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Nuclear respiratory factor 1 promotes spheroid survival and mesenchymal transition in mammary epithelial cells

Oncogene (2018) | Download Citation


Epithelial cells aggregate into spheroids when deprived of matrix, and the proclivity for spheroid formation and survival is a hallmark of normal and tumorigenic mammary stem cells. We show here that Nuclear Respiratory Factor 1 (NRF1) is a spheroid promoter by in silico identification of this transcription factor as highly connected to top shRNA-hits deduced from re-iterative selections for shRNAs enriched in MCF10A spheroids. NRF1-promoted spheroid survival is linked to its stimulation of mitochondrial OXPHOS, cell migration, invasion, and mesenchymal transition. Conversely, NRF1 knockdown in breast cancer MDA-MB-231 cells reduced spheroids, migration, invasion, and mesenchymal marker expression. NRF1 knockdown also reduced tumor burden in mammary fat pads and lungs of orthotopic- or tail vein-transplanted mice. With the Luminal A subtype of breast cancer, higher NRF1 expression is associated with lower survival. These results show that NRF1, an activator of mitochondrial metabolism, supports mammary spheroid survival and tumor development.

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  1. 1.

    Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994;124:619–26.

  2. 2.

    Paoli P, Giannoni E, Chiarugi P. Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta. 2013;1833:3481–98.

  3. 3.

    Vlahakis A, Debnath J. The interconnections between autophagy and integrin-mediated cell adhesion. J Mol Biol. 2017;429:515–30.

  4. 4.

    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.

  5. 5.

    Rayavarapu RR, Heiden B, Pagani N, Shaw MM, Shuff S, Zhang S, et al. The role of multicellular aggregation in the survival of ErbB2-positive breast cancer cells during extracellular matrix detachment. J Biol Chem. 2015;290:8722–33.

  6. 6.

    Mason JA, Hagel KR, Hawk MA, Schafer ZT. Metabolism during ECM detachment: achilles heel of cancer cells. Trends Cancer. 2017;3:475–81.

  7. 7.

    Kim YN, Koo KH, Sung JY, Yun UJ, Kim H. Anoikis resistance: an essential prerequisite for tumor metastasis. Int J Cell Biol. 2012;2012:306879.

  8. 8.

    Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17:1253–70.

  9. 9.

    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100:3983–8.

  10. 10.

    Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res. 2008;10:R52.

  11. 11.

    Dey D, Saxena M, Paranjape AN, Krishnan V, Giraddi R, Kumar MV, et al. Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture. PLoS One. 2009;4:e5329.

  12. 12.

    Au SH, Storey BD, Moore JC, Tang Q, Chen YL, Javaid S, et al. Clusters of circulating tumor cells traverse capillary-sized vessels. Proc Natl Acad Sci USA. 2016;113:4947–52.

  13. 13.

    Grassian AR, Schafer ZT, Brugge JS. ErbB2 stabilizes epidermal growth factor receptor (EGFR) expression via Erk and Sprouty2 in extracellular matrix-detached cells. J Biol Chem. 2011;286:79–90.

  14. 14.

    Kelly DP, Scarpulla RC. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev. 2004;18:357–68.

  15. 15.

    Scarpulla RC. Nuclear control of respiratory chain expression by nuclear respiratory factors and PGC-1-related coactivator. Ann N Y Acad Sci. 2008;1147:321–34.

  16. 16.

    Scarpulla RC. Nuclear control of respiratory gene expression in mammalian cells. J Cell Biochem. 2006;97:673–83.

  17. 17.

    Mattingly KA, Ivanova MM, Riggs KA, Wickramasinghe NS, Barch MJ, Klinge CM. Estradiol stimulates transcription of nuclear respiratory factor-1 and increases mitochondrial biogenesis. Mol Endocrinol. 2008;22:609–22.

  18. 18.

    Tan AS, Baty JW, Dong LF, Bezawork-Geleta A, Endaya B, Goodwin J, et al. Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metab. 2015;21:81–94.

  19. 19.

    LeBleu VS, O’Connell JT, Gonzalez Herrera KN, Wikman H, Pantel K, Haigis MC, et al. PGC-1alpha mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Nat Cell Biol. 2014;16:992–1003. 1001–15

  20. 20.

    Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sanchez N, Marchesini M, et al. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature. 2014;514:628–32.

  21. 21.

    Vais H, Tanis JE, Muller M, Payne R, Mallilankaraman K, Foskett JK. MCUR1, CCDC90A, is a regulator of the mitochondrial calcium uniporter. Cell Metab. 2015;22:533–5.

  22. 22.

    Paupe V, Prudent J, Dassa EP, Rendon OZ, Shoubridge EA. CCDC90A (MCUR1) is a cytochrome c oxidase assembly factor and not a regulator of the mitochondrial calcium uniporter. Cell Metab. 2015;21:109–16.

  23. 23.

    Jeon SM, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature. 2012;485:661–5.

  24. 24.

    Hindupur SK, Balaji SA, Saxena M, Pandey S, Sravan GS, Heda N, et al. Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells. Breast Cancer Res. 2014;16:420.

  25. 25.

    Ng TL, Leprivier G, Robertson MD, Chow C, Martin MJ, Laderoute KR, et al. The AMPK stress response pathway mediates anoikis resistance through inhibition of mTOR and suppression of protein synthesis. Cell Death Differ. 2012;19:501–10.

  26. 26.

    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–15.

  27. 27.

    Qu Y, Han B, Yu Y, Yao W, Bose S, Karlan BY, et al. Evaluation of MCF10A as a reliable model for normal human mammary epithelial cells. PLoS One. 2015;10:e0131285.

  28. 28.

    Oliver D, Ji H, Liu P, Gasparian A, Gardiner E, Lee S, et al. Identification of novel cancer therapeutic targets using a designed and pooled shRNA library screen. Sci Rep. 2017;7:43023.

  29. 29.

    Sims D, Mendes-Pereira AM, Frankum J, Burgess D, Cerone MA, Lombardelli C, et al. High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biol. 2011;12:R104.

  30. 30.

    Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267–73.

  31. 31.

    Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature. 2014;509:487–91.

  32. 32.

    Dey P, Baddour J, Muller F, Wu CC, Wang H, Liao WT, et al. Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer. Nature. 2017;542:119–23.

  33. 33.

    Sullivan LB, Gui DY, Hosios AM, Bush LN, Freinkman E, Vander Heiden MG. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell. 2015;162:552–63.

  34. 34.

    Birsoy K, Wang T, Chen WW, Freinkman E, Abu-Remaileh M, Sabatini DM. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell. 2015;162:540–51.

  35. 35.

    Ivanova MM, Luken KH, Zimmer AS, Lenzo FL, Smith RJ, Arteel MW, et al. Tamoxifen increases nuclear respiratory factor 1 transcription by activating estrogen receptor beta and AP-1 recruitment to adjacent promoter binding sites. FASEB J. 2011;25:1402–16.

  36. 36.

    Mattingly KA, Klinge CM. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells. Arch Toxicol. 2012;86:633–42.

  37. 37.

    Rosenbloom KR, Sloan CA, Malladi VS, Dreszer TR, Learned K, Kirkup VM, et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res. 2013;41:D56–63.

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We thank J. Dai, Q. Wang, Z. Zhang, and G. Cheng for insightful comments on the study, and X. He for technical assistance. Nano-Bio-Chem Centre in Suzhou Institute of Nano-Tech and Nano-Bionics (SINANO) is acknowledged for professional assistance of cell imaging and FACS assay. This study was initiated as a pilot project in the San Diego Systems Biology Center (NIH P50GM085764) that supported G.S., D.Q., O. H., and J.Y.J.W.; D.Q. was also supported by a grant to J.Y.J.W. (NIH R01CA043054). The study of NFR1 was supported by National Natural Science Foundation of China (Grant No. 31471307), Ministry of Science and Technology (MOST) (Grant No. 2014CB965003, and 2017YFA0104301) to G.S., who is also supported by Hundred Talent Program of Chinese Academy of Sciences. The study was also supported by the CAS/SAFEA International Innovation Teams program.

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Author notes

  1. These authors contributed equally: Yuanshuai Zhou, Zhongjuan Xu.

  2. These authors jointly supervised this work: Jean Y. J. Wang, Guangli Suo.


  1. CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu, 215123, China

    • Yuanshuai Zhou
    • , Zhongjuan Xu
    • , Hai Zhang
    • , Tongqian Xiao
    • , Shulan Hou
    •  & Guangli Suo
  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    • Yuanshuai Zhou
    •  & Tongqian Xiao
  3. Division of Hematology/Oncology, Department of Medicine, Moores Cancer Center, University of California, San Diego, School of Medicine, La Jolla, CA, 92093-0820, USA

    • Daniel Quan
    • , Olivier Harismendy
    •  & Jean Y. J. Wang
  4. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 78712, USA

    • Fan Zhang
    •  & Hong Qiao


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The authors declare that they have no conflict of interest.

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Correspondence to Guangli Suo.

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