Metabolomic Analysis Points to Bioactive Lipid Species and Acireductone Dioxygenase 1 (ADI1) as Potential Therapeutic Targets in Poor Prognosis Endometrial Cancer

Metabolomic profiling analysis has the potential to highlight new molecules and cellular pathways that may serve as potential therapeutic targets for disease treatment. In this study, we used an LC-MS/MS platform to define, for the first time, the specific metabolomic signature of uterine serous carcinoma (SC), a relatively rare and aggressive variant of endometrial cancer (EC) responsible for 40% of all endometrial cancer-related deaths. A metabolomic analysis of 31 ECs (20 endometrial endometrioid carcinomas (EECs) and 11 SCs) was performed. Following multivariate statistical analysis, we identified 232 statistically different metabolites among the SC and EEC patient samples. Notably, most of the metabolites identified (89.2%) were lipid species and showed lower levels in SCs when compared to EECs. In addition to lipids, we also documented metabolites belonging to amino acids and purine nucleotides (such as 2-Oxo-4-methylthiobutanoic acid, synthesised by acireductone dioxygenase 1 (ADI1) enzyme), which showed higher levels in SCs. To further investigate the role of ADI1 in SC, we analysed the expression protein levels of ADI1 in 96 ECs (67 EECs and 29 SCs), proving that the levels of ADI1 were higher in SCs compared to EECs. We also found that ADI1 mRNA levels were higher in p53 abnormal ECs compared to p53 wild type tumours. Furthermore, elevated ADI1 mRNA levels showed a statistically significant negative correlation with overall survival and progression-free survival among EEC patients. Finally, we tested the ability of ADI1 to induce migration and invasion capabilities in EC cell lines. Altogether, these results suggest that ADI1 could be a potential therapeutic target in poor-prognosis SCs and other Ecs with abnormal p53 expression.This study was funded by the Instituto de Salud Carlos III (ISCIII) through projects PI20/00502, CP19/00025, CB16/12/00231, PI16/00692, PI18/00573, PI21/00672, CP17/00063 and PI18/00795; and by the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovación y Universidades, RTI2018-099200-BI00), co-funded by the European Regional Development Fund (ERDF) as part of the “A way to make Europe” programme and the European Social Fund (ESF) as part of the “Investing in Your Future” programme. This study was also supported by the “Xarxa de Bancs de Tumors de Catalunya” and sponsored by “Pla Director d’Oncologia de Catalunya (XBTC)”, “IRBLleida Biobank” (B.0000682) and “Plataforma Biobancos” PT20/00021. We also thank the Generalitat of Catalonia: Agency for Management of University and Research Grants (2017SGR1368 and 2017SGR696) and the “Asociación Española Contra el Cáncer” (AECC; Grupos Estables 2018 and LABAE19004LLOB). M.J. is a Serra Húnter Fellow. N.E. (MS19/00025) and D.L-N. (MS17/00063) are recipients of a Miguel Servet research scheme (co-funded by the ESF program “Investing in Your Future”). C. M-L. holds a predoctoral fellowship from the Generalitat de Catalunya (2020FI_B2 00099) and the predoctoral fellowship “Ajuts 2021 de Promoció de la Recerca en Salut-9a edició” from IRBLleida/Diputació de Lleida. IRBLleida is a CERCA Program/Generalitat of Catalonia

Estudio del papel de ARID1A en el desarrollo, progresión y microambiente tumoral del cáncer de endometrio

El càncer d'endometri (Endometrial carcinoma, CE) és el càncer ginecològic més freqüent i representa el sisè tipus de càncer més comú en les dones de països industrialitzats. A més a més, la seva incidència a nivell mundial està incrementant cada any. Tot i que la detecció primerenca del CE, generalment, te bon pronòstic, aproximadament un 20% dels casos són detectats en estadis avançats d'alt grau, els quals normalment, ja són invasius i/o metastàtics i presenten un mal pronòstic. Aquest motiu juntament amb l’escassetat de tractaments efectius son els principals motius de mort de les pacients amb CE. Per això, avui dia, continua sent un repte el desenvolupament de noves estratègies terapèutiques contra aquest tipus de tumors. Actualment se sap que els tumors no només estan compostos per cèl·lules tumorals, sinó que es tracta d'un complex microambient tumoral (tumor microenvironment, TME) conformat per una població cel·lular heterogènia, factors solubles i components de la matriu extracel·lular (Extracellular matrix, ECM), que posseeix evidents influències en comportaments agressius del tumor. Concretament, els fibroblasts associats al càncer (cancer associated fibroblasts, CAFs) són un dels components més importants d'aquest TME, realitzant una gran varietat de funcions pro-tumorigèniques, àmpliament implicades en el desenvolupament i la progressió tumoral. Les mutacions de pèrdua de funció d'ARID1A (domini d'interacció ric en AT de la proteïna A1) acompanyades d'una pèrdua d'expressió de la proteïna són sovint observades als CE. Tanmateix, els mecanismes moleculars lligats a aquest canvi genètic i involucrats en la iniciació, el manteniment i/o la progressió del CE, han estat escassament estudiats i compresos. En aquest context, aquesta tesi pretén analitzar i entendre la funció de la pèrdua d'expressió d'ARID1A al llarg del procés de la tumorigènesi endometrial i la seva implicació en l'activació del TME. En aquest estudi mitjançant l'ús d'una àmplia varietat de models in vitro i in vivo, com ara: línies cel·lulars, cultius primaris 3D i models murins xenògrafs o transgènics es demostra que la pèrdua d'ARID1A no és suficient per liderar la iniciació de la tumorigènesi endometrial. Tanmateix, la pèrdua d’ARID1A, en un context de CE prèviament establert, promou l'acceleració de la progressió tumoral i el desenvolupament de les metàstasis a través de la sobreexpressió i activació de la histona deacetilasa 6 (HDAC6). Aquests nivells incrementats de HDAC6 promouen la proliferació tumoral i la fugida apoptòtica, mitjançant l'evasió del punt de control del cicle cel·lular G2/M i de la via de senyalització del dany al DNA ATM/ATR. A més, també indueix la promoció de programes de la transició epiteli mesènquima (epithelial mesenchymal transition, EMT) mitjançant la hiperactivació de la via de les MAPK. En concordança amb aquests resultats, la inhibició d'HDAC6 pot restablir la vulnerabilitat apoptòtica i revertir els fenotips migratoris i invasius de les cèl·lules de CE amb alteracions d'ARID1A. Aquests resultats indiquen que la inhibició de l'activitat de l’HDAC6 és una potencial estratègia terapèutica per a les pacients que pateixen CE amb mutacions en ARID1A i diagnosticades en estadis avançats. A més, en aquesta tesi també es descriu que les cèl·lules de CE amb pèrdua d'ARID1A, a través de la secreció de CXCL16 i la conseqüent activació de l'eix CXCR6/CXCL16, tenen la capacitat de promoure l'activació del TME. Aquests canvis afecten tant a l'agressivitat de les cèl·lules tumorals veïnes amb expressió salvatge d'ARID1A, com en el reclutament i l'activació de les cèl·lules de l’estroma. Aquests resultats queden corroborats amb el fet que tant la inhibició de CXCL16, com la inhibició de CXCR6, poden revertir els efectes observats en les cèl·lules tumorals i estromals. Aquestes observacions situen a l'eix CXCR6/CXCL16 com una possible nova via terapèutica contra el TME pro-tumorogènic de les pacients amb tumors de CE con alteracions d'ARID1A.El cáncer de endometrio (Endometrial carcinoma, CE) es el cáncer ginecológico más frecuente, y representa el sexto tipo de cáncer más común de las mujeres en los países desarrollados. Además, su incidencia se está viendo incrementada cada año a nivel mundial. Pese a que la detección temprana del CE, generalmente, posee un buen pronóstico, aproximadamente un 20% de los casos son detectados en estados avanzados de alto grado, los cuales normalmente ya son invasivos y/o metastáticos, presentando un mal pronóstico. Este motivo, junto con la falta de tratamientos efectivos son los principales motivos de muerte en las pacientes con CE. Por lo que, hoy en día, sigue siendo un reto el desarrollo de nuevas estrategias terapéuticas contra este tipo de tumores. En la actualidad se sabe que los tumores no están compuestos solamente de las células tumorales, sino que se trata de un complejo microambiente tumoral (tumor microenvironment, TME) conformado por una población celular heterogénea, factores solubles y componentes de la matriz extracelular (Extracellular matrix, ECM), que posee evidentes influencias en comportamientos agresivos del tumor. Concretamente, los fibroblastos asociados al cáncer (cancer associated fibroblasts, CAFs) son uno de los mayores componentes de éste TME, realizando una gran variedad de funciones pro-tumorogénicas ampliamente implicadas en el desarrollo y progresión tumoral. Las mutaciones de pérdida de función de ARID1A (dominio de interacción rico en AT de la proteína A1) acompañadas de una pérdida de expresión de proteína, son frecuentemente observadas en los CE. Sin embargo, los mecanismos moleculares ligados a este cambio genético e involucrados en la iniciación, mantenimiento y/o progresión del CE, han permanecido escasamente estudiados y comprendidos. En este contexto, esta tesis pretende analizar y entender la función de la perdida de expresión de ARID1A a lo largo del proceso de la tumorogénesis endometrial y su implicación en la activación del TME. En este estudio, mediante el empleo de una amplia variedad de modelos in vitro e in vivo, tales como: líneas celulares, cultivos primarios 3D y modelos murinos xenógrafos o transgénicos, se demuestra que la pérdida de ARID1A no es suficiente per se para liderar la iniciación de la tumorogénesis endometrial. Sin embargo, la pérdida de ARID1A en un contexto de CE previamente establecido, promueve la aceleración de la progresión tumoral y el desarrollo de metástasis a través de la sobreexpresión y activación de la histona deacetilasa 6 (HDAC6). Estos niveles incrementados de HDAC6 promueven la proliferación tumoral y la fuga apoptótica, mediante la evasión del punto de control del ciclo celular G2/M y de la vía de señalización del daño al DNA ATM/ATR. Además, también induce la promoción de programas de transición epitelio mesénquima (epithelial mesenchymal transition, EMT), mediante la hiperactivación de la vía de las MAPK. En concordancia con estos resultados, la inhibición de HDAC6, puede reestablecer la vulnerabilidad apoptótica y revertir los fenotipos migratorios e invasivos de las células de CE con alteraciones de ARID1A. Estos resultados indican que la inhibición de la actividad de HDAC6 es una potencial estrategia terapéutica para las pacientes que sufren CE con mutaciones en ARID1A diagnosticadas en estadios avanzados. Además, en esta tesis también se describe que las células de CE con pérdida de ARID1A, por medio de la secreción de CXCL16 y la consecuente activación del eje CXCR6/CXCL16, poseen la capacidad de promover la activación del TME. Afectando tanto a la agresividad de las células tumorales vecinas con expresión salvaje de ARID1A, como al reclutamiento y activación de las células estromales. Estos resultados quedan corroborados con el hecho de que tanto la inhibición de CXCL16, como la inhibición de CXCR6, puede revertir los efectos observados en las células tumorales y estromales. Estas observaciones sitúan al eje CXCR6/CXCL16 como una novedosa vía terapéutica contra el TME pro-tumorogénico de las pacientes con tumores de CE con alteraciones de ARID1A.Endometrial carcinoma (EC) is the most common gynecological cancer, and represents the sixth most common type of cancer in women in developed countries. Moreover, its incidence is increasing every year worldwide. Although early detection of EC generally has a good prognosis, approximately 20% of cases are detected in advanced high-grade stages, which are usually already invasive and/or metastatic, presenting a poor prognosis. This, together with the lack of effective treatments, are the main reasons for death in patients with SC. Therefore, nowadays, the development of new therapeutic strategies against this type of tumors continues to be a challenge. It is now known that tumors are not only composed of tumor cells, but also of a complex tumor microenvironment (TME) made up of a heterogeneous cell population, soluble factors and components of the extracellular matrix (ECM), which has obvious influences on aggressive tumor behavior. Specifically, cancer associated fibroblasts (CAFs) are one of the major components of this TME, performing a variety of pro-tumorogenic functions widely implicated in tumor development and progression. Loss-of-function mutations of ARID1A (AT-rich interaction domain of A1 protein) accompanied by a loss of protein expression are frequently observed in ECs. However, the molecular mechanisms linked to this genetic change and involved in the initiation, maintenance and/or progression of EC have remained poorly studied and understood. In this context, this thesis aims to analyze and understand the function of the loss of ARID1A expression throughout the process of endometrial tumorogenesis and its implication in the activation of TME. In this study, using a wide variety of in vitro and in vivo models, such as: cell lines, 3D primary cultures and xenograft or transgenic murine models, it is demonstrated that loss of ARID1A is not sufficient per se to lead the initiation of endometrial tumorogenesis. However, loss of ARID1A in a previously established EC context promotes the acceleration of tumor progression and the development of metastases through overexpression and activation of histone deacetylase 6 (HDAC6). These increased levels of HDAC6 promote tumor proliferation and apoptotic escape through evasion of the G2/M cell cycle checkpoint and the ATM/ATR DNA damage signaling pathway. In addition, it also induces the promotion of epithelial mesenchymal transition (EMT) programs through hyperactivation of the MAPK pathway. Consistent with these results, HDAC6 inhibition can restore apoptotic vulnerability and reverse the migratory and invasive phenotypes of EC cells with ARID1A alterations. These results indicate that inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from EC with ARID1A mutations diagnosed at advanced stages. Furthermore, this thesis also describes that EC cells with loss of ARID1A, through secretion of CXCL16 and consequent activation of the CXCR6/CXCL16 axis, possess the ability to promote the activation of TME. Affecting both the aggressiveness of neighboring tumor cells with wild-type ARID1A expression and the recruitment and activation of stromal cells. These results are corroborated by the fact that both CXCL16 inhibition and CXCR6 inhibition can reverse the effects observed in tumor and stromal cells. These observations position the CXCR6/CXCL16 axis as a novel therapeutic avenue against pro-tumorogenic EMT in EC tumor patients with ARID1A alterations

Small-Molecule Inhibitors (SMIs) as an Effective Therapeutic Strategy for Endometrial Cancer

Endometrial cancer (EC) is the sixth most common cancer in women. A continued number of low-risk EC patients at diagnosis, as well as patients diagnosed with advanced-stage disease, will experience an aggressive disease. Unfortunately, those patients will present recurrence or overt dissemination. Systemic cytotoxic chemotherapy treatment on advanced, recurrent, or metastatic EC patients has shown poor results, with median survival rates of less than one year, and median progression-free survival rates of four months. Therefore, the search for innovative and alternative drugs or the development of combinatorial therapies involving new targeted drugs and standard regimens is imperative. Over the last few decades, some small-molecule inhibitors have been introduced in the clinics for cancer treatment, but only a few have been approved by the Food and Drug Administration (FDA) for EC treatment. In the present review, we present the current state and future prospects of small-molecule inhibitors on EC treatment, both alone and in combination

Small-Molecule Inhibitors (SMIs) as an Effective Therapeutic Strategy for Endometrial Cancer

Patients diagnosed with endometrial cancer (EC), the most common gynaecological malignancy in women worldwide, cope with a disease associated with poor prognosis and limited treatment options after first-line therapy when it reaches an advanced or metastatic stage. Lately, small-molecule inhibitors have emerged as an alternative targeted therapy, renewing hope in the fight against this disease. The aim of this review is to shed light into the current state and future prospects of small-molecule inhibitors on EC treatment by summarizing the extensive number of clinical trials that have been performed during the last years, and to provide a comprehensive up-to-date document with the most remarkable results. Despite the great effort researchers are making to improve the molecular characterization of tumours, to unravel the underlying mechanism of EC progression, and to increase the efficacy of targeted therapy, we might say that there is still a long way to pave to efficiently treat EC patients. Endometrial cancer (EC) is the sixth most common cancer in women. A continued number of low-risk EC patients at diagnosis, as well as patients diagnosed with advanced-stage disease, will experience an aggressive disease. Unfortunately, those patients will present recurrence or overt dissemination. Systemic cytotoxic chemotherapy treatment on advanced, recurrent, or metastatic EC patients has shown poor results, with median survival rates of less than one year, and median progression-free survival rates of four months. Therefore, the search for innovative and alternative drugs or the development of combinatorial therapies involving new targeted drugs and standard regimens is imperative. Over the last few decades, some small-molecule inhibitors have been introduced in the clinics for cancer treatment, but only a few have been approved by the Food and Drug Administration (FDA) for EC treatment. In the present review, we present the current state and future prospects of small-molecule inhibitors on EC treatment, both alone and in combinatio

ARID1A-deficient cells require HDAC6 for progression of endometrial carcinoma

AT-rich interactive domain-containing protein 1A (ARID1A) loss-of-function mutation accompanied by a loss of ARID1A protein expression is frequently observed in endometrial carcinomas. However, the molecular mechanisms linking these genetic changes to the altered pathways regulating tumour initiation, maintenance and/or progression remain poorly understood. Thus, the main aim of this study was to analyse the role of ARID1A loss of function in endometrial tumorigenesis. Here, using different endometrial in vitro and in vivo models, such as tumoral cell lines, 3D primary cultures and metastatic or genetically modified mouse models, we show that altered expression of ARID1A is not enough to initiate endometrial tumorigenesis. However, in an established endometrial cancer context, ARID1A loss of function accelerates tumoral progression and metastasis through the disruption of the G2/M cell cycle checkpoint and ATM/ATR-mediated DNA damage checkpoints, increases epithelial cell proliferation rates and induces epithelial mesenchymal transition through the activation of histone deacetylase 6 (HDAC6). Next, we demonstrated that the inhibition of HDAC6 function, using the HDAC6-specific inhibitor ACY1215 or by transfection with HDAC6 short hairpin RNA (shRNA), can reverse the migratory and invasive phenotype of ARID1A-knockdown cells. Further, we also show that inhibition of HDAC6 activity causes an apoptotic vulnerability to etoposide treatments in ARID1A-deficient cells. In summary, the findings exposed in this work indicate that the inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from ARID1A-mutant endometrial cancer diagnosed in advanced stages.This work has been funded by the Instituto de Salud Carlos III (ISCIII) through the projects PI20/00502, CP19/00025, CB16/12/00231, PI16/00692, PI18/00573 CP17/00063, PI18/00795 and PI21/00672, by the Ministry of Economy and Competitiveness (MINECO) through the project RTC-2017-6261-1 (Cofounded by European Regional Development Fund (ERDF) ‘a way to make Europe’ and ESF ‘Investing in your future’) and the Fundacion DEXEUS Salud de la Mujer PV100-41. We also thank the Grups consolidats de la Generalitat de Catalunya (2017SGR1368) and the Asociación Española Contra el Cáncer (AECC; Grupos Estables 2018: GCTRA1804MATI and LABAE19004LLOB). NE (MS19/00025) and DL-N (MS17/00063) are recipients of a Miguel Servet research scheme (Cofounded by the European Social Fund (ESF), ‘investing in your future’). CM-L holds a predoctoral fellowship from Generalitat de Catalunya (2020FI_B2 00099) and a predoctoral fellowship ‘Ajuts 2021 de Promoció de la Recerca en Salut-9ª edició’ from IRBLleida/Diputació de Lleida. MA-V holds a fellowship from CIBERONC ‘Ayudas para contratos de inciación a la investigación’. We also thank the CERCA programme/Generalitat de Catalunya for institutional support

RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing

Abstract RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation