Salinomycin kills cancer stem cells by sequestering iron in lysosomes

ABSTRACT Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to

conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide

evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs _in vitro_ and _in vivo_, by

accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron

loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis.

These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells. Access through your

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LYSOSOMAL TRPML1 CHANNEL ELIMINATES BREAST CANCER STEM CELLS BY TRIGGERING FERROPTOSIS Article Open access 27 May 2024 MTOR INHIBITION SUPPRESSES SALINOMYCIN-INDUCED FERROPTOSIS IN BREAST

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access 05 January 2024 REFERENCES * Nieto, M. N., Huang, R. Y.-J., Jackson, R. A. & Thiery, J. P. EMT: 2016. _Cell_ 166, 21–45 (2016). CAS  PubMed  Google Scholar  * Tam, W. L. &

Weinberg, R. A. The epigenetics of epithelial-mesenchymal plasticity in cancer. _Nat. Med._ 19, 1438–1449 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Pattabiraman, D. R. &

Weinberg, R. A. Tackling the cancer stem cells–what challenges do they pose? _Nature Rev. Drug Discov._ 13, 497–512 (2014). CAS  Google Scholar  * Kelly, P. N., Dakic, A., Adams, J. M.,

Nutt, S. L. & Strasser, A. Tumor growth need not be driven by rare cancer stem cells. _Science_ 317, 337 (2007). CAS  PubMed  Google Scholar  * Quintana, E. et al. Efficient tumour

formation by single melanoma cells. _Nature_ 456, 593–598 (2008). CAS  PubMed  PubMed Central  Google Scholar  * Gupta, P. B. et al. Identification of selective inhibitors of cancer stem

cells by high-throughput screening. _Cell_ 138, 645–659 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Morel, A.-P. et al. Generation of breast cancer stem cells through

epithelial–mesenchymal transition. _PLoS ONE_ 3, e2888 (2008). PubMed  PubMed Central  Google Scholar  * Germain, A. R. et al. Identification of a selective small molecule inhibitor of

breast cancer stem cells. _Bioorg. Med. Chem. Lett._ 22, 3571–3574 (2012). CAS  PubMed  Google Scholar  * Hartwell, K. A. et al. Niche-based screening identifies small-molecule inhibitors of

leukemia stem cells. _Nat. Chem. Biol._ 9, 840–848 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Paulus, E. F., Kurz, M., Matter, H. & Vértesy, L. Solid-state and solution

structure of the salinomycin-sodium complex: stabilization of different conformers for an ionophore in different environments. _J. Am. Chem. Soc._ 120, 8209–8221 (1998). CAS  Google Scholar

  * Lu, D. et al. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. _Proc. Natl Acad. Sci. USA_ 108, 13253–13257 (2011). CAS  PubMed

  Google Scholar  * Yue, W. et al. Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance. _Autophagy_ 9, 714–729

(2013). CAS  PubMed  PubMed Central  Google Scholar  * Naujokat, C. & Steinhart, R. Salinomycin as a drug for targeting human cancer stem cells. _J. Biomed. Biotechnol._ 2012, 950658

(2012). PubMed  PubMed Central  Google Scholar  * Huczyński, A. et al. Antiproliferative activity of salinomycin and its derivatives. _Bioorg. Med. Chem. Lett._ 22, 7146–7150 (2012). PubMed

  Google Scholar  * Borgström, B. et al. Synthetic modification of salinomycin: selective _O_-acylation and biological evaluation. _Chem. Commun._ 49, 9944–9946 (2013). Google Scholar  *

Huang, X. et al. Semisynthesis of SY-1 for investigation of breast cancer stem cell selectivity of C-ring-modified salinomycin analogues. _ACS Chem. Biol._ 9, 1587–1594 (2014). CAS  PubMed 

Google Scholar  * Borgström, B., Huang, X., Chygorin, E., Oredsson, S. & Strand, D. Salinomycin hydroxamic acids: synthesis, structure, and biological activity of polyether ionophore

hybrids. _ACS Chem. Med. Lett._ 7, 635–640 (2016). Google Scholar  * Shi, Q. et al. Discovery of a 19F MRI sensitive salinomycin derivative with high cytotoxicity towards cancer cells.

_Chem. Commun._ 52, 5136–5139 (2016). CAS  Google Scholar  * Borgström, B., Huang, X., Hegardt, C., Oredsson, S . & Strand, D. Structure-activity relationships in salinomycin:

cytotoxicity and phenotype selectivity of semi-synthetic derivatives. _Chem. Eur. J._ 23, 2077–2083 (2017). PubMed  Google Scholar  * Nishi, M. et al. Induction of cells with cancer stem

cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors. _Oncogene_ 33, 643–652 (2014). CAS  PubMed  Google Scholar  * Minta, A. & Tsien, R.

Y. Fluorescent indicators for cytosolic sodium. _J. Biol. Chem._ 264, 19449–19457 (1989). CAS  PubMed  Google Scholar  * Charafe-Jauffret, E. et al. ALDH1-positive cancer stem cells predict

engraftment of primary breast tumors and are governed by a common stem cell program. _Cancer Res._ 73, 7290–7300 (2013). CAS  PubMed  Google Scholar  * Rodriguez, R. et al.

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. _Nat. Chem. Biol._ 8, 301–310 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Larrieu, D.,

Britton, S., Demir, M., Rodriguez, R. & Jackson, S. P. Chemical inhibition of NAT10 corrects defects of laminopathic cells. _Science_ 344, 527–532 (2014). CAS  PubMed  PubMed Central 

Google Scholar  * Abell, N. S., Mercado, M., Cañeque, T., Rodriguez, R. & Xhemalce, B. Click quantitative mass spectrometry identifies PIWIL3 as a mechanistic target of RNA interference

activator enoxacin in cancer cells. _J. Am. Chem. Soc._ 139, 1400–1403 (2017). CAS  PubMed  Google Scholar  * Cañeque, T. et al. Synthesis of marmycin A and investigation into its cellular

activity. _Nat. Chem._ 7, 744–751 (2015). PubMed  PubMed Central  Google Scholar  * Pantopoulos, K., Porwal, S. K., Tartakoff, A. & Devireddy, L. Mechanisms of mammalian iron

homeostasis. _Biochemistry_ 51, 5705–5724 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Asano, T. et al. Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and

iron-replete cells. _Mol. Cell Biol._ 10, 2040–2052 (2011). Google Scholar  * Hirayama, T., Okuda, K. & Nagasawa, H. A highly selective turn-on fluorescent probe for iron(II) to

visualize labile iron in living cells. _Chem. Sci._ 4, 1250–1256 (2013). CAS  Google Scholar  * Li, T. et al. Salinomycin induces cell death with autophagy through activation of endoplasmic

reticulum stress in human cancer cells. _Autophagy_ 9, 1057–1068 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Dixon, S. J. & Stockwell, B. R. The role of iron and reactive

oxygen species in cell death. _Nat. Chem. Biol._ 10, 9–17 (2014). CAS  PubMed  Google Scholar  * Boya, P. & Kroemer, G. Lysosomal membrane permeabilization in cell death. _Oncogene_ 27,

6434–6451 (2008). CAS  PubMed  Google Scholar  * Aits, S. & Jäättelä, M. Lysosomal cell death at a glance. _J. Cell Sci._ 126, 1905–1912 (2013). CAS  PubMed  Google Scholar  * Galluzzi,

L., Bravo-San Pedro, J. M. & Kroemer, G. Organelle-specific initiation of cell death. _Nat. Cell. Biol._ 16, 728–736 (2014). CAS  PubMed  Google Scholar  * Dixon, S. J. et al.

Ferroptosis: an iron-dependent form of nonapoptotic cell death. _Cell_ 149, 1060–1072 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Yang, W. S. et al. Regulation of ferroptotic

cancer cell death by GPX4. _Cell_ 156, 317–331 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Conrad, M., Angeli, J. P. F., Vandenabeele, P. & Stockwell, B. R. Regulated

necrosis: disease relevance and therapeutic opportunities. _Nat. Rev. Drug. Discov._ 15, 348–366 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Yang, W. S. & Stockwell, B. R.

Ferroptosis: death by lipid peroxidation. _Trends Cell Biol._ 26, 165–176 (2016). CAS  PubMed  Google Scholar  * Cao, J. Y. & Dixon, S. J. Mechanisms of ferroptosis. _Cell. Mol. Life

Sci._ 73, 2195–2209 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Torti, S. V. & Torti, F. M. Iron and cancer: more ore to be mined. _Nat. Rev. Cancer_ 13, 342–355 (2013). CAS 

PubMed  PubMed Central  Google Scholar  * Takebe, N. et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. _Nature Rev. Clin. Oncol._ 12, 445–464 (2015).

CAS  Google Scholar  * West, N. R., Murray, J. I. & Watson, P. H. Oncostatin-M promotes phenotypic changes associated with mesenchymal and stem cell-like differentiation in breast

cancer. _Oncogene_ 33, 1485–1494 (2014). CAS  PubMed  Google Scholar  * Schonberg, D. L. et al. Preferential iron trafficking characterizes glioblastoma stem-like cells. _Cancer Cell_ 28,

441–455 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Pinnix, Z. K. et al. Ferroportin and iron regulation in breast cancer progression and prognosis. _Sci. Transl. Med._ 2, 43ra56

(2010). PubMed  PubMed Central  Google Scholar  * Yamane, K. et al. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. _Mol. Cell_ 25,

801–812 (2007). CAS  PubMed  Google Scholar  * Yamamoto, S. et al. JARID1B is a luminal lineage-driving oncogene in breast cancer. _Cancer Cell_ 25, 762–777 (2014). CAS  PubMed  PubMed

Central  Google Scholar  * Greer, E. L. & Shi, Y. Histone methylation: a dynamic mark in health, disease and inheritance. _Nat. Rev. Genet._ 13, 343–357 (2012). CAS  PubMed  PubMed

Central  Google Scholar  * Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. _Cell_ 153, 692–706 (2013). CAS  PubMed  PubMed Central 

Google Scholar  * Tsai, Y.-P. et al. TET1 regulates hypoxia-induced epithelial-mesenchymal transition by acting as a co-activator. _Genome Biol._ 15, 513 (2014). PubMed  PubMed Central 

Google Scholar  * Hu, X. et al. Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. _Cell Stem Cell_ 14, 512–522 (2014).

CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the CNRS, INSERM and SATT IDF Innov for generous funding. Research in the R.R. laboratory is supported by the

European Research Council (grant number 647973), Fondation pour la Recherche Médicale (grant reference AJE20141031486), Emergence Ville de Paris and Ligue Contre le Cancer. A.Ha. is funded

by the Fondation de France. We acknowledge the PICT-IBiSA@Pasteur Imaging Facility of Institut Curie, member of the France-BioImaging national research infrastructure. We thank P. Le Bacon

for assistance with high-resolution microscopy, J.-F. Gallard, N. Birlirakis and C. Gaillet for assistance with NMR spectroscopy and J. Poupon for electrothermal atomic absorption

spectrometry experiments. We thank A. Puisieux for providing us with HMLER cells and V. Mitz for mammary tissues obtained from reduction mammoplasty. AUTHOR INFORMATION Author notes * Trang

Thi Mai, Ahmed Hamaï and Antje Hienzsch: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Institut Curie, PSL Research University, Chemical Cell Biology Group, 26

rue d'Ulm, Paris Cedex 05, 75248, France Trang Thi Mai, Tatiana Cañeque, Sebastian Müller, Verónica Acevedo & Raphaël Rodriguez * CNRS UMR3666, Paris, 75005, France Trang Thi Mai, 

Tatiana Cañeque, Sebastian Müller, Verónica Acevedo & Raphaël Rodriguez * INSERM U1143, Paris, 75005, France Trang Thi Mai, Tatiana Cañeque, Sebastian Müller, Verónica Acevedo & 

Raphaël Rodriguez * Institut de Chimie des Substances Naturelles, UPR2301, 1 Avenue de la Terrasse, Gif-sur-Yvette Cedex, 91198, France Trang Thi Mai, Antje Hienzsch, Tatiana Cañeque & 

Raphaël Rodriguez * Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR8253, Université Paris Descartes-Sorbonne Paris Cité, 14 rue Maria Helena Vieira Da Silva, Paris Cedex 14, 75993,

France Ahmed Hamaï, Christine Leroy, Amandine David, Patrice Codogno & Maryam Mehrpour * ABX advanced biochemical compounds, Heinrich-Glaeser-Str. 10-14, Radeberg, D-01454, Germany Antje

Hienzsch * Aix Marseille Université, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Equipe Oncologie Moléculaire labellisée ‘Ligue contre le cancer’, Marseille, 13009, France Julien

Wicinski, Olivier Cabaud, Christophe Ginestier, Daniel Birnbaum & Emmanuelle Charafe-Jauffret * Department of Microbiology, Yokohama City University School of Medicine, Yokohama,

236-0004, Japan Akihide Ryo Authors * Trang Thi Mai View author publications You can also search for this author inPubMed Google Scholar * Ahmed Hamaï View author publications You can also

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Scholar * Raphaël Rodriguez View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.R. conceptualized the study and designed ironomycin. R.R.,

T.T.M., M.M., A.Ha. and P.C. designed the experiments and analysed the data. T.T.M., A.Hi., A.Ha. and M.M. performed the experiments unless stated otherwise. A.Hi. and T.C. synthesized Sal

derivatives and performed NMR experiments. J.W., O.C., C.G., D.B. and E.C.-J. provided PDX data. A.Ha., C.L. and A.D. provided MCF-7 tumour data. S.M. and V.A. provided assistance with cell

imaging. A.R. provided iCSCL-10A2 cells. R.R. wrote the manuscript with contributions from T.C., S.M., A.Hi., A.Ha. and M.M. CORRESPONDING AUTHORS Correspondence to Maryam Mehrpour or

Raphaël Rodriguez. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary information

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sequestering iron in lysosomes. _Nature Chem_ 9, 1025–1033 (2017). https://doi.org/10.1038/nchem.2778 Download citation * Received: 21 April 2016 * Accepted: 03 April 2017 * Published: 16

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