From circulating tumour cells to metastases: The role of Cyclin D1 and characterisation of new biomarkers

La metàstasi és un procés molt complex que està regulat per un elevat nombre de variables i senyalitzacions, entre altres la composició del microambient tumoral. Els objectius d'aquesta tesi són estudiar el paper de la via Ciclina D1 (oncogen) / RB1 (supressor tumoral) i del microambient tumoral en la disseminació, l'establiment i el creixement de les cèl·lules metastàtiques. Per treballar aquests objectius hem utilitzat eines genètiques i proteòmiques en un model de metàstasi pulmonar en ratolí. Els nostres resultats mostren que Ciclina D1 promou la supervivència i l'establiment de les cèl·lules tumorals disseminades (DTCs) independentment de RB1. Per contra, aquesta ciclina afavoreix el creixement dels nòduls de forma RB1-dependent. A més, hem detectat que el fluid intersticial tumoral (TIF) de pulmons metastàtics està enriquit en les proteïnes del citoesquelet Fascina i Adseverina. Finalment, hem comprovat que l'Adseverina és important per a la supervivència i l'establiment de les DTCs i el creixement dels nòduls metastàtics, el que implica que podria servir de marcador o diana terapèutica."La metástasis es un proceso muy complejo que está regulado por un elevado número de variables y señalizaciones, entre otros la composición del microambiente tumoral. Los objetivos de esta tesis son estudiar el papel de la vía Ciclina D1 (oncogén) / RB1 (supresor tumoral) y del microambiente tumoral en la diseminación, establecimiento y crecimiento de las células metastáticas. Para trabajar estos objetivos, hemos usado herramientas genéticas y proteómicas en un modelo de metástasis pulmonar en ratón. Nuestros resultados muestran que Ciclina D1 promueve la supervivencia y establecimiento de las células tumorales diseminadas (DTCs) independientemente de RB1. Por contra, esta ciclina favorece el crecimiento de los nódulos de forma RB1-dependiente. Además, hemos detectado que el fluido intersticial tumoral (TIF) de pulmones metastáticos está enriquecido con las proteínas del citoesqueleto Fascina y Adseverina. Finalmente, hemos comprobado que Adseverina es importante para la supervivencia y establecimiento de las DTCs y el crecimiento de los nódulos metastáticos, lo que implica que podría usarse como marcador o diana terapéutica.Metastasis is a highly complex procedure regulated by many variables and signals, such as the tumour microenvironment composition. This thesis aims are to study the role of the Cyclin D1 (oncogene) / RB1 (tumour suppressor) pathway and the tumour microenvironment in the dissemination, establishment and growth of metastatic cells. To study this, we used genetic and proteomic tools in a lung mice model of metastasis. Our results showed that Cyclin D1 promote disseminated tumour cells (DTCs) survival and establishment in an RB1-independent way. In contrast, this cyclin favours nodules growth in an RB1-dependent way. Moreover, we have detected that tumour interstitial fluid (TIF) of metastatic lungs is enriched in cytoskeleton proteins such as Fascin and Adseverin. Finally, we confirmed that Adseverin is important for DTCs survival and establishment and the growth of the metastatic nodule, which implies that it could be useful as a therapeutic target

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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas

Altres ajuts: This work was funded by the Spanish Ministry of Innovation and Science MICINN (PID2019-104859GB-I00) and by Generalitat de Catalunya (2017-SGR-569). This work was supported by IRBLleida Biobank (B.0000682) and PLATAFORMA BIOBANCOS PT20/0021. "The proteomics analyses were performed in the IJC Proteomics Unit. The IJC Proteomics Unit is part of the Spanish Platform of Molecular and Bioinformatics Resources (ProteoRed), Instituto de Salud Carlos III (PT13/0001)." For detailed "Experimental procedures" for publication, please, contact the Proteomics Unit. Special thanks to Eddie Chalecki lluMme. M. R.-S. is the recipient of a TALENT grant from Lleida Institute for Biomedical Research-Dr Pifarré Foundation supported by Diputació de Lleida. M. G.-V. (FPU17/00229) was supported by a predoctoral fellowship from Ministerio de Educación, Cultura y Deportes.Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets

Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression

The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved