The role of activated leukocyte cell adhesion molecule (ALCAM) in endometrial cancer progression and dissemination

El carcinoma endometrial (CE) es el tumor infiltrante mas comun del tracto genital femenino y el cuarto en la mujer en paises desarrollados, siendo la tipologia endometrioide (CEE) la mas frecuente. El 80% de las pacientes son diagnosticadas cuando el tumor sigue localizado en el utero, presentado una supervivencia a los 10 anos que alcanza el 80%. Sin embargo, un numero pequeno aunque relevante de estas pacientes recurren. Por ello, es de vital importancia encontrar marcadores para predecir la recidiva en esas pacientes para las cuales no se conoce un factor de riesgo suficientemente robusto. El 20% restante de las pacientes son diagnosticadas en una etapa mas avanzada y presentan invasion miometrial, siendo este proceso uno de los factores de riesgo mas relevantes y significativamente asociado a la recurrencia. La progresion del cancer combina multiples procesos que implican a moleculas de adhesion celular. Su expresion aberrante se relaciona con: perdida de adhesion celula-celula, disgregacion de las celulas tumorales de la lamina propia y adquisicion de un fenotipo invasivo y migratorio. ALCAM, miembro de la superfamilia de las inmunoglobulinas, participa en interacciones homotipicas y heterotipicas entre celulas adyacentes. Su expresion se ha relacionado con la progresion de distintos tipos de cancer, aunque su funcion sigue sin conocerse. Esta tesis se centra en dos objetivos: (i) el estudio de ALCAM como marcador de recurrencia en CEE y su funcion en la progresion tumoral; (ii) la caracterizacion de los mecanismos moleculares asociados a ALCAM en el frente superficial e invasivo del tumor y en el proceso de infiltracion miometrial. Un estudio multicentrico retrospectivo en 174 tumores primarios de CEE evidencio que ALCAM es un marcador independiente de recurrencia en los estadios tempranos de la enfermedad. Ademas, el silenciamiento de ALCAM in vitro e in vivo mostro una disminucion en la migracion celular, la invasion y una reduccion significativa del tamano tumoral primario asi como de las metastasis loco-regionales. Analisis de expresion genica evidenciaron las funciones mas alteradas asociadas a la inhibicion de ALCAM: la motilidad, la invasion, y las funciones de ensamblaje y organizacion celular. FLNB, TXNRD1 y LAMC2, se validaron como genes efectores implicados en las funciones mediadas por ALCAM. Por otro lado, se estudio la expresion de ALCAM en el frente superficial e invasivo del tumor en 116 tumores primarios de CEE, asi como su relacion con marcadores epiteliales y mesenquimales relevantes en el CE. Se utilizo un anticuerpo que reconocia la region extracelular de ALCAM, permitiendo la deteccion de la proteina no-clivada en la superficie celular. Se observo que mientras en el area superficial, ALCAM-extracelular correlaciona con el complejo E-cadherina/?-catenina, en el invasivo, esta correlacion se establece con los marcadores mesenquimales COX-2, MMP-9 y ETV5. La correlacion negativa significativa entre ALCAM y MMP-9 se observa en tres cohortes especificas, tumores moderadamente-pobremente diferenciados, tumores con invasion miometrial >50% y tumores ETV5-positivos, todos ellos asociados con un peor pronostico. Ademas, en el frente invasivo, la negatividad de ALCAM-extracelular resulto un marcador pronostico independiente de invasion miometrial. Junto a estos hallazgos a nivel tisular, se confirma en aspirados uterinos el papel de ALCAM-soluble como marcador de infiltracion miometrial y una correlacion significativa positiva de las expresiones de MMP-9 y ALCAM-soluble para estos tumores no bien diferenciados, indicando la existencia de un clivaje de la proteina. Los modelos in vitro e in vivo demostraron, mediante la recuperacion de la expresion de la proteina entera, su participacion en la adhesion y la migracion celular colectiva, sugiriendo que la invasion tumoral requiere la existencia de un “switch” dinamico y adaptativo entre la expresion de ALCAM en la superficie celular y su clivaje por MMP-9. Endometrial carcinoma (EC) is the most common infiltrative tumour of the female genital tract and the fourth most common women cancer in developed western countries. Around 80% of patients are diagnosed when the tumour is still confined to the uterus, which is usually associated with a good prognosis. In fact, the early stages of endometrioid endometrial cancer (EEC) present a 10-year survival of 80%. However, a small but consistent number of these patients relapse. Therefore, it is of vital importance to find markers to predict recurrence for those patients for whom a sufficiently robust risk factor is still not known. The other 20% of patients, diagnosed at a more advanced stage of the disease, present myometrial invasion that is one of the most relevant risk factors, being strongly correlated with an increase in the rate of recurrence. Cancer progression combines multiple processes, which involve cell adhesion molecules. Aberrant expression of these molecules has been related to loss of cell-cell adhesion, disaggregation of tumour cells from the lamina propria, and acquisition of an invasive and migratory phenotype. ALCAM is a member of the immunoglobulin superfamily, which participates in homotypic and heterotypic interactions between adjacent cells. In the last years, ALCAM expression has been related to the tumorigenesis of many cancers, but its function still remain unclear. Under this scenario, this thesis has focused on two specific objectives: (i) The study of ALCAM as a marker of recurrence in EEC and its function in tumour progression; (ii) The characterization of the molecular mechanisms associated with ALCAM in the superficial and the invasive front of the tumour and its relation to myometrial infiltration. A retrospective multicentre study in 174 EEC primary tumours evidenced that ALCAM is an independent marker of recurrence in early stage EEC. Moreover, ALCAM knockdown in vitro and in vivo experiments showed a decreased in cell migration, cell invasion and a reduced primary tumour size and metastatic local spread. Microarray gene expression analysis of Hec1A ALCAM-depleted cells demonstrated that motility, invasiveness, cellular assembly and organization were the most associated deregulated functions. FLNB, TXNRD1 and LAMC2, were validated as downstream effector genes involved in ALCAM-mediated cell migration. In addition, we focused on the role of ALCAM in two tumour areas (superficial and invasive front) in 116 EEC primary tumours and its correlation with a set of important epithelial and mesenchymal EC markers. For that, we used an antibody that specifically recognised its extracellular region, allowing us to detect the presence of full-ALCAM (non-cleaved protein) at the cell surface. While at the superficial tumour extracellular-ALCAM was correlated with the epithelial E-Cadherin/?-catenin complex, at the invasive tumour ALCAM correlated with the mesenchymal markers COX-2, MMP-9 and ETV5 mesenchymal markers. Interestingly, the inverse correlation between ALCAM and MMP-9 was only significant in the moderately-poorly differentiated tumours, in patients presenting a myometrial invasion >50% and in the ETV5-positive tumours cohorts, all the scenarios related to poor prognosis. Moreover, at the invasive front extracellular ALCAM-negativity was an independent prognostic marker of myometrial invasion. Together with these findings at tissue level, we confirmed that in the moderately-poorly differentiated patients, soluble ALCAM detected in uterine aspirates was significantly increased in tumours presenting myometrial invasion and positively correlated to MMP-9 expression, suggesting that ALCAM decrease at the invasive front of primary tumours is caused by protein shedding. In vitro and in vivo models evidenced that ALCAM participates in cell-cell adhesion and collective cell migration, and suggest that to invade in the frontier between tissues-restricted carcinoma and disseminated tumour cells, a dynamic and adaptive switch between ALCAM expression at the cell surface and ALCAM cleavage by MMP-9 might take place.