8 Death Receptor Mutations

From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ

149

8 Death Receptor Mutations

Sug Hyung Lee, ^MD , ^P h D , Nam Jin Yoo, ^MD , ^P h D ,

and Jung Young Lee, ^MD , ^P h D ,

SUMMARY

It is generally believed that human cancers may arise as the result of an accumulation of mutations in genes and subsequent clonal selection of variant progeny with increas- ingly aggressive behaviors. Also, among the remarkable advances in our understanding in cancer biology is the realization that apoptosis has a profound effect on the malignant phenotypes. Along with these, compelling evidence indicates that somatic mutations in the genes encoding apoptosis-related proteins contribute to either development or pro- gression of human cancers. In this chapter, we present an overview of the death receptor pathway and its dysregulation in cancers. We then review the current knowledge of death receptor mutations that have been detected in humans.

INTRODUCTION

Programmed cell death through apoptosis plays a fundamental role in a variety of physiological processes, and its deregulation contributes to many diseases, including autoimmunity, cancer, acquisition of drug resistance in tumors, stroke, progression of some degenerative diseases, and acquired immunodeficiency syndrome (AIDS) (1–4).

Apoptosis is an active cell-suicide process executed by a cascade of molecular events involving a number of membrane receptors and cytoplasmic proteins (1–4). Although many pathways for activating caspases may exist, only two, the intrinsic pathway and the extrinsic pathway, have been demonstrated in detail (3). The extrinsic pathway can be induced by members of the tumor necrosis factor (TNF) receptor family, such as TNF receptor 1 (TNFR1) and Fas (5–7). These proteins recruit adaptor proteins, including FADD, to their cytosolic death domains, which then bind caspases-8 and -10 (8–12). The intrinsic pathway can be induced by release of cytochrome c from mitochondria (13–15).

In the cytosol, cytochrome c binds and activates apaf-1, allowing it to bind and activate

caspase-9 (14,15). Active initiator caspases of the extrinsic pathway (caspases-8 and -10)

and the intrinsic pathway (caspase-9) have been shown to directly cleave and activate the effector protease, caspase-3 (16,17). Also, though commonly viewed as separate path- ways and capable of functioning independently, cross-talk can occur between these path- ways at multiple levels, depending on the repertoire of apoptosis-modulating proteins expressed (18–21).

APOPTOSIS AND CANCER

Several lines of evidence indicate that tumorigenesis is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal cells into malignant phenotypes (22). The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as subtle as point mutations and as obvious as changes in chromosome complement (23). In the multistep tumorigen- esis model, mutations in key cellular genes produce a series of acquired capabilities that allow the developing cancer cell to grow unchecked in the absence of growth-stimulating signals, while overcoming growth-inhibitory signals and host immune responses (22–

25). They also allow the tumor to replicate indefinitely, maintain an oxygen and nutrient supply, and invade adjacent and distant tissues (26–29). Finally, the ability of cells to evade apoptosis is also an essential hallmark of cancer (22,30,31).

Since the discovery of bcl-2 as an oncogene that promotes cell survival, it has been widely acknowledged that antiapoptotic genetic lesions are necessary for tumors to arise (32,33). Clonal expansion and tumor growth are the results of the deregulation of intrinsic proliferation (cell division) and cell death (apoptosis). The evidence is mounting, from studies in mouse models and cultured cells, as well as from descriptive analyses of tumor tissues along the multistep carcinogenesis (22,23). Enhanced cell survival is needed at several steps during tumorigenesis: deregulated oncogene expression not only leads to accelerated proliferation, but concomitantly induces apoptosis, which needs to be sup- pressed for the transformed cell to survive and multiply (34). The tumor cells can persist in a hostile environment. For example, sufficient nutrition for every tumor cell becomes restricted; starvation of tumor cells from cytokines usually leads to apoptotic cell death (26,27). Finally, defective apoptosis facilitates metastasis (22). To metastasize, a tumor cell must acquire the ability to survive in the bloodstream and invade a foreign tissue.

Normally, this process is prevented by the propensity of epithelial cells to die in suspen- sion, or in the absence of appropriate tissue survival. During metastasis, cancer cells can ignore restraining signals from neighbors and survive detachment from the extracellular matrix (29). Hence, loss of apoptosis can impact tumor development, progression, and metastasis. Loss of apoptosis is also a significant impediment to anticancer therapy. It is now well established that anticancer agents induce apoptosis, and the disruption of apoptotic machineries reduces treatment sensitivity (35). The mutations that favored tumor development dampen the response to chemotherapy, and treatment might select more refractory clones.

DEATH RECEPTORS

Cell surface death receptors transmit an apoptosis signal on binding of a specific death

ligand (2). The best known family of death receptors is represented by tumor necrosis

factor receptors (TNFRs), Fas (CD95, Apo-1), and TNF-related apoptosis-inducing

ligand receptors (TRAIL-Rs) (36-41). The receptors’ ligands comprise another related family that includes TNF, CD95 ligand (FasL/CD95L), and TRAIL (2). Each of the ligands is synthesized as a membrane-associated protein and shares a characteristic 150- amino-acid region towards the C-terminus by which each ligand interacts with its cognate receptor (2). For the most part, these ligands exist as trimeric or multimeric membrane- bound proteins that may function to induce receptor aggregation.

The Fas-FasL system has been recognized as a major pathway for the induction of apoptosis in cells and tissues (1,2). Fas is a member of the death receptor subfamily of the TNFR superfamily (37–39). Ligation of Fas by either agonistic antibody or by its natural ligand transmits a death signal to the target cells, potentially triggering apoptosis.

Fas has three cystein-rich extracellular domains and an intracellular death domain (DD) essential for signaling (37–39).

The death domain, a name deriving from its ability to recruit downstream effectors that can induce apoptosis, is present in the cytoplasmic tail of all death receptors (1). The death domain is a protein interaction module consisting of a compact bundle of six _-helices (42). Death domains bind each other, probably forming oligomers of unknown stoichiom- etry. Stimulation of Fas results in aggregation of its intracellular death domain, leading to the recruitment of two key signaling proteins that, together with the receptor, form the death-inducing signaling complex (DISC) (43). FADD/MORT-1 (8) couples through its C-terminal death domain to crosslinked Fas receptors and recruits caspase-8 (9) through its N-terminal death effector domain (DED) to the DISC. Caspase-10 (Mch4/FLICE2) is a caspase homologous to caspase-8 and is present as an inactive proenzyme, comprising a prodomain that contains two DEDs to allow caspase-10 to interact with the DED of FADD and a catalytic protease domain that can be further processed to give a large and a small subunit (44). The local aggregation of the procaspases-8 and -10 is sufficient to allow autoprocessing or transprocessing to produce active caspases-8 and -10, which can subsequently activate downstream executioners, such as caspases 3 and 7 (44).

Five receptors have been identified for TRAIL, including two apoptosis-inducing receptors (death receptor 4/TRAIL-R1 and DR5/TRICK-2/TRAIL-R2/KILLER-DR7), two decoy receptors (DcRs) (TRID/DcR1 and TRUNDD/DcR2), and osteo-progeterin.

TRAIL induces apoptosis through DR4 and DR5 requiring FADD, caspase-8 and caspase- 10, just like CD95-mediated cell killing (45).

DEATH RECEPTOR MUTATIONS

Fas, DR5, and DR4 are widely expressed in normal and neoplastic cells (46,47), but

the expression of these proteins does not necessarily predict susceptibility to killing

(48,49). This can reflect the presence of inhibiting mechanisms of death receptor-medi-

ated apoptosis. Fas-mediated apoptosis can be blocked by several mechanisms, including

the production of soluble Fas (50), the lack of cell-surface Fas expression (51–53), the

overexpression of inhibitory proteins in signal transduction pathways such as Fas-asso-

ciated phosphatase-1 (54) and FLICE- inhibitory protein (FLIP) (55), and the mutation

of the primary structure of Fas (56–65). TRAIL-induced apoptosis can be blocked by

several mechanisms, including the expression of decoy receptors for TRAIL (10), the

loss of TRAIL receptor expression (5), the overexpression of inhibitory proteins in signal

transduction pathways such as FLIP (7), and the mutation of the primary structure of DR4

and DR5 (11).

Germline Mutation of Death Receptors

The consequences of naturally occurring mutants of Fas/FasL have been well demon- strated in both mice and humans. lpr and gld are mutations in mice of Fas and FasL, respectively (56,66). Because the Fas/FasL system is involved in the apoptotic process that occurs during cell muturation, lpr and gld mutations result in the development of lymphadenopathy and autoimmune diseases in the mice (67). To date, two lpr mutations are known: lpr and lpr

. The mouse Fas gene consists of over 70kb, and is split by 9 exons (56). The restriction mapping of the Fas gene from lpr mice has revealed the insertion of an early transposable element (ETn) of 5.4 kb in intron 2. The ETn is a mouse endogenous retrovirus, of which about 1,000 copies can be found in the mouse genome. The ETn has long terminal repeat (LTR) sequences at both the 5' and 3' termini. This LTR sequence contains a polyadenylation signal, and transcription terminates at this area.

Furthermore, insertion of ETn into an intron of a mammalian expression vector reduced the expression efficiency in mammalian cells. These data indicate that the lpr mice have a defect in the expression of Fas due to insertion of ETn in intron 2. Unlike the lpr mice, lpr

mice express full-length Fas mRNA as abundantly as wild-type mice (56). However, the Fas mRNA carries a point mutation (T to A) in the death domain. This mutation results in an amino acid change, from isoleucine to asparagine, and abolishes the ability of Fas to transduce the cell-death signal to cells.

The autoimmune lymphoproliferative syndrome (ALPS; sometimes called the Canale–

Smith syndrome) arises in early childhood and can have fatal complications (44,51–67).

It is associated with prominent nonmalignant lymphadenopathy, hepatosplenomegaly, and autoimmune manifestations. Underlying ALPS are heritable mutations in genes that regulate lymphocyte survival by triggering programmed death of lymphocytes, or apoptosis. By far, the most common form of ALPS is that associated with heterozygous Fas mutations (ALPS type Ia) (51–67); ALPS is inherited in an autosomal dominant fashion. The region of the Fas gene most often mutated is the death domain. These mutations are predicted to result in early termination (frameshift insertions and deletions;

amino acid changes to stop codons) or in single amino-acid substitutions (missense mutations) that disrupt the three-dimensional structure of the death domain. Patients with mutations of the FasL (67) or caspase-10 (44) gene are referred to as ALPS type Ib or II patients, respectively, whereas the remaining patients with the same symptoms but with- out identified mutations in genes involved in the induction of apoptosis are diagnosed as having ALPS type III (44).

Interestingly, the lpr mice have been reported to have spontaneous development of plasmacytoid tumors (69), and some ALPS patients have been reported to have malig- nancies (59,60), including multiple tumor development in one patient (59). Although it is not clear whether the tumors that occurred in ALPS patients arose as a result of Fas mutations, it is conceivable that Fas mutation might have influences on tumor develop- ment in these patients.

Somatic Mutation of Fas Gene

The key role of the Fas system in negative growth regulation has been studied mostly

within the immune system, and the germline inactivation of the Fas system exhibits

phenotypic abnormalities mainly in the lymphoid system (2). Thus, mutational analyses

of the Fas gene have been focused on hematopoietic tumors. However, there is mounting

evidence that disruption of the Fas system frequently occurs in nonlymphoid malignan- cies as well (48,50–52), and Fas gene mutations have been reported both in hematopoi- etic and nonhematopoietic tumors (62–65,70–80). Mutational analysis of the Fas gene was performed in a number of human cancers. Fas mutation occurred in 0–65% of hematopoietic malignancies and 0–28% of nonhematopoietic malignancies (Table 1).

The dominant negative effect of monoallelic mutations within the death domain is likely attributable to the trimerization of the Fas receptor on the cell surface. The death domain is a highly conserved region that is required and sufficient for the transduction of the death signal (37–39). Given the functional importance of this region, it is not surprising that approx 60% of somatic mutations in lymphoid or solid tumors involve this region.

F^AS MUTATIONS IN HEMATOPOIETIC T^UMORS

The somatic mutation of Fas was first reported in multiple myelomas (62). Multiple myelomas harbor Fas mutations at a frequency of 10% (5/48). All of the mutations identified were located in the death domain of the Fas antigen. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lpr

mouse. In child- hood T-lineage acute lymphoblastic leukemias, two Fas mutations were observed in exon 3 and the AP-2-binding region of the promotor (63). The mutation in exon 3 caused a 68Pro

trast, no Fas mutations could be detected in childhood B-lineage acute lymphoblastic leukemias (81), although most of the leukemias were resistant to Fas-mediated apoptosis.

Adult T-cell leukemia (ATL) is an aggressive neoplasm of activated T-lymphocytes, and human T-lymphotropic virus type I (HTLV-I) was found to be the causative virus of this tumor (82). Tamiya et al. (64) reported that one Fas-negative ATL showed two types of aberrant transcripts: one had a 5-bp deletion and a 1-bp insertion in exon 2, and the other transcript lacked exon 4. These mutations caused the premature termination of both alleles, resulting in the loss of expression of surface Fas antigen. Also, analysis of 35 Fas- positive ATL cells revealed a mutation that lacked exon 4 (64).

Lymphoma is another type of hematopoietic malignancy that has been reported to have

Fas mutations. Grønbæk et al. (65) analyzed 150 cases of non-Hodgkin’s lymphoma

(NHL), and identified 16 tumors (11%) with Fas gene mutations. Fas mutations were

identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas,

2 (6%) follicle center-cell lymphomas, 1 (50%) anaplastic large-cell lymphoma, and 1

unusual case of B-cell chronic lymphocytic leukemia. They observed that missense

mutations within the death domain of the receptor were associated with retention of the

wild-type allele, indicating a dominant-negative mechanism, whereas missense muta-

tions outside the death domain were associated with allelic loss. Of note, 10 of 13 evaluable

patients with the Fas mutations showed features suggestive of autoimmune disease,

suggesting a link between Fas mutation, cancer, and autoimmunity. Another suggestive

commonality between Fas mutation, cancer, and autoimmunity is observed in thyroid

lymphoma (72), which is supposed to arise from active lymphoid cells formed in the

preceding autoimmune chronic lymphocytic thyroiditis. Mutations of the Fas gene were

detected in 3 (27.3%) of 11 cases of autoimmune chronic lymphocytic thyroiditis and 17

Table 1

Summary of the Somatic Mutations of Fas Gene in Human Cancers

Type of human cancers Frequency Reference

Multiple myeloma 5/48 (10.4%) 62

Childhood T-lineage leukemia 2/81 (2.5%) 63 Childhood B-lineage leukemia 0/32 (0%) 81

Adult T-cell leukemia 2/47 (4.3%) 64

Non-Hodgkin’s lymphoma 16/150 (10.6%) 65

Hodgkin’s lymphoma 2/10 (20%) 75

Mycosis fungoides 6/44 (13.6%) 71

Thyroid lymphoma 17/26 (65.4%) 72

Cutaneous T-cell lymphoma 13/22 (59%) 73

Nasal NK/T-cell lymphoma 7/14 (50%) 74

Large granular lymphocyte leukemia 0/11 (0%) 85 Marginal zone B-cell lymphomas 0/27 (0%) 86

Malignant melanoma 3/44 (6.8%) 76

NSCLC 5/65 (7.7%) 77

Bladder transitional cell carcinoma 12/43 (28%) 78

Gastric cancer 5/43 (11.6%) 79

Gastric cancer 2/20 (10%) 87

Colon cancer 2/20 (10%) 87

Colon cancer 0/24 (0%) 93

Burn scar-related SCC 3/21 (14.3%) 80

Conventional cutaneous SCC 0/50 (0%) 80

Breast cancer 0/58 (0%) 89

Hepatocellular carcinoma 0/50 (0%) 90

Hepatoblastoma 0/23 (0%) 91

Ovary cancer 0/8 (0%) 92

Glioblastoma multiforme 0/23 (0%) 95

Abbreviations: SCC, squamous cell carcinoma; NSCLC, non-small-cell lung cancer.

(65.4%) of 26 of thyroid lymphoma. Of note, all of the mutations involve the alteration

of exon 9 that encodes the death domain. Fas mutations have also been detected in T-cell

lymphoma, including cutaneous T-cell lymphoma (74), mycosis fungoides (72), and the

nasal natural killer (NK)/T-cell lymphoma (74). Doorn et al. (73) analyzed cutaneous

T-cell lymphomas, a group of clinically heterogeneous malignancies of mature skin-

homing T–cells, and identified a novel mutation of the Fas gene that displays retention

of intron 5 in 13 of 22 patients (59%). Two of these 13 tumors were found to have

additional missense mutations. Dereure et al. (71) described the presence of six point

mutations of the coding sequence of the Fas gene in 6 of 44 patients (13%) with mycosis

fungoides, a cutaneous T-cell lymphoma. Nasal NK/T-cell lymphoma (NKTCL) is a

clinical condition of lethal midline granuloma that shows necrotic, granulomatous lesions

in the upper respiratory tract, especially in the nasal cavity. Takakuwa et al. (74) reported

mutations of the Fas gene in 7 (50%) of 14 NKTCLs which comprised four frameshift, two

missense, and one silent mutations. All of the mutations involved the alteration of

exon 9.

Hodgkin and Reed–Sternberg (H/RS) cells in classical Hodgkin’s disease (HD) are thought to be derived from preapoptotic germinal center B-cells (83). Single microm- anipulated H/RS cells from 10 cases of HD were analyzed for somatic mutations within the CD95 gene, and two cases displayed Fas mutation within the 5' region in one case and within exon 9 in another case (75).

In contrast to the aforementioned reports of Fas mutations, some reports described the negative correlation between hematologic tumor pathogenesis and death receptor muta- tions. Rozenfeld-Granot et al. (84) screened somatic mutation at the death domains of Fas, FADD, TNFR, TRADD, and RIP, in the promoter region of Fas and in the protease domain of caspase-10, in a larger variety of hematological malignancies (31 chronic lymphocytic leukemias, 28 chronic myelogenous leukemias, 8 essential thrombo- cythemias, 6 acute lymphocytic leukemias, 6 acute myeloblastic leukemias, 3 hairy-cell leukemias, 3 Burkitt’s lymphomas, 3 polycythemia veras, 2 myelofibroses, and 2 chronic myelomonocytic leukemias), but could not find any mutations in any of the malignancies.

Also, no mutation could be detected in marginal zone B-cell lymphomas and large granu- lar lymphocyte lymphoma (85,86).

FAS MUTATIONS IN NONHEMATOPOIETIC TUMORS

Fas mutations in nonhematopoietic tumors have been detected in non-small-cell lung cancers (NSCLC) (77), malignant melanomas (76), transitional cell carcinomas of the urinary bladder (78), gastric carcinomas (79,87), colon carcinomas (87), burn scar-related squamous cell carcinomas (80), and testicular germ-cell tumors (88), while no Fas muta- tions have been detected in breast carcinomas (89), hepatocellular carcinomas (90), hepatoblastomas (91), ovarian cancers (92), and colon cancers (93). Lee et al. and Shin et al. at the same laboratory have reported the majority of these mutations (77–80,90,91).

They analyzed the allelic status of the Fas gene as well as Fas mutations by using intragenic polymorphic markers.

In the NSCLCs, five tumors (7.7%) were found to have Fas mutations, which were all missense mutations (77). Four of the five mutations identified were located in the cyto- plasmic region (death domain) and one mutation was located in the transmembrane domain. In the cutaneous malignant melanomas, three tumors (6.8%) were found to have Fas mutations, which were all missense variants and identified in the death domain (76).

A particularly high incidence of Fas mutations was detected in bladder cancer (28%) (78). Ten of the 12 identified mutations were located in the death domain and 8 of these 10 mutations showed an identical G to A transition at bp 993 (codon 251), indicating a potential mutation hotspot in bladder cancers. Burn scar-related squamous cell carci- noma, a skin tumor that is more aggressive and carries a poorer prognosis than conven- tional cutaneous squamous cell carcinoma, was reported to have a Fas mutation, whereas conventional cutaneous squamous cell carcinoma was not (80). Microsatellite mutator phenotype (MMP) plays an important role in developing gastrointestinal cancer (94). In the coding region of Fas (codons 133–135), there is a mononucleotide track (TTTTTTT).

In the gastric and colon cancers with MMP, 10% of the cancers showed mutations in this repeat (87).

The incidence of Fas mutation in nonhematopoietic tumors seems to be tissue-type

specific. In the reports that analyzed Fas mutations in hepatocellular carcinomas (90),

hepatoblastomas (91), breast carcinomas (89), ovary carcinomas (92), colon carcinomas

(93), glioblastoma multiformes (95), and conventional squamous cell carcinomas (80), the investigators could not detect any Fas mutations.

Mutations of TRAIL Receptor Genes

Mutational analyses of TRAIL receptors have been performed less widely in human cancers than analyses of Fas. Mutations of the death domain-containing receptors TRAIL- R1 and TRAIL-R2 have been found in different human cancers (Table 2) (96–101).

TRAIL-R2 mutation was observed in 5% of head and neck cancer (96), 10.6% of NSCLC (97), 7% of gastric adenocarcinoma (99), 1% of hepatocellular carcinoma (101), 5.1% of non-Hodgkin’s lymphoma (100), and 11.7% of metastatic breast cancers (98). Of note, all TRAIL-R2 mutations were detected only in the death domain sequences of these genes, except for one in the splice site. The vast majority of mutations consisted of missense alterations; the remaining ones consisted of nonsense, splice-site, and silent mutations.

By contrast, TRAIL-R2 mutation was not detected in colorectal cancers (102), breast cancers (103), and NSCLC (104) by other researchers. A site-directed mutagenesis strat- egy and functional analysis of TRAIL-R2 mutations derived from NSCLC (105), breast cancer (98), gastric adenocarcinoma (99), and head/neck cancer (96) provided novel insights into the functional significance of specific structural determinants within the death domain of TRAIL-R2. It has been shown that death domain mutations displayed divergent phenotypes. TRAIL-R2 mutation appears to be involved in the pathogenesis of breast cancers. A mutational analysis of the death domain of TRAIL-R2 in breast cancer in a Korean population revealed mutations in tumors with metastasis to regional lymph nodes (98). No mutations have been identified in any of the node-negative tumors. Trans- fection studies showed loss of apoptotic function in these mutants.

TRAIL-R1 mutation has been analyzed in several tumors, including breast cancer (98) and non-Hodgkin’s lymphoma (100). A mutational analysis of the death domain of TRAIL-R1 in non-Hodgkin’s lymphoma revealed mutations in two tumors (1.7%) (100).

The same research group also detected TRAIL-R1 mutations in 8.8% of tumors with metastasis to regional lymph nodes (98). In these two studies, analysis of TRAIL-R1 mutation was performed only in the death domain. Seitz et al. analyzed the death domain of TRAIL-R1 for the detection of mutations in breast cancers, but they did not detect any (103). They also screened for TRAIL-R2 and TRAIL-R4 mutations in the same series of breast cancers, but they did not find any (103). Next, they analyzed individuals from breast cancer families for the detection of TRAIL-R2 germline mutations (103). One alteration has been found in the Kozak consensus motif at position –4 with respect to the translation initiation AUG. Pai et al. (96) also found a germline TRAIL-R2 mutation in a head/neck cancer, and observed loss of growth-suppressive function in the cell lines overexpressed with the tumor-derived TRAIL-R2 mutant. In summary, it appears that TRAIL receptor mutation occurs occasionally in sporadic tumors as well as in familial tumors, and that it may contribute to cancer development and progression.

Death Receptors as Tumor Suppressor Genes

The development of human tumors results from clonal expansion of genetically modi-

fied cells that have acquired selective growth advantage through accumulated alterations

of proto-oncogenes and tumor suppressor genes (23). Inactivation of tumor suppressor

gene is frequently accompanied by loss of portions of the chromosome on which the

Table 2

Summary of Mutations of TRAIL-R1 and TRAIL-R2 Genes in Cancers

Gene Type of human cancers Frequency References TRAIL-R1 Non-Hodgkin’s lymphoma 2/117 (1.7%) 100

TRAIL-R1 Breast cancer 3/57 (5.3%) 98

TRAIL-R1 Breast cancer 0/115 (0%) 103

TRAIL-R2 Head/neck cancer 2/60 (3.3%) 96

TRAIL-R2 NSCLC 11/104 (10.6%) 97

TRAIL-R2 Non-Hodgkin’s lymphoma 6/117 (5.1%) 100

TRAIL-R2 Breast cancer 4/57 (7.0%) 98

TRAIL-R2 Breast cancer 0/115 (0%) 103

TRAIL-R2 Gastric cancer 3/43 (7.0%) 99 TRAIL-R2 Hepatocellular carcinoma 1/100 (1%) 101

TRAIL-R2 Colon cancer 0/41 (0%) 102

TRAIL-R4 Breast cancer 0/115 (0%) 103

Abbreviation: NSCLC, non-small-cell lung cancer.

tumor suppressor gene resides (23). Deletions and rearrangements of chromosome 10q24, where the Fas gene resides, have been reported in many types of human tumors, raising the possibility of the presence of tumor suppressor genes in this region (106). TRAIL-R1, -R2, -R3 and -R4 genes are mapped to chromosome 8p21-22 (93), suggesting that all of these genes have arisen by tandem duplication. Such duplications may reflect the relative instability of the chromosomal region. Allelic losses of chromosome 8p21-22 have been reported as a frequent event in several cancers (106). These data strongly indicate that chromosome 8p21-22 may harbor one or more tumor suppressor genes, and suggest that TRAIL-R2 gene might be one of the candidate tumor suppressor genes in this region.

Because inaction of death receptors might result in deficient apoptotic signaling, TRAIL- receptor genes and the Fas gene are candidate tumor suppressor genes in these regions.

Many of the studies on mutational analysis of death receptors analyzed the allelic status of these genes, as well as their mutational status. Using the intragenic polymor- phisms in the Fas gene, Lee et al. and Shin et al. have found loss of heterozygosity (LOH) with a range from 27 to 35% according to the tumor type (76–78). The LOH in the tumors with Fas mutation was detected in 38 to 100% according to the tumor type (76–78).

Regarding TRAIL-receptor genes, LOH was observed in from 23 to 65%, and LOH in the

tumors with TRAIL-receptor mutations was detected in 75 to 90% (97,98,100). Higher

incidence of the LOHs of Fas and TRAIL receptor genes in the tumors with the mutations

indicates that these genes might be tumor suppressor genes in these loci. Also, the pres-

ence of LOH of Fas and TRAIL receptor genes in the tumors without the mutations

suggests that there may be other tumor suppressor genes, such as DMBT1 in 10q25-26,

in these loci (107). The authenticity of a tumor suppressor gene is most clearly established

by the identification of inactivating germline mutations that segregate with tumor predis-

position, coupled with the identification of somatic mutations inactivating the wild-type

allele in cancers arising from a germline mutation. At the current stage, the data may not

be sufficient to call the death receptors tumor suppressor genes; the death receptor genes

should be considered most appropriately as candidate tumor suppressor genes until additional data are available.

CONCLUSIONS

Mutations in apoptosis genes contribute to the pathogenesis of human tumors. The examples of mutated death receptor genes result in reduced apoptosis. However, muta- tion is only one mechanism of apoptosis dysregulation. Alterations in the expression of apoptosis genes in tumor cells, by known or still unknown mechanisms, may also be involved in the pathogenesis of diseases. The identification of alterations in death recep- tor genes contributes to the understanding of the function of the molecules involved, offers novel molecular tools for diagnosis, and reveals potential targets for therapeutic intervention.

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