. Therefore, elucidating the mechanisms of TRAIL resistance in normal cells or tissues is also an important step in TRAIL cancer therapy. It is reported that expression of the decoy receptors is attributed to TRAIL resistance in normal human cells. 20,21 However, accumulative data have already shown that the decoy receptor expression is not the only mechanism of TRAIL resistance of normal cells. For examples, DR5 receptor agonistic antibodies are capable of inducing apoptosis in DR5-expressed cancer cells, but not in normal human fibroblast cells and human hepatocytes. 91 Further studies show that the cancerous tissues express higher levels of DR5 mrna and protein than do normal cells or tissues. As these DR5 receptor agonistic antibodies have high specificity for binding and activation of DR5 rather than other TRAIL receptors, such as DR4, DcR1, and DcR2, it is obvious that low expression of DR5 contributes to the resistance to the DR5 receptor agonistic antibodies. 91 Furthermore, when DR5 is upregulated by either MYC oncogene or doxorubicin, the normal cells can become sensitive to either the DR5 receptor agonistic antibodies or TRAIL. 24,92 In most of the normal cells tested, DR4 shows much lower expression than that of DR5, which may also reduce TRAIL sensitivity. 91 In general, current data suggest that, in certain normal cells or tissues, the ratio of expression between real death receptor DR4 and DR5, and decoy receptors DcR1 and DcR2, may determine the TRAIL sensitivity. 20,21,24,91,92 Liver toxicity is a major concern for application of TRAIL gene therapy to human cancer patients. Although the human liver toxicity could be caused by special TRAIL protein preparation, such as tagged histidine or leucine, 93 the full length of TRAIL protein can readily induce liver toxicity. 2 However, the ratio of expression between death receptors DR4 and DR5, and decoy receptors DcR1 and DcR2, does not correlate with the sensitivity of hepatocytes to TRAIL, 94 suggesting that other molecules in the signaling pathway of TRAILinduced apoptosis may determine the fate of normal cells in response to TRAIL. In fact, in addition to the death receptors, overexpression of cflip and IAPs has also been reported to play a major role in protection of certain human normal cells, such as melanocytes and lung and foreskin fibroblasts, from TRAIL-induced apoptosis. 24,95 However, no cflip and IAP expression knockdown experiments are performed in these reports. Therefore, much has to be done before we can completely picture the mechanisms of TRAIL resistance in normal human cells or tissues. Future prospects The concept of using recombinant TRAIL genes and proteins for cancer therapy has attracted much attention in anticancer research. Nevertheless, the potential for hepatotoxicity and the resistance of many tumor cells to TRAIL protein, recombinant or otherwise, has prevented this approach from entering clinical trials. Although TRAIL-mediated systemic toxicity might be prevented by targeted TRAIL gene therapy or other approaches, characterizing the mechanisms of resistance and developing strategies to overcome that resistance are still critical for future successes. Ongoing progress in molecular biology and cancer biology will certainly shed further light on TRAIL-mediated death signal transduction from the membrane to the nucleus, as well as the molecular events that stop this transduction. 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