Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance

Abstract

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

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Figure 1: Establishing an EMT lineage tracing system in triple-transgenic mice.
Figure 2: The EMT lineage tracing system reports EMT in tumour cells with high fidelity.
Figure 3: mir-200 inhibition of EMT in tri-PyMT cells did not impact lung metastasis.
Figure 4: EMT tumour cells are resistant to chemotherapy.
Figure 5: miR-200 overexpression abrogates CTX resistance.

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References

  1. 1

    Bastid, J. EMT in carcinoma progression and dissemination: facts, unanswered questions, and clinical considerations. Cancer Metastasis Rev. 31, 277–283 (2012)

  2. 2

    Kalluri, R. & Weinberg, R. A. The basics of epithelial–mesenchymal transition. J. Clin. Invest. 119, 1420–1428 (2009)

  3. 3

    Scheel, C. & Weinberg, R. A. Phenotypic plasticity and epithelial–mesenchymal transitions in cancer and normal stem cells? Int. J. Cancer 129, 2310–2314 (2011)

  4. 4

    Mani, S. A. et al. The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715 (2008)

  5. 5

    Gal, A. et al. Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis. Oncogene 27, 1218–1230 (2008)

  6. 6

    Hennessy, B. T. et al. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res. 69, 4116–4124 (2009)

  7. 7

    Gao, D. et al. Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition. Cancer Res. 72, 1384–1394 (2012)

  8. 8

    Lin, E. Y. et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am. J. Pathol. 163, 2113–2126 (2003)

  9. 9

    Guy, C. T., Cardiff, R. D. & Muller, W. J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol. Cell. Biol. 12, 954–961 (1992)

  10. 10

    Bhowmick, N. A. et al. TGF-β signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848–851 (2004)

  11. 11

    Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007)

  12. 12

    Xue, C., Plieth, D., Venkov, C., Xu, C. & Neilson, E. G. The gatekeeper effect of epithelial–mesenchymal transition regulates the frequency of breast cancer metastasis. Cancer Res. 63, 3386–3394 (2003)

  13. 13

    Okada, H., Danoff, T. M., Kalluri, R. & Neilson, E. G. Early role of Fsp1 in epithelial–mesenchymal transformation. Am. J. Physiol. 273, F563–F574 (1997)

  14. 14

    Gunasinghe, N. P., Wells, A., Thompson, E. W. & Hugo, H. J. Mesenchymal–epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer. Cancer Metastasis Rev. 31, 469–478 (2012)

  15. 15

    Cabeźon, T. et al. Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer. Int. J. Cancer 121, 1433–1444 (2007)

  16. 16

    Guy, C. T. et al. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc. Natl Acad. Sci. USA 89, 10578–10582 (1992)

  17. 17

    Troeger, J. S. et al. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Gastroenterology 143, 1073–1083 (2012)

  18. 18

    Dumont, N. et al. Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers. Proc. Natl Acad. Sci. USA 105, 14867–14872 (2008)

  19. 19

    Park, S. M., Gaur, A. B., Lengyel, E. & Peter, M. E. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 22, 894–907 (2008)

  20. 20

    Gregory, P. A. et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nature Cell Biol. 10, 593–601 (2008)

  21. 21

    Singh, A. & Settleman, J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741–4751 (2010)

  22. 22

    Zhang, Y., Toy, K. A. & Kleer, C. G. Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition. Mod. Pathol. 25, 178–184 (2012)

  23. 23

    Creighton, C. J. et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc. Natl Acad. Sci. USA 106, 13820–13825 (2009)

  24. 24

    von Minckwitz, G. Docetaxel/anthracycline combinations for breast cancer treatment. Expert Opin. Pharmacother. 8, 485–495 (2007)

  25. 25

    Lau, C. K. et al. An Akt/hypoxia-inducible factor-1α/platelet-derived growth factor-BB autocrine loop mediates hypoxia-induced chemoresistance in liver cancer cells and tumorigenic hepatic progenitor cells. Clin. Cancer Res. 15, 3462–3471 (2009)

  26. 26

    Xiao, Z. M., Wang, X. Y. & Wang, A. M. Periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway. Biotechnol. Appl. Biochem. 62, 401–406 (2015)

  27. 27

    Yamada, D. et al. Role of crosstalk between interleukin-6 and transforming growth factor-beta 1 in epithelial-mesenchymal transition and chemoresistance in biliary tract cancer. Eur. J. Cancer 49, 1725–1740 (2013)

  28. 28

    Yao, Z. et al. TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer. Proc. Natl Acad. Sci. USA 107, 15535–15540 (2010)

  29. 29

    Russo, J. E. & Hilton, J. Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells. Cancer Res. 48, 2963–2968 (1988)

  30. 30

    Tam, W. L. & Weinberg, R. A. The epigenetics of epithelial–mesenchymal plasticity in cancer. Nature Med. 19, 1438–1449 (2013)

  31. 31

    Trimboli, A. J. et al. Direct evidence for epithelial–mesenchymal transitions in breast cancer. Cancer Res. 68, 937–945 (2008)

  32. 32

    Yu, M. et al. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339, 580–584 (2013)

  33. 33

    Korpal, M. et al. Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization. Nature Med. 17, 1101–1108 (2011)

  34. 34

    Gupta, P. B. et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138, 645–659 (2009)

  35. 35

    Diessner, J. et al. Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1). Cell Death Dis. 5, e1149 (2014)

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Acknowledgements

This work was supported by a grant from the US Department of Defense CDMRP LCRP (LC110643). K.F. is supported by a fellowship from the NIH (1 F31 CA186510-01). V.M. was supported by the National Cancer Institute sub-award (U54 CA149196-05) and WCMC Meyer Cancer Center Pilot Funding. This work was also supported by funds from The Neuberger Berman Foundation Lung Cancer Research Center; the Arthur and Myra Mahon Donor-Advised Fund; the Liz Claiborne and Art Ortenberg Foundation; the Douglas & Katherine McCormick Family Foundation, the R. & M. Goldberg Family Foundation; the P. & C. Collins Fund; the Eliot Stewart ‘Wren’ Fund; the William and Shelby Modell Family Foundation Trust; and generous funds donated by patients in the Division of Thoracic Surgery to N.K.A. The funding organizations played no role in experimental design, data analysis or manuscript preparation.

Author information

D.G., K.R.F. and V.M. designed the experiments. K.R.F. and D.G. performed the experiments. A.D., S.L., H.C., T.E.R. and S.R. provided technical support with experiments and animal work. L.T.V. and N.K.A. made critical comments to improve the study design. J.S., F.L. and S.T.C.W. performed RNA-sequencing analysis. J.T. and R.F.S. generated the Vim-Cre transgenic mice. K.R.F., D.G. and V.M. wrote and edited the manuscript with input from the other authors. All authors discussed the results and conclusions drawn from them.

Correspondence to Vivek Mittal or Dingcheng Gao.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of the primary tumour and lung metastasis of tri-PyMT mice.

a, b, Sections of primary tumours (a) and lungs (b) from tri-PyMT mice were immunostained for E-cadherin (E-cad, top), vimentin (Vim, middle) and CD45 (bottom) in white pseudo-colour. Representative images are shown (n > 5 mice). Note the co-localization of PyMT with RFP, and CD45 with GFP (as indicated by arrows), in both primary tumours and lung metastases.

Extended Data Figure 2 Characterization of the primary tumour and lung metastasis of tri-Neu mice.

Sections of primary tumours (left panel) and lungs (right panel) from tri-Neu mice were immunostained for CD45, Neu, E-cadherin, and vimentin (in white pseudo-colour). Representative images are shown (n > 5 mice). Note that both primary tumours and lung metastases are largely composed of epithelial RFP+ tumour cells.

Extended Data Figure 3 Characterization of the primary tumour and lung metastasis of tri-PyMT/Vim mice.

Tri-PyMT/Vim mice were obtained by crossing MMTV–PyMT, vimentin–Cre and Rosa26–RFP–GFP transgenic mice. a, b, Sections of primary tumours (a) and lungs (b) were immunostained for PyMT, E-cadherin and vimentin (in white pseudo-colour). Representative images are shown (n > 5 mice). Note that both primary tumours and lung metastases are largely composed of epithelial RFP+ tumour cells.

Extended Data Figure 4 Characterization of tri-PyMT cells.

a, EMT of tri-PyMT cells with TGF. RFP+ tri-PyMT cells were sorted by flow cytometry and cultured in medium containing 2% FBS with or without TGF-β1 (2 ng ml−1) for 3 days. Plot shows quantification of the percentage of GFP+ cells analysed by flow cytometry (n = 2 biological replicates). b, Cell migration assay of tri-PyMT cells. The tracing plots show the movement of individual RFP+ and GFP+ cells in 10 h of live imaging. Quantification plot (right panel) showed the average distance that RFP+ and GFP+ cells have moved during the time frame (n > 20, *P < 0.01). c, Relative expression of epithelial, mesenchymal and tumour markers in sorted RFP+ and GFP+ tri-PyMT cells as determined by qRT–PCR with Gapdh as the internal control. n = 2 individual experiments. d, EMT of tri-PyMT cells is reported by fluorescent marker switch. Flow cytometry plot shows E-cadherin (E-cad) and E-cadherin+ (E-cad+) subpopulations of tri-PyMT cells (upper panel). Of the E-cad and E-cad+ subsets, the populations were further dissected according to innate fluorescence (lower panel). Numbers indicate the percentage of GFP+, RFP+, or transitioning (Q2) cells in the parental E-cad or E-cad+ subsets, respectively.

Extended Data Figure 5 Establishing an orthotopic model with sorted RFP+ tri-PyMT cells.

a, Flow cytometry plots show tri-PyMT cells before and after sorting for RFP+ cells. Numbers indicate the percentage and purity of RFP+ cells used for establishing orthotopic breast tumours in mice. b, Schematic of the orthotopic breast tumour model with sorted RFP+ tri-PyMT cells. Cells are injected into the mammary gland of wild-type mice to generate primary breast tumours, resection of primary tumour at 4 weeks and lung metastases evaluation in another 4 weeks. c, Characterization of tumour cells in the primary tumour, disseminated tumour cells (DTCs) and tumour cells in the lung metastasis of the tri-PyMT orthotopic model. Sections of primary tumours and lungs from tri-PyMT orthotopic mice were immunostained for E-cadherin and vimentin (in white pseudo-colour). Essentially all RFP+ tumour cells are detected as E-cad+/Vim, while the scattered GFP+ tumour cells in the primary tumour are E-cad/Vim+ (as indicated by arrows in the top panel). Representative images are shown (n = 8). d, Plot shows the percentage of GFP+ cells out of total tumour cells (GFP+ plus RFP+, n = 6).

Extended Data Figure 6 Characterization of EMT status of orthotopic tri-Vim–PyMT primary tumours.

a, b, Sections of tri-Vim–PyMT orthotopic primary tumours (a) and metastatic lung (b) were immunostained for E-cadherin and vimentin (in white pseudo-colour). As expected, RFP+ tumour cells are entirely E-cadherin-positive and vimentin-negative, GFP+ tumour cells are vimentin-positive and E-cadherin-negative, and lung metastases are epithelial and RFP+.

Extended Data Figure 7 Dissemination of tri-PyMT cells in vivo.

a, Disseminated tumour cells are RFP+ and epithelial. RFP+ tri-PyMT cells were injected into the fat pad of mice. The fluorescence of the primary tumour, circulating tumour cells in the blood and disseminated tumour cells in the lung were analysed by flow cytometry. The flow cytometry plots depicted are the enumeration of RFP+ and GFP+ cells. b, The ratios of detected RFP+ versus GFP+ cells are shown in the chart (n = 4 mice). c, Relative expression of miR-200-family microRNAs in tri-PyMT control and miR-200-expressing cells. n = 2 individual experiments. d, Relative expression of EMT markers and tumour markers in tri-PyMT control and mir-200-expressing cells as determined by qRT–PCR with Gapdh as the internal control, n = 2 individual experiments.

Extended Data Figure 8 Effects of CTX therapy on primary tumours.

a, Quantification of primary tumour growth after 2 weeks of CTX therapy. For tumour growth data see accompanying Source Data. b, Proliferation status of primary tumour cells as detected by Ki67 staining in control mice and after 2 weeks of CTX therapy. c, Level of apoptosis in primary tumours as detected by active caspase-3 staining in control mice and after 2 weeks of CTX therapy. d, e, Representative images of Ki67 (d) and active caspase-3 staining (e) (white pseudo-colour) of primary tumours in control mice and CTX-treated mice. Scale bars, 50 μm. f, Proliferation status of RFP+ and GFP+ primary tumour cells as detected by Ki67 staining in control and CTX-treated mice. g, Level of apoptosis in RFP+ and GFP+ primary tumours as detected by active caspase-3 staining in control and CTX-treated mice. h, Percentage of GFP+ tumour cells in control and CTX-treated primary tumours. n = 3 mice for all figures described above. Quantification performed using ImageJ software. Source data

Extended Data Figure 9 EMT tumour cells are resistant to CTX treatment both in vitro and in vivo.

a, b, Long-term CTX treatment in vitro results in a GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 μM). Fluorescent imaging (a) and flow cytometry (quantified, b, n = 3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to untreated control cells c, d, EMT status of lung nodules in competitive survival assay. Representative fluorescent images of tri-PyMT lung metastases in untreated control lungs (c) and CTX-treated lungs (d), depicting RFP+ and GFP+ tumour cells. Immunostaining showing E-cadherin (E-cad) or vimentin (Vim) in white pseudo-colour. White arrow indicates GFP+ tumour cells with epithelial phenotypes (E-cad+/Vim), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad/Vim+). Nuclei were counter-stained with DAPI. n = 5.

Extended Data Figure 10 Gene expression profile analysis of RFP+ and GFP+ tri-PyMT cells.

RFP+ and GFP+ tri-PyMT cells were sorted by flow cytometry and subjected to transcriptomic analysis by RNA-sequencing. a, Heat map of differentially expressed genes (adjusted P < 0.05) from RNA-seq of sorted RFP+ and GFP+ tri-PyMT cells, biologically duplicated. Genes that are established epithelial markers (Group 1) include Cdh1 (which encodes E-cad), Dsp, Epcam, Fgfbp1, Krt18, Krt19, Ocln, Tjp3, Krt14 and Tjp2; the mesenchymal markers (Group 2) include Cdh2 (which encodes N-cad), Col23a1, Col3a1, Col5a1, Col6a2, Fsp1, Mmp3, Wnt5a and Zeb1. b, Cell cycle (left panel) and chemoresistance-related (right panel) genes alternatively regulated in RFP+ and GFP+ cells. c, GFP+ tri-PyMT cells were also sorted from CTX-treated (4 μM) samples. Interestingly, a branch of genes related to drug metabolism were significantly elevated in CTX-treated GFP+ cells. Group 1 genes are drug transporters including Abcb1a, Abcb1b and Abcc1. Group 2 genes are phase I drug-metabolizing enzymes including Adh7, Aldh1a1, Aldh1a3, Aldh1l1, Aldh1l2, Aldh2, Aldh3a1, Aldh3a2, Aldh3b2, Aldh4a1, Cyp1a1, Cyp2f2, Cyp2j6, Ptgs1 and Ptgs2. Group 3 genes are phase II drug metabolizing enzymes including Aox1, Blvrb, Ces2e, Ces2f, Ces2g, Chst1, Ephx1, Fmo1, Gpx2, Gsta3, Gsta4, Gstm2, Gsto1, Gstp1, Gstt3, Maoa, Mgst1, Mgst2, Nat6, Nat9, Nqo1, Pon3, Ugt1a6a and Ugt1a7c. d, Aldehyde dehydrogenase (ALDH) activity assay. Cell lysates were prepared from flow cytometry-sorted RFP+ and GFP+ tri-PyMT cells. ALDH activity in samples was measured by OD at 450 nm in a kinetic mode (every 3 min for 60 min). Representative result from two independent experiments depicted. e, EMT tumour cells (GFP+ cells) showed resistance to multiple commonly used chemotherapies. Tri-PyMT cells were subjected to treatment with CTX (8 μM), doxorubicin (Dox, 2 μM), paclitaxel (Taxol, 10 μM) and fluorouracil (5FU; 1.6 μM) for 3 days. Flow cytometry analysis of apoptotic cells was performed after Annexin staining. The percentage of dead cells (Annexin+) in RFP+ and GFP+ cells, respectively, was quantified. n = 2 biological replicates.

Supplementary information

Supplementary Figures

This file contains Supplementary Figure 1, which shows the un-cropped gels for Figure 2d. (PDF 75 kb)

Supplementary Tables

This file contains the RNA-Sequencing data. The raw RNA sequencing data was deposited in the NIH GEO repository under the accession number GSE72480. (XLSX 53 kb)

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Fischer, K., Durrans, A., Lee, S. et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472–476 (2015). https://doi.org/10.1038/nature15748

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