Antitumor Effects of Ral-GTPases Downregulation in Glioblastoma.

Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.This work was funded by the Catalan Government—AGAUR (2017 SGR-569), Ministerio de Ciencia e Innovaciön (PID2019-104859GB-I00; RTI2018-094739-B-I00; PID2019-104734RB-I00), and by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC). T Cemeli (FPU13/06590), M.Guasch (FPU17/00229), R. Navaridas (FPU18/04480), and M. Ribes (TALENT-IRBLleida) were supported by a pre-doctoral fellowship from Ministerio de Educación, Cultura y Deportes, and from Diputació de Lleida

Aplicación de los Péptidos de Penetración Celular y de la herramienta de edición génica CRISPR/Cas9 en la modelización in vivo de carcinomas uterinos

l càncer d'endometri (CE) és el càncer ginecològic més comú als països desenvolupats i la seva incidència està augmentant a tot el món. Els factors que han contribuït a aquesta tendència són una població més envellida i la disminució de la pràctica d’histerectomies benignes. Si bé és cert que la majoria dels càncers d'endometri es curen mitjançant la histerectomia, les pacients que tenen una malaltia avançada presenten un pronòstic dolent. La necessitat del tractament del càncer d’endometri continua, avui en dia, sent un repte. També ho és la necessitat d’una comprensió més profunda de la diversitat genètica d’aquesta malaltia, així com la dels impulsors que en desenvolupen els diferents estats patogènics. Tot això ha permès el desenvolupament i l’estudi de diversos models murins per tal d'entendre les complexitats d'aquest tipus de tumor. A més, els avenços en el camp de la biologia molecular han possibilitat el desenvolupament de noves estratègies quimioterapèutiques dirigides. Pten i p53, que tenen funció supressora de tumors, són dos dels gens amb major nombre d'alteracions genètiques en CE. Així, l'objectiu principal d'aquest treball és aconseguir la supressió específica i única de la funció d’aquests gens en les cèl·lules epitelials endometrials. En aquest estudi s'han establert diversos models per al futur estudi de la implicació d'ambdues mutacions en l'inici i la progressió de tumors ginecològics, principalment del CE. Els primers models que es descriuen s'han desenvolupat mitjançant el sistema Cre/loxP, que permet la inducció temporal de l'ablació gènica amb tamoxifè utilitzant la línia Cre:ERT com a font de recombinasa. En el segon model s’ha utilitzat la recombinasa fusionada a un pèptid de penetració cel·lular (CPP), que genera l'ablació gènica in situ a les cèl·lules en les quals s'ha administrat. Finalment, hem creat un mètode per generar mutacions a demanda en cèl·lules epitelials endometrials mitjançant el sistema d'edició gènica CRISPR/Cas9, a través de l'administració in vivo de ribonulceoproteïna (RNP) a l'úter del ratolí amb la tècnica de l’electroporació. Emprant aquest tercer model no només som capaços de generar mutacions en Pten i p53, sinó que també podem generar mutacions en altres gens implicats en la carcinogènesi endometrial.El cáncer de endometrio (CE) es el cáncer ginecológico más común en los países desarrollados y su incidencia está aumentando en todo el mundo. Los factores que han contribuido a esta tendencia son una población más envejecida y la disminución de la práctica de histerectomías benignas. Si bien es cierto que la mayoría de los cánceres de endometrio se curan mediante la histerectomía, las pacientes que padecen una enfermedad avanzada presentan un pronóstico malo. La necesidad del tratamiento del cáncer de endometrio sigue, hoy en día, siendo un reto. También lo es la necesidad de una comprensión más profunda de la diversidad genética de esta enfermedad, así como la de los impulsores que desarrollan sus diferentes estados patogénicos. Todo esto ha permitido el desarrollo y el estudio de varios modelos murinos para entender las complejidades de este tipo de tumor. Además, los avances en el campo de la biología molecular han posibilitado el desarrollo de nuevas estrategias quimioterapéuticas dirigidas. Pten y p53, que tienen función supresora de tumores, son dos de los genes con mayor número de alteraciones genéticas en CE. Así, el principal objetivo de este trabajo es conseguir la supresión específica y única de la función de estos genes en las células epiteliales endometriales. En este estudio se han establecido varios modelos para el futuro estudio de la implicación de ambas mutaciones en el inicio y progresión de tumores ginecológicos, principalmente del CE. Los primeros modelos que se describen se han desarrollado mediante el sistema Cre/loxP, que permite la inducción temporal de la ablación génica con tamoxifeno utilizando la línea Cre:ERT como fuente de recombinasa. En el segundo modelo se ha utilizado la recombinasa fusionada a un péptido de penetración celular (CPP), que genera la ablación génica in situ en las células en las que se ha administrado. Por último, hemos creado un método para generar mutaciones a demanda en células epiteliales endometriales mediante el sistema de edición génica CRISPR/Cas9, a través de la administración in vivo de ribonulceoproteína (RNP) en el útero del ratón con la técnica de la electroporación. Empleando este tercer modelo no sólo somos capaces de generar mutaciones en Pten y p53, sino que también podemos generar mutaciones en otros genes implicados en la carcinogénesis endometrial.Endometrial cancer (EC) is the most common gynecologic cancer in developed countries and its incidence is increasing worldwide. Factors that have contributed to this trend are an aging population and the decline in the practice of benign hysterectomies. While it is true that most endometrial cancers are treated by hysterectomy, patients with advanced disease have a poor prognosis. The need for treatment of endometrial cancer remains a challenge today. So is the need for a deeper understanding of the genetic diversity of this disease, as well as that of the drivers that develop its different pathogenic states. All this has allowed the development and study of several murine models to understand the complexities of this type of tumor. In addition, advances in the field of molecular biology have enabled the development of new-targeted chemotherapeutic strategies. Pten and p53, which have tumor suppressor functions, are two of the genes with the highest number of genetic alterations in SC. Thus, the main objective of this work is to achieve specific and unique suppression of the function of these genes in endometrial epithelial cells. In this study, several models have been established for the future study of the involvement of both mutations in the initiation and progression of gynecological tumors, mainly EC. The first models described have been developed using the Cre/loxP system, which allows the temporary induction of gene ablation with tamoxifen using the Cre:ERT line as a source of recombinase. In the second model, recombinase fused to a cell penetrating peptide (CPP) has been used, which generates in situ gene ablation in the cells in which it has been administered. Finally, we have created a method to generate on-demand mutations in endometrial epithelial cells using the CRISPR/Cas9 gene editing system through in vivo administration of ribonulceoprotein (RNP) in the mouse uterus using the electroporation technique. Using this third model, we are not only able to generate mutations in Pten and p53, but we can also generate mutations in other genes involved in endometrial carcinogenesis

Lack of extracellular matrix switches TGF-β induced apoptosis of endometrial cells to epithelial to mesenchymal transition

The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-β in normal versus tumoral cells.Supported by grants and PID2019-104734RB-I00 from Spanish Ministerio de Ciencia, Innovación y Universi-dades, Grupos estables de la Asociación Española Contra el Cancer, AECC

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