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CD44 regulates epigenetic plasticity by mediating iron endocytosis

Abstract

CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial–mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.

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Fig. 1: CD44 mediates Hyal-dependent iron endocytosis.
Fig. 2: CD44-mediated iron endocytosis prevails in the mesenchymal state of cells.
Fig. 3: EMT is characterized by a redox signature that implicates iron.
Fig. 4: Nuclear iron is a rate-limiting regulator of epigenetic plasticity.
Fig. 5: Targeting iron homeostasis interferes with the maintenance of mesenchymal cells.
Fig. 6: Reciprocal endocytic–epigenetic regulation that involves iron.

Data availability

All data are available in the manuscript or the Supplementary Information. Mass spectrometry data have been deposited at the ProteomeXchange Consortium (PRIDE Archive) with identifiers PXD011447 and PXD012862. ChIP-seq and RNA-seq data are available on the National Center for Biotechnology Information website with accession reference GSE121664. Source data are provided with this paper.

Code availability

Code employed for ChIP-seq and RNA-seq data analyses are available on Github at https://github.com/nservant/EMTiron.

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Acknowledgements

We thank A. Puisieux, C. Hivroz and S. Dogniaux for providing us with HMLER and primary human T cells, the PICT-IBiSA@Pasteur Imaging Facility of Institut Curie, a member of the France-BioImaging national research infrastructure for the use of microscopes and SIMS, C. Gaillet for assistance with NMR spectroscopy, J.-L. Guerquin-Kern for assistance with SIMS sample preparation, the ICP-MS platform at the Institut de Physique du Globe de Paris, G. Arras for assistance with mass spectrometry data analysis, S. Durand and G. Kroemer for providing access to the metabolomics platform and P. Legoix for NGS sample preparation. The R.R. research group is funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 647973), the Fondation Charles Defforey-Institut de France and Ligue Contre le Cancer (Equipe Labellisée). R.R. and D.L. are supported by Region IdF for NMR and MS infrastructures. The Institut de Physique du Globe de Paris is supported by the IPGP multidisciplinary program PARI and Paris–Region IdF (SESAME grant agreement no. 12015908). High-throughput sequencing was performed by the ICGex NGS platform of Institut Curie, supported by ANR-10-EQPX-03 (Equipex), ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche (Investissements d’Avenir program) and by the Cancéropole IdF and the SiRIC-Curie program—SiRIC Grant (INCa-DGOS-4654).

Author information

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Authors

Contributions

R.R. conceptualized the study and directed the research. R.R., S.M. and F.S. designed the experiments. T.C. performed NMR spectroscopy and synthesized the clickable iron chelators. A.V. synthesized the iron(ii)-specific fluorescent probes. S.M. produced knock out cell lines and performed the experiments in relation to iron endocytosis, which included western blotting, cell imaging, RNA interference, flow cytometry and ICP-MS. T.-D.W. performed SIMS imaging. A.L. performed RT-qPCR and subcellular fractionation experiments. F.S. prepared the samples for quantitative proteomics, metabolomics and next generation sequencing. B.L. and D.L. carried out quantitative proteomics. E.C.-J. and C.G. provided tumour samples and performed cell sorting. A.D., C.V. and S.B. provided assistance with the NGS library preparation. N.S. performed bioinformatics analysis. R.R., S.M. and F.S. interpreted the data and wrote the article.

Corresponding author

Correspondence to Raphaël Rodriguez.

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R.R. is a founder, shareholder and serves on the scientific advisory board of SideROS.

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Extended data

Extended Data Fig. 1 CD44-mediates iron endocytosis in distinct cell lines and primary cells.

a, Fluorescence microscopy of RhoNox-M-positive vesicles colocalizing with internalized Hyal-FITC or TF-647 (left panels). Scale bars, 10 μm. n = 3 biologically independent experiments for MDA-MB-468, U2OS and HT1080 cell lines and n = 1 for primary cancer cells and the LNCaP cell line. Flow cytometry of CD44 (right panels). b, Fluorescence microscopy of RhoNox-M-positive vesicles colocalizing with internalized Hyal-FITC in primary human T cells (left panel). Scale bar, 2 μm. n = 3 biologically independent experiments. Flow cytometry of CD44 (right panel). Bars and error bars, mean values ± s.d.

Extended Data Fig. 2 CD44-mediated iron uptake is Hyal-dependent in distinct cell lines and primary cells.

a, Flow cytometry of RhoNox-M fluorescence in HMLER CD44 and TFRC ko clones and a MCF7 CD44 ko clone treated with Hyal. b, Flow cytometry of RhoNox-M fluorescence in primary cancer cells and primary T cells treated with Hyal. n = 1. c, Flow cytometry of RhoNox-M fluorescence in MDA-MB-468 cells treated with Hyal of varying molecular mass. d, and e, Flow cytometry of RhoNox-M in MDA-MB-468, primary lung CTC, HMLER CD44high and MCF7 cells treated with Hyal, hyaluronidase or blocking antibodies. n = 1. HMM Hyal (0.6-1 MDa) was used in a, b, d. Data representative of n = 3 biologically independent experiments for a and c.

Extended Data Fig. 3 CD44-mediated iron endocytosis prevails in the mesenchymal state of cells.

a, Time course flow cytometry of CD44 and TfR1 at plasma membrane of MCF7 and HMLER CD44low cells treated with OSM or TGF-β as indicated. Data representative of n = 3 biologically independent experiments. b, Fluorescence microscopy of RhoNox-M-positive vesicles colocalizing with internalized Hyal-FITC or TF-647 in cells treated as indicated for 72 h. Scale bars, 10 μm. n = 3 biologically independent experiments for MCF7 cells and n = 1 for primary cells. Bars and error bars, mean values ± s.d. Unpaired t-tests, two-sided.

Extended Data Fig. 4 Quantitative analysis of proteins and metabolites in cells undergoing EMT.

a, Gene Ontology-term enrichment heatmap of proteins ranked by molecular function illustrating a bias towards proteins with oxidoreductase activity being increased in the mesenchymal cell state. n = 3 biologically independent experiments. Statistical analysis, Material and Methods. b, Western blots of selected proteins. Data representative of n = 3 biologically independent experiments. c, Heatmap of metabolites in cells treated with EGF for 60 h. n = 4 technical replicates. MDA-MB-468 cells throughout the figure and treated with EGF for 72 h unless stated otherwise.

Extended Data Fig. 5 Genome-wide analysis of histone marks in cells undergoing EMT.

a, H3K9me2 ChIP-seq profiles for selected genes. n = 3 biologically independent experiments. b, ChIP-qPCR of selected genes. n = 2 biologically independent experiments. ce, ChIP-seq profiles of H3K4me3, H3K27me3 and H3K9me3 for selected genes. n = 2 biologically independent experiments. MDA-MB-468 cells were used throughout the figure and treated with EGF for 72 h.

Extended Data Fig. 6 Pharmacological targeting of iron-regulated processes.

a, Western blots of H3K9me2 in cells co-treated with OSM and DFO or TGF-β and DFO as indicated. n = 1 for primary cells. b, Western blots of proteins whose genes are regulated by H3K9me2 in cells co-treated with OSM and DFO or TGF-β and DFO as indicated. n = 1 for primary cells. c, Molecular structure of clickable deferasirox (cDFX) (left), fluorescence microscopy of labelled cDFX and the mitochondrial component Cyt c (right). Scale bar, 10 μm. d, αKG quantification assay of MDA-MB-468 cells co-treated with EGF and deferasirox (DFX). n = 3 technical replicates. e, Western blots of CD44 and H3K9me2 in MDA-MB-468 cells co-treated with EGF and DFX. Data representative of n = 3 biologically independent experiments throughout the figure unless stated otherwise. Bars and error bars, mean values ± s.d.

Supplementary information

Supplementary Information

Materials and Methods, Supplementary References, Table 5 and original western blots.

Reporting Summary

Supplementary Table 1

Quantitative label-free proteomics.

Supplementary Table 2

Quantitative metabolomics.

Supplementary Table 3

Quantitative mass spectrometry analysis of histone marks.

Supplementary Table 4

ChIP-seq and RNA-seq analyses.

Source data

Source Data Fig. 1

Statistical Source Data.

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Source Data Fig. 2

Statistical Source Data.

Source Data Fig. 2

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Source Data Fig. 3

Statistical Source Data.

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Source Data Fig. 5

Statistical Source Data.

Source Data Fig. 5

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Source Data Extended Fig. 3

Statistical Source Data.

Source Data Extended Fig. 4

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Source Data Extended Fig. 6

Statistical Source Data

Source Data Extended Fig. 6

Unprocessed western blots.

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Müller, S., Sindikubwabo, F., Cañeque, T. et al. CD44 regulates epigenetic plasticity by mediating iron endocytosis. Nat. Chem. 12, 929–938 (2020). https://doi.org/10.1038/s41557-020-0513-5

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