Abstract

Accumulating data suggest that metastatic dissemination often occurs early during tumour formation, but the mechanisms of early metastatic spread have not yet been addressed. Here, by studying metastasis in a HER2-driven mouse breast cancer model, we show that progesterone-induced signalling triggers migration of cancer cells from early lesions shortly after HER2 activation, but promotes proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by increased HER2 expression and tumour-cell density involving microRNA-mediated progesterone receptor downregulation, and was reversible. Cells from early, low-density lesions displayed more stemness features, migrated more and founded more metastases than cells from dense, advanced tumours. Notably, we found that at least 80% of metastases were derived from early disseminated cancer cells. Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination.

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References

  1. 1.

    , & Metastasis awakening: targeting dormant cancer. Nat. Med. 19, 276–277 (2013)

  2. 2.

    & Metastasis awakening: the challenges of targeting minimal residual cancer. Nat. Med. 19, 274–275 (2013)

  3. 3.

    , , , & Polychemotherapy for early breast cancer: an overview of the randomised clinical trials with quality-adjusted survival analysis. Lancet 358, 277–286 (2001)

  4. 4.

    et al. Treatment with trastuzumab for 1 year after adjuvant chemotherapy in patients with HER2-positive early breast cancer: a 4-year follow-up of a randomised controlled trial. Lancet Oncol. 12, 236–244 (2011)

  5. 5.

    et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 360, 683–689 (2002)

  6. 6.

    et al. From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc. Natl Acad. Sci. USA 100, 7737–7742 (2003)

  7. 7.

    Parallel progression of primary tumours and metastases. Nat. Rev. Cancer 9, 302–312 (2009)

  8. 8.

    et al. Systemic spread is an early step in breast cancer. Cancer Cell 13, 58–68 (2008)

  9. 9.

    et al. EMT and dissemination precede pancreatic tumor formation. Cell 148, 349–361 (2012)

  10. 10.

    et al. Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma. J. Clin. Invest. 120, 2030–2039 (2010)

  11. 11.

    et al. Hematogenous and lymphatic tumor cell dissemination may be detected in patients diagnosed with ductal carcinoma in situ of the breast. Breast Cancer Res. Treat. 131, 801–808 (2012)

  12. 12.

    et al. Disseminated tumor cells in the bone marrow of patients with ductal carcinoma in situ. Int. J. Cancer 129, 2522–2526 (2011)

  13. 13.

    & Tumor metastasis: molecular insights and evolving paradigms. Cell 147, 275–292 (2011)

  14. 14.

    et al. Revealing progesterone’s role in uterine and mammary gland biology: insights from the mouse. Semin. Reprod. Med. 23, 22–37 (2005)

  15. 15.

    , , & Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc. Natl Acad. Sci. USA 100, 9744–9749 (2003)

  16. 16.

    , , & Progesterone receptor isoforms A and B: temporal and spatial differences in expression during murine mammary gland development. Endocrinology 146, 3577–3588 (2005)

  17. 17.

    Progesterone signalling in breast cancer: a neglected hormone coming into the limelight. Nat. Rev. Cancer 13, 385–396 (2013)

  18. 18.

    et al. Enrichment of a population of mammary gland cells that form mammospheres and have in vivo repopulating activity. Cancer Res. 67, 8131–8138 (2007)

  19. 19.

    , , & HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene 27, 6120–6130 (2008)

  20. 20.

    et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007)

  21. 21.

    & Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy. Proc. Natl Acad. Sci. USA 93, 15180–15184 (1996)

  22. 22.

    et al. The landscape of cancer genes and mutational processes in breast cancer. Nature 486, 400–404 (2012)

  23. 23.

    et al. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 107, 885–892 (2006)

  24. 24.

    et al. Combined genome and transcriptome analysis of single disseminated cancer cells from bone marrow of prostate cancer patients reveals unexpected transcriptomes. Cancer Res. 74, 7383–7394 (2014)

  25. 25.

    Framework models of tumor dormancy from patient-derived observations. Curr. Opin. Genet. Dev. 21, 42–49 (2011)

  26. 26.

    et al. Genomic characterization of brain metastases reveals branched evolution and potential therapeutic targets. Cancer Discov. 5, 1164–1177 (2015)

  27. 27.

    et al. Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. Genes Dev. 14, 650–654 (2000)

  28. 28.

    et al. The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse. Dev. Biol. 328, 127–139 (2009)

  29. 29.

    et al. Steroid hormone receptor status of mouse mammary stem cells. J. Natl. Cancer Inst. 98, 1011–1014 (2006)

  30. 30.

    et al. Progesterone induces adult mammary stem cell expansion. Nature 465, 803–807 (2010)

  31. 31.

    et al. Targeting RANKL to a specific subset of murine mammary epithelial cells induces ordered branching morphogenesis and alveologenesis in the absence of progesterone receptor expression. FASEB J. 24, 4408–4419 (2010)

  32. 32.

    , , , & Breast cancer mortality after a diagnosis of ductal carcinoma in situ. JAMA Oncol. 1, 888–896 (2015)

  33. 33.

    et al. Gene expression analysis of immune-mediated arrest of tumorigenesis in a transgenic mouse model of HER-2/neu-positive basal-like mammary arcinoma. Am. J. Pathol. 166, 1205–1216 (2005)

  34. 34.

    et al. HER2-positive circulating tumor cells in breast cancer. PLoS One 6, e15624 (2011)

  35. 35.

    et al. Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc. Natl Acad. Sci. USA 104, 13450–13454 (2007)

  36. 36.

    et al. Multiple oncogenic mutations and clonal relationship in spatially distinct benign human epidermal tumors. Proc. Natl Acad. Sci. USA 107, 20780–20785 (2010)

  37. 37.

    et al. High frequency of BRAF mutations in nevi. Nat. Genet. 33, 19–20 (2003)

  38. 38.

    , , , & Activation of Akt-1 (PKB-α) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res. 64, 3171–3178 (2004)

  39. 39.

    et al. MYC suppresses cancer metastasis by direct transcriptional silencing of αV and β3 integrin subunits. Nat. Cell Biol. 14, 567–574 (2012)

  40. 40.

    et al. Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres. EMBO J. 30, 1841–1851 (2011)

  41. 41.

    et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998)

  42. 42.

    & Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein. Cell Growth Differ. 11, 395–408 (2000)

  43. 43.

    et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501 (2003)

  44. 44.

    , , & Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protoc. Cell Biology Chapter 3, Unit 3.22 (2006)

  45. 45.

    , , & A miR-155-dependent microRNA hierarchy in dendritic cell maturation and macrophage activation. FEBS Lett. 588, 632–640 (2014)

  46. 46.

    et al. The UCSC genome browser database: extensions and updates 2013. Nucleic Acids Res. 41, D64–D69 (2013)

  47. 47.

    , & Analysis of cDNA microarray images. Brief. Bioinform. 2, 341–349 (2001)

  48. 48.

    et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002)

  49. 49.

    , , & A stepwise framework for the normalization of array CGH data. BMC Bioinformatics 6, 274 (2005)

  50. 50.

    & Normalization of cDNA microarray data. Methods 31, 265–273 (2003)

  51. 51.

    et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015)

  52. 52.

    et al. Breaking the waves: improved detection of copy number variation from microarray-based comparative genomic hybridization. Genome Biol. 8, R228 (2007)

  53. 53.

    & A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 23, 657–663 (2007)

  54. 54.

    et al. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 12, R41 (2011)

  55. 55.

    et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc. Natl Acad. Sci. USA 96, 4494–4499 (1999)

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Acknowledgements

We thank T. Perry for his critical reading of the manuscript. This work was supported by grants from the DFG (Kl 1233/2-1, KL 1233/3-1, KL 1233/10-1 (C.A.K.); Me2064/4-1, (G.M.); SP 938/2-1 (R.S.); INST 89/341-1 FUGG (TissueFAX)); the Dr Josef Steiner Foundation and ERC (322602) to C.A.K.; the SWCRF, CA109182, CA196521, CA163131, BC132674 (J.A.A.-G.) F31 CA183185 (K.L.H.) BC112380 (M.S.S.), NIH 1S10RR024745 Microscopy CoRE at ISMMS and by a donation from A. Jungmayer.

Author information

Author notes

    • Milan M. S. Obradović
    • , Maria Soledad Sosa
    •  & Lahiri Kanth Nanduri

    Present addresses: Tumor Heterogeneity, Metastasis and Resistance, Department of Biomedicine, University of Basel, University Hospital Basel, CH-4031 Basel, Switzerland (M.M.S.O.); Department of Pharmacological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York 10029, USA (M.S.S.); Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany (L.K.N.).

Affiliations

  1. Experimental Medicine and Therapy Research, University of Regensburg, 93053 Regensburg, Germany

    • Hedayatollah Hosseini
    • , Milan M. S. Obradović
    • , Lahiri Kanth Nanduri
    • , Carolin Ehrl
    • , Matthias Maneck
    • , Nina Patwary
    • , Gundula Haunschild
    • , Miodrag Gužvić
    • , Christian Reimelt
    •  & Christoph A. Klein
  2. Project group ‘Personalized Tumour Therapy’, Fraunhofer Institute for Toxicology und Experimental Medicine, 93053 Regensburg, Germany

    • Martin Hoffmann
    • , Christian Werno
    •  & Christoph A. Klein
  3. Division of Hematology and Oncology, Department of Medicine, Department of Otolaryngology, Department of Oncological Sciences, Tisch Cancer Institute, Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York 10029, USA

    • Kathryn L. Harper
    • , Maria Soledad Sosa
    •  & Julio A. Aguirre-Ghiso
  4. Institute of Immunology, University of Regensburg, 93053 Regensburg, Germany

    • Melanie Werner-Klein
  5. Department of Statistical Bioinformatics, Institute of Functional Genomics, University of Regensburg, 93053 Regensburg, Germany

    • Michael Grauvogl
    •  & Rainer Spang
  6. Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany

    • Norbert Eichner
    •  & Gunter Meister
  7. Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany

    • Florian Weber
  8. Department of Gynecology and Obstetrics, University of Tübingen, 72076 Tübingen, Germany

    • Andreas D. Hartkopf
    • , Florin-Andrei Taran
    •  & Sara Y. Brucker
  9. Department of Gynecology and Obstetrics, University of Düsseldorf, 40225 Düsseldorf, Germany

    • Tanja Fehm
  10. Department of Gynecology and Obstetrics, University Munich, 80337 Munich, Germany

    • Brigitte Rack
  11. Department of Gynecology and Obstetrics, University Medical Center Regensburg, 93053 Regensburg, Germany

    • Stefan Buchholz

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Contributions

C.A.K. and H.H. designed and evaluated core experiments. H.H. performed core experiments. M.M.S.O., C.W., L.K.N., C.E., C.R. and M.Gu. helped with in vivo, in vitro and primary culture experiments. M.H., M.M., M.Gr., R.S. and H.H. performed bioinformatic and statistical analyses. M.W.-K., K.L.H., M.S.S. and F.W. performed staining and pathological analysis. N.E. and G.M. performed miRNA sequencing and analysis. G.H., N.P., A.K.H., F.-A.T., S.Y.B., B.R., S.B. and T.F. performed DCC analysis or collected patient data. C.A.K. and H.H. wrote the manuscript with input from J.A.A.-G.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Christoph A. Klein.

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    Supplementary Information

    This file contains Supplementary Figure 1, uncropped western blot gels and Supplementary Tables 1-11.

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    Supplementary Data 1

    This file contains microarray analyses.

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    Supplementary Data 2

    This file contains miRNA-Seq analyses.

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https://doi.org/10.1038/nature20785

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