34 Apoptosis in Melanoma

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Apoptosis in Melanoma

Approaches to Therapy

Heike Röckmann and Dirk Schadendorf

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Summary

Apoptosis deficiency seems to be involved in the high resistance of melanoma to therapeutic treat- ment. This has come into focus because the cytotoxic effects of chemotherapeutic agents via apoptosis are known. Extensive investigations have been made analyzing the role of alterations in the apoptotic pathway in melanoma. The molecular changes affect antiapoptotic, as well as proapoptotic, processes and survival signals and involve various molecules. These mechanisms are also discussed in light of their use in further therapeutical strategies. Actually, a number of these findings have already been employed to test their therapeutical applicability in melanoma treatment. Furthermore, two concepts have been translated from the cell system via animal models into clinical trials.

Key Words: Melanoma; apoptosis; apoptosis deficiency; death receptors; caspases; Bcl-2-family; p53.

INTRODUCTION

Most chemotherapeutic drugs act through induction of apoptosis (programmed cell death). In 1972, Kerr et al. described an experimentally induced killing of tumor cells that involved a coordinated cell disintegration following typical morphological changes, coining the term apoptosis (1). The cytotoxic effect of chemotherapeutic agents appears mainly contributed by apoptosis (2,3). The weak response of metastatic melanoma to anticancer agents gives rise to the hypothesis that the chemoresistance of this malig- nancy is caused by raising its apoptotic threshold (2). Inactivation of apoptosis is a

“hallmark of cancer,” an obligate ritual in the malignant transformation of benign cells (4). As a result, these cells enhance their chances of survival and increase their resistance to chemotherapy (3). Indeed, Staunton et al. (5) demonstrated a constitutive low level of spontaneous apoptosis in melanoma cells compared with other malignant cell types. An intensive search for cell death factors altered in melanoma has been made. It has been

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From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ

shown that, indeed, various molecular changes in cell-death control in melanoma are present, and three types can be distinguished: activation of antiapoptotic processes, inactivation of proapoptotic effectors, and reinforcement of survival signals.

PROGRAMMED CELL-DEATH PATHWAYS

Apoptosis (programmed cell death) represents a complex genetic program consisting of several pathways that are summarized in Fig. 1. Tremendous efforts have been made to discover and describe the molecular mechanisms of apoptosis, which are discussed elsewhere in detail (6). Briefly, depending on cell type and stimulus, a complex net of sensor and regulator proteins is activated and balanced and there is no unique linear or defined pathway. However, to simplify, two main well-characterized caspase-activating cascades that regulate apoptosis are currently known.

One cascade is triggered from the cell surface by death receptors and the other is initiated by changes of mitochondrial membrane integrity (7). Oligomerization of sur- face receptors is followed by recruitment of adapter molecules, such as Fas-associated protein with death domain (FADD) and the initiator caspases-8 and -10 (8) into the death-inducing signaling complex (DISC). The subsequent autocatalytic cleavage of procaspase-8 or -10 is followed by activation of effector caspases (e.g., caspase-3) (9) and induction of specific endonucleases, resulting in DNA fragmentation (10,11). These two pathways converge with the activation of effector caspases and induction of specific endonucleases, resulting in DNA fragmentation and cleavage of nuclear proteins essen- tial for nuclear and cellular structure, DNA-repair, and DNA-replication (12,13).

The family of death receptors include CD95 (Fas/APO-1), tumor necrosis factor (TNF)-R1, TNF-receptor apoptosis-inducing ligand receptor (TRAIL-R)1 and TRAIL-R2, DR3 (death receptor 3), and DR6 (reviewed in Locksley et al., ref. 14). Among them, the CD95 receptor, TRAIL-R1 and TRAIL-R2 are the most efficient mediators of apoptosis.

Several studies suggest that death receptor–ligand interaction is involved in tumor sen- sitivity toward chemotherapeutic drugs (15–17). The extrinsic pathway is regulated on multiple levels, whereas the death-inducing signaling complex can also recruit negative and positive regulators of caspase-8 (18,19). Furthermore, in type II cells, the initial activation of caspase-8 is not sufficient. Here, caspase-8 cleaves the Bcl-2 family mem- ber, BID, which translocates to the mitochondrial membrane and activates the intrinsic pathway (20). By this mechanism, the death receptor and the mitochondrial pathway is connected.

The intrinsic pathway involves mitochondrial release of cytochrome-c, which binds apoptotic protease activating factor (Apaf)-1, and, in the presence of adenosine triphos- phate (ATP), coordinates a series of conformational changes that allow the oligomeriza- tion of Apaf-1 into a ring-like complex, referred to as the “apoptosome” (21). The apoptosome binds and activates caspase-9 into the complex (9,22,23), which, in turn, recruits and activates effector caspases (e.g., caspase-3). These are considered as execu- tors of apoptosis.

There are various additional points of control that can modulate apoptosis after

cytochrome-c release. The family of inhibitors of apoptosis (IAP), containing X-chro-

mosome-linked IAP (XIAP), neuronal IAP (NIAP), melanoma (ML)-IAP, and survivin,

can interfere with the formation of the apoptosome and activation of the downstream

caspases (Figs. 1 and 2). IAPs are inhibited by other proapoptotic factors released by the

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Fig. 1. The apoptotic pathway. In this simplified scheme, the extrinsic pathway is initiated by oligomerization of death receptors after binding of the ligand and recruitment of initiator caspases (caspase-8 and caspase-10). The intrinsic pathway involves the translocation of proapoptotic Bcl-2 family members to mitochondria and release of cytochrome-c into the cytosol, oligomerization of apoptotic protease activating factor-1 (Apaf-1) in a complex with caspase-9, and the subsequent activation of caspase-3. Receptor-initiated signals can be transduced through the mitochondrial pathway, for example, through cleavage of Bid (for details see page 608).

Fig. 2. Caspase regulatory proteins. The inhibitor of apoptosis (IAP) gene family encodes a group of structurally related proteins (e.g., IAP-1, IAP-2, X-chromosome-linked IAP, survivin, and melanoma IAP) that are able to suppress caspase function. They are thought to directly inhibit certain caspases and are controlled by further inhibitory proteins of IAP, Smac (second mitochon- drial activator of caspases)/Diablo (direct IAP-binding protein with low pI).

mitochondria, the second mitochondrial activator of caspases (Smac)/Diablo and Omi/

OtrA (24). Furthermore, p53 can modulate the expression of apoptotic effectors, and heat-shock proteins can also regulate the formation of the apoptosome.

APOPTOSIS DEFICIENCY AND MELANOMA

Wild-type p53 promotes cell cycle arrest and apoptosis in response to DNA-damaging drugs most likely by controlling transcriptional regulation of target genes, such as Bcl-2 and Bax (25,26) (Fig. 1), and acts, therefore, as a tumor suppressor. p53 mutations are common genetic alterations in human cancer. Melanoma cell lines expressing wild-type p53 exhibit a higher response to anticancer agents than melanoma with mutant p53 (27).

Mutation of p53 was associated with metastatic potential (28). Abnormal phosphoryla- tion of p53 by Ck2 kinase was associated with melanoma resistance to radiotherapy (29).

A phase I dose-escalation study of single intratumoral injection of a replication-defec- tive adenoviral expression vector containing p53 was performed by Dummer et al. in patients with metastatic melanoma. In this study, the therapeutic approach was proven safe, feasible, and biologically effective (30).

Another very common specific gene defect in human melanoma is mutation of acti- vated ras (31). Ras proteins are regulators of multiple signal pathways that control cell growth, differentiation, and apoptosis. The assumed mechanism by which the activated ras oncogene is involved in drug resistance is an upregulation of Bcl-2 expression (32).

A decreased CD95 surface expression in ras transfectants was demonstrated in mela-

noma cell lines (33). Indeed, overexpression of activated mutants of N-ras increase cisplatin resistance in melanoma cells and in a SCID (severe combined immunodeficient) mouse model (34). Improved understanding of the molecular mechanisms of Ras processing and membrane targeting provided important tools for the development of molecules that inhibit the association of Ras with the inner cell membrane. Such molecules include inhibitors of farnesyltransferase, prenyl-CAAX protease, methyl- transferase, and inhibitors, such as trans-farnesyl thiosalicylic acid (Fig. 3). Farnesyl thiosalicylic acid, a Ras antagonist, suppresses melanoma growth in vitro and in vivo through a combination of cytostatic and proapoptotic effects (35). Furthermore, a recent study evaluated the effect of farnesyl thiosalicylic acid treatment in combination with dacarbazine on established human melanoma xenografts grown in mice. A significant tumor growth paralleled by an acceptable toxicity profile was shown in a mouse system (36) (Fig. 3).

B-Raf, a Ras effector molecule, was found to be mutated in 66% of human melanomas (37). Previous studies indicated that wild-type B-Raf may inhibit apoptosis downstream of cytochrome-c release by activating nuclear factor (NF)-NB (38).

Extensive efforts have been made in analyzing the involvement of death receptors in melanoma cell death. Downregulation, loss, and mutation of CD95/Fas receptor in melanoma have been described, resulting in triggering resistance to CD95L/Fas ligand (39,40). A respective correlation to clinical response was demonstrated by Mouawad et al. (41). They reported that melanoma patients with low clinical response to various drugs (cisplatin, recombinant interleukin-2, and interferon-D) exhibited a significant increase of soluble sCD95 and sCD95L in the plasma after drug treatment, whereas in the plasma of responders, no changes in sCD95 or sCD95L levels were observed.

Various studies demonstrated that, in contrast to the CD95 receptor, TRAIL-R2 is widely expressed in melanoma. Furthermore, melanoma cell lines were shown to undergo apoptosis after exposure with recombinant TRAIL very readily, whereas melanocytes did not demonstrate such a high TRAIL sensitivity (42). This study also showed that TRAIL-R expression is not predictive of sensitivity. There are some known agents available to upregulate TRAIL-R expression, such as cisplatin, betulinic acid, CD437 retinoid, and TNF-D (overviewed in Hersey et al., ref. 43). Furthermore, Griffith et al.

demonstrated increased apoptosis in melanoma cells after TRAIL-expressing adenovi- ral infection as a possible therapeutic approach (44). Sensitivity toward death-receptor signaling in melanoma cells was also increased using a soluble NF-NB inhibitor (45).

Bcl-2 appears to influence response to chemotherapy by inhibiting apoptosis induc- tion by many cytostatic drugs, including alkylating agents, topoisomerase inhibitors, antimetabolites, and others. The high expression of Bcl-2 in human melanoma and other tumors has been correlated with resistance to chemotherapy and decreased survival (46).

Furthermore, Raisova et al. showed that a low Bax:Bcl-2 ratio might be characteristic for

drug-resistant melanoma cells (47). Additionally, the oncogenic potential of Bcl-2 seems

to be involved in melanoma angiogenesis through vascular endothelial growth factor

(VEGF) mRNA stabilization and hypoxia-inducible factor (HIF)-mediated transcrip-

tional activity (48). C-Myc low-expressing compared with high-expressing melanoma

lines demonstrated an increased cisplatin sensitivity (49). In a human melanoma

xenograft in SCID mouse models, the improvement of chemosensitivity of dacarbazine

in combination with bcl-2 oligo–antisense (Augmerosen, Gentasense, G-3139) could be

demonstrated (50). Furthermore, Heere-Ress et al. demonstrated recently how antisense

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Fig. 3. Ras proteins carry C-terminal lipid modifications, through which they associate with cellular membranes and which are essential for their transforming activity. After initial cytosolic farnesylation, Ras proteins translocate to intercellular membranes. Here they undergo successive removal of the terminal –AXX residues, methylation, and further modification by S-acetylation. (Reproduced with permission from ref. 86.)

oligonucleotides reduced Bcl-X

expression and enhanced the chemosensitivity of melanoma cell lines to cisplatin (51). A recent study showed that the simultaneous downregulation of Bcl-2 and Bcl-X

expression and induction of apoptosis by antisense oligonucleotides in melanoma cells of different clinical stage may provide an additional clinical benefit (52).

Antisense oligonucleotides are short, single-stranded nucleotides that bind comple- mentary to their respective target mRNA, thus, inhibiting translation and initiating deg- radation of the targeted mRNA. Antisense oligonucleotides directed against specific genes, such as Bcl-2, associated with neoplastic progression are currently being evalu- ated in several clinical trials. Therefore, mouse systems (50) have been translated into clinical phase I/IIa studies, in which 6 of 14 treated patients showed an antitumor response associated with low Bcl-2 levels and increased apoptosis in melanoma biopsies (82).

Recently, a worldwide phase III study recruited 770 patients with metastatic melanoma to compare the efficiency of the standard chemotherapy dacarbazine alone and dacarbazine plus Bcl-2 antisense. The final analysis is currently being performed.

Antisense molecules for Bcl-X

and c-Myc are available, and clinical application can be expected.

Caspase proteases are initially synthesized as precursor proteins with little or no enzymatic activity. The cleaved proteins are the primary apoptotic executers, which act in a cascade ultimately leading to the cleavage of substrates that produce the character- istic features of apoptosis. Caspase proteases are cleaved by upstream molecules (such as caspases, FADD ([Fas-associated protein with death domain], or Apaf-1) and are further controlled by a variety of proteins that directly interact with proteases: IAPs and FLICE (FADD-like interleukin-1-E-converting enzyme inhibitory proteins (FLIPs).

Caspase inhibition is achieved by proteins of the IAP family (Fig. 2), which are struc-

turally related by their baculovirus IAP repeat domain. In melanoma, two members of

the IAP family (survivin and ML-IAP/Livin) and FLIP have been associated with tumor

progression. One of the best-characterized members in melanoma is survivin, which

exerts its effect by directly inhibiting caspases and was found in melanoma only (cell

lines, metastatic lesions, and invasive melanoma) compared with melanocytes (54). In

contrast to other apoptotic inhibitors, survivin expression is cell-cycle dependent. In

early mitosis, survivin, linked to the microtubules of the mitotic spindle, inhibits the

activation of caspase 3 (55). A recent study by Gradilone et al. showed a significant

correlation between survivin expression and outcome of sentinel lymph node-positive

melanoma patients (56). Antisense oligonucleotides directed against this molecule were

shown to induce spontaneous apoptosis in melanoma in vitro (54). This was proven by

the same group by a phosphorylation-defective Thr34 baculoviral IAP repeat (BIR)

mutant (the relevant domain in IAPs), which prevented tumor formation and slowed the

growth of established tumors in a melanoma xenograft model (57), suggesting that

therapeutic targeting of survivin might also be beneficial in patients with recurrent or

metastatic melanoma. An additional IAP molecule has been discovered in melanoma by

two other groups. ML-IAP/livin is expressed in developed tissues, including melano-

cytes, but is predominantly overexpressed in melanoma (58,59). ML-IAP acts directly on

the mitochondrial pathway by directly inhibiting caspase-9, caspase-3, and the

proapoptotic factor, Smac/Diablo (60). Furthermore, this molecule was identified as a

possible target for immune-mediated tumor destruction in melanoma (58,61). Further

studies found that transfection of an antisense construct against livin could trigger

apoptosis specifically in melanoma cell lines expressing livin mRNA. This was associ- ated with an increase in DNA fragmentation and in DEVD-like caspase activity (58).

A mitochondria-derived activator of caspase (Smac/Diablo) construct was shown in vitro to sensitize melanoma to TRAIL-induced apoptosis as a very selective tool inhib- iting IAP (62). There are strong reasons why caspase inhibitors can be considered char- acteristic of the drug-resistant phenotype in melanoma cells; caspase inhibitors enhance the ability of the melanoma cells to resist apoptosis and present a rewarding therapeutic target.

FLIP was shown to inhibit caspase-8 and, therefore, to inhibit apoptosis induced by receptor-associated cytokines (e.g., TNF-D, CD95L/Fas ligand, TRAIL). FLIP was shown to be overexpressed in melanoma and associated with resistance (63). Neverthe- less, endogenous levels of FLIP do not necessarily correlate with drug response in melanoma patients (40,64).

Apaf-1 represents an essential downstream molecule to induce apoptosis. Soengas et al. reported cases of drug-resistant malignant melanomas in which Apaf-1 expression was impaired (65). Here, cytostatic drugs induced cytochrome-c release, but failed to induce caspase-9 activation. This could be reversed by reactivating Apaf-1 with the demethylating agent, 5-aza-2-deoxycytidine, in vitro.

Various studies suggest that increased antiapoptotic, heat-shock protein (HsP) ex- pression, such as HsP70 and HsP90, may be related to drug resistance (66,67). HsP70 antagonizes the caspase-independent apoptotic-inducing factor (AIF) (68). Hsp70 may also interfere with Apaf-1, by inhibiting the oligomerization and/or by blocking the recruitment of caspase-9 to the apoptosome (69). HsP70 was shown to be upregulated in primary melanoma and cell lines (70,71), and an association with resistance to ultra- violet radiation was seen (72). Clinical trials using 17-AAG for Hsp90 inhibition in advanced cancer are currently ongoing (73).

Neef et al. identified the human pleckstin-homology-like domain family A, member 1 (PHLDA1)/TDAG51 gene, which was shown to be downregulated in metastatic mela- noma and associated with apoptosis resistance (74). PHLDA1 expression was associated with reduced cell growth, cloning efficiency, and colony formation, and increased basal apoptosis. Chemosensitivity to doxorubicin and camptothecin was enhanced. Moreover, recently, a group of human melanoma cell populations that are heterogeneously susceptible to C2-ceramide-mediated apoptosis was identified (75). Studies with these melanoma cells revealed a correlation between ceramide-mediated apoptosis and D-NMAPPD, a ceramide analog, confirming the effect of this inhibitor on ceramide signaling in human melanoma cells. These findings suggest ceramidase inhibitors as a potential new therapeutical class of antiproliferative and cytostatic drugs.

NF-NB is a transcription factor linked at the crossroads of life and death (Fig. 4). It functions as a modulator of inflammation, angiogenesis, differentiation, cell cycle, ad- hesion, migration, and survival (76). NF-NB has been recognized as an possible potential target in cancer treatment (77). In melanoma cells, NF-NB can be affected by upregulation of the NF-NB subunit, p50 or Rel A, or downregulation of the NF-NB inhibitor, INB (78).

Subsequently, all target molecules downstream of NF-NB regulation are affected.

Therefore antiapoptotic factors, such as c-myc and TNF receptor-associated factor

(TRAF)-2, are frequently upregulated in melanoma. In melanoma, gene-transfer

approaches targeting NF- NB disruption have been used, inactivating the NF-NB subunit,

Rel A (79),and overexpressing I NB ( 80).

Table 1 Summary of Therapeutic Approaches in Melanoma From In Vitro Studies to Clinical Trialsa Target and experimental settingReference Clinical trials bcl-2

Antisense oligonucleotides combined (plus dacarbazine) Jansen et al., 2000

Intratumoral injection Dummer et al., 2000

Overexpression in SCID-mouse model Jansen et al., 1997

Farnesyl thiosalicylic acid combined with dacarbazine in an human mela- noma xenograft mouse model Halaschek-Wiener et al., 2003

Smalley et al., 2002

Antisense oligonucleotides Jansen et al., 1998

Adenoviral vector Cirielli et al., 1995

Transient transfection of C85A mutant Grossman et al., 2001

Adenoviral vector (plus cyclin D) Sauter et al., 2002

Antisense oligonucleotides Heere-Ress et al., 2002

Simultaneous downregulation by antisense oligonucleotides Olie et al., 2002

Antisense oligonucleotides Grossman et al., 1999

Conditional expression T

A mutant (plus cisplatin) Olie et al., 2002

Adenoviral vector Cirielli et al., 1995

Ribozyme Ohta et al., 1996

Antisense Jansen et al., 1997

Retrovirus, 5aza2dc (plus adriamycin) Soengas et al., 2001

Antisense oligonucleotide Kasof et al., 2001

Antisense oligonucleotide McNulty et al., 2001

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Fig. 4. The nuclear factor (NF)-NB pathway. In the context of cell death control, NF-NB modu- lates the expression of survival factors.

Apoptosis deficiency appears to be strongly associated with the specific drug-resis- tant phenotype (81). In this study, cisplatin resistance has been associated with reduced caspase-9 activity and cytochrome-c release paralleled by normal DNA fragmentation, whereas no apoptotic events could be induced in etoposide-resistant melanoma cells (81).

Recently, tremendous efforts have been made in identifying new strategies and drug targets inhibiting apoptosis to support melanoma treatment (Table 1). Various investi- gations in mouse systems could prove the benefit of strategies interfering with apoptotic mechanisms in melanoma. Nevertheless, until now, these strategies could only be trans- ferred in a few clinical trails.

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