miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma

Journal name:
Nature Medicine
Volume:
16,
Pages:
1134–1140
Year published:
DOI:
doi:10.1038/nm.2227
Received
Accepted
Published online

Abstract

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′ untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.

At a glance

Figures

  1. The p53 3[prime] UTR contains binding sites for miR-380-5p, a developmentally restricted miRNA.
    Figure 1: The p53 3′ UTR contains binding sites for miR-380-5p, a developmentally restricted miRNA.

    (a) Alignment of human, mouse, rat and hamster p53 3′ UTRs, identifying a highly conserved 104-bp region. The predicted miR-380-5p binding sites are indicated in red. (b) Northern blot of miR-380 using total RNA from mouse embryonic, human fetal and adult tissues. (c) Quantitative RT-PCR (qRT-PCR) analysis of miR-380-5p expression in normal brain, embryonic carcinoma (P19) and mouse ES cells. (d) qRT-PCR analysis showing miR-380-5p expression in ES cells differentiated to the neuronal lineage. (e) Immunofluorescent staining for Sox1 (green) in cultures of neural progenitor cells. Scale bar, 65 μm. (f,g) Immunofluorescent staining for an early neuronal marker, Tuj1 (red), and a marker of astrocytes, GFAP (green), in differentiated cultures of neurons (f) and astrocytes (g). Scale bars: 150 μm (f); 100 μm (g). In c and d, error bars depict s.d.; in d, independent biological replicates indicated by separate bars. *P < 0.0002.

  2. miR-380-5p is required for ES cell survival.
    Figure 2: miR-380-5p is required for ES cell survival.

    (a) Activity of a miR-380 reporter either alone or after transfection with a control LNA (LNA-Ctrl) or an LNA directed against miR-380-5p (LNA-380). (b) Left, amount of ES cell death 24h after transfection of wild-type (WT) or Trp53−/− ES cells with LNA-Ctrl or LNA-380. Right, cell images 24 h post transfection with LNA-Ctrl or LNA-380. (c) qRT-PCR analysis of relative miR-380-5p expression in WT, Trp53−/− and Dgcr8−/− ES cells. (d) Western blots showing p53 induction and PARP cleavage (indicated by the arrow) after knockdown of miR-380-5p by LNA-380 compared to LNA-ctrl–transfected wild-type, Trp53−/− and Dgcr8−/− ES cells that were ultraviolet irradiated (UV). In a, b and d, results are from at least three independent experiments (performed in triplicate in c). In ac, error bars depict s.d. *P < 0.00002; **P < 0.00007; ***P < 0.0004; NS, not significant. Scale bars, 200 μm.

  3. miR-380-5p targets p53 and decreases cell death after genotoxic stress.
    Figure 3: miR-380-5p targets p53 and decreases cell death after genotoxic stress.

    (a) Western blot showing p53 expression in MCF10a cells transfected with either a scrambled (SC) miRNA or miR-380-5p. (b) Quantification of p53 protein in MCF10a cells transfected with SC miRNA or miR-380-5p with or without UV treatment, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (c) qRT-PCR analysis of TP53 mRNA in MCF10a cells after expression of miR-380, normalized to GAPDH levels. (d) Luciferase activity of TP53 3′UTR reporters after expression of miR-380. (e,f) Relative cell death of MCF10a cell populations stably expressing miR-380 or SC 24 h after treatment with ultraviolet light (e) or cisplatin (f). Error bars depict s.e.m. (b,c) or s.d. (df). a,b and df are results from at least three independent repeats; in c experiments were performed in triplicate. *P < 0.0009, **P < 0.03, ***P < 0.002, ****P < 0.05, *****P < 0.03, ******P < 0.001.

  4. miR-380 prevents oncogene-induced senescence and increases tumor incidence in a mouse mammary cancer model.
    Figure 4: miR-380 prevents oncogene-induced senescence and increases tumor incidence in a mouse mammary cancer model.

    (a) The incidence of palpable mammary tumors (1 tumor per mouse) arising from cells infected with the indicated miRNA-encoding retrovirus plus HRASV12 after 6 weeks, control group with empty vector or expressing scrambled control (vector/SC). n = 15; miR-125b, n = 10; miR-380, n = 15; p53 shRNA, n = 17. (b) qRT-PCR of mature miR-380-5p in tumors arising from cells infected with HRASV12 and miR-380 or p53 shRNA virus. MCF10a cells that stably express miR-380 or a scrambled miRNA are shown as controls. (c) Immunohistochemical staining of mammary tumors. SA-β-gal, senescence-associated β-galactosidase. (d) qRT-PCR for p21waf1 expression normalized to GAPDH in tumors arising from cells infected with HRASV12 and p53 shRNA or miR-380 retrovirus compared to the primary MMECs. In b and d, error bars depict s.d.; each column represents a separate tumor. Scale bars, 100 μm.

  5. miR-380-5p is expressed in mouse and human neuroblastoma and is associated with poor outcome in subjects with MYCN amplification.
    Figure 5: miR-380-5p is expressed in mouse and human neuroblastoma and is associated with poor outcome in subjects with MYCN amplification.

    (a) qRT-PCR for miR-380-5p expression in tumors and neuroendocrine ganglion (SCG) tissue from wild type and transgenic mice. (b) miR-380-5p expression detected by qRT-PCR in primary human neuroblastoma samples taken before chemotherapy. miR-380-5p expression was normalized to U6 small nuclear 2 RNA, normal human brain expression (indicated by red dashed line); 'low' and 'high' designate the lowest quartile of miR-380-5p expression and the remainder, respectively. (c) Kaplan-Meier survival curves of event-free survival (EFS) in subgroups of subjects with neuroblastoma according to relative expression level of miR-380-5p, all with MYCN amplification (n = 22). Subjects were dichotomized around the lower quartile of miR-380-5p expression. High: n = 6, mean miR-380-5p expression = 3.19, s.e.m. = 0.83. Low: n = 16, mean miR-380-5p expression = 0.49, s.e.m. = 0.13. P = 0.004. In a, error bars depict s.d., *P < 0.02, **P < 0.02.

  6. Treatment with miR-380-5p antagonist induces p53-dependent cell death in neuroblastoma cells and decreases tumor growth in vivo.
    Figure 6: Treatment with miR-380-5p antagonist induces p53-dependent cell death in neuroblastoma cells and decreases tumor growth in vivo.

    (a) Western blots showing p53 and p21waf1 induction and PARP cleavage after knockdown of miR-380-5p by LNA-380 compared to control LNA in NBL-WS cells (left) but not TP53-mutant BE(2)C cells (right). Arrow indicates cleaved PARP. (b) Images of NBL-WS cells 24 h after mock transfection (mock) or treatment with the indicated LNAs; an LNA directed against let7e (LNA-let7e) was included as an additional control. Scale bars, 200 μm. (c) MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay showing that knockdown of miR-380-5p by LNA-380 induces rapid loss of cell viability in NBL-WS cells (left) but not BE(2)C cells (right). Dox, doxorubicin treatment. (d) Tumor size after systemic treatment with miR-380 antagonist (anti-miR380) for 3 weeks; mass depicted is the weight of the kidney (indicated by dashed red line) plus associated tumor (n = 5 mice for each treatment). (e) Representative images of kidneys and associated neuroblastoma tumor mass from two different mice for each treatment group. Scale bars, 1 cm. In ac, results are representative of at least three independent experiments, error bars depict s.e.m. *P < 0.01.

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Author information

  1. These authors contributed equally to this work.

    • Alexander Swarbrick &
    • Susan L Woods

Affiliations

  1. Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

    • Alexander Swarbrick,
    • Alexander Shaw,
    • Yuwei Phua &
    • Akira Nguyen
  2. St. Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, Australia.

    • Alexander Swarbrick,
    • Yuwei Phua &
    • Thomas Preiss
  3. G.W. Hooper Research Foundation, University of California–San Francisco, San Francisco, California, USA.

    • Susan L Woods
  4. Division of Genetics & Population Health, Queensland Institute of Medical Research, Brisbane, Queensland, Australia.

    • Susan L Woods
  5. Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia.

    • Alexander Shaw &
    • Thomas Preiss
  6. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia.

    • Alexander Shaw &
    • Thomas Preiss
  7. Department of Medicine, University of California–San Francisco, San Francisco, California, USA.

    • Asha Balakrishnan,
    • Peter Lengyel &
    • Andrei Goga
  8. Biomedical Sciences Program, University of California–San Francisco, San Francisco, California, USA.

    • Yvan Chanthery,
    • Lionel Lim,
    • Robert L Judson,
    • Noelle Huskey &
    • Christopher S Hackett
  9. Children's Cancer Institute Australia for Medical Research, Sydney, New South Wales, Australia.

    • Lesley J Ashton,
    • Michelle Haber &
    • Murray D Norris
  10. Department of Urology, University of California–San Francisco, San Francisco, California, USA.

    • Robert Blelloch
  11. Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research and Center for Reproductive Sciences, University of California–San Francisco, San Francisco, California, USA.

    • Robert Blelloch
  12. Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.

    • Albert Chetcuti
  13. Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas, USA.

    • Christopher S Sullivan
  14. Regulus Therapeutics, San Diego, California, USA.

    • Eric G Marcusson
  15. Department of Neurology, University of California–San Francisco, San Francisco, California, USA.

    • William Weiss
  16. Department of Pediatrics, University of California–San Francisco, San Francisco, California, USA.

    • William Weiss
  17. Center for Neuroscience, University of California–Davis, Davis, California, USA.

    • Noelle L'Etoile
  18. Helen Diller Cancer Center, University of California–San Francisco, San Francisco, California, USA.

    • Andrei Goga
  19. Liver Center, University of California–San Francisco, San Francisco, California, USA.

    • Andrei Goga

Contributions

S.L.W., A. Swarbrick and A.G. conceived and designed the experiments, discussed the results and wrote the manuscript. A. Swarbrick, S.L.W., A. Shaw, Y.P., A.N., A.G., R.L.J., C.S.S., C.S.H., P.L., A.B., N.H., Y.C. and L.L. performed experiments. L.J.A. and M.D.N. performed statistical analysis of the human neuroblastoma data set. A.C. provided human samples and clinical data, and E.G.M. provided anti-miRs for in vivo studies. M.H., T.P., W.W., N.L. and C.S.S. supervised experiments or experimental design.

Competing financial interests

E.G.M. is an employee and shareholder of Regulus Therapeutics.

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