Understanding Melanoma Signaling Networks as the Basis for

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     Understanding Melanoma Signaling Networks as the
     Basis for Molecular Targeted Therapy
     Keiran S.M. Smalley1

     Despite years of research, there has been little improvement in survival for patients with disseminated
     melanoma. Recent work has identified mutations in BRAF and NRAS, leading to constitutive mitogen-activated
     protein kinase (MAPK) pathway as well as constitutive activity in the phosphoinositide 3-kinase (PI3K)/protein
     kinase B (AKT) pathway, as being critical events in melanoma growth and progression. In the current review, we
     discuss how these complex mutational and signaling profiles can be understood using a network biology
     approach, and suggest how an understanding of the key signaling nodes involved in progression and survival
     will lead to improvements in melanoma therapy.
     Journal of Investigative Dermatology (2010) 130, 28–37; doi:10.1038/jid.2009.177; published online 2 July 2009

     INTRODUCTION                                         now identified many of the activating                 2001). The interactions between the
     Melanoma is the deadliest form of skin               mutations responsible for melanoma                    various signaling molecules involved in
     cancer. It arises from the malignant                 development/progression, and attempts                 melanoma progression are best viewed
     transformation of melanocytes, the                   are now underway to integrate these                   as a series of links between a number
     pigmented cells of the skin. Incidence               vast data sets (Davies et al., 2002; Lin              of interconnected nodes, and the sum
     of melanoma continues to increase at                 et al., 2008). It is becoming clear that              total of all of these nodal links make up
     an alarming rate with the National                   melanoma constitutes a heterogeneous                  the melanoma cell signaling network.
     Institutes of Health predicting 62,480               group of tumors, with different patterns              What these network maps show is that,
     new cases of melanoma in the year                    of oncogenic mutation, overexpression,                although most signaling nodes are
     2008, with 8,420 of these patients                   and genomic amplification. In this                    connected by a few links, there are a
     succumbing to their disease. From a                  review, we aim to shed some light on                  few nodes with a great number of links,
     therapeutic standpoint, early detection              the complexity and functional redun-                  which are called hubs. One feature of
     of melanoma is vitally important, with               dancy within the melanoma signaling                   these networks is their robustness,
     the surgical removal of early stage                  network, and will discuss how combi-                  meaning that the removal of one node
     (non-metastatic) lesions constituting a              nation-targeted therapy approaches                    from the network will not impair its
     cure. Once disseminated, melanoma                    may be required to achieve maximal                    function. In melanoma therapy terms,
     becomes a considerable clinical pro-                 therapeutic benefit.                                  this means that attacking the network
     blem associated with median survival                    Although most studies to date have                 at one nodal point (for example,
     times of o6 months. There are cur-                   focused on the role of individual                     inhibiting MAPK/extracellular signal
     rently no FDA (the Food and Drug                     signaling pathways, the complexity of                 regulated kinase (ERK) kinase (MEK))
     Administration)-approved agents avail-               the signaling observed in melanoma                    is unlikely to make the system fail, as
     able that have been shown to alter the               calls for a network biology approach.                 there will be other network connec-
     natural history of metastatic disease in             The use of network modeling in bio-                   tions that allow the cell to circumvent
     large randomized clinical trials. With               logy is a relatively new concept                      the node under attack. Examples from
     this in mind, a huge amount of research              (Barabasi and Oltvai, 2004). It has been              other biological systems tell us that
     has been undertaken to increase our                  used most successfully in simple model                attacking the network at the most
     understanding of melanoma biology,                   organisms, such as yeast, in which                    connected points, for example, the
     with the hope of discovering new                     two-hybrid screens have led to the                    network hubs, can lead to system
     targets for therapeutic intervention.                generation of complete maps of pro-                   failure. It is expected that the therapeu-
     High throughput genomic screens have                 tein–protein interactions (Jeong et al.,              tic targeting of the critical melanoma

      The Molecular Oncology Program and Comprehensive Melanoma Research Center, The Moffitt Cancer Center, Tampa, Florida, USA
     Correspondence: Dr Keiran S.M. Smalley, The Moffitt Cancer Center and Research Institute, Molecular Oncology Program, Comprehensive Melanoma Research
     Center, 12902 Magnolia Drive, Tampa, Florida 33612, USA. E-mail: Keiran.Smalley@moffitt.org
     Abbreviations: AKT, protein kinase B; CDK, cyclin-dependent kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK,
     MAPK/ERK kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog
     Received 27 January 2009; revised 24 March 2009; accepted 25 April 2009; published online 2 July 2009

28   Journal of Investigative Dermatology (2010), Volume 130                                                   & 2010 The Society for Investigative Dermatology
                                                                                                                   KSM Smalley
                                                                                                     Molecular Targeted Therapy

signaling hubs will lead to apoptosis        melanoma specimens have continuous          with activating NRAS mutations are
and tumor regression.                        hyperactivity in the MAPK pathway           also known to stimulate MAPK signal-
                                             (Cohen et al., 2002). Although MAPK         ing through CRAF rather than BRAF
MUTATIONS THAT ACTIVATE THE                  activity in melanoma cells can arise        (Dumaz et al., 2006).
MAPK PATHWAY                                 through autocrine growth factor stimu-          Acquisition of the BRAF V600E
Of all the signaling pathways that are       lation (Nesbit et al., 1999), N-cadherin-   mutation seems to be an early event
constitutively activated in melanoma,        based homotypic cell–cell adhesion          in melanoma development with a high
the most attention has been focused on       (Li et al., 2001), and melanoma cell–-      percentage of melanocytic nevi also
the mitogen-activated protein kinase         matrix adhesion, it is more commonly        found to harbor the mutation (Pollock
(MAPK) pathway (Satyamoorthy et al.,         activated after the acquisition of an       et al., 2003). Interestingly, the presence
2003; Sharma et al., 2006; Smalley           activating oncogenic mutation. The          of the BRAF V600E mutation alone is
et al., 2006; Solit et al., 2006). The       first such MAPK-activating mutation to      not sufficient to oncogenically trans-
MAPK pathway is primarily involved in        be reported in melanoma was in NRAS         form nevi cells into melanoma. Instead,
the regulation of cell growth. Under         (Padua et al., 1984, 1985). Mutations in    the forced expression of BRAF V600E
physiological situations, stimulation of     NRAS have since been identified in          in primary human melanocytes leads
the pathway occurs after the interaction     15–20% of all melanomas, and are            to an irreversible growth arrest charac-
of growth factors with their respective      most commonly the result of the sub-        teristic of senescence (Michaloglou
cell surface receptors and the transmis-     stitution from leucine to glutamine at      et al., 2005). Pathological studies have
sion of these signals through the small      position 61 (Brose et al., 2002; Davies     confirmed these in vitro findings, and
GTPase, RAS (Robinson and Cobb,              et al., 2002). A role for RAS mutations     showed that most nevi are growth
1997). When active in its GTP-bound          in melanoma initiation has been con-        arrested and express many markers of
state, RAS activates a number of down-       firmed in animal models, wherein the        senescence (Michaloglou et al., 2005).
stream effectors, one of which is the        introduction of mutated HRAS leads to       This process, termed ‘‘oncogene-
RAF family of serine/threonine kinases.      melanoma in transgenic mice lacking         induced senescence,’’ is thought to be
There are three isoforms of RAF,             expression of the cyclin-dependent          an important mechanism in protecting
namely, A-Raf, BRAF, and CRAF (also          kinase (CDK) inhibitor p16INK4A (Chin       cells from malignant change. The
called Raf-1). Once activated, RAF           et al., 1997). The most common muta-        phenomenon has been best character-
stimulates the MAPK cascade, resulting       tion to be reported in melanoma thus        ized in fibroblasts and describes a state
in the sequential activation of MEK1         far is in BRAF, the serine–threonine        in which the acquisition of inappropri-
and MEK2, which in turn activates            kinase located downstream of NRAS           ate growth signals, such as those from
ERK1 and ERK2 (Crews et al., 1992;           (Davies et al., 2002). Although the         mutated BRAF, result in the increased
Kyriakis et al., 1992). Once activated,      precise mutational frequency varies         expression of the ARF/p53/p21 axis
the ERKs either activate cytoplasmic         according to the study, it seems that       as well as the CDK inhibitor, p16
targets or migrate to the nucleus,           at least 60% of all melanomas harbor        (Sharpless and DePinho, 2005). As the
where they phosphorylate transcription       activating mutations in the BRAF gene.      loss/mutation of the p16 gene is a
factors.                                     So far, over 50 distinct mutations in       common event in some inherited forms
    In melanocytes, the MAPK pathway         BRAF have been identified (Garnett          of melanoma (Hayward, 2003), it was
is activated by growth factors released      and Marais, 2004). Of these, the BRAF       initially thought that the acquisition of
from the local microenvironment, such        V600E mutation, resulting from a            BRAF mutations occurred in tandem
as stem cell factor, a-melanocyte-           valine to glutamic acid substitution, is    with p16 inactivation. Interestingly, this
stimulating hormone, and hepatocyte          by far the most common and accounts         seems not to be the case in melanoma
growth factor (Mattei et al., 1994). Not     for over 80% of all reported BRAF           development, with the expression of
all of these growth factors use the same     mutations (Davies et al., 2002; Wan         p16 in nevi being rather mosaic and
mechanism to stimulate the MAPK              et al., 2004). Most of the transforming     irregular in appearance (Michaloglou
pathway. The receptor tyrosine kinases,      activity of the BRAF V600E mutation is      et al., 2005). Follow-up in vitro studies
such as stem cell factors/c-KIT, stimulate   thought to result from the stimulation of   confirmed these histological findings
RAS directly, whereas a-melanocyte-          the MAPK pathway (Davies et al., 2002).     and showed that small-interfering RNA
stimulating hormone—which signals                It is worth noting that not all BRAF    knockdown of p16 in melanocytes did
through G-protein coupled melano-            mutations reported are at the V600E         not lead to malignant transformation
cortin receptors—requires the prior          position, and there exist rare sub-         when combined with the BRAF V600E
activation of adenylate cyclase. Under       groups of melanomas with D594G              mutation, nor was the introduction of
physiological conditions, these growth       and G469E BRAF mutations (Smalley           BRAF V600E alone found to activate
factors only induce a weak stimulation       et al., 2009). These mutants typically      the ARF/p53/p21 axis (Michaloglou
of the MAPK pathway that is insuffi-         show low intrinsic BRAF kinase activity     et al., 2005). Recently, a genome-wide
cient to induce melanocyte prolifera-        and instead transactivate CRAF to           RNAi (RNA interference) screen has
tion. In most melanoma cells, the            stimulate their MAPK signaling (Wan         showed that a secreted protein, insulin
situation is very different and it has       et al., 2004). In common with low-          growth factor binding protein-7, in-
been shown that 490% of clinical             activity BRAF mutants, melanomas            duced senescence in melanocytes after

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     KSM Smalley
     Molecular Targeted Therapy

                                             c KIT
                                                                                                             BRAF inhibitors (Solit et al., 2006;
                                                                                                             Haass et al., 2008; Tsai et al., 2008;
                                                                                                             Smalley et al., 2009).
                                                                                                                 In addition to its role in prolifera-
                                                                                                             tion, the MAPK pathway contributes
                                                                                                             to a number of other oncogenic
                                                                                                             processes. Melanomas are known to
                                                                                                             be exceptionally resistant to apoptosis
                                                                                                             (Soengas and Lowe, 2003) and it seems
                                                                                                             likely that constitutive MAPK activity,
                                                                                                             in part, regulates the apoptotic res-
                                                                                                             ponse (Figure 2). The induction of
                                                                                                             tumor necrosis factor-related apop-
                                         CRAF         BRAF
                                                                                                             tosis-inducing ligand in melanoma
                                                                                                             proceeds through the release of Smac/
                                             MEK                                 AKT                mTORC2   DIABLO from the mitochondria (Zhang
                                                                                                             et al., 2003), and can be suppressed
                                                                Fox03                                        by constitutive MAPK. Constitutive
                                                                                                             MAPK activity also suppresses apoptosis
                                                                p27                                          through the ribosomal S6 kinase-
                                                                                                             mediated inactivation of the pro-apop-
                                                             Cyclin D1                                       totic protein BAD (Eisenmann et al.,
                                                                                                             2003). More recent studies have shown
                                       p16         CDK4
                                                                          CDK2               MITF            that the BRAF V600E mutation decrea-
                                                                                                             ses the levels of the pro-apoptotic
                                                                                   Cyclin E                  Bcl-2 family member BIM through an
                                                             Cell Cycle   S
                                                                                                             MAPK-mediated mechanism (Cartlidge
                                                                                                             et al., 2008). Constitutive MAPK acti-
                                                                G2                                           vity may also regulate angiogenesis
                                                                                                             through the control of hypoxia induci-
     Figure 1. Sample scheme showing how the BRAF/MEK/ERK and PI3K/AKT signaling pathways                    ble factor-1a expression (Kumar et al.,
     intersect to regulate cell cycle entry and proliferation.                                               2007). Furthermore, downregulation
                                                                                                             of V600E BRAF with small-interfering
                                                                                                             RNA significantly decreases the
     the introduction of the BRAF V600E                        CDK4 and CDK6, which interact with            amount of vascular endothelial growth
     mutation (Wajapeyee et al., 2008).                        cyclin D1, as well as by CDK2, which          factor produced by melanoma cells
     Mechanistically, the autocrine secre-                     interacts with cyclins A/E (Sherr, 1994).     (Sharma et al., 2005). This raises the
     tion of insulin growth factor binding                     Constitutive MAPK activity increases          possibility that melanoma-associated
     protein-7 acts through a negative feed-                   cyclin D1 and downregulates p27               angiogenesis is partially mediated
     back loop to suppress MAPK signaling                      expression in melanoma cells (Bhatt           by the MAPK pathway. There is also
     in melanocytes, leading to the induc-                     et al., 2005). As would be expected,          evidence that BRAF/MAP kinase signal-
     tion of senescence (Wajapeyee et al.,                     inhibition of either BRAF or MEK in           ing activity may contribute to the
     2008). In contrast, melanoma cells are                    melanoma cell lines using pharmaco-           metastatic spread of melanoma cells
     able to escape this tumor-suppressive                     logical inhibitors leads to a profound        through the regulation of b3-integrin
     mechanism by silencing their expres-                      G1-phase cell cycle arrest. Indeed,           expression and control of matrix
     sion of insulin growth factor binding                     the BRAF inhibitors, SB590885 (King           metalloproteinase expression (Woods
     protein-7.                                                et al., 2006), AZ-628 (Montagut et al.,       et al., 2001; Benbow et al., 2002; Ge
        Since the discovery of activating                      2008), and PLX-4720, (Tsai et al.,            et al., 2002; Huntington et al., 2004).
     BRAF and NRAS mutations in melano-                        2008) as well as the MEK inhibitors,          Consistent with an anti-metastatic role
     ma, the work of many laboratories                         U0126 (Smalley et al., 2007), CI-1040         for inhibitors of the MAPK pathway,
     has now unraveled the role of MAPK                        (Solit et al., 2006), PD0325901, and          recent studies have shown that U0126
     signaling in tumor progression. The                       AZD6244 (Haass et al., 2008), all have        treatment suppressed lung metastasis
     best-characterized role for MAPK sig-                     cytostatic effects on melanoma cells          in a mouse melanoma model (Sharma
     naling in melanoma is in the regulation                   harboring the BRAF V600E mutation. It         et al., 2006). There are also possibilities
     of cell growth, particularly at the G1                    is also suggested that the presence of a      that BRAF/MAPK signaling may allow
     cell cycle checkpoint (Figure 1).                         BRAF V600E mutation, rather than an           melanoma cells to escape immune
     Progression through the G1 restriction                    NRAS or low-activity BRAF mutation,           surveillance through suppression of
     point into the S-phase is driven by                       predicts for sensitivity to both MEK and      their highly immunogenic pigmentation

30   Journal of Investigative Dermatology (2010), Volume 130
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                                                                                                                Molecular Targeted Therapy

                                      c KIT
                                                                                                   activation of receptor tyrosine kinases
                                                                                                   and associated adaptor proteins that
                                                                                                   bind to the SH2 domain of the p85
                                                                                                   subunit. The p110 domain can also
                                                                                                   be recruited and activated after the
                                                                                                   activation of RAS. After membrane
                                                                                                   recruitment and activation, PI3K then
                                                                                                   phosphorylates the PIP2 (phosphatidyl-
                                                                                                   inositol-4,5,bisphosphate) ring at the
                                                                                                   30 position, converting PIP2 to PIP3
                                                   Inhibitors                                      (phosphatidylinositol-3,4,5,trisphosphate).
                                        RAS                           PI3K                         Once generated, PIP3 recruits and
                                                                                 PTEN              activates the downstream serine–
                                                                                                   threonine kinases, PDK1 and AKT
                                        MEK                                                        (Figure 1). One of the most critical
                                                   Imatinib                                        regulators of AKT is the phosphatase
                                                                                                   and tensin homolog (PTEN), which
                                                                                                   degrades the products of PI3K, thereby
                                                                                                   preventing the activation of AKT. The
                                                                                                   mechanism by which the PI3K pathway
                                                                                                   is activated in melanoma is not fully

                                          BAD                                                      elucidated, but may involve the loss
                                                                                                   of expression or functional inactivation
                                                                                                   of PTEN. Typically, PTEN expression
                                      Bcl-2                                     JAK
                    MITF                                                                           is lost in up to 30% of melanoma
                                Bim                                                                cell lines and 10% of human tumor
                                                                                                   material. There is also evidence of
                                                                                                   inactivating PTEN mutations in 410%
                                                            r   ia                                 of short-term melanoma cell cultures
                 Cytochrome C                               Mcl-1                                  (Lin et al., 2008). PTEN can also
                                                                                                   function as a haploinsufficient tumor
                                                                                                   suppressor, and it is known that allelic
                                                                                                   loss of PTEN occurs in at least 58% of
                            Caspase cleavage
                                                                                                   melanoma metastases (Birck et al.,
                                                                                                   2000). The AKT family consists of three
Figure 2. Sample scheme showing how the BRAF/MEK/ERK and PI3K/AKT signaling pathways               members, AKT1–3 (Brazil et al., 2002),
intersect to regulate apoptosis.                                                                   which exhibit different expression
                                                                                                   patterns depending on the cell type.
                                                                                                   Of these, 43–50% of melanomas have
antigens (Kono et al., 2006). It has been            3-kinase (PI3K)/protein kinase B (AKT)        selective constitutive activity in AKT3
further shown that treating melanoma                 pathway (Stahl et al., 2004; Tsao et al.,     (Stahl et al., 2004). There is evidence
cells with either U0126 or an RNAi to                2004). Activation of the PI3K/AKT             that overexpression of AKT3 may occur
V600E BRAF mutation reduces the                      pathway in melanoma occurs through            as a result of copy number increases
release of immunosuppressive cyto-                   either paracrine/autocrine growth fac-        in the long arm of chromosome 1
kines from melanoma cells (Sumimoto                  tors or the loss of expression and/or         (Thompson et al., 1995; Bastian et al.,
et al., 2006) as well as reversing the               mutation of negative pathway regula-          1998). Another mechanism for PI3K/
suppressive effects of melanoma cell                 tors. In particular, the insulin-like         AKT pathway activation in melanoma
culture supernatants on dendritic cell               growth factor-I is known to aid the           is through the acquisition of activating
activation.                                          growth of early-stage melanoma cells,         E17K mutations in AKT3, but this is
                                                     at least in part, through the activation of   thought to be relatively rare (Davies
ACTIVATION OF THE PI3K/AKT                           PI3K/AKT (Satyamoorthy et al., 2002).         et al., 2008). AKT has a critical role in
PATHWAY                                              PI3K exists as multiple isoforms, with        cancer development through its ability
Activation of the MAPK pathway does                  type 1a PI3K being the most frequently        to regulate apoptosis through the direct
not account for all aspects of mela-                 activated in cancer (Yuan and Cantley,        phosphorylation of BAD as well as it
noma progression and there is evidence               2008). Structurally, PI3K forms a het-        effects many other pathways, including
that other signaling pathways may be                 erodimer consisting of a p85 regulatory       the stimulation of ribosomal S6 kinase,
equally important. The most widely                   subunit and a p110 catalytic subunit. It      the inhibition of forkhead signaling,
studied of these is the phosphoinositide             is recruited to the membrane after the        and the inhibition of glycogen synthase

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     KSM Smalley
     Molecular Targeted Therapy

     kinase-3 (Datta et al., 1997; Robertson,        as the regulatory proteins, rictor and       mutations, again showing a require-
     2005) (Figure 2).                               mSin-1. The two different mTORC              ment for dual MAPK and PI3K/AKT
         The PI3K/AKT signaling has a role in        complexes have opposite effects on           pathway activity (Tsao et al., 2004;
     melanoma initiation, with recent stu-           AKT signaling, with mTORC1 suppres-          Davies et al., 2008; Lin et al., 2008).
     dies showing that AKT transforms                sing AKT signaling and mTORC2                AKT3 seems to cooperate with the
     melanocytes under hypoxic conditions            directly activating AKT through a phos-      BRAF V600E mutation in melanoma
     through a mechanism involving Notch             phorylation event at Ser473 (Sarbassov       initiation by suppressing MAPK signal-
     signaling (Bedogni et al., 2005, 2008).         et al., 2004). There is also evidence        ing activity through phosphorylation at
     Another study has shown a requirement           that mTORC1 inhibition may lead to           Ser364/428 of BRAF (Cheung et al.,
     for constitutive AKT activity in the            increased PI3K/AKT signaling through         2008). It is therefore suggested that
     BRAF V600E mutation-mediated devel-             the upregulated expression of the            AKT3’s ability to downregulate MAPK
     opment of melanoma (Cheung et al.,              insulin-like growth factor-I adaptor         activity may allow early-stage melano-
     2008). Consistent with the role of PI3K/        protein IRS2 (Tamburini et al., 2008).       ma cells to escape from oncogene-
     AKT signaling in melanoma progres-              Thus, there has been some concern that       induced senescence. The strongest
     sion, inhibition of the pathway, using          the clinical use of mTORC1 inhibitors        proof of concept for the cooperation
     either PI3K inhibitors or RNAi’s against        may lead to a rebound increase in AKT        between the BRAF V600E mutation and
     AKT3, reduces melanoma growth and               signaling. mTOR signaling is known to        PI3K signaling in melanoma develop-
     induces apoptosis (Stahl et al., 2004).         be active in melanoma, with immuno-          ment is derived from the mouse mod-
     Although small molecule inhibitors of           histochemical studies showing the            eling work of Martin McMahon and
     both PI3K and AKT are now available,            constitutive phosphorylation of p70 S6       Marcus Bosenberg (Dankort et al.,
     the clinical development of PI3K inhi-          kinase in a panel of metastatic mela-        2009). These authors have elegantly
     bitors is more advanced, with three             noma samples (Karbowniczek et al.,           shown that the conditional expression
     inhibitors (BEZ-235, BGT-226, and               2008). Further work has shown that           of melanocyte-specific mutated BRAF
     PX-866) currently undergoing phase I            rapamycin has growth inhibitory acti-        alone led to the development of
     evaluation. A recent pre-clinical               vity across a small panel of human           benign melanocytic hyperplasia but
     study on PX-866, which is a modified            melanoma cell lines, and that this may       not melanoma. Full transformation to
     analog of wortmannin, showed signifi-           synergize with either sorafenib or           melanoma occurred only when the
     cant inhibition of growth and inva-             bevacizumab (Molhoek et al., 2005,           BRAF-V600E mutation was activated
     sion under three-dimension melanoma             2008). Although initial clinical trials of   in the presence of PI3K/AKT activity
     spheroid culture conditions (Howes              rapamycin analogs in melanoma were           after suppression of PTEN expression
     et al., 2007). The PI3K/AKT pathway             essentially negative (Margolin et al.,       (Dankort et al., 2009).
     is known to regulate proliferation of           2005), there are preliminary indica-             The melanoma network is highly
     melanoma cells, through the regulation          tions that rapamycin may have activity       interconnected with a great deal of
     of cell cycle entry at the G1 checkpoint        in melanoma patients in combination          functional redundancy built into it
     (Smalley et al., 2007) and through the          with carboplatin and paclitaxel (Meier       (sample network shown in Figure 3).
     inactivation of GSK-3b, leading to              et al., 2009).                               This is particularly true with regard to
     cyclin D1 degradation (Diehl et al.,                                                         the fact that inhibition of either PI3K or
     1998) (Figure 1).                               BOTH PI3K/AKT AND BRAF/AKT                   MEK alone can inhibit melanoma
         Another signaling cascade inti-             SIGNALING ARE REQUIRED FOR                   growth and both the PI3K/AKT and
     mately linked to the PI3K/AKT pathway           MELANOMA PROGRESSION                         MEK signaling pathways regulate apop-
     is the mammalian target of rapamycin            It is clear from pre-clinical studies that   tosis at some level (Stahl et al., 2004;
     (mTOR) pathway. mTOR signaling                  the BRAF/MAPK and the PI3K/AKT               Smalley et al., 2006, 2007; Solit et al.,
     involves the activity of two signaling          pathways have overlapping functions          2006; Tran et al., 2008). Studies have
     complexes, mTORC1 and mTORC2.                   with regard to melanoma progression          already shown that single-agent MEK
     Structurally, the mTORC1 complex                (Figures 1 and 2). Analysis of melano-       inhibition is relatively ineffective at
     consists of mTOR as well as the                 ma lines and human melanoma sam-             inducing melanoma regression in both
     regulatory proteins, raptor and mLST8.          ples have shown that mutational              pre-clinical models (Haass et al.,
     Activation of the mTORC1 signaling              profiles are favored that simultaneously     2008) and in early-phase clinical trials
     complex leads to the phosphorylation            promote both MAPK and PI3K/AKT               (Rinehart et al., 2004; Lorusso et al.,
     and activation of p70 S6 kinase and             signaling (Tsao et al., 2000, 2004). As      2005; Adjei et al., 2008). The disap-
     to the eukaryotic initiation factor, 4E-        activating NRAS mutations can stimu-         pointing clinical activity seen after
     binding protein (Reiling and Sabatini,          late both the MAPK and PI3K/AKT              single-agent MEK inhibition parallels
     2006). Increased activity in these two          pathways, they are rarely found in           with that observed with single-agent
     downstream pathway components                   concert with BRAF mutations and these        sorafenib (Eisen et al., 2006). However,
     leads to increased protein translation          mutations are mutually exclusive. Like-      there is some suggestion of benefit
     and cell proliferation (Figure 1). In           wise, BRAF mutations are frequently          when sorafenib is combined with
     contrast, the mTORC2 complex also               found in combination with either PTEN        either carboplatin/paclitaxel or dacar-
     consists of mTOR and mLST8, as well             loss/inactivation or activating AKT3         bazine (McDermott et al., 2008). The

32   Journal of Investigative Dermatology (2010), Volume 130
                                                                                                                                                                                                    KSM Smalley
                                                                                                                                                                                Molecular Targeted Therapy


                                        RAS                     PI3K                PTEN                        Imatinib         C-KIT
                                 CRAF           BRAF                                                                                         RAS                    PI3K             PTEN
                                                                    AKT                                                MST-2         CRAF           BRAF
                        NFKB                                                                mdm2
                                       MEK            mTOR                                                                                                              AKT
                                                                                                                        NFKB                                                                mdm2
                                                                                                                                            MEK          mTOR

                                        ERK            cycD     GSK3b                       p53
                      STAT3                                                                                                                  ERK           cycD     GSK3b                   p53
                                                                                   p27                                               RSK
                        MCL-1                   BIM           BAD                                                                                                                p27
                                                                                                                       MCL-1                       BIM            BAD

                                                                            CDK4                                                                                              CDK4
                                                  Bcl-2                                                                                              Bcl-2
                                                                                         CDK2                                                                                          CDK2
                                   MITF                                                                                                    MITF

                                                                                                      BRAF inhibitor

                                                                                         RAS                      PI3K                PTEN

                                                                    MST-2      CRAF               BRAF
                                                                    NFKB                                                                      mdm2
                                                                                         MEK             mTOR

                                                                                         ERK             cycD     GSK3b                       p53

                                                                MCL-1                           BIM             BAD



Figure 3. Sample melanoma signaling networks. (a) A simplified view of the melanoma signaling network. Nodes shown in green indicate points where there
are either activating oncogenic mutations or increases in protein expression. Some of the nodes are highly interconnected, such as AKT and BAD, and may
represent hubs within the network. It is likely that the real melanoma signaling network is far more interconnected and complex than the scheme indicated
above. Points of network attack are shown in (b) Imatinib treatment or in (c) BRAF inhibitor treatment.

mechanism by which BRAF/MEK inhi-                              et al., 2008b). Others have shown that                                                    can easily switch MAPK signaling
bition enhances response to chemo-                             the melanocyte survival factor, MITF                                                      from BRAF to CRAF is suggestive of
therapy in melanoma is currently under                         (microphthalmia-associated transcrip-                                                     a mechanism by which resistance to
investigation (Smalley and Flaherty,                           tion factor), is also amplified in a small                                                BRAF inhibitors may be acquired.
2009).                                                         sub-group of BRAF V600E-mutated                                                               There is also redundancy at the level
    The signaling pathways active in                           melanomas, and that this in turn                                                          of melanoma survival, with BRAF/MEK
melanoma cells are interconnected                              regulates CDK2 and Bcl-2 expression                                                       inhibition being associated with the
through multiple feedback loops; there-                        (McGill et al., 2002; Du et al., 2004;                                                    induction of a G1-phase cell cycle
fore, inhibition of one pathway often                          Garraway et al., 2005).                                                                   arrest but little apoptosis (Verhaegen
leads to the activation of a parallel                             The melanoma signaling network is                                                      et al., 2006). This is a surprising finding
pathway (Figure 3). This is best char-                         also highly robust, with BRAF-V600E-                                                      given that BRAF signaling directly
acterized in the case of mTOR inhibi-                          mutated melanoma cells rapidly deve-                                                      regulates the expression of the pro-
tion, which leads to a rebound increase                        loping resistance to BRAF inhibition                                                      apoptotic protein, BIM, in melanoma
in MAPK signaling through a PI3K-                              in vitro (Montagut et al., 2008). Rather                                                  cells, and that the inhibition of MEK
mediated mechanism (Carracedo et al.,                          than this resulting from an acquired                                                      or BRAF can lead to an increase in
2008). These kinds of reciprocal                               genetic change in the melanoma cells,                                                     its expression (Cartlidge et al., 2008)
relationships between the signaling                            resistance instead results from a simple                                                  (Figure 2). In this instance, it seems that
pathways active in melanoma argue                              switching within the MAPK pathways                                                        the high level of Bcl-2 expression
strongly in favor of combined targeted                         so that the signal is routed from                                                         that is often observed in melanoma
therapy strategies. Recent work from                           BRAF to CRAF (Figure 3). Studies on                                                       counteracts the pro-apoptotic activity
our laboratory has identified other sites                      melanoma cell lines harboring NRAS                                                        of BIM (Cragg et al., 2008). This
of redundancy within the melanoma                              mutations have already shown that                                                         redundancy can be easily overcome
signaling network, with cyclin D1                              melanomas can use CRAF as a mecha-                                                        when combination therapy approaches
amplifications being found in concert                          nism to activate downstream MAPK                                                          are used. Recent work has shown
with BRAF V600E mutations in minor                             signaling (Dumaz et al., 2006). The                                                       that the combined administration of
sub-groups of melanomas (Smalley                               presence of a network connection that                                                     a MEK inhibitor with a BH3 family

                                                                                                                                                                                                   www.jidonline.org   33
     KSM Smalley
     Molecular Targeted Therapy

     (Bcl-2 family) mimetic, ABT-737, leads          pathway activity, which likely accounts     constitutive activity in many signaling
     to massive apoptosis induction (Cragg           for the apoptosis observed (Jiang et al.,   pathways, some anti-apoptotic activity
     et al., 2008).                                  2008) (Figures 2 and 3).                    and others pro-apoptotic (Sharma and
         The challenge for melanoma thera-               The findings from studies on c-KIT-     Settleman, 2006). When the activity of
     pists is to identify the key signaling          dependent melanomas suggest that            the driving oncogene is disrupted using
     hubs in the melanoma network to                 multiple signaling pathways need to         a pharmacological inhibitor, the cells
     ensure that the cell proliferation/             be inhibited simultaneously. Most           typically undergo apoptosis. There
     survival network fails. There are clearly       studies to date have focused on either      seems to be a temporal component to
     examples in cancer biology, in which            the combination of MEK and PI3K             this apoptosis induction in that the
     the identification and pharmacological          signaling or on BRAF/MEK and mTOR           pro-survival signaling activity, such as
     targeting of the correct network hub            signaling. Under three-dimension cell       AKT and MAPK, tends to decrease
     can lead to network failure (for exam-          culture and xenograft conditions, in        quickly after targeted therapy treat-
     ple, apoptosis) and impressive clinical         which drug resistance is increased,         ment, whereas the pro-apoptotic sig-
     results. These approaches have been             single-agent treatment with either an       nals driven through the stress-activated
     best characterized for tumors, in which         MEK or PI3K inhibitor is ineffective        protein kinases, p38 and JNK, tend
     survival and progression are driven by a        at blocking growth, whereas the dual        to persist for many hours after drug
     single oncogenic event that regulates           combination is highly effective             treatment (Johnson et al., 2000; Parmar
     the entire signaling network, for               (Bedogni et al., 2004; Smalley et al.,      et al., 2004). The combined inhibition
     example Bcr-Abl in chronic myeloid              2006). Likewise, the liposomal admin-       of BRAF/MEK and PI3K/AKT signals
     leukemia (Druker et al., 2001). Similar         istration of an RNAi against BRAF is        could be highly effective in melanoma,
     observations have been made for the             relatively weak at suppressing the          in part, by allowing the pro-apoptotic
     minor sub-group of acral lentiginous/           growth of melanoma xenografts alone,        signals present to dominate.
     mucosal melanomas that harbor acti-             but markedly potentiated when com-             We stand today at the crossroads
     vating c-KIT mutations. In these tumors,        bined with an small-interfering RNA         of melanoma therapy. A greater knowl-
     c-KIT signaling does seem to be the             against AKT3 (Tran et al., 2008).           edge about the complexity and
     critical network hub, and a limited             There is evidence of synergistic pro-       redundancy that lies within the multi-
     number of patients with c-KIT-mutated           apoptotic activity between sorafenib/       ple pathways responsible for melano-
     melanomas have shown impressive                 MEK inhibitors and rapamycin in             ma progression is opening up exciting
     responses to the c-KIT inhibitor, im-           pre-clinical models of melanoma             new avenues for investigation. Already
     atinib mesylate (Gleevec, Novartis, East        (Molhoek et al., 2005; Meier et al.,        a whole array of molecular targeted
     Hanover, NJ) (Curtin et al., 2006; Hodi         2007; Lasithiotakis et al., 2008). From a   agents     against    BRAF      signaling
     et al., 2008; Lutzky et al., 2008). There       mechanistic standpoint, the combina-        (PLX-4032, RAF-265), MEK signaling
     are also reports of a mucosal melano-           tion of MEK/mTOR inhibition reduces         (AZD6244,        PD0325901),         AKT
     ma patient harboring a V560D muta-              the expression of Mcl-1 and Bcl-2           (GSK690693), and mTOR (CCI-779,
     tion in c-KIT showing a complete                leading to enhanced levels of apoptosis     RAD-001) are undergoing clinical
     response to sorafenib (Quintas-Carda-           (Lasithiotakis et al., 2008).               evaluation. It is hoped that continued
     ma et al., 2008). It is currently unclear           As melanomas also exhibit constitu-     investigation will lead to the long
     whether the presence of high levels of          tive activity in multiple other pathways,   needed breakthrough in the treatment
     c-KIT receptor protein or an activating         including nuclear factor kappa B, Src,      of melanoma.
     mutation predicts for effective response        JAK/STAT3, Wnt, hedgehog, and Notch
     to imatinib. A recent phase II clinical         (Yang and Richmond, 2001; Niu et al.,
                                                                                                 CONFLICT OF INTEREST
     trial of 21 melanoma patients reported          2002; Balint et al., 2005; Stecca et al.,   The author state no conflict of interest.
     one clinical response in a patient with         2007), the identification of combina-
     acral lentiginous melanoma exhibiting           tion therapies for melanoma will re-        ACKNOWLEDGMENTS
     high c-KIT receptor expression but no           quire a systematic screening approach,      We thank Dr Liliana Cantini for assistance with
     evidence of c-KIT mutation (Kim et al.,         in which pairwise combinations of           the figures. This work was supported by grants
     2008).                                          signal transduction inhibitors are          from the Melanoma Research Foundation, the
                                                                                                 American Cancer Society Institutional Research
         From a mechanistic standpoint, it           tested, in turn, across panels of mela-     Grant no. 93-032-13.
     seems that c-KIT-dependent melano-              noma cell lines with defined patterns of
     mas still require MEK signaling; how-           genetic mutation. This synthetic lethal
     ever, MEK inhibition is only cytostatic         approach will identify the critical pairs
     in these cell lines, in which inhibition        of signaling hubs that need to be
                                                                                                 Adjei AA, Cohen RB, Franklin W, Morris C,
     of c-KIT signaling leads to apoptosis           targeted in melanoma for maximal                Wilson D, Molina JR et al. (2008) Phase I
     (Smalley et al., 2008a). Further studies        therapeutic effect. An increased under-         pharmacokinetic and pharmacodynamic
     have shown that treating c-KIT-depen-           standing of the temporal regulation of          study of the oral, small-molecule mitogen-
                                                                                                     activated protein kinase kinase 1/2 inhibitor
     dent melanomas with imatinib leads to           melanoma signaling can also be used
                                                                                                     AZD6244 (ARRY-142886) in patients
     the simultaneous inhibition of MAPK,            to therapeutic advantage. Tumors                with advanced cancers. J Clin Oncol 26:
     PI3K/AKT, and JAK/STAT signaling                that show ‘‘oncogene addiction’’ have           2139–46

34   Journal of Investigative Dermatology (2010), Volume 130
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                                                                                                                          Molecular Targeted Therapy

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