Biology subjects

Dupuy, J.-W.

Publications and source records attributed to Dupuy, J.-W..

16 recordsLinked to original sources

bioRxiv2025

p190A/ARHGAP35 and p190B/ARHGAP5 proteins in endometrial cancer, a novel cancer-relevant paralog interplay

Endometrial cancer is one of the main gynecological malignancies worldwide, with an estimated 320, 000 new cases annually. Several studies highlight ARHGAP35 as a significantly mutated gene in these tumors. It encodes for the protein p190RhoGAP-A (p190A), which is a major regulator of the small GTPase family of proteins. ARHGAP5 is a paralog of ARHGAP35 that encodes the protein p190RhoGAP-B (p190B). By analyzing human endometrial cancer samples, we found a co-occurrence of mutations in ARHGAP35 and ARHGAP5 genes and we reported that both are less expressed at the mRNA level in tumoral samples compared to healthy tissues. We were interested in understanding the impact of p190A/B under-expression in endometrial cancer and the relationship between the two paralogs. To do so, we have used CRISPR/Cas9 technology to generate HEC-1-A knockout cells for p190A and p190B. We showed that removal of each paralog led to a similar actin remodeling phenotype with the formation of Cross-Linked Actin Networks (CLANs), dependent on the Rho/ROCK pathway. Moreover, proteomic analysis of p190A and p190B knockout cells highlighted similar affected cell functions. Finally, our study demonstrates a synthetic lethality between p190A and p190B where removal of both paralogs is deleterious in endometrial cancer cells, unveiling a potential actionable vulnerability.

cancer biology↗

bioRxiv2025

Glycerol metabolism triggers trypanosome differentiation into transmissible forms in mammalian tissue-like conditions

In the mammalian bloodstream, Trypanosoma brucei, the parasite responsible for sleeping sickness, proliferates as slender forms before undergoing quorum-sensing (QS)-mediated differentiation into cell cycle-arrested stumpy forms (stumpy-QS), a transition that regulates parasitaemia and primes parasites for tsetse fly transmission. Beyond the bloodstream, T. brucei also occupies extravascular, adipose-rich tissues such as the skin, a potential reservoir for transmission. Here, we identify an alternative slender-to-stumpy differentiation pathway driven by glycerol, a host metabolite abundant in adipose-rich tissues, that could resolve the long-standing paradox of successful parasite transmission from human hosts during chronic infection despite low parasitaemia. We show that high (10 mM) and non-physiological glycerol concentrations under low glucose conditions (0.5 mM) induce differentiation, generating distinct stumpy-Glyc forms that resemble stumpy-QS parasites but have an extended lifespan. Under conditions mimicking dermal tissue interstitial fluids (4 mM glucose, 0.25 mM glycerol), we show that glycerol promotes the emergence of proliferative intermediate forms that retain transmission potential and can differentiate into fly host-specific procyclic forms in vitro and within tsetse flies. These findings open the door for reevaluation of the model of T. brucei transmission and supports a dominant role for adipocyte-derived glycerol in the skin in sustaining parasite transmission.

microbiology↗

bioRxiv2025

Tunable 3D Alveolosphere Model from Human Alveolar Cells: A Breakthrough Tool to Explore Emphysema Pathophysiology

RationaleThree-dimensional (3D) culture models such as alveolosphere, provide a unique tool to study emphysema mechanisms. Reducing the heterogeneity of alveolospheres which are currently mostly grown in Matrigel remains challenging. ObjectivesTo develop a tunable and reproducible 3D-alveolosphere model exclusively from human primary type II alveolar epithelial cells (AEC2) for modeling and understanding emphysema. MethodsAEC2 cells (HTII-280+) were isolated from 52 smoker and non-smoker lung samples and cultured in preformed photopolymerized hydrogel microwells of adjustable shape and size. Topological and phenotypic characterization were performed at Day (D)1, 7 and 14. Lamellar bodies (LB) were quantified using artificial intelligence (AI)-based image analysis of transmission electron microscopy (TEM) serial block face images. Emphysema was modeled through chronic exposure to 1 and 5% cigarette smoke extract (CSE) during 5 consecutive days. Measurements and Main Results3D-alveolospheres were maintained in culture for 14 days with central lumen formation observed from D7 to D14. Presence of tight junctions (TEM imaging and ZO-1 immunostaining) suggested epithelial barrier formation. AEC1 markers (p2xr4, pdpn) appeared progressively from D1 to D14 while AEC2 markers (abca3, sftpa, sftpc) persisted over time. TEM images indicated surfactant synthesis (LB, lipid bodies) and AI-driven LB quantification showed a decrease in the proportion of LB-containing cells over time. CSE exposure led to cell death, architectural disorganization, oxidative stress and inflammation. ConclusionThis standardized and adjustable 3D-alveolosphere model from human primary AEC2, reproduced key native alveolar features. CSE exposure provides an opportunity to appropriately study the pathophysiological pathways involved in emphysema.

cell biology↗

bioRxiv2025

Cancer cells transfer invasive properties through microRNAs contained in collagen-tracks

Invasion is a prerequisite for metastasis formation. During tumor development, the extracellular matrix (ECM) is remodeled in part through overexpression of type I collagen, increasing tumor microenvironment stiffness, and facilitating cancer dissemination. During breast cancer cell migration, we observed membrane debris left behind, attached to the collagen fibrils, along the migration path. We named these structures collagen-tracks. These collagen-tracks can be deposited in 3D matrices in vitro and in vivo and their formation is stimulated by the interaction between the ECM and matrix receptors, such as the discoidin-domain receptor (DDR1). However, they are different from structures already known to be involved in cell-cell communication such as exosomes and migrasomes, due to their specific nucleic acid and protein contents. When deposited by highly invasive breast cancer cells, internalized collagen-tracks reprogram non-invasive cells into highly pro-metastatic ones by inducing a partial epithelial-mesenchymal transition (EMT). This cell reprogramming is dependent on specific miRNAs present in the collagen-tracks, that are necessary to promote ECM degradation, increase cell motility and invasiveness. Collagen-tracks thus represent a new form of cell-cell communication important for driving tumor invasion that could be targeted to prevent metastasis.

cell biology↗

bioRxiv2025

Proteomic profiling of advanced hepatocellular carcinoma identifies predictive signatures of response to treatments

PurposeHepatocellular carcinoma (HCC) is the most common form of liver cancer with a bad prognosis in case of advanced HCC, only eligible for palliative systemic therapies. After a decade of exclusive sorafenib monotherapy, with a response rate of <10%, the advent of immunotherapies represents a revolution in HCC. The combination of atezolizumab/bevacizumab is recommended as the first-line systemic treatment, with a response rate around 30%. However, there are currently no predictive factors for response to these treatment options. Experimental DesignWe profiled, by high-resolution mass spectrometry-based proteomics combined with machine learning analysis, a selected cohort of fixed biopsies of advanced HCC. We grouped subjects according to their objective response to treatments, corresponded to a tumor regression vs tumor progression at 4 months after treatment. ResultsWe generated a proteome database of 50 selected HCC samples. We compared the relative protein abundance between tumoral and non-tumoral liver tissues from advanced HCC patients treated. The clear distinction of these two groups for each treatment is based on deregulation for 141 protein or 87 for atezolizumab/bevacizumab and sorafenib treatment, respectively. These specific proteomic signatures were sufficient to predict the response to treatment, and revealed biological pathways involved in treatments resistance. Particularly, we validated a shift in tumor cell metabolism with an immunosuppressive environment involved in the resistance to atezolizumab/bevacizumab combination. ConclusionsWe performed an in-depth analysis of quantitative proteomic data from HCC biopsies to predict the treatment response to advanced HCC giving the ability to optimize patient management.

cancer biology↗

bioRxiv2024

Role of the clathrin adaptor AP-1 in cell wall integrity and virulence factor secretion in the plant pathogen Botrytis cinerea

In filamentous fungi, the secretory pathway plays a key role in polarized growth through the dlivery of vesicles carrying enzymes and components necessary to cell wall synthesis and fungal nutrition. In pathogenic species, it moreover supports the delivery of virulence factors. In metazoa and yeast, the formation of several secretory vesicles occurs at the Golgi apparatus and endosomes through a complex and partially characterized machinery involving clathrin and adaptor proteins such as AP-1. Data on the molecular mechanisms controlling this process are still scarce in filamentous fungi. Using a mutant under-expressing the {beta}-subunit of the heterotetrameric AP-1 clathrin adaptor complex, we investigated the importance of intracellular vesicle trafficking in the phytopathogenic fungus Botrytis cinerea. The mutant exhibits pleiotropic developmental defects, an abnormality in cell wall integrity associated with a mislocalization of the plasma-membrane chitine synthase BcCHSIIIa. Moreover, the AP-1 mutant is affected in the secretion of hydrolytic enzymes and displays a severe defect in pathogenicity. This study confirms the importance of the AP1/clathrin machinery in the secretory pathway of B. cinerea.

microbiology↗

bioRxiv2024

Src promotes tumor cell invasion by hijacking the translation machinery

The Src oncogene controls cancer cell invasiveness by promoting invadosome formation and extracellular matrix degradation (ECM). Invadosomes are enriched in the eukaryotic translation initiation factor 3 (eIF3) complex associated with a local mRNA translation activity mandatory for their maintenance. Here, we show that Src regulates mRNA translation by controlling the expression of eIF3 subunits. Among them, eIF3h/e/d are essential for invadosome formation and ECM degradation. We demonstrate that Src controls the canonical mTOR/eIF4E and the non-canonical eIF3d cap-dependent translation initiation pathways. We show that both pathways are necessary for invadosome formation and their ECM degradation function. Finally, we highlighted a correlation between Src and eIF3h/e/d overexpression, which is associated with poor prognosis in hepatocellular carcinoma (HCC) patients and controls the ECM degradation and invasive properties of HCC cells. These findings identify Src as a major regulator of translation initiation pathways, which leads to invadosome formation, ECM degradation and tumor cell invasion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/606119v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1804203org.highwire.dtl.DTLVardef@16ddae8org.highwire.dtl.DTLVardef@13ecca5org.highwire.dtl.DTLVardef@1ed685a_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cell biology↗

bioRxiv2023

Repression of exosome secretion by mutated β-catenin contributes to immune escape in hepatocellular carcinoma

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of {beta}-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic {beta}-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of {beta}-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated {beta}-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated {beta}-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of {beta}-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic {beta}-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of {beta}-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

cancer biology↗

bioRxiv2023

Loss of RND3/RHOE controls entosis through LAMP1 expression in hepatocellular carcinoma

Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after Rnd3 silencing. We demonstrated that this process depends on RhoA/ROCK pathway, but not on E-cadherin. The proteomic profiling of entotic cells allowed us to identify LAMP1 as a protein upregulated by Rnd3 silencing and implicated not only in the degradation final stage of entosis, but also in the full mechanism. Moreover, we found a positive correlation between the presence of entotic cells and the metastatic potential of tumors in human patient samples. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for entosis analysis in medicine research as a novel therapeutic target.

cancer biology↗

bioRxiv2023

CXCR4 blockade alleviates pulmonary and cardiac outcomes in early COPD

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disease lacking effective treatment. Focusing on early COPD should help to discover disease modifying therapies. We aimed to examine the role of the CXCL12/CXCR4 axis in early COPD from both human samples and murine models. Blood samples and lung tissues of early COPD patients and controls were obtained in order to analyse CXCL12 and CXCR4 levels. To generate an early COPD model, ten-week-old male C57BL/6J mice were exposed to cigarette smoke (CS) for 10 weeks and intranasal instillations of polyinosinic-polycytidylic acid (poly(I:C)) for the last 5 weeks to mimic exacerbations. CXCR4 expressing cells number was increased in the blood of patients with COPD, as well as in the blood of exposed mice. Lung CXCL12 expression was higher in both early COPD patients and exposed mice. Exposed mice presented mild airway obstruction, peri-bronchial fibrosis and right heart thickening. The density of fibrocytes expressing CXCR4 was increased in the bronchial submucosa of these mice. Conditional inactivation of CXCR4 at adult stage as well as pharmacological inhibition of CXCR4 with plerixafor injections improved lung function, reduced inflammation, and protected against CS and poly-(I:C)-induced airway and cardiac remodeling. CXCR4-/- and plerixafor-treated mice also had less CXCR4-expressing circulating cells and a lower density of peri-bronchial fibrocytes. We demonstrate that targeting CXCR4 has beneficial effects in an animal model of early COPD and provide a framework to translate these preclinical findings to clinical settings in a drug repurposing approach. Clinical relevanceWe demonstrate that CXCL12/CXCR4 axis plays an important role in the pathogenesis of early COPD. Inhibition of this axis improves lung function and cardiac tissue remodeling, supporting the future use of CXCR4 inhibitors to slow down the progression of the disease.

pathology↗

bioRxiv2023

Novel axonemal protein ZMYND12 is required for flagellum function and male fertility

Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66 and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous ZMYND12 variants. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

genetics↗

bioRxiv2022

The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins

The large majority of mitochondrial proteins is synthesized in the cytosol and then imported to the organelle. To ensure proper mitochondrial functions, the quality of these proteins needs to be guaranteed. Here, we show that the E3 ubiquitin ligase F-box/LRR-repeat protein 6 (FBXL6) participates to the quality of these mitochondrial proteins at the level of the cytosolic translation. We found that lack of FBXL6 has severe effects including mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism and inhibited cell cycle progression in oxidative conditions. FBXL6 was found to interact specifically with ribosomal-associated quality control proteins and chaperones involved in the regulation of newly synthesized proteins and also it preferentially binds newly synthesized mitochondrial ribosomal proteins. Consistently, deletion of the RQC protein, NEMF or HSP70-family chaperone HSPA1A impedes FBXL6 interaction with its substrate. In addition, cells lacking FBXL6 display altered degradation of defective mitochondrial ribosomal protein containing C-terminal alanyl-threonyl extension.

cell biology↗

bioRxiv2021

The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. ImportanceUnusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

microbiology↗

bioRxiv2020

Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei

The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout ({Delta}pepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the {Delta}pepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the {Delta}pepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the {Delta}pepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.

microbiology↗

bioRxiv2020

The infection cushion: a fungal weapon of plant-biomass destruction

O_LIGrey mold disease affects fruits, vegetables and ornamental plants around the world, causing considerable losses every year. Its causing agent, the necrotrophic fungus Botrytis cinerea, produces infection cushions (IC) that are compound appressorial structures dedicated to the penetration of the plant tissues. C_LIO_LIA microarray analysis was performed to identify genes up-regulated in mature IC. The expression data were supported by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and mutagenesis of two candidate genes. C_LIO_LI1,231 up-regulated genes and 79 up-accumulated proteins were identified. They highlight a secretion of ROS, secondary metabolites including phytotoxins, and proteins involved in virulence: proteases, plant cell wall degrading enzymes and necrosis inducers. The role in pathogenesis was confirmed for two up-regulated fasciclin genes. DHN-melanin pathway and chitin deacetylases genes are up-regulated and the conversion of chitin into chitosan was confirmed by differential staining of the IC cell wall. In addition, up-regulation of sugar transport and sugar catabolism encoding genes was found. C_LIO_LIThese results support a role for the B. cinerea IC in plant penetration and suggest other unexpected roles for this fungal organ, in camouflage, necrotrophy or nutrition of the pathogen. C_LI

microbiology↗

bioRxiv2019

Counteracting resistance to targeted therapy in melanoma by inhibiting discoidin domain receptors

Anti-BRAF plus anti-MEK are used as first-line treatment of patients with metastatic melanomas harboring BRAF V600E mutation. The main issue with targeted therapy is acquired cellular resistance. In 70% of acquired resistance, melanoma cells switch their phenotype and become more aggressive and invasive. The molecular signature of this phenotype is MITF low, AXL high associated with actin cytoskeleton reorganization. After this switch, resistant cells present an anarchic cell proliferation due to MAP kinase pathway hyper-activation. We demonstrate that resistant cell lines presenting phenotype switching overexpress DDR1 and DDR2. We show that DDR2 inhibition induces a decrease in AXL and reduces actin stress fiber formation. Once this phenotype switching is acquired, we report that both DDRs promotes tumor cell proliferation, but only DDR2 can over-activate the MAP kinase pathway in resistant invasive cells in vitro and in vivo. Therefore, DDRs inhibition could be a promising strategy for countering this resistance mechanism. SignificanceOur results show that DDR2 is a relevant target in melanoma resistance. DDR2 is required at the beginning of resistance for melanoma cell phenotype switching to occur. After phenotype switching, DDRs promote tumor cell proliferation of resistant invasive melanoma cells, but only DDR2 is able to over-activate the MAP kinase pathway. We put forward dasatinib (a DDR inhibitor) as a potential second-line treatment after targeted dual therapy for resistant patients overexpressing DDRs.

cancer biology↗