Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320

Journal name:
Nature Cell Biology
Volume:
14,
Pages:
159–167
Year published:
DOI:
doi:10.1038/ncb2396
Received
Accepted
Published online

Abstract

PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten–miR-320–Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.

At a glance

Figures

  1. miR-320 is a PTEN target in fibroblasts that suppresses tumour growth.
    Figure 1: miR-320 is a PTEN target in fibroblasts that suppresses tumour growth.

    (a) Expression of selected microRNAs was validated by qrtPCR in three sets of primary Pten+/+ or Pten−/− MMFs. Relative miR expression level (normalized to 18S ribosomal RNA) is shown as mean±s.d. miR-296-5p is an example of a miR whose expression did not change. n=4. (b) Gene profiling was carried out (n=2) on three types of cell from mammary glands with PtenLoxP/LoxP (Pten+/+) or Fspcre;PtenLoxP/LoxP (Pten−/−) as indicated. The heat map shows the gene signature with 314 genes changed in fibroblasts, 919 genes changed in epithelial cells and 323 genes changed in endothelial cells (>2-fold and P<0.05). (c) Representative images of tumours before and after excision from mice injected with DB7 cells admixed with Pten+/+ (n=7) or Pten−/− (n=16) MMFs (left panel), and DB7 cells admixed with Pten−/− MMFs or Pten−/−cells re-expressing miR-320 (n=9) (right panel). Tumour weights were quantified after four weeks, and the data are expressed as mean±s.d. All three groups were tested by analysis of variance and subsequent adjusted pairwise comparisons were carried out. These comparisons were significant: Pten+/+ versus Pten−/− MMFs, P=5.58×10−6; Pten+/+ versus Pten−/− miR-320 MMFs, P=0.022215; Pten−/− versus Pten−/− miR-320 MMFs, P=0.007976. (d) Epithelial tumour-cell proliferation measured by BrdU incorporation in xenografts composed of DB7 cells admixed with Pten−/− or Pten+/+ MMFs transfected with either negative control (NC), miR-320 (320, upper right panel) or anti-miR-320 (a320, lower right panel); n=4 for each group. The epithelial/fibroblast composites were collected five days after subcutaneous growth in mice. BrdU injection was carried out 3h before collecting the tissue. Scale bars, 25μm. Data are expressed as mean±s.d. *P<0.05. (e) Angiogenesis determined using CD31 staining to detect blood vessels in nascent tumours composed of DB7 cells and the same Pten−/− or Pten+/+ MMFs transfected with negative control (NC), miR-320 (320, upper panels) or anti-miR-320 (a320, lower panels), as above; n=4 for each group. The epithelial/fibroblast mixtures were grown for five days subcutaneously in mice. Representative micrographs showing merged images are shown (green, CD31; blue, 4,6-diamidino-2-phenylindole, DAPI). Scale bars, 50μm. Data are expressed as mean±s.d. *P<0.05.

  2. miR-320 and PTEN are co-expressed in human tumour stroma.
    Figure 2: miR-320 and PTEN are co-expressed in human tumour stroma.

    (a) Representative images of miR-320 in situ hybridization (ISH) staining in paraffin-imbedded, human normal (control) and breast cancer tissue; scrambled control (SC) was carried out on adjacent normal breast tissue. Images were captured with the Nuance multispectral system (blue channel shown). Scale bars, 50μm. The percentage of positive cells per random field was determined in normal and carcinoma TMA sections from 126 matched patient samples (Methods); results are shown as mean±s.d. on the graph. P=1.76×10−15. (b) Representative multispectral images of human breast carcinoma TMA samples stained for both miR-320 (ISH staining, blue) and PTEN (IHC, red). Upper panels, low magnification; lower panels, high magnification. Scale bars, 50μm. Co-localization of miR-320 and PTEN signals determined from the merged image was converted to fluorescent (yellow) signal using the Nuance multispectral system and is indicated by the arrows.

  3. Pten and miR-320 in fibroblasts control tumour-cell migration and growth.
    Figure 3: Pten and miR-320 in fibroblasts control tumour-cell migration and growth.

    (a) Left: representative images of DB7 mammospheres in the presence of DMEM/fetal bovine serum medium (Con) (n=6) or conditioned medium (CM) produced by either Pten+/+ (n=4) or Pten−/− (n=6) MMFs. Scale bars, 100μm. The insets in all panels are magnified ×2.5. Right: quantification of migratory zones indicated by arrows expressed as mean area of migration ±s.d., *P<0.01. (b) Left: representative images of DB7 mammospheres in the presence of conditioned medium from Pten−/− MMFs expressing miR negative control (Pten−/−NC) (n=12) or miR-320 precursor (Pten−/−320) (n=12). Scale bars, 200μm. Right: quantification of migratory zones indicated by arrows expressed as mean area of migration ±s.d., *P<0.01. (c) Left: representative images of BrdU incorporation in DB7 cells in the presence of conditioned medium from MMFs expressing miR negative control (Pten−/−NC) (n=4) or with miR-320 precursors (Pten−/−320) (n=4). RFP, red fluorescent protein. Scale bars, 100μm. Right: quantification of BrdU incorporation. Data expressed as mean±s.d., *P<0.01. (d) Left: representative images of tube-like formation of endothelial cells grown on Matrigel in the presence of conditioned medium from MMFs expressing miR negative control (Pten−/−NC) (n=3) or with miR-320 precursor (Pten−/−320) (n=3). Right: quantification of tube formation expressed as mean±s.d., *P<0.01. Scale bars, 50μm. (e) Top: representative images of BrdU incorporation by endothelial cells in the presence of conditioned medium from MMFs expressing miR negative control (Pten−/−NC) (n=3) or with miR-320 precursors (Pten−/−320) (n=3). Scale bars, 100μm. Bottom: quantification of BrdU incorporation. Data expressed as mean±s.d., *P<0.01.

  4. miR-320 regulates the secretome of MMFs.
    Figure 4: miR-320 regulates the secretome of MMFs.

    (a) Pten+/+ and Pten−/− MMFs were left untreated (lanes 1 and 2) or were transiently transfected with miR negative control (NC), miR-320 precursors (320) or anti-miR-320 precursor (anti-320), respectively. Conditioned media were examined by Western blotting with antibodies against the indicated proteins; thrombospondin 2 (THBS2) serves as an internal control. (b) Representative images of DB7 mammospheres in the presence of conditioned medium from Pten−/− MMFs expressing siRNA negative control (NC) (n=4) or siRNA against Mmp9 (n=4). MMP9 downregulation was verified by Western blot using MMP9 antibody. MMP2 was used as a loading control. Scale bars, 200μm. The insets in all panels are magnified ×2.5. Quantification of migratory zones (indicated by arrows) expressed as mean area of migration ±s.d., *P<0.01 (bar graphs). (c) Representative images of DB7 mammospheres in the presence of conditioned medium from Pten−/−MMFs precleared with IgG (n=4) or IgG precoupled with α-MMP9 (n=4). Western blots using MMP9 antibody in precleared and input control samples were carried out as a control. Scale bars, 200μm. The insets in all panels are magnified ×2.5. Quantification of migratory zones (indicated by arrows) expressed as mean area of migration ±s.d., *P<0.01 (bar graphs). (d) Representative images of BrdU incorporation in endothelial cells in the presence of conditioned medium from Pten−/− MMFs expressing siRNA negative control (NC) (n=3) or siRNA against Emilin2 (n=3). EMILIN2 downregulation was verified by Western blot using EMILIN2 antibody. Scale bars, 100μm. Quantification of BrdU incorporation expressed as mean±s.d., *P<0.01. (e) Representative images of BrdU incorporation in endothelial cells in the presence of conditioned medium from Pten−/−MMFs precleared with IgG (n=3) or IgG precoupled with α-EMILIN2 (n=4). Western blots using EMILIN2 antibody in precleared and input control samples were carried out as a control. Scale bars, 100μm. Quantification of BrdU incorporation expressed as mean±s.d., *P<0.01. Full-length blots are presented in Supplementary Fig. S7a.

  5. Identification and characterization of Ets2, Mmp9 and Emilin2 as bona fide targets of miR-320.
    Figure 5: Identification and characterization of Ets2, Mmp9 and Emilin2 as bona fide targets of miR-320.

    (a) Pten+/+ and Pten−/− MMFs were left untreated (lanes 1 and 2) or were transiently transfected with miR negative control (NC), miR-320 precursors (320) or anti-miR-320 (anti-320), respectively. Nuclear extracts were blotted with anti-ETS2 antibody or anti-PTEN antibody; anti- β-actin antibody was used as a loading control. (b) COS7 cells were transfected with full-length cDNA (5′-UTR–open reading frame–3′-UTR) of Ets2 (Ets2+UTR) wild type (wt) or mutated in miR-320 seeding region (mut) expression vector and co-transfected with either miR negative control (NC) or miR-320 precursors (320). Cell lysates or conditioned medium were blotted with anti-ETS2 antibody; anti- β-actin antibody was used as a loading control. (c) COS7 cells were transfected with full-length cDNA (5′-UTR–open reading frame–3′-UTR) of Mmp9 (Mmp9+UTR) (left panel) or Emilin2 (Emilin2+UTR) (right panel) and co-transfected with either miR negative control (NC) or miR-320 precursors (320). Cell lysates or conditioned medium were blotted with anti-MMP9 or anti-EMILIN2 antibody; anti- β-actin antibody was used as a loading control. (d) Pten−/− MMFs were transiently transfected either with siRNA negative control (NC) or siRNA against Akt1, Mapk8/Jnk1, Mapk9/Jnk2 or the combination of Mapk8/Jnk1 and Mapk9/Jnk2. AKT and JNK phosphorylation status was verified by Western blot using anti-phospho-specific antibodies, and compared with total kinase levels using non-discriminating antibodies. Tubulin was used as a loading control. Expression of miR-320 was validated by qrtPCR (n=3). Mean relative miR expression level ±s.d. is shown. Full-length blots are presented in Supplementary Fig. S7b–d.

  6. The miR-320 secretome profile separates human breast normal and cancer stroma (tumour type, TT) and predicts patient outcome.
    Figure 6: The miR-320 secretome profile separates human breast normal and cancer stroma (tumour type, TT) and predicts patient outcome.

    (a) Heat map showing the differential expression in human tumour versus normal stroma of the 40 human orthologues from the 54-factor mouse secretome that were retrieved from the McGill stromal microarray (GSE4823). The P value indicates the ability of the 40-gene signature to partition normal and tumour stroma when compared with 10,000 random permutations (Methods). (b) Kaplan–Meier curves of high- and low-risk groups based on expression of the 40-gene secretome signature present in the GSE9014 dataset. Expression of the 40-gene secretome signature correlates with poor patient outcomes. The permutation P value of the log-rank test statistic between risk groups is based on 1,000 permutations. (c) Heat map showing the differential expression in tumour versus normal stroma of 13 human orthologues from the 20 ETS2-target genes that were retrieved from the McGill breast cancer stroma microarray (GSE4823). The P value indicates the ability of the mouse 17-gene signature to partition normal and tumour stroma as above (Methods). (d) Kaplan–Meier survival curves of high- and low-risk groups based on expression of the 13-gene ETS2-related secretome signature. Expression of the subset of secretome genes directly regulated by ETS2 (13/20 genes in GSE4823) correlates with poor patient outcome. The permutation P value of the log-rank test statistic between risk groups is based on 1,000 permutations.

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Author information

Affiliations

  1. Tumor Microenvironment Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA

    • A. Bronisz,
    • G. Leone &
    • M. C. Ostrowski
  2. Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA

    • A. Bronisz,
    • J. A. Wallace,
    • A.S. Merchant,
    • H. Mathsyaraja,
    • R. Srinivasan,
    • C. K. Martin,
    • F. Li &
    • M. C. Ostrowski
  3. Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University Medical Center and James Comprehensive Cancer Center, Columbus, Ohio 43210, USA

    • J. Godlewski,
    • M.O. Nowicki,
    • S. E. Lawler &
    • E. A. Chiocca
  4. Department of Molecular Genetics, College of Biological Sciences, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA

    • A. J. Trimboli,
    • F. Li,
    • T. Pécot &
    • G. Leone
  5. Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio 43210, USA

    • A. J. Trimboli,
    • T. Pécot &
    • G. Leone
  6. Center for Biostatistics, Office of Health Sciences, The Ohio State University, Columbus, Ohio 43210, USA

    • L. Yu &
    • S. A. Fernandez
  7. The Ohio State University Computer Science and Engineering, The Ohio State University Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA

    • T. Pécot
  8. Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA

    • T. J. Rosol
  9. McGill Centre for Bioinformatics, McGill University, Montreal, Quebec, H3A0B1, Canada

    • S. Cory &
    • M. Hallett
  10. Department of Biochemistry, Rosalind and Morris Goodman Cancer Center University, Montreal, Quebec, H3A0B, Canada

    • S. Cory,
    • M. Hallett &
    • M. Park
  11. Department of Oncology, McGill University, Quebec, H3A 1A1, Canada

    • M. Park
  12. Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Internal Medicine, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA

    • M. G. Piper &
    • C. B. Marsh
  13. Department of Surgery, School of Medicine, The Ohio State University, Columbus, Ohio 43210, USA

    • L. D. Yee
  14. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA

    • R. E. Jimenez
  15. Department of Pathology, The Ohio State University Medical Center and James Comprehensive Cancer Center, Columbus, Ohio 43210, USA

    • G. Nuovo

Contributions

M.C.O. and G.L. designed and supervised this study, analysed data and helped write and edit the manuscript. A.B. and J.G. designed and carried out experiments, collected and analysed data and co-wrote the paper. J.A.W., H.M., R.S., A.J.T. and F.L. assisted technically with experiments, and collected and analysed data. M.O.N. assisted with fluorescent and confocal microscopy and immunohistochemistry. L.Y. and S.A.F. contributed to the statistical analyses of data and writing the manuscript. A.S.M., S.C., M.H., M.P. and T.P. contributed to the analysis and comparison of mouse and human profile data. M.G.P. and C.B.M. contributed to the analysis of microRNA data and writing the manuscript. C.K.M., L.D.Y., R.E.J., G.N. and T.J.R. contributed to the histopathological analysis of human samples and writing the manuscript. S.E.L. and E.A.C. contributed to the data analysis and writing the manuscript.

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The authors declare no competing financial interests.

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