Programa de D octorado en Biociencias Moleculares, Facultad Medicina . Universidad Autónoma de Madrid ROLE OF EMILIN1 DURING MELANOMA PROGRESSION AND LYMPH NODE PRE - METASTATIC NICHE FORMATION Ana Isabel Amor López Madrid, 2019 Role of EMILIN1 during melanoma progression and lymph node pre - metastatic niche formation Doctoral Thesis submitted to the Universidad Autónoma de Madrid for the degree of Doctor of Philosophy by M.Sci. in Molecular Biomedicine, Ana Isabel Amor López Univers idad Autónoma de Madrid, Department of Biochemistry, Faculty of Medicine Thesis Director Héctor Peinado Selgas Microenvironment and Metastasis Group, Spanish National Cancer Research Centre (CNIO) Madrid, 2019 Dr. Héctor Peinado Selgas, head of Microenvironment and Metastasis Group in the Spanish National Cancer Research Centre (CNIO) CERTIFIES: That Ms. Ana Isabel Amor López, Master in Neuroimaging from King´s College of London has completed her Doctoral Thesis “Role of EMILIN1 during melanoma progression and lymph node pre - metastatic niche formation” and meets the necessary requirements to obtain the PhD Degree in Molecular Biosciences. To this purpose, she will defend her Doctoral Thesis at the Universidad Autónoma de Madrid. The Doct oral Thesis has been carried out under my direction and hereby I authorize it to be defended to the appropriate Thesis Tribunal. I hereby issue this certificate in Madrid, on 23 th of October 2019. Héctor Peinado Selgas PhD Thesis Director This Thesis, submitted for degree of Doctor of Philosophy at the Universidad Autónoma de Madrid, has been completed in the M icroenvironment and Metastasis Laboratory at the Spanish National Cancer Research Centre (CNIO), under the supervision of Dr. Héctor Peinado Selgas. This work was supported by the following grants and fellowships: Beca Excelencia Severo Ochoa PhD Fellowship, 20 14 call - Ana Isabel Amor López. Ministerio de Ciencia, Innovación y Universidades. Proyecto GRUPOS COORDINADOS ESTABLES DE INVESTIGACIÓN, Asociación Española contra el cáncer (AECC). SAF2014 54541 - R. MINECO A mi familia SUMMARY 1 2 Summary __________________________________________________________________________ ____ It has been described that melanoma - derived exosomes have a role in lymph node metastasis reinforcing lymphangiogenesis and extracellular matrix remodeling. Several studies have demonstrated that melanoma - derived exosomes home in sentinel lymph nodes promoting gene expression changes that favor metastasis. Recent data from our laboratory support a role for melanoma - derived exosomes in establishing the lymph node pre - metastatic niche formation due to specific cargo . In this thesis, we postulated that lymph node microenvironment acts as a selective pressure selecting specific phenotypes for metastasis. As such, we wanted to analyze if lymph node metastatic melanoma cells present a specific signature that may favor th eir survival in lymph nodes. In our analysis , we have found a specific signature of genes over - expressed and proteins hyper - secreted in exosomes from a mouse melanoma lymph node metastatic model. Out of these candidates, EMILIN1 was selected due to its rel evance in lymphatic vessel functionality. We have found that EMILIN1 impacts negatively on cell proliferation and migration of melanoma cells. Overall, our data support that EMILIN1 acts as a tumour suppressor - like protein both intrinsically and extrinsically and its function is inactivated by its degradation and secretion in exosomes. Importantly, our in vivo studies demonstrate that its overexpression reduced primary tumour growth and metastasis in mouse melanoma models. Analysis in human melanoma showed that it s expression is maintained along melanoma progression but cells expressing high levels of EMILIN1 are reduced in metastatic lesions. Overall, our analysis suggests a novel mechanism involved in the inactivat ion of EMILIN1 in melanoma f avouring tumour progression. 3 4 RESUMEN 5 6 Resumen _______________________________________________ _______________________________ Se ha descrito que los exosomas derivados del melanoma tienen un papel en la metástasis a los ganglios linfáticos , reforzando tanto la linfangiogénesis como la remodelación de la matriz extracelular. Varios estudios han demostrado que los exosomas de melanoma anidan en los ganglios linfáticos centinela s , promoviendo cambios en la expresión génica favorec iendo la metástasis de l melanoma. D atos recientes obtenid os en nuestro laboratorio , respaldan que los exosomas derivados de células de melanoma participan activamente en la formación de l nicho pre - metastásico en los ganglios linfáticos debido a una firma molecular específic a . En esta tesis, postulamos que el mic roambiente de los ganglios linfáticos actúa como una presión selectiva , que selecciona fenotipos específicos que están favorecidos para metastatizar eficientemente . Por ello, en esta tesis hemos analizado si células de melanoma murino derivadas de metástasis en los ganglios linfáticos , presentan una firma molecular específica que favorece su metástasis . En nuestro análisis hemos seleccionado genes sobre - expresados y proteí nas hiper - secretad a s en un modelo de rivado de metástasis a ganglios linfáticos. De estos candidatos, se seleccionó EMILIN1 debido a su relevancia en la funcionalidad de los vasos linfáticos. En nuestros estudios, hemos podido ver que EMILIN1 reduce la proliferación celular y la migración de células de melanoma. En resumen , nuestros datos respaldan que EMILIN1 actúa como una proteína con propiedades similares a los supresor es tumorales , tanto intrínseca como extrínsecamente, y su función se inactiva por su degradación y secreción en los exosom as. Es importante destacar que nuestros estudios in vivo demuestran que su sobre - expresión conduce tanto a un a reducción del tamaño tumoral como de la metástasis. El análisis inmunohistoquímico llevado a cabo en muestras de pacientes de melanoma , mostró que su expresión se mantiene a lo largo de la progresión del melanoma, pero las células que expresan altos niveles de EMILIN1 se reducen en las lesiones metastásicas. En general, nuestro estudio demuestra un nuevo mecanismo implicado en la inactiva ción de EMILIN1 en el melanoma que favorece la progresión tumoral . 7 8 INDEX 9 10 Index SUMMARY ................................ ................................ ................................ ................................ ..... 1 RESUMEN ................................ ................................ ................................ ................................ ...... 5 INDEX ................................ ................................ ................................ ................................ ............. 9 ABBREVIATIONS ................................ ................................ ................................ ......................... 15 1. INTRODUCTION ................................ ................................ ................................ ...................... 21 1.1. The term “metastasis” ................................ ................................ ................................ ............ 23 1.1.1. Main steps involved in metastasis ................................ ................................ .................... 23 1.2. Pre - metastatic niche concept ................................ ................................ ................................ 25 1.2.1. Pre - metastatic niche formation ................................ ................................ ........................ 26 1.3. Involvement of extracellular vesicles in metastasis ................................ ................................ 27 1.3.1. Exosomes in PMN initiation and evolution ................................ ................................ ....... 29 1.3.2. Exosomes in lymph node metastasis ................................ ................................ ............... 29 1.4. Melanoma ................................ ................................ ................................ .............................. 31 1.4.1. From melanocytes to melanoma ................................ ................................ ...................... 31 1.4.2. Melanoma subtypes ................................ ................................ ................................ ......... 32 1.4.3. Metastasis in melanoma ................................ ................................ ................................ .. 32 1.5. Extracellular matrix remodelling during metastasis ................................ ................................ 33 1.5.1. Molecular composition of ECM and its role in tissue homeostasis ................................ ... 33 1.5.2. ECM changes in PMN ................................ ................................ ................................ ..... 34 1.5.2.1. ECM changes in Lymph nodes ................................ ................................ .................. 35 1.6. EMILIN/Multimerin Family ................................ ................................ ................................ ...... 36 1.6.1. EMILIN1 ................................ ................................ ................................ .......................... 37 1.6.1.1. Role of EMILIN1 gC1q in physiology ................................ ................................ ......... 37 1.6.1.2. Role of EMILIN1 in pathological conditions ................................ ............................... 38 2. OBJETIVES ...............................