Faculté de Pharmacie
Ecole Doctorale enSciences Pharmaceutiques
New prognosis markers and new targets for therapy in high risk
melanoma: Evaluation of TYRP1 as a melanoma prognostic marker and
its regulation by miRNA(s)
Petra EL HAJJ
Thèse en cotutelle présentée en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques- Université Libre de Bruxelles
Docteur en Sciences et Technologie- Université Libanaise
Promoteurs et co-promoteur:
Prof. Ghanem Elias GHANEM (Laboratoire d’Oncologie et de Chirurgie Expérimentale, ULB) Prof. Bassam BADRAN (Service d’Immunologie, Faculté des Sciences, UL)
Prof. Fabrice JOURNE (Laboratoire d’Oncologie et de Chirurgie Expérimentale, ULB)
Composition du jury :
Prof. Jean-Michel KAUFFMANN- Président (Faculté de Pharmacie, ULB) Prof. Véronique MATHIEU- Secrétaire (Faculté de Pharmacie, ULB)
Prof. Wissam FAOUR - Rapporteur (School of Medicine, Lebanese American University) Prof. Nader HUSSEIN – Examinateur (Faculté de Sciences, UL) Prof. Pierre DUEZ (Faculté de Médecine et de Pharmacie, UMONS)
Prof. J.C. GARCIA BORRON (School of Medicine, University of Murcia)
Année Académique 2014-2015
Ecole Doctorale des
2 ACKNOWLEDGMENT
I wish to thank everyone who helped me complete this dissertation. Without their continued efforts and support, I would have not been able to bring my work to a successful completion.
First and foremost I offer my sincerest gratitude to my supervisor, Prof. Ghanem Ghanem, for the continuous support of my PhD study and research, for his patience, motivation, support, not to mention his immense knowledge whilst providing me with an excellent atmosphere for doing research. One simply could not wish for a better or friendlier supervisor.
Also, I would like to thank my supervisor, Prof. Bassam Badran who undertook to act as my supervisor despite his many other academic and professional commitments. His wisdom, knowledge has been invaluable on an academic level.
My sincere thanks also go to Dr. Fabrice Journe who showed me the road and helped to get me started. He was always available for my questions and gave generously of his time and vast knowledge. He always knew where to look for the answers to obstacles while leading to the right source.
My Deepest gratitude also goes to Dr. Renato Morandini who helped me develop my background in cell culture, transfection, blotting and informatics issues.
My gratitude also goes to all members of the jury; prof. Garcia Borron, prof. Pierre Duez, prof. Jean-Michel Kauffmann, Dr. Véronique Mathieu, Dr. Wissam Faour and Dr. Nader Hussein who, despite their many duties, agreed to judge this thesis.
Many thanks to all workers in the laboratory of oncology and experimental surgery (LOCE), Jules Bordet Institute, for showing me a good manipulation of the work. They added truly memorable experience for me. My research would not have been possible without their helps.
4
SUMMARY
Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumor and is often adjusted during disease progression.
Our study aimed to extend our initial observations in skin metastases and to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node (LN) metastases of stages III and IV melanoma patients. TYRP1 is a melanosomal enzyme that shares structural similitude with tyrosinase, a key enzyme regulating pigmentation.
TYRP1 mRNA expression from 104 lymph node metastases was quantified by real-time PCR and normalized to S100 calcium binding protein B (S100B) mRNA expression to correct for tumor load. TYRP1/S100B ratios were calculated and median was used as cut-off value. TYRP1/S100B mRNA ratios were correlated to clinical follow-up and histopathological characteristics of the primary lesion.
A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free and overall survival, increased Breslow thickness and presence of ulceration of the primaries. Moreover, high TYRP1/S100B was of better prognostic value for overall survival than Breslow thickness and ulceration of the primaries and it was well conserved during disease progression with respect to high/low TYRP1 groups.
We found that high TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavorable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients. Its conserved expression further supports its use as a target for therapy.
Second, by evaluating TYRP1 protein expression by immunohistochemistry (IHC) in skin and LN metastases, we showed that TYRP1 protein was not detected in half of tissues expressing mRNA and in contrast to mRNA, it was not associated with survival, suggesting a post-transcriptional regulation.
6
RESUME
L’espérance de vie des patients atteints de mélanome à haut risque ne peut être prédite d’une façon fiable en se basant sur les analyses d’histopathologies de la lésion primitive et est souvent ajustée durant la progression de la maladie. Notre étude vise à élargir nos observations initiales au niveau des métastases cutanées et d’évaluer la valeur pronostique de tyrosinase related protein 1 (TYRP1) dans les métastases ganglionnaires des patients atteints de mélanome de stades III et IV. TYRP1 est une enzyme mélanosomale qui partage des similitudes structurelles avec la tyrosinase, l'enzyme clé de la mélanogenèse.
L’expression de l'ARNm de TYRP1 a été quantifiée dans 104 métastases ganglionnaires par PCR en temps réel et normalisée par rapport à l’expression de l’ARNm de S100B (marqueur reconnu du mélanome) pour corriger l’expression de TYRP1 suivant la charge tumorale de l’échantillon. Le rapport TYRP1/S100B a été calculé et la médiane a été utilisée en tant que valeur seuil. Ensuite nous avons étudié la relation entre les valeurs de TYRP1/S100B, le suivi clinique et les caractéristiques histopathologiques de la tumeur primitive.
Un rapport élevé de l’ARNm TYRP1/S100B corrélait significativement avec une survie sans récidive et une survie globale plus courtes, avec une épaisseur de Breslow plus élevée et avec la présence d'une ulcération au niveau de la tumeur primitive. En outre, une expression élevée de TYRP1/S100B était de meilleure valeur pronostique pour la survie globale que l'épaisseur de Breslow et l'ulcération des primitifs. De plus, cette expression est bien conservée au cours de la progression de la maladie par rapport aux groupes de TYRP1 bas/élevé.
Nous avons constaté qu’une expression élevée de TYRP1/S100B dans les métastases de patients atteints de mélanome est associée à un résultat clinique défavorable et une survie courte. Menée sur des patients atteints d'un mélanome à haut risque de récidive, cette première étude a suggéré que l'ARNm de TYRP1 dans les métastases pourrait servir de biomarqueur pour affiner le pronostic initial des patients surtout ceux ayant des lésions primitives de localisation inconnues ou non évaluables et peut permettre une gestion différente des deux groupes de patients. Son expression conservée au cours de la progression de la maladie est en faveur de son utilisation comme cible thérapeutique.
7 des tissus exprimant bel et bien l'ARNm correspondant et qu’elle, contrairement à l'ARNm, n’était pas associée à la survie.
8
AIMS
Cutaneous melanoma is the less common (4%) and the deadliest form of skin cancer; it causes the majority of deaths related to skin cancer (75%), with an increasing incidence in the last decades1. Melanoma is an extremely heterogeneous disease with a poor clinical outcome even in patients with in situ primary lesions or thin melanomas2.
Treatment options for advanced melanoma are limited and rarely curative. Early detection and complete surgical excision of melanoma are the best strategies to reduce mortality. However, about 15% of patients diagnosed with primary melanoma develop distant metastases3. Although long-term survival for patients with advanced melanoma is low, it is highly variable4. The variability in survival of patients with stage III (39-70% for 5-year survival) and stage IV melanoma (33-62% for 1-year survival)5 points to an insufficient understanding of the heterogeneity of the disease and warns of difficulties to select patients who could benefit from treatments.
The currently used staging system for melanoma5, based on histopathological and clinical criteria, such as Breslow tumor thickness, mitotic rate, lymph node status, and ulceration, is limited in its ability to provide a precise prognosis , upon first diagnosis, and is often adjusted during disease progression. Indeed, a large number of patients with similar or even identical clinical features have a variable outcome4.
In this context, various groups identified genes and proteins associated with patient outcomes6 or suggested the use of serum and tissue markers in order to refine the prognosis of patients with high risk melanoma, to ensure adequate follow-up, and to predict the possible benefit from a therapy. However, despite many attempts to identify complementary molecular markers predicting clinical outcome in melanoma, most studies consist in small series requiring additional evaluation in larger populations and thus, have not been shown to improve the current AJCC staging, preventing their translation into routine clinical assessment.
Thus, it is of importance to establish new prognostic markers for high risk melanoma and to classify melanoma that has already metastasized into categories that more accurately predict patient survival, especially in melanoma patients where prognostic factors at diagnosis cannot be evaluated clinically (namely unknown or ulcerated primaries consisting 15-20% of all patients) and in metastases of thin melanomas in order to refine prognosis.
9 (DMFS) and overall survival (OS) and correlated with Breslow thickness suggesting that TYRP1 can be of a prognostic value particularly useful when pathology parameters at the primary lesion are lacking7.
Our first aim was to investigate whether a prognostic relevance or any potential correlation exist between TYRP1 in lymph node (LN) metastases of stages III and IV melanoma patients and pathological features of the primaries. For that, the expression of TYRP1 mRNA was measured by RT-qPCR in 104 LN melanoma metastases. Since LN biopsies may contain variable amounts of stroma and tumor tissue, we related TYRP1 values to those of S100B mRNA. We calculated correlations between TYRP1/S100B mRNA expression and disease-free survival (DFS), OS, and conventional histopathological parameters such as Breslow thickness, ulceration and lymph node involvement. Then, we compared the prognostic value of TYRP1/S100B to those of Breslow thickness and ulceration.
In addition, we checked for TYRP1 protein expression by IHC in a panel of paraffin-embedded biopsies from skin and LN metastases and found a lack of association with patient survival. We also reported discrepancies with the TYRP1 mRNA levels measured by real-time PCR in corresponding frozen tissues in about 50% of the cases where mRNA was expressed but not the protein, suggesting post-transcriptional regulation(s). In this context, Li et al. suggested that miR-155 acts as a “rheostat” to optimize TYRP1 expression for local adaptation to differential UV radiation along the latitudes8. The authors showed that the 3’-UTR of TYRP1 mRNA contains three putative miR-155 binding sites, among which two are polymorphic (SNPs rs683/rs910) inducing or disrupting regulation by miR-155.
10
TABLE OF CONTENTS
Acknowledgment
Summary .…….……….……….………4
Resume .……….………….………6
Aim of the thesis .………...………8
Table of contents .……….…….………..10
List of Abbreviations ……….……….15
Part I. Introduction...……….………..18
Chapter 1. Melanogenesis...……….………18
1. The human skin ...……….……….18
1.1 Skin anatomy...………18
1.2 Skin pigmentation...……….………19
1.2.1Melanocytes...………19
1.2.1.1Melanocyte development: Melanocytic origin and embryonic development from neural crest through melanoblasts to melanocytes...……….. ………..20
1.2.2 Melanin: the pigment ………...……….……….…………...20
1.2.2.1 Melanin types...………..………….……….21
1.2.2.2 Phenotypic diversity of pigmentation...….………21
1.2.3 Melanogenesis and Melanosome...………..………22
1.2.3.1 Biogenesis and maturation of melanosomes...…..……….22
1.2.3.2 Transfer of melanosomes...………....………..22
1.2.4 Triggers of melanogenesis...………..………..23
1.2.5 Enzymes of melanogenesis and melanin synthesis...……..………...……….24
Chapter 2. Melanoma...………..………..27
1 Overview; Cutaneous melanoma...………….………...………..27
2 Stages of melanoma progression...……… ….…….………...27
2.1 Common Acquired/congenital melanocytic nevus...……….………….………..27
2.2 Dysplastic nevus ...……….……….……….……….28
2.3 Radial Growth Phase...……….….……….………28
2.4 Vertical Growth Phase...……….………….……….……….29
2.5 Metastatic Melanoma...………..………...29
2.5.1 Sites of metastasis ...……….………..29
11
3 Classification of cutaneous malignant melanoma...……..……….32
4 Risk factors for melanoma...………..………...…….33
4.1 Environmental risk factors...……….………….………...………...33
4.2 Phenotypic risk factors...……….……….………...……….34
4.3 Genetic risk factors...……….………....………..35
5 Epidemiology of melanoma...………..………..36
6 Pathology...……….….……….………..38
6.1 Role of receptor tyrosine kinases...………….……….39
c-KIT...……….…….………..39
AKT Pathway and PTEN in melanoma progression...………….………..………40
Mitogen activating protein kinase (MAPK) pathway ...……….…………...………..40
6.2 Cell cycle …………...…….……….……….………..41
P53...……….42
6.3 MITF...………...……….……….………43
6.4 Growth factor dependence in melanoma development...…...……..….….………43
6.5 Cadherin expression in melanoma progression...…………...………..…………..43
7 Diagnosis...……….……….………...43
7.1 Diagnosis of primary melanoma...………..………...…………..43
7.2 Sentinel lymph node biopsy...………..………...………….44
7.3 Imaging tests...……….………….………44 8 Therapy...……….………..……….45 8.1 Surgery...…….……….……..…….………..45 8.2 Radiotherapy...………...………...…………...46 8.3 Chemotherapy...………..….………...……….46 8.4 Immuno-therapy...………...………...………..47 8.4.1 Interleukin-2 therapy...…………..……….47 8.4.2 Interferon-α therapy...…………..………..48 8.4.3 anti-CTLA-4 therapy...…………..……….48 8.4.4 anti-PD1 therapy...………..………...49 8.5 Combination therapy...………..……….………...………50
8.6 Targeted therapies for melanoma treatment...………..…..………..50
8.6.1 Targeting the RAS/RAF/MAPK Pathway in Melanoma...………...………….50
Resistance to BRAF inhibitors ………...……..……….51
8.6.2 Targeting the PI3K/AKT Pathway in Melanoma...……...……..……….…………..51
9 Prognostic factors in malignant melanoma...………….………...53
12
9.2 Progression markers………..………55
Prognosis for melanoma of unknown primaries……….………...56
9.3 Blood markers and the need of new biomarkers in metastatic patients…….………...57
9.4 Tissue biomarkers in metastatic patients……….……….….60
Chapter 3. Tyrosinase related protein1, TYRP1...………...………...61
1 TYRP family...……….………..61
2 TYRP1 gene...……….61
3 Synthesis and Transport of TYRP1...………62
3.1 TYRP1 cellular localization...………..63
3.2 Regulation of TYRP1 transcription and role of MITF...…..………..64
4 Roles of TYRP1in pigmentation...……….………64
4.1 In mammals………...64
4.1.1 TYRP1 catalase acticity………..…………..…………..………65
4.2 In men ...……….………...65
4.2.1 TYRP1 regulation of tyrosinase activity...……..….…..……..……….66
4.2.2 TYRP1 and oxidative stress...………..….………..………..67
5 TYRP1 in melanoma...………...67
5.1 TYRP1 gene variants and melanoma risk...………..67
5.2 TYRP1 as a target for therapy...………...………68
5.2.1 Preclinical studies. ...……….………...68
5.2.2 Clinical trials...……….……….69
5.3 TYRP1 expression and melanoma progression...………….………70
Chapter 4. MicroRNAs...………72
1. Overview...………72
1.1 miRNA Discover...………..….………72
1.2 miRNA Biogenesis...………72
1.3 miRNA mechanisms of action...………..73
1.4 miRNA Nomenclature and Annotation...………74
1.5 Target Prediction and Algorithms...……….74
1.6 miRNAs in Cancer...………..………..75
2. miRNAs and melanoma...……...………..75
2.1 miRNAs as diagnostic and prognostic biomarker...………..….………77
13
3. miR-155 in melanoma and its role in regulating TYRP1...……..………..78
4. miRNAs and pigmentation………..………81
Part II. Material and methods...………..………83
1. Patients and tissue collection...………..……….83
2. Melanoma cell lines and cell culture conditions...………85
3. Cell lysis and Western blot analysis...………..………..86
4. RNA extraction...……….……….………..86
4.1 Assessment of RNA integrity|...………88
4.2 Reverse Transcriptase Reaction...……….88
5. Polymerase Chain Reaction (PCR) ...……….………..……….88
5.1 mRNA Real-Time PCR...………..……….……..………..………...88
5.2 Primers choice for TYRP1...………..………...89
5.3 miRNA real time PCR...……....……….……….………. 90
5.4 SNP Genotyping...……….………92
5.5 PCR specificity and efficiency...….……….……….93
5.6 Choice for endogenous control...…….………….……….94
5.6.1 S100B in melanoma tissues...……….……….……….94
5.6.2 18s in cell lines...……….……….95
5.6.3 RNU44 control for microRNA(s) ...………96
6. Cell transfection...………..………..………...96
7 Stimulation of melanoma cell pigmentation………...……….………99
8 Taqman Low Density Array for miRNA profiling and MicroRNAs target prediction….……….99
9. Immunohistochemistry...……….………...………100
10. Statistical analyses...……….……….………..………...100
Part III. Results and Discussion...…...………..………102
1. Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients..……….…………..………102
Summary...………..………103
2. SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma...………….………105
14 3. Differential expression profiles of microRNAs involved in malignant melanocyte pigmentation 109
Summary………..110
4. General discussion...………..………...………..111
Part IV. Conclusions and Perspectives...………..………118
15
LIST OF ABBREVIATIONS
AJCC – American Joint Committee on Cancer ALM – Acral lentinigous melanoma
APC – antigen presenting cells
ARNm – acide ribonucleotide messager bp – base pair
BCC – Basal cell carcinoma Bcl-2 – B-cell lymphoma 2
bFGF – basic fibroblast growth factor
BRAF – V-raf murine sarcoma viral oncogene homolog B1 cAMP – cyclic adenosine monophosphate
CDKN2A – Cyclin dependent kinase inhibitor 2A cDNA – Complementary DNA
CREB – cAMP responsive-element-binding protein CT – Cycle threshold
CTL – cytotoxic T lymphocyte
CTLA-4 – Cytotoxic T-lymphocyte associated antigen 4 Da – Dalton
DCT – dopachrome tautomerase DFS – disease-free survival
DHICA – dihydroxyindole carboxylic acid DMFS – distant metastasis free survival DNA – deoxyribonucleic acid
dNTP – Deoxyribonucleotide triphosphate DTIC – dacarbazine
ER – endoplasmic reticulum
ERK – Extracellular signal-regulated kinases FDA – food and drug administration
FFPE – formalin-fixed paraffin embedded FGF – fibroblast growth factor
HGF – hepatocyte growth factor HR – Hazard Ratio
Ig – Immunoglobulin
16 IL – interleukin
INF – interferon KDa – kilodalton
LDH – Lactate dehydrogenase
LMM – Lentigus malignant melanoma LN – Lymph node
mAb – monoclonal Antibody
MAPK – Mitogen activated protein kinase MC1R – Melanocortin-1 Receptor
MEK – MAPK/ERK kinase
MIA – Melanoma inhibitory activity miRNA/miR – microRNA
MITF – Microphthalmia-associated transcription factor mRNA – messenger RNA
mlph – melanophilin
MSH – Melanocyte Stimulating Hormone/Melanotropin NM – Nodular melanoma
nt – nucleotide
OCA3 – Oculocutaneous Albinism type 3 OS – Overall survival
PCR – Polymerase chain reaction PD-1 – Programmed cell death 1 PDGF – Platelet‐derived growth factor PDL-1– Programmed death-ligand 1 PFS – Progression-free survival
PI3K – Phosphatidylinositide 3-kinase
PMEL – Premelanosome protein also known as silver locus protein homolog (SILV), PMEL17 and gp100
Pre-miRNA – Precursor miRNA Pri-miRNA – Primary miRNA
PTEN – Phosphatase and tensin homolog RAS – Rat Sarcoma viral oncogene homolog RAF – Rapid Accelerated Fibrosarcoma RB – retinoblastoma protein
RGP – radial growth phase
17 RNA – Ribonucleic Acid
RNase – Ribonuclease RT – reverse transcription
RT-qPCR – reverse transcription quantitative polymerase chain reaction RTK – Receptor tyrosine kinases
S100 – S100 calcium binding protein SCC – Squamous cell carcinoma SCF – stem cell factor
SLN(B)– Sentinel lymph node (biopsy) snoRNA – small nucleolar RNA
SNP – single nucleotide polymorphisms
SRC – V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene Homolog SSM – Superficial spreading Melanoma
TYRP1 – Tyrosinase Related Protein 1 TYRP2 – Tyrosinase Related Protein 2 UTR – untranslated region
UVR – ultraviolet radiation
18
Part I. Introduction
Chapter One: Melanogenesis
1. The human skin
The skin is the largest organ of the body, making up 16% of body weight, with a surface area of 1.5-2m2. The skin is a dynamic organ as cells of the outer layers are continuously replaced by inner cells moving up to the surface9.
It constitutes an important physical barrier to the environment while providing protection against micro-organisms, ultraviolet radiation (UVR), toxic agents and mechanical insults. It is associated with various afflictions, including developmental defects, autoimmune disorders, allergies and cancer.
1.1.Skin anatomy
The skin consists of three layers: the epidermis, the dermis and subcutis. Hair, nails, sebaceous, apocrine sweat glands are regarded as derivatives of skin10 (Figure 1).
The epidermis is the outer part of the skin; an avascular layer, mainly composed of keratinocytes (80٪) constantly migrating from the basal layer to the outer skin surface over a period of about 26 days, the keratinocytes provide cohesion to the epithelium, form a barrier from the exterior and protect against excessive light exposure11. The epidermis is composed as well as smaller populations of pigment producing melanocytes containing melanosomes (5-10٪) and mechanosensory Merkel cells. The immune system is present in the epidermis as the migratory Langerhans cells (4–8%) serve as antigen-presentation to lymphocytes and intraepidermal T-cells.
Beneath the epidermis is the dermoepidermal junction with the basal layer, and underlying this is the dermis, which mainly consists of supportive connective tissue with dermal fibroblasts and a complex network of vessels, nerves, eccrine glands and hair follicles.
19 Figure 1: The three layers of the skin: Epidermis, dermis and subcutaneous tissue. Cross section of the epidermal melanin units of the skin showing a melanocyte in the basal layer of the epidermis surrounded by keratinocytes. (Adapted from Melanoma Anatomy © 2008 Terese Winslow, U.S.)
1.2 Skin pigmentation (Melanogenesis)
The epidermal units of the skin, composed of a melanocyte surrounded by keratinocytes (Figure 1) and regulated by a closed paracrine system, are responsible for melanin production and distribution, a process called melanogenesis.
1.2.1 Melanocytes
Melanocytes are dendritic cells found in the epidermis, in the inner ear, in the uveal tract, in the leptomeninges and in the hair follicles. Melanocytes in the epidermis are dispersed along the basal layer at the dermoepidermal junction. They produce the pigment melanin within melanosomes, organelles that are transferred to surrounding keratinocytes and hair follicle cells.
20 synthesis is largely influenced by UV radiation, as well as by signals from neighboring keratinocytes and to a lesser degree from dermal fibroblasts12.
1.2.1.1 Melanocyte development: Melanocytic origin and embryonic development from neural crest through melanoblasts to melanocytes
Melanocytes originate in neural crest as a bipotential glial-melanocyte lineage progenitor that develops into an unpigmented precursor cell called the melanoblast. Melanoblasts migrate to different destinations, including the basal layer of the epidermis and hair follicles. Their migration, proliferation, and differentiation into melanocytes depend on mediators produced by cells of the dorsal neural tube, ectoderm and keratinocytes, such as the family of glycoproteins WNT, endothelin 3 (EDN3), and stem cell factor (SCF) which binds the c-Kit receptor tyrosine kinase in melanocytes and melanoblasts13.
A number of studies have identified genes, such as MITF, c-Kit, and snail/slug that are important for melanocyte development: melanoblast survival is dependent on MITF through its transcriptional upregulation of the anti-apoptotic gene bcl-2. In addition, slug appears to be required to initiate neural crest migration, whereas, c-Kit is involved in melanoblast expansion, survival and migration14,15.
In the epidermis and in response to UVR, keratinocytes secrete factors that regulate melanocyte survival, differentiation, proliferation and motility, stimulating melanocytes to produce melanin and resulting in the tanning response. Thereby, melanocytes have a key role in protecting our skin from the damaging effects of UVR, however, UVR and oxidative stress may create genetic mutations in the melanocytes capable of inducing malignant transformations.
1.2.2 Melanin: the pigment
Melanin is the primary determinant of skin, hair, and eye color. It defines an important human phenotypic trait and has a critical role in photoprotection due to its ability to dissipate 99.9% of absorbed UVR16.
21
1.2.2.1 Melanin types
Two types of melanin are synthesized within melanosomes: eumelanin, a brown-black insoluble polymer and pheomelanin red-yellow soluble polymer formed by the conjugation of cysteine or glutathione (Figure 2).
A B
Figure 2: the base units of eumelanin (A) and pheomelanin (B) (Wakamatsu, K. & Ito, S. Advanced chemical methods in melanin determination. Pigment Cell Res. 15, 174–183, 2002).
Eumelanin is the major type in individuals with dark skin and hair and is more efficient in
photoprotection. Eumelanin polymers comprise numerous cross-linked 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) polymers. The two types are recognized as black and brown.
Pheomelanin is predominantly found in individuals with red hair and skin phototypes I and II, in whom skin tumors are more common. It is particularly concentrated in the lips, nipples, glans of the penis, and vagina. Pheomelanin is a cysteine-containing red-brown polymer of benzothiazine units. It differs from eumelanin in that its oligomer structure incorporates benzothiazine and benzothiazole units that are produced, instead of DHI and DHICA, when the amino acid L-cysteine is present18.
1.2.2.2 Phenotypic diversity of pigmentation
22 numerous, and elongated, resulting in delayed degradation in keratinocytes and consequently in increased visible pigmentation13.
1.2.3 Melanogenesis and Melanosome
1.2.3.1 Biogenesis and maturation of melanosomes
Melanin synthesis occurs in melanosomes, lysosome-related organelles of melanocytes, in the presence of melanogenic enzymes (tyrosinase, TYRP1, TYRP2) and components of the fibrillar matrix that bind to melanin.
In these organelles, melanin is synthesized along four maturation stages12 (Figure 3).
Stage I melanosomes in eumelanogenesis and pheomelanogenesis are common and derive from the late endosomes. In the stage II of maturation, premelanosomes are ellipsoidal in eumelanogenesis and contain well-organized lamellae/filaments riche in Pmel17, tyrosinase is not active yet. In stage II and III, Melanogenic enzymes from trans-Golgi are incorporated into melanosomes which will then become electron-dense because of melanin deposition and the important activity of tyrosinase (stage III). In stage IV, melanosomes become amorphous, fully matured and melanized12.
In contrast, pheomelanosomes are always spherical and contain only granular materials in all four stages of melanosomal maturation. Stage IV melanosomes are ready to be transported to the surrounding keratinocytes.
1.2.3.2 Transfer of melanosomes
When melanin synthesis is completed, melanosomes move bi-directionally from the perinuclear area towards melanocyte dendrites, in a movement controlled by microtubule proteins (kinesin, dynein). This transport ends with melanosomes binding actin filaments through a complex formed by myosin Va, Rab27a, and melanophilin (mlph) (Figure 3).
23 Figure 3: Biogenesis, maturation and transfer of melanosome secreting eumelanin. Melanosome stages I through IV are indicated. Non‐pigmented stage I melanosomes contain Pmel17 sorted into intralumenal vesicles. Stage II melanosomes are characterised by Pmel17 undergoing proteolytic cleavage, forming intralumenal fibrillar striations. In stage II and III, melanogenic enzymes from trans-Golgi are incorporated into melanosomes which will become electron-dense. In stage IV, melanosomes become amorphous, fully matured and melanized. Stage IV melanosomes are ready to be transported to the surrounding keratinocytes. (Wasmeier, C, et al. Melanosomes at a glance. J. Cell Sci. 121, 3995–3999, 2008).
1.2.4 Triggers of melanogenesis
The regulation of melanogenesis is achieved by keratinocytes that produce, in response to UV radiation, from the Pro-opiomelanocortin (POMC) the melanocyte stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) both agonists of melanocortin 1 receptor (MC1-R) a member of G-protein receptors that predominates in melanocytes. Agonist-bound MC1R activates adenylyl cyclase, inducing cyclic AMP production, which leads to phosphorylation of cAMP responsive-element-binding protein (CREB) transcription factor family members by PKA. CREB, in turn, transcriptionally activates microphthalmia transcription factor (MITF) gene promoting eumelanogenesis, by binding to the CRE element upstream of the human MITF transcription initiation site20. In addition to CREB, the expression of the MITF protein is regulated by other transcription factors and mediators produced by keratinocytes and fibroblasts including sex-determining region Y-box (SOX9)21.
24 melanosomal matrix protein Pmel17, which are important in melanosome transport, and regulates the anti-apoptotic protein (bcl-2) of melanocytes, which is often expressed in melanomas13 (Figure 4).
Figure 4: The MC1R signaling pathway. The POMC precursor is cleaved to give the α-MSH/ACTH ligands for the MC1R receptor which activates adenylate cyclase (AC) through a heterotrimeric G-protein complex. AC catalyzes the production of cAMP. Elevated cAMP triggers the phosphorylation/activation of the CREB transcription factor. In melanocytes, one critical CREB target is MITF that transactivates genes important for pigmentation/differentiation. (Adapted from Chin, L., et al. Malignant melanoma: genetics and therapeutics in the genomic era. Genes Dev. 20, 2149–2182, 2006)
1.2.5 Enzymes of melanogenesis and Melanin synthesis
Upon exposure of the skin to UV radiation, melanogenesis is enhanced by the activation of MITF and hence by the key enzyme of melanogenesis tyrosinase. Tyrosinase has an inner melanosomal domain with a catalytic region (approximately 90% of the protein), followed by a short transmembrane domain and a cytoplasmic domain. Histidine residues present in the catalytic portion bind copper ions and are required for tyrosinase activity.
25 Following the formation of dopaquinone, the eumelanin and pheomelanin pathways diverge. In the eumelanin pathway, dopaquinone is converted to leucodopachrome and dopachrome. Dopachrome is either spontaneously converted to dihydroxyindole or is enzymatically converted to 5,6-dihydroxyindole-2-carboxylic acid via enzymatic conversion by dopachrome tautomerase (DCT also called TYRP2). DHICA can be converted to indole-5,6- carboxylic acid quinone by TYRP1 (role not sure). The polymerization of indoles and quinones leads to eumelanin formation (Figure 5).
Figure 5: Biochemical pathways leading to the synthesis of Eu- and pheomelanin. TYR : tyrosinase; TRP; tyrosinase related protein; dopa : 3,4-dihydroxyphenylalanin; DHICA : 5,6-dihydroxyindole-2-carboxylic acid; DHI : 5,6-dihydroxyindole; ICAQ : indole-5,6-hydroquinone-2-5,6-dihydroxyindole-2-carboxylic acid; IQ, indole-5,6-quinone; HBTA, Alanyl-hydroxy-benzothiazine. (Chang, T.-S. An Updated Review of Tyrosinase Inhibitors. Int. J. Mol. Sci. 10, 2440–2475, 2009)
There are two tyrosinase-related proteins, TYRP1 and TYRP2, present in the membrane of melanosomes. The role of TYRP1 in eumelanin synthesis is not yet clarified; it is possible that it has a role in the activation and stabilization of tyrosinase13.
26 pheomelanin (figure 5). The type and ratio of melanin produced (eu/pheo) depends on several different availability of cysteine22.
While tyrosinase plays a major role in the biogenesis of both eumelanosomes and pheomelanosomes, tyrosinase-related proteins are exclusively expressed by eumelanosomes.
Eumelanin is photoprotective and acts as a scavenger to reactive oxygen species, unlike pheomelanin that is more photolabile and can cause DNA damage after UVR exposure23.
27
Chapter Two: Melanoma
1 Overview; Cutaneous melanoma
Cutaneous melanoma is the most aggressive form of skin cancer. It arises from the melanocytes of the skin. Because malignant melanoma (MM) is a melanocyte-derived cancer; it can be found in all organs that harbor melanocytes such as the ears, the eyes, the mucosal membranes (nose, oral cavity, anorectal mucosa and the genitourinary mucosa), the central nervous system (leptomeningeal melanoma) and in the gastrointestinal tract.
We will be focusing throughout this work on cutaneous melanoma that will be referred hereinafter as melanoma or malignant melanoma.
Cutaneous melanoma causes the majority of skin cancer related deaths; In fact, while it represents only around 4% of all skin cancers, melanoma accounts for nearly 80% of skin cancer related deaths. Recent statistics are alarming, showing that the worldwide incidence of melanoma is increasing at a rate of 3‐7% per year. Cutaneous melanoma is among the most common types of cancer in young adults. The most common sites of cutaneous melanoma are the trunk (43.5%), extremities (33.9%), acral sites (11.9%), and head and neck (10.7%)24
cutaneous melanoma can arise de novo in the skin (about 70%) or have a common nevus or a clinically atypical nevus as a precursor lesion (in about 30%)25. As long as the cutaneous melanoma grows in the epidermis the tumor is characterized as in situ, but when it grows down in the dermis it is invasive with a potential to metastasize. Cutaneous melanoma can be classified according to location, stage and progression and are defined in 5 stages.
2 Stages of melanoma progression (Figure 6)
2.1 Common Acquired/congenital melanocytic nevus
The common acquired melanocytic nevus is a benign growth, it is the earliest hyperplastic melanocytic lesion and is common in humans. These nevi are usually recognizable as brown areas on the skin of varying size and shape usually smaller than about 5 millimeters wide and are round or oval. This pigmented nevus, called a mole, develops as a proliferation of melanocytes which are clumped into nests rather than being placed singly along the basement membrane26.
28 underlying dermis; they are flat and brown to black. They can be compound nevi, which are a mixture of junctional and intradermal proliferation which are slightly raised and brown to black (Beauty marks are usually compound nevi), also, they can be intradermal nevi which are in the dermal layer, most are raised and flesh-colored (not pigmented).
Melanocytic nevi can also be classified by age of appearance. There are two classes: the congenital nevi, which appear within the first six months after birth, and the acquired nevi, which appear when the patient is over 1 year of age. The difference and clinical importance of this classification is that congenital nevi are thought to more likely become malignant in life27. This can be related to the fact that congenital nevi, whilst clustered at the dermal-epidermal junction, go deeper into the dermis than acquired nevi and can invade blood vessels, nerves and erector pili muscles26. Melanocytic nevi show higher relative risk for SSM and nodular melanoma.
2.2 Dysplastic nevus
The dysplastic nevus (DN) (atypical mole or atypical melanocytic nevus), usually a compound nevus with cellular and architectural dysplasia, has increased abnormal growth compared to the melanocytic nevi. DN may occur within a preexisting benign nevus or in a new location. It is often large usually more than 5 millimeters wide and flat and tends to have irregular borders and coloration. Dysplastic nevi are commonly found on the trunk. The risk of developing DN is estimated to be around 7-18 % in a lifetime28.
People who have many dysplastic nevi (atypical mole syndrome) have a greater risk than others to develop melanoma. It is estimated that 22-36 % of malignant melanomas originate as DN. However, most dysplastic nevi do not turn into melanoma28.
2.3 Radial Growth Phase
The third stage, radial growth phase (RGP) called in situ melanoma, is the first recognizable malignant stage. During RGP the cells remain confined to the epidermis before invading the dermis passing through the basal lamina, however, some types of melanoma (nodular melanoma) arise directly in the dermis11. Morphologically, RGP consist of atypical, rounded (epithelioid) or spindle (lentiginous) shaped cells, which initially aggregate in the epidermis and form nests above the basal membrane.
29
2.4 Vertical Growth Phase
The fourth stage is the vertical growth phase (VGP) in which the melanoma cells escape the control of keratinocytes and pass through the basal lamina to invade as an expanding mass the dermal layer and the basement membrane29. They establish a close network with fibroblasts and are able to acquire growth factors and grow without anchorage with a high competence for metastasis.
Not all melanomas pass through each of these individual phases, RGP or VGP can both develop directly from isolated melanocytes or nevi, and both can progress directly to metastatic malignant melanoma30.
2.5 Metastatic Melanoma
Vertical growth phase can progress to the most aggressive form of cutaneous melanoma characterized by extensive vascularization and invasion; metastasis. As the tumor continues to expand deeper into the dermis and reaches the subcutaneous fat tissue, the risk of metastasis is high and systemic metastases are likely to occur. Cells that are shed from the primary lesion infiltrate the circulatory and lymphatic system, and migrate to new sites where they adhere to the walls of the capillary and invade new organs.
At the secondary site, micrometastases can survive for several years before they become proliferative, stimulate angiogenesis, and begin to form a metastatic tumor.
Advanced stage melanomas are described by the presence of regional or distant metastases.
2.5.1 Sites of metastasis:
Three routes of metastasis include in-transit and satellite metastases, regional lymph node metastases and distant metastases, including distant skin metastases.
30 Figure 6: Histopathological progression from a normal melanocyte into malignant melanoma. The benign common/acquired nevus phase is characterized by limited proliferation of structurally normal melanocytes. When the cells enter the dysplastic nevi phase, the lesions present irregular borders, asymmetry and increased diameter. Starting in the radial-growth phase, cells acquire the ability to proliferate and spread laterally across the basement membrane. In the subsequent vertical-growth phase, lesions invade through the basement membrane into the lower dermis layer of the skin. The final step of the progression to melanoma implies that the cells can successfully spread to other areas of the body, proliferate and form distant metastases (Adapted from Miller, et al. Melanoma. N. Engl. J. Med. 355, 51–65, 2006).
In any area of skin where there is a penetrating melanoma, there will be a nearby sentinel node to which it drains. The sentinel lymph node is the very first lymph node to receive drainage from melanoma and is the one most likely to contain melanoma cells if any lymph nodes are involved.
31 Table 1: Frequency of melanoma metastases to other organ
Teresa Petrella, et al. Canadian Perspective on the Clinical Management of Metastatic Melanoma. NE Oncology Issue – 2012.
2.5.2 Causing events of metastasis
The actual initiating event of metastasis and how tumor cells reach lymphatic vessels is not yet fully elucidated. Different studies attempted to understand the initiating events: One concept suggests that the fusion of cancer cells with macrophages or other migratory bone marrow-derived cells underlies metastasis by activating pathways related to epithelial–mesenchymal transition, such as snail/slug31.
Tumor cells that invade the extracellular matrix reach lymphatic vessels. Cells then flow within the lymphatics to reach the subcapsular sinus of the lymph nodes. Neoplastic cells may secrete cytokines that induce growth of lymphatics towards the tumor or within the tumor, for example, the binding of the melanoma secreted VEGF-C to VEGF-R3 expressed on lymphatic endothelial cells, induce the production of lymphatic vessels31.
Melanocytes are located in the epidermis, a milieu of decreased oxygen concentration which is thought to be responsible for their possible neoplastic transformation through stabilization of hypoxia-inducible factor HIF-1α and the increase expression of CXCR-4 on melanoma cells. In addition, the skin is abundant in elastin and elastin-derived peptides, such as VGVAPG or VAPG, that bind to receptors on melanoma: galectin-3, integrin αVβ3 and the elastin-binding protein which lead to the expression of more CXCR-4 that will be recognized by stromal cell-derived factor-1 (SDF-1) secreted by target organs, therefore melanoma cells become more aggressive31.
32 metastases. On the other hand, melanomas expressing integrin α4β1 develop lymph node metastases31.
One study showed that of all cancers, cutaneous melanoma is the most common type to metastasize to the submucosa of the small intestine; this migration is thought to be directed by CCL25, a cytokine produced by the epithelium of the small intestine that attracts CCR9-bearing melanoma cells31.
In addition, melanoma cells develop mechanisms to escape from the immune system. In one example, melanoma cell proliferation is normally inhibited by cytokines, such as IL-2, TNF-α and IL-6. Metastatic melanomas can develop resistance to these cytokines through the modification of the receptor of oncostatin M; an IL-6-related cytokines. This resistance is due to the histone hypoacetylation at the promoter rendered this receptor less responsive to oncostatin M31.
3 Classification of cutaneous malignant melanoma
Primary melanoma lesions typically present as any of four main subtypes: superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo malignant melanoma (LMM) and acral lentiginous melanoma (ALM).
SSM is the most common primary cutaneous melanoma subtype (70%), it usually displays a prominent lateral spread throughout the epidermis and while most of the SSM arise de novo on chronically UV exposed skin especially at an early age (predominantly on the trunk and lower extremities)33, a quarter are associated with dysplastic nevi, indeed most nevus-associated primary melanomas are SSM25. SSM initially displays a radial growth phase; it then progresses to the vertical phase of growth. SSM usually occurs over a period of 7 years.
SSM lesions are variable in shape and color, usually flat but may become irregular and elevated in later stages; the lesions average 2 cm in diameter, with variegated colors. Diagnosis of SMM mainly occurs in patients in their 4th or 5th decades and is equally prevalent in both men and women33.
33 blue-black nodules but may lack pigment in some circumstances, the nodule can extend down into the skin as far as subcutaneous tissues35. NM affects men mainly in their 5th decade.
LMM is less common (10%) and the least aggressive melanoma type. it usually occurs in older patients, on chronically sun-exposed skin such as on the head and neck. This subtype is histologically characterized by the confluent growth of atypical melanocytes along the dermal-epidermal junction with frequent protrusions into the dermis35.
ALM accounts for less than 5% of the total case numbers and is the most common type of melanoma in dark skinned people (35%). This type is most commonly found on the palms of the hands and soles of the feet or around the big toenail. Melanomas from acral lentiginous frequently harbor activating mutations and/or increased copy number in the KIT tyrosine kinase receptor gene, which are very rare in the more common cutaneous tumors36. Unlike other forms of melanoma, ALM is not associated with UV exposure.
In addition to these major subtypes, there are several rarer variants of malignant melanoma including desmoplastic, nevoid, verrucous, amelanotic melanoma.
Amelanocytic melanomas which do not contain any pigment, account for 2% of all malignant melanoma cases. They usually go undiagnosed due to their uncharacteristic appearance. For example desmoplastic melanoma, a rare amelanocytic melanoma variant, is usually not diagnosed due to it being mistaken as scar tissue37.
4 Risk factors for melanoma 4.1 Environmental risk factors
Several risk factors for the development of cutaneous melanoma have been identified. The most well-known and commonly associated being lifetime exposure to ultraviolet radiation (UVR). UVR has damaging effects on the skin via direct and indirect mechanisms, such as the formation of cyclobutane pyrimidine dimers, gene mutations, immunosuppression and oxidative stress. Both UVA (λ=320-400nm) and UVB (λ=280-320nm) radiations damage skin and cause skin cancer; however, UVB rays are a more potent cause of melanoma.
34 sessions. A meta-analysis by Boniol et al. showed an overall summary relative risk (RR) of 1.2 (95% CI 1,08- 1,34) of melanoma development in ‘ever use’ of sunbeds and a 1,8% increase of risk for each additional session of sunbed use per year. In a subgroup analysis of subjects who first used sunbeds at an age below 35 years, the summary RR rose to 1.87 (95% CI 1,41-2,48) indicating a higher melanoma risk with an early onset of tanning bed exposure40. The International Agency of Cancer Research recommends restricted access for minors and young adults to indoor tanning facilities.
4.2 Phenotypic risk factors
Red hair, fair skin, blue eyes, poor tanning ability and freckling are phenotypic features associated with increased cutaneous melanoma risk. These characteristics are strongly correlated with skin photosensitivity. Red hair carries the highest relative risk (RR = 3.64, 2.56–5.37) compared to dark hair. Patients with high-density freckling have double the risk compared with patients with little or no freckling41 (Table 2).
It is no longer accepted that a nevus is usually the precursor lesion. The presence of pre-existing nevi at the tumor site is unlikely, suggesting that the majority of lesions arises de novo (70٪). The lifetime risk of any single nevus in a 20- year-old person transforming into melanoma by the age of 80 years is 1/3000 for men and 1/11 000 for women42.
Table 2: Risk factors for melanoma. Environmentally related
risk factors
Phenotypically related risk factors
Genetically related risk factors
UVR: sunshine, sunbeds Red hair, fair skin, blue eyes, freckles
Pigmentary genes polymorphisms (MC1R, tyrosinase, MITF…)
Family history of melanoma Mutation in CDKN2A, PTEN genes
Multiple common nevi Family and personal history of melanoma
35 However, increased number of both common melanocytic and/or atypical nevi increase the risk for melanoma; in their meta-analysis, Gandini et al showed that patients with a high number of common nevi (> 100) carried a sevenfold increased risk for melanoma, compared with those with low numbers (0–15 common nevi) and that the presence of even a single atypical nevus conferred a high risk of melanoma, increasing six-fold with presence of five atypical nevi43. Also, a family history of melanoma increases the risk for melanoma; 8-12% of all melanoma patients have a hereditary component44.
4.3 Genetic risk factors
Heritable factors play an important role in cutaneous melanoma predisposition and a family history of melanoma is associated with a significant twofold increased risk of melanoma45 (Table 2).
Several pigmentary gene polymorphisms favoring pale skin and red hair color phenotype have been associated with increased risk of melanoma. One example is the MC1R loss-of-function polymorphisms; MC1R loss causes a shift of melanogenesis from the photoprotective eumelanin to pheomelanin, resulting in a phenotypic spectrum of red hair color, pale skin and freckles46 and MC1R variants increase the risk of sporadic cutaneous melanoma in darker-pigmented Caucasians47.
In addition, a series of genome wide association studies on pigmentary phenotypes and skin cancer risks have implicated several genetic risk loci affecting pigmentation48. MITF variant was also associated with a higher nevus count and additional risk factors38.
Approximately, 5-10% of melanoma occurs in families with hereditary melanoma predisposition including multiple cases in family, multiple primary cancers in one individual, and melanoma diagnosis at a young age (< 40 years). Inherited mutation of genes with a critical role in cell cycle occurs in 30–40% of familial melanomas. Examples of these genes are cyclin dependent kinase inhibitor 2A (CDKN2A) (autosomal dominant) and Phosphatase and Tensin homolog (PTEN). Defects in these genes in familial melanoma principally are homozygous deletions49.
CDKN2A is located on chromosome 9p21. The mutation of this gene has been found in 25% to 40% of familial melanomas and affects two tumor-suppressor proteins, p16INK4A and p14ARF both involved in regulation of cell cycle progression and induction of senescence (Figure 11).
36 Other risk factors include a personal history of non-melanoma skin cancer and immunosuppression related to organ transplantation, Xeroderma pigmentosum, lymphoproliferative disease or human immunodeficiency virus infection/ AIDS. Exposure to heavy metals and insecticides has also been reported44,45.
5 Epidemiology of melanoma
In recent decades, incidence rates for cutaneous melanoma have been increasing in most fair-skinned populations worldwide. Annually, this increase in incidence rate varies among populations, ranging from 3% to 7%, equal to a doubling of rates every 10–20 years38. The calculated life-time risk for new-borns of developing melanoma being is 1:50.
Over the past twenty years, cutaneous melanoma has become a more prevalent cancer, indicating an influence of changing environmental risk factors50; people are spending more time outside, in direct contact with the sun.
The incidence of cutaneous melanoma varies worldwide with latitude and altitude, with generally higher incidence reported nearer to the equator and at higher altitude. Populations in Australia and New Zealand show the highest incidence rates (Figure 7) likely because they are subject to a combination of a high risk predisposition (fair-skin) and environmental factors (intensive exposure to UV light)38.
Figure 7: Geographic variability in melanoma incidence (Ferlay J, et al. GLOBOCAN 2002 Cancer Incidence, Mortality and Prevalence Worldwide. IARC CancerBase No. 5, version 2.0 IARCPress, Lyon. 2004)
Some epidemiologic data showed:
37 at diagnosis was 60 years in males and 55 years in females (Belgian cancer registry for 2008). In 2012, melanoma constituted the 7th most frequent tumor in males (3.9%) and the 5th most frequent in females (6%). However, it was an uncommon cause of cancer death in males (1.1%) and females (1.3%) (Belgian cancer registry for 2012) (Figure 8).
In addition, in Lebanon, in 2013, melanoma constituted 0.8% whereas other skin cancers constituted 8.6 % (10 times more) of total cancer registry (ministry of public health, Lebanon).
Figure 8: The ten most frequently occurring tumors by sex, Belgium 2012 (Belgian cancer registry, 2012)
Cutaneous melanoma affects a younger patient population than many other malignancies. The median age at diagnosis is 62 years of age and each death related to melanoma corresponds to about 19 years of life lost, one of the highest for any cancer.
38
6 Pathology
Three major events are required for melanoma progression, including clonal expansion of cells harboring initial mutation, acquisition of additional mutations allowing cells to overcome senescence, and acquisition of mutations which result in suppression of apoptosis51. A number of relevant oncogenes or tumor suppressor genes have been found in association with melanoma development (Figure 9).
Initial mutations in melanocytes, such as activating mutations in BRAF, NRAS, or KIT would lead to hyperproliferation. In normal cells, this over-activation triggers p16INK4A-mediated senescence. Additional mutations, such as inactivation of retinoblastoma protein (Rb) pathways or activation of telomerase, are required to enable cells to overcome senescence and to become proliferative and immortalized.
Lesions with changes described above, are early malignant melanocytic lesions in the RGP of melanoma (in situ) (Figure 9).
Additional changes are required to allow melanoma cells to escape keratinocyte dependence and invade deeper layers of the skin, a stage that characterizes the VGP of melanoma progression. This stage requires mutations that actively suppress apoptosis. Genetic alterations consistent with advanced melanomas include loss of phosphatase and tensin homolog (PTEN), RAS and RAF activating mutations and β‐catenin activation52
.
Finally, vascularisation and melanoma growth, seen at later metastatic stages, are regulated by several factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet‐derived growth factor (PDGF)52
.
Each factor will be detailed dependent on the pathway it is involved in.
39
6.1 Role of receptor tyrosine kinases
Receptor tyrosine kinases (RTK) play a pivotal role in the normal regulation of all basic cellular functions, including cell proliferation, differentiation, migration and survival. RTKs are trans-membrane polypeptides that contain both an extra-cellular ligand binding domain and a cytoplasmic tyrosine kinase domain, which has the ability to regulate signaling via a number of key pathways, including the Ras/MAPK and the PI3K/AKT pathways. In addition, RTKs can stimulate the production of growth factors such as FGF and PDGF leading to angiogenesis and matrix degradation.
c-Kit
c-kit is a receptor tyrosine kinase; it is stimulated by cytokine ligand stem cell factor (SCF). Until recently it was believed that C-Kit expression was lost with melanoma progression. However recent studies have shown that C-Kit is overexpressed in a small percentage of melanoma patients53. Patients who have mutated c-Kit are generally not BRAF mutated. These data suggest important differences between melanomas on a molecular level based on their site and constitutional and genetic factors that are not yet entirely clear54.
AKT Pathway and PTEN in melanoma progression
Although PI3K itself is rarely mutated or overexpressed in melanoma, activation of downstream signaling components, e.g. AKT, have been implicated in melanoma progression. Increased phosphorylated AKT was detected in primary and metastatic melanoma. The AKT pathway stimulates cell cycle progression by controlling G1 progression, cell proliferation and inhibition of apoptosis55.
PTEN, a tumor suppressor gene, encodes phosphatase with dual specificity. The lipid phosphatase activity of PTEN can down-regulate the AKT pathway55. Moreover, the protein phosphatase activity of PTEN can inhibit MAPK signaling. Loss or mutation of PTEN is associated with activation of the AKT pathway and has been found in approximately 30-50% of melanoma cell lines54 (Figure 10).
Mitogen activating protein kinase (MAPK) pathway
40 differentiation and survival through activation of various signaling pathways. In melanocytes, this pathway is activated by growth factors such as SCF, FGF and HGF. Stimuli of the RAS family of proto-oncogenes cause the activation of the RAF family of serine/threonine kinases (e.g. BRAF, CRAF and ARAF). RAF then phosphorylates the MAPK kinase MEK, which can lead to activation of the MAPKs, ERK1 and ERK254. ERKs relay proliferative or survival signals through phosphorylation of a variety of cytoplasmic targets, such as prosurvival ribosomal S6 kinase (p90rsk) or proapoptotic bcl-2 interacting mediator of cell death (BIM); cytoskeletal targets, such as microtubule-associated proteins 2 and 4 (MAP 2/4); and nuclear transcription factors, such as c-MYC, c-FOS54 (Figure 10).
41 BRAF, a key player in the pathway, is mutated in 60-70 % of melanoma cases (somatic mutation, no germinal mutation described). All BRAF mutations are within the kinase domain, with a single substitution (V600E) accounting for 80%. This V600E mutant possesses 10.7 fold kinase activity versus wild-type BRAF56. Other common BRAF mutations in melanoma, found in the same codon, are V600K (about 16% of mutations in melanoma) and V600D/R (3% of all mutations). These less common variants are found at slightly higher rates in melanomas arising in older patients57. All of these V600 mutations result in a mutant form of the BRAF protein that is constitutively active without the need for activation signals from growth factors through cell surface tyrosine kinase receptors. The result is uncontrolled proliferation, enhanced invasiveness, and resistance to apoptosis. BRAF mutations are early events in melanomagenesis: 80% of dysplastic nevi harbor the mutation57.
V600E BRAF also contributes to neoangiogenesis by stimulating autocrine VEGF secretion58. Thus, BRAF is implicated in several aspects of melanoma induction and progression.
Some groups have reported that despite the high prevalence of BRAF mutations in nevi, RGP has a low frequency of mutations that increases upon the transition to VGP59 and as melanoma metastasize60. Also, studies have demonstrated that melanomas with the highest degree of BRAF mutations were those with intermittent sun exposure54.
RAS mutations, particularly NRAS, are also associated with melanoma. Studies have found that approximately 10-12 % of all melanoma mutations are Ras mutations54. The most common mutation is the Q61L61. Activating NRAS mutations have been correlated with chronic sun exposure62. In the majority of cases, NRAS and BRAF mutations are mutually exclusive63.
6.2 Cell cycle
Senescence is a key cellular protection mechanism against cancer because it halts aberrant cell proliferation. P16INK4a/Rb signaling is a key regulator of melanocyte senescence and, consequently, to override senescence melanoma cells must inactivate this pathway.
The cyclin-dependant kinases CDKS constitute a group of serine/threonine kinases that are responsible for driving progression of the cell cycle through a series of sequential phosphorylation events involving target proteins critical to nuclear transcription that become operative after their activation 51,53.
42 damaged DNA. p16 mutations are found in both hereditary malignant melanoma and in sporadically developed melanomas54. Somatic mutations in this gene can be point mutation, deletion, promoter methylation, for example CC→TT translocation; a hallmark of UVR-induced mutagenesis65,66.
Figure 11: The CDKN2A locus (A). Both p16Ink4a and p14Arf proteins are encoded by the CDNK2A gene. These two proteins start from different first exons but share some coding sequence in two different reading frames. (B) p16Ink4a binds directly to cyclin-dependent kinases Cdk4 and Cdk6 blocking the assembly of catalytically active cyclinD-Cdk complexes. By phosphorylating members of the pRb family, these Cdk complexes enable the transcription of genes that are under the control of the E2F family of transcription factors. Elevated expression of p16Ink4a causes a G1-phase cell cycle arrest that is dependent on functional pRb. p14Arf stabilizes and enhances p53 level by inhibiting Mdm2-mediated p53 ubiquitination and degradation through the proteasome. p53 accumulation leads to either cell cycle arrest or apoptosis (Lomas, J. The genetics of malignant melanoma. Front. Biosci. Volume, 5071, 2008)
p53:
43
6.3 MITF
MITF gene amplification is seen in approximately 10% of primary cutaneous melanomas and 20% of metastatic tumors, but not in benign nevi, and disruption of MITF is lethal to melanoma cells with these amplifications. In metastatic melanoma patients, amplification of MITF was associated with decreased 5-year survival rates and resistance to chemotherapy68.
6.4 Growth factor dependence in melanoma development
Melanocytes are known to produce basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). When melanocytes undergo transformation to melanoma cells they show an increase in growth factor receptors and cytokine receptors69. bFGF not only plays a role in the survival of melanoma cells but is also involved in regulating motility by up-regulating serine proteases and matrix metalloproteinases70.
HGF acts through its tyrosine kinase receptor (C-Met) present on melanocytes, HGF stimulates proliferation and motility of melanoma cells through the disruption of adhesion between melanocytes and keratinocytes via down regulation of E-cadherin66.
Paracrine growth factors such as vascular endothelial growth factor (VEGF) are associated with modulation of the microenvironment. Because of its strong angiogenic properties, VEGF can stimulate endothelial cell growth, migration and invasion70.
6.5 Cadherin expression in melanoma progression
The genetic and cellular differences that result in progression from RGP to VGP are not fully understood, however, one factor crucial to the progression to melanoma is a change in cadherin expression. Cadherins are a family of cell surface glycoproteins (type 1 transmembrane proteins) that promote calcium dependent cell-cell adhesion. E-cadherin acts as a mediator between keratinocytes and normal melanocytes; it plays a key role in melanoma development and is lost during melanoma metastasis. Upon losing expression of E-cadherin, cells increase mobility and invasiveness and keratinocytes no longer control melanocytes69.
7 Diagnosis
7.1 Diagnosis of primary melanoma
