Cellular Pathways Leading To Malignant Melanoma Biology Essay

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Each year in the UK the incidence of a form of skin cancer malignant melanoma is more than 10,400 people, within this figure there are more women diagnosed with this cancer than men. Malignant melanoma is less common though more severe then non-melanoma skin cancer and is the 6th most common cancer overall in the UK (when non-melanoma skin cancer is excluded)(. Melanoma risks and causes.2009){{166 Anonymous 2009}}. Furthermore according to the World Health Organisation, the number of melanoma cases worldwide is increasing faster than that of any other type of cancer(Kuphal & Bosserhoff 2009)

{{167 Kuphal, S. 2009}}. One main factor causes the risk of developing melanoma, Ultra-violet light. Other risk factors related to sun exposure include moles, skin colour and freckles, sunburn, sun exposure, sun-beds, sun-screen and where you were born (e.g. hot countries such as Australia or Israel increases risk)(. Melanoma risks and causes.2009){{166 Anonymous 2009}}.

The alarming incidence of malignant melanoma and its high number of risk factors is not only due to the hostility of the tumour but is due to the current shortage of effective therapeutic interventions. In the last few years, detailed understanding of cellular events leading to the malignant melanoma has greatly developed and so this is the key focus of this eassay.

Malignant melanoma develops from melanocytes, melanoma tumour progression is illustrated in Fig. 1. The role of melanocytes is to produce melanin (in hair follicles) which provides protection against skin damage from the sun. The role of melanocytes is regulated by transcription factors, extracellular ligands, transmembrane receptors and intracellular signalling molecules. Melanoma is known as a malignant cancer and has highly metastasis properties. The metastatic cascade (Fig.1a), highlights the events from a primary tumour to a metastatic tumour and so the these cellular events will be discussed with respect to the development of malignant melanoma.) With respect to these events changes from the melanocyte to from a metastatic melanoma is shown in Fig. 1b.

Figure. 1. a. metastatic cascade4. b. Melanoma Tumor Progression. From{{206 Natalil, P.G. 1993}}



Weinburg and Hanahan (2000) identified six characteristics of malignant cells which distinguished them from normal cells (Fig.2). These characteristics include sustained angiogenesis, invasion and metastasis (both of which are involved in tumour metastasis and invasion), growth signal autonomy, evasion of inhibitory signals, evasion of apoptosis and unlimited replicative potential (these last four Hallmarks of cancer stated contribute to an increase in cell number). These alterations in cell

Figure. 2. Acquired Capabilities of Cancer cells{{168 Hanahan, H. 2000}}.

physiology (e.g. melanocytes) dictate the malignant growth of melanoma from melanocytes. To note, the cellular events have been discussed with respect to melanoma development in the metastatic cascade however there are some cellular pathways contribute to various hallmarks of cancer (and so the necessary order of the metastasis cascade has not always been maintained (as specified in my essay).

Loss of differentiation of melanocytes

Loss of differentiation of melanocytes is a key early cellular event that leads to malignant cancer. MIFT is a protein that is essential for the differentiation of melanocytes and is important in the survival and development of melanomas. Fig.3 illustrates the transcription of MIFT in the nucleus by various pathways. WNT signalling increase β-catenin which interacts with TCF/LEF family proteins in the nucleus to activate MIFT transcription (Sekulic et al. 2008). The melancortin pathway increases transcription of MIFT through adenylate cyclase. Though, phosphorylation of MIFT enables MIFT to carry out its activity to increases the transcription of genes involved in pigmentation, survival of melanocytes(Sekulic et al. 2008) {{196 Sekulic, A. 2008}} and genes which encode markers for melanoma progression (Yasumoto et al. 1994) .

Figure. 3. MITF signalling. a. Melancortin pathway, α-MSH binds to MC1R, which activates adenylate cyclase to stimulate binding of CREB1 to the MITF promoter. b. MAPK-mediated MITF phosphorylation enables higher transcriptional activity of MIFT c. WNT signalling; WNT acts through Frizzled to suppress GSK3B, to increase β-catenin levels which binds lymphocyte enhancer factor (LEF) and Tcell transcription factor (TCF) type transcription factors, activating the transcription MIFT gene. Increased MIFT stimulates transcription of Tyr, Tryp1 (pigmentation), Mlana, Silv (markers) and Bcl2 (survival).{{196 Sekulic, A. 2008}}

Loss of cell adhesion and migration of melanoma cells

In malignant melanoma there are alterations in cell adhesion. In melanoma rather then controlling cell migration, cell adhesion contributes to invasion, and cell signalling (Sekulic et al. 2008) . Normally melanocytes associate with keratinocytes through cell-cell adhesion, E-cadherin expression is upregulated to form tight junctions, adherens, desmosomes and induce epithelial cell polarity. The expression of E-cadherin is mediated by WNT signalling (Gottardia et al. 2001) (fig.3). Poser I et al (2001) demonstrated in malignant melanoma progression loss of E-cadherin is achieved through the upregulation of the transcription factor Snail, causing cells to undergo the epithelial-mesenchymal transition (to lose their polarity and cell adhesion). Gottardi J et al (2001) postulated increased levels of N-cadherin is attained through decreased levels of E-cadherin{{217 Gottardia, C.J. 2001}}, maybe due to E-cadherin releasing N-cadherin from the actin cytoskeleton. Fig. 3 N-cadherin is then able to induce the expression of target genes MIFT and CCND1 (contributes to proliferation).

Witze ES et al demonstrated migration and invasion of melanoma from the primary site into surrounding tissue, lymph and blood is induced by cell polarity. The presence of a cytokine gradient (CXCL12), Wnt5a recruits actin, myosin IIB and frizzled 3 at the cell peripherally to cell orientation, polarity, and directional movement in melanoma(Witze et al. 2008).

Cell cycle changes leading to proliferation in malignant melanoma

A key feature of malignant melanoma is enhanced cell proliferation at the early and latter stages of the metastatic cascade even in the absence of external stimuli, this suggests a disruption in normal cell cycle control may contribute to the development of malignant melanoma. This particular characteristic of melanoma looks at the hallmark of growth signal autonomy at which cancerous cells escape all checkpoints of the cell cycle despite not being able to meet these checkpoints. Figure 4 illustrates the regulation of the cell cycle and apoptosis in particular by the p53 and pRb pathways.

Figure 4. p53 and pRb pathways and their regulation of the cell cycle and apoptosis.

Rb pathway a. cdk4/6/cyclinD hyperpohosphorylates pRb to cause its release from E2F to allow G1/S progression. b. p16INK4A binds to cdk4/6 inhibiting cdk4/6/cyclinD, hypophosphorlyated Rb bound to E2F causing cell cycle arrest. c. p27 binds cdk4/6 inhibiting cdk4/6/cyclinD and cdk2/cyclinE.

P53 PATHWAY; d. DNA damage causes elevated levels of p53 as p14ARF inhibits Mdm2 function, phosphorylated p53 binds p21CIP1. e. p21CIP1 inhibits Cdk2 and so inhibits cdk2/cyclinE formation and the phosphorylation of Rb to inhibit cell cycle progression. Adapted from {{191 Li, W. 2006}}

During cell cycle progression cdk4/6 complexed with cyclin D drives entry into the cell cycle by phosphorylating (and inactivation) of the retinoblastoma protein (pRb) to cause its hyperphosphorylation. pRb causes the release of transcription factor E2F, and the expression of E2F induces the expression of its target genes and allows cell cycle progression. p16INK4a is a cyclin-dependent kinase inhibitor (CKI) from the INK4 family which inhibits the formation of cdk4/6 and cyclin D. As a consequence pRb is hypo-phosphorylated and remains bound to E2F (suppressing the activity of E2F) resulting in cell cycle arrest (Fig.4), and so p16INK4a is a tumour suppressor. p27 is a CKI though from the cip/kip family and plays an important role in regulating progression through G1 and entrance into the S phase of the cell cycle. This is achieved by inhibiting the activation of cdk4/cyclin D and cdk2/cycle E (Fig.4)(Polyak et al. 1994)(Coats et al. 1996). Inactivation of the pRb, or p16INK4a (tumour suppressor), cdk4 mutation or overexpression of cyclin D have been found in various melanoma cell lines and so disrupts the G1 to S phase cell cycle checkpoint. The function of pRb in malignant melanomas is inhibited by hyperphosphorylation(Castellano et al. 1997). The mechanisms underlying this include constant phosphorylation of pRb by deregulated cdks(Ranade et al. 1995)(Quelle et al. 1995), mutations in the Rb gene itself(Horowitz et al. 1990)(Horowitz et al. 1989), or viral transforming protein such as the human papillomavirus E7 protein(Whyte et al. 1988) (Dyson et al. 1989)the adenoviral E1A protein(Peeper & Zantema 1993) or the SV40 large T-antigen(Dyson et al. 1990).

The cell cycle protein p53 is branded 'Guardian of the Genome'. Normally cellular levels of p53 is low (as it is bound by the ubiquitin ligase Mdm2 which ubiquitinates p53 and targets it for degradation). Though in response to DNA damage (Fig.4) , p53 is found in higher levels in the cell as p14ARF (a CKI from the INK4 family) inhibits Mdm2 activity and so levels of p53 stabilised. p53 is activated in the nucleus upon its phosphorylation (by Chk2), it then targets p21cip1 which inhibits CDK2 and DNA polymerase(Bartkova et al. 1996) and so inhibits the replication of damaged DNA and allows DNA repair to occur. Apoptosis may occur depending on the severity of damage (Fig.4).

TP53 gene mutations are likely to be rare in melanoma when compared to the frequency of this mutation in other cancers(Li et al. 2006) Those TP53 mutations that do occur in melanoma may be UV related as melanomas in patients with Xeroderma Pigmentosum indicate high frequencies of TP53 mutations (~60%)(Spatz et al. 2001). Furthermore, TP53 gene mutations in melanoma involves C:G substituted with T:A pairs(Albino et al. 1994). Mutations in p53 and pRb to inactive these proteins prevent senescence and enable cancer cells to undergo rapid growth and continue dividing and these characteristics are those conserved in the hallmark Limitless replicative potential.

The CDKN2A locus (Fig.5) on chromosome 9 (9p21) was identified as potential tumour suppressor, due to the gene structure of CDKN2A which encodes for the cyclin-dependent kinase inhibitors p14ARF and p16INK4A(Sekulic et al. 2008). Mutations within this locus establish CDKNA2 as a major melanoma susceptibility locus in familial melanoma(Hussussian et al. 1994).

Fig. 5 The CDKN2A locus encoding two overlapping but very different cyclin dependent inhibitors: p14ARF and p16INK4A Adapted from {{196 Sekulic, A. 2008}}

Most CDKN2A mutations affect p16INK4A alone or in combination with p14ARFsuggesting p16INK4A mutation is more susceptible(Sekulic et al. 2008). In a large majority of cultured melanoma cell lines the p16 gene is either mutated or deleted (the rate averaging ~70% across numerous studies)(Castellano et al. 1997)(Walker et al. 1998). Possible mechanisms for this include, homozygous deletion(Peng et al. 1995) loss of heterozygosity(Funk et al. 1998, Flores et al. 1996), intragenic mutation(Smith-Sorensen & Hovig 1996), hypermethylation of promoter regions(Van der Velden et al. 2001)(Merbs & Sidransky 1999) and microsatellite instability(Matsumura et al. 1998). Complete loss of expression of p16 in melanoma has been shown in almost 50% of primary melanoma(Funk et al. 1998). The gradual loss of p16 involved in melanoma development has been demonstrated in both sporadic and familial melanoma at protein(Reed et al. 1995, Sparrow et al. 1998, Keller-Melchior et al. 1998) and mRNA levels. Loss of p16 activity (tumour suppressor) means melanoma fails to undergo cell cycle arrest. Reports of mutations in p14ARF alone suggests that p14ARF could also be sufficient to give a melanoma phenotype though the independent roles of p16INK4A and p14ARF in the development of melanomas is still being determined(Rizos et al. 2001, Harland et al. 2005).

A very small amount of mutations in some melanomas involve CDK4, substituting arginine to cysteine (R24C), although this blocks binding of p16INK4A to CDK4 it conserves CDK4-CCND1 interaction resulting in this complex to be constitutively active. Moreover familial melanoma patients tend to only have p16INK4A mutations or CDK4R24C mutations, but not both suggesting these occur in a single pathway and only one 'hit' is necessary for pathway activation(Sekulic et al. 2008). As mutations in these CKI inhibitors involve cancerous cells escaping cell cycle checkpoints and avoiding differentiation inducing signals after G0 phase of the cell cycle and so proceeding to G1, these are characteristics acquired by cancerous cells under the hallmark insensitivity to anti-growth signals.

Cell signalling changes in melanoma

Various signalling pathways are important in the development of malignant melanoma, most importantly their regulatory mechanism are being uncovered and so these key signalling pathways hold potential therapeutic targets.

Cellular proliferation and survival is achieved through the MAPK pathway and PI3K/AKT pathway (Fig.6) this contributes to the development of malignant melanoma.

MAPK pathway

In the MAPK pathway, Fig. 6a growth factor receptors are linked to cellular effects through various kinases which then signals to the following cellular processes important for malignant melanoma development; proliferation, survival, differentiation and angiogenesis. The three isoforms of RAS include HRAS, KRAS, and NRAS. ARAF, BRAF, and RAF1 (CRAF) are the three isoforms of RAF. The two isoforms of MEK include MEK1 and MEK2 and the isoforms of MAPK are MAPK3 and MAPK1.

Figure. 6 (a) MAPK pathway; receptor stimulation activates RAS, enabling a complex formation between RAS and RAF. Activated RAF phosphorylates MEK, activated MEK phosphorylates MAPK, and activated MAPK transduces signals that regulate cellular processes in melanoma. (b) PI3K/AKT pathway; receptor stimulation activates PI3K which generates PIP3 which causes AKT to be phosphorylated. Activated AKT causes inhibition of apoptosis, survival gene transcription, cell cycle progression, protein translation, and cell growth and proliferation. Adapted from {{196 Sekulic, A. 2008}}

Over-activation of the MAPK pathway is achieved by constitutively active BRAF (Fig. 7) due to a T1796A transversion which results in the substitution mutation; V600E, this occurs in 70% of melanoma cases(Davies et al. 2002). Recent data suggested IGFBP7 inhibits BRAF-mediated proliferation in melanocytes, however in melanoma constitutively active (mutated BRAF) and loss of IGFBP7 expression therefore causes uncontrolled proliferation. This clarifies how BRAF mutations can induce either senescence or malignancy, depending on the cellular context of IGFBP7.(Wajapeyee et al. 2008)(Pollock et al. 2003).

Interestingly, Goodall J et al demonstrated Brn-2 expression is controlled by Wnt/β-catenin signalling pathway, siRNA inhibition of Brn2 expression in melanoma cells over-expressing β-catenin causes decreased proliferation(Goodall et al. 2004a) Additional work carried out by Goodall J et al. demonstrated that high expression levels of the protein Brn-2, stimulated by a constitutively active BRAF mutant (V599E) causes an increase in proliferation in melanomas. Furthermore endogenous Brn-2 expression is inhibited by BRAF-specific siRNA to cause a decrease in proliferation(Goodall et al. 2004b).

In melanomas lacking BRAF mutation there are mutations common in NRAS (Fig.7) (~15-30% of melanoma)(Panka et al. 2006) a missense mutation causing leucine to be substituted for glutamate at position 61 causes constitutively active MAPK pathway(Sekulic et al. 2008). A key point regarding NRAS and BRAF is melanomas found on the skin without chronic skin exposure from the sun have mutations in NRAS or BRAF suggesting their role in malignant melanoma to be critical. RAF depends on heat shock protein 90 (hsp90) for folding and activity, currently work with 17-AAG which acts to disrupt hsp90 and thus disrupt RAF activity in melanoma cells is ongoing. Sorafenib is a multikinase inhibitor that targets RAF kinases and VEGF-R and is currently in Phase 3 trails(Sekulic et al. 2008).

GTP-RAS to phosphorylate and activate CRAF inducing the MAPK pathway(Panka et al. 2006)(Davies et al. 2002) through constitutively produced HGF and FGF. Natal. P.G et al (2003) demonstrated the expression of C-MET proto-oncogene which encodes HGF-R was high in malignant melanoma(Natalil et al. 1993) Fig.7). Furthermore in cultured melanoma cells ERK phosphorylation is achieved through an autocrine loop involving the interaction of secreted growth factors with their respective membrane RTK(Panka et al. 2006) (Fig 6). MAPK pathway to cause proliferation occurs at both early and later stage of metastatic cascade.

Figure 7. Mechanisms of apoptosis induced by MAPK inhibition. Adapted from{{204 Panka, D.J. 2006}}

Mechanisms of Apoptosis induced by MAPK inhibition

There are two major mechanisms at which apoptosis is induced by MAPK inhibition. During MAPK signalling the ERK substrate ribosomal S6 kinase (p90rsk) phosphorylates the pro-apoptotic protein BAD on Ser75. This phosphoserine lies within a consensus sequence that binds to 14-3-3. This protein sequesters BAD in the cytoplasm inhibiting its pro-apoptotic actions (Fig.7). The presence of an MEK inhibitor means ERK and p90rsk are inactive and so BAD is dephosphorylated and is able to dissociate from 14-3-3 and translocate to the mitochondria where it is able to bind to BCL-2 and BCL-XL and so inhibiting their anti-apoptotic properties and inducing apoptosis(Robertson 2005).

During the MAPK signalling cascade, active ERK1 and ERK2 phosphorylate BIM (a pro-apoptotic protein), the phospho-protein BIM is targeted to the proteosome where it is degraded. Therefore, inhibition of the MAPK pathway results in accumulation of BIM, which is then able to translocate to the mitochondria and associate and inhibit the pro-apoptotic function of BAX in doing so apoptosis is induced(Panka et al. 2006).

PI3K/AKT pathway

Fig.6b illustrates the PI3K/AKT pathway, this pathway activates survival gene transcription, inhibits apoptosis, cell cycle progression, protein translation and cell growth and proliferation(Sekulic et al. 2008). Proliferation here occurs at late stages of the metastatic cascade. PI3K activity can be inhibited by a phosphatase PTEN to reduce PIP3 levels, lowering AKT activity. Furthermore, PTEN upregulates the CKI p21KIP1 to arrest the cell cycle(Wu et al. 2003). In melanocytes normal apoptotic signalling is maintained, through PTEN expression which inhibits AKT activation. Studies of cultured melanoma cells have found deletions or mutations of PTEN in up to 60% of melanoma cell lines(Stahl et al. 2003) Recent studies demonstrate the deregulation of AKT signaling pathway in 43-67% melanomas through decreased PTEN activity, increases PIP3 levels to cause AKT activation to promote melanoma cell proliferation and survival. This is achieved through the intrinsic apoptotic pathway by pAKT forming a complex with Hsp90 and Hsp27 to phosphorylate BAD which then forms a complex with 14-3-3 to inhibit the pro-apoptotic protein BAX. AKT also activate FOXO1 which abrogates apoptosis through pro-survival genes(The American Society for Clinical Investigation 2010). Recently AKT3 activity was found predominant in melanoma tumour progression (VGP) and so targeting this isoform expression (siRNA knockdown) or activity (PTEN expression) can pose as an anti-cancer strategy in melanoma(Robertson 2005). This is characteristic of the hallmark, evasion of apoptosis involves cancerous cells to abrogate apoptosis upon sensors to induce survival signals and abolish death signals.

In malignant melanoma enhanced tumourgenesis, growth, chemoresistance, invasion, migration and cell cycle deregulation is achieved through interaction between the MAPK and PI3K/AKT pathways. With particular focus on PI3K oncogenic RAS binds and activates PI3K whereas PTEN acts to inhibit PI3K activity.(Sekulic et al. 2008) Tsao H et al. demonstrated PTEN mutations to inactive PTEN and NRAS activating mutations in HS597 melanoma cell lines are rare(Tsao et al. 2000), this is because loss of function PTEN and gain of function RAS are functionally overlapping leading to stabilised levels of PI3K.

Melanoma invasion and metastasis

Iida J et al (2007) demonstrated melanoma invasion and metastasis is promoted through MMP-1 (ECM degrading enzyme enabling migration) and activation of TGF-β (facilitate invasion and growth survival)(Iida & McCarthy 2007)

{{221 Iida, J. 2007}}. This point is characteristic of the hallmark tissue invasion and metastasis (which is dependent on limitless replicative potential, sustained angiogenesis, evasion of inhibitory signals, evading apoptosis and growth signal autonomy.

Melanoma-vascular endothelial cell adhesion

During melanocyte differentiation in melanotypic nevus, loss of epithelial-mesenchymal- transition (and cell adhesion) and promotion of invasion through decreasing levels of E-cadherin and increasing levels of integrins and N-cadherin(Meier et al. 1998). During early stages of the metastatic cascade cell adhesion is stimulated through the interaction of Mel-CAM, and integrins and CD44 (fig 8A). Melanoma cell progression from RGP to VGP increases the expression of αVβ3, α4β1, α2β1, MT1-MMP and MMP-2 and to promote adhesion between melanoma and vascular endothelial cell interaction(Meier et al. 1998). This occurs just before extravasation during the metastatic cascade.



Figure.8b A. melanoma-melanoma via Mel-CAM binding to unknown ligand and L1 interacting with αVβ3 integrin and CD44 (which requires prior activation) bindings to hyaluronate.

B. melanoma-endothelial; Mel-CAM on endothelial cell surface binds to ligand this acts in concert with αVβ3 integrin binding to PECAM-1 (on endothelial cell surface) and VLA-4 integrin binding to V-CAM-1 on endothelial cell surface. Adapted from {{223 Meier, F. 1998}}

Angiogenesis of malignant melanoma

MAPK hyperphosphorylation by MEK accounts for ~90% of cases of melanoma(Cohen et al. 2002). With reference to this, Xianhe Bai et al. (2003) demonstrated that the constitutively active MAPKK leads to tumourgenesis of melanoma, through the phosphorylation and activation of MAPK in malignant melanoma in comparison to benign melanocytic nevi (non-progressive and mild disease lacking malignant properties of cancer) (Fig.9). In this study the angiogenic

Figure.9. Immunohistochemistry staining nuclei for phosphor-MAPK on an atypical nevus (A), and on a malignant melanoma B (40x magnification). Adapted from {{205 Xianhe Bai, G.B. 2003}}

switch was also induced through increased production of the proangiogenic factors (VEGF, MMP-2 and MMP-9), which consequently lead to angiogenesis(Xianhe Bai et al. 2003). This finding favours malignant melanoma in retaining the acquired hallmark sustained angiogenesis and is able to occur both at the early and late stages of the metastatic cascade.

To conclude the degree of cellular pathways that lead to malignant melanoma from melanocytes branch from critical signalling pathways (e.g. MAPK and PI3K/AKT) used physiologically for cell survival and development. However inhibition of inhibitors e.g. CKI to act against this pathway and inhibiton or stimulation of key components of these pathways (e.g. by loss or gain -of function mutations), means that melanomas have cleverly evolved these pathways in this way to gain characteristics of the Hallmarks of cancer. Research into these pathways proves never-ending; I appreciate cross-talk between pathways are important in the survival of these cancerous cells various cellular molecules within the cellular events that lead to melanoma are involved in contributing to the different stages of the metastatic cascade. Although little has been discussed here, therapeutic strategy e.g with particular focus on components of the apoptotic pathway in melanoma cells has shown to be successful, also gene therapy of melanoma to transfer tumour suppressor genes, and inactivate oncogenes expression has known to be feasible and safe(Sotomayor et al. 2002)

{{224 Sotomayor, M.G. 2002}}. Though our knowledge on cellular events leading to malignant melanoma is moving much faster than the therapeutic intervention which is partially responsible for the high incidence of malignant melanoma.

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