Role of RKIP and Pirin in the malignant progression of cutaneous melanoma. New diagnosis and prognosis biomarkers

DOCTORAL THESIS ROLE OF RKIP AND PIRIN IN THE MALIGNANT PROGRESSION OF CUTANEOUS MELANOMA. NEW DIAGNOSIS AND PROGNOSIS BIOMARKERS CRISTINA PENAS LAGO LEIOA, 2022 Supervisors: Maria Dolores Boyano López Carmen Álvarez Domínguez (cc)2022 CRISTINA PENAS LAGO (cc by-nc-nd 4.0) II III Abbreviation Description ACTB Actin beta AJCC American Joint Committee on Cancer AJCC American Joint Committee on Cancer AKT Protein Kinase B ALM Acral lentigo melanoma ANOVA variance analysis BCL-3 B-cell lymphoma 3-encoded protein BIC Bayesian Information Criterion BPs Biological Processes BRAF B-Raf Proto-Oncogene, Serine/Threonine Kinase BSA Bovine Serum Albumin Cdna complementary DNA c-MYC Myc proto-oncogene, bhlh transcription factor c-Rel, REL Proto-Oncogene, NF-KB Subunit CSD Cumulative sun damage CT computed tomography DEGs Differentially expressed genes DEPC diethylpyrocarbonate DMEM Dulbecco's Modified Eagle Medium DMSO dimethyl sulfoxide DNA Deoxyribonucleic acid DTT Dithiothreitol E2F1 E2f transcription factor 1 EAPC Estimated Annual Percentage Change ECL Enhanced chemiluminescence ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid EGFP Enhanced Green Fluorescent Protein EMT Epithelial-to-mesenchymal transition ERK1 Mitogen-Activated Protein Kinase 3 ERK2 Mitogen-Activated Protein Kinase 1 FBS Fetal bovine serum FDR False discovery rate FFPE Formaldehyde Fixed Paraffin Embedded GAPDH Glyceraldehyde 3-phosphate dehydrogenase GEO Gene expression omnibus GFP Green fluorescence protein GO Gene Ontology GPCR G protein-coupled receptors GSK3β Glycogen synthase kinase-3 beta H&E hematoxylin and eosin staining HEMn-DP Human epidermal melanocytes, neonatal, darkly pigmented HEMn-LP Human epidermal melanocytes, neonatal, lightly pigmented HEMn-MP Human epidermal melanocytes, neonatal, moderately pigmented HMB-45 anti-melanosoma, HMB45 HRP Horseradish Peroxidase IGF-1Rs Insulin-like growth factor I receptor IHC immunohistochemistry IV IKK Inhibitor Of Nuclear Factor Kappa B Kinase JAK Janus Kinase JARID1B Lysine Demethylase 5B gene KEGG Kyoto Encyclopedia of Genes and Genomes KLF4 Kruppel Like Factor 4 KRAS KRAS Proto-Oncogene, GTPase LM Lentigo melanoma LMM Lentigo malignant melanoma LUM Lumican MAPK Mitogen-activated protein kinase MART-1/MelanA Melanoma Antigen Recognized by T Cells MEK Mitogen-Activated Protein Kinase Kinase 1 MIB1 MIB E3 Ubiquitin Protein Ligase 1 miR-21 MicroRNA 21 MITF Melanocyte Inducing Transcription Factor MOI Multiplicity of Infection mRNA messenger Ribonucleic Acid NANOG Nanog homeobox NCSC neural-crest stem-cell NF1 neurofibromin 1 NFKB nuclear factor-kappaB NGS Next Generation Sequencing NM Nodular melanoma NRAS NRAS Proto-Oncogene, GTPase NTRK2 Neurotrophic Receptor Tyrosine Kinase 2 OCT4 POU class 5 homeobox 1 OIS Oncogene-induced senescence p100/p52 Nuclear Factor Kappa B Subunit 2 p105/p50 Nuclear Factor Kappa B Subunit 1 PBS Phosphate buffer saline PCR Polymerase chain reaction PI3K phosphatidylinositol-3-kinases PIR Pirin PKC Protein Kinase C PMEL o gp-100 premelanosome protein PTEN Phosphatase And Tensin Homolog RAF Raf-1 Proto-Oncogene, Serine/Threonine Kinase RelA (p65) RELA Proto-Oncogene, NF-KB Subunit RelB RELB Proto-Oncogene, NF-KB Subunit RGP radial growth proliferation RIN RNA Integrity Number RIPA Radioimmunoprecipitation assay buffer RIPA Radioimmunoprecipitation Assay RKIP Raf kinase Inhibitor protein RNA ribonucleic acid RNU6-2 endogenous reference small nuclear ribonucleic acid (snRNA) B RPKM Reads per kilobase of exon model RPMI Roswell Park Memorial Institute Medium RPS15 Ribosomal Protein S15 gene RPS15 Ribosomal Protein S15 V RTKs receptor tyrosine kinases RT-qPCR Real time quantitative polymerase chain reaction S100 S100 Calcium Binding Protein SDS Sodium dodecyl sulfate shRNA Short hairpin RNA SNAIL Snail transcriptional repressor SOX2 SRY-box transcription factor 2 SRA Sequence read archive SRCCA Spearman’s rank correlation coefficient SSM Superficial spread melanoma STAT Signal Transducer And Activator Of Transcription TAK1 Mitogen-Activated Protein Kinase Kinase Kinase 7 TBST Tris-buffered saline with Tween 20 TBST Tris-buffered saline THY-1 THY-1 cell surface antigen TMM Trimmed mean of M values TNFR1 TNF Receptor Superfamily Member 1A TYR tyrosinase UV Ultraviolet VGP vertical growth proliferation WHO World Health Organization XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide ZEB Zinc Finger E-box-binding homeobox VI VII VIII IX Figure 1. Temporal incidence/mortality trends of Melanoma Figure 2. Melanoma incidence Figure 3. Melanoma mortality Figure 4. Skin phototypes Figure 5. Cross-section of skin’s layers Figure 6. Biologic events and molecular changes in the progression of melanoma Figure 7. Melanoma key signaling pathways Figure 8. ABCDE criteria for the early detection of melanoma Figure 9. Histological subtypes of melanoma and clinical-pathological correlations Figure 10. Distribution of melanoma cell lines and primary cultures of melanocytes analysed in a two-component plot by Varimax orthogonal rotation Figure 11. RKIP and Pirin expression in cell culture of melanocytic cells by RT-qPCR and Western blot Figure 12. Immunostaining for Pirin in three representative melanoma biopsies (negative, low and high) Figure 13. Workflow overview of transduction protocol with lentiviral particles and transfection protocol with lipofectamine and overexpression plasmids. Figure 14. Workflow overview of wound healing assay Figure 15. Workflow overview of the protein detection process by western blot Figure 16. RNA integrity numbers (RINs) obtained with Agilent 2100 Bioanalyzer Figure 17. Library profile qualification by Agilent High Sensitivity DNA Kit Figure 18. Mapped reads per conting analysed by bamtool stats bioinformatics tool Figure 19. Distribution of the samples according to diagnosis, tumour location and histological subtype, AJCC stages, Breslow Index expressed in mm and evolution of patients diagnosed in AJCC I and II stages X Figure 20. RKIP expression pattern distribution according to diagnosis, histological type and progession of melanoma patients Figure 21. Statistical analyses of Raf Kinase Inhibitor Protein (RKIP) expression in FFPE biopsies from patients Figure 22. Representative images of manually scored RKIP staining categories in FFPE biopsies from patients Figure 23. Representative images of Pirin staining in FFPE biopsie from a melanoma hematoxylin-eosin staining and imunohistochemistry of Pirin expression Figure 24. Pirin expression pattern distribution according to diagnosis, histological type and progresion of melanoma patients Figure 25. Statistical analyses of Pirin expression in FFPE biopsies from patients Figure 26. Modulation of RKIP expression in A375 and MelHO primary melanoma cell lines Figure 27. Modulation of RKIP expression in MeWO and A2058 metastatic melanoma cell lines Figure 28. RNA Sequencing data quality of RKIP downregulated HEMn-LP Figure 29. RNA Sequencing data and analysis after RKIP downregulation in HEMnLP Figure 30. RKIP as a key regulator of NANOG expression in melanoma Figure 31. Functional assays after Pirin upregulation in melanoma cells Figure 32. RNA Sequencing data quality of Pirin downregulated HEMn-LP Figure 33. RNA Sequencing data analysis and validation after Pirin downregulation in HEMn-LP Figure 34. PIR as a key regulator of JARID1B expression in melanoma XI XVIII (58). La inhibición de Pirina se ha asociado con la capacidad migratoria de las células de melanoma (122) y también se ha propuesto como un inhibidor de la senescencia celular aunque se sabe poco sobre los mecanismos que subyacen a este efecto (135). La combinación de todos estos cambios genéticos conduce a que los melanocitos normales adquieran diversos fenotipos malignos. Así, se ha descrito un equilibrio entre los fenotipos proliferativos e invasivos en las células de melanoma, lo cual permite que las células sean capaces de mostrar una plasticidad de, lo que se ha llamado, fenotipo adaptativo. De acuerdo con esta propuesta de plasticidad fenotípica, los fenotipos estables de melanoma se definen por transcriptomas de melanocitos diferenciados o melanocitos de ciclo lento o ‘slow-cycling cells’ con marcadores de célula madre o ‘stem-like cell makers’. Las propiedades asociadas al perfil ‘stem-like cell’ pueden explicar la persistencia de ciertas células tumorales tras el tratamiento con fármacos. Uno de los genes relacionados con la regulación del mantenimiento del estado desdiferenciación es NANOG (70). Así, por ejemplo, durante la formación de esferas en el melanoma, se incrementa la expresión de NANOG (74), y se ha visto su implicación en la regulación de la transición epitelio-mesenquimatosa, proceso clásicamente ligado al aumento de la motilidad de las células cancerosas favoreciendo la diseminación de la enfermedad (75). Por otro lado, el fenotipo asociado a ciclo proliferativo lento o ‘slow cycling’ también se ha relacionado con una subpoblación de células que mantiene la supervivencia de las células tumorales (191). Entre los reguladores maestros del ciclo celular retardado se encuentra JARID1B/KDM5B, o enzima desmetilasa de histonas específica de lisinas. Varios estudios han proporcionado evidencia de que JARID1B es un supresor de tumores en el melanoma maligno, ya que sus niveles de expresión están regulados a la baja e inhibe la proliferación celular de manera dependiente de Rb (82-84). Dado que la plasticidad del fenotipo está estrechamente relacionada con el inicio, la progresión y la resistencia a la terapia del melanoma, es de gran interés identificar cómo se generan estas transiciones de fenotipo a fin de identificar nuevos enfoques terapéuticos. En otro orden de cosas, los melanomas también muestran una amplia variedad a nivel histológico, pudiendo mostrar características epiteliales, hematológicas, mesenquimatosas y neurales, que a menudo pueden dificultar el diagnóstico de la enfermedad (96). De hecho, se ha desarrollado una variedad de marcadores inmunohistoquímicos a fin de facilitar la labor del personal clínico de los servicios de dermatología, como el marcador S-100, que sigue siendo el marcador más sensible para las lesiones melanocíticas. Otros marcadores, como HMB-45, MART-1/Melan-A, tirosinasa y MITF son relativamente sensibles, aunque no tanto como S-100. Actualmente, estos biomarcadores utilizados para facilitar el diagnóstico de melanoma permiten diferenciar tumores melanocíticos de otros tipos de tumores, pero no se ha demostrado que alguno de ellos sea predictivo de supervivencia para pacientes con neoplasias melanocíticas. Con el desarrollo de tratamientos más novedosos y específicos, los biomarcadores tumorales son cada día más importantes de cara a su utilización en nuevas estrategias terapéuticas (106,107). XIX En estudios de proteómica diferencial previos realizados por nuestro grupo de investigación, observamos que RKIP y Pirina eran dos proteínas cuya expresión difería significativamente entre melanocitos de piel y células de melanoma. Por ello, consideramos que presentaban gran potencial para ser utilizados como biomarcadores del melanoma maligno. Hipótesis y objetivos En base a todo lo expuesto anteriormente, en esta tesis se ha mantenido la hipótesis de que RKIP y Pirina son proteínas que pueden ser excelentes biomarcadores para el diagnóstico y pronóstico del melanoma cutáneo además de desempeñar un papel importante en la etiopatogenia del melanoma cutáneo. Así, para determiner la veracidad de esta hipótesis, nos planteamos los siguientes objetivos específicos: 1. Validar la expresión de RKIP y Pirina como marcadores de diagnóstico y pronóstico en melanoma. 2. Evaluar el papel de RKIP y Pirina en las funciones biológicas de los melanocitos de piel. 3. Determinar en que medida RKIP y Pirina contribuyen a la progression metastásica del melanoma maligno. Material y métodos Nuestra estrategia metodológica para valorar el primer objetivo se basó en un estudio retrospectivo en una cohorte pacientes con melanoma en el que se examinó la expresión de las proteínas RKIP y Pirina en cortes histológicos de biopsias de melanoma mediante inmunohistoquímica (IHC). El seguimiento clínico de los pacientes abarcó de 18 meses a 5 años. Posteriormente, se realizó una correlación con los datos clinicopatológicos y la progresión metastásica. Para evaluar el segundo objetivo sobre las funciones de las proteínas RKIP y Pirina en la biología de los melanocitos, realizamos una evaluación de los cambios transcripcionales tras el silenciamiento de la expresión de ambos genes de forma independiente en células de melanocitos sanos. Los controles y los melanocitos RKIP o PIR silenciados se examinaron mediante secuenciación de ARN. Luego, los resultados del análisis in silico de enriquecimiento de ontología génica se validaron en líneas celulares de melanoma a las que se les ha sobreexpresado RKIP o Pirina, respectivamente. Para abordar el tercer objetivo, se utilizaron líneas celulares de melanoma humano primario y metastásico. Tanto la expresión de RKIP como la de Pirina fueron moduladas por plásmidos de forma XX independiente y se realizaron ensayos funcionales de la capacidad proliferativa, migración e invasión. Además, se determinaron y validaron dianas moleculares de RKIP y Pirina mediante co-transfecciones y determinaciones moleculares. Resultados y discusión El melanoma maligno es una forma de cáncer de piel que es extremadamente letal. Para garantizar un tratamiento adecuado y un resultado exitoso, es esencial un diagnóstico oportuno y preciso del melanoma maligno. En este sentido, las alteraciones moleculares en la patogenia del melanoma son objeto de una investigación muy activa, lo que ha llevado a la identificación de oncogenes y genes supresores de tumores asociados a esta enfermedad para desarrollar enfoques terapéuticos que se necesitan con urgencia. Por ello, el primer objetivo de este trabajo ha sido estudiar el valor potencial de RKIP y Pirina como marcadores de melanoma, lo cual se analiza en el Capítulo 1 de la sección de Resultados a través de un estudio inmunohistoquímico en una cohorte de 314 pacientes (75 nevus y 239 melanoma). Respecto a RKIP, de manera general, la expression intramuestra de esta proteina fue homogénea, observándose un marcaje citoplasmático. El análisis univariante de la comparación por grupos, mostró una diferencia estadísticamente significativa entre la alta expresión que mostraban las biopsias de nevi (94% de los casos) frente a los melanomas (51% de los casos). Además, mediante un análisis de regresión logística (en el que se incluyeron la edad y el sexo como covariables) se observó una asociación lineal, es decir, mayores niveles de proteína se correlacionaban significativamente con una mayor probabilidad de que las biopsias fuesen identificadas como nevus. Por otro lado, tanto el análisis univariante como el multivariante confirmaron una diferencia significativa entre la expresión de RKIP entre las biopsies de nevi y las de melanoma diagnosticados en estadios tempranos (estadios I y II, según AJCC 8ª Ed.). Al analizar su potencial como marcador pronóstico, aunque no se observaron diferencias significativas entre la expression de RKIP y el desarrollo de metastasis, altos niveles de RKIP se correlacionaron con un grosor de Breslow más bajo en muestras de todos los estadios de melanoma. Según la bibliografía existente, varios estudios han demostrado que los niveles de RKIP son bajos en una gran variedad de cánceres y que, además, apenas se expresa en las metastasis (115,148-150,164). En el caso del melanoma, en concreto, se ha observado una disminución de RKIP en melanoma uveal y una baja expression en melanomas cutáneos, tanto primarios como metastásicos (52, 165). Aunque estos estudios son interesantes, se realizaron con cohortes pequeñas de pacientes, y, además, comparaban los nivele de expresión de RKIP entrebiopsias de tumores primarios y biopsias tomadas en sitios metastásicos (52,134,148). Aún así, estos resultados muestran un claro silenciamiento de RKIP en relación a la malignidad en las células tumorales, aunque no investigaron la posible utilidad de la expresión de RKIPcomo marcador pronóstico de buenaa o mala evilución. Según nuestros resultados, parece que la tinción de RKIP mediante inmunohistoquímica tiene utilidad como marcador d diagnóstico para pacientes de melanoma. XXI De forma similar se correlacionó la expresión de Pirina con los datos clínicos. En primer lugar, mencionar que la tinción intramuestra, a diferencia que en el caso de RKIP, fue heterogénea, observándose células con marcaje sólo nuclear, sólo citoplasmático o ambos. Este patron heterogéneo no mostró ninguna relación con el tipo histológico, el estadio tumoral o la progresión del melanoma. Al realizar la comparación por grupos, se observó que el 80% de los nevi mostraban una alta expresión de Pirina frente al 60% de los melanomas. En relación al tipo histológico, los y las pacientes con melanoma de extension supercial (MES) mostraron una expresión de Pirina similar con independencia de su evolución. Sin embargo, en el caso del melanoma nodular (MN), quienes eventualmente desarrollaron enfermedad metastásica mostraron niveles de pirina más altos en sus biopsias primarias en una proporción significativamente mayor que aquellos que permanecieron libres de enfermedad. Por otro lado, se evaluó la utilidad de Pirina como marcador pronóstico temprano, utilizando únicamente las biopsias de pacientes con melanoma en estadio temprano (estadios I y II, según AJCC 8ª Ed.), sin embargo, no se observó una asociación directa entre la expresión de Pirin y el hecho de permanecer libre de enfermedad o desarrollar metástasis durante el seguimiento. Aún así, dado que nuestros datos de melanomas primarios tempranos eran heterogéneos, se podría esperar que otros factores de riesgo potenciales pudieran estar enmascarando la asociación entre la expresión de Pirin y la probabilidad de metástasis. Por ello, se realizón un análisis de las diferencias en un escenario multivariado que incluía la expresión de Pirin como el efecto de interés, y la edad, el sexo y la profundidad de Breslow como posibles covariantes. En este caso, un nivel alto de Pirina se asoció significativamente con una mayor probabilidad de metástasis según el modelo de regresión logística. Complementariamente se llevó a cabo un análisis de Factores de Bayes, que resultó ser positivo, indicando que la alta expresión de Pirin en una biopsia implica que sea 10 veces más probable que se desarrolle metástasis. Finalmente, un análisis de Cox mostró que pacientes con una expresión más alta de Pirin tenían más del doble de probabilidad de desarrollar metástasis temprana en comparación con aquellos que expresaban bajos niveles de Pirina. Según lo descrito hasta la fecha en relación a Pirina, parece estar involucrada en la regulación de varios procesos celulares, incluida la inhibición proteínas quinasa, funciones antioxidantes y cofactor transcripcional (30,82,116). Además, se ha demostrado que Pirin puede desempeñar un papel en la tumorigénesis a través de su participación en la regulación de la proliferación celular y la progresión maligna (182). Por otro lado, Lucciulli y colaboradores describieron una deslocalización de Pirina desde el núcleo hasta el citoplasma en un subconjunto de muestras de pacientes con melanoma cuando las compararon con las biopsias de nevi. Además, observaron una correlación positiva entre los niveles citoplasmáticos de Pirin y la progresión del melanoma (135). En nuestro caso, hemos podido establecer que su determinación mediante inmunohistoquímica, junto con el índice de Breslow, podría ser útil como un indicador de pronóstico ya que pacientes con altos niveles de expresión mostraron menor tiempo de supervivencia libre de enfermedad. En otro orden de cosas, hay que tener en cuenta que la incapacidad para comprender los mecanismos que subyacen a la metástasis, la cual conduce a la mayoría de las muertes relacionadas con el cáncer, plantea un problema importante para el desarrollo de métodos de diagnóstico y pronóstico así como de terapias efectivas. Debido a esto, centramos nuestra atención en el papel de RKIP y Pirina en la biología XXII de los melanocitos normales y malignos. Para ello, se comenzó por el silenciamiento de ambas proteínas de forma independiente en melanocitos primarios sanos y el posterior análisis de su transcriptome mediante Secuenciación de ARN. Se completaron los estudios con análisis moleculares y funcionales en líneas de melanoma. Los hallazgos relacionados con los cambios transcripcionales producidos por el silenciamiento de RKIP en melanocitos nomales se recogen en el Capítulo 2 de la sección de Resultados. Lo primero que llamó nuestra atención fue que estas células mostraron modificaciones a nivel de expresión génica asociadas a la firma genética del cancer. Concretamente, se observaron alteraciones de los procesos celulares íntimamente relacionados con la transformación maligna de las células, como el desarrollo y la diferenciación. Esto iría en corcondancia con la expresión más alta de RKIP encontrada en melanocitos diferenciados de lesiones de nevus cuando se compara con muestras de melanoma en el Capítulo 1. Más específicamente, se encontró que más del 70% de los genes expresados diferencialmente que se incluyeron en esta sección, desarrollo y diferenciación, eran dianas putativas de NANOG, un factor de transcripción relacionado con la troncalidad o ‘stemness’ (70). Los ensayos de cotransfección de plásmidos para la sobrexpresión de RKIP junto con plásmidos del promotor de NANOG asociado a GFP montaron que la presencia de RKIP produjo una disminución de la activación del promotor NANOG, lo cual que apunta hacia una relación funcional entre la expresión de RKIP y NANOG. En este contexto, también hemos encontrado que la expresión de miR-21, una diana aguas abajo de NANOG (78) y relacionado con la transición epitelio-mesénquima, fue significativamente menor en las células que sobreexpresaban RKIP. Estas mismas células mostraton un aumento de la capacidad migratoria tanto en el test de la herida como a través de filtros con matriz de colágeno. En línea con estos resultados, Lee et al. (173) notaron una gran cantidad de interferencias entre las vías reguladas por RKIP y aquellas bajo el control de los principales factores de transcripción de tallo (es decir, OCT4, KLF4, SOX2 y NANOG) y propusieron RKIP como un regulador del estado de diferenciación de las células. En conjunto, nuestros resultados sugieren que RKIP regula los estados diferenciados en las células melanocíticas a través del factor de transcripción NANOG. Por otro lado, tal y como se observa en el Capítulo 3 de la sección de Resultados, el transcriptoma de los melanocitos con Pirina silenciada reveló un enriquecimiento de genes involucrados en la transición G1/S, la organización de la matriz extracellular, la proliferación, la migración y diferenciación celular. Uno de los reguladores del ciclo celular es JARID1B/KDM5B, una histona desmetilasa específica de Lisina. Aunque las células de melanoma que expresan JARID1B representan solo una pequeña proporción de las células en las poblaciones de melanoma primario y metastásico (187), nuestro conjunto de datos de RNA-seq y el análisis in silico de enriquecimiento de factores de transcripción encontraron que JARID1B podría regular más de 100 de los genes diferencialmente expresadas tras el silenciamiento de Pirina en los melanocitos. Así, los experimentos de cotransfección mostraron una disminución de la activación del promotor de JARID1B después de la sobreexpresión de Pirina, lo que sugiere una relación funcional entre la expresión de Pirina y JARID1B. Además, demostramos que la sobreexpresión de Pirina en las dos líneas celulares de melanoma metastásico estudiadas condujo a una XXIII disminución significativa en la expresión del gen JARID1B y de sus genes diana E2F1 y c-MYC (81,141). En relación a esto, se observó una bajada en la proliferación de las líneas de melanoma que sobreexpresaban Pirina, lo cual concuerda con los bajos niveles de expresión de JARID1B, E2F1 y cMYC obtenidos en los ensayos de cotransfección. Así, en el contexto de nuestro estudio, hemos podido determinar que la capacidad proliferativa de las células de melanoma depende de la interacción de Pirina con JARID1B, quien está involucrada en la organogénesis, la función de las células madre y el desarrollo del cancer (80,81). Con todo ello, mantenemos la Tesis de que en la génesis del melanoma cutáneo pueden estar implicados multiples mecanismos celulares. En nuestro studio concreto, la expression de RKIP y Pirina dibujan escenarios distintos en un nevus frente al melanoma. La expression de RKIP en los melanocitos de los nevi inhibiría la expression de NANOG y sus díanas moleculares, manteniendo el estado diferenciado de los melanocitos. Además, la expression de Pirina modularia la proliferación a través de JARID1B y sus dianas moleculares como E2F1. Por su parte, en el melanoma, la ausencia de RKIP induciría un panorama diferente, ya que la expression de NANOG favorecería la adquisición del fenotipo invasivo y en este contexto, las células tumorales con ciclo lento inducido por JARID1B podría actuar como un estímulo para entrar de nuevo en ciclo y favorecer la formación de metastasis. XXIV Conclusiones En nuestra hipótesis inicial planteábamos que RKIP y Pirina eran proteínas que desempeñaban un papel en la etiopatogenia del melanoma cutáneo, lo que las convertía en excelentes biomarcadores para el diagnóstico y pronóstico del melanoma cutáneo. Para demostrar esto, investigamos su aplicación potencial como marcadores de melanoma y su papel en las células melanocíticas. En base a los resultados presentados en esta tesis, se pueden extraer las siguientes conclusiones: La detección inmunohistoquímica de RKIP en biopsias de melanoma puede ser una herramienta útil para el diagnóstico de melanoma. La detección inmunohistoquímica de Pirina junto con el índice de Breslow podría usarse como marcador pronóstico en estadios tempranos (I-II) del melanoma. La baja expresión de RKIP en melanocitos humanos condujo a una firma transcripcional asociada con el cáncer, que incluía una desregulación de genes relacionados con la pigmentación y los procesos de desarrollo y diferenciación. RKIP parece estar involucrado en el mantenimiento del estado de diferenciación de los melanocitos al regular negativamente el factor de transcripción NANOG y sus dianas moleculares, como miR-21. La baja expresión de de Pirina en melanocitos humanos condujo a un perfil transcripcional caracterizado por una desregulación en la organización de la matriz extracelular, la migración, la proliferación y la respuesta a interferón tipo II. Pirina ejerce un efecto antiproliferativo en las células de melanoma a través de la regulación del factor de transcripción JARID1B y sus genes diana, incluidos E2F1 y cMYC. A partir de estos resultados, en su conjunto, mantenemos la Tesis de la implicación de ambas proteínas RKIP y Pirina en la tumorigénesis y progresión maligna del melanoma cutáneo y podrían ser la base del diseño de nuevas estrategias terapéuticas para el tratamiento de la enfermedad metastásica del melanoma. XXV XXVI XXVII Abstract Melanoma is an extremely lethal skin cancer that arises as a result of the malignant transformation of melanocytes. The incidence of this pathology worldwide has increased in the last 30 years in greater proportion than the rest of the cancer, between 4-6% yearly, and shows substantial disparities between populations. To ensure appropriate treatment and a successful outcome, a timely and accurate diagnosis and prognosis of malignant melanoma is essential. Because of this, molecular alterations in the pathogenesis of melanoma are the subject of more active research. In terms of histology, melanoma show a wide variety of characteristics including epithelial, hematological, mesenchymal, and neural features, which can often make the diagnosis of the disease challenging. As new biomarkers candidates, in this thesis we examined the expression of RKIP (Raf Kinase Inhibitor Protein) and Pirin. RKIP has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancer, and Pirin originally was considered to act as a transcriptional co-factor, but it has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. However, these studies were performed with a small cohort of patients, so a larger study is required for further evaluation of this marker's diagnostic or prognostic value. In this context, this doctoral thesis’ goals were to evaluate the potential value of RKIP and Pirin as melanoma markers and their implication on melanocytic cell biology. Regarding RKIP, immunohistochemistry analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. In relation to Pirin, the immunohistochemistry multivariate analysis revealed that early melanoma with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. On the other hand, transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. To summarize, the results obtained in this thesis support the diagnostic utility of RKIP staining due to the significantly lower RKIP protein levels in melanoma samples, even at early stages (I–II) of the disease, and the use of Pirin staining along with the Breslow index as a prognostic marker at early stages (I-II) of melanoma. Moreover, we propose that RKIP could play a role in the maintenance of the differentiation state by negatively regulating NANOG gene expression and, that Pirin could play an important role in modulating the proliferative state of melanoma cells by regulating JARID1B gene expression. 6 7 8 Etiological factors for malignant melanoma Factors contributing to the development of cutaneous melanoma can be grouped into those specific to the individual (e.g., skin phototype, genetic predisposition) and those specific to the environment. In terms of grading melanoma risks, ultraviolet radiation would be among the most significant. In addition to sunlight, ultraviolet light can also be produced by artificial lighting systems, such as tanning beds (18). The wavelengths of this light range from 200 to 400nm, with the UVB wavelength being the most carcinogenic to the skin (19-20). By absorbing these waves, the melanocytes are compromised in their ability to repair DNA (21-22). Carcinogenesis is consequently caused by the accumulation of genetic alterations (23-24). Our ability to tan is one of our natural defenses against solar radiation damages, such as sunburn. Every individual has a unique ability to adjust to the sun, which is determined by their skin phototype. Figure 4 illustrates the six types of skin phototypes. As a result of their light skin, blond or red hair, freckles, blue eyes, and a light complexion, people with light skin phototypes are at greater risk of developing melanoma. (25,26). Additionally, it may be important to consider the age at which tanning began and the duration of the tanning behavior (27) because prolonged radiation exposure can cause skin burns that cause the genetic changes listed in the previous paragraphs to accumulate over time. Accordingly, there is a direct correlation between sunburns and melanoma development (25, 28). 9 As described in the epidemiology section, most phenotypic skin characteristics and sun exposure intensity are geographically determined, implying that the incidence of melanoma is also dependent on geographical location (Figure 2). Accordingly, the highest incidence rates have been reported in Australia and New Zealand, followed by Northern Europe (29). Additionally, people with phototypes I and II tend to have pigmented nevi than those with other phototypes. Pigmented nevi are skin lesions characterized by the benign proliferation of melanocytes. Approximately 15% of melanoma derives from this lesion (30). Therefore, if a person has multiple pigmented nevi that vary in color and shape, they are at a higher risk of melanoma. It is crucial to consider an individual's personal history in this context. It is essential to evaluate an individual's unique history in this context (31). Melanoma is one of the most immunogenic cancer, with spontaneous remission occurring in roughly 10% to 35% of cases (32). Consequently, immunodeficiency has also been shown to be associated with an increased risk of developing cancer (33). Additionally, almost 15% of melanoma occurs in patients with a family history, and a subgroup of these patients have germline mutations associated with melanoma predisposition (34-35). Despite this, the magnitude of this genetic factor is still unclear, which is why it is recommended that people with a family history maintain a careful examination of their skin and more frequent consultations with dermatologists (36). Exposure to carcinogenic substances is another risk factor. A study has indicated that herbicides have been associated with a greater likelihood of developing acral melanoma (palms of the hands and feet). 10 This study revealed a higher incidence of acral melanoma in people who have used these herbicides at home is higher than in people who do not use them (37). Histopathology of melanoma Melanoma development Malignant melanoma develops from melanocytes, melanin-producing cells. In addition to growing in the skin (95% of cases), melanoma can also develop in uvea and mucous membranes due to its embryonic origin at the neural crest. Skin Among all the organs of the body, the skin is the largest. As a barrier against physical, chemical, and biological agents, it provides the first level of protection, prevents water loss, and regulates body temperature. The human skin comprises three layers: the epidermis, dermis, and subcutaneous tissue (38) (Figure 5a). In what has come to be known as the epidermis, the outermost layer is a stratified epithelium made up mainly of keratinocytes (representing 95% of the total cells in this layer), which migrates progressively from the epidermal basement membrane to the skin surface and forms several well-defined layers along the way. In the following order, the basal layer appears followed by the spinous stratum, the granular stratum, and finally, the stratum corneum (Figure 5b). 11 Another cell type in this layer is Langerhans cells, which function as part of the immune system, looking for antigens within their surroundings to activate an immune response, or Merkel cells, which are closely associated with terminal filaments of cutaneous nerves and are responsible for sensation (39-40). Besides the epidermis, melanocytes are also present in that basal layer, having a ratio of one melanocyte per ten basal cells. As previously stated, the melanocytes produce the pigment melanin, which is packaged into cellular vesicles known as melanosomes, and delivered into the cytoplasm of the keratinocytes (40) to protect the body from the harmful effects of ultraviolet radiation. A layer of tissue called the dermis lies beneath the epidermis. Blood vessels, nerves, glands, and hair follicles are found within this thick layer of connective tissue, which is tightly attached to the epidermis by the basement layer. It is connected to the underlying organs, such as bones or muscles, via the subcutaneous layer or hypodermis, which consists of loose connective tissue that contains variable amounts of adipose cells (cells that store fat), depending upon the area of the body and its nutritional requirements. Melanoma skin cancer According to the above description, skin melanoma arises from the malignant transformation of melanocytes in the epidermis. Among the molecular alterations, these cells undergo are mutations of genes responsible for regulating the cell cycle, differentiation, adhesion, signaling, and apoptosis. It involves acquiring diverse phenotypic features by normal melanocytes that lead to the development of a malignant phenotype. In the Clark model, one of the most popular, the significance of the histopathological changes related to the progression of melanoma is highlighted (Figure 6). The first phenotypic shift in melanocytes is the development of benign nevi. Despite disrupting the control of growth in these cells, the growth of a nevus is limited, and rarely progress to cancer (41). Oncogenesinduced cell senescence may be responsible for the absence of progression, in which oncogenes stimulate cell growth. From a molecular perspective, excessive activation of the mitogen-activated protein kinase (MAPK) signaling pathway promotes the development of melanoma cells. Activation of this pathway is caused by mutations in NRAS, which accounts for approximately 15 percent of melanoma, or mutations in BRAF, which account for about 50 percent of melanoma (42). 12 Cytologic atypia, which may develop from preexisting benign lesions or as a new tumor, seems to be the next step towards melanoma following the Clark model. This stage of the disease progression involves molecular abnormalities that affect cell growth, DNA repair, and susceptibility to cell death. Next is the radial-growth phase, which refers to tumors that grow laterally along with the epidermis and do not metastatic spread. The condition can last for years, and cancer can be surgically removed with a high success rate. Manifestations of invasive behavior in Clark's model occur in the vertical-growth phase when melanoma cells penetrate the basement membrane and grow intradermally as expanding nodules. Even though this progression model is viewed as a linear stepwise process, many melanoma tumors may not adhere to it in an orderly manner. For example, RGP and VGP melanoma may result from existing nevi lesions or develop spontaneously from normal melanocytes (43). In both cases, cancer spreads because of the uncontrolled growth of malignant tumor cells due to genetic mutations that cause neoplastic transformation and enable them to escape inhibitory signals. Several hallmarks of cancer characterize this process. 13 It has been shown that a comprehensive set of molecular pathways is involved in the initiation, proliferation, survival, progression, and invasion of a tumor. In this way, MAPK, PI3K, and NFkB signaling pathways interconnect significantly during melanomagenesis (Figure 7). Essentially, stimulation of G protein-coupled receptors (GPCR) results in the activation of PKC protein. As a result, activated PKC stimulates the MAPK pathway. Additionally, the receptor tyrosine kinases (RTKs), activated through the binding of extracellular growth factor ligands, also mediate the activation of RAS protein, the top member of the MAPK cascade. Simultaneously, RTK turns on the PI3K path. PI3K may also be activated by GPCRs, IGF-1Rs, and RAS. Briefly, both MAPK and PI3K/AKT pathways mediate cell survival and proliferation. As part of the TNF-alpha pathway (canonical NFkB pathway), binding of the cytokine to its receptor TNFR1 results in activation of TAK1. TAK1 promotes the aggregation of a downstream kinase complex, IKK. In response to the phosphorylation of IkB by the IKK complex, NFkB is released. As a result, this element is translocated to the nucleus, leading to the activation of genes involved in cell survival and anti-apoptosis. MAPK Pathway It is well known that mitogen-activated protein kinase (MAPK) is an important signal transduction pathway for various physiological processes, including cell proliferation, differentiation, development, migration, apoptosis, and transformation (45). It is particularly relevant to the development of melanoma. In a normal situation, the MAPK cascade is regulated by scaffolding and regulatory proteins. One of that regulators is Raf Kinase Inhibitor Protein (RKIP), also known as phosphatidylethanolamine binding protein 1 (PEBP1) (46). Raf-1 and MEK are required for the phosphorylation of MEK and the subsequent phosphorylation cascade, and RKIP disrupted the physical interaction of Raf-1 and MEK proteins, reducing the path activation (46). Two mechanisms deregulate the pathway: the gain-of- 14 function mutations, which make the RAS and RAF proteins consistently present in the cell in their activated state irrespective of external stimuli, and the ineffectiveness of natural inhibitors, such as RKIP. Concerning the first question, among the mutations most likely to be found in skin cancer is the BRAFV600 mutation of the BRAF gene (47). In addition, mutations affect the KRAS protein (mutant KRASQ61) and the neurofibromin 1 (NF1) protein. All of these mutations result in activation of the MAPK pathway and, consequently, in the proliferation and survival of cells (48). Regarding the second one, it has been described a loss of RKIP expression in melanoma. Published data report the role of RKIP in regulating cell proliferation, migration, and invasion capability (4951), by mechanisms leading to Ras-ERK1/2 and NFκB pathways inhibition. Nevertheless, there is little known about the pathways regulated by RKIP in normal melanocytes nor its role in the malignant transformation of this type of cell. Some studies (51,52) describe a gradual reduction of malignancy-related RKIP levels in melanoma patients, but these were performed with a small cohort of patients, so it would require a more extensive study to establish the real diagnostic or prognostic value of this marker in melanoma. PI3K-AKT Pathway The phosphatidylinositol-3-kinases (PI3Ks) are a family of lipid kinases involved in many cellular processes, including cell survival and growth, differentiation, proliferation, transcription, and translation. Besides transducing signals from growth factors and cytokines, the pathway is a major downstream effector of RTKs and G-protein-coupled receptors (GPCRs) (Figure 9). An essential component of this signaling pathway is AKT, which transmits signals by phosphorylating different downstream effector targets, influencing critical cellular processes, such as apoptosis, DNA repair, cell cycle, glucose metabolism, cell growth, and motility, invasion, and angiogenesis (53). There are negative regulators to prevent persistent and long-term activation of PI3K-AKT signaling. PTEN is a central regulator of this signaling pathway. When PTEN is missing, AKT is constitutively activated, resulting in malignant melanoma tumor development (54,55). NFkB Pathway The nuclear factor-kappaB (NFκB) is a pleiotropic transcription factor that regulates several genes involved in many critical pathways that allow for a wide range of physiological functions such as immune responses, inflammatory responses, development, and cancer initiation and progression (56). The NFkB family comprises five members identifiable by their conserved Rel homology domains, the part of the proteins that control DNA binding: RelA (p65), RelB, c-Rel, p100/p52, and p105/p50. 15 Regulation of NFkB family members occurs primarily through binding them by IkB proteins (57). Among the members of the IkB family is the proto-oncoprotein Bcl-3, which is predominantly located in the nucleus. It has been shown that Bcl-3 and Pirin, a member of the cupin superfamily, form a stable complex that enhances Bcl3-p50’s ability to bind DNA, inducing transcription of various target genes involved in cell survival and antiapoptosis (58). The effects of ultraviolet irradiation on skin cells include promoting inflammatory responses and cytokines, many of which have NFkB as a downstream target or effector. If the presence of NFkB is sustained, it may result in the augmentation of pro-inflammatory mediators, which may damage tissues, leading to organ dysfunction and eventually cancer (59). There are different mechanisms through which the activation of NFkB may occur due to upstream deregulation of MAPK and PI3K-AKT signaling pathways. In melanoma cells, these changes increase proliferation and resistance to apoptosis (60). Melanoma heterogeneity and plasticity Malignant melanoma is one of the most frequently mutated cancer. However, as described in the preceding section, driver mutations in genes such as BRAF or NRAS occur early in the course of melanoma development. In contrast, a hierarchical pattern has not been detected between mutations associated with metastasis, suggesting that transcriptional programs are involved in melanoma progression (61). Two predominant transcriptional programs have been identified in cultured melanoma cells based on gene expression analysis: proliferative’ phenotype or ‘invasive’ phenotype. Interestingly, the two phenotypes are not determined by genetic mutations, and transcriptional activity can reprogramme a phenotype. There is a balance between both phenotypes regulated by transcriptional master regulators, which enable cells to be capable of adaptive phenotype plasticity (61). One evidence for adaptive phenotype plasticity can be found in single-cell analyses of melanoma biopsies, which have detected populations of cells with invasive or proliferative phenotypic states and single cells with transitional phenotypic forms. Based on this phenotype plasticity hypothesis, stable melanoma phenotypes are defined by transcriptomes relating to differentiated melanocytes or slowcycling cells with neural-crest stem-cell (NCSC) markers (61). Tumor persistence seems to rely on a particular subset of cells with these acquired “stem cell-like” properties (62,63). Like embryonic stem cells (ESC), this subpopulation of tumor stem-like cells can grow intensively and infiltrate local tissue (52, 62-69). The self-renewal capability of embryonic stem cells is regulated by pluripotency-related transcription factors such as Nanog homeobox (NANOG), POU class 5 homeobox 1 (OCT4), and SRYbox transcription factor 2 (SOX2) (70) which are also aberrantly expressed in many malignant human tumors (71–73). 22 Molecular features of melanocytic lesions The morphological criteria for determining atypia are often disagreeable and subject to inter-observer variability, particularly in non-conventional diagnoses lesions. Considering these challenges, the World Health Organization (WHO) incorporated known molecular pathways into its latest classification of melanocytic tumors, introducing the concept of "intermediate" lesions (97). This multidimensional classification suggests that the conventional approach of identifying melanocytic tumors as either benign or malignant may no longer be adequate. WHO 2018 identifies nine categories or pathways leading to melanoma, each driven by genetic factors (Table 3). As a result of these molecular signatures' heterogeneity, two critical implications emerge: first, it emphasizes the need for individualization of melanoma diagnosis, prognosis, and treatment; second, it offers a broad range of potential biomarkers and novel putative therapeutic targets. In dermatology, the following antigens/antibodies are commonly used to diagnose melanoma (106,107): The frequency of S100 expression in malignant cutaneous melanoma is approximately 95%. Several factors can influence its expression, including too much or too little fixation time, previously frozen tissue, and enzymatic pretreatment with trypsin. Antibodies can be polyclonal or monoclonal, and both identify melanocytosis in a cytoplasmic and nuclear manner. Among the many antigens detected by anti-S100, A6 is expressed by some melanocytotic lesions and may also be helpful when detecting neurotheliomas. The Melanoma Antigen Recognized by T Cells-1 (MART1) is one of the most important melanocytic markers. Two different antibodies detect the antigen (Melan-A and A-103), expressed by a wide range of benign and malignant melanocytic lesions. Consequently, it is of great value in detecting melanocytic differentiation. On the other hand, a diagnosis of desmoplastic melanoma is unlikely if this marker is highly expressed in a spindle cell melanocytic lesion. 23 Table 3. Classification of melanoma based on 2018 world health organization classification (97) UV exposure Categories Melanoma subtype Key molecular genes Low-CSD melanoma Pathway I Superficial spreading melanoma BRAFV600 mut TERT mut CDKN2A mut PTEN mut NRAS mut TP53 mut High-CSD melanoma Pathway II Lentigo maligna melanoma NRAS mut TP53 mut KIT mut CDKN2A mut TERT mut PTEN mut BRAFnon-V600Emut Pathway III Desmoplastic melanoma NF1 mut NRAS mut NFKBIE mut PIK3CA mut Low or no UV /CSD melanoma Pathway IV Spitz melanoma ALK rearr CDKN2A mut NTRK1 rearr HRAS mut NRTK3 rearr Pathway V Acral melanoma KIT mut ALK rearr NRTK3 rearr CDKN2A mut CCND1amp TERT mut NRAS or BRAF mut Pathway VI Mucosal melanoma KIT mut SF3B1 mut CDKN2A mut CCND1 amp CDK4 mut MDM2 amp NRAS or BRAF mut Pathway VII Melanoma arising in congenital nevi NRAS mut BRAFV600E mut Pathway VIII Melanoma arising in blue nevi GNA11 mut GNAQ mut CYSLTR2 mut SF3B1 mut BAP1 mut EIFAX mut Pathway IX Uveal melanoma GNA11 mut GNAQ mut CYSLTR2 mut PLCB4 mut BAP1 mut EIFAX mut SF3B1 mut May occur in any or most of the pathways Nodular melanoma Abbreviations: amp, amplification| CSD, cumulative sun damage | mut, mutation | rearr,rearrangement The anti-HMB45 antibody detects the premelanosome protein (PMEL o gp-100) in melanoma and junctional nevi. The expression of gp100 in primary cutaneous melanoma differs from that of nevi since it is often distributed in patchy patterns throughout the dermis. This pattern is also found in nevoid melanoma. It is especially useful in detecting the pattern of nevi maturation. Accordingly, superficial type Amelanocytes (epithelioid cells located within the epithelium or close to the epithelium and predominantly pigmented) express neuronal markers and gp100. In contrast, deeply located type C melanocytes (spindle cells) express schwannian markers. However, it has been proven to be insensitive to desmoplastic malignant melanoma. Proliferation markers such as MIB1 (detected by anti-Ki67 antibody) are expressed in proliferating cells. Similar to gp100, its pattern of expression indicates whether or not a tissue has matured. A small percentage of cells in both common and dysplastic nevi exhibit reactivity, which is typically located at the dermalepidermal junction or in more superficial dermis compartments. Melanoma, on the other 24 hand, display a random pattern of immunoreactivity and typically have a proliferative fraction of over 10%, particularly near the edge of the lesion. The enzyme tyrosinase (TYR) participates in melanogenesis and therefore is relatively specific for the differentiation of melanocytic cells. However, it also exhibits a lower sensitivity to detect desmoplastic melanoma. In the manner described, the tissue markers S100, MART-1, and gp100/HMB45 can be used to differentiate melanoma from other types of cancer, but none of these markers can accurately distinguish non-malignant melanotic lesions from malignant melanoma, nor are they able to stratify melanoma patients by their risk of progression. 25 26 27 Hypothesis and objectives Background Although cutaneous melanoma account for less than 5% of all skin cancer, they are the deadliest due to their propensity to metastasize and a lack of effective treatment at advanced stages. Currently, the diagnosis and prognosis of malignant melanoma are mainly based on the evaluation of biopsies of skin lesions removed from patients. Clinicians determine a melanoma patient's prognosis based mainly on the Breslow and the presence of ulceration and sentinel nodes (108). It has been reported that 10% of melanoma recurrences within five years of follow-up have occurred in the early stages (I and II according to the 8th edition of AJCC) (109). In this context, it has been recently found in a study involving 784 patients that 53.8% of patients with metastatic melanoma had an initial stage of I-II (110). Therefore, it would appear that conventional risk markers are not detecting many early melanoma with the potential to metastasize. The most critical aspect of reducing melanoma deaths is identifying markers that can be used for early stratification of melanoma, particularly those with a worse course. Related to the latter group, there is a need to understand the biology of thin melanoma that is ultimately lethal. In previous work by our research group, the differential proteomic analysis that included melanoma and primary melanocyte lines highlighted several novel candidate markers of cutaneous melanoma (111). In the principal component analysis, where mRNAs and proteins of RKIP and PIR were analyzed showed the highest significance in terms of expression between healthy primary melanocytes (HEMnMP, HEMn-DP, and HEMm-LP) and melanoma cells (JSG, HT144, Hs-294t, A375, Mel-HO, WM298B, 1205lu, Mel-Juso, RMPI7951, and Colo-800) (Figure 10). 28 In its original description, RKIP was an inhibitor of MAPK or ERK1/2 pathways. However, later studies revealed its additional role as a regulator of other signaling cascades, such as GPCR, GSK3b, and NFkB (46,112). It functions as a signaling switch in essential processes such as differentiation, proliferation, and survival of cells. The deregulation of this protein has been associated with a wide variety of diseases, including cancer (113-115). As far as PIR/Pirin is concerned, it was initially described as a ubiquitously expressed nuclear protein with a putative function as a transcriptional cofactor (116). Over time, several studies have revealed that this protein plays an essential role in processes such as cell cycle regulation (117-119), inflammatory response (120,121), migration regulation, and epithelial-mesenchymal transition regulation (122-126). Based on our results quantifying mRNA levels by RT-PCR, RKIP expression was generally reduced in melanoma cell lines compared to primary normal melanocytes (Figure 11a). We found that the protein level of RKIP was also consistently reduced in melanoma cell lines with no change in primary cell lines (A375, Colo-800, WM793B, Mel-Ho, and Mel-JUSO) or metastatic cell lines (1205Lu, A2058, Hs294t, HT-144, MeWO, and RMPI7951) (Figure 11b). A lower expression of the PIR gene was also observed in melanoma cell lines compared to melanocytes (Figure 11c). However, comparing melanoma cell lines, it was found that PIR mRNA levels were more heterogeneous than in the RKIP study. As a result, we observed a much lower amount of Pirin protein in all melanoma cell lines than in melanocytes (Figure 11d). Consequently, these markers were licensed under a European Patent (No. EP3051291. Method of Diagnosis and prognosis of cutaneous melanoma). 29 Hypothesis According to our previous results, we maintain the hypothesis that RKIP and Pirin are proteins that could play a role in the etiopathogenesis of cutaneous melanoma, making them excellent biomarkers for diagnosis and prognosis of cutaneous melanoma. 30 Objectives To achieve the hypothesis, we set the following specific objectives: 31 38 the proliferative rate and cell viability and chromosomal and metabolic alterations, which can generate disturbances in the analyses carried out. Therefore, cell cultures were tested for mycoplasma every three months to eliminate any contaminated cultures. The commercial Venor® GeM One-Step Test (11-8025, Minerva Biolabs, USA) was used, which detects ten mycoplasma species by PCR. For the analysis, the supernatant of culture media that has been in culture for at least 48 hours was used. Modulation of gene expression Gene expression analysis studies how genes are transcribed to synthesize functional gene products: RNA or proteins. The study of the regulation of these pathways provides information on normal cellular processes, such as cell differentiation, or pathological processes, such as tumorigenesis. Silencing by transduction with lentiviral particles Lentiviral vectors are a high transduction efficacy and safe method for gene delivery into hard-totransfer cells, such as primary melanocytes. Moreover, lentiviral particles can be employed in standard Biosafety Level 2 tissue culture facilities, as they are replication-incompetents. Normal primary melanocyte cell line HEMnLP was transduced with lentiviral particles for gene silencing following the manufacturer’s instruction with minor modifications (Figure 13a). Briefly, 24 hours before viral infection, cells were seeded in a 6-well plate. In our case, we did not use polybrene® because it was toxic for our primary cells. Polybrene® is a polycation that neutralizes charge interactions to increase the binding between the pseudoviral capsid and the cellular membrane. The ratio of the number of transducing lentiviral particles to the number of cells (Multiplicity of Infection, MOI) used in these experiments was of 2 MOI of lentiviral particles, directly added to each well and were incubated overnight. Specifically, the shRNA specified in Table 6 was used.The day after, the medium with the lentiviral particles was replaced, and a fresh medium was added. Two days after that, the cells were selected with 5 µg/mL of Puromicine (P8833, Sigma-Aldrich Quimica, S.A., Spain) to get stable cell lines. Table 6. Lentiviral particles specifictions Gene Reference Supplier Transduction efficiency control copGFP Control Lentiviral Particles (sc-108084) Santa Cruz Biotechnology Inc., USA Transduction scramble control Control shRNA Lentiviral Particles (sc-108080) Santa Cruz Biotechnology Inc., USA RKIP | PEBP1 PEBP1 shRNA Lentiviral Particles (sc-36430-V) Santa Cruz Biotechnology Inc., USA PIR PIR shRNA Lentiviral Particles (sc-61359-V) Santa Cruz Biotechnology Inc., USA 39 40 Overexpression by tranfection with plasmid Transfection plasmid is widely used to achieve efficient gene transfer in easy-to-transfer cells, such as melanoma cells. Due to that, A2058 and MeWO metastatic melanoma cell lines were transfected with overexpressing plasmid for RKIP or PIR using Lipofectamine 2000 (11668019, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions (Figure 13b). Briefly, after seeding cells were 70–90% confluent the transfection reagent mix was added. This mix included Opti-MEM® Medium, Lipofectamine 2000 and plasmid of interest. The Table 7 includes the information of the plasmid used in this study. All of the transfection experiments were performed with 500 ng of each plasmid. The mix was incubated during 15-20 min and then was transferred to the seeded cells. All the experimental assays were performed at least after 24 hours of transfection and a scramble plasmid was used as control. Functional assays Proliferation assays Cell proliferation can be used to assess normal cell health, to measure responses to toxic insult, or as a prognostic and diagnostic tool in several cancer. The cell viability was determined using the standardized XTT kit (Roche Molecular Biochemicals, USA). This assay is based on the ability of viable cells to transform the yellow XTT tetrazolium salt into orange formazan in the presence of a reducing reagent. The process occurs only in viable, metabolically active cells. The amount of formazan and the orange dye formed is easily quantifiable using a plate spectrophotometer by measuring the absorbance at 490 nm. For this assay, cells were seeded in flat-bottomed 96-well plates with the appropriate density according to the cell line in 100 µL of culture medium and left overnight in the oven to adhere to the well. After adding the reducing reagent and XTT mixture to the wells in a 1:50 ratio, the plate was incubated for 4 hours in the incubator. The absorbances were read at 490 nm utilizing a plate spectrophotometer BioTek Table 7. Plasmids specifications Gene Reference Supplier scramble pCMV-mycDKK OriGene Technologies, Inc, USA RKIP PEBP1 pCMV-PEBP1myc-DDK OriGene Technologies, Inc, USA PIR pCMV-PIRmyc-DKK OriGene Technologies, Inc, USA Nanog promoter PL-SINNanog-EGFP #21321 Addgene, USA Jarid1B promoter EWI024-FP pLUJARID1B1B promoterEGFP Kerafast Inc., USA 41 Synergy HTX (Agilent Technologies, Inc, USA). Cell viability was calculated as the percentage of cell viability with respect to control cells as follows: (sample absorbance/control absorbance) * 100. Migration assays Cell migration occurs during critical physiological processes and is dysregulated in pathological situations, such as cancer metastasis and inflammation. For migration capacity assays (Figure 14), after seeding the cells in 24-well plates, the monolayers were incubated with 0.5 µg/mL of Mitomycin C for 2 hours. Then they were scraped with a sterile plastic micropipette tip. The wound closure was observed over 48 hours, and the photos were taken at 0, 24, and 48 hours with a light microscope. Then, each group's migration percentage was calculated according to the control. Moreover, the transwell active migration assay was performed using Type I-Collagen coated inserts with 6.5-mm-diameter polycarbonate filters (8-μm pore size). Cells (1 × 104) suspended in 200 μL of DMEM without FBS were seeded in the top chambers. The bottom chambers were filled with 300 μl of DMEM containing 10% FBS (as a chemoattractant). Cells were allowed to migrate overnight. The nonmigrated cells on the upper surface of the filter were carefully and thoroughly removed with cotton swabs. Migrated cells were fixed with a mix of cold 4% paraformaldehyde plus 2% ethanol and stained with crystal violet. Five images per insert were taken using a compound optical microscope, and migrated cells were quantified by ImageJ software. Results were expressed as the average number of migrated cells per well obtained from three separate experiments done in triplicate. 42 Data analysis In all quantitative tests performed (proliferation and migration assays), the SPSS version 26 program was used. Fisher exact test was applied to check the normality of the data. To compare differences between the two groups, Student's t-test was performed. P<0.05 was considered statistically significant, P<0.01 was considered highly statistically significant, and P>0.05 was considered statistically insignificant. Molecular analysis Protein analysis Protein extraction Melanoma cells and primary melanocytes were harvested by trypsinization, washed with PBS and lysed in RIPA lysis buffer (80 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP 40, 0.5% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail (Sigma-Aldrich Quimica S.A., Spain) for 15 minutes on ice. Lysates were then cleared by centrifugation at 1500 xg for 5 minutes. Total protein concentration was determined using the bicinchoninic acid assay. Protein quantification Protein quantification was performed by preparing a straight standard with increasing known concentrations of Bovine Serum Albumin (BSA). To prepare the samples, each eppendorf was adequately named and mixed 5 L of each sample with 45 L of Milli-Q H2O. Then, knowing that the relation between bicinchoninic acid and cupric sulfate had to be 50:1, 1mL of this mix was poured into each eppendorf, and all samples were incubated at 37oC in darkness for about 30 minutes. Finally, the absorbance was measured at 562 nm in a spectrophotometer. Protein detection by western blotting Western blot is a widely used analytical technique for the study of proteins. This method allows the detection of a single protein within a biological sample, specifically with an antibody that recognizes a unique epitope on the protein of interest. Protein detection using this technique involves a first step in separating the proteins based on their molecular weight, transferring them to a membrane, and subsequently labeling them with the antibody of interest. 43 SDS polypolyacrylamide gel electrophoresis The SDS polypolyacrylamide gel electrophoresis technique (SDS-PAGE) allows the separation of denatured proteins on gels with a polypolyacrylamide matrix. The gels were prepared by polymerization of polyacrylamide, taking into account the percentage of this polymer concentration. The higher the percentage of polyacrylamide, the smaller the pore size and the better the resolution of low molecular mass proteins. On the contrary, if a protein with high molecular weight is to be detected, the percentage of polyacrylamide must be lowered to obtain larger pores that allow a better resolution of large proteins. In this study, 12% of polyacrylamide concentration was used. Table 8. Percentage of polypolyacrylamide used depending on the protein of interest % polyacrylamide 15% 10% 7.5% 5% KDa range 12-43 16-68 36-94 57-212 The gels are composed of two different parts (Table 9):  Stacking gel: It has large pores that allow the proteins to migrate freely and get stacked at the interface between stacking and running gel. The purpose is that proteins start migrating at the same time.  Running gel: The resolving part of the gel, in which proteins run according to their molecular weight. 44 Table 9. Composition of the stacking and running gel Stacking gel Running gel 4% 7.5% 10% 12% 15% 30% Acry-bis 1 mL 30% Acry-bi 3.8 mL 5 mL 6 mL 7.5 mL 0.5M Tris-HCL (pH6.8) 0.75 mL 2M Tris-HCL (pH8.8) 3 mL 3 mL 3 mL 3 mL H2O miliQ 5.75 mL H2O miliQ 9 mL 7 mL 6 mL 4.5 mL 20% SDS 37.5 mL 20%SDS 75 mL 75 mL 75 mL 75 mL 10%APS 62.5 mL 10%APS 75 mL 75 mL 75 mL 75 mL TEMED 6.25 mL TEMED 5 mL 5 mL 5 mL 5 mL Every sample contained 2 µL of Dithiothreitol (DTT, which breaks down hydrogen bonds) and 4 µL of Laemmli 1X loading buffer (60 mM Tris-HCl pH 6.8, 2% p/v SDS, 10% glycerol, 0.002% blue bromophenol, 1mM DTT and bidistilled water up to 20µL). Then, samples were boiled at 95oC for 5 minutes and loaded with 7 µL of molecular weight ladder Precision Plus ProteinTM Dual Colors Standards of BioRad and 20 µL of each sample. The assembly was covered with the electrophoresis buffer (Table 10). Then, electrophoresis was at 100 V for 10 minutes; and then at 180 V for around 45 minutes. Table 10. Composition of 1L of the electrophoresis buffer (5X) Tris (tris(hydroxymethyl)aminomethane) 15.15 g Glycine 72.1 g Sodium dodecyl sulphate 5 g Milli-Q H2O Up to 1 L Transference This step involves transferring the proteins from the gel to a nitrocellulose membrane (Whatman GmbHGE Healthcare, Dassel, Germany) by applying voltage. For that, an electroblotting cassette containing a small pillow, two thin filters, the gel with proteins, the nitrocellulose membrane, and another two filters plus a small pad was assembled and placed on the electrodes in the blotting unit with transfer buffer (Table 11). The transference conditions were 3 hours at 300 mA. To verify a successful transfer, the nitrocellulose membrane was incubated for 2 minutes in Ponceau red solution. This stain binds reversibly to the positively charged functional groups of the protein (amino group) and the non‐polar regions. After checking the transference, the membrane was washed extensively in water until the dye was gone. 45 Table 11. Composition of 1L of the transfer buffer Tris (tris(hydroxymethyl)aminomethane) 5.8 g Glycine 29 g Sodium dodecyl sulphate 1 g Metanol 200 mL Milli-Q H2O Up to 1 L Immunostaining The blots were incubated with PBS containing 5% Bovine Serum Albumin and 0.1% Tween-20 for 1 hour to block nonspecific binding and then incubated with an appropriate dilution of primary antibody at 4oC overnight (Table 12). The membrane was washed with TBST three times (10 min/each time), then incubated with goat anti-mouse Horseradish Peroxidase (HRP) conjugated secondary antibody for 2 hours at room temperature. Finally, proteins were visualized by enhanced chemiluminescence using the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA). The following table includes the antibody details used in this study. Table 12. Details of sources and concentrations of antibodies used for western blot in this study Antibody name Dilution Reference Recombinant Anti-RKIP antibody 1:5000 Abcam, UK ab76582 Anti-Pirin rabbit polyclonal antibody 1:1000 Thermo Fisher Scientifc, USA PA5-29777 Anti-gamma Tubulin antibody 1:1000 Abcam, UK ab 11321 Goat F(ab')2 Anti-Mouse IgG(H+L), Human ads-HRP 1:8000 SouthernBiotech, USA 1032-05 Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP 1: 10000 Abcam, UK ab6721 Gene expression analysis RNA extraction and quantification Total RNA from cultured cells was isolated using the RNeasy Mini kit (Qiagen Inc, Germany). The entire process was carried out on the ice and with sterile material. After collecting the cells, the number of cells was counted, and 1 x 107 cells were separated since RNA extraction was performed from 1 x 107 cells. They were centrifuged at 300 xg for 5 minutes, and the PBS was removed. The pellet was resuspended with 600 µL of a solution consisting of 1 ml of RLT buffer and 10 µL of - mercaptoethanol. The sample was passed ten times through a syringe with a 20G needle for complete and homogeneous cell lysis. Then, the 70% ethanol addition step is followed by the transfer of 700 µL of the sample to an RNeasy separation column. After centrifuging it at 1500 xg for 15 seconds, the eluate was discarded, and the column was placed in the same collecting tube. This step was repeated as often as necessary until the entire sample was collected. Then 700 µL of RW1 buffer was added to the 46 column to clean it, and it was centrifuged at 1500 xg for 15 seconds. The eluate was again discarded, and the treatment with DNAse was carried out, for which 80 µL of a solution containing 10 µL of DNAse and 70 µL of RDD buffer were added. It was incubated for 15 minutes at room temperature (2030oC), and then 700 µL of buffer RW1 was added and centrifuged for 15 seconds at 1500 xg. The eluate was discarded, and the column was transferred to a new collecting tube. Two washes were then carried out with 500 µL of RPE buffer each, centrifuging at 1500 xg for 15 seconds in the first wash and 2 minutes in the second. Finally, the column was transferred to a 1.5 mL Eppendorf tube, and the RNA was eluted by adding 50 µL of DEPC water (Ambion Inc., USA) by centrifugation at 1500 xg for 1 minute. Samples were correctly labeled, quantified by NanoDrop (Thermo Fisher Scientific, USA), and stored at -80oC until use to ensure RNA integrity. Real Time quantitative Polimerase Chain Reaction (RTqPCR) For each sample, cDNA was synthesized from 1 µg total RNA using the iScriptTM cDNA Synthesis kit (Bio-Rad, USA) according to the manufacturer’s instruction. The reaction mixture contained 0.1 µL cDNA from the reverse transcription reaction, together with forward and reverse specific primers and iQTM SYBR® Green Supermix (Bio-Rad, USA) in a final reaction volume of 20 µL. Quantitative realtime RT-PCR assays were carried out using an iCycler PCR platform (Bio-Rad, USA). The PCR reaction began with heating at 95oC for 10 min, followed by 45 cycles of desnaturation at 95oC for 30 sec, annealing at the corresponding temperature for each gene (56-61oC) for 20 sec and extension at 72oC for 30 sec. Each assay included a negative control consisting of the absence of cDNA. Expression data were generated from 2 amplification reactions with samples and controls run in triplicate. Optical data obtained by real-time PCR were analyzed using the MyiQ Single-Color Real-Time PCR Detection System Software v.1.0 (Bio-Rad, USA). The expression of three different housekeeping genes (ACTB, GAPDH, and RPS15) also was analyzed to normalize expression data using the Gene Expression Macro Software Version 1.1 (Bio-Rad Laboratories, Hercules, CA, USA), where the relative expression values were computed by the comparative Ct method (132,133). The sequences of primers used are specified in the following Table 13. 47 Table 13. RTqPCR primers’ sequences Gene name Forward primer Reverse primer ACTB 5'-AGATGACCCAGATCATGTTTGAG-3' 5'-GTCACCGGAGTCCATCACG-3' c-MYC 5'-GCTCCTGGCAAAAGGTCAG-3' 5'-GTTGTGCTGATGTGTGGAGAC-3' E2F1 5'-TGACATCACCAACGTCCTTGA-3' 5'-CTGTCGGAGGTCCTGGGTC-3' GAPDH 5'-CCTGTTCGACAGTCAGCCG-3' 5'-CGACCAAATCCGTTGACTCC-3' JARID1B 5'-GACTGGGACAACAGAACCT-3' 5'-TGGACTAACACCATGGAGG-3' LUM 5'-AACTGCCCTGAAAGCTACCC-3' 5'-AGCCACTGCAGATCAGTTACA-3' NTRK2 5′-CTCCCGGAATTGGGTTGGAG-3′ 5′-GGGGCGCAGATTCCTTGTTA-3′ PIR 5'-GGAGCCTCAGTACCAGGAACT-3' 5'-CTTGGACTTTATTCCCAGGGC-3' RKIP 5′-AATAGACCCACCAGCATTTCG-3′ 5′-TGCCACTGCTGATGTCATTG-3′ RPS15 5'-CGACCAAATCCGTTGACTCC-3' 5'-CGGGCCGGCCATGCTTTACG-3' THY-1 5'-GTTTGACCAGGAAAGCAGCG-3' 5'CTCTTGGGAGCTTGGGACAG-3' ZEB1 5′-GTGCAGTTACACCTTTGCA-3′ 5′-CACATGTCTTTGATCTCTTCCT-3′ Has-miR-215p - 5′-UAGCUUAUCAGACUGAUGUUGA-3′ Has_RNU6-2 - 5′-CGCTTCGGCAGCACATATACTA-3′ Gene expression levels results were analysed using the SPSS version 26 program. Fisher exact test was applied to check the normality of the data. To compare differences between two groups, Student's t-test was performed. P<0.05 was considered statistically significant, P<0.01 was considered highly statistically significant, and P>0.05 was considered statistically insignificant. For detection of mature miRNA, cDNA was prepared in a reverse transcription reaction using miScript HiSpec Buffer from the miScript II RT Kit (Qiagen Inc, Hilden, Germany). Oncogene miR-21 was detected by RT-qPCR. The primers used were Has-miR-21-5p (MS00009079, Qiagen Inc, Hilden, Germany) and Has-RNU6-2 (MS00033740, Qiagen Inc, Hilden, Germany) as reference mature miRNA. The RT-qPCR assay was conducted under the following conditions: Stage 1: 15 min at 95 °C; Stage 2: 60 cycles of 15 s at 94 °C, 30 s at 55 °C, 30 s at 70 °C, 1 s at 72; and Stage 3: 5 s at 95 °C, 1 min at 65 °C. The real-time fluorescence intensity was monitored at each cycle of the third stage. Light Cycler® 480 II Real-Time PCR System (Roche, Basilea, Switzerland) was used to perform the reaction. RNA sequencing analysis Second generation sequencing (Next Generation Sequencing or NGS) is used to analyze the presence of genes as well as the quantification of their expression globally throughout the entire transcriptome. The complete protocol included an RNA extraction step, followed by its quantification as well as its purity and integrity analysis. Subsequently, libraries were prepared and bioinformatic analysis was carried out after sequencing. 54 55 56 57 58 59 Chapter 01 Potential value of RKIP and Pirin proteins as melanoma diagnostic and prognostic markers Characteristics of the patients enrolled in the study During this project, a total of 314 patients (154 females and 160 males) were enrolled who had been diagnosed by pathologists with a nevus or malignant melanoma. The clinicopathological features of the group are shown in Table 19. Our collection included 75 nevi and 239 melanoma. Regardless of the diagnosis, the incidence of nevi and melanoma was equal among men and women (Figure 19a). Patients diagnosed with nevus ranged in age from 24 to 78 years old, with the median age being 50 years old, while those diagnosed with melanoma ranged in age from 23 to 87 years old, with the median age being 57 years old (Table 19). Table 19. Clinical and pathological data from nevus and melanoma patients N (%) N (%) NEVUS 75 HISTOLOGICAL SUBTYPE Age at diagnosis (years, range) Sex - Male - Female 56 (24-78) 28 (37) 47 (63) SSM NM ALM LMM LM Others ND 102 (43) 53(22) 21(9) 9(4) 3 (1) 12 (5) 39 (16) MELANOMA 239 AJCC STAGES AT DIAGNOSIS Age at diagnosis (years) Sex - Male - Female 57 (23-87) 131 (55) 108 (45) In situ IA IB IIA IIB IIC IIIA IIIB IIIC IV 34 (14) 46 (19) 54 (23) 32 (13) 15 (6) 24 (10) 9 (4) 11 (5) 6 (3) 8 (3) LOCALIZATION DISEASE EVOLUTION Head and neck Trunk Upper limb Lower limb Acral Others ND 43(18) 74(31) 24 (01) 69 (29) 21 (9) 5 (2) 3 (1) Disease-free Metastasis 147 (62) 92 (38) 60 Analyzing data according to the body location of the tumors, the subgroup of women had a higher incidence of tumors associated with the lower extremities. In contrast, the subset of men had more melanoma attributed to the head and neck and the trunk (Figure 19b). There were also collected data concerning the subtype of melanoma. As shown in Figure 19c, the superficial spreading melanoma proved to be the most frequent subtype across both sexes (43%), followed by the nodular melanoma, which accounted for 22% of all cases. The remaining samples were diagnosed as lentigo maligna (1%), lentigo malignant melanoma (5%), and acral melanoma (9%). Under the category "others" (12%), low-frequency melanoma, such as those of mucosal origin, were grouped. 61 A notable aspect of this study is how many patients included in it were diagnosed at an early stage of their disease (86%) according to AJCC's staging system (8th edition). As shown in Figure 19d, melanoma were found in 14% of cases as in situ melanoma, 42% as stage I melanoma, and 30% as stage II melanoma. Patients diagnosed in stages III and IV constituted 14% of the total. In connection with that, the samples according to the Breslow index, which is used to determine the thickness of tumors, was 41% for tumors that had a thickness of less than a millimeter and 30% for tumors with a thickness between 1 millimeter and 4 millimeters (Figure 19e). Furthermore, 62% of all melanoma cases analyzed in this study remained disease-free. In comparison, 38% of patients were diagnosed at an advanced stage or developed metastasis during follow-up (the inclusion criteria for this group required a minimum tracking period of two years). This study did not observe a difference in sex-related outcomes, with the same number of men and women in the two groups (Figure 19f). Differential expression of RKIP protein between nevi and melanoma biopsies Two independent reviewers evaluated RKIP staining at negative, low, and high expression. A high proportion of nevus samples expressed RKIP, whereas a large number of melanoma samples lost this protein expression (Figure 20a). Among those with melanoma, most samples analyzed correspond to the histological subtypes of superficial spreading melanoma and nodular melanoma. As shown in Figure 20b, the pattern of RKIP expression does not differ between the groups based on histology and malignant progression. When analyzing the distribution based on the stage at diagnosis, more samples are marked as negative in patients with stages I and II who have metastasized, as well as in those with stages III and IV (Figure 20c). 62 Representative IHC images of most common lesions (compound nevus, superficial spreading melanoma, and nodular melanoma) are shown in Figure 21a, while the statistical analyses are summarized in Figure 21b–e. Nevi samples exhibited higher positivity for RKIP staining compared with the whole cohort of melanoma samples (Figure 21b); 94% of nevi samples were positive for RKIP whereas only 51% of melanoma cases presented positive staining. Interestingly enough, in situ melanoma, characterized by an excellent prognosis upon surgical removal, exhibited a strong positive RKIP expression in almost 80% of cases. Of note, RKIP staining displayed minimum intrasample variation and a cytoplasmic localization. Univariate analysis confirmed the statistical significance of observed differences among nevi and the entire set of melanoma samples (q < 0.001) (Figure 21b) and both, univariate and multivariate analysis, provided statistical evidence for a different level of expression of RKIP in nevi and melanoma at early stages (AJCC 8th I/II) (Figure 21c). Moreover, by means of a logistic regression analysis (Nevus = 0, Melanoma = 1) to control for age and sex as covariates (Figure 21c), a polynomial contrast expansion in RKIP demonstrates that this association is linear (β = −2.288, q < 0.001), i.e., linear increments in protein levels correlate significantly with a larger probability of the biopsies of being identified as nevus, while quadratic effects tend to be moderate and non-significant (β = 0.465, q = 0.218). 63 70 71 Chapter 02 RKIP regulates melanocyte differentiation by modulating the stemness-related transcription factor As a signaling switch, RKIP plays a role in essential processes such as differentiation, proliferation, and cell survival. Moreover, deregulation of RKIP expression has been associated with a wide variety of disorders such as neurological diseases, diabetes, altered spermiogenesis and cancer (113-115). With a general loss of RKIP expression in tumor tissues and its demonstrated ability to influence pathways leading to tumor cell proliferation or an invasive phenotype (65-67, 134), a more comprehensive analysis of RKIP-dependent pathways on primary cells is necessary to determine profile alterations that may influence cellular transformation or promote an aggressive phenotype. Thus, in this work, we examined the role of RKIP in the biology of primary melanocytes and malignant melanoma cells. Involvement of RKIP on malignancy-related properties of melanoma cells To study the involvement of RKIP in the pathogenesis of melanoma, we modified the endogenous RKIP levels expression on A375 and MelHo primary melanoma cell lines and A2058 and MeWo metastatic melanoma cell lines. Downregulation of endogenous RKIP was accomplished by RKIP shRNA lentiviral particles, while RKIP-overexpressing plasmids were used to increase cellular RKIP levels. Downregulation by shRNA led to a decrease of up to the 70-80% on the endogenous RKIP mRNA level on selected primary melanoma cells lines (Figure 26a) which was also consistent with a reduction on the protein percentage (Figure 26b). Reduction of endogenous RKIP by lentiviral silencing did not alter proliferation capability of A375 and MelHO cells (Figure 26c). By contrast, the RKIP-downregulated primary melanoma cells showed a significantly increase in motility, assessed both by wound healing and collagen-coated transwell assays (Figure 26d-e). 72 To reinforce our data regarding the involvement of RKIP expression in melanoma cell motility, MeWO and A2058 metastatic melanoma cell lines were transfected with a RKIP-overexpressing plasmid resulting in a 5 and 15-fold increase of RKIP-mRNA level, respectively (Figure 27a). In addition, we detected a concomitant elevation of intracellular RKIP-protein percentage (Figure 27b). Consistent with our previous results in primary melanoma, the increase on cellular RKIP expression level led to a decrease in the migration capability of melanoma cells (Figure 27c). Surprisingly, both 73 analyzed cell lines showed different behavior on the active migration assay (Figure 27d); thus, while no differences were observed in MeWO cells, RKIP overexpression clearly diminished the capacity of A2058 cells to pass through a collagen-based barrier. Of note, basal collagen-through migration activity of MeWO cells was significantly lower than that of A2058 cells (data not shown). Briefly, cellular RKIP levels were inversely correlated to the migration capability of both, primary and metastatic melanoma cell lines, while no major effect was detected on cellular proliferation. 74 Transcriptome modulation by RKIP downregulation in HEMnLP cells With the aim of elucidating the molecular mechanisms whereby RKIP could modulate processes related to cellular malignancy, RKIP was downregulated in primary melanocytes (HEMn-LP) by the above described shRNA lentiviral particles. Infection resulted in a 70-80% reduction of RKIP mRNA and 40% of protein level (Figure 28). Two independent replicates of control (shCTR) and RKIP knockdown (shRKIP) HEMn-LP samples were subjected to RNA sequencing. The first part of the analysis focused on the identification of a set of differentially expressed genes between shCTR and shRKIP HEMn-LP based on standard threshold Log2FC≥1, p-value≤ 0.05 and False Discovery Rate (FDR) ≤0.05. The resulting 224 differentially expressed genes were used for monitoring the functions and pathways that were mainly affected in melanocytes due to the decreased RKIP expression. The set of genes with modified expression were roughly equally divided into over- (113) and under-expressed genes (111). The Log2FC, p-values and FDR for each gene are detailed in Annex B: Table S1. In order to gain insight into the functional characteristics of detected changes, overand under-expressed genes after RKIP silencing were subjected to pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG) (Figure 29a) and Gene Ontology (GO) (Figure 29b) enrichment analyses. RKIP knockdown on HEMn-LP cells displayed a transcriptional misregulation in the GO term ‘cancer gene signature’ (pvalue < 0.001). 75 Moreover, the set of genes with altered expression upon endogenous RKIP reduction showed an enrichment in a variety of essential processes including developmental pigmentation, proliferation and developmental and cell differentiation (p-value < 0.05; Figure 29b, Annex C: Table S3). Interestingly, RKIP knockdown led to the downregulation of essential melanocyte-pigmentation genes such as PMEL (Melanocytic linage-specific antigen, 2-fold decrease, p value 0.0003, FDR 0.04), MLANA (Melanoma Antigen recognized by T-cells, 8-fold decrease, p value 0.001, FDR 0.02), GPR143 (G-Protein Coupled Receptor 143,11-fold decrease, p value 10-5, FDR 0.01) and TYRP1 (Tyrosinase‐related protein 1, 5fold decrease, p value 10-6, FDR 0.007). On the other hand, only KIT (proto-oncogene KIT,) was upregulated among the deregulated genes belonging to the developmental pigmentation group (2.3-fold increase, p value 0.0001, FDR 0.02). 76 Development and differentiation showed the best FDR value among significantly enriched biological processes. This signature encompassed 83 genes which represented 37% of the total altered gene-set and included HOX family members, proto-oncogene KIT, proto-oncogene MYC, ZEB1 and Thy-1 cell surface antigen (THY-1), among others (Annex C: Table S3). We focused on genes belonging to this process due to the statistical robustness of this group on our data set as well as to the intimate link among this particular process and the cellular migration-capability. As shown in Figure 29c-d, downregulation of endogenous RKIP led to an increase on the expression of selected genes, validating the RNA Seq data. Interestingly, neurotrophic receptor tyrosine kinase 2 (NTRK2), ZEB1 and THY-1 are not only implicated in developmental processes, as they also known regulators of cellular migration. Based on our previous results that implicated RKIP on the migration capability of melanoma cells, we made use of RKIP overexpression to analyze the effect on ZEB1, NTRK2 and THY-1 transcription. RKIP-driven transcriptional repression was confirmed by RT-qPCR for ZEB1 and THY-1 in both cell lines (A2058 and MeWO) while NTRK2 revealed a cell type-dependent response (Figure 29d). Taking together, RKIP revealed the capacity to modulate genes involved in essential processes (e.g. Development and differentiation) and to repress genes with described roles in cellular migration. NANOG as a putative transcription factor regulated by RKIP RKIP has no described function as a direct transcriptional regulator. Thus, observed transcriptional alterations imply the presence of yet unknown transcription factors or regulators downstream RKIP. We focused on deregulated genes belonging to development and differentiation and conducted an in silico approach in order to detect potential transcription factors acting between RKIP and its downstream modulated genes. Seventy-one percent of genes in this category were putative targets of NANOG transcription factor (Figure 30a, Annex C: Table S4, Table S5). NANOG is a transcription factor involved in the maintenance of stemness and often linked to cancer aggressiveness (Figure 30b). Therefore, we wonder whether RKIP could somehow modulate NANOG expression. To analyze this point, we made use of a construct encoding the NANOG promoter attached to the Enhanced Green Fluorescent Protein (EGFP) coding sequence, and cells were cotransfected with either empty plasmid (pCTR) or RKIP-coding plasmid (pRKIP). Activation of NANOG promoter was determined as the percentage of cells expressing EGFP. As shown in Figure 30c, increased RKIP expression led to a significant decrease on NANOG promoter activation; a similar effect was observed in both cell lines. The gene miR-21 is a described target for NANOG (78). To further validate the implication of RKIP in NANOG regulation, we determined miR-21 transcription level upon RKIP overexpression. As shown in Figure 30d, the expression of miR-21 was significantly lower on RKIP-overexpressing cells. 77 These findings point towards the involvement of NANOG downstream RKIP in the regulation of gene expression. 78 79 86 (Figure 31c) were in agreement with this molecular analysis, since both c-MYC and E2F1 are proteins involved in cell proliferation and cell cycle regulation. In that case, the cells overexpressing Pirin showed a decreased proliferation compared to control cells. These data are consistent with the downregulation of JARID1B gene expression. Together, these results suggest that PIR might modulate melanoma proliferation by targeting the slowcycling transcriptional regulator JARID1B. 87 88 89 Discussion RKIP and Pirin as biomarkers for melanoma diagnosis and prognosis Melanoma is an extremely lethal form of skin cancer. A timely and accurate diagnosis of malignant melanoma is fundamental to ensuring appropriate treatment and a successful outcome. However, histologically, melanoma exhibits a wide range of features, which include epithelial, hematologic, mesenchymal, and neural characteristics (96). In some cases, this makes the diagnosis of the disease difficult. Classically, IHC-mediated routine identification of melanocytic lesions include the use of melanocyte and melanoma markers, like tyrosinase (TYR) and tyrosinase-related proteins (TYRP1 and DCT), gp100 and Melan-A (142); nonetheless, the utility of a combined immunohistochemical analysis including Bcl-2, nuclear S100A4, Ki67 and MITF to improve the risk stratification of early-stage malignant melanoma patients has been recently reported (143). Due to that, the molecular alterations involved in the pathogenesis of melanoma represent a topic of active research, which has enabled the identification of disease-associated key, oncogenes, and tumor suppressor genes providing a scientific foundation for urgently needed therapeutic approaches (144,147). Based on our previous results from a comparative proteomic analysis between melanocytes and melanoma cell lines results, we selected RKIP and Pirin as candidates for melanoma biomarkers. As a consequence, in this thesis, we focused on the analysis of RKIP and Pirin expression by immunohistochemistry in melanoma patient biopsies. Regarding RKIP, several studies have shown this protein exhibits low expression levels in various tumors and it is often absent in metastases (115,134, 148,149, 150-164). In agreement, decreased RKIP expression has been associated with metastatic uveal melanoma while low levels of RKIP were detected on both metastatic as well as non-metastatic cutaneous melanoma biopsies (52,165). These studies, although interesting, were carried out with small cohorts of patients. In addition, in those studies claiming the association among low RKIP expression and metastasis, decreased RKIP expression was assessed by comparison of primary tumors and biopsies at metastatic sites (52,134,148). Results obtained from the aforementioned works revealed a clear malignancy-related silencing of RKIP on tumor cells, although they did not analyze the possible predictive role of RKIP. Our study, including 75 nevi and 239 samples of malignant melanoma, allowed deepening on the diagnostic and prognostic 90 value of this protein. Of note, all melanoma biopsies were obtained from the primary lesion, which may explain the lack of statistical RKIP-staining differences among stage I–IV melanoma. Also, the cohort size of stage III and IV melanoma patients was small (when compared to stage I–II patients) and we cannot discharge its effect when analyzing all stages together. In relation to Pirin expression, it seems to participate in the regulation of different cellular processes, acting as a protein kinase inhibitor, antioxidant or putative transcriptional co-factor (143,147,165-167). Some evidence has implicated Pirin in tumorigenesis by promoting cell proliferation and malignant progression of several cancer (168,169). As RKIP, to evaluate the impact of Pirin on melanoma tumorigenesis, we first analyzed the expression of Pirin by immunohistochemistry in the consecutives slides and in the same way as in RKIP study. In both nevus and melanoma groups, strong Pirin expression was observed and significant differences were detected among benign and malignant lesions, with homogenously strong Pirin expression in benign melanocytes from nevi relative to the heterogeneous expression in malignant melanoma. Looking for new metastatic biomarkers we focused on early stage melanoma (stage I–II according to AJCC 8th edition) in order to evaluate RKIP and Pirin usefulness to discriminate among patients with good and bad evolution of the disease. Here, our results agree with previous studies on the diagnostic capability of RKIP staining, as melanoma samples exhibited an overall decrease in staining when compared with benign lesions (i.e., nevi). Unfortunately, RKIP staining was not able to distinguish stage I–II patients with a favorable evolution of the disease from those who eventually developed metastasis. Nevertheless, it is worth mentioning the association among strong RKIP staining and lower Breslow index across all melanoma stages (stage I–IV) suggesting that RKIP may not determine tumor malignancy but may be related to the primary tumor position or progress through the skin. In the case of Pirin staining, based on multivariate analyses by Logistic Regression and Cox models, including age and the Breslow index as co-variates, strong Pirin expression was significantly associated with a risk of metastasis, suggesting its importance as a prognostic marker. To summarize, our study supports the diagnostic utility of RKIP staining due to the significantly lower RKIP protein levels in melanoma samples, even at early stages (I–II) of the disease. Additionally, Pirin staining along with the Breslow index seem to be a prognostic marker at early stages (I-II) of melanoma, since high Pirin protein levels are associated with a more significant probability of metastasis, as well as a shorter time until this clinical end-point. 91 RKIP Regulates Differentiation-Related Features in Melanocytic Cells Delving into the study of RKIP protein as regards the pathogenesis of melanoma, we carried out molecular and functional assays using melanocytes and melanoma cell lines. In accordance with our histopathological results and previously published studies (51, 152), we found that both RKIP mRNA and protein expression were significantly lower in melanoma cell lines than in primary cultures of melanocytes with the exception of the Mel-HO cell line; this cell line exhibited RKIP mRNA level similar to that observed on melanocytes but a reduced protein content that suggests the involvement of a post-transcriptional mechanism limiting translation. Of note, RKIP has been described as a target for several microRNAs able to regulate cellular protein level (112). Several authors have suggested that RKIP may not have a significant role in primary tumors but that instead, this protein could play an important role as a metastatic suppressor (115,134, 148,149, 150164). In this sense, and despite the described role for RKIP in the regulation of the MAPK/ERK pathway, RKIP has been implicated on the invasive behavior of malignant melanoma cells but not on their proliferative capability (51). Moreover, Schoentgen and Jonic (68) described the involvement of RKIP on the cortical actin organization during the membrane changes that happen during tumor cell migration. In agreement with previous studies, we confirmed the implication of RKIP on the motility of malignant melanoma cells as RKIP expression was inversely correlated with the migration capability of both, primary and metastatic melanoma cell lines. Nevertheless, modulation of the cellular RKIP level did not show an influence on the proliferative activity of melanoma cells. Therefore, considering the relevance of cellular motility on tumor metastasis, these results support a role for RKIP loss in melanoma dissemination. To define the cellular mechanisms regulated by RKIP that could explain the selective force favoring a decreased presence of this protein on melanoma when comparing with benign lesions (i.e., nevi), RKIP gene was silenced using lentivirus in primary melanocytes and RNA sequencing were performed to analyze the transcriptome changes derived from RKIP modulation. The transcriptome of melanocytes after RKIP silencing revealed a transcriptional misregulation in cancer gene signature. Among others, this signature included altered expression pattern of the oncogenes KIT, BCL3, MAF, MYC, MYCL, HOXA9, CDC25B, and PIM1. In our data, all of them showed a two to five-fold increase, supporting the role for RKIP like a tumor suppressor gene (170). Interestingly enough, downregulation of RKIP expression on melanocytes resulted in the alteration of cellular processes intimately linked to malignant transformation of cells, such as development and differentiation. Moreover, developmental pigmentation, a process specifically linked to the melanocytic 92 lineage, was also enriched. According to our RNA-seq data, RKIP would be a repressor of KIT and an inducer of TYRP1, MLANA, and PMEL gene expression, among others. TRYP1, MLANA, and PMEL are among the best-known transcriptional targets of the master melanogenic regulator microphthalmia-associated transcription factor (MITF) (171) and it would be of interest to further analyze the possible crosstalk among RKIP and MITF. In addition, data indicate that RKIP represents a brake for the EMT process, by regulating the expression of genes such us ZEB1, THY-1 and NTRK2. Scientific evidence demonstrates that in a heterogeneous tumor mass, those cells responsible for drugresistance, recurrence and metastasis contain characteristics of stem cells, that is, the ability to selfrenew and differentiate in any cell type of the tumor mass (172). In this work, after silencing of RKIP in HEMn-LP melanocytes, more than 70% of the differential expression genes belonging to development and differentiation were found to be putative targets of NANOG. NANOG has been identified as one of the crucial inducers of this stem cell-like state type (70) and is aberrantly expressed in many types of tumors (71-73). We observed that transient forced-increase of RKIP expression in metastatic melanoma cells led to the decrease of NANOG promoter activation pointing towards a functional relationship among RKIP and NANOG expression. In line with these results, Lee et al. (173) noticed a high amount of crosstalks between pathways regulated by RKIP and those under the control of main stemness transcription factors (i.e., OCT4, KLF4, SOX2, and NANOG) and proposed RKIP as a regulator of the differentiation state of cells. This hypothesis would be in agreement with the stronger RKIP expression found in differentiated melanocytes from nevi lesions when comparing with melanoma samples. In addition to the maintenance of the stemness, NANOG has been also implicated in the EMT (69,76) and by regulating the expression of ZEB1 and THY-1 among other genes (78). In fact, EMT and development of stemness properties are often closely related processes (174). As previously mentioned, these two genes are among those with deregulated expression in our RNA-seq study. ZEB1 is one of the major activators of the EMT program and increasing evidence places ZEB1 also as an important regulator of differentiation, proliferation, DNA damage response and cell survival (175). Interestingly, ZEB1 is among the transcription factors driving the early hybrid EMT state and hybrid EMT states (i.e., states with intermediate characteristics among fully epithelial and fully mesenchymal cells) have been linked to collective cells migration and highest metastatic potential (174). This result, together with the observed modulation of the cellular migration capacity driven by RKIP, are in line with the rapid RKIP diminution observed on malignant lesions, even at early stages, as well as the association among low Breslow index and presence of RKIP. In fact, capacity of a tumor to deepen on the skin requires the acquisition of characteristics as those blocked by RKIP. On the other hand, THY-1 is a protein implicated in the endothelium transvasation of melanoma cells during metastasis spreading (139). These results could be indicating the implication of RKIP loss in the plasticity required for the intraand extravasation during melanoma metastasis. In this context, we have also found that the expression of miR-21 was significantly lower on RKIP-overexpressing cells. miR-21 is a known target for NANOG (78) and an important inducer of EMT affecting migration and invasion capability (176-178) suggesting 93 a possible role for this onco-miRNA in melanoma malignancy (78,179). These results point towards the involvement of NANOG downstream RKIP in the regulation of gene expression related to malignant phenotype of melanoma cells. To summarize, we propose that RKIP could play a role in the maintenance of the differentiation state by negatively regulating NANOG gene expression although further research would be required for a better description of the underlying mechanism. Pirin dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression The cellular activity of Pirin has mainly been studied in terms of extracellular matrix (ECM) tumorigenicity (82,125,180) and thus, despite its broad distribution, there is little information regarding the role of Pirin in non-transformed cells and tissues (116,181). To better understand the role of Pirin in a melanocytic context, we firstly studied PIR/Pirin expression in primary melanocytes and melanoma cell lines by RT-qPCR and in Western Blots, demonstrating that primary melanocytes exhibit generally stronger and more homogenous Pirin expression than melanoma cell lines, which had significantly lower expression and more heterogeneity among the different cell lines analyzed. We studied proliferation and migration of metastatic melanoma cells in which Pirin was overexpressed and we found this upregulation did not modify migration but rather, it did induce a significant decrease in the proliferation rate of both the melanoma cell lines studied. In this context, controversial results have been found in different tumors. For example, in DLD1 colorectal cancer cells Pirin does not affect viability or migration (126), whereas knocking down Pirin in breast cancer cells was seen to significantly dampen in vitro proliferation and decrease xenograph tumor growth in mice (182). In melanoma, Pirin has been related to an inhibition of migration (122), and it has been proposed to be an inhibitor of melanocyte senescence (135) and a malignant biomarker (183). In accordance with the antiproliferative activity observed when Pirin is overexpressed in melanoma cells, the transcriptomic analysis following PIR-silencing in primary melanocytes here revealed an enrichment of genes involved in the negative regulation of cell proliferation, the G1/S transition and extracellular matrix organization and positive regulation of cell migration (Annex C: Table S6). Furthermore, deleterious mutations in the PIR gene were recently identified in breast cancer that could affect protein structure, stability and function (182). These results could explain the discrepancies found when studying different cancer or different tumor cell lines. On the other hand, melanoma heterogeneity was recently proposed to be due to the co-existence of different melanoma cell phenotypes and adaptive phenotype plasticity given that transcriptional reprogramming could drive melanoma progression (184). 94 Transcriptional reprogramming has been detected at different stages of melanoma, with enhanced mesenchymal traits in circulating melanoma cells and proliferative features in metastatic tumors (184). Hence, cells with different phenotypes may interact in a cooperative manner and contribute to successful metastatic progression (61,185). In recent years, attention is being paid to epigenetic regulation in melanoma (85), which led to the description of JARID1B as an epigenetic regulator implicated in the transcriptional reprogramming of several tumor cells and in tumor heterogeneity (186). Although JARID1B expressing melanoma cells represent only a small proportion of the cells in the primary and metastatic melanoma populations (187), the RNA-seq dataset and the transcription factor enrichment analysis found that JARID1B could target more than 100 of the DEGs identified. Furthermore, co-transfection experiments showed a decrease of JARID1B promoter activation after Pirin overexpression, pointing to a functional relationship between Pirin and JARID1B expression. In addition, we demonstrated that the overexpression of Pirin in both the metastatic melanoma cell lines studied led to a significant decrease in JARID1B gene expression, and that of its target genes E2F1 and c-MYC (81,141). These results may explain the antiproliferative effect of Pirin observed in melanoma cell lines. Indeed, in canine oral melanoma cell lines JARID1inhibitors drive anti-proliferative activity and overcame cisplatin resistance (188). From our data, we believe that in normal melanocytes Pirin expression could regulate the rate of proliferation through JARID1B and the E2F1 pathway, although the expression of other genes favors melanoma tumors acquiring an invasive phenotype through the expression of genes related to the epithelial-mesenchymal transition (69,76,78,189). Indeed, Pirin is functionally associated with several proteins involved in cytoskeleton reorganization, such as WASF2 and NCKAP1, which could explain the link between Pirin overexpression and malignant progression (122, 190). In this cell context, the delay in cell cycle progression produced by JARID1B downregulation could stimulate tumor cells to re-enter the cell cycle, increasing proliferation. Tumor cells with a slow-cycling phenotype may be metabolically active and highly aggressive, with increased potential to grow and metastasize (84,191,192). We propose that Pirin could play an important role in modulating the proliferative state of melanoma cells by regulating JARID1B gene expression. However, further research will be necessary to better understand the mechanisms underlying this phenomenon, which could shed light on useful therapeutic strategies for these tumors. Finally, if we approach all the results obtained from this thesis as a whole, we can think that both systems RKIP/NANOG and Pirin/JARID1B could be working together, so it would be facing two scenarios. On the one hand, benign melanocytes from nevus, in which the high expression RKIP maintenance the differentiation state blocking the stemness transcription factor NANOG. Additionally, high Pirin expression may be regulating the cell cycle through JARID1B and E2F1 expression. In the other hand, melanoma cells, in which the absent of RKIP expression produces a different panorama. 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