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International audience; In the environment, populations are exposed to different kinds of ionizing radiation. Little is known about their modes of action on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, byexposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified, and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defence response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study, therefore, brought a body of evidence on the effects of HTO observed at the molecular level on zebrafish larvae. Furtherstudies could investigate if the effects persist in adult organisms.

Background:Pancreatic Ductal AdenoCarcinoma (PDAC), the most frequent pancreatic cancer, is a deadly cancer since often diagnosed late and resistant to current therapies. A high proportion of PDAC patients are affected by cachexia induced by the tumor. This cachexia, characterized by loss of muscle mass and strength, contributes to patient frailty and poor therapeutic response. We showed that mitochondrial metabolism is reprogrammed in PDAC tumors and constitutes a vulnerability, opening novel therapeutic avenues. The objective of the present work was to investigate the molecular mechanisms underlying mitochondrial remodeling in PDAC cachectic skeletal muscle.Methods:Our study focused on the gastrocnemius muscle of genetically-engineered mice developing spontaneously a PDAC associated with cachexia (KIC GEMM). We compared KIC mice developing a pancreatic tumor in 9-10 weeks to control littermates. We did an integrative study combiningin vivofunctional analyses by non-invasive Magnetic Resonance, andex-vivohistology, Seahorse, RNA-sequencing, and proteomic mass spectrometry and western blotting analyses.Results:The cachectic KIC PDAC mice show a severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophic muscles show decreased respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways at the molecular level. Increased expression of multiple reactive oxygen species (ROS) defense genes suggests oxidative stress prone to affect mitochondrial macromolecules and homeostasis. Interestingly, multiple genes and proteins involved in DNA metabolism pathways, such as DNA damage, degradation of DNA, nucleotide synthesis, and folate pathway were found altered in sarcopenic mitochondria. While the number of mitochondria was not changed per cell, the mitochondrial mass was decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis.ConclusionsWe unveiled that mitochondrial alterations in skeletal muscle play a central role in PDAC-induced cachexia. Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrates and protein, but concern also lipids, ROS and nucleic acids. Our data provide a reference framework to guide towards the most relevant molecular markers that would be affected at the start of tumor development and could be targets in the clinic to limit PDAC- induced cachexia at early stages of the pathology.

International audience; A hallmark of Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, is its ability to metabolize host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogs, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyCyne) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogs. We confirmed that the CyC bind covalently to the catalytic Ser114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6-2.6 Å. The identification of mycobacterial enzymes directly captured by the CyCyne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life-cycle.

In pancreatic ductal adenocarcinoma (PDAC), signaling from stromal cells is implicated in metastatic progression. Tumor-stroma cross-talk is often mediated through extracellular vesicles (EVs). We previously reported that EVs derived from cancer-associated stromal fibroblasts (CAFs) that are abundant in annexin A6 (ANXA6 + EVs) support tumor cell aggressiveness in PDAC. Here, we found that the cell surface glycoprotein and tetraspanin CD9 is a key component of CAF-derived ANXA6 + EVs for mediating this cross-talk. CD9 was abundant on the surface of ANXA6 + CAFs isolated from patient PDAC samples and from various mouse models of PDAC. CD9 colocalized with CAF markers in the tumor stroma, and CD9 abundance correlated with tumor stage. Blocking CD9 impaired the uptake of ANXA6 + EVs into cultured PDAC cells. Signaling pathway arrays and further analyses revealed that the uptake of CD9 + ANXA6 + EVs induced mitogen-activated protein kinase (MAPK) pathway activity, cell migration, and epithelial-to-mesenchymal transition (EMT). Blocking either CD9 or p38 MAPK signaling impaired CD9 + ANXA6 + EV–induced cell migration and EMT in PDAC cells. Analysis of bioinformatic datasets indicated that CD9 abundance was an independent marker of poor prognosis in patients with PDAC. Our findings suggest that CD9-mediated stromal cell signaling promotes PDAC progression.

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that β‐hydroxybutyrate (βOHB) is an alternative cell‐intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG‐CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage‐independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while βOHB stimulates metastatic dissemination to the liver. These findings suggest that βOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.

NUPR1 binds to PARP1 and inhibits PARP1 activity in pancreatic cancer cells, with the NUPR1 inhibitor ZZW-115 abolishing the protective effect of NUPR1, inducing strong hyperPARylation and cell death by mitochondrial dysfunction. Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD(+)/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD(+) precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.

Upregulation of the developmental Wnt/planar cell polarity pathway is observed in many cancers and is associated with cancer development at early and late stages. We recently showed that PRICKLE1 and VANGL2, two core Wnt/PCP components, are overexpressed in triple negative breast cancer and associated with poor prognosis. PRICKLE1 is a cytoplasmic protein phosphorylated by the poorly described serine/threonine kinase MINK1 which triggers its localization at the plasma membrane, a key step for its function. Knockdown experiments have demonstrated that MINK1 and PRICKLE1 contribute to TNBC cell motility and spreading in vitro and in vivo. However, the identity of MINK1 substrates and the role of MINK1 enzymatic activity in this process have not yet been addressed issues.We carried out a phosphoproteomic strategy and identified novel MINK1 substrates including LL5β. LL5β is a membrane scaffold molecule that anchors microtubules at the cell cortex through its association with the plus-end MT proteins CLASPs to trigger focal adhesion disassembly. LL5β is a prominent member of the MINK1-PRICKLE1 protein complex and is directly phosphorylated by MINK1 that promotes its interaction with CLASP. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in the protein complex assembly and localization, and cell migration. Analysis of gene expression data show that the concomitant up-regulation of PRICKLE1 and LL5β mRNA levels encoding MINK1 substrates is associated with a poor metastasis-free survival for TNBC patients. Altogether, our results suggest that MINK1 may represent a potential target in TNBC.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton.SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of the Wnt signaling pathways and is strongly implicated in cancer development at early and late stages (PMID: 28718442 and PMID: 30952630). Upregulation of the Wnt/PCP components is observed in many cancers and is associated with cancer progression. However, how their molecular functions are regulated remains an open question. Recent data from our laboratory showed that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in Triple Negative Breast Cancers (TNBC) and associated with poor prognosis (PMID: 27184734 and 26754771). Prickle1 is a cytoplasmic protein phosphorylated by the serine/threonine kinase Mink1, which triggers Prickle1 localization at the plasma membrane and regulates its activity (PMID: 22037766). Activation of this axis contributes to breast cancer cell motility and metastatic spreading in vitro and in vivo (PMID: 27184734). To extend our knowledge of the Mink1 substrates, we carried out a phosphoproteomic strategy and identified LL5β. LL5β is a membrane scaffold molecule that anchors microtubules (MTs) at the cell cortex through its association with CLASP, a plus-end MT protein, to trigger focal adhesion disassembly. We found that LL5β is a prominent member of the Prickle1-associated protein complex and harbors a consensus motif for Mink1 phosphorylation. At the molecular level, we demonstrated a two-step phosphorylation cascade carried out by Mink1 that phosphorylates sequentially Prickle1 to mediate its association with LL5β, then LL5β to promote its interaction with CLASP. Finally, analysis of gene expression data suggests that the concomitant up-regulation of Prickle1 and LL5β mRNA levels is associated with a poor metastasis-free survival for TNBC patients. In an effort to counteract the Mink1-Prickle1-LL5β prometastatic pathway, we selected a Mink1 inhibitor that recapitulates all the phenotypes observed following the knockdown of Mink1 expression. This chemical compound is currently under investigation in preclinical assays using xenografted TNBC cell lines and patient-derived xenografts in nude mice. Citation Format: Avais Daulat, Monica Silveira Wagner, Malgorzata Kowalczewska, Pascal Finetti, Rémy Castellano, Stéphane Audebert, François Bertucci, Luc Camoin, Jean-Paul Borg. The serine-threonine kinase Mink1 as a potential therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2290.

Summary Interfaces between cells with distinct genetic identities elicit signals to organize local cell behaviors driving tissue morphogenesis. The Drosophila embryonic axis extension requires planar polarized enrichment of myosin-II powering oriented cell intercalations. Myosin-II levels are quantitatively controlled by GPCR signaling, whereas myosin-II polarity requires patterned expression of several Toll receptors. How Toll receptors polarize myosin-II and how this involves GPCRs remain unknown. Here, we report that differential expression of a single Toll receptor, Toll-8, polarizes myosin-II through binding to the adhesion GPCR Cirl/latrophilin. Asymmetric expression of Cirl is sufficient to enrich myosin-II, and Cirl localization is asymmetric at Toll-8 expression boundaries. Exploring the process dynamically, we reveal that Toll-8 and Cirl exhibit mutually dependent planar polarity in response to quantitative differences in Toll-8 expression between neighboring cells. Collectively, we propose that the cell surface protein complex Toll-8/Cirl self-organizes to generate local asymmetric interfaces essential for planar polarization of contractility., Graphical abstract, Highlights • Asymmetric expression of a single Toll receptor leads to Myo-II polarization • The adhesion GPCR Cirl binds to Toll-8 mediating Toll-8-induced Myo-II polarization • Toll-8 boundaries generate a Cirl interfacial asymmetry that can polarize Myo-II • Differences in Toll-8 levels lead to interdependent Toll-8 and Cirl planar polarity, Lavalou, Mao et al. report that Toll-8 controls myosin-II planar polarity in Drosophila embryos and wing discs via a physical interaction with the GPCR Cirl/latrophilin. They show that Toll-8 expression boundaries generate a Cirl interfacial asymmetry and propose that it is a potential signal leading to myosin-II polarization.

Identification of protein networks becomes indispensable for determining the function of a given protein of interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and maintenance of cell architecture. In the present chapter, we present two complementary approaches (immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these two approaches, we identified several PDZ domain containing proteins. Some of them were found with both approaches, and some were specifically identified using peptide-based purification procedure. This exemplifies advantages and differences of both strategies to identify PDZ interactions.

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells., T and B cells continually recirculate between blood and secondary lymphoid organs. The RHO GTPase activating protein ARHGAP45 regulates naïve T cell deformability, motility, and chemotaxis, and thereby regulates key trafficking steps of T and B cells in vivo.

International audience; Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

In acute myeloid leukemia (AML), the stromal microenvironment plays a prominent role in promoting tumor cell survival and progression. Although widely explored, the crosstalk between leukemic and stromal cells remains poorly understood. Syntenin, a multi-domain PDZ protein, controls both the trafficking and signaling of key molecules involved in intercellular communication. Therefore, we aimed to clarify the role of environmental syntenin in the progression of AML. Byin vivoapproaches in syngeneic mice, we demonstrate that a syntenin-deficient environment reprograms AML blasts to survive independently of the stroma. Up-regulation of EEF1A2 in the blasts controls this gain of cell survival. Furthermore, usingex vivoco-culture systems, we show that syntenin-deficient bone marrow stromal cells (BMSC) enhance the survival of different types of AML cells, including patient samples, and suffice to educate syngeneic AML, recapitulating micro-environmental effects observedin vivo.We establish that syntenin-deficiency causes an increase of eIF5A and autophagy-related factors in BMSC, and provide evidence that the inhibition of autophagy prevents syntenin-deficient BMSC to stimulate AML survival. Altogether, these findings indicate that host-syntenin in the BM microenvironment acts as a repressor of AML aggressiveness.Key points-A syntenin-deficient host reprograms AML blasts, enhancing total protein synthesis and cell survival pathways-Autophagy in the syntenin-deficient microenvironment is responsible for the gain of AML cell survival

Identification of protein networks becomes indispensable for determining the function of a given protein of interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and maintenance of cell architecture. In the present chapter, we present two complementary approaches (immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these two approaches, we identified several PDZ domain containing proteins. Some of them were found with both approaches, and some were specifically identified using peptide-based purification procedure. This exemplifies advantages and differences of both strategies to identify PDZ interactions.

It is important to understand the biogenesis of exosomes, extracellular vesicles of endosomal origin controlling cell-to-cell communication. We previously reported that Phospholipase D2 (PLD2) supports late endosome (LE) budding and the biogenesis of syntenin-dependent exosomes. Here, we reveal that PLD2 has a broader generic effect on exosome production. Combining gain- and loss-of-function experiments, proteomics, microscopy and lipid-binding studies with reconstituted liposomes mimicking LE, we show that: (i) PLD2 activity controls the recruitment of MVB12B to LE and the exosomal secretion of ESCRT-I; (ii) loss-of-MVB12B phenocopies loss-of-PLD2, similarly affecting LE budding, the number of exosomes released and exosome loading with cargo; (iii) MVB12B MABP domain directly interacts with phosphatidic acid, the product of PLD2. We therefore propose that PLD2 and phosphatidic acid support ESCRT-I recruitment to LE for the formation of exosomes. This work highlights a major unsuspected piece of the molecular framework supporting LE and exosome biogenesis.

First described inDrosophila melanogaster, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse,Xenopusand zebrafish embryos. The regulated and poorly understood traffic of Vangl2 to the plasma membrane is a key event for its function in development. Here we report on the molecular and functional characterization of a novel 569-amino acid N-terminally extended Vangl2 isoform, Vangl2-Long, that arises from an alternative non-AUG translation initiation site, lying 144 base pair upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, includingDrosophila melanogaster, this N-terminal extension is conserved in all vertebrate Vangl2 sequences and confers a subcellular localization in the Golgi apparatus, probably as a result of an extended retention time in this organelle. Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2 and we show that its down-regulation leads to severe PCP-related phenotypes inXenopusembryos, including shorter body axis and neural tube closure defects. Altogether, our study unveils a novel level of complexity in Vangl2 expression, trafficking and function.

Invasion/metastasis axis is the major driver of cancer mortality. By maintaining tissue cohesion, E-cadherin is considered as a protective marker for tumoral progression. However, recent studies suggested that the appearance of hybrid epithelial-mesenchymal (E/M) cells still expressing E-cadherin is favourable for the establishment of metastasis. This hypothesis is consistent with the observation that most pancreatic ductal adenocarcinomas (PDAC) are invasive and express E-cadherin in primary tumour and metastases. By using a data constituted by a series of patient-derived xenografts (PDX) from the PaCaOmics multi-centric clinical trial, we show that E-cadherin expression is not associated with stages of the pathology, prognosis, and overall survival in PDAC. The role of E-cadherin in PDAC aggressiveness was tested bothin vitroand in cancer cell implantation models. We show that E-cadherin is a key component of membrane protrusions implicated in the extracellular matrix remodelling and degradation, called invadopodia. E-cadherin downregulation in a pancreatic model of E/M hybrid cells reveals that E-cadherin downregulates Arp2/3 complex expression. As this complex is essential for branched actin structure, E-cadherin depletion strongly impaired invadopodia formation. On the other hand, we demonstrate that E-cadherin interacts with the membrane protease MT1-MMP at the in-vadopodial membrane. E-cadherin could be recycled back to the invadopodial membrane simultaneously with MT1-MMP. Indeed, both Rab7 vesicle-dependant and/or a Rab11 vesicle-dependant pathway are required for both E-cadherin and MT1-MMP trafficking. This new localization of E-cadherin and its implication in cell invasion shines a new light on hybrid EMT features in tumoral invasion.

International audience; Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains the deadliest infectious disease worldwide with 1.5 million deaths in 2018, of which about 15% are attributed to resistant strains. Another significant example is Mycobacterium abscessus (M. abscessus), a nontuberculous mycobacteria (NTM) responsible for cutaneous and pulmonary infections, representing up to 95% of NTM infections in cystic fibrosis (CF) patients. M. abscessus is a new clinically relevant pathogen and is considered one of the most drugresistant mycobacteria for which standardized chemotherapeutic regimens are still lacking. Together the emergence of M. tb and M. abscessus multi-drug resistant strains with ineffective and expensive therapeutics, have paved the way to the development of new classes of anti-mycobacterial agents offering additional therapeutic options. In this context, specific inhibitors of mycobacterial lipolytic enzymes represent novel and promising antibacterial molecules to address this challenging issue. The results highlighted here include a complete overview of the antibacterial activities, either in broth medium or inside infected macrophages, of two families of promising and potent anti-mycobacterial multi-target agents, i.e. oxadiazolone-core compounds (OX) and Cyclophostin & Cyclipostins analogs (CyC); the identification and biochemical validation of their effective targets (e.g., the antigen 85 complex and TesA playing key roles in mycolic acid metabolism) together with their respective crystal structures. To our knowledge, these are the first families of compounds able to target and impair replicating as well as intracellular bacteria. We are still impelled in deciphering their mode of action and finding new potential therapeutic targets against mycobacterial-related diseases.

International audience; Mycobacterium abscessus (M. abscessus), a rapidly growing mycobacterium, is an emergent opportunistic pathogen responsible for chronic bronchopulmonary infections in individuals with respiratory diseases such as cystic fibrosis. Most treatments of M. abscessus pulmonary infections are poorly effective due to the intrinsic resistance of this bacteria against a broad range of antibiotics including anti-tuberculosis agents. Consequently, the number of drugs that are efficient against M. abscessus remains limited. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs impair extracellu-lar M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or act intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., compound iBpPPOX, via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis. Among them, the Ag85C protein has been confirmed as a vulnerable target of iBpPPOX. This study clearly emphasizes the potential of the OX derivatives to inhibit the extracellular and/or intracellular growth of M. abscessus by targeting various enzymes potentially involved in many physiological processes of this most drug-resistant mycobacterial species.

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4-TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine. ispartof: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol:117 issue:11 pages:5913-5922 ispartof: location:United States status: published

Publiher's version (útgefin grein), Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape., We thank D. Isnardon and M. Rodriguez (CRCM Microscopy Platform) for support and S.A. Courtneidge, K.J. Roux and C. Lachaud for sharing reagents. This work was supported by Canceropôle Provence-Alpes-Côte d’Azur (PACA), Institut National du Cancer, Conseil Régional PACA and Site de Recherche Intégrée sur le Cancer (SIRIC). Proteomic analyses were performed at the mass spectrometry facility of Marseille Proteomics supported by IBISA (Infrastructures Biologie Santé et Agronomie), Plateforme Technologique Aix-Marseille, Canceropôle PACA, Région Sud Provence-Alpes-Côte d’Azur, Fonds Européen de Développement Régional (FEDER) and Plan Cancer.

19 pags., 6 figs., 4 tabs., Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR., This work was supported by La Ligue Contre le Cancer, INCa, Canceropole PACA and INSERM to JI; Fondation ARC to PS. La Ligue Contre le Cancer (Equipe Labellisée) to VG and JI; Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017) to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, ‘Investing in your future’) (PI15/00663 and PI18/00343) to OA; Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to AVC, RTI2018-097991-BI00 to JLN); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to AVC, and Digestive Pathology Group B25_17R to OA); Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd); Fondation de France to PSC; China Scholarship Council to WL and CH; Programme XU GUANGQI to YX and JI; and National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities (106112017CDJQJ468823) to YX. Part of this work was performed using the France-BioImaging infrastructure supported by the Agence Nationale de la Recherche (ANR-10-INBS-04-01, call “Investissements d’Avenir”).

International audience; Among the more than 160 PDZ containing proteins described in humans, the cytoplasmic scaffold Scribble stands out because of its essential role in many steps of cancer development and dissemination. Its fame has somehow blurred the importance of homologous proteins, Erbin and Lano, all belonging to the LRR and PDZ (LAP) protein family first described twenty years ago. In this review, we will retrace the history of LAP family protein research and draw attention to their contribution in cancer by detailing the features of its members at the structural and functional levels, and highlighting their shared-but also differentimplication in the tumoral process.