Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Plakins are involved in the regulation of centrosome position in polarized epithelial cells.

BACKGROUND INFORMATION: The control of epithelial cell polarity is key to their function. Its dysregulation is a major cause of tissue transformation. In polarized epithelial cells,the centrosome is off-centred toward the apical pole. This asymmetry determines the main orientation of the microtubule network and intra-cellular traffic. However, the mechanism regulating centrosome positioning at the apical pole of polarized epithelial cells is still poorly undertood. RESULTS: In this study we used transcriptomic data from breast cancer cells to identify molecular changes associated with the different stages of tumour transformation. We correlated these changes with variations in centrosome position or with cell progression along the epithelial-to-mesenchymal transition (EMT), a process that involves centrosome repositioning. We found that low levels of epiplakin, desmoplakin and periplakin correlated with centrosome mispositioning in cells that had progressed through EMT or tissue transformation. We further tested the causal role of these plakins in the regulation of centrosome position by knocking down their expression in a non-tumorigenic breast epithelial cell line (MCF10A). The downregulation of periplakin reduced the length of intercellular junction, which was not affected by the downregulation of epiplakin or desmoplakin. However, down-regulating any of them disrupted centrosome polarisation towards the junction without affecting microtubule stability. CONCLUSIONS: Altogether, these results demonstrated that epiplakin, desmoplakin and periplakin are involved in the maintenance of the peripheral position of the centrosome close to inter-cellular junctions. They also revealed that these plakins are downregulated during EMT and breast cancer progression, which are both associated with centrosome mispositioning. SIGNIFICANCE: These results revealed that the down-regulation of plakins and the consequential centrosome mispositioning are key signatures of disorganised cytoskeleton networks, inter-cellular junction weakening, shape deregulation and the loss of polarity in breast cancer cells. These metrics could further be used as a new readouts for early phases of tumoral development.

PSL Chemical Biology Symposia: Recent Progress in Ferroptosis.

This symposium is the 5th PSL (Paris Sciences & Lettres) Chemical Biology meeting (2015, 2016, 2019, 2023, 2024) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition hosted around 150 participants and was focused on the burgeoning field of ferroptosis, its mechanism and implications in health and disease. While not initially planned, it was felt that the next large Ferroptosis venue (CSHA, China) would not happen before late 2024. A discussion involving Conrad, Birsoy, Ubellacker, Brabletz and Rodriguez next to lake Como in Italy sponsored by the DKFZ, prompted us to fill in this gap and to organize a Ferroptosis meeting in Paris beforehand.

Spatial Transcriptomics Reveal Pitfalls and Opportunities for the Detection of Rare High-Plasticity Breast Cancer Subtypes.

Breast cancer is one of the most prominent types of cancers, in which therapeutic resistance is a major clinical concern. Specific subtypes, such as claudin-low and metaplastic breast carcinoma (MpBC), have been associated with high nongenetic plasticity, which can facilitate resistance. The similarities and differences between these orthogonal subtypes, identified by molecular and histopathological analyses, respectively, remain insufficiently characterized. Furthermore, adequate methods to identify high-plasticity tumors to better anticipate resistance are lacking. Here, we analyzed 11 triple-negative breast tumors, including 3 claudin-low and 4 MpBC, via high-resolution spatial transcriptomics. We combined pathological annotations and deconvolution approaches to precisely identify tumor spots, on which we performed signature enrichment, differential expression, and copy number analyses. We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia public databases for external validation of expression markers. By focusing our spatial transcriptomic analyses on tumor cells in MpBC samples, we bypassed the negative impact of stromal contamination and identified specific markers that are neither expressed in other breast cancer subtypes nor expressed in stromal cells. Three markers (BMPER, POPDC3, and SH3RF3) were validated in external expression databases encompassing bulk tumor material and stroma-free cell lines. We unveiled that existing bulk expression signatures of high-plasticity breast cancers are relevant in mesenchymal transdifferentiated compartments but can be hindered by abundant stromal cells in tumor samples, negatively impacting their clinical applicability. Spatial transcriptomic analyses constitute powerful tools to identify specific expression markers and could thus enhance diagnosis and clinical care of rare high-plasticity breast cancers.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Iron-Sensitive Prodrugs That Trigger Active Ferroptosis in Drug-Tolerant Pancreatic Cancer Cells.

Persister cancer cells represent rare populations of cells resistant to therapy. Cancer cells can exploit epithelial-mesenchymal plasticity to adopt a drug-tolerant state that does not depend on genetic alterations. Small molecules that can interfere with cell plasticity or kill cells in a cell state-dependent manner are highly sought after. Salinomycin has been shown to kill cancer cells in the mesenchymal state by sequestering iron in lysosomes, taking advantage of the iron addiction of this cell state. Here, we report the chemo- and stereoselective synthesis of a series of structurally complex small molecule chimeras of salinomycin derivatives and the iron-reactive dihydroartemisinin. We show that these chimeras accumulate in lysosomes and can react with iron to release bioactive species, thereby inducing ferroptosis in drug-tolerant pancreatic cancer cells and biopsy-derived organoids of pancreatic ductal adenocarcinoma. This work paves the way toward the development of new cancer medicines acting through active ferroptosis.

Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.

Numerous epithelial-mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties, and therapy-associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that EMT-associated transcription factors (EMT-TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT-TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor-specific molecular vulnerabilities, which result in selective destruction of cancer cells. Here we review recent advances, providing novel insights into the role of EMT in the DDR and repair pathways, with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.

Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer.

BACKGROUND: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. METHODS: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. RESULTS: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. CONCLUSION: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus.

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases, although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology, we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus.

Stiffness measurement is a biomarker of skin ageing in vivo.

Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin ageing is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin ageing. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through ageing in these models, can be a new biomarker of skin ageing, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin ageing in vivo.

Structure-activity relationship study: Mechanism of cyto-genotoxicity of Nitropyrazole-derived high energy density materials family.

Stringent toxicological tests have to be performed prior to the industrial development of alternative chemicals particularly high energy dense materials (HEDMs) such as explosives. The properties (e.g., power, stability) of these compounds are constantly being improved, the current axis of research being the nitration of nitrogen heterocycles leading to HEDMs such as nitropyrazole-derived molecules. However, except for 3,4,5-trinitropyrazole (3,4,5-TNP), which was shown to be highly toxic in mice, the toxicological impact of these HEDMs has so far not been investigated. Furthermore, as industrials are strongly advised to develop alternative safety testing assays to in vivo experiments, we herein focused on determining the cytotoxic and genotoxic effects of seven Nitropyrazole-derived HEDMs on three rodent cell lines (mouse embryonic BALB/3T3 clone A31 cells, Chinese hamster ovary cells CHO-K1 and mouse lymphoma L5178Y TK +/- clone (3.7.2C) cells), two human fibroblast lines (CRC05, PFS04062) and on the human hepatic HepaRG model (both in proliferative and differentiated cells). A stronger cytotoxic effect was observed for 1,3-dinitropyrazole (1, 3-DNP) and 3,4,5-TNP in all cell lines, though differentiated HepaRG cells clearly displayed fewer likely due to the metabolism and elimination of these molecules by their functional biotransformation pathways. At the mechanistic level, the sub-chronic cytotoxic and genotoxic effects were linked to ROS/RNS production (experimental assays), HA2.X and to transcriptomic data highlighting the increase in DNA repair mechanisms.

Toxicokinetics and tolerance of a high energy material 3,4,5-trinitropyrazole (TNP) in mice.

The high-energy compound 3,4,5-trinitropyrazole (TNP) was developed as an alternative to other less energetic and more sensitive explosive materials, in particular 1-methyl-2,4,6-trinitrobenzene (TNT). However, the level of toxicity of TNP remains understudied. Here using an in vivo CD1 mouse model, we mimicked an acute exposure (24 h) to TNP, given either orally or intravenously, and determined the maximum administrable doses (190 mg/kg and 11 mg/kg, respectively), as well as the lethal dose for 50% (LD50) of female or male mice (390 mg/kg for both) treated intravenously with TNP alone. Several metabolites including nitroso-dinitro-pyrazole, hydroxylamino-dinitro-pyrazole, hydroxyl-dinitro-pyrazole and amino-dinitro-pyrazole were identified in urine. TNP is quickly metabolized and eliminated via urine as two main amino-dinitro-pyrazole metabolites. A comparison of the transcriptomic effects of TNP and TNT after 10 days exposure enabled us to demonstrate no major induction of transcripts involved both in cell death mechanisms (apoptosis, necrosis, autophagy) and physiological pathways (glycolysis, ATP production). Finally, subchronic exposure to TNP was replicated in female mice, fed 16.8-52.8 mg/kg/day of TNP for one month, to study the impact on cellular functions. Although blood TNP levels remained high, a lower rate of TNP accumulation in the liver and lungs were observed than during an acute exposure. Conversely, cellular stress functions explored using the RT2 Profiler™ PCR Array Mouse Molecular Toxicology PathwayFinder remained unaltered after this chronic exposure. These findings demonstrate that TNP can be rapidly eliminated in vivo without accumulating in vital organs.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

A 13-gene expression-based radioresistance score highlights the heterogeneity in the response to radiation therapy across HPV-negative HNSCC molecular subtypes.

BACKGROUND: Radiotherapy for head and neck squamous cell carcinomas (HNSCC) is associated with a substantial morbidity and inconsistent efficacy. Human papillomavirus (HPV)-positive status is recognized as a marker of increased radiosensitivity. Our goal was to identify molecular markers associated with benefit to radiotherapy in patients with HPV-negative disease. METHODS: Gene expression profiles from public repositories were downloaded for data mining. Training sets included 421 HPV-negative HNSCC tumors from The Cancer Genome Atlas (TCGA) and 32 HNSCC cell lines with available radiosensitivity data (GSE79368). A radioresistance (RadR) score was computed using the single sample Gene Set Enrichment Analysis tool. The validation sets included two panels of cell lines (NCI-60 and GSE21644) and HPV-negative HNSCC tumor datasets, including 44 (GSE6631), 82 (GSE39366), and 179 (GSE65858) patients, respectively. We finally performed an integrated analysis of the RadR score with known recurrent genomic alterations in HNSCC, patterns of protein expression, biological hallmarks, and patterns of drug sensitivity using TCGA and the E-MTAB-3610 dataset (659 pancancer cell lines, 140 drugs). RESULTS: We identified 13 genes differentially expressed between tumor and normal head and neck mucosa that were associated with radioresistance in vitro and in patients. The 13-gene expression-based RadR score was associated with recurrence in patients treated with surgery and adjuvant radiotherapy but not with surgery alone. It was significantly different among different molecular subtypes of HPV-negative HNSCC and was significantly lower in the "atypical" molecular subtype. An integrated analysis of RadR score with genomic alterations, protein expression, biological hallmarks and patterns of drug sensitivity showed a significant association with CCND1 amplification, fibronectin expression, seven hallmarks (including epithelial-to-mesenchymal transition and unfolded protein response), and increased sensitivity to elesclomol, an HSP90 inhibitor. CONCLUSIONS: Our study highlights the clinical relevance of the molecular classification of HNSCC and the RadR score to refine radiation strategies in HPV-negative disease.

Destabilization of the TWIST1/E12 complex dimerization following the R154P point-mutation of TWIST1: an in silico approach.

BACKGROUND: The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. RESULTS: Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. CONCLUSIONS: Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes.

Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells.

DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.

Tif1γ is essential for the terminal differentiation of mammary alveolar epithelial cells and for lactation through SMAD4 inhibition.

Transforming growth factor ß (TGFß) is widely recognised as an important factor that regulates many steps of normal mammary gland (MG) development, including branching morphogenesis, functional differentiation and involution. Tif1γ has previously been reported to temporally and spatially control TGFß signalling during early vertebrate development by exerting negative effects over SMAD4 availability. To evaluate the contribution of Tif1 γ to MG development, we developed a Cre/LoxP system to specifically invalidate the Tif1g gene in mammary epithelial cells in vivo. Tif1g-null mammary gland development appeared to be normal and no defects were observed during the lifespan of virgin mice. However, a lactation defect was observed in mammary glands of Tif1g-null mice. We demonstrate that Tif1 γ is essential for the terminal differentiation of alveolar epithelial cells at the end of pregnancy and to ensure lactation. Tif1 γ appears to play a crucial role in the crosstalk between TGFß and prolactin pathways by negatively regulating both PRL receptor expression and STAT5 phosphorylation, thereby impairing the subsequent transactivation of PRL target genes. Using HC11 cells as a model, we demonstrate that the effects of Tif1g knockdown on lactation depend on both SMAD4 and TGFß. Interestingly, we found that the Tif1γ expression pattern in mammary epithelial cells is almost symmetrically opposite to that described for TGFß. We propose that Tif1γ contributes to the repression of TGFß activity during late pregnancy and prevents lactation by inhibiting SMAD4.

Metastasis: a question of life or death.

The metastatic process is highly inefficient--very few of the many cells that migrate from the primary tumour successfully colonize distant sites. One proposed mechanism to explain this inefficiency is provided by the cancer stem cell model, which hypothesizes that micrometastases can only be established by tumour stem cells, which are few in number. However, recent in vitro and in vivo observations indicate that apoptosis is an important process regulating metastasis. Here we stress that the inhibition of cell death, apart from its extensively described function in primary tumour development, is a crucial characteristic of metastatic cancer cells.