notch and noxa-related pathways in melanoma cells

Notch and NOXA-Related Pathways in Melanoma Cells

Brian J. Nickoloff,� Mary J. C. Hendrix,w Pamela M. Pollock,z Jeffrey M. Trent,z Lucio Miele,yand Jian-Zhong Qin��Department of Pathology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University, Chicago, Illinois, USA; wChildren’s Memorial Institute forEducation and Research, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA; zTranslational Genomics Research Institute, Phoenix,Arizona, USA; yDepartment of Biopharmaceutical Sciences and Cancer Center, University of Illinois at Chicago, Chicago, Illinois, USA

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch

expression are associated with various malignancies in which Notch may function as an oncogene or less com-

monly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing

stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the

Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for

selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines

are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-

secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can

block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability

in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without

subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma

cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance

of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin.

Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination

with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective

killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to

the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity

(e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies

in cutaneous oncology.

Key words: apoptosis/gamma secretase inhibitor/melanocytes/melanoma/Notch/NOXAJ Investig Dermatol Symp Proc 10:95 –104, 2005

The Notch signaling pathway plays a key role in cell fatedecisions during development, and also regulates cell-to-cell interactions involving stem cell behavior, cell prolifera-tion, differentiation, and death (Artavanis-Tsakonas et al,1999). Notch was originally described by Thomas HuntMorgan in 1917 because Drosophila-bearing mutations inNotch had a distinctive phenotype in which the structure ofthe wing blades was irregular in shape (Morgan, 1917). DrMorgan received the 1933 Nobel Prize for his discoveriesconcerning the role played by the chromosome in heredity.Note how, compared with the smooth edge of normalwings, hemizygous Notch deficiency imparts a serrated-likeappearance to the edge of the wing (Fig 1A). The actualgene responsible for producing the distinctive phenotypewas cloned in 1985, and it was recognized that this se-quence encoded a cell surface receptor (Wharton et al,1985). Focusing on Notch by investigative skin biologists isnot a coincidence, as homozygous Notch deficiency (Notchnull genotype) characteristically causes a ‘‘neurogenic phe-

notype’’ in which the number of neurons in Drosophila wasincreased secondary to a decrease in epidermal cells. Thisis due to unrestrained differentiation of neuroectodermal cellstoward the neural phenotype (Artavanis-Tsakonas et al, 1999).Thus, Notch signaling has continued to attract interest by cu-taneous biologists since it first received notoriety in 1937, whenPoulson initially described this neurogenic cell fate switch in-volving Drosophila’s ectodermal cuticle (Poulson, 1937).

Besides its role as a highly evolutionarily conservedpathway regulating epithelial development, Notch signalinghas also been implicated beyond the epidermis to includehair follicles and feathers (Kopan and Weintraub, 1993; Po-well et al, 1998; Viallet et al, 1998). In mammals, the Notchfamily consists of four different Notch receptors and severalNotch ligands (Lai, 2004). The Notch receptors are desig-nated as Notch-1 through Notch-4, and the different ligandsinclude Jagged-1, Jagged-2, and Delta-like (DLL1, DLL3,DLL4—see Fig 1B). The four known Notch receptors differby the number of epidermal growth factor-like (EGF-like)repeats in the extracellular domain, as well as by the lengthof the intracellular domain. The cell fates regulated by Notchsignaling include renewal of stem cells, and control of pro-liferation, differentiation, and apoptosis (Fig 1B).Abbreviation: GSI, gamma-secretase inhibitor

Copyright r 2005 by The Society for Investigative Dermatology, Inc.

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Figure 1C presents an overview of Notch signaling with asimplified portrayal of intracellular biochemical events thatparticipate in Notch signaling (Hansson et al, 2004). Theaffinity of Notch ligands for their cognate receptors is mod-ulated by glycosylation and fucosylation of the extracellularsubunits (Nickoloff et al, 2003). Regulated proteolysis playsa major role in Notch signaling. Notch receptors are pro-duced as large single-chain precursors, which are cleavedby serine proteases related to furin in the trans-Golgi togenerate calcium-stabilized heterodimeric receptors con-sisting of an extracellular subunit and a trans-membranesubunit. Once a cell expressing a Notch receptor is stim-ulated by an adjacent cell via a Notch ligand on the cellsurface, the extracellular subunit is trans-endocytosed inthe ligand-expressing cell. The remaining transmembranesubunit undergoes two consecutive enzymatically mediatedcleavages. The first activating cleavage event is mediatedby a metalloprotease-dependent TNF-a converting enzyme(TACE) belonging to the disintegrin and metalloproteasefamily (Mumm et al, 2000) that cleaves the extracellularportion of the transmembrane subunit (Brou et al, 2000).This step is rapidly followed by a second cleavage in thetransmembrane domain requiring a g-secretase/presenilin-1complex (De Strooper et al, 1999; Okochi et al, 2002) togenerate an intracellular truncated version of the receptordesignated as NIC (Fig 1). Very recently, it was discoveredthat g-secretase cleavage requires and is preceded bymono-ubiquitination and endocytosis (Gupta-Rossi et al,2004). Thus, the rate of cleavage of Notch-1 is finely mod-ulated by multiple post-translational modifications and cel-lular compartmentalization events.

The truncated or intracellular form of the Notch-1 recep-tor (e.g., NIC-1) can then translocate to the nucleus, where itforms a multimeric complex with a highly conserved tran-scription factor (CBF1), and other transcriptional co-activa-tors that influence the intensity and duration of Notchsignals (Jarriault et al, 1995; Lai, 2002). CBF-1 exists as arepressor in the absence of Notch-1, forming a complex

with adaptor protein SKIP and one or more co-repressors,including SMRT (signaling mediator of retinoid and thyroidreceptor), N-CoR and/or CIR (Nickoloff et al, 2003), andhistone deacetylase 1 (HDAC1). Notch-1 dissociates theco-repressors and HDAC1 from CBF-1, instead recruitingco-activator MAML1 (Mastermind in Drosophila), and hi-stone acetyltransferases P/CAF, GCN5, and/or p300. Theultimate result is activation of transcription at promoterscontaining CBF-1-responsive elements. Since many of thegenes regulated in this manner are themselves transcriptionfactors, this process can ultimately result in induction orrepression of numerous genes. Thus, the ultimate outcomefor Notch signaling is to either stimulate or repress varioustarget genes (Lai, 2004). Three of the most commonly stud-ied target genes for Notch signaling include Hairy-Enhancerof split (HES), and HES-related proteins (HERP/HEY) as wellas p21WAF1, although cross-talk between Notch and theStat3 signaling pathway has been demonstrated as well(Maier and Gessler, 2000; Rangarajan et al, 2001b; Kawa-mata et al, 2002; Kamakura et al, 2004).

In addition to this ‘‘classical’’ pathway of Notch signaling,other pathways are beginning to emerge. The role of Notch-associated protein Deltex remains poorly understood. Del-tex, of which there are several mammalian isoforms, bindsto the ankyrin repeats of Notch and has been proposed toact as a downstream Notch mediator involved in JNK in-hibition, as a nuclear mediator of Notch signaling, and as afeedback inhibitor of Notch signaling (Nickoloff et al, 2003).More recently, convincing evidence has been presentedthat Notch-1 acts as a kinase activation scaffold, forming acomplex with Src-family protein tyrosine kinase Lck andphosphoinositide-3-kinase (PI3K). This process leads toactivation of the PI3K-AKT pathway (Sade et al, 2004) andmay be responsible for survival signals mediated by Notchactivation. There is considerable evidence of cross-talk be-tween the Notch and NF-kB pathways, but the moleculardetails remain unclear. Transcriptional induction of all fiveNF-kB subunits (Cheng et al, 2001) as well as more rapid

Figure 1Historical perspective and overview ofNotch signaling. (A) Phenotypic appear-ance of Drosophila wings in normal, wildtype as well as in flies bearing hem-izygous Notch mutation. Note the serrat-ed, rather than smooth contour, on thewing edges in the mutant fly wings as firstdescribed by Thomas Morgan in 1917. (B)Diverse roles for Notch signaling that in-fluence various cell fate decisions includ-ing four different Notch receptors andmultiple Notch ligands. (C) Simplifiedoverview of Notch signaling pathway, inwhich the activation of the Notch receptorin the plasma membrane results in regu-lated proteolysis involving a g-secretase/presenilin complex to generate a truncat-ed intracellular form (e.g., NIC) that trans-locates to the nucleus and functions as atranscription factor to influence gene ex-pression including HES and other targets(see text for further explanation).

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activation of NF-kB (Nickoloff et al, 2002; Palaga et al, 2003;Jang et al, 2004) have been described.

Given the multitude of cell fate-regulatory processesmodulated by Notch signaling and the number of cell fatedecisions and post-developmental influences that Notchsignaling mediates in a diverse array of cell types, it is notsurprising that Notch plays a complex role in pathophysi-ology, and that multiple positive and negative regulatorycircuits tightly control the activity of Notch receptors. In thenext section, we review the role of Notch signaling in aphysiological context (i.e., epidermal keratinocyte differen-tiation), as well as in pathology, with special emphasis onderegulation of Notch signaling in various neoplastic con-ditions (Axelson, 2004).

Notch Signaling as a Proto-Oncogene orTumor Suppressor

Over the past two decades, many processes have beenidentified in which Notch signaling is operative in normalcells to maintain tissue homeostasis. Furthermore, with re-gard to tumorigenesis, Notch may play a role as either atumor suppressor or as an oncogene (Radtke and Raj,2003). In this section, we briefly review the role of Notchsignaling in the differentiation of epidermal keratinocytes(Fig 2A), as well as the evidence that persistent activation ofNotch signaling can produce malignancies such as human T

cell acute lymphoblastic leukemias (Fig 2B). In general,Notch signaling tends to delay and restrict differentiationpathways, thereby maintaining cells in an undifferentiatedstate (Varnum-Finney et al, 1998). This influence on cellfates contributes to stem cell renewal for several tissue sitesincluding the central nervous system, and the bone marrow(Hansson et al, 2004).

An important exception to this general rule is the epi-dermal keratinocyte, because we and others have clearlydemonstrated that activation of Notch signaling promotesepidermal keratinocyte differentiation (Lowell et al, 2000;Rangarajan et al, 2001b; Nickoloff et al, 2002). Character-izing expression patterns for Notch-1 in human tissues hasrevealed that in the skin, Notch-1 was expressed by hairfollicles, sebaceous glands, and sweat glands (Baldi et al,2004). In the epidermis, Notch-1 appears to first becomeapparent in the suprabasal layers corresponding to the cellsundergoing initial differentiation (Nickoloff et al, 2002). Allfour Notch receptors are expressed in the epidermis, withNotch-2 being more basal than Notch-1, and Notch-3 and -4 being more superficial than Notch-1. Notch ligand Delta-1is expressed in ‘‘epidermal stem cells’’ (Lowell et al, 2000),and Jagged-1 is expressed throughout non-cornified layers(Nickoloff et al, 2002). Expression of Notch receptors andligands abruptly disappears in cornified layers. Rangarajanet al (2001b) showed that in murine keratinocytes, Notch-1induces cell cycle arrest and early differentiation, whereasinhibiting late differentiation. Using a submerged epidermal

Figure 2Opposite roles of Notch-1 receptor signaling in keratinocytes as a tumor suppressor, or in T-ALL cells as a proto-oncogene. (A) As Notch-1receptor activation triggers growth arrest and differentiation in normal epidermal keratinocytes, it can function as a tumor suppressor. Presumablyhighly regulated interaction between Jagged-1-expressing keratinocytes can deliver a physiological stimulus induced by Notch-1 receptors totrigger differentiation in healthy skin. (B) Either a chromosomal translocation producing enhanced levels of activated Notch-1 receptors, or pointmutations in the Notch-1 receptor that enhance g-secretase-mediated activation of Notch-1 receptors, generate sustained Notch signaling cul-minating in tumorigenesis. (T cell acute lymphoblastic leukemia, T-ALL). Although a chromosomal translocation is rare, occurring in approximately10% of patients with T-ALL, the point mutations are observed in over 50% of T-ALL patients (Weng et al, 2004).

PATHWAYS TO DISCOVER NOVEL ANTI-CANCER AGENTS IN DERMATOLOGY 9710 : 2 NOVEMBER 2005

equivalent model system, we demonstrated that activationof Notch signaling (very likely, all four Notch receptors insequence) is necessary and sufficient for triggering terminaldifferentiation, including cornification (Nickoloff et al, 2002).

Thus, in the skin, Notch-1 signaling could function inkeratinocytes as a tumor suppressor because of its ability totrigger induction of differentiation (Fig 2A). The precise rolesof the other three Notch receptors in epidermopoiesis re-main unknown and are presently under investigation. Addi-tional and more direct evidence that Notch-1 can functionas a tumor suppressor is derived from in vivo studies inwhich the long-term consequences of Notch-1 deficiencywere characterized using a chemical carcinogenesis proto-col (Nicolas et al, 2003). Using a tissue-specific, gene-tar-geting approach, Notch-1 �/� mice were found to haveenhanced b-catenin signaling and increased frequency ofskin tumors resembling basal cell carcinoma and squamouscell carcinoma. This is consistent with the anti-proliferativerole of Notch-1 in murine keratinocytes. Whether Notch-1 isa bona-fide tumor suppressor in the epidermis, i.e., whetherfunctional inactivation of Notch-1 is required for BCC orSCC progression in humans, is not clear. Selective loss ofNotch-1 in advanced cervical cancer and cervical cancercell lines had been reported (Talora et al, 2002), but others(Lathion et al, 2003) have been unable to reproduce thesefindings, and we (Weijzen et al, 2003) have shown that notonly is Notch-1 expressed in advanced cervical cancer cellsand clinical specimens, but it is necessary for survival ofcervical cancer cells through its anti-apoptotic activity.

Thus, it appears that the role of Notch-1 in keratinocytesis not so simple (Kadesch, 2004), as one group has reportedthat forced overexpression of activated Notch-1 can par-ticipate with HPV oncoproteins E6 and E7 to transform pri-mary human keratinocytes (Lathion et al, 2003), and there isother evidence that Notch-1 inhibits apoptosis in immortal-ized keratinocytes (Rangarajan et al, 2001b) and in cervicalcancer cells (Nair et al, 2003). The work of Lathion et al shedsome light on this apparent contradiction. These authorsshowed that intracellular Notch-1 expressed from a verystrong (i.e., cytomegalovirus) promoter at very high levelsinhibits the expression of E6 and E7 HPV oncoproteins, asreported by Talora et al. When expressed in more moderateamounts from a Rous sarcoma virus promoter, however,intracellular Notch did not inhibit E6 or E7 expression, andtransformed keratinocytes in cooperation with them. Thesefindings underscore two crucial characteristics of Notchsignaling, concentration dependence and context depend-ence. The effects of exogenously overexpressed Notch re-ceptors do not necessarily correspond to those of thespontaneously expressed proteins. Additionally, the co-presence of other oncogenic alterations (such as inactiva-tion of retinoblastoma (Rb) and p53 by viral oncoproteins)affects the outcome of Notch signaling. This context de-pendence has relevance to the concept of synthetic lethalityin that it suggests possible molecular explanations for dif-ferential sensitivity of neoplastic versus normal cells toNotch inhibition. In this case, inactivation of Rb and p53results in the disabling of the G1 restriction point, thus lim-iting the effects of p21 induction by Notch-1, whilesynergizing with the anti-apoptotic signals mediated byNotch-1. It should be noted that with the possible exception

of T-cells, intracellular Notch-1 (and Notch-2) transformscells of several lineages in the co-presence of viral onco-proteins that inactivate Rb and p53 (Capobianco et al, 1997;Jeffries and Capobianco, 2000; Rangarajan et al, 2001b;Bocchetta et al, 2003). It should be kept in mind that the endresult of Notch signaling is dictated by the cellular context,and the signal strength and duration, which in turn are reg-ulated by post-translational modifications and pathwaysthat influence receptor recycling and membrane availability(Panin and Irvine, 1998). In the following paragraph, webriefly review evidence linking excessive or sustained Notchreceptor activation to its proto-oncogene activity in othercell types.

A recent report documented the presence of point mu-tations in strategic sites within Notch-1 that ultimately pro-duced a receptor that was more readily processed by g-secretase, leading to a sustained Notch-1 receptor signa-ling in approximately 50% of the cases of T cell acutelymphoblastic leukemias (Weng et al, 2004). This makes itthe most frequently mutated oncogene in this disease(Fig 2B). Earlier, a chromosomal translocation (t 7; 9) wasidentified in a rare ( � 10%) subset of T-ALL patients thatalso resulted in elevated Notch-1 receptor signaling (Re-ynolds et al, 1987; Ellisen et al, 1991). Using a mouse mod-el, forced overexpression of the truncated form of thehuman Notch-1 gene in the hematopoietic compartmentalso produced T cell leukemia (Pear et al, 1996). This con-sisted of CD4þ , CD8þT-cells, and appeared after a laten-cy period, suggesting that other mutations are required.Besides T-ALL, there are many other examples of deregu-lated expression of wild-type Notch receptors in humantumors including: head and neck carcinomas, renal carci-nomas, endometrial carcinomas, mesothelioma, breast,lung, and pancreatic carcinomas, as well as chronic lymph-oid and a subset of acute myeloid leukemias, anaplasticlarge cell non-Hodgkin lymphomas, and a subtype of Hodg-kin lymphoma (Nickoloff et al, 2003). As regards interactionsbetween Notch and other oncogenic pathways of relevanceto investigative skin biologists, it should be noted thatcross-talk involving the Wnt signaling pathway as well asthe Sonic-Hedgehog pathway has been observed (Theluet al, 2002). Both Wnt- and Hedgehog-mediated signalingare of increasing interest to investigators studying carcin-ogenesis-related pathways (Beachy et al, 2004). In the nextsection, a brief review of the mechanism by which Ras andNotch signaling can influence the neoplastic phenotype inmelanoma will be presented.

Ras and Notch Signaling in Melanoma

Prior to the discovery of the aforementioned activating pointmutations in Notch-1 found in T-ALL patients, several lab-oratories had focused on the interaction between Rassignaling and the Notch pathway (Fig 3). It was discoveredthat oncogenic Ras could enhance Notch signaling by atleast two distinct, but complementary, pathways (Weijzenet al, 2002). The first way in which oncogenic Ras can en-hance Notch signaling is by increasing the level of Notchligands such as Delta-1, and the second mechanism isby increasing the levels of the g-secretase/presenilin 1


complex (Nickoloff et al, 2003). We are focusing on Ras inthis review because of the importance of Ras signaling inthe pathogenesis of melanoma (Herlyn et al, 2002). It alsoappears that Ras-mediated transformation requires thepresence of intact Notch signaling, and if such Notch re-ceptor signaling is disrupted, the ability of Ras to transformcells is significantly reduced (Weijzen et al, 2002; Kiaris et al,2004). Although the precise mechanistic details by whichNotch signaling can contribute to tumorigenesis remain tobe defined, several interesting insights have been gained byfocusing on cell survival pathways. It appears that Notchsignaling can enhance the survival of cells by interactingwith the PKB/AKT (Rangarajan et al, 2001a), as well as NF-kB pathways (Cheng et al, 2001).

The molecular and genetic pathways contributing to thetransformation of normal melanocytes to melanomas arethe subject of intense investigation (Herlyn et al, 2002). Oneimportant reason for this interest is that the incidence of

melanoma is rapidly increasing, as is the mortality from thisdeadly neoplasm (Buzzell and Zitelli, 1996; Houghton andPolsky, 2002). Figure 4 provides a representative phase-contrast microscopic appearance of normal proliferatinghuman melanocytes, as well as a panel of ten different me-lanoma cell lines. This collection of melanoma cells includesearly-passage (o50 passages; RJ002L, BO-003) as well asmany late-passage cells (450 passages; C8161, MUM2B,MUM2C, C81-61, OCM1A, SK-Mel 5, SK-Mel 100, and SK-Mel 147). Note that normal melanocytes have elongatedcytoplasmic processes resembling neuronal dendrites,whereas the melanoma cell lines appear either epithelioidor spindle shaped and display a tendency to pile up as theyproliferate. As can be appreciated by the differences in theappearance of the cultured cells, morphology does not pre-dict Notch expression as detailed in Figs 4 and 5. CEM cellsgrow in suspension and are also portrayed in Fig 4 as theyrepresent a cell line with markedly enhanced Notch-1 levelsas mentioned earlier for T-ALL cells (Weng et al, 2004).

Abnormalities involving several important proteins havebeen uncovered in the pathogenesis of melanoma includ-ing: inactivation of p16INK4a, cyclin-dependent kinase inhib-itor (CDK4/6) with suppression of Rb protein, silencing ofthe pro-apoptotic protein APAF-1, activating mutations in B-Raf and N-Ras, and constitutive activation of mitogenic re-ceptor signaling pathways including basic fibroblast growthfactor receptor (Reed et al, 1995; Buzzell and Zitelli, 1996;Dracopoli and Fountain, 1996; Davies et al, 2002; Alsinaet al, 2003; Soengas and Lowe, 2003; Tucker and Goldstein,2003). There may also be deregulation of E2F transcriptionalactivity, as well as alterations in the Wnt pathway involvingWnt5a (Halaban et al, 2000; Weeraratna et al, 2002). Two ofthe most common molecular lesions in melanomas, namely,activation of Ras signaling and inactivation of the G1checkpoint, can potentially result in enhanced Notch signa-ling (Ras) and co-operate with the transforming activity ofNotch-1. As discussed earlier, whereas there are multipleNotch receptors and ligands, due to space constraints, thisreview will focus predominantly on Notch-1 receptor acti-vation in melanoma as a paradigm for understanding therole of Notch signaling in carcinogenesis.

Since melanocytes arise from the neural crest, and be-cause Wnt as well as Notch developmental signaling cas-cades regulate central nervous system stem cell dynamics,it was possible that Notch signaling may be activated inmelanoma cells, as well as in various neural crest-derivedbrain tumors (Fan et al, 2004). In melanocytes and me-lanoma cells, one of the first steps in determining whetherNotch signaling may have been relevant in the pathogenesisof melanoma is to assess the relative levels and state ofactivation for Notch receptors in both melanocytes as wellas melanoma cells (Fig 5). In two earlier reports, we initiallyused an immunohistochemical staining approach, and al-though we could not detect any of the four different Notchreceptors to be expressed by melanocytes in normal ordiseased skin, enhanced immunoreactivity was detected forall four different Notch receptors in melanoma lesions (Hen-drix et al, 2002; Nickoloff et al, 2003). Interest in Notchsignaling in melanoma derived from the observation thatsome aggressive melanoma cell lines displayed a stemcell-like plasticity, and the question as to a role for Notch

Figure 3Role of cross-talk between oncogenic Ras and Notch receptors inmelanoma, and portrayal of therapeutic targets derived from thissignaling pathway. Note that there are at least two mechanisms bywhich activated Ras can enhance Notch signaling: either by inducingNotch ligand expression, or by enhancing g-secretase activity. Tran-scriptional targets for Notch signaling in tumor cells include Hes-1,STAT3, NF-kB, and p21. Therapeutic approaches for interrupting Notchsignaling include use of GSI (g-secretase inhibitor), or a genetic silenc-ing approach featuring RNAi targeting individual Notch receptors (seetext for further explanation).


signaling as an underlying molecular mechanism for thetrans-differentiation between melanoma cells and end-othelial cells arose from such studies (Hendrix et al, 2004).Given the role of Notch signaling in regulating vasculatureformation during embryogenesis (Gridley, 2001; Uytten-daele et al, 2001), the enhanced levels of Notch receptors inmelanoma were consistent with this developmental-relatedtheme for melanoma pathogenesis. To extend our immuno-staining results, a polyclonal rabbit-derived antibody de-tecting the activated state of Notch-1 (Abcam, Cambridge,Massachusetts) was used to probe whole-cell extracts. Noactivated Notch-1 was detected by western blot analysis inany of the proliferating normal human melanocytes derivedfrom ten different foreskins (Fig 5). Using ten different me-lanoma cell lines, however, activated Notch-1 was detectedin all of the cell lines, albeit at variable levels of overex-pression and irrespective of their morphology (spindleshaped or epithelioid) as shown in Fig 4. The T-ALL cellline, CEM, was used as a positive control as it containsabundant activated Notch-1. As regards the overexpressionof Notch receptors that we have detected in human me-lanomas, another group using a microarray approach hasalso independently verified enhanced Notch mRNA levels inmelanoma (Hoek et al, 2004), and our recently completedreal-time quantitative polymerase chain reaction analysis ofthese ten melanoma cell lines confirmed elevated levels forall four Notch receptor mRNAs compared with normal pro-liferating melanocytes.1

Concept of Synthetic Lethality

As one considers the biology of oncogenic events, it is im-portant to note that, in general, most cancer-associatedgrowth-promoting signals are directly coupled to increasedcell death (Hanahan and Weinberg, 2000). Thus, initial onc-ogenic events can only have a net tumor growth effect whenthey are complemented by, or cooperate with, anti-apopto-tic pathways in tumor cells. Identification of this specificapoptotic vulnerability in melanoma can then be targeted toachieve selective killing of malignant cells while sparingnon-transformed (i.e., normal) cells (Hartwell et al, 1997).This approach has been termed synthetic lethality becauseit emphasizes combining a drug that does not affect normalcells that contain wild-type alleles, with induction of death inmalignant cells containing a specific genetic alteration thatrenders the tumor cell susceptible to induction of apoptosis(Reddy and Kaelin Jr, 2002). Figure 6 provides an overviewof these concepts including the pharmacogenomic strategyas applied to synthetic lethality in melanoma. The term‘‘pharmacogenomic’’ is used here not to indicate the studyof inter-individual genetic variability in drug susceptibility,but to indicate the genetic basis for differential drug sus-ceptibility between normal cells and cancer cells.

As portrayed in Fig 6, a normal cell is depicted with aproperly balanced set of proteins regulating proliferationand apoptosis, but following proto-oncogene activation(e.g., c-myc activation), there is enhancement in both pro-liferation and apoptosis such that no net tumor is formed.Following an additional genetic alteration such as increasedBcl-xL, NF-kB, or AKT, the enhanced survival pathway

Figure 4Phase-contrast microscopic appearance of proliferating human melanocytes derived from normal skin, and a panel of melanoma cell linesderived from patients with metastatic melanoma. (Magnification � 85) (A) Normal human melanocytes. (B–K) Melanoma cell lines. (L) T-ALL cellline: CEM cells. Note that normal human melanocytes were derived from neonatal foreskins and grown in standard medium (Qin et al, 2004).Melanoma cell lines and CEM cells were grown in RPMIþ10% FCS. All cells were maintained at 371C in a humidified incubator with 5% CO2.

1B. J. Nickoloff, A. Weeraratna, unpublished observations,2004.


overcomes the pro-apoptotic tendency to shift the balancein favor of tumor formation. There are several examples inwhich an oncogenic genetic alteration can sensitize thetumor cells to induction of apoptosis including: activationand deregulation of MYC that leads to either sustained E2F-1 levels that trigger caspase activation and apoptosis, orenhanced death receptor (DR5)-mediated susceptibility(Wang et al, 2004). Another relevant example is the activa-tion of Ras/Src that is accompanied by sensitization to lys-osomal cysteine protease, cathepsin B (Fehrenbacher et al,2004). By using agents that can exploit the hypersensitivityof malignant cells, a pharmacogenomics approach that isanalogous to the concept of genetic synthetic lethality canbe envisioned for a molecularly targeted therapeutic appli-cation. In this review, data are presented indicating thatNotch receptor activation is abnormal in melanoma cells,and in the next section focus is directed toward the use ofan agent initially selected to block Notch signaling. This lineof inquiry led to the discovery of a novel apoptotic pathwayfeaturing induction of a BH3-only pro-apoptotic proteinknown as NOXA.

Use of gamma-secretase inhibitor (GSI) toInduce NOXA in Melanoma Cells

Since all four different Notch receptors appear to be acti-vated in melanoma lesions (Nickoloff et al, 2003), we de-cided to use a GSI that could simultaneously inhibit allNotch receptor pathways (Weijzen et al, 2003). GSI is a

tripeptide aldehyde that resembles other agents developedfor the treatment of Alzheimer’s disease (Wolfe, 2001). Thechemical structure of GSI is depicted in Fig 6. Since the b-amyloid peptide in senile plaques requires a cleavage stepsimilar to Notch receptor processing, many such inhibitorsare already available, and some are in clinical trials that willprovide valuable patient tolerability and safety data for fu-ture drug development in melanoma patients (Josien, 2002).According to a published report (Qin et al, 2004), whenproliferating melanocytes or melanoma cells were exposedto GSI, the melanocytes underwent a G2/M growth arrest,but the melanoma cells underwent rapid and prominentapoptosis, as summarized in the bottom portion of Fig 6.

Next, studies were designed to define the mechanism ofaction for the selective apoptotic response of GSI in me-lanoma cells (Qin et al, 2004). To identify the apoptoticpathway, both melanocytes and melanoma cells were stud-ied, and the kinetics of induction of apoptosis following ad-dition of GSI to melanoma cells was determined. Using apanel of five different melanoma cell lines, including an ear-ly-passage metastatic melanoma cell line (RJ002L) or fourdifferent late-passage melanoma cell lines (C8161, MUM2B,SK-Mel-28, SK-Mel-100), the induction of apoptosis be-came detectable by Annexin V-FITC staining and flowcytometry approximately 6–8 h after exposure to GSI. Todetermine whether this apoptosis required new protein syn-thesis of pro-apoptotic factor(s), or could be attributed tothe loss of survival factor(s), melanoma cells were pre-treated with cycloheximide (1 mg per mL; 1 h) to block pro-tein synthesis, and then exposed to GSI. The inhibition ofnew protein synthesis almost completely blocked the GSI-mediated induction of apoptosis. This result strongly point-ed toward induction of a pro-apoptotic signal following GSI,so whole-cell extracts were prepared before and at varioustime intervals (1, 3, 6, 18, 24 h) following GSI exposure.Western blots were probed for numerous survival and apo-ptotic proteins, and it was discovered that the most rapid(approximately 3 h) and prominent pro-apoptotic proteininduced by GSI was the BH3-only protein, NOXA. AnotherBH3-only protein, Bim, was also induced but at later timepoints (18, 24 h). Western blots for the melanoma cells re-vealed constitutive levels of the important apoptotic pro-teins Bax and Bak, as well as high levels for survivalproteins Bcl-2, Mcl-1, and survivin.

One reason why it took such a long time to discover thebasis for induction of NOXA was previous reports implyingthat p53 was necessary for NOXA induction (Qin et al,2004). Since several of the melanoma cell lines containedmutations in both p53 alleles, the initial survey of candidateproteins did not include p53-inducible proteins such asNOXA. But, once the rapid and prominent induction ofNOXA was detected using two different antibodies thatcorrectly detected a single protein of the correct molecularmass, we next asked four important questions. First, wasGSI-induced NOXA p53 dependent? Second, did GSI in-duce NOXA in normal melanocytes? Third, did other agentsused to treat melanoma or other cancers induce NOXA?Fourth, did blocking NOXA induction reduce the apoptoticresponse in melanoma cells? To determine whether p53was required for NOXA induction, we initially reducedp53 levels using an siRNA approach and while inhibiting

Figure 5Status of Notch-1 receptor levels and activation in normal humanmelanocytes and melanocytes and melanoma cell lines. Westernblot analysis of ten different normal human melanocyte cultures and tendifferent human melanoma cell lines examined for activated Notch-1receptors (acute T cell lymphoblastic leukemia line; CEM cells contain-ing activating point mutations in Notch-1 as a positive control). Des-ignation of normal melanocytes represents ten different neonatalforeskins, as follows: lane 1, MC-07; lane 2, MC-08; lane 3, MC-06;lane 4, MC-04; lane 5, MC-05; lane 6, MC-010; lane 7, MC-011; lane 8,MC-012, lane 9, MC-013; and lane 10, MC-014. Lane numbers des-ignated for the melanoma cell lines are as follows: lane 1, C8161; lane2, MUM2B; lane 3, RJ-002L; lane 4, BO-003; lane 5, MUM2C; lane 6,C81-61; lane 7, OCM1A; lane 8, SK-Mel 5, lane 9, SK-Mel 100; and lane10, SK-Mel 147. Whole-cell extracts (50 mg protein per lane) were pre-pared as previously described (Qin et al, 2004), and western blottingwas performed using: anti-Notch-1 (Abcam ab8925), anti-actin (SantaCruz Biotechnology, Santa Cruz, California) antibodies.


GSI-induced levels of two p53-dependent proteins (GAD-D45, MDM2), no reduction in GSI-induced NOXA was ob-served. Next, we used two other cell lines known to be p53null (e.g., PC-3 prostate cancer cells, SAOS-2 osteogenicsarcoma cells), as well as verified that two of our melanomacell lines (MUM2B, SK-Mel-28) were p53 null, and observedthat whereas no p53 protein was detected, GSI could stillinduce NOXA and trigger cell death. When normal prolifer-ating melanocytes were treated with GSI, no NOXA wasinduced, although the cells underwent a prominent G2/Mgrowth arrest. Treatment of melanoma cells with genotoxicagents such as adriamycin (1 mg per mL), etoposide (10 mgper mL), or UV light (30 mJ per cm2), induced a slight apo-ptotic response, but no expression of NOXA. Finally, whenan antisense oligonucleotide targeting NOXA was used toprevent NOXA induction, the apoptotic response to GSI wasreduced by approximately 30%–50% depending on the cellline utilized. Taken together, these results strongly support-ed the conclusion that GSI was inducing apoptosis in me-lanoma cell lines by engaging a previously uncharacterizedapoptotic pathway that was, in part, dependent on the BH3-only protein, NOXA. Importantly, GSI caused striking celldeath and NOXA induction in melanoma xenografts as well,without causing observable toxicity to nude mice. This sug-gests that it may be possible to identify a therapeutic win-dow for use of GSI in melanoma.

The final aspect of this study was to further delineate thesequence of molecular events occurring in the melanomacells once NOXA was induced. As NOXA is a BH3-onlyprotein, it can bind the pro-survival proteins Mcl-1, Bcl-2,and Bcl-x, thus facilitating interaction between the pro-apoptotic proteins Bax and Bak located in the mitochondrialmembrane (Fig 7). Evidence for this interaction leading tomitochondrial dysfunction included release of cytochromeC from the mitochondria into the cytoplasm. Once cyto-chrome C is released, it can facilitate creation of the apo-ptotic complex in which APAF-1 is recruited, leading tocaspase 9 activation, followed by activation of caspase 3,cleavage of PARP and other ‘‘end-stage’’ substrates, andculminating in DNA degradation and cell death. Interesting-ly, whereas APAF-1 levels were barely detectable in severalmelanoma cell lines, and the cells had high levels of anti-apoptotic factors (e.g. Bcl-2, Mcl-1, survivin), the inductionof NOXA was sufficient to overcome all of these obstaclesto trigger a prominent apoptotic response in all of the me-lanoma cell lines irrespective of their p53 status.

Further studies are required to more fully determinewhether other stimuli (besides GSI) can trigger NOXA in-duction in a p53-independent fashion in melanoma cells,and to elucidate the mechanism responsible for the inabilityof GSI to induce NOXA in normal melanocytes. It will alsobe necessary to determine whether GSI induction of NOXA

Figure 6Overview of pharmacogenomic strategy for synthetic lethality including use of gamma-secretase inhibitor (GSI) to achieve selective killingof melanoma cells, but not normal melanocytes. The initial proto-oncogenic activation (portrayed by c-myc) within a normal cell (green highlights)induces both enhanced proliferation that is offset by enhanced apoptosis (portrayed by increased death receptor-DR5 levels) yielding no net tumorformation (yellow highlights). When additional genetic alterations are present (portrayed by increased Bcl-x, NF-kB, or AKT), the balance betweenproliferation and apoptosis is tipped toward enhanced survival pathways leading to tumor formation (red highlights). An ideal selective chemo-therapeutic agent such as GSI (N-benzyloxycarboxy Leu-Leu-Nle-CHO) can trigger growth arrest, but not apoptosis in normal melanocytes,whereas GSI induces prominent killing of melanoma cell lines (Qin et al, 2004). An important molecular determinant for selective killing of melanomacells versus melanocytes is the ability of GSI to induce the proapoptotic BH3-only protein NOXA in melanoma cells, but not in normal melanocytes.One potentially important characteristic of melanoma cells is their activation of Notch receptors relative to melanocytes, although the specific linkbetween Notch signaling and GSI-mediated induction of NOXA remains to be defined. This figure is derived from several publications includingHanahan and Weinberg (2000), Hartwell et al (1997) and Wang et al (2004).


results from inhibition of Notch signaling alone, given thatg-secretase has numerous substrates, including: ErbB4,syndecan, CD44, and others (Qin et al, 2004).

Conclusions

Taken together, these results indicate that Notch receptoractivation is a characteristic of melanoma cells, which dis-tinguishes the malignant cells from normal melanocytes.Moreover, by using an agent that was initially chosen totarget the activation of all Notch receptors, selectiveapoptosis of melanoma cells could be achieved while spar-ing normal melanocytes. The selective killing of melanomacells was attributed, in part, to induction of NOXA, as GSItriggered the pro-apoptotic BH3-only protein NOXA exclu-sively in melanoma cells and not melanocytes. Moreover,the use of an antisense oligonucleotide targeting NOXAsignificantly reduced the GSI-induced apoptosis in me-lanoma cells.

Future studies are indicated to determine the precisemechanism whereby Notch receptor-mediated signaling

pathways provide a survival advantage to melanoma cells.It will be important to elucidate the relationship between re-duction in Notch signaling, and engagement of the apopto-tic machinery in melanoma cells. Finally, devising additionalreagents that can selectively target NOXA induction in me-lanoma cells, while sparing normal melanocytes, will pro-vide new therapeutic options for patients with melanoma.

This work was supported by NIH grants CA 59702, CA 80318 (M. J. C.H.), PO1 CA 27502 (P. M. P., J. M. T. , B. J. N.), CA 84065 (L. M.), andPO1 CA 59327 (BJN). Larry Stennett and Barb Bodner performed thetissue culture and western blot analysis. Lynn Walter and Brian Bonishprepared the text and figures.

DOI: 10.1111/j.1087-0024.2005.200404.x

Manuscript received December 15, 2004; revised February 28, 2005;accepted for publication March 9, 2005

Address correspondence to: Dr Brian J. Nickoloff, MD, PhD, Director,Oncology Institute, Loyola University Medical Center, Cardinal Bernar-din Cancer Center Room 301, 2160 S. First Ave, Building 112, May-wood, IL 60153-5385, USA. Email: [email protected]

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