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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
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
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
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
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.
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.
SLUG and Truncated TAL1 Reduce Glioblastoma Stem Cell Growth Downstream of Notch1 and Define Distinct Vascular Subpopulations in Glioblastoma Multiforme
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
Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance
Abstract
CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM
Simple Summary
Abstract
Integrative Analysis to Identify Genes Associated with Stemness and Immune Infiltration in Glioblastoma
Integrative Analysis to Identify Genes Associated with Stemness and Immune Infiltration in Glioblastoma
Abstract
A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration
doi: 10.1038/s41467-021-23034-x.
A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration
Abstract
At numerous locations of the body, transition zones are localized at the crossroad between two types of epithelium and are frequently associated with neoplasia involving both type of tissues. These transition zones contain cells expressing markers of adult stem cells that can be the target of early transformation. The mere fact that transition zone cells can merge different architecture with separate functions implies for a unique plasticity that these cells must display in steady state. However, their roles during tissue regeneration in normal and injured state remain unknown. Here, by using in vivo lineage tracing, single-cell transcriptomics, computational modeling and a three-dimensional organoid culture system of transition zone cells, we identify a population of Krt17+ basal cells with multipotent properties at the squamo-columnar anorectal junction that maintain a squamous epithelium during normal homeostasis and can participate in the repair of a glandular epithelium following tissue injury.
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
Simple Summary
Abstract
Autophagy in Osteosarcoma Cancer Stem Cells Is Critical Process which Can Be Targeted by the Antipsychotic Drug Thioridazine
Olivier Camuzard , Marie-Charlotte Trojani , Sabine Santucci-Darmanin , Sophie Pagnotta , Véronique Breuil, Georges F Carle , Valérie Pierrefite-Carle
Cancer stem cells (CSCs) represent a minor population of cancer cells with stem cell-like properties which are able to fuel tumor growth and resist conventional treatments. Autophagy has been described to be upregulated in some CSCs and to play a crucial role by maintaining stem features and promoting resistance to both hostile microenvironments and treatments. Osteosarcoma (OS) is an aggressive bone cancer which mainly affects children and adolescents and autophagy in OS CSCs has been poorly studied. However, this is a very interesting case because autophagy is often deregulated in this cancer. In the present work, we used two OS cell lines showing different autophagy capacities to isolate CSC-enriched populations and to analyze the autophagy in basal and nutrient-deprived conditions. Our results indicate that autophagy is more efficient in CSCs populations compared to the parental cell lines, suggesting that autophagy is a critical process in OS CSCs. We also showed that the antipsychotic drug thioridazine is able to stimulate, and then impair autophagy in both CSC-enriched populations, leading to autosis, a cell death mediated by the Na+/K+ ATPase pump and triggered by dysregulated accumulation of autophagosomes. Taken together, our results indicate that autophagy is very active in OS CSCs and that targeting this pathway to switch their fate from survival to death could provide a novel strategy to eradicate these cells in osteosarcoma.
Cancers, 2020 Dec 7;12(12):3675
doi: 10.3390/cancers12123675.
CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies
Dongrui Wang, Briana C Prager, Ryan C Gimple, Brenda Aguilar, Darya Alizadeh, Hongzhen Tang, Deguan Lv, Renate Starr, Alfonso Brito, Qiulian Wu, Leo J.Y Kim, Zhixin Qiu, Peng Lin, Michael H. Lorenzini, Behnam Badie, Stephen J Forman, Qi Xie, Christine E Brown and Jeremy N. Rich
Cancer Discov; Published OnlineFirst December 16, 2020; doi:10.1158/2159-8290.CD-20-1243
Dependence receptors: new targets for cancer therapy
Dependence receptors: new targets for cancer therapy
Morgan Brisset 1 2, Mélodie Grandin 1 2, Agnès Bernet 3, Patrick Mehlen 3, Frédéric Hollande 1 2
1 Department of Clinical Pathology, Victorian Comprehensive Cancer Centre, The University of Melbourne, Melbourne, Vic., Australia.
2 University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, Melbourne, Vic., Australia.
3 Apoptosis, Cancer and Development Laboratory, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Centre Léon Bérard, Université de Lyon, Lyon, France.
Abstract
Dependence receptors are known to promote survival and positive signaling such as proliferation, migration, and differentiation when activated, but to actively trigger apoptosis when unbound to their ligand. Their abnormal regulation was shown to be an important feature of tumorigenesis, allowing cancer cells to escape apoptosis triggered by these receptors while promoting in parallel major aspects of tumorigenesis such as proliferation, angiogenesis, invasiveness, and chemoresistance. This involvement in multiple cancer hallmarks has raised interest in dependence receptors as targets for cancer therapy. Although additional studies remain necessary to fully understand the complexity of signaling pathways activated by these receptors and to target them efficiently, it is now clear that dependence receptors represent very exciting targets for future cancer treatment. This manuscript reviews current knowledge on the contribution of dependence receptors to cancer and highlights the potential for therapies that activate pro-apoptotic functions of these proteins.
Direct and Indirect Regulators of Epithelial-Mesenchymal Transition (EMT)-mediated Immunosuppression in Breast Carcinomas
ERK-Mediated Loss of miR-199a-3p and Induction of EGR1 Act as a “Toggle Switch” of GBM Cell Dedifferentiation into NANOG- and OCT4-Positive Cells
Fabien Almairac , Laurent Turchi , Nathalie Sakakini , David Nicolas Debruyne , Sarah Elkeurti , Elisabet Gjernes , Beatrice Polo , Laurence Bianchini , Denys Fontaine , Philippe Paquis , Herve Chneiweiss , Marie-Pierre Junier , Patrick Verrando , Fanny Burel-Vandenbos , Thierry Virolle.
There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here, we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas (GBM) and that have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated “toggle switch” that promotes pluripotency marker expression and stem-like features in GBM cells. SIGNIFICANCE: This study defines an ERK-mediated molecular mechanism of dedifferentiation of GBM cells into a stem-like state, expressing markers of pluripotency
doi: 10.1158/0008-5472.CAN-19-0855. Epub 2020 May 4.
FTO-Mediated Cytoplasmic m6Am Demethylation Adjusts Stem-Like Properties in Colorectal Cancer Cell
Sébastien Relier 1, Julie Ripoll 2, Hélène Guillorit 1 3, Amandine Amalric 1, Cyrinne Achour 4 5, Florence Boissière 6, Jérôme Vialaret 7 8, Aurore Attina 7 8, Françoise Debart 9, Armelle Choquet 1, Françoise Macari 1, Virginie Marchand 10, Yuri Motorin 10, Emmanuelle Samalin 1 6, Jean-Jacques Vasseur 9, Julie Pannequin 1, Francesca Aguilo 4 5, Evelyne Lopez-Crapez 6, Christophe Hirtz 7 8, Eric Rivals 11, Amandine Bastide 12, Alexandre David 13 14
- 1IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France.
- 2LIRMM, Univ. Montpellier, CNRS, Montpellier, France.
- 3Stellate Therapeutics, Paris, France.
- 4Wallenberg Centre for Molecular Medicine (WCMM), Umea University, Umea, Sweden.
- 5Department of Medical Biosciences, Umea University, Umea, Sweden.
- 6ICM, Montpellier, France.
- 7IRMB-PPC, Univ. Montpellier, INSERM, CHU Montpellier, CNRS, Montpellier, France.
- 8INM, Univ. Montpellier, INSERM, Montpellier, France.
- 9IBMM, CNRS, Univ. Montpellier, ENSCM, Montpellier, France.
- 10Université de Lorraine, IMoPA UMR7365 CNRS-UL and UMS2008/US40 IBSLor, UL-CNRS-INSERM, BioPole, Vandoeuvre-les-Nancy, France.
- 11LIRMM, Univ. Montpellier, CNRS, Montpellier, France. rivals@lirmm.fr.
- 12IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France. amandine.bastide@igf.cnrs.fr.
- 13IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France. alexandre.david@igf.cnrs.fr.
- 14IRMB-PPC, Univ. Montpellier, INSERM, CHU Montpellier, CNRS, Montpellier, France. alexandre.david@igf.cnrs.fr.
Abstract
Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2′-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.
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