Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling

    Roads to Melanoma: Key Pathways and Emerging Players in Melanoma Progression and Oncogenic Signaling Jasmina Paluncic, Zaklina Kovacevic, Patric J. Jansson, Danuta Kalinowski, Angelica Merlot, Michael L.-H. Huang, Hiu Lok, Sumit Sahni, Darius J.R. Lane, Des R. Richardson PII: DOI: Reference:

S0167-4889(16)30015-5 doi: 10.1016/j.bbamcr.2016.01.025 BBAMCR 17795

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

10 November 2015 27 January 2016 29 January 2016

Please cite this article as: Jasmina Paluncic, Zaklina Kovacevic, Patric J. Jansson, Danuta Kalinowski, Angelica Merlot, Michael L.-H. Huang, Hiu Lok, Sumit Sahni, Darius J.R. Lane, Des R. Richardson, Roads to Melanoma: Key Pathways and Emerging Players in Melanoma Progression and Oncogenic Signaling, BBA - Molecular Cell Research (2016), doi: 10.1016/j.bbamcr.2016.01.025

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ACCEPTED MANUSCRIPT INVITED REVIEW TO BIOCHIMICA BIOPHYSICA ACTA – MOL. CELL RES.

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Oncogenic Signaling

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Roads to Melanoma: Key Pathways and Emerging Players in Melanoma Progression and

Jasmina Paluncic, Zaklina Kovacevic, Patric J. Jansson, Danuta Kalinowski, Angelica Merlot,

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Michael L.-H. Huang, Hiu Lok, Sumit Sahni, Darius J. R. Lane* and Des R. Richardson*

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*Co-corresponding and Co-senior Authors

Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute,

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University of Sydney, Sydney, New South Wales, 2006, Australia.

To whom correspondence should be addressed: Dr. D.R. Richardson, Molecular Pharmacology and Pathology Program, Department of Pathology, Blackburn Building, University of Sydney, Sydney, New South Wales, 2006, AUSTRALIA. Email: [email protected]. Ph: +612-9036-6548; FAX: +61-2-9036-6549; Dr. D.J.R. Lane, Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building, University of Sydney, Sydney, New South Wales, 2031 AUSTRALIA. Ph: +61-2-9351-6144; FAX: +61-2-9036-6549; Email: [email protected]. 1

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Abstract

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Melanoma has markedly increased worldwide during the past several decades in the Caucasian

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population and is responsible for 80% of skin cancer deaths. Considering that metastatic melanoma is almost completely resistant to most current therapies, and is linked with a poor patient prognosis,

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it is crucial to further investigate potential molecular targets. Major cell-autonomous drivers in the pathogenesis of this disease include the classical MAPK (i.e., RAS-RAF-MEK-ERK), WNT and

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PI3K signaling pathways. These pathways play a major role in defining the progression of melanoma, and some have been the subject of recent pharmacological strategies to treat this

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belligerent disease. This review describes the latest advances in the understanding of melanoma

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progression and the major molecular pathways involved. In addition, we discuss the roles of

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emerging molecular players that are involved in melanoma pathogenesis, including the functional

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role of the melanoma tumor antigen, p97/MFI2 (melanotransferrin).

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ACCEPTED MANUSCRIPT 1. General Introduction: Melanoma Melanoma is one of the most aggressive and treatment-resistant human cancers, having increased radically worldwide during the past several decades in the Caucasian population [1]. With an

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overall mortality rate of around 20%, it is responsible for the 80% of skin cancer-related deaths

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worldwide, which is largely due to its propensity to metastasize to other organs [1, 2]. In fact,

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metastatic melanoma is the 4th most common cancer in Australia and the 6th most common cancer in the USA [3, 4]. Although early-stage melanoma can be effectively treated with surgery, metastatic

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melanoma is highly resistant to conventional therapies [3].

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Unfortunately, the annual incidence rates of melanoma worldwide are increasing rapidly each year, and at a greater rate than any other major cancer [1]. According to recent studies, 1- and 2- year

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survival rates in patients with metastatic melanoma were ~25% and 10%, respectively, with a

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median overall survival of 6 months [1]. There is a consensus that ultraviolet (UV)-induced DNA

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damage is a major etiological factor in the pathogenesis of these tumors [5]. Indeed, UVA (wavelength range: 315 to 400 nm) and UVB (wavelength range: 280 to 315 nm) promote deleterious effects on biomolecules and can also trigger DNA damage through UV-induced reactive

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oxygen species (ROS) production, which directly damages DNA, causing genetic alterations [6]. UVB is thought to be more carcinogenic than UVA, as it induces the formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts and development of oxidative stress [7], while UVA is generally is found to produce mainly oxidative stress [8]. Considering these multiple effects, UV radiation may be conducive to melanoma development via combined genotoxic and mitogenic effects in melanocytes [6].

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ACCEPTED MANUSCRIPT 1.1 Melanoma Progression: From Melanocytes to Melanoma 1.1.1 Melanocyte development: an overview Melanoma originates from neural-crest derived melanocytes, which are pigment-producing cells

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located in various anatomic sites, such as the base layer (stratum basale) of the skin‟s epidermis, the

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uvea of the eyes, the inner ear, meninges, bones and heart [9]. During embryonic development,

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multipotent trunk neural crest cells undergo lineage specification to form melanocyte precursors (viz., melanoblasts) [10]. After migration and proliferation between the somites and ectoderm, and

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later invasion into the ectoderm, melanoblasts differentiate into melanocytes [11]. Following the maturation of melanocytes, melanin production starts in special organelles, namely melanosomes

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(Fig. 1), via a biochemical process known as melanogenesis, which involves tyrosine oxidation products that are polymerized to form melanin [12]. Finally, the mature melanosomes (filled with

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granules of melanin) are transported to keratinocytes and the life cycle of melanocytes (hair

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melanocytes) ends with eventual cell death [12].

The transformation of melanocytes into melanoma cells is a multi-step process that begins with the horizontal or radial growth phase, which then progresses towards the invasive phenotype [10].

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Indeed, the development of melanoma from melanocytes occurs via a stepwise mechanism involving clonal succession and acquisition of deleterious genomic alterations [3]. Following the vertical growth phase, the tumor cells invade deeply into the dermis/hypodermis, and eventually penetrate the endothelium of capillaries and enter the blood stream, allowing them to form distant metastases [10]. Integral to the progression of metastatic melanoma is a complex series of molecular events that include multiple and sequential genetic lesions (e.g., driver mutations) that result in an invasive phenotype.

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ACCEPTED MANUSCRIPT 1.1.2 Melanomagenesis and the tumor microenvironment It should be noted that while this review focuses on melanoma cell-autonomous signaling, including major genetic alterations and key signaling pathways, it is well described that melanomagenesis is a

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multi-step process involving the interaction of environmental, genetic, and host factors [13]. Indeed,

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although key genetic drivers are required for melanomagenesis (discussed further in Section 1.1.3),

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they are not sufficient for melanoma development and progression to occur. Importantly, key microenvironmental factors are known to play vital roles in modulating the transformation process,

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thereby increasing or decreasing the likelihood of melanomagenesis and progression [13].

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The composition of the melanoma tumour stroma can wary widely between tumors from different patients [13]. Moreover, the tumor stroma in which melanoma cells grow and proliferate is a

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complex and dynamic microenvironment. This includes interactions with the extracellular matrix,

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microvasculature, fibroblastic cells and changing concentrations of growth factors, cytokines,

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nutrients (e.g., glucose) and key metabolic gases (e.g., oxygen) [13]. All of these factors are active participants in melanomagenesis and the progression towards metastatic disease. Moreover, an understanding of the interplay between the melanoma microenvironment and key genetic drivers

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that are involved in cell-autonomous regulation of melanomagenesis will provide new and effective therapeutic targets. A detailed discussion of this field is outside the scope of this review, and we direct readers to several excellent reviews [13-16].

1.1.3 Some key ‘driver’ mutations in melanoma development Genome-wide sequencing approaches have identified remarkable genetic complexity in melanoma, comprising thousands of mutations, deletions, amplification, translocations and changes in DNA methylation within a single tumor [13]. Despite this abundance and complexity of genetic lesions, many of which are so-called „passenger‟ mutations, several key genetic alterations in melanoma

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ACCEPTED MANUSCRIPT tumorigenesis have been identified that are known as „driver‟ mutations (for recent reviews, see [3, 14]).

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Perhaps the best studied oncogenic mutation in melanoma is that of v-Raf murine sarcoma viral

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oncogene (BRAF) that encodes a serine/threonine protein kinase, which acts in the RAS-RAF-

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MEK-ERK mitogen-activated protein kinase (MAPK) pathway (for review see: [3]). Activating mutations in BRAF occur in >50% of melanomas, with the most common mutation leading to a Val-

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to-Glu substitution at position 600 (p.V600E; [3]). While the wild-type BRAF kinase is typically activated by KRAS, the p.V600E mutation confers BRAF with unregulated kinase activity, leading

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to constitutive activation of MEK that drives the growth-promoting extracellular signal-regulated kinase (ERK) pathway [3]. Interestingly, melanomas that possess wild-type BRAF typically have

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mutations in key genes encoding upstream proteins of the MAPK pathway, including NRAS, KIT,

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GNAQ, and GNA11 [3]. This observation strongly suggests the centrality of over-activation of the

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RAS-RAF-MEK-ERK pathway in melanoma pathogenesis. Moreover, activating BRAF mutations are frequently found in benign and dysplastic nevi, and the activation of BRAF leads to nevus development through a process known as oncogene-induced senescence [3]. Despite the over-

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activation of BRAF in these nevi, there is the induction of cell cycle arrest resulting from the expression of the key cyclin-dependent kinase inhibitor, p16INK4A [15] (see below for further discussion). Nonetheless, BRAF mutations are considered to be common early stage genetic mutations in melanoma progression [3]. However, activating mutations in BRAF alone are not sufficient to cause melanoma. Indeed, additional genetic alterations in BRAF-mutant cells are required to elicit a fully cancerous phenotype [14].

One such further genetic lesion that cooperates with activating BRAF mutations, and represents another high-penetrance genetic alteration in melanoma, is the loss or inactivating mutation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus [16, 17]. Notably, this locus is located on 6

ACCEPTED MANUSCRIPT chromosome 9p21 and encodes two tumor suppressor proteins, p16INK4A and p14ARF (differing in post-translational modifications), which both function to arrest the cell cycle. Importantly, the CDKN2A locus is the most frequently deleted region in melanoma [10], occurring in 16-41% of

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sporadic melanomas and with high penetrance in familial melanoma [10, 14]. As alluded to above,

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p16INK4A is a key negative regulator of the cell cycle that binds to and inhibits the cyclin-dependent

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kinase (CDK4), thereby activating the retinoblastoma (RB) protein (i.e., by preventing its phosphorylation) and preventing cell cycle progression at the G1-S checkpoint [18]. On the other

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hand, p14ARF acts as a positive regulator of p53 by inhibiting its major negative regulator, the nuclear-localized E3 ubiquitin ligase, murine double minute 2 (MDM2) [10, 14]. For this reason, an

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inactivation of CDKN2A through deletion, promoter silencing or mutation leads to uncontrolled cell

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proliferation [10].

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Interestingly, sustained expression of oncogenic BRAFV600E in melanocytes induces the expression of p16INK4A, which does not appear to involve telomere attrition and loss of replicative potential

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[15]. This so-called “oncogene-induced senescence” barrier in melanocytes promotes nevus development and is controlled by the p16INK4A-cyclin D/CDK4-RB cell cycle checkpoint [3, 15]. In

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normal melanocytes, the induction of p16INK4A by the MAPK pathway typically involves the activation of microphthalmia-associated transcription factor (MITF) [19]. Crucially, amplification of the MITF locus is found in 20-30% of melanomas, and is frequently accompanied by activating BRAF mutations and p16INK4A loss [19]. Such observations underscore the importance of this oncogene-induced senescence checkpoint as a gatekeeper of melanoma progression [18, 20].

Another genetic lesion associated with melanoma is amplification or mutations in CDK4, which is associated with a small number of cases of familial melanoma [21, 22]. CDK4 is located on chromosome 12q13 and is the binding partner of p16INK4a. Mutations of CDK4 are often found in its binding domain, making this protein incapable of binding to functional p16 INK4A [21, 22]. Indeed, 7

ACCEPTED MANUSCRIPT the CDK4 pathway is dysregulated in most melanomas as a result of hyper-activation of ERK or loss of p16INK4A (see above; [3]). Both high-penetrance genes, CDKN2A and CDK4, are involved in cell cycle control, as both mutations, which affect p16INK4A and CDK4, respectively, disturb the

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G1/S phase check point [21, 22].

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It is worth emphasizing here that, although promotion of the cell cycle by activation of the RASRAF-MEK-ERK pathway is a major contributor to melanoma pathogenesis and progression

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(discussed further in section 1.2.1), other dysregulated signaling pathways also play a significant etiological role. Indeed, due to the large number of driver genes involved in melanoma progression

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[3], subsequent sections of this review will deal primarily with the major signaling pathways that are dysregulated in melanoma. For more comprehensive reviews of the driver mutation “landscape”

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in melanoma, we refer readers to recent reviews on this topic (see [3, 14]).

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1.2 Key Oncogenic Signaling Pathways Involved in Melanoma Cancer results from abnormal cellular growth and proliferation, which is uncoordinated with that of the normal tissues around it and is caused by a combination of genetic and epigenetic modifications

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that lead to neoplastic transformation [23]. In the past years, several key molecular pathways have been uncovered that are involved in melanoma onset, progression and proliferation [24]. Notably, melanoma metastasis is typically linked with an activation of signaling pathways that are responsible for embryogenesis [25]. The following section will expound the most crucial oncogenic signaling pathways involved in this disease.

1.2.1 MAPK Signaling Pathway The MAPK signal transduction pathway has been the focus of intense investigation in oncology, as it is known to regulate cell growth, proliferation, differentiation, migration and apoptosis [26]. In

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ACCEPTED MANUSCRIPT the field of melanoma biology, the MAPK pathway has been of interest since the discovery of frequent activating mutations of the BRAF kinase in melanoma and nevi [27].

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Initiated either by receptor tyrosine kinases (RTKs) binding to their cognate ligands, or integrin

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adhesion of the extracellular matrix and the cell membrane, MAPK signaling involves the

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transmission of activation signals via Rat sarcoma (RAS) GTPase, which is associated with the inner leaflet of the plasma membrane (Fig. 2) [27]. In its active GTP-bound state, RAS can bind and

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activate RAF (RAF-1, BRAF and ARAF) by a complex sequence of events [26]. The signaling cascade culminates in MEK1/2 dual-specificity protein kinases phosphorylating and activating the

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ERK1 and ERK2 MAPKs, which are then able to translocate to the nucleus and regulate several transcription factors (Fig. 2) [26, 28, 29]. As a result, gene expression patterns are altered by an

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increased expression of nuclear transcription factors, such as c-MYC and MITF etc., leading to cell

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proliferation [26, 28, 29]. Additionally, MAPK signaling can lead to increased expression of cyclin

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D1, which then interacts with CDK4/6 to promote phosphorylation and inhibition of the retinoblastoma family of transcriptional repressors, ultimately leading to the enhancement of E2F-

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dependent transcription of S-phase genes [30].

More recently, and as alluded to above, BRAF has attracted special interest, as it was found to be mutated in 66% of malignant melanomas [26, 31]. Importantly, the V600E somatic missense mutation in BRAF results in a conformational change in the activation loop between kinase subdomains VII and VIII. This mutation induces a constitutive and unregulated activation of kinase activity that leads to hyper-activation of MAPK signaling [26, 29]. Interestingly, this mutation is not a typical UV “fingerprint” mutation as it is more common in melanomas occurring on intermittently sun-exposed skin than melanomas in unexposed or chronically sun-damaged skin (acral and mucosal melanomas), and is often accompanied by amplification of the mutant allele [23]. These and other observations suggest that complex genetic interactions promote BRAF 9

ACCEPTED MANUSCRIPT mutations rather than physicochemical mechanisms [32] and further indicate that BRAF mutations are normally lethal unless a certain genetic and biochemical microenvironment allows those cells to

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survive and proliferate [26].

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1.2.2 WNT Signaling Pathway

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The WNT signaling pathway controls crucial processes, such as cell fate determination, cell polarity, proliferation and migration [33]. The WNT family of proteins are secreted glycoproteins

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that bind to the seven-transmembrane Frizzled receptor (FZD) proteins to initiate intracellular signal transduction [33]. Following the binding of WNT to FZD, three possible signaling pathways are

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activated: (i) a canonical β-catenin-dependent pathway; (ii) a non-canonical β-catenin-independent pathway for cell polarity signaling; and (iii) a WNT-dependent, protein kinase-C (PKC)-dependent

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pathway (Figs. 3,4) [34].

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Abnormal activation of the WNT pathway is considered to be one of the key signaling cascades that results in melanoma development [35]. Indeed, the canonical and non-canonical WNT signaling cascades are thought to affect different stages of tumor progression, as the canonical pathway

[35].

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contributes to melanoma formation, while the non-canonical pathways is involved in metastasis

1.2.2.1 The Canonical WNT Pathway The key role of this pathway is the accumulation and translocation of the adherens junction complex molecule, β-catenin, into the nucleus, where it cooperates with other transcription factors to activate WNT target gene expression (Fig. 3) [33]. As part of its normal activity, β-catenin functions as an anchor to the actin cytoskeleton by directly linking it with a single-span, transmembrane glycoprotein, namely E-cadherin, via α-catenin [33]. In fact, β-catenin is, in general, 10

ACCEPTED MANUSCRIPT bound at the cell membrane to the intracellular domain of E-cadherin as part of the adherens junction complex [36].

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In the absence of WNT signaling (Fig. 3A), a β-catenin destruction complex comprised of Axin,

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adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1α

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(CK1α) causes hyper-phosphorylation of β-catenin by casein kinase and GSK3β, targeting it for ubiquitination and subsequent degradation by the proteasome [33]. Binding of WNT (Fig. 3B) to its

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receptor, FZD, and to the co-receptor low-density-lipoprotein-related proteins 5/6 (LRP5/6), leads to the release of β-catenin from E-cadherin and the stabilization of β-catenin within the cytoplasm

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[33]. As a result, β-catenin then translocates into the nucleus, where it acts as a co-activator of the TCF/LEF transcription factors to up-regulate WNT-responsive genes, including c-MYC, cyclin D1

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and ZEB-1, which promote proliferation, cell cycle progression and inhibition of E-cadherin

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expression, respectively, in melanoma and other cancers [37-39].

The non-canonical pathway can be divided into two separate branches: the planar cell polarity (PCP) pathway and the WNT/Ca2+ pathway, the latter of which is often denoted as the β-catenin-

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independent pathway (Fig. 4) [33]. The PCP pathway is distinguished by its regulation of the actin cytoskeleton (for a review see [33]). However, a detailed discussion of these additional WNT pathways is beyond the scope of this review, due to the uncertainty of their role in melanoma. For further information of the non-canonical pathways, we refer readers to the following reviews[40] [33, 40].

1.2.3 WNT Signaling and its Role in Melanoma Although WNT signaling is considered to be an important driver of oncogenesis in diverse cancers, the precise role of WNT/β-catenin signaling in melanoma remains unclear [35, 41, 42]. 11

ACCEPTED MANUSCRIPT Nevertheless, immunohistochemical detection of nuclear β-catenin has provided evidence for the activation of the canonical WNT/β-catenin pathway in a subclass of primary melanomas, suggesting a role for this pathway in melanoma development [41]. Additionally, constitutively activated

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canonical WNT signaling via β-catenin acts synergistically with the MAPK cascade, with both

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signaling pathways resulting in the induction of melanoma formation and development [43].

Normally, WNT signaling is required for the development of melanocytes from their neural crest

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precursors [44]. This is mediated by β-catenin-dependent up-regulation of transcriptional targets, such as the homeobox gene, MITF, which encodes the transcription factor, MITF, that promotes

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pigment cell formation [44]. MITF modulates the expression of several cell-cycle progression and differentiation genes, such as CDK2, as well as the expression of melanogenic proteins [45]. Hence,

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it is recognized as the master regulator of melanocyte development, survival and function [45, 46].

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In addition, β-catenin increases the growth and proliferation of melanoma cells via up-regulating

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the POU domain transcription factor, BRN2, which has been implicated in controlling proliferation of melanoma cells [47].

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An interesting recent study by Grossmann and colleagues has provided a functional connection between the canonical WNT signaling pathway and the non-canonical pathway via its ligand WNT5A in melanoma metastasis [48]. Non-canonical WNT signaling contributes to tumor metastasis after its activation by WNT5A [49]. As part of this mechanism, the binding of the WNT5A ligand to the FZD4-LRP6 receptor complex at the cell surface of melanoma cells leads to activation of the small GTPase, ARF6, which releases β-catenin from its interaction with Ncadherin, thereby increasing: (i) the pool of free β-catenin; (ii) β-catenin-mediated transcription, and (iii) invasion. WNT5A signals via protein kinase C (PKC) and calcium-dependent enzymes independently of β-catenin, leading to melanoma cell migration and metastasis [48]. In fact, WNT5A gene over-expression is commonly linked with high grade melanomas [50, 51]. 12

ACCEPTED MANUSCRIPT Interestingly, WNT5A can promote the canonical WNT pathway by interacting with FZD and LRP6 to increase β-catenin signaling [48]. However, while this latter induction of canonical WNT signaling by WNT5A has been demonstrated in non-melanoma cell types, such as HEK293 and

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mouse L cell lines, it is yet to be examined in melanoma [52]. Notably, Weeraratna and colleagues

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have established that WNT/PKC stimulates cell motility via the regulation of genes involved with

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the epithelial to mesenchymal transition (EMT), including up-regulation of E-cadherin and downregulation of vimentin and SNAIL in melanoma cells [51] (see 1.2.5 for further discussion of the

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EMT in melanoma).

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1.2.4 GPCRs in Melanoma

In addition to the key WNT receptor FZD, it is interesting to briefly consider the role in melanoma

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of other G-protein-coupled receptor (GPCR) family members, to which FZD belongs [57]. Their

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major role involves amongst others, the regulation of physiological functions, such as

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neurotransmission, immune responses and blood pressure regulation [57]. However, GPCRs also play a key role in proliferating cells, contributing to inflammation, angiogenesis, normal cell growth, cancer, etc. There is increasing evidence, that GPCRs are involved in tumorigenesis and

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metastatic progression of melanoma by playing a significant role in tumor-induced angiogenesis and tumor metastasis [57]. Several GPCRs are involved in melanoma, such as melanocortin type 1 receptor (MC1R), endothelin receptors (EDNR), WNT/FZD, chemokine receptor (CXCR) and protease-activated receptor-1, thrombin receptor (PAR-1). For further reviews on the role of GPCRs in melanoma, we refer readers to the following recent review [57].

1.2.4.1 Exosomes and vesicular trafficking in melanoma Increasing evidence suggests that another key process involved in melanoma is altered vesicular trafficking, and in particular, the release of exosomes that play an important role in intercellular communication [7]. Exosomes are able to carry a host of molecules including transfer mRNA, 13

ACCEPTED MANUSCRIPT miRNA and proteins, and are involved in various biological functions, including protein and genetic intercellular exchange, immune regulation and modulation of therapy response that have an important role in melanoma progression [9, 10]. Notably, it has been demonstrated that WNT5A

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induces the release of exosomes filled with pro-angiogenic and immunosuppressive factors from

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malignant melanoma cells [7, 11]. Therefore, transfer of oncogenes may control the activity of

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signaling pathways and further play an active role in tumor growth and metastasis [7, 11]. Released by melanoma cells, exosomes may contribute to anti-tumor immune response by damaging

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macrophage and dendritic cell functions and moreover, their transfer of proteins and microRNA to multiple cell types, such as endothelial cells, fibroblasts and bone marrow progenitor cells leads to

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the generation of metastatic microenvironment [7].

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Importantly, the role of exosomes in transporting miRNAs is crucial to the crosstalk between

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melanoma cells and their microenvironment and promotes proliferation of malignant cells and their

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metastatic and invasive capabilities. For a recent review of this intriguing topic, please see [58].

In summary, WNT signaling in melanoma is extremely complex, and requires the differential

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involvement and coordinated expression of multiple intracellular molecules and interacting pathways throughout the progression of the disease. As such, although WNT signaling is a key driver of melanoma progression, the precise functions of the WNT pathways in melanoma remain to be fully elucidated.

1.2.5 PI3K Signaling Pathway Another signaling pathway known to be crucially involved in melanoma is the phosphatidylinositol3-kinase (PI3K) pathway [53]. Belonging to a conserved group of lipid kinases, PI3K is involved in various functions within the cell, such as cell growth, proliferation and metabolism [54]. There are three subgroups of these enzymes: class 1, 2 and 3, with class 1 being the most extensively 14

ACCEPTED MANUSCRIPT characterized [55]. PI3Ks are canonically activated by insulin growth factor 1 (IGF-1) binding to the IGF-1 receptor [55]. The central role of class 1 PI3Ks is the catalysis of phosphatidylinositol4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), which then functions as

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a docking site for 3-phosphoinositide-dependent protein-kinase 1 (PDK1) to activate the protein

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kinase [54].

PDK1 is able to phosphorylate the serine-threonine-specific kinase, AKT (also known as protein

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kinase B), leading to its activation [54]. The active p-AKT then has the potential to phosphorylate up to 100 target proteins [54]. One of these substrates is GSK3β, which is inhibited when

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phosphorylated by p-AKT [56]. Indeed, as a result of GSK3β inhibition, free β-catenin is able to accumulate and translocate to the nucleus, where it can up-regulate expression of oncogenic genes,

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such as c-MYC and cyclin D1 (Fig. 5) [33]. In addition, phosphorylation of ERK by p-AKT causes

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increased expression of c-MYC by activating the mammalian target of rapamycin (mTOR) [57].

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Through these regulatory mechanisms, p-AKT elicits a strong anti-apoptotic effect and promotes cancer progression [58].

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1.2.5.1 PI3K Signaling Pathway and its Role in Melanoma Oncogenic RAS, which is involved in MAPK signaling (see above), is also known to be a positive upstream regulator of the PI3K pathway [59]. Hence, activation of RAS transcription leads to downstream activation of two different, but interconnected pathways: the RAF-MEK-ERK pathway and the PI3K-AKT signaling pathway, the latter of which leads to activation of AKT and its downstream targets [29]. While both cause cell proliferation, dissemination and survival, the PI3KAKT signaling pathway also promotes anabolism, whereas the RAF-MEK-ERK pathway is more active in proliferation and invasion [29, 60].

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ACCEPTED MANUSCRIPT As a PI3K pathway inhibitor, the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), catalyzes the de-phosphorylation reaction of PIP3 back to PIP2 via its lipid phosphatase activity, resulting in reduced p-AKT levels and inhibition of the PI3K signaling pathway (Fig. 5;

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[55]. Moreover, PTEN is able to target and dephosphorylate other proteins, such as focal adhesion

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kinase (FAK), resulting in inhibition of focal adhesions and a decrease in cellular migration [61,

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62]. Interestingly, PTEN is also thought to interact with MAPK signaling by dephosphorylating

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adapter proteins, leading to decreased MEK1/2 and ERK1/2 phosphorylation [63].

Uncontrolled signaling through the PI3K pathway results from PTEN loss/inactivation [64] and

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notably, the loss of tumor suppressor genes on chromosome 10, including PTEN, is detected in 30% to 60% of non-inherited melanomas [65]. Importantly, a loss of PTEN expression has been observed

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in 30% to 50% of established melanoma cell lines and 5% to 20% of primary melanomas [63].

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Notably, both BRAF and PTEN mutations have been found to occur concomitantly at high rates in

and PTEN [27].

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melanoma, while N-RAS was found to be mutated in mutual exclusivity with mutations in BRAF

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1.2.6 Metabolic reprogramming and vesicular trafficking in melanoma Melanomagenesis and the development of metastatic disease is associated with distinct changes in cellular metabolism, particularly changes in energy metabolism that involve a shift from mitochondrial oxidative phosphorylation to cytoplasmic aerobic glycolysis (known as the “Warburg effect”) [3]. While glycolysis is a mitochondria-independent process, oxidative phosphorylation depends on intact mitochondria to generate ATP [1]. Although the metabolic switch to aerobic glycolysis classically results in the less efficient utilization of glucose, the benefits of a more rapid production of ATP, as well as increases in levels of certain biosynthetic intermediates is required to meet the melanoma cells‟ increased bioenergetic and biosynthetic demands [3]. Besides assuring energy supply for rapidly proliferating cells, aerobic glycolysis is important for invasive and 16

ACCEPTED MANUSCRIPT metastatic cells [3]. Indeed, studies have demonstrated that some cancer-types with KRAS and BRAF mutations have increased glucose uptake activities and glycolytic rates [4]. These cells can

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survive better under hypoglycemic conditions than non-mutated cell lines [4].

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One of the key regulators of the switch to aerobic glycolysis is hypoxia inducible factor 1α (HIF1α), which can be induced in response to a number of cellular stressors, including hypoxia and oxidative stress [72]. Normally, HIF1α is a key part of an adaptive response to low oxygen levels that leads to

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increased expression of glucose transporters and glycolytic enzymes, as well as a down-regulation

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of mitochondrial energy metabolism [3, 72]. The effects of HIF1α can be partially offset by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) [3, 73]. Interestingly, MITF, which is a key regulator of melanocyte differentiation, also plays a crucial role

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as a regulator of mitochondrial metabolism via the regulation of PGC1-α. This appears to occur, at least in part, through the ability of MITF to the transcriptionally activate PGC1-α, which suppresses

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aerobic glycolysis by promoting mitochondrial oxidative phosphorylation through enhancing mitochondrial biogenesis and respiration [73]. Under normal conditions, PGC1-α expression is low,

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but becomes strongly increased when there is a high metabolic or energetic demand [7]. PGC1-αhigh cells produce more ATP, are more resistant to genotoxic chemotherapeutics and generate less lactate than PGC1-α-low cells [66]. Furthermore, the increase of mitochondrial mass via PGC1-α alters tumor cell metabolism to oxidative phosphorylation [7, 8].

Importantly, activation of the MAPK pathway via BRAFV600E acts to negatively regulate MITF, thereby suppressing the effects of PGC1-α. Moreover, this oncogenic driver also acts to increase the expression of HIF1α via indirect stimulation of mTOR [3]. Activation of BRAFV600E also leads to phosphorylation of the protein LKB1, thereby suppressing a key energetic-stress checkpoint

17

ACCEPTED MANUSCRIPT comprised of LKB1 and its target, AMPK [74]. This results in a lack of responsiveness of cells to

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high AMP/ATP ratios [3].

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Overactivation of PI3K-AKT signaling is also critical in the metabolic reprogramming that occurs

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in melanoma, which appears to involve the AKT-dependent activation of mTOR and its downstream target, HIF1α [3]. In the case of the WNT pathway, the increased expression of c-MYC that results from hyper-activation of canonical WNT signaling is known to lead to a host of changes

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in cellular energy and carbon metabolism, many of which overlap with those changes induced by

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HIF1α [75]. However, whether this facet of metabolic reprogramming is a significant contributor to metabolic reprogramming in melanoma remains to be determined. For more information on

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metabolic reprogramming in melanoma, please see the following recent review [3].

1.2.7 The Epithelial-to-Mesenchymal Transition and Melanoma

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A crucial enabling process that is involved in melanoma development and progression is the EMTlike phenotype switching. Under normal physiological conditions, the EMT is vital during

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development, and is characterized by rapid and often reversible changes in cellular phenotype [67]. It is not only required for embryonic development, tissue remodeling and wound repair, but is also initially responsible for cell migration, invasion and metastasis [67-69]. During the EMT, epithelial cells lose their cell-cell adherens junctions, reorganize their cytoskeleton, alter their polarity and gain mesenchymal characteristics via a switch in expression from keratin- to vimentin-type filaments (Fig. 6) [67]. In association with the over-expression of N-cadherin, the mesenchymal marker vimentin is also increased in cancer cells undergoing the EMT [70]. Moreover, vimentin is an intermediate filament and plays a multifunctional role in cancer cell motility and mesenchymal cell shape [71].

18

ACCEPTED MANUSCRIPT Having migrated to their target destinations, it is likely that tumor cells may revert to their original epithelial phenotype through a process known as mesenchymal-epithelial transition (MET), leading to the formation of secondary cancers (Fig. 7). This reverse process illustrates an inherent plasticity

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of the epithelial phenotype [67].

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E-Cadherin, a single-span, trans-membrane glycoprotein expressed in all epithelia, has been demonstrated to act as a tumor suppressor through its ability to bind in trans to E-cadherin from

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neighbouring cells through homotypic interactions, thereby promoting cell to cell adhesion and preventing metastasis (Fig. 8) [72]. Furthermore, E-cadherin forms a complex with β-catenin, p120

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and α-catenin, which in turn connect this complex with the actin cytoskeleton, allowing the cell to be anchored to the neighbouring cells and to maintain the epithelial phenotype. However, loss of E-

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cadherin releases free β-catenin into the cytoplasm, which is then able to act as a coactivator of the

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TCF/LEF transcription factors by entering the nucleus (Fig. 5) [33]. The down-regulation of Ecadherin is accompanied by an increase of mesenchymal neural cadherin (N-cadherin), in what is

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termed the “cadherin switch”, which alters cell adhesion by facilitating cell migration and invasion [67]. N-cadherin is connected to the cytoskeleton via α- and β-catenin and also interacts with p120

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catenin, signaling mediators and receptor tyrosine kinases (RTKs) [67]. Moreover, in contrast to Ecadherin, which is ubiquitously expressed, N-cadherin is only expressed in neural tissue and muscle under normal conditions [67, 73]. Interestingly, through this “cadherin switch”, transitioning cells obtain a greater affinity for mesenchymal cells, while losing their association with epithelial cells via homotypic N-cadherin interactions [67]. Considering that these latter interactions are weaker than the homotypic E-cadherin interactions, this further facilitates cell migration and invasion [67]. Studies have demonstrated that this switch is more evident in metastatic than in primary tumors, leading to the hypothesis that N-cadherin itself can promote tumor progression and metastasis [70]. In fact, the decrease in E-cadherin during tumor development was observed in several different tumor types, such as malignant melanoma, and is correlated with poor patient prognosis [74]. 19

ACCEPTED MANUSCRIPT While the expression of the E-cadherin gene, CDH1, can be downregulated by promoter hypermethylation in a subset of pancreatic cancers [75], several inducible transcriptional factors are

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well-described to suppress expression of CDH1 by binding to the promoter region via their

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carboxyterminal zinc fingers (for reviews, see [76] and [67]). These include the SNAIL family

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members, SNAIL1 and SNAIL2 (also known as SLUG), which are homologous zinc finger transcriptional repressors that function to activate the EMT [77, 78]. This group of EMT-inducing

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transcription factors repress epithelial genes, such as E-cadherin, by binding to E-box DNA in the promoter region of CDH1 [67]. However, while acting as a direct repressor of genes involved in

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maintenance of the epithelial phenotype, SNAIL proteins can directly up-regulate the expression of genes involved in promoting a mesenchymal phenotype [76]. In fact, besides acting as a suppressor

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of E-cadherin, SNAIL is also well-known to regulate several aspects of the EMT phenotype,

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including the down-regulation of epithelial markers, such as claudins, occludins and cytokeratins,

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and the up-regulation of matrix metalloproteinase (MMP) levels and expression of mesenchymal cell/fibroblast makers, such as fibronectin and vitronectin (Fig. 6) [77]. Importantly, MMPs can target transmembrane proteins, such as stromelysin-1, leading to the release of the extracellular

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domain of E-cadherin, resulting in loss of the adherens junction [79].

SNAIL1 and SLUG can be induced by a variety of signaling pathways, including bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signaling, as well as RAS-dependent signaling through the RAF-MEK-ERK and PI3K-AKT pathways [80]. In the case of melanoma, loss or inactivity of PTEN correlated with a switch from E-cadherin to N-cadherin expression during melanoma progression that could be reversed by reintroduction of PTEN or pharmacologic blockade of PI3K activity [81]. This cadherin switch in melanoma cells was found to be mediated by the PI3K-dependent transcriptional up-regulation of SNAIL, and another Ecadherin transcriptional repressor, twist-related protein 1 (TWIST1) [81]. Moreover, a recent study 20

ACCEPTED MANUSCRIPT has indicated that PI3K-AKT signaling in melanoma can lead to up-regulation of SLUG expression, which appears to be driven by osteonectin (also known as SPARC), a secreted extracellular matrix-

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associated factor that promotes the EMT [82].

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Activation of NRAS and BRAF has recently been found to mediate an EMT-like phenotype switch in late-stage melanoma that relies on TWIST1, another transcriptional repressor, ZEB1, and Ecadherin loss, and results in enhanced invasion [6]. TWIST1 is a crucial basic helix loop helix

including prostate [83], breast

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transcription factor in embryological morphogenesis, and is over-expressed in various tumor-types, and gastric [84] cancers, and acts to repress E-cadherin and

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simultaneously activate mesenchymal gene expression by inducing N-cadherin expression [67, 85]. Intriguingly, a recent study by Caramel and colleagues indicates that a switch in expression of

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embryonic EMT-inducing transcription factors drives the development of malignant melanoma, and

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predicts a poor outcome in melanoma patients [86]. Strikingly, SLUG and ZEB2 are expressed in

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normal melanocytes where they act as onco-suppressive proteins by activating MITF-dependent differentiation. However, in response to NRAS or BRAF activation in late stage melanoma, TWIST1 and ZEB1 are preferentially expressed, leading to the promotion of a reversible de-

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differentiation and neoplastic transformation [86].

In conclusion, the WNT-signaling pathway is one of the key regulators that drives EMT and plays an important role in the promotion of melanoma progression and EMT-like phenotype switchdependent metastasis [87].

2. Other Pathways and Molecules Under Investigation in Melanoma Pathogenesis The exemplified key molecular pathways described above are involved in melanoma onset, progression and proliferation [24]. In addition to these oncogenic pathways, a number of molecules 21

ACCEPTED MANUSCRIPT including p53, c-KIT and melanoma tumor antigen p97 (melanotransferrin; MFI2/MTf) also play

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vital roles in melanoma progression.

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2.1 p53 and melanoma

As discussed above, the most crucial DNA-damaging stress in melanomagenesis is UV radiation [27]. In fact, the cellular stress caused by UV light compromises the fidelity of DNA replication,

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genomic stability, chromosome repair and cell division [88-90]. The tumor suppressor, p53, is a master regulator of the cellular stress response to DNA damage, and can function as a transcription

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factor to up-regulate genes that accelerate DNA repair and inhibit cell cycle progression, or, in the case of extensive DNA damage, can initiate cell senescence or apoptosis [91]. However, p53 is

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frequently found to be inactivated or mutated in most human cancers [92, 93]. Interestingly, it has

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been estimated that nearly 90% of human melanomas contain inactivated, functionally defective or

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hypo-expressed p53, although the mechanisms leading to this inactivation and/or hypo-expression are varied [94-96]. A recent study has indicated that p53 function in melanoma may be able to be restored by simultaneously blocking the p53 E3 ligase, MDM2, and the p53 inhibitor, inhibitor of

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apoptosis-stimulating protein of p53 (iASPP) [94]. Indeed, this study demonstrated that reactivation of p53 by this strategy, together with inhibition of BRAFV600E, induced apoptosis and suppressed melanoma growth [94].

2.2 c-KIT and melanoma c-KIT is a receptor tyrosine kinase for the stem cell factor (SCF), and is essential for proliferation, melanocyte development, differentiation, survival, and migration [97]. Interestingly, its downstream effectors include MAPK and PI3K cascades [97] and MITF [98]. SCF on the other hand is responsible for increased melanin production in normal melanocytes, and hence, their survival is 22

ACCEPTED MANUSCRIPT dependent on c-KIT signaling [97]. Pathogenic mutations of KIT have been found in a large number of tumors and notably, these mutations were found in 39% of mucosal melanomas, 36% of acral melanomas, 28% of melanomas on chronically sun damaged skin, but 0% of melanomas on skin

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without sun damage, suggesting an active role of UV radiation in mutations of KIT [99].

2.3 p97/melanotransferrin and melanoma

Another molecule linked with melanoma development is one of the first tumor antigens described

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for this tumor-type [100], namely MFI2/MTf. Indeed, MFI2 plays a vital role in cell proliferation

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and migration, endothelial migration and angiogenesis, differentiation and tumorigenesis (Fig. 9) [101]. To date, the function of MFI2 remains unclear, but several investigations have suggested its involvement in physiological and pathological processes, including arthritis [102], chondrogenesis

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[103], Alzheimer‟s disease [104, 105], eosinophil differentiation [106], angiogenesis [107] and plasminogen activation [108]. A soluble and actively secreted form of MFI2 (sMFI2) has been

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reported to be increased in patients with Alzheimer‟s disease or arthritis and may modulate angiogenesis, cell migration and plasminogen activation [107-110]. Interestingly, it has also been

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demonstrated that MFI2 can interact with an unidentified member of the lipoprotein receptor-related protein (LRP) family, followed by the internalization of MFI2 [108, 111], although the molecular function of this process remains unclear.

The highest levels of MFI2 expression are found in malignant melanoma cells, although it was also found in small quantities in normal tissues [112, 113]. Selectively down-regulating MFI2 expression resulted in inhibition of migration and invasion, proliferation and growth of melanoma in vitro [114]. Furthermore, silencing of MFI2 expression in melanoma cells resulted in considerably decreased cell proliferation, melanoma xenograft growth, DNA synthesis, cellular migration and reduced tumor initiation in nude mice [101]. In more recent studies, it has been shown that the down-regulation of Mfi2 in mice results in up-regulation of myocyte enhancer factor 23

ACCEPTED MANUSCRIPT 2a (Mef2a), transcription factor 4 (Tcf4) and glutaminase (Gls), whereas the apolipoprotein D (Apod) gene was down-regulated [101, 102]. Both, Mef2a and Tcf4 are linked with cell proliferation and survival and are known transcription factors [115, 116]. The Mef2a transcription factor is found

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to be strongly expressed in muscle tissues and is involved in fetal cardiac development, MAPK

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signaling and calcium signaling [115-119]. Interestingly, the Tcf4 gene product is a transcription

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factor that physically interacts with β-catenin and is crucially involved in the WNT signaling pathway [120], as well as the regulation of melanocyte differentiation [119]. Moreover, Tcf4 is

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highly expressed in some cancers and plays an important role in cell proliferation [116].

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Recent observations suggest that melanoma cells can drive the progression of the disease by “switching” between proliferative and invasive states (potentially utilizing EMT and MET

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reversals) that involves WNT signaling [121]. This phenotypic switching behavior was found to

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crucially involve changes in the expression of TCF4, and another β-catenin-binding transcription

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factor, LEF1 [121]. Specifically, LEF1 was preferentially expressed in differentiated and rapidly proliferating melanoma cells, while TCF4 was preferentially expressed in de-differentiated and invasive melanoma cells [121]. Taken together, such findings are strongly suggestive of a role for

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MFI2 in modulating the WNT signaling pathway.

In contrast, the MFI2-regulated molecules, Apod and Gls, show differential expression and encode proteins that are important in proliferation, namely Apod and glutaminase, respectively [101]. In fact, Apod inhibits proliferation and is linked with transporting sterols, steroids and arachidonic acid for tissue repair [122, 123]. Conversely, glutaminase has been established to be highly expressed in mitochondria of tumor cells immediately before the maximum rate of proliferation is attained [124-126]. Hence, the down-regulation of Apod and up-regulation of Gls by Mfi2 deletion in mice supports a role for this latter protein in proliferation [101].

24

ACCEPTED MANUSCRIPT At present, metastatic melanoma is almost completely resistant to available therapies and is linked with poor patient prognosis. Hence, a new and expanded knowledge of novel prognostic markers that can affect melanoma metastasis, such as MFI2, may be crucial for the development of new and

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3. Current Drugs and Drug Targets in Melanoma

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more effective treatment strategies.

Since most of the activating mutations (>70%) in the BRAF gene are V600E and because mutated

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BRAF leads to activation of the MAPK signaling pathway, much of the current research has focused on inhibiting the constitutively active BRAF protein kinase in melanoma patients [127, 128].

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Melanoma in the metastatic stage (stage III and IV) is exceptionally resistant to many types of treatments, and hence, new therapeutics that are able to target metastatic melanoma are crucial, as

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these patients have very low survival rates (6-10 months) [32].

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Before 2011, neither administration of the cytokine, IL-2, nor the chemotherapeutic agent, dacarbazine (DTIC), were able to improve the overall survival rate (OR) of melanoma patients [32]. While DITC is limited by an insufficient OR of 8 months and low response rate (RR; 5-15 %),

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high-dose IL-2 is limited by a low RR of 6-10% as well as a short duration of response in most patients and is associated with severe side effects [129]. Since 2011, several novel anti-melanoma agents have received FDA approval, and these include: (1) vermurafenib, which targets melanomas with mutated BRAFV600E; (2) ipilimumab, a therapeutic anti-CTLA-4 monoclonal antibody; and (3) the pegylated form of interferon α-2b (PEG-IFN), currently used as adjuvant treatment for stage III melanoma [130]. Additionally, besides vermurafenib, other agents, such as dabrafenib and trametinib have been approved for melanomas with the BRAF V600E mutation [32].

Mutated BRAF leads to activation of the MAPK signaling pathway and therefore, concentrated research has focused on inhibiting BRAF in melanoma patients [127, 128]. However, due to the off25

ACCEPTED MANUSCRIPT target effects induced via inhibition of wild-type BRAF, the results were unsatisfactory [128]. Only vermurafenib was capable of silencing mutant BRAFV600E without interfering with the wild-type BRAF [128]. As such, vermurafenib represents an excellent model for successful targeted antiV600E

mutation [128, 131]. Nevertheless, as a highly

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cancer therapy for patients with the BRAF

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selective mutant BRAFV600E inhibitor, the single use of vermurafenib is not as efficient in patients

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who have mutations other than V600E (e.g., V600K, which is present in 5-20% patients with

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melanoma), and hence, these patient have a correspondingly low RR [128, 132].

Immune check point inhibitors are also emerging as highly promising novel drug targets for

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melanoma [32, 133, 134]. As a negative regulator of T-cells, the T-cell receptor, programmed cell death-1 (PD-1), acts to limit T-cell cytotoxic activity via interacting with its two ligands, PD-L1 and

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PD-L2, which can be highly expressed by melanoma cells [32]. Hence, this modulator is an

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interesting target for investigations of innovative immune-based therapy drugs by developing agents

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that can block these receptors, thereby leading to re-activation of T-cells, and hence, regenerated anti-tumor activity [32, 133]. In fact, malignant skin cancer cells use this inhibitory mechanism to prevent effective anti-tumor responses by “turning off” T-cell activation and furthermore, PD-1 is

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present on the surface of activated T, pro-B, NK cells and monocytes [32]. With a high affinity for PD-1, Nivolumab and pembrolizumab (monoclonal immunoglobulin G4 antibodies) act to prevent PD-1 interaction with its ligands (PD-L1 and PD-L2) in the tumor microenvironment [135, 136]. In fact, the expression of PD-L1 on cancer cells may be enhanced by BRAFV600E signaling via the MAPK pathway, which stimulates production of IL-1 and PD-1 ligands that depress T-cell activity and promote tumor growth [32].

To date, nivolumab and pembrolizumab have both shown high activity in multiple clinical trials, inducing responses in 20-40% of patients, with less than 10% of patients experiencing a grade 3/4 adverse event [32]. Moreover, nivolumab and pembrolizumab are both linked with an appropriate 26

ACCEPTED MANUSCRIPT tolerance and have a long-standing safety profile [32]. Notably, PD-1 blockade occurs in melanomas without mutated BRAF, highlighting the emerging importance of the PD-1/PD-L1

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pathway in malignant melanoma [32].

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As one of the major key pathways involved in melanoma, PI3K is known to cause cell proliferation,

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anabolism, dissemination and tumor survival [29, 60]. Hence, investigations have focused on PI3K signaling targets in melanoma. Due to the fact that mTOR is a serine/threonine kinase downstream

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of AKT that modulates angiogenesis, protein synthesis and cell cycle progression [64], mTOR inhibitors may be rational therapeutic agents in melanoma [27, 64]. In fact, studies undertaken by

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Karbowniczek et al. demonstrated that mTOR activation is strongly associated with melanocytic lesions in vivo [137]. Moreover, the inhibition of mTOR prevented the proliferation of melanoma-

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derived cell lines [137]. The agents, Temsirolismus (CCI-779; Wyeth-Ayerst, Madison, NJ),

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Everolismus (Novartis) and AP-23573 (Ariad Pharmaceuticals, Cambridge, MA), are among the

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current mTOR inhibitors with clinical potential [27]. In a two-stage phase II trial with the singleagent, Everolismus, 7 of 20 patients (35%) demonstrated stable disease at 16 weeks in stage IV

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melanoma [27].

Considering the importance of deregulated WNT signaling activation in the pathogenesis of melanoma, a range of new strategies in terms of cancer pharmacopoeia have been assessed. In fact, since the late 1990s, intense interest in developing effective WNT pathway inhibitors [138, 139] has led to non-steroidal anti-inflammatory drugs (NSAIDS) and vitamin derivatives that directly or indirectly suppress the dysregulated WNT pathway [140]. However, no effective treatment has resulted.

In order to overcome single line treatment resistance and hence, treating melanoma successfully, there is an emerging paradigm shift in treatment strategies from single target towards a multifaceted 27

ACCEPTED MANUSCRIPT combination therapy strategy could be beneficial, rather than targeting only one oncogenic driver [35]. One example of these strategies is maximizing the efficacy of BRAF inhibitors by combining them with the MEK inhibitor, Trametinib, as several resistance mechanisms of BRAF inhibitors

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lead automatically to MEK activation [35]. Hence, these combination therapies may provide strong

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inactivation of the MAPK signaling pathway [35, 150].

As most of the more recently developed anti-cancer drugs focus on highly specific molecular targets

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to modulate an explicit abnormal pathway, it is not surprising that these drugs result in treatment failure [141]. In this case, selection pressure by a single target chemotherapeutic results in tumor

dysregulated

and

involve

both

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clones that demonstrate resistance [141]. In diseases such as cancer, where multiple pathways are independent

and

overlapping

molecular

targets,

a

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polypharmacological strategy that focuses on a range of key molecules may be more beneficial and

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may prevent cancer cells from adapting and developing [142]. This is especially important as cancer

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is a disease that progresses with some molecular targets becoming more crucial than others at

3.1

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different stages [143].

Possible

Novel

Strategies

for

Melanoma

Treatment:

The

di-2-pyridylketone

thiosemicarbazones

A novel class of agents that demonstrate polypharmacology are the di-2-pyridylketone thiosemicarbazones (DpT series), including the lead compounds of this series, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone 4-cyclohexyl-4-methyl-3thiosemicarbazone (DpC) [141]. Tumors require a greater amount of iron and copper for their rapid growth and proliferation than normal cells [144-146]. Interestingly, the DpT series of agents target these metal ions, leading to the formation of redox active iron or copper complexes that generate 28

ACCEPTED MANUSCRIPT cytotoxic reactive oxygen species (ROS) [147, 148]. Furthermore, it has been demonstrated that the DpT agents are also able to inhibit multiple oncogenic pathways, such as MAPK [149], RAD/ERK [150], PI3K [150, 151], SRC [152], STAT3 [153] and the transforming growth factor-β signaling

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pathways [150]. In addition, these agents have also been shown to inhibit the EMT, that is

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responsible for metastasis [68, 154], while also leading to dysfunctional pro-survival autophagy

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[155, 156]. Moreover, a recent study on Dp44mT demonstrated that this agent could overcome drug resistance mediated by the drug efflux pump, P-glycoprotein (P-gp) [157]. Considering that P-gp is

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an important contributor to multidrug resistance in metastatic melanoma [158], these novel agents

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may be highly effective in treating advanced and multi-drug resistant melanoma.

Notably, Dp44mT and its analogues have shown effective anti-tumor activity in vitro and in vivo

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against numerous aggressive tumor xenografts, such as neuroepithelioma, pancreatic lung

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carcinoma, hepatoma, breast and melanoma [68, 157, 159-167]. In fact, Dp44mT demonstrated more potent activity in vivo than a 16-fold higher dose of the clinically-trialed thiosemicarbazone,

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Triapine® [159, 160]. Moreover, this agent demonstrated marked activity at inhibiting the dissemination of breast cancer cells in vivo [152, 164]. The ligand, DpC, is a newer analogue of the

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DpT series that is highly potent and does not induce cardiotoxicity in mice, even when used at markedly higher doses than Dp44mT [162, 163]. Indeed, studies have demonstrated that Dp44mT causes cardiac fibrosis at high non-optimal doses [159] and also the formation of methemoglobin (metHb) and metmyoglobin (metMb) in vivo in mice [168]. On the contrary, DpC has been demonstrated to significantly reduce these adverse effects and have higher activity than Dp44mT in inhibiting pancreatic tumor growth in vivo in nude mice [169] and in lung cancer cells [170].

Metastasis is a major clinical problem that is responsible for 90% of cancer deaths, and hence, development of drugs that target proteins that inhibit metastasis has become an important therapeutic strategy [143, 171, 172]. One of these proteins is N-MYC downstream regulated gene-1 29

ACCEPTED MANUSCRIPT (NDRG1) [162, 171, 173]. This potent metastasis suppressor has been found to play an important role in decreasing metastases and inhibiting angiogenesis by inhibiting multiple oncogenic pathways that are relevant to melanoma, such as the WNT- and PI3K signaling pathway [68, 174-

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176]. Hence, NDRG1 is a promising therapeutic target for the treatment of cancer [68, 150].

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Interestingly, it has been shown that cellular iron-depletion markedly up-regulates expression of the potent metastasis suppressor, NDRG1, at both the mRNA and protein levels in various cancer cell

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types [177]. Indeed, both Dp44mT and DpC are able to up-regulate NDRG1 expression and also have the ability to increase the phosphorylation of NDRG1 at Serine-330 and Threonine-346 [162].

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These sites are crucial for the anti-tumor activity of NDRG1 in pancreatic cancer [178].

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Considering the positive anti-cancer effects of the DpT compounds, a polypharmacological strategy

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could be highly beneficial in melanoma chemotherapy, decreasing the capability of melanoma cells

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to adjust and escape a therapeutic insult. Hence, this approach may revolutionize drug design

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strategies [141], especially in melanoma.

4. Conclusion

The growing understanding of pathways involved in melanoma progression and development has led to the identification of some interesting new molecules that might be crucial for clinical development of new treatments in the future. However, treatment options for metastatic melanoma, which is responsible for the majority of melanoma related deaths, remain limited. In terms of treatment, a new polypharmacological strategy might be more effective than focusing on one particular pathway or molecular target in melanoma. Indeed, the progression of malignant melanoma involves a very complex series of events, which includes many pathways and key oncogenic targets that interact together. Hence, elucidating the intricate mechanisms that initiate 30

ACCEPTED MANUSCRIPT and drive this disease is crucial and likely to lead to more effective treatment strategies for melanoma.

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Acknowledgements:

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D.J.R.L. thanks the AMP Foundation for a Tomorrow Fund Award, the Sydney Medical School Foundation for Fellowship support, the National Health and Medical Research Council of Australia (NHMRC) for a Peter Doherty Early Career Fellowship, and the Cancer Institute of New South

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Wales (NSW) for an Early Career Research Fellowship. Z.K. and M.L.-H.H. sincerely appreciate

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NHMRC Peter Doherty Early Career Fellowships. D.R.R. thanks the NHMRC for a Senior Principal Research Fellowship and Project Grant funding. P.J.J. sincerely appreciates a Cancer

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Institute NSW Early Career Research Fellowship. D.S.K. thanks the NHMRC for Project Grant

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support, and a NHMRC RD Wright Fellowship.

31

ACCEPTED MANUSCRIPT Figure Legends Figure 1. Schematic of the structure and function of melanocytes. Melanocytes are located at the stratum basale and produce melanin with melanosomes. Melanin itself can be transferred to the

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neighbouring keratinocytes and protects the cells in the skin from the hazardous effect of UV

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radiation.

Figure 2. Schematic of cellular events during MAPK signaling. MAPK signaling can be initiated

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by the activation of receptor tyrosine kinases (RTK; e.g., epidermal growth factor receptor), for example, by growth factors (GF; e.g., epidermal growth factor). Activation of RTKs stimulates the

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conversion of RAS from a GDP-bound state to GTP-bound (i.e., active) via the activity of guanine nucleotide exchange factors (GEF). Activated RAS can then phosphorylate RAF, leading to its

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activation. Once active, RAF can phosphorylate and activate MEK1 and MEK2 that then

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subsequently activate ERK1/2, resulting in the translocation and regulation of several transcription

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factors in the nucleus, such as cyclin D1, c-MYC and MITF. Besides initiation of MAPK signaling,

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RAS-GDP is also able to promote survival through the PI3K signaling pathway, by activating AKT.

Figure 3. Schematic representation of the canonical WNT signal transduction cascade. (A): In absence of WNT ligands, β-catenin is normally bound to E-cadherin as part of the adherens junction complex. Dissociated from the E-cadherin complex, β-catenin is bound by the destruction complex of Axin, GSK3, CK1α and APC in the cytosol. Hyper-phosphorylation of β-catenin by CK1α and GSK3 facilitates its targeted degradation mediated by β-TrCP. (B): WNT stimulation causes inhibition of GSK3 via disheveled protein (DVL), followed by inhibition of the GSK3/Axin/APC complex. Both PI3K and MAPK pathways also contribute to inhibiting the GSK3/Axin/APC complex. DVL itself gets activated by PAR-1, a kinase that phosphorylates DVL directly. As a result, free β-catenin accumulates in the cytoplasm and is then able to translocate to 32

ACCEPTED MANUSCRIPT the nucleus where it complexes with T cell factor (TCF) to mediate transcriptional activation of oncogenic target genes, such as cyclin D1, c-MYC and ZEB-1.

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Figure 4. Schematic representation of the non-canonical WNT signal transduction cascade.

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After the activation of FZD receptors by WNT5A, WNT11 ligands, or rat Fz2 (RFz-2) protein, the

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dissociation of heterotrimeric G-protein into Gα- and Gβ/γ-subunits releases intracellular Ca2+ from the ER. Increased intracellular calcium promotes activation of protein kinase C (PKC) and

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calcium/calmodulin-dependent kinase II (CamKII). On the other hand, CamKII activates the nuclear factor associated with T-Cell (NFAT) transcription factor and also the TGF-β activated

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kinase (TAK1) and Nemo-like kinase (NLK) that antagonizes β-catenin/TCF signaling.

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Figure 5. Schematic of the PI3K signaling pathway and its effects on β-catenin. PI3K

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phosphorylates PIP2 to PIP3, which leads to the phosphorylation of AKT. Active p-AKT then acts like an oncogenic signaling kinase that promotes tumor progression by inhibiting GSK3β and

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allowing β-catenin to accumulate and move to the nucleus. As a result, β-catenin up-regulates the oncogenic expression of c-MYC, cyclin D1 and ZEB-1. Furthermore, p-AKT activates mTOR,

proliferation.

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which then leads to increased levels of c-MYC, a nuclear transcriptional factor that promotes

Figure 6. Schematic of cellular events during EMT-like phentotype switching. The onset of EMT-like phentotype switch in melanoma is associated with loss of epithelial-like cell-cell contacts between melanoma tumoral and/or stromal cells. These involve adherens junctions that are connected to the actin cytoskeleton, tight junctions and gap junctions, which allow direct chemical interactions with neighbouring cells and desmosomes. As a result, the epithelial-like actin architecture reorganizes and matrix metalloproteinases (MMP) are expressed, leading to the

33

ACCEPTED MANUSCRIPT degradation of extracellular matrix (ECM) proteins to release epithelial-like cells from the surrounding tissue and to maintain the mesenchymal-like phenotype.

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Figure 7. Schematic of the development from normal epithelium to secondary carcinomas via

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EMT and MET. During EMT, mesenchymal cells are able to enter the lymph/blood vessel through

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the basement membrane and disseminate to distant organs. In the second step, metastatic cancer cells exit the lymph/blood vessels and enter organs to form micro-metastases, which can transform

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to macro-metastases via the mesenchymal-epithelial transition (MET).

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Figure 8. Schematic of the adherens junction. β-catenin and α-catenin function like a bridge between E-cadherin and the cytoskeleton. The extracellular domains of E-cadherin interact with

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those of the adjacent cells, mediating cell-cell adhesion.

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Figure 9. Summary of the potential function(s) of MFI2. One of the first identified cell surface markers of melanoma is the membrane-bound form of melanoma tumor antigen p97 (mMFI2). MFI2 plays a vital role in cell proliferation and migration, endothelial migration and angiogenesis,

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differentiation and tumorigenesis. A soluble and actively secreted form of MFI2 (sMFI2) has been reported to be increased in patients with Alzheimer‟s disease or arthritis and may modulate angiogenesis, cell migration and plasminogen activation. To date, the mechanism of how MFI2 is involved in melanoma remains unclear and requires further investigation.

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ACCEPTED MANUSCRIPT [151] K.M. Dixon, G.Y. Lui, Z. Kovacevic, D. Zhang, M. Yao, Z. Chen, Q. Dong, S.J. Assinder, D.R. Richardson, Dp44mT targets the AKT, TGF-beta and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells, Br J Cancer, 108

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[152] W. Liu, F. Yue, M. Zheng, A. Merlot, D.H. Bae, M. Huang, D. Lane, P. Jansson, G.Y. Lui, V.

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Richardson, S. Sahni, D. Kalinowski, Z. Kovacevic, D.R. Richardson, The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1,

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Oncotarget, 6 (2015) 8851-8874.

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Novel thiosemicarbazones regulate the signal transducer and activator of transcription 3 (STAT3) pathway: inhibition of constitutive and interleukin 6-induced activation by iron depletion, Mol

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[154] D.J. Lane, T.M. Mills, N.H. Shafie, A.M. Merlot, R. Saleh Moussa, D.S. Kalinowski, Z.

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Kovacevic, D.R. Richardson, Expanding horizons in iron chelation and the treatment of cancer: role of iron in the regulation of ER stress and the epithelial-mesenchymal transition, Biochim Biophys Acta, 1845 (2014) 166-181.

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[155] E. Gutierrez, D.R. Richardson, P.J. Jansson, The anticancer agent di-2-pyridylketone 4,4dimethyl-3-thiosemicarbazone (Dp44mT) overcomes prosurvival autophagy by two mechanisms: persistent induction of autophagosome synthesis and impairment of lysosomal integrity, J Biol Chem, 289 (2014) 33568-33589. [156] S. Sahni, D.H. Bae, D.J. Lane, Z. Kovacevic, D.S. Kalinowski, P.J. Jansson, D.R. Richardson, The metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), inhibits stress-induced autophagy in cancer cells, J Biol Chem, 289 (2014) 9692-9709. [157] P.J. Jansson, T. Yamagishi, A. Arvind, N. Seebacher, E. Gutierrez, A. Stacy, S. Maleki, D. Sharp, S. Sahni, D.R. Richardson, Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone

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ACCEPTED MANUSCRIPT (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp), J Biol Chem, 290 (2015) 9588-9603. [158] M. Colone, A. Calcabrini, L. Toccacieli, G. Bozzuto, A. Stringaro, M. Gentile, M. Cianfriglia,

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[163] D.B. Lovejoy, D.M. Sharp, N. Seebacher, P. Obeidy, T. Prichard, C. Stefani, M.T. Basha, P.C. Sharpe, P.J. Jansson, D.S. Kalinowski, P.V. Bernhardt, D.R. Richardson, Novel secondgeneration di-2-pyridylketone thiosemicarbazones show synergism with standard chemotherapeutics and demonstrate potent activity against lung cancer xenografts after oral and intravenous administration in vivo, J Med Chem, 55 (2012) 7230-7244. [164] W. Liu, F. Xing, M. Iiizumi-Gairani, H. Okuda, M. Watabe, S.K. Pai, P.R. Pandey, S. Hirota, A. Kobayashi, Y.-Y. Mo, K. Fukuda, Y. Li, K. Watabe, N-myc downstream regulated gene 1

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up-regulates the tumor metastasis suppressor gene Drg-1 in prostate and breast cancer, Cancer Res,

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ACCEPTED MANUSCRIPT CONFLICT OF INTEREST:

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D.R.R. is a stakeholder in the companies, Oncochel Therapeutics LLC and Oncochel Therapeutics Pty Ltd that are developing the thiosemicarbazone, DpC, for the treatment of cancer. D.R.R. also consults for Oncochel Therapeutics LLC and Pty Ltd.

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