Especific roles of endolysosomal trafficking in melanoma progression and drug response

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UNIVERSIDAD AUTÓNOMA DE MADRID

Facultad de Ciencias

Departamento de Biología Molecular

Tesis Doctoral

FUNCIONES ESPECÍFICAS DEL TRÁFICO ENDOLISOSOMAL EN LA PROGRESIÓN Y RESPUESTA A TERAPIA

DEL MELANOMA

DIRENA ALONSO CURBELO

Madrid, 2013

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AUTONOMOUS UNIVERSITY OF MADRID Faculty of Science

Department of Molecular Biology

SPECIFIC ROLES OF ENDOLYSOSOMAL TRAFFICKING IN MELANOMA PROGRESSION AND DRUG RESPONSE

A doctoral thesis submitted to the Autonomous University of Madrid for the degree of Doctor of Philosophy in Molecular Biology

Direna Alonso Curbelo

Thesis Director

Dr. María S. Soengas

Melanoma Group (Molecular Pathology Program)

Spanish National Cancer Research Center

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Dr. María S. Soengas, Director of the Molecular Pathology Program and Head of the Melanoma group at the Spanish National Cancer Research Center (CNIO)

CERTIFIES:

That the Doctoral Thesis “Specific roles of endolysosomal trafficking in melanoma progression and drug response” developed by Ms Direna Alonso Curbelo meets the necessary requirements to obtain the PhD Degree in Molecular Biology and, to this purpose, will be presented at the Autonomous University of Madrid. The thesis has been carried out under my direction and hereby I authorize it to be defended to the appropriate Thesis Tribunal.

I hereby issue this certification in Madrid on April 30st 2013.

María S. Soengas PhD Thesis Director

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Dr. Jaime Millán Martínez, Head of group of Cell Biology of Inflammation at the Centro de Biología Molecular Severo Ochoa (CBMSO)

CERTIFIES:

That the Doctoral Thesis “Specific roles of endolysosomal trafficking in melanoma progression and drug response” developed by Ms Direna Alonso Curbelo meets the necessary requirements to obtain the PhD Degree in Molecular Biology and, to this purpose, will be presented at the Autonomous University of Madrid. The thesis has been carried out under my direction and hereby I authorize it to be defended to the appropriate Thesis Tribunal.

I hereby issue this certification in Madrid on April 30st 2013.

Jaime Millán Martínez PhD Thesis Tutor

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The work presented in this doctoral thesis was carried out in the Melanoma Group at the Spanish National Cancer Research Center (CNIO) from June 2008 to June 2013 under the supervision of María S.

Soengas.

This work has been supported by the following fellowships and grants:

 “Formación de Profesorado Universitario” (FPU) PhD Fellowship, awarded by the Spanish Ministry of Science and Education. Direna Alonso Curbelo (2008 – 2012)

 INNPACTO program. María S. Soengas (2012 – 2013)

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“Lo imposible es posible intentarlo”

José Miguel Alonso Fernández-Aceytuno

“The impossible is always possible to be pursued”

José Miguel Alonso Fernández-Aceytuno (1951 – 2004)

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A mi padre

del que tanto aprendí

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Acknowledgements

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Esta tesis representa el final de una etapa que he vivido intensamente y en la que he aprendido muchísimo, tanto a nivel científico como a nivel personal. Y si hoy me encuentro ante esta página en blanco que llenar con mis más sinceros sentimientos de gratitud es gracias al apoyo, a la inspiración, a la energía positiva y a la ayuda incondicional que me habéis dado todos a lo largo de estos años. A todos vosotros, GRACIAS.

GRACIAS Marisol por haber confiado en mis ganas de aprender aquel agosto de 2008 en el que hablamos por primera vez, dándome la gran oportunidad de embarcarme en este apasionante mundo de la ciencia a través de tu laboratorio y del CNIO. Gracias muy especialmente por haber reconocido también las ganas del resto de mis compañeros y construir este grupo de investigación tan estupendo. Y gracias de corazón por tu gran apoyo que no sólo ha hecho posible esta tesis, sino que además me ha abierto las puertas de la siguiente etapa, que espero con muchísima ilusión.

THANKS MELANOMA GROUP! I have no words to express all the gratitude and love I feel for you guys. It has been a real privilege to work with and learn from you all along these years. You have been the best travel companions and my seat belts on this PhD roller coaster. You are the definitely the faces of these last years and one of the most valuable things I take from them. I am sure that in a distant future, when my memories of western blotting and cell line #9 have vanished away, I´ll always remember the awesome time we had together, in and outside the lab. Estela, eres la mejor lab manager, compañera, y

“writing consultant” que se puede tener, pero sobre todo, eres una gran persona y una excelente amiga.

GRACIAS por todo tu cariño, por cuidar siempre de mí. No sabes lo que voy a echar de menos tu risa y la energía positiva que desprendes… Damià, “pseudo-jefe”, contigo di mis primeros pasitos del doctorado y desde entonces no he parado de aprender de ti. Tu capacidad para transformar las ideas en hechos, tu ilusión por mejorar lo que nos rodea, y tu forma de hacerlo, siempre con sonrisa puesta, admirables. Eva, no te imaginas lo importante que ha sido para mí tenerte a mi lado todos estos años.

Tu paciencia, tu forma de hacer, de estar y de ser siempre han sido un gran ejemplo para mí. Gracias también por tu disposición para escuchar y ayudar a los demás, que además creo que son fundamentales para el laboratorio en general. Erica, muchas gracias por tus siempre sabias palabras, capaces de devolver la necesaria dosis de perspectiva a los momentos difíciles. He aprendido muchísimo de ti; de tu fortaleza, de tu optimismo y de tu sinceridad. Lisa, my lab “big sister”, my german “Other Self”, thanks so much for being such a good friend and filling the lab atmosphere with your incombustible inner glow. Your big smile is very small compared to your huge heart. And many THANKS too for the English editing of the thesis! Metehan, I want to thank you very much for all your support, for having so much patience with my incessant questions, for so many great conversations and for always seeking and coming up with an original idea, solution, or strategy to make our lives a lot easier.

Takis, today, here, I am not going to emphasize my admiration to your pipetting muscles. I really want to thank you for always having that friendly “yes” sitting at the tip of your tongue. Thanks also for your constant willingness to help me and others whenever you can. DOC, el flautista de Hamelín más majo y coqueto del CNIO y una pieza clave del laboratorio, muchas gracias por haberme enseñado tanto sobre metástasis, ¡y los mejores lugares de tapas del centro! Tonan, gracias de verdad, no sólo por tu siempre excelente ayuda técnica, que además ha sido FUNDAMENTAL para este trabajo, sino también por todo tu cariño. Eres la gasolina que mantiene rodando el laboratorio (y mi entropía en cierto orden!). He aprendido muchísimo de ti. Gracias! David Sáenz, ¡cómo te he echado de menos estos últimos años en el laboratorio! Existen pocos como tú, con esa entrega incondicional hacia los demás y hacia su trabajo, y con todo eso de buena persona que tienes y que tanto te caracteriza. Lionel y Agi, thanks a lot for everything you taught me and for showing me what persistence in science means. María, ¡eres una crack! Me ha encantado conocerte y descubrir cómo si se quiere, se puede. Si vuelvo del postdoc en Nueva York pareciéndome un poquito más a ti, ya me puedo dar más que por satisfecha. Alicia,

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muchísimas gracias por tu compresión y por todo tu apoyo ;) ¡Estoy segura de que el futuro del pICPEI está en excelentes manos! ¡Suerte con todo! Ángel, el pichichi en geles del labo, muchas gracias por tu excelente ayuda técnica y, sobre todo por encargarte, aún sin hacerlo a conciencia, de mantener el buen rollo en el laboratorio. Raúl, no sabes la penita que me da que no poder coincidir más tiempo contigo en el laboratorio. ¡¿Por qué no llegaste antes?! Bueno, no sé si ya lo sabes, pero es tradición en el laboratorio que los doctorandos de más de 1.90m de altura mantengan SIEMPRE la curiosidad y la ilusión. Daniela, te dejo encargada de que la gente del labo acabe diciendo “SENIO” en lugar de CNIO jeje. Muchísimas suerte con el doctorado, aunque sé que no te hará falta, porque eres buenísima. Napala, thanks SO MUCH for your constant smile and for the English editing of this thesis in record time. Carla, Renata y Carol, thanks for bringing a little closer to the lab the best energy of Brazil (and the brigadeiros!). By the way Marisol, perhaps we should include “cachaça” in the lab´s reagents list! Y a todos los demás que, en algún momento habéis formado parte de este equipo (Iván, Marco, Elisa, Joe, Bobby, Elena, Silvia, Marta…), gracias también por vuestra apoyo

.

MUCHAS GRACIAS también a los miembros de mi Comité de Tesis en el CNIO: Xosé Bustelo, Mirna Pérez-Moreno y Manuel Serrano por haber compartido conmigo toda vuestra experiencia, que ha sido fundamental para el desarrollo de este proyecto así como para mi aprendizaje a nivel científico y personal.

MANY THANKS to the Epithelial Carcinogenesis Group (CNIO) for their support and input during the Monday lab meetings, as well as for being SUCH COOL LAB NEIGHBOURS. THANKS as well to the Lymphoma Group (CNIO), and very especially to Elena Rodriguez, for “adopting” me when I was just about to start the PhD and the Michigan Melanoma group was still moving to the CNIO.

MUCHAS GRACIAS también a nuestros colaboradores del Hospital 12 de Octubre de Madrid: los doctores José Luis Peralto, Pablo Ortiz, y Erica Riveiro por haber hecho posible el estudio de RAB7 en muestras humanas. Ha sido emocionante ver como un proyecto que se inició y se desarrolló en la poyata adquiere una dimensión de realidad, haciendo que esta experiencia sea más enriquecedora y merecedora de todo este esfuerzo.

De la misma manera, quiero dar las gracias a Damià y a su equipo de Bioncotech Therapeutics por intentar que los frutos de la investigación se traduzcan finalmente en una mejora real en la expectativa de vida de pacientes. ¡No existe mejor motivación que ésta para hacer ciencia!

I really want to thank all of my colleagues who actively participated in the RAB7 project. Hopefully, all the effort will soon be rewarded! THANKS to Gonzalo Gómez and Osvaldo Graña (Bioinformatics Groups, CNIO) for your important contribution to this work. In addition, I would also like to thank all the people working at the CNIO Flow Cytometry, Histopathology, Genomics, and Animal Facility Units; the CNIO Tumor Bank; and to José Manuel (from CNIO Information Technologies) for their excellent technical support.

VERY VERY SPECIAL THANKS to Diego Megías and his great Confocal Microscopy Unit team, Ximo Soriano and Manu Pérez for their unconditional help and for being the coolest microscopy guys ever.

Without you guys this thesis would not have been possible!

THANKS to Dr. Reuven Agami (NKI, Amsterdam) and to Dr. Johanna Joyce (MSKCC, NY) for giving me the enriching opportunity of joining their labs as a visiting PhD student. During those months I met great scientists and friends that made my stays in Amsterdam and NY an unforgettable experience: Arnold

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Bos, Carlos Melo, Maritt Terweij, Dominika Bijos, Hayley Moore, Nicolas Leveillé, Carlos Le Sage, and, of course David Ontoso, in Amsterdam; and Sonia Mulero, Chema Carvajal, Alberto Schuhmacher, Joni Van Der Meulen, Silvia Domcke, Lisa Sevenich, Leila Akkari, Hao-Wei Wang, Oakley Olson, Bobby Bowman, Carlos Carmona, Richard Stein, Neils Weinhold, and Nick Gauthier in New York. THANKS SO MUCH FOR MAKING FEEL AT HOME!

Gracias también a los chicos de Mantenimiento de CNIO por ser tan simpáticos y eficaces; así como a Emma y al resto del equipo de la Cafetería del CNIO por alimentarme casi como una madre y por, como no, las tapas de los viernes!

Quiero darle las GRACIAS también a muchos compañeros del CNIO que, con su amistad, con su ayuda, o tan sólo mediante un cruce de sonrisas cómplices por los pasillos, han hecho que mi estancia aquí haya sido tan agradable. GRACIAS muy especialmente a Eva Sánchez, Juanlu, Alba, Ana del Río, Eva Briso, Lina, Laia, Sara Mainardi, Carolina Navas, Dani Martín, Bea H, Daniela, Martina, Javier Leandro, Lara, Marta Shahbazi, Miguel Foronda, Patricia Nieto, y muchos otros (porque sería imposible nombraros a todos) por ser tan majos y los protagonistas de muchos de los recuerdos que me llevo del CNIO. Laura y Bárbara, a vosotras muy en especial, MUCHÍSIMAS GRACIAS por vuestro apoyo incondicional y por regalarme vuestra amistad. Haberme embarcado en el doctorado ya mereció la pena el día que os conocí.

También quiero agradecer el apoyo que he recibido de viejos y nuevos amigos que me han acompañado a lo largo de esta etapa de tesis. Habéis sido mis “gatorades” en esta maratón. MUCHÍSIMAS GRACIAS Marty, Mer y Auro; porque vuestra amistad siempre me ha hecho más feliz, mejor persona y más fuerte. Sois mi mejor equipo. GRACIAS Tere. Me siento muy afortunada por haber compartid carrera, hospital, tesis y casa con una gran persona y amiga como tú. Eres muy grande, que lo sepas! GRACIAS Daniel Movilla por tantos buenos momentos en los que hemos arreglado el mundo y nuestras vidas.

“Redescubrirte” ha sido el mejor regalo del 2012 (Birdybirdybirdy). GRACIAS David Ontoso por ser un amigo sin igual. Hasta la “Dire muerta de hambre” sólo tendría buenas palabras para ti ;). GRACIAS Carlos Gordo y Marc por todo vuestro cariño. GRACIAS también a Helena, Carmen, y a Ángela por ser tan buenas amigas y las mejores compañeras de piso. MUCHAS gracias también a “La Cuadrilla” de Madrid por hacerme sentir como si fuera del “Jesús Maestro”. MUCHAS GRACIAS a Mari Mar y a Paco, por haber formado una familia tan estupenda y por todo esos buenos ratos y ratitos de mesa y sobremesa (y por los tápers de carne picada! jeje).

MUCHAS GRACIAS a mis amigos de Las Palmas, muy especialmente a Lidu, Alfredo, Héctor, Aday, Laura, Nolo, Juan, Laura Merino, y Cris Santana por el día a día y los largos veranos de ayer, y por los

“Encuentros” revitalizadores de hoy. Con vosotros, la distancia no existe.

Y por último quisiera dedicar los últimos agradecimientos a las personas más importantes de mi vida, mis grandes pilares, mis norte-sur-este-y-oeste. ¡MUCHÍSIMAS GRACIAS A MI MARAVILLOSA FAMILIA!

Sois muchos y sólo tengo buenas palabras para cada uno de vosotros. GRACIAS muy especialmente a mis abuelas, por todo vuestro amor y por enseñarme las claves para ser feliz; a Ana Mari, porque para mí eres un gran referente, y a Margarita Curbelo, Cristina Curbelo, Nano y Marina, por haber creído tanto en mí y demostrármelo siempre.

MUCHISÍSISISISIMAS GRACIAS a Jorge y Ana, ¡por ser los mejores hermanos del mundo! No paro de aprender de vosotros, a pesar de ser yo la hermana mayor. ¡Os quiero muchísimo!

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Javi, MUCHAS GRACIAS por haber hecho que estos años hayan sido inolvidables, por dibujarme una sonrisa cada mañana y hacer de la tesis un “paraíso con gastos pagados”. Recuerdo las ganas de empezar el día en Galileo 25 y los trayectos en la “olivita” hacia el CNIO del principio… qué rápido ha pasado el tiempo la verdad, aunque no me extraña, porque estos años no los he medido en días, sino en fines de semana. Muchas gracias por tu enorme apoyo, por hacerme reír tantísimo, por subir el “phD de mi piel”, por tantos buenos momentos y viajes juntos, por creer tanto en mí, por todo tu amor. TANGO QUÉBEC.

PARA MIS PADRES NUNCA TENDRÉ SUFICIENTES PALABRAS DE AGRADECIMIENTO… Papi, te llevo en el corazón, muy cerquita, siempre, a todas partes. Y, aunque mientras escribo estas líneas las lágrimas evidencien la tremenda nostalgia y el vacío irremplazable que siento (porque te echo muchísimo de menos y deseo que pudieras estar aquí con nosotros), el recuerdo de tu incombustible ilusión, de tu siempre optimista mirada hacia el futuro, y de la entereza que te caracterizó hasta el final me da la fuerza para seguir siendo una persona muy feliz. Gracias por enseñarme tanto y por ser una grandísima persona. Mami, a ti te dedico las últimas palabras porque, si en la vida dicen que uno va eligiendo su propio camino, tú eres mi brújula, mi mapa, mi gasolina, mi “airbag” cuando tropiezo, y sobre todo, la mejor compañera y guía de viaje. Un millón de gracias por tu enorme corazón, por tu fuerza, por tu apoyo incondicional, y por tu bien criterio. Gracias también por tus zumos revitalizadores de papaya con naranja y tus palabras sanadoras, y por cuidar tan bien de mí y de Jorge y Ana. ¡Sin duda tus hijos somos los más afortunados del mundo!

Gracias por último a J.S. Bach, y a todos los que desde chiquitita me inculcaron el amor por la MÚSICA, que me ha dado tantos momentos de placer y es mi mejor anestesia.

¡MUCHAS GRACIAS A TODOS POR ACOMPAÑARME EN ESTA ETAPA! Sólo puedo terminar esta etapa de tesis y estos agradecimientos, como diría Sabina: añadiendo al punto final, dos puntos suspensivos…

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"En este proceso mental, precursor del descubrimiento, nada es inútil:

los primeros grosos errores, así como las falsas rutas donde la imaginación se aventura, son necesarios, pues acaban por conducirnos al verdadero camino, y entran, por tanto, en el éxito final, como entran

en el acabado cuadro del artista los primeros informes bocetos."

Santiago Ramón y Cajal (1852-1934)

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ABBREVIATIONS

SUMMARY

RESUMEN

INTRODUCTION

1. THE MELANOMA CHALLENGE: WHERE ARE WE NOW? 29

2. THE CELLULAR ORIGIN OF MELANOMA: THE MELANOCYTE 30

3. CLASSIFICATION OF CUTANEOUS MELANOCYTIC LESIONS 31

3.1 BENIGN MELANOCYTIC LESIONS: NEVI 32

3.2. MALIGNANT MELANOCYTIC LESIONS: MELANOMA 32

4. DEVELOPMENT AND PROGRESSION OF MELANOCYTIC TUMORS 34

4.1 HISTOLOGIC, BIOLOGIC AND GENETIC FEATURES ASSOCIATED WITH MELANOMA PROGRESSION

34

4.2. INTRATUMOR HETEROGENEITY AND MELANOMA-CELL PLASTICITY 39

5. MELANOMA ONCOGENES AND “NON-ONCOGENE” DEPENDENCIES 41

5.1. MELANOMA ONCOGENES: “CLASSICAL” VERSUS “LINEAGE-SPECIFIC” FACTORS 41 5.2. NON-ONCOGENE DEPENDENCIES IN MELANOMA: AUTOPHAGY AND BEYOND 44

6. TREATMENT OF CUTANEOUS MELANOMA 48

OBJECTIVES

OBJETIVOS

MATERIALS AND METHODS

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1. CELLS 61

2. GENE SET ENRICHMENT ANALYSIS (GSEA) IN MULTITUMOR DATASETS 61

3. OLIGONUCLEOTIDE ARRAY CGH (COMPARATIVE GENOMIC HYBRIDIZATION) 62

4. TISSUE MICROARRAYS (TMAS) AND IMMUNOHISTOCHEMISTRY (IHC) 62

5. KAPLAN-MEIER SURVIVAL ANALYSES 62

6. PROTEIN IMMUNOBLOTTING 63

7. IMMUNOFLUORESCENCE AND CONFOCAL-BASED SINGLE-CELL QUANTIFICATION IN TISSUES

63

8. IMMUNOFLUORESCENCE IN FIXED CELLS 64

9. RAB7 EXPRESSION IN MELANOMA “INVASIVE” OR “PROLIFERATIVE” GENE SIGNATURES

65

10. STABLE INHIBITION OF RAB7 FUNCTION 65

11. SITE-DIRECTED MUTAGENESIS AND RAB7 shRNA- RESCUE ASSAYS 66

12. siRNA-MEDIATED GENE SILENCING OF ATG7, RAB7, VPS34, SOX10 AND MITF 66

13. BECLIN1 STABLE RNA INTERFERENCE 67

14. CELL PROLIFERATION AND COLONY FORMATION ASSAYS 67

15. ANIMAL EXPERIMENTS: XENOGRAFT ASSAYS AND MELANOMA MODELS 68

16. MATRIGEL INVASION ASSAYS 68

17. ASSESSMENT OF LYSOSOMAL FUNCTION 69

18. GENERATION OF PEI-COMPLEXED PIC GENERATION OF PEI-COMPLEXED PIC 69

19. DRUG TREATMENTS AND VIABILITY ASSAYS 69

20. FLUID PHASE ENDOCYTOSIS ASSAYS 70

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21. RNA EXTRACTION, RT-PCR AND HIGH THROUGHPUT RNA SEQUENCING 71

22. VISUALIZATION AND QUANTITATIVE ANALYSIS OF CYTOSKELETAL ALTERATIONS (CYTOOCHIPS)

72

23. VIDEO AND FIXED-CELL FLUORESCENCE MICROSCOPY OF ENDOCYTIC AND AUTOPHAGIC TRAFFICKING

73

24. TRANSMISSION ELECTRON MICROSCOPY 73

25. PROTEIN SECRETION ASSAYS 74

26. ONCOGENE-INDUCED SENESCENCE ASSAYS IN PRIMARY HUMAN MELANOCYTES 74

27. STATISTICAL ANALYSES 75

RESULTS

1. LINEAGE-RESTRICTED TRAITS ASSOCIATED WITH THE LYSOSOME IN MELANOMA 79 2. LINEAGE-RESTRICTED OVEREXPRESSION OF RAB7 IN MELANOMA 81

3. MITF-INDEPENDENT OVEREXPRESSION OF RAB7 IN MELANOMA 83

4. LINEAGE-ADDICTION OF MELANOMA CELLS TO RAB7 84

5. MELANOMA CELL MORPHOLOGY AND INVASIVE POTENTIAL CONTROLLED BY RAB7 87 6. RAB7 IS AN EARLY-INDUCED MELANOMA DRIVER TUNED DOWN AT INVASIVE

STAGES OF TUMOR PROGRESSION IN VIVO

89

7. HALTED DEGRADATION OF NON-CANONICAL AUTOPHAGOSOMES AND MACROENDOSOMES IN RAB7-DEPLETED MELANOMA CELLS

91

8. DERAILED VESICLE TRAFFIC BY RAB7 DOWNREGULATION PROMOTES THE SECRETION OF LYSOSOMAL PROTEASES

93

9. GLOBAL CHANGES IN GENE EXPRESSION AND PROTEIN SECRETION PROGRAMS BY MODULATION OF RAB7 LEVELS

95

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10. UPSTREAM REGULATION OF RAB7 BY MELANOCYTE DEVELOPMENTAL PATHWAYS 97 11. REGULATION OF RAB7 EXPRESSION AND FUNCTION BY ONCOGENIC SIGNALING

PATHWAYS IN MELANOMA CELLS

98

12. ACTIVATION OF ONCOGENIC SIGNALING IN NORMAL MELANOCYTES DEREGULATES RAB7 AND ITS ASSOCIATED VESICLE TRAFFICKING PATHWAYS

100

13. ONCOGEN-DRIVEN ACTIVATION OF RAB7 IN VIVO 102

14. MODULATION OF RAB7-ASSOCIATED ENDOLYSOSOMAL VESICLE TRAFFICKING BY TREATMENT WITH ds-RNA-BASED NANOCOMPLEXES

103

15. RAB7-MEDIATED VESICLE TRAFFICKING IS ACTIVELY INVOLVED IN THE ANTI- MELANOMA ACTIVITY OF ds-RNA-BASED NANOCOMPLEXES

104

DISCUSSION

1. LESSONS FROM MULTITUMOR GSEA IN MELANOMA GENE DISCOVERY 111

2. BIOLOGICAL IMPLICATIONS OF MELANOMA-ASSOCIATED TRAITS IDENTIFIED BY GSEA 113 3. CELL LINEAGE AS A DETERMINANT OF RAB7 EXPRESSION AND FUNCTION IN CANCER 115

4. RAB7 EXPRESSION AND FUNCTION IN MELANOMA PROGRESSION 116

5. RAB7 VERSUS MITF AND OTHER LINEAGE-SPECIFIC MELANOMA DRIVERS 119

6. DOWNSTREAM EFFECTOR PATHWAYS OF RAB7 IN MELANOMA CELLS 119

7. ANTITUMOR THERAPEUTIC OPPORTUNITIES TARGETING ENDOLYSOSOMAL PATHWAYS

122

8. PERSPECTIVES 125

CONCLUSIONS

CONCLUSIONES

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REFERENCES

APPENDIX

1. SUPPLEMENTARY TABLES 163

2. SUPPLEMENTARY FIGURE 171

3. SUPPLEMENTARY VIDEO LEGENDS 172

4. PUBLICATIONS 173

5. PRESENTATIONS 173

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Abbreviations

Abbreviations

“Lo bueno, si breve, dos veces bueno”

Baltasar Gracián (1601-1658)

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Abbreviations

AJCC - American Joint Committee on Cancer AFU - Arbitrary Fluorescence Units

AKT - v-Akt murine thymoma viral oncogene homolog ATG - Autophagy-related gene

ATP - Adenosine triphosphate AURKB - Aurora kinase B

BCL2 - B-cell lymphoma 2 BECN1- Beclin1

BRAF - v-Raf murine sarcoma viral oncogene homolog B1 BRN2 - PUO class 3 homeobox 2 (POU3F2)

BSA - Bovine serum albumin CCND1 - Cyclin D1

CCLE - Cancer Cell Line Encyclopedia CDC - Cell division cycle

CDK - Cyclin-dependent kinase

CDKN2A - Cyclin-dependent kinase inhibitor 2A cDNA - Complementary DNA

CEACAM1 - Carcinoembryonic antigen-related cell adhesion molecule 1 CGH - Comparative genomic hybridization

CI - Confidence intervals CM - Conditioned media

CMT2B - Charcot-Marie-Tooth type 2B

Abbreviations

10 CNIO - Centro Nacional de Investigaciones Oncológicas CSC - Cancer stem cell

CQ - Chloroquine

CTLA-4 - Cytotoxic T-lymphocyte antigen-4 CTRL - Control

CTS - Cathepsin

DAPI - 4,6-diamidino-2-phenylindole DFS- Disease Free Survival

DMBA - 7,12-dimethylbenz[a]anthracene DMEM - Dulbecco’s Modified Eagle’s Medium DMSO - Dimethyl sulfoxide

DN - Dominant negative DNA - Deoxyribonucleic acid dsRNA - Double-stranded RNA E2F1 - E2F transcription factor 1 EDNRB - Endothelin receptor type B EDTA - Ethylenediaminetetra-acetic acid EGFR - Epidermal growth factor receptor EIPA - 5-(N-ethyl-N-isopropyl) amiloride EMT - Epithelial-to-mesenchymal transition ER - Endoplasmic reticulum

Abbreviations

ERK - ERK, extracellular signal-regulated kinase ETV1 - Ets variant 1

FACS - Fluorescence-activated cell sorting FBS - Fetal Bovine Serum

FDA - US Food and Drug Administration FDR - False discovery rate

FGF - Fibroblast growth factor FGM - Fibroblast growth medium

FYCO1 - FYVE and coiledcoil domain containing 1 GAP - GTPase-activating protein

GAPDH - Glyceraldehydes‐3‐phosphate dehydrogenase GDP - Guanosine diphosphate

GEF - Guanine nucleotide exchange factor GFP - Green fluorescent protein

GLI2 - Glioma-associated oncogene family member-2 GO - Gene ontology

GNAQ - Guanine nucleotide binding protein (G protein), q polypeptide GSEA - Gene Set Enrichment Analysis

GTP - Guanosine 5'-triphosphate

GTPase - Guanine nucleotide triphosphatase

H&E - Hematoxylin and eosin

Abbreviations

12 HOPs - Homotypic fusion and protein sorting complex HR - Hazard ratio

HRAS - v-Ha-ras harvey rat sarcoma viral oncogene homolog HRP – Horseradish peroxidase

HSP70 - 70-kDa heat shock protein IF- Immunofluorescence

IFNα – Interferon-alpha IgG – Immunoglobulin G IHC - Immunohistochemistry IL - Interleukin

INH - Inhibitor kDa - Kilodalton

KEGG - Kyoto Encyclopedia of Genes and Genomes KGM - Keratinocyte growth medium

KIT - v‐kit Hardy‐Zuckerman 4 feline sarcoma viral oncogene homologue LAMP - Lysosomal membrane protein

LC3 - Microtubule-associated protein 1 light chain 3 LDH - Lactate dehydrogenase

LTR - Lysotracker

3-MA - 3-Methyladenine

MAPK - Mitogen-activated protein kinase MC1R - Melanocortin-1 receptor

Abbreviations

MDA-5 - Melanoma differentiation-associated protein 5 MEF - Mouse embryonic fibroblasts

MEK - mitogen-activated protein/extracellular signal-regulated kinase kinase MET - met proto-oncogene (hepatocyte growth factor receptor)

MGM -Melanocyte growth medium miRNA - microRNA

MITF - Microphthalmia-associated transcription factor MMP - Matrix metalloproteinase

mRNA - Messenger RNA

MSRC - Matrix screening remote control mTOR - Mammalian target of rapamycin MUT - Mutated/mutant

MVB - Multivesicular bodies

MYC - v-Myc myelocytomatosis viral oncogene homolog NCCN - National Comprehensive Cancer Network NCI - National Cancer Institute

NEDD9 -Neural precursor cell expressed, developmentally down-regulated 9 NF1 - Neurofibromatosis Type 1

NRAS - v-Ras neuroblastoma viral oncogene homolog NT - Non treated

OIS - Oncogene Induced Senescence

Abbreviations

14 ORP1L - OSBP (oxysterol-binding protein) related protein OS - Overall Survival

P - Probability values PAX3 -Paired box-3

PBS - Phosphate-Buffered Saline

PBS-T Phosphate-Buffered Saline with Tween PCR - Polymerase chain reaction

PD1 - Programmed death 1

PDL1 - Programmed cell death 1 ligand PEI - Polyethyleneimine

PET-CT -Positron emission tomography - computed tomography PFA - Paraformaldehyde

PGC1α - PPARGC1A

PI3K - phosphoinositide-3 kinase

PI3KC3 - Class III type phosphoinositide 3-kinase pIC - Polyinosine-polycytidylic acid

[pIC]^PEI - Polyinosine-polycytidylic acid complexed with polyethyleneimine PKC - Protein kinase C

PTEN - Phosphatase and tensin homolog

qRT- PCR - Real-time reverse transcription polymerase chain reaction RAB - Ras-related in brain

Abbreviations

Rabring7 - Rab7-interacting ring-finger protein RAC1 - Ras-related C3 botulinum toxin substrate 1 RAS - at sarcoma viral oncogene homolog

RB - Retinoblastoma

RILP - Rab7-interacting lysosomal protein RGP - Radial-growth Phase

RNA - Ribonucleic acid RNAi - RNA interference

RECIST - Response Evaluation Criteria In Solid Tumors RT - Room Temperature

RTK - Receptor tyrosine kinase

SA-β-Gal - Senescence-associated β-galactosidase SAHF - Senescence-associated Heterochromatin Foci SD - Standard deviation

SDS - Sodium dodecyl sulfate

SEM - Standard error of estimate of mean value shRAB7 - RAB7 shRNA

shCtrl - Control shRNA shRNA - Short hairpin RNA siRNA - Small interfering RNAs SMO - Smoothened

Abbreviations

16

SNARE - Soluble N-ethylmaleimide-sensitive factor attachment protein receptor SOX10 - SRY-box-containing gene 10

TBC1D15 - TBC1 domain family, member 15 TBC1D16 - TBC1 domain family, member 16 TF - Transcription factor

TFDP1 - Transcription factor Dp-1 TGFα - transforming growth factor-alpha TGFβ - transforming growth factor-beta TGN - Trans-Golgi network

TNM - Tumor-Node-Metastasis TMA -Tissue Microarrays TP53 - tumor protein 53

TRP2 - Tyrosinase-related protein 2 TYR - Tyrosinase

UV - Ultraviolet

UVRAG - UV radiation resistance-associated gene; Vps, vacuolar protein sorting VEGF - vascular endothelial growth factor

VGP - Vertical-growth Phase VPS34 - Vacuolar protein sorting 34 WB - Western blotting

WHO - World Health Organization

Abbreviations

WNT - Wingless‐type MMTV integration site family WT - Wild Type

Summary

18

Abbreviations

Summary

Summary

20

Summary

Melanoma was first described as a tumor entity in 1806, and it has since remained a prime example of a heterogeneous, aggressive and treatment-resistant malignancy. Despite great progress made in the understanding of the molecular basis underlying melanoma initiation and progression, the field still lacks clinically relevant biomarkers, consensus on metastatic progression mechanisms and effective treatments for the management of advanced stages. Consequently, this PhD thesis was set to: (1) identify new genes driving melanoma pathogenesis, (2) characterize their role in tumor initiation and progression, and (3) use this information for the development of novel therapeutic strategies. We focused on the study of lineage-specific traits as a strategy to identify novel factors that might be inherently and distinctively altered in melanoma. Mining of multi-tumor gene expression data sets identified a cluster of lysosomal genes that is uniquely enriched in melanoma cells and that distinguishes this tumor type from over 35 malignancies. Within this cluster, we demonstrated a dependency of melanoma cells on the GTPase RAB7, which was observed to maintain cell proliferation in a tumor type- selective manner. In contrast to classical melanoma-associated oncogenes such as BRAF, whose depletion blocks both cell proliferation and invasion, tuning down RAB7 favored the transition to metastatic stages. RAB7 levels were found to affect melanoma cell phenotype by modulating the fate of PI3K-driven vesicles, which instead of being directed towards the lysosome for degradation, accumulated and were diverted into secretory pathways when RAB7 expression was tuned-down. The outcome of derailed RAB7-regulated vesicle traffic translated into melanoma-cell selective changes in gene expression profiles, cytoskeletal reorganization, and secretion modulators of extracellular proteolysis and matrix remodeling. Importantly, we found RAB7 to be expressed independently of MITF, the best known lineage-specific melanoma oncogene known to date. Instead, we identified that, in melanoma cells, RAB7 levels are controlled by both SOX10, an early driver of the melanocytic lineage, and PI3K signaling, which is frequently activated during tumor initiation. These results were revealed by computational methods, live microscopy, histological and functional analyses of human biopsies, cell lines and mouse models. Moreover, the clinical relevance of these results was demonstrated in follow- up studies of patient prognosis. Finally, here we demonstrated that tumor-cell specific features of RAB7- dependent vesicle traffic have the potential to be exploited therapeutically. Specifically, we found a novel strategy (based on dsRNA-based nanocomplexes) to promote an efficient self killing of melanoma cells by inducing a massive mobilization of autophagosomes, endosomes, and lysosomes, and the subsequent activation of apoptotic caspases. Together, the results of this PhD thesis underscore a unique lineage-restricted wiring of endolysosomal pathways that actively contributes to melanoma progression and serves as a tractable vulnerability that can be pursued for drug development.

Summary

22

Summary

Resumen

Resumen

24

Resumen

El melanoma se describió por primera vez como una entidad tumoral en 1806, y desde entonces, se mantiene como ejemplo de neoplasia agresiva, heterogénea y quimiorresistente. A pesar de avances notables en la compresión de las bases moleculares de la progresión del melanoma, no se dispone de biomarcadores con suficiente valor pronóstico. Del mismo modo, no existe un consenso sobre los mecanismos que subyacen al proceso de metástasis, ni se han desarrollado tratamientos eficaces para las fases avanzadas de la enfermedad. Por todo ello, esta tesis doctoral se ha centrado en: (1) identificar nuevos genes esenciales para el desarrollo del melanoma, (2) definir su regulación y su función en la progresión tumoral, y (3) utilizar esta información para el desarrollo de nuevas estrategias terapéuticas.

En particular, nos centramos en el estudio de características específicas de linaje celular con el fin de identificar nuevos factores pro-oncogénicos inherentes al melanoma. El análisis de perfiles de expresión génica de diversos tipos tumorales reveló que las muestras de melanoma presentan un enriquecimiento selectivo de genes codificantes de proteínas lisosomales, que distingue a este tipo de cáncer de más de otros 35 tipos tumorales distintos. Dentro de esta huella genética, identificamos la GTPasa RAB7 como un nuevo gen esencial para el mantenimiento de la capacidad proliferativa de estas células tumorales. A diferencia de “oncogenes” clásicos como BRAF, cuya inactivación inhibe tanto la proliferación como la invasión tumoral, la reducción en los niveles de RAB7 favorece la transición a estadios metastásicos.

Encontramos que esta doble función oncogénica de RAB7 se debe a su capacidad para regular el destino final (degradación o reciclaje) de vesículas citoplasmáticas inducidas por rutas oncogénicas que activan PI3K. La desregulación de tráfico vesicular controlado por RAB7 produce cambios globales en los perfiles de expresión génica de las células de melanoma, afectando a genes implicados en rutas de señalización clave en cáncer. Además, afecta al citoesqueleto y la secreción de factores involucrados en la remodelación de la matriz extracelular. Por otro lado, determinamos que RAB7 se expresa y actúa de manera independiente de MITF, el oncogén específico de melanoma mejor conocido hasta el momento.

En cambio, demostramos que la expresión selectiva de RAB7 en las células de melanoma está controlada específicamente por SOX10, el factor más apical en la diferenciación melanocítica, y por la vía de señalización dePI3K, activada frecuentemente durante la iniciación tumoral. El papel de RAB7 en la progresión del melanoma se determinó mediante estudios en líneas celulares humanas, biopsias clínicas y modelos animales. Además, la relevancia clínica de estos datos se determinó en estudios de seguimiento a 10 años, en los que se demostró que los niveles de expresión de RAB7 determinan el riesgo de desarrollo de metástasis en pacientes. Finalmente, demostramos que las rutas de tráfico vesicular dependientes de RAB7 que están específicamente activadas en células tumorales pueden constituir nuevas dianas terapéuticas. En concreto, desarrollamos una estrategia (basada en

Resumen

26

nanopartículas de ARN de doble cadena) para inducir la autodestrucción de las células tumorales a través de la movilización de macroendosomas, autofagosomas y lisoaomas, y la posterior activación de caspasas apoptóticas. En conjunto, los resultados de esta tesis doctoral han revelado una regulación y activación de la maquinaria endolisosomal que se establece de forma específica en el melanoma, contribuyendo a la progresión de esta enfermedad y que, por otro lado, también confiere una vulnerabilidad a las células tumorales que puede ser explotada con fines terapéuticos.

Summary

Introduction

Resumen

28

Introduction

1. THE MELANOMA CHALLENGE: WHERE ARE WE NOW?

Malignant melanoma is a cancer that arises from specialized pigment-producing cells, the melanocytes, which predominantly reside in the skin¹. This tumor type is characterized by having an intrinsic capacity to metastasize^{2, 3} and an unyielding resistance to chemotherapy⁴. Thus, despite accounting for only a small proportion of skin cancer cases (less than 5%), melanomas are responsible for over 80% of skin cancer related deaths^{5, 6}. During the last 30 years, the number of new melanoma cases has strikingly increased worldwide^{5, 7, 8}, becoming an unsolved public health problem in many parts of the globe⁹. In the USA, 1 in 35 men and 1 in 54 women are expected to develop melanoma during their lifetime, a probability that places this tumor type as the fifth and seventh most frequently occurring cancers in males and females, respectively⁵.

The increasing incidence and persistent resistance of melanoma to treatment has sparked many efforts aimed at elucidating the etiology and pathogenesis of this disease, as well as developing improved therapies. To date, these efforts have resulted in important scientific milestones (reviewed in ¹⁰). These range from comprehensive genomic analyses^11-13 to the discovery of new promising antitumoral drugs^14-

16. In addition, early detection and prevention campaigns have effectively increased awareness about this disease, consequently improving patient survival in countries with high-incidence rates, such as Australia, the United States, and Northwestern Europe^17-19.

Despite this extensive scientific progress, melanoma is still a paradigm of aggressiveness in human cancer. So far, this tumor is only curable by surgical resection at very early stages⁵, and the median overall survival of patients with metastatic disease rarely surpasses one year^{16, 20-22}. Genetic complexity¹², histopathological and biological heterogeneity^23,

24, and the inherent ability of melanoma cells to circumvent emerging targeted therapy16, 25, 26

are some of the main challenges that complicate the attainment of a cure for

Fig. 1 Age-adjusted Melanoma Death Rates per Sex, European Union, 1975 – 2006. Rates per 100,000 population. Source: Ref. ²⁷

Introduction

30

metastatic melanoma. Consequently, and in contrast to most cancer types (which have shown decreasing mortality rates during the last three decades), melanoma remains one of the few exceptions currently exhibiting an increasing trend in mortality (Fig. 1), especially among Caucasian individuals of 50 years of age and older^{5, 27}. The challenge, therefore, persists.

2. THE CELL OF ORIGIN OF MELANOMA: THE MELANOCYTE

Melanomas arise from the malignant transformation of melanocytes. These cells are located primarily in the skin¹, the largest organ of the human body²⁸ . As depicted in Fig. 2, the skin is comprised of three main layers: i) the outer layer, the epidermis, mostly composed of keratinocytes; ii) the middle layer, the dermis, containing fibroblasts, immunocompetent mast cells and macrophages, and structures such as blood and lymph vessels, hair roots and sweat glands;

and (iii) the most inner layer, the subcutaneous layer, mostly composed of fatty tissue^29-31. Specifically, melanocytes reside along the basal layer of the epidermis and in the hair follicles³². Through dendritic

projections, each melanocyte establishes contacts with about 36 keratinocytes, forming the so-called epidermal-melanin unit^{29, 33}.

Epidermal and follicular melanocytes derive from highly motile neural crest progenitors that migrate to the skin during early embryonic stage³⁴. Once differentiated, melanocytes are the manufacturers of melanin pigment, which they transfer to neighbouring keratinocytes within specialized membrane- bound organelles termed melanosomes^{29, 35}. By producing and delivering melanin to keratinocytes, melanocytes provide photoprotection, thermoregulation, and the visible pigmentation of the skin and hair. More importantly, as melanin functions as an absorptive pigment, melanocytes provide protection against ultraviolet (UV) damage to the skin and the underlying tissues^{36, 37}. The function and survival of melanocytes is highly dependent on neighbouring cells (such as epidermal keratinocytes and dermal fibroblasts) as well as on external signals from the environment (such as UV irradiation)^{38, 39}. Alterations

Fig. 2. The skin architecture. At the top, the close-up shows melanocytes in the basal layer of the epidermis, surrounded by keratinocytes (basal cells)

Source: National Cancer Institute website

(http://www.can cer.gov)

Introduction

of these cutaneous melanocytes can give rise to benign and malignant proliferative disorders (nevi and malignant melanoma, respectively) as detailed in the following section.

In addition to the skin, melanocytes can also be found in extracutaneous tissues of the body, such as pigmented tissues of the eye⁴⁰, the leptomeninges⁴¹, the inner ear^{42, 43}, mucosal surfaces from respiratory, gastrointestinal and genitourinary tracts⁴⁴, and the heart^{45, 46}. Malignant transformation of these melanocytes results in noncutaneous forms of melanoma, which account for about 5% of all malignant melanocytic tumors⁴⁷. These include ocular melanomas⁴⁸, leptomeningeal melanomas⁴⁹, and mucosal melanomas^{50, 51}, among others. The anatomic location of melanocytes is emerging as a key factor that defines developmental patterns, morphology, function, and gene expression profile in these cells^{23, 32}. Consequently, the impact of the anatomic location on the epidemiological, clinical, histopathological, and genetic differences between cutaneous and noncutaneous melanomas is currently being studied²³.

3. CLASSIFICATION OF CUTANEOUS MELANOCYTIC LESIONS

Cutaneous melanocytic tumors encompass a variety of lesions that display a heterogeneous spectrum of clinical, histopathological and molecular presentations. As this heterogeneity can be observed even at the early onset of the lesions, melanocytic tumors have been classified into multiple subtypes^{23, 52, 53}. 3.1. BENIGN MELANOCYTIC LESIONS: NEVI

Nevi (commonly known as moles) are indolent clonal proliferations of melanocytes^{6, 52}. Although there is still no universal consensus on a coherent classification scheme for nevi^54-56, the conventional system grossly divides nevi according the time of onset (congenital or acquired) and histopathology (junctional, compound, or dermal)⁵⁷. Congenital nevi are those present at birth, or that appear shortly thereafter⁵⁸. Acquired nevi, in contrast, start to appear after 6^th months of age, and increase in number until a peak during the third decade of life⁵⁹. These can be subdivided into junctional, dermal, and compound nevi, according to the histologic location of the melanocytic nests within the skin: in the dermal-epidermal junction, in the dermis, or both in the epidermis and the dermis, respectively^{57, 59}. Junctional or compound acquired nevi exhibiting architectural and cytological atypia are termed dysplastic nevi⁵², and they often occur in a familial manner⁶⁰. These and other clinical and histopathological criteria are the

Introduction

32 basis of the current World Health Organization (WHO) classification of benign nevi, which recognizes different categories, such as common acquired nevi, congenital nevi, spitz nevi and blue nevi, among others⁵² (see examples in Fig. 3). Importantly, it has been demonstrated that the clinicopathologic heterogeneity of nevi correlates with the presence of activating mutations in specific oncogenes (Fig. 3)^61-66. These activating mutations are also found in malignant melanoma. However, in the case of benign nevi, operant senescence pathways (see section 4) are thought to prevent malignant transformation of melanocytes.

The distinction of different types of nevi is clinically relevant for various reasons. First, most nevi remain benign for decades⁶⁷. However, specific subtypes, such as dysplastic or large congenital nevi are considered to be potential precursors of melanoma^68-72 and mark individuals with an increased risk of melanoma development^73-77. Nevertheless, the extent to which melanocytic nevi can transform into melanoma cells is controversial^{60, 78}. Secondly, nevi can be pathologically complex and mimic histological features of melanomas, therefore resulting in misdiagnosis. In fact, misdiagnosis of melanoma is the second most common reason for cancer malpractice claims in the United States^79-82. Therefore, main efforts in the field are oriented to define and validate molecular biomarkers that accurately distinguish benign nevi from malignant melanomas^83-85.

3.2. MALIGNANT MELANOCYTIC LESIONS: MELANOMA

Melanomas are the result of malignant transformation of melanocytes^{1, 6}. Since their first description as an independent disease entity by Dr. René Laennec in 1806^{86, 87}, it has become clear that melanomas are, in fact, markedly heterogeneous^{23, 53, 88}. For decades, clinical and histological features have been the basis for melanoma classification^{23, 53}. Currently, with the advent of molecular profiling techniques,

Fig. 3. Representative subtypes of nevi and their most frequently mutated oncogene. Sources: Refs. ^61-66

Most commonly mutated oncogene Clinicopathologic

subtype of nevi

Common acquired

Spitz

Congenital

Blue

BRAF

HRAS

NRAS

GNAQ

Introduction

these classification schemes are being redefined⁸⁹. An overview of different classifications of melanoma is presented below.

Clinicopathological classification

The site of presentation and histologic growth pattern have been traditionally used to classify cutaneous melanomas into four major subtypes: superficial spreading, lentigo malignant, acral-lentiginous, and nodular melanomas^90-94. Table S1 (Appendix) shows the key defining clinical and histopathological features of these melanoma subtypes. The WHO classification⁵² includes these frequent melanomas and more uncommon ones: namely, desmoplastic melanoma⁹⁵, naevoid melanoma⁹⁶, melanomas arising from a blue naevus⁹⁷, melanomas arising in a congenital nevi⁹⁸, melanoma of the childhood⁹⁹, and persistent melanoma¹⁰⁰, all of which differ in their specific clinical and/or histological presentation.

It was originally suggested that the major subtypes of cutaneous melanoma were associated with characteristic biologic behaviors and different patient outcomes^91-93. However, more complex analyses of larger datasets demonstrated no significant difference in overall survival between subtypes when tumors of equivalent thickness were compared^{101, 102}. Consequently, most, if not all, current guidelines for melanoma staging and treatment are

formulated as if it were a single disease entity23, 103, 104. However, as detailed below, classification schemes for melanoma are currently being redefined and are expected to gain significant clinical relevance in the coming years.

Emerging clinicogenetic classifications

Comprehensive genomic studies11, 105-108

have revealed that distinct genomic profiles do in fact associate well with the classical clinicopathological features distinguished above; specifically, with the

anatomical site of presentation and the Fig. 4. Melanoma clinicopathologic subtypes and their most frequently mutated oncogenes. Adapted from Ref. ¹⁰⁹

Commonly mutated oncogenes Clinicopathologic subtypes

of melanoma Superficial spreading

melanoma Lentigo maligna

melanoma

Acral melanoma

Nodular melanoma

BRAF 59-78%

NRAS 3-22%

BRAF 40-60%

KIT 16-28%

NRAS 15-29%

BRAF 12-23%

KIT 9-36%

NRAS 8-15%

BRAF 43-68%

NRAS 12-31%

Uveal melanoma

Mucosal melanoma

GNAQ 50%

KIT 1-76%

KIT 15-39%

NRAS 5-15%

BRAF 3-11%

Introduction

34

degree of sun damage. A brief summary of some of the most commonly mutated genes found in each melanoma subtype is depicted in Fig. 4¹⁰⁹. These genomic studies have been highly relevant from a basic and translational point of view. They have provided molecular evidence supporting the long-suspected heterogeneity of the clinicopathological melanoma subtypes, setting the basis for the recognition of putative divergent routes for melanomagenesis thought to result from a complex relationship between melanoma and sun exposure23, 110, 111. In addition, these studies have led to the redefinition of melanoma classification schemes, which are expected to gain significant relevance in the clinical management of future melanoma patients23, 88, 89, 112-114. The precise number of clinicogenetic melanoma subtypes and their definitive defining criteria are still, however, under determination^{23, 115}, and will most likely evolve along with the development of additional technological advances and emerging concepts.

4. DEVELOPMENT AND PROGRESSION OF MELANOCYTIC LESIONS

Despite the great progress made in the clinicopathologic and molecular classification of malignant melanoma, it is clear that even within each subgroup, lesions can display notable intra- and inter-tumor heterogeneity¹¹⁶. As presented below, this additional level of melanoma heterogeneity has important biological and clinical implications as it derives from, but also fosters, cancer progression^117-120.

4.1. HISTOLOGIC, BIOLOGIC AND GENETIC FEATURES ASSOCIATED WITH MELANOMA PROGRESSION

Cancer progression has been conceptualized as a multistep process whereby normal cells accumulate genetic alterations that enable tumor growth and metastatic dissemination¹²¹.

In the case of melanocytic neoplasia, different histologic lesions are thought to reflect different steps of this process6, 122, 123. This was first recognized by Dr. Clark and colleagues in the mid 1980´s, proposing a landmark model for melanoma progression comprised of five different clinicopathologic steps: i) benign nevus, characterized by an increased number of nested melanocytes; ii) dysplastic nevus, a benign lesion with random and discontinuous cytologic atypia; iii) radial-growth phase (RGP) melanoma, a malignant lesion in which tumor cells grow restricted to the epdiermis; iv) vertical-growth phase (VGP) melanoma, defined by the presence of nodular dermal invasion; and v) metastatic melanoma, distinguished by the presence of melanoma cells growing at sites different from the site of origin¹²².

Introduction

The traditional multi-step model for melanoma progression implies a transition from a benign (nevi) to malignant (melanoma) lesion^{6, 122}. However, this concept has raised controversy^{60, 78}, as up to 80% of melanomas lack histological signs of a pre-existing nevus69, 124-128. This has prompted the definition of a revised model for melanoma progression (Fig.

5)^{129, 130}, which theorizes melanoma as developing de novo, i.e. directly from normal melanocytes or precursor cells, although the contribution of melanocyte stem cells or non- pigment producing melanoblasts to melanomagenesis remains poorly characterized^{131, 132}.

Despite this controversy, and as detailed below, it has been widely demonstrated that nevi, RGP, VGP, and metastatic melanomas reflect distinct molecular and biologic characteristics associated with the malignant and metastatic potential of melanocytic tumors6, 123, 133.

Nevi and melanocyte oncogene-induced senescence (OIS)

As mentioned above, nevi are the benign counterpart of melanomas6, 123, 130, 134. They harbor activating mutations in oncogenes such as BRAF, NRAS, or HRAS⁶⁶, but their malignant degeneration is thought to be prevented by the activation of fail-safe mechanisms, the best characterized being oncogene-induced senescence (OIS)67, 135, 136. OIS was described and proposed as a barrier to tumorigenesis more than a decade ago, in a study in which the overexpression of oncogenic HRAS was found to trigger an irreversible arrest in primary human and rodent fibroblasts¹³⁷. This premature form of senescence is mediated by tumor suppressor pathways, primarily p16(INK4a)/Rb and p19(ARF)/p53/p21 (reviewed in ref. ¹³⁸). Not surprisingly, these pathways are commonly inactivated in many cancer types, including melanoma^{135, 139}. Dysplastic nevi, classically considered precursors of melanoma6, 60, 122, and familial forms of melanoma⁶⁷ also harbor genetic aberrations in these tumor suppressor pathways.

Normal Skin

Bening Nevus Dysplastic

Nevus

RGP

VGP

Metastatic Melanoma

?

?

Fig. 5. Models for melanoma progression.

Adapted from Ref. ¹³⁰

Introduction

36

Ultimately, OIS induces phenotypic and molecular changes that have come to be regarded as “markers”

of the process, and have been instrumental in identifying novel tumor suppressors and oncogenes^{135, 140,}

141. These changes include: senescence-associated β-galactosidase activity (SA-β-Gal); morphological changes; increased expression of p16, ARF, p21 or p53; senescence-associated heterochromatin foci (SAHF); DNA damage; decreased Ki-67 proliferation marker; and the absence of gross telomere shortening, among others67, 135, 142.

Studies in human cells, and in mice and fish in vivo, have reinforced the concept of active OIS blunting the transformation of melanocytes^143-145. 136, 144-149. Curiously, the expression of oncogenic BRAF, HRAS, and NRAS in primary human melanocytes triggers distinct types of OIS^{143, 150}. For example, OIS driven by HRAS (and not by BRAF) is associated with a massive cytosolic vacuolization (see Fig. 6) and an induction of the Unfolded Protein Response (UPR), an adaptive intracellular signaling pathway that responds to metabolic stress, oxidative stress, and inflammatory response pathways (reviewed in ¹⁵¹)¹⁴³. Moreover, different from other human and murine cells, p53, p21^CIP/WAF, p16^INK4A, and p14^ARF are not essential drivers of OIS in melanocytic cells¹⁵².

Importantly, human nevi can manifest features of OIS, such of SA-β-Gal, giant and multinucleate cells, decreased levels of the proliferative marker Ki67, and high levels of p16136, 144-149. However, the specificity of the association of some of these OIS markers to benign, but not malignant, melanocytic tumors has been debated145, 153-156. This raises the need to better define bona fide markers of senescence in vivo⁷⁸. These definitions could hopefully serve as the gold standard for the correct distinction between nevi and melanomas. Moreover, the precise genetic determinants of the different subtypes of nevi have yet to be determined.

Fig. 6. Differential OIS programmes induced by HRAS^G12V and BRAFB^600E in primary human melanocytes. Both oncogenes result in the induction of positive SA-β-Gal staining (bue), but BRAFV^600E-expressing melancoytes do not exhibit the characteristic cytosolic vacuolization of their HRAS^G12V counteraparts. Adapted from Ref. ¹⁴³

HRAS^G12V BRAF^V600E

Normal

Introduction

RGP melanoma and tumor initiation

One of the early events in the pathogenesis of melanoma is the activation of the mitogen-activated protein kinase phosphatase (MAPK) and/or phosphoinositide-3 kinase (PI3K) pathways (mainly by mutations in BRAF or NRAS but also in upstream receptor tyrosine kinases such as KIT or ERBB4)^{157, 158} . However, activation of these pathways is not sufficient to promote the malignant transformation of melanocytes^{159, 160}. The development of radial growth phase (RGP) of melanoma requires the acquisition of additional genetic mutations by melanocytes, that prevent or bypass the OIS barrier, and/or cooperate in malignant transformation¹⁶¹. Via these additional genetic aberrations, RGP melanoma cells acquire the ability to actively proliferate; however, they do so within the epidermis because they are still keratinocyte-dependent for survival and are not yet tumorigenic nor invasive⁶.

The identification of the genetic combinations that synergize with oncogenic BRAF or NRAS to successfully promote melanoma initiation has been the subject of active investigation in the last decade.

Extensive research using in vitro and/or in vivo experimental models of melanomagenesis has yielded the identification of a handful of initiating genetic alterations, mainly the loss of tumor suppressors such as CDKN2A^{162, 163}, PTEN^164-166, TP53^{167, 168}, RB1¹⁶⁸ or NF1¹⁶⁹, and the activation of additional oncogenes, such as AKT3¹⁷⁰ and MITF¹⁷¹, shown to cooperate with oncogenic BRAF in the malignant transformation of melanocytic cells. Importantly, these driving genetic aberrations have been identified in human melanoma biopsies, albeit at different relative frequencies¹⁶⁰ (see Table 1 in section 5). Still, the onset and underlying mechanisms driving these molecular changes are not yet completely understood^172-175. For example, PTEN loss has been shown to promote both initiation and metastatic progression in experimental melanoma models164-166, 176, 177. However, it is not clear whether PTEN loss is an early or late event in human melanomas12, 175, 178, 179. Thus, there is a remaining need to better delineate the increasing list of melanoma tumor suppressors and oncogenes within the initiation and/or progression of the human disease.

VGP melanoma and the acquisition of the competency to metastasize

During the vertical growth phase (VGP), melanoma cells acquire the competency to invade. They become immortal and tumorigenic, can escape from the anchorage to surrounding keratinocytes, and