Sprouty1 is a broad mediator of cellular senescence

Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms

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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

ER alpha-mediated repression of pro-inflammatory cytokine expression by glucocorticoids reveals a crucial role for TNF alpha and IL1 alpha in lumen formation and maintenance

Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNF alpha and IL1 alpha in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNF alpha and IL1 alpha expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor alpha (ER alpha) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ER alpha-regulated pro-inflammatory cytokine expression in lumen formation and maintenance

FLIP is frequently expressed in endometrial carcinoma and has a role in resistance to TRAIL-induced apoptosis

The FLICE-inhibitory protein (FLIP) plays a key role in the regulation of apoptosis triggered by death ligands. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in some types of tumor but not in others. To assess the possible role of FLIP in apoptosis resistance in endometrial carcinoma, we performed an immunohistochemical study on a tissue microarray composed of 95 endometrial carcinomas. We found positive signals in 43% of the cases, as well as a significant difference in FLIP expression between stage I and II tumors. Moreover, we observed that endometrial carcinoma cell lines Ishikawa and KLE did not undergo apoptosis after TRAIL treatment. Cotreatment of these cells with the inhibitor of transcription actinomycin D resulted in a dramatic decrease in cell viability and induced activation of caspase-8. These events coincided with downregulation of FLIP mRNA and protein. Inhibitors of caspase-8 or overexpression of FLIP completely blocked apoptosis induced by actinomycin D plus TRAIL cotreatment. More importantly, downregulation of endogenous FLIP expression by specific siRNAs sensitized endometrial carcinoma cells to TRAIL-induced apoptosis in the absence of actinomycin D. Taken together, our results suggest for the first time a critical role for FLIP in the regulation apoptosis triggered by TRAIL in endometrial carcinoma cells

Abnormalities in the NF-κB family and related proteins in endometrial carcinoma

The NF-κB family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. A tissue microarray was constructed from paraffin wax-embedded blocks from 95 endometrial carcinomas (EC), previously studied for microsatellite instability, as well as for alterations in PTEN, k-RAS and betacatenin. Immunohistochemical evaluation included members of the NF-κB (p50, p65, p52, c-Rel, Rel-B) and the IκB (IκBα, IκBβ, IκBε, Bcl-3) families, as well as putative targets of NF-κB such as Flip, Bcl-xL, Cyclin D1, and oestrogen and progesterone receptors. Results were correlated with the clinical and pathological data. Nuclear immunostaining for members of the NF-κB family was frequent in EC (p50, 20%; p65, 16.5–21.9%; p52, 9.3%; cRel, 48.9%; Rel-B, 15.7%); and it correlated with negativity for members of the IκB family in some cases. There was a statistically significant association between immunoreaction for p50 and p65 (p = 0.006), suggesting activation of the so-called ‘classic form’ of NF-κB, similar to that described in breast cancer. Bcl-3 nuclear immunostaining was detected in 60.7% of cases. The vast majority of p52-positive tumours showed Bcl-3 nuclear immunoreaction (p = 0.038). Immunostaining for putative targets of NF-κB was as follows: Bcl-xL, 76.2% (p = 0.001); Flip 43.0%; Cyclin D1, 64.79%. p65 immunostaining correlated with increased immunoreaction for steroid hormone receptors. No correlation was found between NF-κB nuclear pattern and the presence of microsatellite instability, or alterations in PTEN, kRAS, or beta-catenin. These results suggest that the NF-κB and IκB families of genes may be important in endometrial carcinogenesis, by controlling apoptosis and cell proliferation