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

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-201

Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples

Altres ajuts: The present work has been also funded by the "Fonds National de la Recherche du Luxembourg" (FNR) via the PEARL-CPIL program to BD, and an AFR grant to AL (PDR 2013-2, Project Reference 6835664). The authors would like to acknowledge the work of all clinicians that have participated in the recruitment of clinical samples. We thank the patients for their willingness to participate in the study.About 30% of endometrial cancer (EC) patients are diagnosed at an advanced stage of the disease, which is associated with a drastic decrease in the 5-year survival rate. The identification of biomarkers in uterine aspirate samples, which are collected by a minimally invasive procedure, would improve early diagnosis of EC. We present a sequential workflow to select from a list of potential EC biomarkers, those which are the most promising to enter a validation study. After the elimination of confounding contributions by residual blood proteins, 52 potential biomarkers were analyzed in uterine aspirates from 20 EC patients and 18 non-EC controls by a high-resolution accurate mass spectrometer operated in parallel reaction monitoring mode. The differential abundance of 26 biomarkers was observed, and among them ten proteins showed a high sensitivity and specificity (AUC > 0.9). The study demonstrates that uterine aspirates are valuable samples for EC protein biomarkers screening. It also illustrates the importance of a biomarker verification phase to fill the gap between discovery and validation studies and highlights the benefits of high resolution mass spectrometry for this purpose. The proteins verified in this study have an increased likelihood to become a clinical assay after a subsequent validation phase

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets gamma-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism offat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.Peer reviewe

Generation and integrated analysis of advanced patient-derived orthoxenograft models (PDOX) for the rational assessment of targeted therapies in endometrial cancer

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments

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

El carcinoma endometrial (CE) es el tumor infiltrante más común del tracto genital femenino y el cuarto en la mujer en países desarrollados, siendo la tipología endometrioide (CEE) la más frecuente. El 80% de las pacientes son diagnosticadas cuando el tumor sigue localizado en el útero, presentado una supervivencia a los 10 años que alcanza el 80%. Sin embargo, un número pequeño 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 más avanzada y presentan invasión miometrial, siendo este proceso uno de los factores de riesgo más relevantes y significativamente asociado a la recurrencia. La progresión del cáncer combina múltiples procesos que implican a moléculas de adhesión celular. Su expresión aberrante se relaciona con: pérdida de adhesión célula-célula, disgregación de las células tumorales de la lámina propia y adquisición de un fenotipo invasivo y migratorio. ALCAM, miembro de la superfamilia de las inmunoglobulinas, participa en interacciones homotípicas y heterotípicas entre células adyacentes. Su expresión se ha relacionado con la progresión de distintos tipos de cáncer, aunque su función sigue sin conocerse. Esta tesis se centra en dos objetivos: (i) el estudio de ALCAM como marcador de recurrencia en CEE y su función en la progresión tumoral; (ii) la caracterización de los mecanismos moleculares asociados a ALCAM en el frente superficial e invasivo del tumor y en el proceso de infiltración miometrial. Un estudio multicéntrico retrospectivo en 174 tumores primarios de CEE evidenció que ALCAM es un marcador independiente de recurrencia en los estadios tempranos de la enfermedad. Además, el silenciamiento de ALCAM in vitro e in vivo mostró una disminución en la migración celular, la invasión y una reducción significativa del tamaño tumoral primario así como de las metástasis loco-regionales. Análisis de expresión génica evidenciaron las funciones más alteradas asociadas a la inhibición de ALCAM: la motilidad, la invasión, y las funciones de ensamblaje y organización celular. FLNB, TXNRD1 y LAMC2, se validaron como genes efectores implicados en las funciones mediadas por ALCAM. Por otro lado, se estudió la expresión de ALCAM en el frente superficial e invasivo del tumor en 116 tumores primarios de CEE, así como su relación con marcadores epiteliales y mesenquimales relevantes en el CE. Se utilizó un anticuerpo que reconocía la región extracelular de ALCAM, permitiendo la detección de la proteína no-clivada en la superficie celular. Se observó que mientras en el área superficial, ALCAM-extracelular correlaciona con el complejo E-cadherina/β-catenina, en el invasivo, esta correlación se establece con los marcadores mesenquimales COX-2, MMP-9 y ETV5. La correlación negativa significativa entre ALCAM y MMP-9 se observa en tres cohortes específicas, tumores moderadamente-pobremente diferenciados, tumores con invasión miometrial >50% y tumores ETV5-positivos, todos ellos asociados con un peor pronóstico. Además, en el frente invasivo, la negatividad de ALCAM-extracelular resultó un marcador pronóstico independiente de invasión miometrial. Junto a estos hallazgos a nivel tisular, se confirma en aspirados uterinos el papel de ALCAM-soluble como marcador de infiltración miometrial y una correlación 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 proteína. Los modelos in vitro e in vivo demostraron, mediante la recuperación de la expresión de la proteína entera, su participación en la adhesión y la migración celular colectiva, sugiriendo que la invasión tumoral requiere la existencia de un “switch” dinámico y adaptativo entre la expresión 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/β-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

Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro

Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determinant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited to genes involved in the maintenance of the identity of neural progenitors. We combined analysis of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression are associated to shorter overall survival of neuroblastoma patients, even when those patients with the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets gamma-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism offat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.Peer reviewe