Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-ß activation.

Metastasis is the major cause of cancer death, and the development of therapy resistance is common. The tumor microenvironment can confer chemotherapy resistance (chemoresistance), but little is known about how specific host cells influence therapy outcome. We show that chemotherapy induces neutrophil recruitment and neutrophil extracellular trap (NET) formation, which reduces therapy response in mouse models of breast cancer lung metastasis. We reveal that chemotherapy-treated cancer cells secrete IL-1ß, which in turn triggers NET formation. Two NET-associated proteins are required to induce chemoresistance: integrin-αvß1, which traps latent TGF-ß, and matrix metalloproteinase 9, which cleaves and activates the trapped latent TGF-ß. TGF-ß activation causes cancer cells to undergo epithelial-to-mesenchymal transition and correlates with chemoresistance. Our work demonstrates that NETs regulate the activities of neighboring cells by trapping and activating cytokines and suggests that chemoresistance in the metastatic setting can be reduced or prevented by targeting the IL-1ß-NET-TGF-ß axis.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

COVID-19 patients age, comorbidity profiles and clinical presentation related to the SARS-CoV-2 UK-variant spread in the Southeast of France.

The variant 20I/501Y.V1, associated to a higher risk of transmissibility, emerged in Nice city (Southeast of France, French Riviera) during January 2021. The pandemic has resumed late December 2020 in this area. A high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze modifications in clinical profile and outcome traits. We performed an observational study in the University hospital of Nice from December 2020 to February 2021. We analyzed data of sequencing of SARS-CoV-2 from the sewage collector and PCR screening from all positive samples at the hospital. Then, we described the characteristics of all COVID-19 patients admitted in the emergency department (ED) (n = 1247) and those hospitalized in the infectious diseases ward or ICU (n = 232). The UK-variant was absent in this area in December, then increasingly spread in January representing 59% of the PCR screening performed mid-February. The rate of patients over 65 years admitted to the ED decreased from 63 to 50% (p = 0.001). The mean age of hospitalized patients in the infectious diseases ward decreased from 70.7 to 59.2 (p < 0.001) while the proportion of patients without comorbidity increased from 16 to 42% (p = 0.007). Spread of the UK-variant in the Southeast of France affects younger and healthier patients.

Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing.

BACKGROUND: Wastewater surveillance was proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were based on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. METHODS: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. FINDINGS: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. INTERPRETATION: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

A new long noncoding RNA (lncRNA) is induced in cutaneous squamous cell carcinoma and down-regulates several anticancer and cell differentiation genes in mouse.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively) is associated with the up-regulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one consisting of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically down-regulating the expression of genes of the late cornified envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16 Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.

Elucidation of IgH 3' region regulatory role during class switch recombination via germline deletion.

In mature B cells, class switch recombination (CSR) replaces the expressed constant Cµ gene with a downstream C(H) gene. How the four transcriptional enhancers of the IgH 3' regulatory region (3'RR) control CSR remains an open question. We have investigated IgG1 CSR in 3'RR-deficient mice. Here we show that the 3'RR enhancers target the S(γ1) acceptor region (and poorly the S(µ) donor region) by acting on epigenetic marks, germline transcription, paused RNA Pol II recruitment, R loop formation, AID targeting and double-strand break generation. In contrast, location and diversity of S(µ)-S(γ1) junctions are not affected by deletion of the 3'RR enhancers. Thus, the 3'RR controls the first steps of CSR by priming the S acceptor region but is not implicated in the choice of the end-joining pathway.

MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

Can the microRNA signature distinguish between thyroid tumors of uncertain malignant potential and other well-differentiated tumors of the thyroid gland?

The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.

Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions.

BACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.

Relationships between early inflammatory response to bleomycin and sensitivity to lung fibrosis: a role for dipeptidyl-peptidase I and tissue inhibitor of metalloproteinase-3?

RATIONALE: Different sensitivities to profibrotic compounds such as bleomycin are observed among mouse strains. OBJECTIVES: To identify genetic factors contributing to the outcome of lung injury. METHODS: Physiological comparison of C57BL/6 (sensitive) and BALB/c (resistant) mice challenged by intratracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration. MEASUREMENTS AND MAIN RESULTS: Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first corresponded to dipeptidyl-peptidase I (DPPI), a cysteine peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponded to tissue inhibitor of matrix metalloproteinase-3, which also inhibits members of the ADAM (a disintegrin and metalloproteinase) family, such as the tumor necrosis factor-converting enzyme. In functional studies performed in the bleomycin-induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of polymorphonuclear neutrophil-derived serine proteases and tumor necrosis factor-alpha-dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme. CONCLUSIONS: This study highlights the importance of the early inflammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and tissue inhibitor of matrix metalloproteinase-3 may play in this process.

An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.