Deciphering the Action of Neuraminidase in Glioblastoma Models

Nathalie Baeza-Kallee,Raphaël Bergès,Victoria Hein ,Stéphanie Cabaret ,Jeremy Garcia ,Abigaëlle Gros,Emeline Tabouret,Aurélie Tchoghandjian ,Carole Colin andDominique Figarella-Branger

Int. J. Mol. Sci. 2023, 24(14), 11645; https://doi.org/10.3390/ijms241411645

Abstract

Glioblastoma (GBM) contains cancer stem cells (CSC) that are resistant to treatment. GBM CSC expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity and tumorigenesis of GBM CSC. Our aim was to characterize the resulting effects of neuraminidase that removes A2B5 in order to target GBM CSC. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3 and GBM CSC lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography–mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM CSC lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM CSC lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Reuniting philosophy and science to advance cancer research

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Biological Reviews (2023)

Thomas Pradeu, Bertrand Daignan-Fornier, Andrew Ewald, Pierre-Luc Germain, Samir Okasha, Anya Plutynski, Sébastien Benzekry, Marta Bertolaso, Mina Bissell, Joel S. Brown, Benjamin Chin-Yee, Ian Chin-Yee, Hans Clevers, Laurent Cognet, Marie Darrason, Emmanuel Farge, Jean Feunteun, Jérôme Galon, Elodie Giroux, Sara Green, Fridolin Gross, Fanny Jaulin, Rob Knight, Ezio Laconi, Nicolas Larmonier, Carlo Maley, Alberto Mantovani, Violaine Moreau, Pierre Nassoy, Elena Rondeau, David Santamaria, Catherine M. Sawai, Andrei Seluanov, Gregory D. Sepich-Poore, Vanja Sisirak, Eric Solary, Sarah Yvonnet, Lucie Laplane

ABSTRACT

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Mitochondria Transfer from Mesenchymal Stem Cells Confers Chemoresistance to Glioblastoma Stem Cells through Metabolic Rewiring

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Jean Nakhle , Khattar Khattar , Tülin Özkan , Adel Boughlita, Daouda Abba Moussa , Amélie Darlix , Frédérique Lorcy, Valérie Rigau , Luc Bauchet, Sabine Gerbal-Chaloin , Martine Daujat-Chavanieu , Floriant Bellvert , Laurent Turchi , Thierry Virolle , Jean-Philippe Hugnot , Nicolas Buisine , Mireille Galloni , Valérie Dardalhon , Anne-Marie Rodriguez , Marie-Luce Vignais
Cancer Research Communications (2023) 3 (6): 1041–1056.

Abstract:

Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ.

Significance:

Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

Disruption of lineage integrity as a precursor to breast tumor initiation

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Trends In Cell Biology

DOI: https://doi.org/10.1016/j.tcb.2023.03.010

Abstract

Increase in lineage infidelity and/or imbalance is frequently observed around the earliest stage of breast tumor initiation. In response to disruption of homeostasis, differentiated cells can partially lose their identity and gain cellular plasticity, a process involving epigenome landscape remodeling. This increase of cellular plasticity may promote the malignant transformation of breast tumors and fuel their heterogeneity. Here, we review recent studies that have yield insights into important regulators of lineage integrity and mechanisms that trigger mammary epithelial lineage derail, and evaluate their impacts on breast tumor development.

ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR

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Denoulet M, Brulé M, Anquez F, Vincent A, Schnipper J, Adriaenssens E, Toillon RA, Le Bourhis X, Lagadec C.

Bioinformatics. 2023 Jan 1;39(1):btad008. doi: 10.1093/bioinformatics/btad008. PMID: 36629453; PMCID: PMC9846423.

Abstract

Motivation

Nowadays, epigenetic gene regulations are studied in each part of the biology, from embryonic development to diseases such as cancers and neurodegenerative disorders. Currently, to quantify and compare CpG methylation levels of a specific region of interest, the most accessible technique is the bisulfite sequencing PCR (BSP). However, no existing user-friendly tool is able to analyze data from all approaches of BSP. Therefore, the most convenient way to process results from the direct sequencing of PCR products (direct-BSP) is to manually analyze the chromatogram traces, which is a repetitive and prone to error task.

Results

Here, we implement a new R-based tool, called ABSP for analysis of bisulfite sequencing PCR, providing a complete analytic process of both direct-BSP and cloning-BSP data. It uses the raw sequencing trace files (.ab1) as input to compute and compare CpG methylation percentages. It is fully automated and includes a user-friendly interface as a built-in R shiny app, quality control steps and generates publication-ready graphics.

Availability and implementation

The ABSP tool and associated data are available on GitHub at https://github.com/ABSP-methylation-tool/ABSP.

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of primary and metastatic hepatocellular carcinoma

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Lozano A., Souche FR., Chavey C., Dardalhon V., Ramirez C., Vegna S., Desandre? G., Riviere A., , Zine El Aabidine A., Fort P., Akkari L., Hibner U. , Gregoire D.

eLife; 12:e76294

 

Abstract:

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Production of CRISPRi-engineered primary human mammary epithelial cells with baboon envelope pseudotyped lentiviral vectors

Sonia Pastor 1, Julien Wicinski 2, Emmanuelle Charafe-Jauffret 2, Els Verhoeyen 3, Geoffrey Guittard 4, Christophe Ginestier 5

STAR PROTOCOL 2023 Jan 19;4(1):102055

doi: 10.1016/j.xpro.2023.102055

 

Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022).1.

Challenges in glioblastoma research: focus on the tumor microenvironment

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Bikfalvi A1,da Costa CA2,Avril T3,Barnier JV4,Bauchet L5,Brisson L1,Cartron PF6,Castel H7,Chevet E3,Chneiweiss H8,Clavreul A9,Constantin B10,Coronas V10,Daubon T11,Dontenwill M12,Ducray F13,Enz-Werle N12,Figarella-Branger D14,Fournier I15,Frenel JS7,Gabut M13,Galli T16,Gavard J6,Huberfeld G17,Hugnot JP18,Idbaih A19,Junier MP8,Mathivet T1,Menei P9,Meyronet D20,Mirjolet C21,Morin F7,Mosser J3,Moyal EC22,Rousseau V4,Salzet M15,Sanson M19,Seano G23,Tabouret E24,Tchoghandjian A24,Turchi L25,Vallette FM6,Vats S16,Verreault M19,& Virolle T25

Trends in Cancer, 16 Nov 2022, 9(1):9-27
DOI: 10.1016/j.trecan.2022.09.005 PMID: 36400694 

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Basal-like mammary carcinomas stimulate cancer stem cell properties through AXL-signaling to induce chemotherapy resistance

Garyfallia Pantelaiou-Prokaki, Oliver Reinhardt, Nadine S. Georges, David J. Agorku, Olaf Hardt, Evangelos Prokakis, Iga K. Mieczkowska, Wolfgang Deppert, Florian Wegwitz, Frauke Alves

 

MOLECULAR CANCER BIOLOGY

Abstract

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.

A new natural killer cell-specific gene signature predicting recurrence in colorectal cancer patients

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Carolyn Shembrey , Momeneh Foroutan , Frédéric Hollande 

Front Immunol. 2023 Jan 6;13:1011247. doi: 10.3389/fimmu.2022.1011247

Abstract

The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which underlie NK cell sensitivity. Here, a novel CRC-specific NK cell gene signature that infers NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. In contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the signature includes genes representative of NK cells of different maturation states, activation status and anatomical origin. Our signature also accurately discriminates murine NK cells, demonstrating the applicability of this geneset when mining datasets generated from preclinical studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.

CD44v3 is a marker of invasive cancer stem cells driving metastasis in gastric carcinoma

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Julie Giraud, Lornella Seeneevassen, Benoit Rousseau, Damien Bouriez, Elodie Sifré, Alban Giese, Tra Ly Nguyen, Camille Tiffon, Yannick Lippi, Lamia Azzi-Martin, Julie Pannequin, Armelle Ménard, Emilie Bessède, Cathy Staedel, Francis Mégraud, Geneviève Belleannée, Philippe Lehours, Caroline Gronnier, Pierre Dubus & Christine Varon 

Gastric Cancer (2022)

Abstract

Background

Cancer stem cells (CSCs) are at the origin of tumour initiation and progression in gastric adenocarcinoma (GC). However, markers of metastasis-initiating cells remain unidentified in GC. In this study, we characterized CD44 variants expressed in GC and evaluated the tumorigenic and metastatic properties of CD44v3+?cells and their clinical significance in GC patients.

Methods

Using GC cell lines and patient-derived xenografts, we evaluated CD44+?and CD44v3+?GC cells molecular signature and their tumorigenic, chemoresistance, invasive and metastatic properties, and expression in patients-derived tissues.

Results

CD44v3+?cells, which represented a subpopulation of CD44+?cells, were detected in advanced preneoplastic lesions and presented CSCs chemoresistance and tumorigenic properties in vitro and in vivo. Molecular and functional analyses revealed two subpopulations of gastric CSCs: CD44v3+?CSCs with an epithelial-mesenchymal transition (EMT)-like signature, and CD44+/v3– CSCs with an epithelial-like signature; both were tumorigenic but CD44v3+ cells showed higher invasive and metastatic properties in vivo. CD44v3+?cells detected in the primary tumours of GC patients were associated with a worse prognosis.

Conclusion

CD44v3 is a marker of a subpopulation of CSCs with metastatic properties in GC. The identification of metastasis-initiating cells in GC represents a major advance for further development of anti-metastatic therapeutic strategies.

Emerging Roles of Hedgehog Signaling in Cancer Immunity

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Alessandro Giammona, Enrica Crivaro, Barbara Stecca

Int. J. Mol. Sci. 2023, 24(2), 1321; https://doi.org/10.3390/ijms24021321

Abstract

Hedgehog–GLI (HH) signaling plays an essential role in embryogenesis and tissue homeostasis. Aberrant activation of the pathway through mutations or other mechanisms is involved in the development and progression of numerous types of cancer, including basal cell carcinoma, medulloblastoma, melanoma, breast, prostate, hepatocellular and pancreatic carcinomas. Activation of HH signaling sustains proliferation, suppresses cell death signals, enhances invasion and metastasis, deregulates cellular metabolism and promotes angiogenesis and tumor inflammation. Targeted inhibition of the HH pathway has therefore emerged as an attractive therapeutic strategy for the treatment of a wide range of cancers. Currently, the Smoothened (SMO) receptor and the downstream GLI transcriptional factors have been investigated for the development of targeted drugs. Recent studies have revealed that the HH signaling is also involved in tumor immune evasion and poor responses to cancer immunotherapy. Here we focus on the effects of HH signaling on the major cellular components of the adaptive and innate immune systems, and we present recent discoveries elucidating how the immunosuppressive function of the HH pathway is engaged by cancer cells to prevent immune surveillance. In addition, we discuss the future prospect of therapeutic options combining the HH pathway and immune checkpoint inhibitors.

Stochasticity and Drug Effects in Dynamical Model for Cancer Stem Cells

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Ludovico Mori, Martine Ben Amar

Cancers 2023, 15(3), 677; https://doi.org/10.3390/cancers15030677

Abstract

The Cancer Stem Model allows for a dynamical description of cancer colonies which accounts for the existence of different families of cells, namely stem cells, highly proliferating and quasi-immortal, and differentiated cells, both undergoing cellular processes under numerous activated pathways. In the present work, we investigate a dynamical model numerically, as a system of coupled differential equations, and include a plasticity mechanism, of differentiated cells turning into a stem state if the stem concentration drops low. We are particularly interested in the stability of the model once we introduce stochastically evolving parameters, associated with environmental and cellular intrinsic variabilities, as well as the response of the model after introducing a drug therapy. As long as we stay within the characteristic time scale of the system, defined on the base of the needed time for the trajectories to converge on stable states, we observe that the system remains stable for the main parameters evolving stochastically according to white noise. As for the drug treatments, we discuss a model both for the kinetics and the dynamics of the substance in the organism, and then consider the impact of different types of therapies in a few particular examples, outlining some interesting mechanisms, such as the tumor growth paradox, that possibly impact the outcome of therapy significantly.

A2B5 Expression in Central Nervous System and Gliomas

Dominique Figarella-Branger , Carole Colin , Nathalie Baeza-Kallee , Aurélie Tchoghandjian

A2B5 IgM recognizes c-series gangliosides with three sialic acids. The aim of this review was to focus on A2B5 expression in the central nervous system and gliomas. In brain development, A2B5+ cells are recorded in areas containing multipotent neural stem cells (NSC). In adults, A2B5+ cells persist in neurogenic areas and in white matter where it identifies oligodendrocyte precursor cells (OPCs) but also cells with NSC properties. Although the expression of A2B5 has been widely studied in culture, where it characterizes bipotential glial progenitor cells, its expression in vivo is less characterized mainly because of technical issues. A new interest was given to the NSCs and OPCs since the discovery of cancer stem cells (CSC) in gliomas. Among other cell surface molecules, A2B5 has been identified as an accurate marker to identify glioma CSCs. We and others have shown that all types of gliomas express A2B5, and that only A2B5+ cells, and not A2B5- cells, can generate a tumor after orthotopic implantation in immunocompromised animals. Moreover, A2B5 epitope expression is positively correlated with stemness and tumor growth. This review highlights that A2B5 is an attractive target to tackle glioma CSCs, and a better characterization of its expression in the developing and adult CNS will benefit to a better understanding of gliomagenesis.

ROBO3s: a novel ROBO3 short isoform promoting breast cancer aggressiveness

Marcel Werner, Anna Dyas, Iwan Parfentev, Geske E Schmidt, Iga K Mieczkowska , Lukas C Müller-Kirschbaum , Claudia Müller , Stefan Kalkhof , Oliver Reinhardt , Henning Urlaub, Frauke Alves, Julia Gallwas, Evangelos Prokakis, Florian Wegwitz

Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.

Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity

A. Huyghe, G. Furlan, J. Schroeder, E. Cascales, A. Trajkova, M. Ruel, F. Stüder, M. Larcombe, Y. Bo Yang Sun, F. Mugnier, L. De Matteo, A. Baygin, J. Wang, Y. Yu, N. Rama, B. Gibert, J. Kielbassa, L. Tonon, P. Wajda, N. Gadot, M. Brevet, M. Siouda, P. Mulligan, R. Dante, P. Liu, H. Gronemeyer, M. Mendoza-Parra, J. M. Polo & F. Lavial

Nature Cell Biology volume 24, pages1350–1363 (2022)

 

Abstract

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.

Multivariate Analysis of RNA Chemistry Marks Uncovers Epitranscriptomics-Based Biomarker Signature for Adult Diffuse Glioma Diagnostics

S. Relier; A. Amalric; A. Attina; I.B. Koumare; V. Rigau; F. Burel Vandenbos; D. Fontaine; M. Baroncini; J.P. Hugnot; H. Duffau; L. Bauchet; C. Hirtz*; E. Rivals* & A. David*

Anal. Chem. 2022

One of the main challenges in cancer management relates to the discovery of reliable biomarkers, which could guide decision-making and predict treatment outcome. In particular, the rise and democratization of high-throughput molecular profiling technologies bolstered the discovery of “biomarker signatures” that could maximize the prediction performance. Such an approach was largely employed from diverse OMICs data (i.e., genomics, transcriptomics, proteomics, metabolomics) but not from epitranscriptomics, which encompasses more than 100 biochemical modifications driving the post-transcriptional fate of RNA: stability, splicing, storage, and translation. We and others have studied chemical marks in isolation and associated them with cancer evolution, adaptation, as well as the response to conventional therapy. In this study, we have designed a unique pipeline combining multiplex analysis of the epitranscriptomic landscape by high-performance liquid chromatography coupled to tandem mass spectrometry with statistical multivariate analysis and machine learning approaches in order to identify biomarker signatures that could guide precision medicine and improve disease diagnosis. We applied this approach to analyze a cohort of adult diffuse glioma patients and demonstrate the existence of an “epitranscriptomics-based signature” that permits glioma grades to be discriminated and predicted with unmet accuracy. This study demonstrates that epitranscriptomics (co)evolves along cancer progression and opens new prospects in the field of omics molecular profiling and personalized medicine.

Genome-wide RNA interference screen in cancer stem cell

Guillaume Pinna 1, Marie Vandamme 1, Celia Rouault 2, Emmanuelle Charafe-Jauffret 2, Christophe Ginestier 3

  • 1Plateforme ARN interférence (PARi), Université Paris Cité & Université Paris-Saclay, Inserm, iRCM/IBFJ CEA, Stabilité Génétique Cellules Souches et Radiations, Fontenay-aux-Roses, France.
  • 2CRCM, Inserm, CNRS, Institut Paoli-Calmettes, Aix-Marseille Université, Epithelial Stem Cells and Cancer Lab, Equipe labellisée LIGUE contre le cancer, Marseille, France.
  • 3CRCM, Inserm, CNRS, Institut Paoli-Calmettes, Aix-Marseille Université, Epithelial Stem Cells and Cancer Lab, Equipe labellisée LIGUE contre le cancer, Marseille, France. Electronic address: christophe.ginestier@inserm.fr.

Tumor heterogeneity represents a major hurdle for therapy. This cellular heterogeneity is mainly sustained by different subpopulations of tumorigenic cells, the so-called cancer stem cells (CSCs). CSCs burden is associated with disease progression and patient poor prognosis. In this context, deciphering molecular mechanisms regulating stemness is a key step in the development of new therapeutic strategy. Here, we provide a detailed protocol for high-throughput screening (HTS) strategy to detect modulators of CSC proportion. It is based on a miniaturized ALDEFLUOR-probed CSC assay quantitated by high-content imaging, that allows monitoring the changes in CSC proportions in response to gene silencing. Gene loss-of-function is achieved by transfecting a genome-wide RNA interference library. These genome-wide HTS strategies could lead to the identification of new therapeutic approaches in the treatment of various cancers.

XIST loss impairs mammary stem cell differentiation and increases tumorigenicity through Mediator hyperactivation

Laia Richart 1, Mary-Loup Picod-Chedotel 2, Michel Wassef 1, Manon Macario 2, Setareh Aflaki 1, Marion A Salvador 2, Tiphaine Héry 1, Aurélien Dauphin 1, Julien Wicinski 2, Véronique Chevrier 2, Sonia Pastor 3, Geoffrey Guittard 3, Samuel Le Cam 1, Hanya Kamhawi 2, Rémy Castellano 4, Géraldine Guasch 2, Emmanuelle Charafe-Jauffret 5, Edith Heard 6, Raphaël Margueron 7, Christophe Ginestier 8

Cell, 2022 May 13;S0092-8674(22)00532-3

Summary

X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.

Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

Guillaume Jacquemin Annabelle Wurmser Mathilde Huyghe Wenjie Sun Zeinab Homayed Candice Merle Meghan Perkins Fairouz Qasrawi Sophie Richon Florent Dingli Guillaume Arras Damarys Loew Danijela Vignjevic Julie Pannequin Silvia Fre

E-Life2022 May 11;11:e76541.

Abstract

Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context. We identify the glycoprotein thrombospondin-1 (THBS1) as the essential factor that mediates non-cell-autonomous morphological and transcriptional responses. Importantly, Thbs1 is associated with bad prognosis in several human cancers. This study reveals the THBS1-YAP axis as the mechanistic link mediating paracrine interactions between epithelial cells in intestinal tumours.

Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

Feliz Morel, Á.J.; Hasanovic, A.; Morin, A.; Prunier, C.; Magnone, V.; Lebrigand, K.; Aouad, A.; Cogoluegnes, S.; Favier, J.; Pasquier, C.; Mus-Veteau, I.

Pharmaceutics 2022, 14, 988.

Abstract

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

Bansard L, Bouvet O, Moutin E, Le Gall G, Giammona A, Pothin E, Bacou M, Hassen-Khodja C, Bordignon B, Bourgaux JF, Prudhomme M, Hollande F, Pannequin J, Pascussi JM, Planque C.

2022 Feb 22;S2213-6711(22)00094-7

Abstract

Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

Abstract

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells’ (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.

HDAC8 suppresses the epithelial phenotype and promotes EMT in chemotherapy-treated basal-like breast cancer

Abstract:

Background

Basal-like breast cancer (BLBC) is one of the most aggressive malignant diseases in women with an increased metastatic behavior and poor prognosis compared to other molecular subtypes of breast cancer. Resistance to chemotherapy is the main cause of treatment failure in BLBC. Therefore, novel therapeutic strategies counteracting the gain of aggressiveness underlying therapy resistance are urgently needed. The epithelial-to-mesenchymal transition (EMT) has been established as one central process stimulating cancer cell migratory capacity but also acquisition of chemotherapy-resistant properties. In this study, we aimed to uncover epigenetic factors involved in the EMT-transcriptional program occurring in BLBC cells surviving conventional chemotherapy.

Results

Using whole transcriptome data from a murine mammary carcinoma cell line (pG-2), we identified upregulation of Hdac4, 7 and 8 in tumor cells surviving conventional chemotherapy. Subsequent analyses of human BLBC patient datasets and cell lines established HDAC8 as the most promising factor sustaining tumor cell viability. ChIP-sequencing data analysis identified a pronounced loss of H3K27ac at regulatory regions of master transcription factors (TFs) of epithelial phenotype like Gata3, Elf5, Rora and Grhl2 upon chemotherapy. Interestingly, impairment of HDAC8 activity reverted epithelial-TFs levels. Furthermore, loss of HDAC8 activity sensitized tumor cells to chemotherapeutic treatments, even at low doses.

Conclusion

The current study reveals a previously unknown transcriptional repressive function of HDAC8 exerted on a panel of transcription factors involved in the maintenance of epithelial cell phenotype, thereby supporting BLBC cell survival to conventional chemotherapy. Our data establish HDAC8 as an attractive therapeutically targetable epigenetic factor to increase the efficiency of chemotherapeutics.

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.

Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.

In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance

Abstract

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5?-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

SLUG and Truncated TAL1 Reduce Glioblastoma Stem Cell Growth Downstream of Notch1 and Define Distinct Vascular Subpopulations in Glioblastoma Multiforme

Sophie Guelfi 1 2, Béatrice Orsetti 3, Virginie Deleuze 4, Valérie Rigau 5 6, Luc Bauchet 6 7, Hugues Duffau 6 7, Bernard Rothhut 2, Jean-Philippe Hugnot 6 8

Affiliations

  • 1VIB-KU Leuven Center for Cancer Biology, 3000 Leuven, Belgium.
  • 2Institut des Neurosciences de Montpellier, University of Montpellier (UM), Institut National de la Santé et la Recherche Médicale (INSERM), 34091 Montpellier, France.
  • 3Institut de Recherche en Cancérologie de Montpellier, University of Montpellier (UM), INSERM, 34298 Montpellier, France.
  • 4Institut de Génétique Moléculaire de Montpellier, University of Montpellier (UM), Centre National de la Recherche Scientifique (CNRS), 34293 Montpellier, France.
  • 5Department of Pathology and Oncobiology, Hôpital Gui de Chauliac, 34295 Montpellier, France.
  • 6Institut de Génomique Fonctionelle, University of Montpellier (UM), CNRS, INSERM, 34094 Montpellier, France.
  • 7Neurosurgery Department, Hôpital Gui de Chauliac, 34295 Montpellier, France.
  • 8Department of Biology, University of Montpellier (UM), CEDEX 5, 34095 Montpellier, France.

Abstract

Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-?1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.

The Diverse Applications of Pancreatic Ductal Adenocarcinoma Organoids

The Diverse Applications of Pancreatic Ductal Adenocarcinoma Organoids

Ronnie Ren Jie Low, Wei Wen Lim , Paul M. Nguyen , Belinda Lee , Michael Christie, Antony W. Burgess ,Peter Gibbs ,Sean M. Grimmond ,Frédéric Hollande andTracy L. Putoczki

Cancers 2021, 13(19), 4979

Simple Summary

Patients diagnosed with pancreatic cancer have very few treatment options. In order to identify new treatment opportunities, and develop new drugs for clinical use, appropriate model systems that take into account the complexities of a tumor are required. In this review, we summarize the current and emerging opportunities to accurately model pancreatic cancer using organoid technologies. We highlight the need for continued development of these complex model systems in order to inform personalized treatment.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies. While immortalized cancer cell lines and genetically engineered murine models have increased our understanding of PDAC tumorigenesis, they do not recapitulate inter- and intra-patient heterogeneity. PDAC patient derived organoid (PDO) biobanks have overcome this hurdle, and provide an opportunity for the high throughput screening of potential new therapies. This review provides a summary of the PDAC PDO biobanks established to date, and discusses how they have advanced our understanding of PDAC biology. Looking forward, the development of coculturing techniques for specific immune or stromal cell populations will enable a better understanding of the crosstalk that occurs within the tumor microenvironment, and the impact of this crosstalk on treatment response.

Integrative Analysis to Identify Genes Associated with Stemness and Immune Infiltration in Glioblastoma

Neerada Meenakshi Warrier,Prasoon Agarwal and Praveen Kumar 

Abstract

It is imperative to identify the mechanisms that confer stemness to the cancer cells for more effective targeting. Moreover, there are not many studies on the link between stemness characteristics and the immune response in tumours. Therefore, in the current study involving GBM, we started with the study of BIRC5 (one of the rare genes differentially expressed in normal and cancer cells) and CXCR4 (gene involved in the survival and proliferation of CSCs). Together, these genes have not been systematically explored. We used a set of 27 promoter methylated regions in GBM. Our analysis showed that four genes corresponding to these regions, namely EOMES, BDNF, HLA-A, and PECAM1, were involved with BIRC5 and CXCR4. Interestingly, we found EOMES to be very significantly involved in stemness and immunology and it was positively correlated to CXCR4. Additionally, BDNF, which was significant in methylation, was negatively correlated to BIRC5

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM

Simple Summary

In the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity.

Abstract

Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.