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Contribution of three-dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer

Oncogenevolume 37pages40134032 (2018) | Download Citation

Abstract

Hepcidin is a peptide hormone that negatively regulates iron efflux and plays an important role in controlling the growth of breast tumors. In patients with breast cancer, the combined expression of hepcidin and its membrane target, ferroportin, predict disease outcome. However, mechanisms that control hepcidin expression in breast cancer cells remain largely unknown. Here, we use three-dimensional breast cancer spheroids derived from cell lines and breast cancer patients to probe mechanisms of hepcidin regulation in breast cancer. We observe that the extent of hepcidin induction and pathways of its regulation are markedly changed in breast cancer cells grown in three dimensions. In monolayer culture, BMPs, particularly BMP6, regulate hepcidin transcription. When breast cancer cells are grown as spheroids, there is a >10-fold induction in hepcidin transcripts. Microarray analysis combined with knockdown experiments reveal that GDF-15 is the primary mediator of this change. The increase in hepcidin as breast cells develop a three-dimensional architecture increases intracellular iron, as indicated by an increase in the iron storage protein ferritin. Immunohistochemical staining of human breast tumors confirms that both GDF-15 and hepcidin are expressed in breast cancer specimens. Further, levels of GDF-15 are significantly correlated with levels of hepcidin at both the mRNA and protein level in patient samples, consistent with a role for GDF-15 in control of hepcidin in human breast tumors. Inclusion of tumor-associated fibroblasts in breast cancer spheroids further induces hepcidin. This induction is mediated by fibroblast-dependent secretion of IL-6. Breast cancer cells grown as spheroids are uniquely receptive to IL-6-dependent induction of hepcidin by tumor-associated fibroblasts, since IL-6 does not induce hepcidin in cells grown as monolayers. Collectively, our results suggest a new paradigm for tumor-mediated control of iron through the control of hepcidin by tumor architecture and the breast tumor microenvironment.

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References

  1. 1.

    Tonik SE, Shindelman JE, Sussman HH. Transferrin receptor is inversely correlated with estrogen receptor in breast cancer. Breast Cancer Res Treat. 1986;7:71–6.

  2. 2.

    Daniels TR, Bernabeu E, Rodriguez JA, Patel S, Kozman M, Chiappetta DA, et al. The transferrin receptor and the targeted delivery of therapeutic agents against cancer. Biochim Biophys Acta. 2012;1820:291–317.

  3. 3.

    Wrba F, Ritzinger E, Reiner A, Holzner JH. Transferrin receptor (TrfR) expression in breast carcinoma and its possible relationship to prognosis. An immunohistochemical study. Virchows Arch A Pathol Anat Histopathol. 1986;410:69–73.

  4. 4.

    Wang W, Deng Z, Hatcher H, Miller LD, Di X, Tesfay L, et al. IRP2 regulates breast tumor growth. Cancer Res. 2014;74:497–507.

  5. 5.

    Pinnix ZK, Miller LD, Wang W, D’Agostino R Jr., Kute T, Willingham MC, et al. Ferroportin and iron regulation in breast cancer progression and prognosis. Sci Transl Med. 2010;2:43ra56.

  6. 6.

    Zhang S, Chen Y, Guo W, Yuan L, Zhang D, Xu Y, et al. Disordered hepcidin-ferroportin signaling promotes breast cancer growth. Cell Signal. 2014;26:2539–50.

  7. 7.

    Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090–3.

  8. 8.

    Abboud S, Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem. 2000;275:19906–12.

  9. 9.

    Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 2000;403:776–81.

  10. 10.

    McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell. 2000;5:299–309.

  11. 11.

    Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance. J Clin Invest. 2007;117:1933–9.

  12. 12.

    Andriopoulos B Jr, Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet. 2009;41:482–7.

  13. 13.

    Lee P, Peng H, Gelbart T, Wang L, Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6. Proc Natl Acad Sci USA. 2005;102:1906–10.

  14. 14.

    Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods. 2007;4:359–65.

  15. 15.

    Kim JB, Stein R, O’Hare MJ. Three-dimensional in vitro tissue culture models of breast cancer-- a review. Breast Cancer Res Treat. 2004;85:281–91.

  16. 16.

    Bissell MJ, Hall HG, Parry G. How does the extracellular matrix direct gene expression? J Theor Biol. 1982;99:31–68.

  17. 17.

    Debnath J, Brugge JS. Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer. 2005;5:675–88.

  18. 18.

    Weigelt B, Bissell MJ. Unraveling the microenvironmental influences on the normal mammary gland and breast cancer. Semin Cancer Biol. 2008;18:311–21.

  19. 19.

    Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol. 2012;180:599–607.

  20. 20.

    Liu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A, et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc. 2017;12:439–51.

  21. 21.

    Soule HD, Vazguez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst. 1973;51:1409–16.

  22. 22.

    Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood. 2006;108:3204–9.

  23. 23.

    Arman A, Auron PE. Interleukin 1 (IL-1) induces the activation of Stat3. Adv Exp Med Biol. 2003;534:297–307.

  24. 24.

    Caldenhoven E, van Dijk T, Raaijmakers JA, Lammers JW, Koenderman L, De Groot RP. Activation of the STAT3/acute phase response factor transcription factor by interleukin-5. J Biol Chem. 1995;270:25778–84.

  25. 25.

    Yang CH, Murti A, Pfeffer LM. STAT3 complements defects in an interferon-resistant cell line: evidence for an essential role for STAT3 in interferon signaling and biological activities. Proc Natl Acad Sci USA. 1998;95:5568–72.

  26. 26.

    Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, et al. Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene. 2001;20:2499–513.

  27. 27.

    Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature. 2009;461:109–13.

  28. 28.

    Coloff JL, Murphy JP, Braun CR, Harris IS, Shelton LM, Kami K, et al. Differential glutamate metabolism in proliferating and quiescent mammary epithelial cells. Cell Metab. 2016;23:867–80.

  29. 29.

    Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V. Cell-cell adhesion and cytoskeleton tension oppose each other in regulating tumor cell aggregation. Cancer Res. 2015;75:2426–33.

  30. 30.

    Perou CM, Jeffrey SS, van de Rijn M, Rees CA, Eisen MB, Ross DT, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci USA. 1999;96:9212–7.

  31. 31.

    Nieman MT, Prudoff RS, Johnson KR, Wheelock MJ. N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J Cell Biol. 1999;147:631–44.

  32. 32.

    Torti FM, Torti SV. Regulation of ferritin genes and protein. Blood. 2002;99:3505–16.

  33. 33.

    Hentze MW, Caughman SW, Rouault TA, Barriocanal JG, Dancis A, Harford JB, et al. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987;238:1570–3.

  34. 34.

    Arosio P, Levi S. Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage. Biochim Biophys Acta. 2010;1800:783–92.

  35. 35.

    Theil EC, Tosha T, Behera RK. Solving biology’s iron chemistry problem with ferritin protein nanocages. Acc Chem Res. 2016;49:784–91.

  36. 36.

    Longati P, Jia X, Eimer J, Wagman A, Witt MR, Rehnmark S, et al. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer. 2013;13:95.

  37. 37.

    Yu M, Lin G, Arshadi N, Kalatskaya I, Xue B, Haider S, et al. Expression profiling during mammary epithelial cell three-dimensional morphogenesis identifies PTPRO as a novel regulator of morphogenesis and ErbB2-mediated transformation. Mol Cell Biol. 2012;32:3913–24.

  38. 38.

    Francia G, Man S, Teicher B, Grasso L, Kerbel RS. Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents. Mol Cell Biol. 2004;24:6837–49.

  39. 39.

    Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, et al. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J Cell Sci. 2017;130:203–18.

  40. 40.

    Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, et al. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci USA. 1997;94:11514–9.

  41. 41.

    Welsh JB, Sapinoso LM, Kern SG, Brown DA, Liu T, Bauskin AR, et al. Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum. Proc Natl Acad Sci USA. 2003;100:3410–5.

  42. 42.

    Yalcin MM, Altinova AE, Akturk M, Gulbahar O, Arslan E, Ors Sendogan D, et al. GDF-15 and hepcidin levels in nonanemic patients with impaired glucose tolerance. J Diabetes Res. 2016;2016:1240843.

  43. 43.

    Yilmaz H, Cakmak M, Darcin T, Inan O, Bilgic MA, Bavbek N, et al. Can serum Gdf-15 be associated with functional iron deficiency in hemodialysis patients? Indian J Hematol Blood Transfus. 2016;32:221–7.

  44. 44.

    Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee YT, et al. High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med. 2007;13:1096–101.

  45. 45.

    Winand FJ, et al. GDF15 and hepcidin as prognostic factors in patients with prostate cancer. J Mol Biomark Diagn. 2014;5:199.

  46. 46.

    Li C, Wang J, Kong J, Tang J, Wu Y, Xu E, et al. GDF15 promotes EMT and metastasis in colorectal cancer. Oncotarget. 2016;7:860–72.

  47. 47.

    Li YL, Chang JT, Lee LY, Fan KH, Lu YC, Li YC, et al. GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway. Oncotarget. 2017;8:1508–28.

  48. 48.

    Wrana JL. Regulation of Smad activity. Cell. 2000;100:189–92.

  49. 49.

    Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61–70.

  50. 50.

    Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012;21:309–22.

  51. 51.

    Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT, et al. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol. 2007;1:84–96.

  52. 52.

    Martin KJ, Patrick DR, Bissell MJ, Fournier MV. Prognostic breast cancer signature identified from 3D culture model accurately predicts clinical outcome across independent datasets. PLoS ONE. 2008;3:e2994.

  53. 53.

    Horning JL, Sahoo SK, Vijayaraghavalu S, Dimitrijevic S, Vasir JK, Jain TK, et al. 3-D tumor model for in vitro evaluation of anticancer drugs. Mol Pharm. 2008;5:849–62.

  54. 54.

    Nath S, Devi GR. Three-dimensional culture systems in cancer research: focus on tumor spheroid model. Pharmacol Ther. 2016;163:94–108.

  55. 55.

    Fairlie WD, Moore AG, Bauskin AR, Russell PK, Zhang HP, Breit SN. MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation. J Leukoc Biol. 1999;65:2–5.

  56. 56.

    Schober A, Bottner M, Strelau J, Kinscherf R, Bonaterra GA, Barth M, et al. Expression of growth differentiation factor 15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain. J Comp Neurol. 2001;439:32–45.

  57. 57.

    Park YJ, Lee H, Lee JH. Macrophage inhibitory cytokine-1 transactivates ErbB family receptors via the activation of Src in SK-BR-3 human breast cancer cells. BMB Rep. 2010;43:91–6.

  58. 58.

    Sasahara A, Tominaga K, Nishimura T, Yano M, Kiyokawa E, Noguchi M, et al. An autocrine/paracrine circuit of growth differentiation factor (GDF) 15 has a role for maintenance of breast cancer stem-like cells. Oncotarget. 2017;8:24869–81.

  59. 59.

    Meynard D, Kautz L, Darnaud V, Canonne-Hergaux F, Coppin H, Roth MP. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet. 2009;41:478–81.

  60. 60.

    Pickl M, Ries CH. Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene. 2009;28:461–8.

  61. 61.

    Aboussekhra A. Role of cancer-associated fibroblasts in breast cancer development and prognosis. Int J Dev Biol. 2011;55:841–9.

  62. 62.

    Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, Huang H, et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell. 2004;6:17–32.

  63. 63.

    Whiteside TL. The tumor microenvironment and its role in promoting tumor growth. Oncogene. 2008;27:5904–12.

  64. 64.

    Marques O, Porto G, Rema A, Faria F, Cruz Paula A, Gomez-Lazaro M, et al. Local iron homeostasis in the breast ductal carcinoma microenvironment. BMC Cancer. 2016;16:187.

  65. 65.

    Bruzzese F, Hagglof C, Leone A, Sjoberg E, Roca MS, Kiflemariam S, et al. Local and systemic protumorigenic effects of cancer-associated fibroblast-derived GDF15. Cancer Res. 2014;74:3408–17.

  66. 66.

    Tesfay L, Clausen KA, Kim JW, Hegde P, Wang X, Miller LD, et al. Hepcidin regulation in prostate and its disruption in prostate cancer. Cancer Res. 2015;75:2254–63.

  67. 67.

    Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA, Ries LA, et al. US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status. J Natl Cancer Inst. 2014;106:dju055.

  68. 68.

    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747–52.

  69. 69.

    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001;98:10869–74.

  70. 70.

    Cooke KS, Hinkle B, Salimi-Moosavi H, Foltz I, King C, Rathanaswami P, et al. A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates. Blood. 2013;122:3054–61.

  71. 71.

    Wilkinson Jt, Pietsch EC, Torti SV, Torti FM. Ferritin regulation by oxidants and chemopreventive xenobiotics. Adv Enzym Regul. 2003;43:135–51.

  72. 72.

    Nguyen DH, Zhou T, Shu J, Mao J. Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity. https://doi.org/10.1038/protex.2013.097.

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Acknowledgements

This work was supported in part by NCI R01CA188025 (SVT), NCI R01CA171101 (FMT), and NCI F32CA214030 (AK). We thank Li Chen and Drs. Nathaniel Dyment and David Rowe for assistance in image acquisition, Tara L Arvedson (Amgen, Thousand Oaks, CA) for a generous gift of anti-ferroportin antibody and Xiaohong Wang for her assistance in histological sectioning.

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Author notes

    • Daniel Kita

    Present address: Alexion Pharmaceuticals, New Haven, CT, USA

    • David Lemler

    Present address: Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA

Affiliations

  1. Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, USA

    • Nicole Blanchette-Farra
    • , Lia Tesfay
    • , David Lemler
    •  & Suzy V. Torti
  2. Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA

    • Daniel Kita
    •  & Kevin P. Claffey
  3. Center for Quantitative Medicine, University of Connecticut Health Center, Farmington, CT, USA

    • Anna Konstorum
  4. Department of Pathology, University of Connecticut Health Center, Farmington, CT, USA

    • Poornima Hegde
  5. Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA

    • Frank M. Torti

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The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Suzy V. Torti.

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https://doi.org/10.1038/s41388-018-0243-y