Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

Nature Cell Biology volume 18, pages 431–442 (2016)Cite this article

Subjects

Abstract

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signalling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that the lncRNA NBR2 (neighbour of BRCA1 gene 2) is induced by the LKB1–AMPK pathway under energy stress. On energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy-stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumour development in vivo. NBR2 is downregulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, the results of our study uncover a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to understand further the regulation of kinase signalling by lncRNAs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Energy stress induces NBR2 expression through the LKB1–AMPK pathway.
Figure 2: NBR2 regulates AMPK–mTORC1 signalling under energy stress.
Figure 3: NBR2 regulates cell proliferation, apoptosis and autophagy in response to energy stress.
Figure 4: NBR2 inhibits tumour development.
Figure 5: Energy stress induces NBR2 interaction with AMPK.
Figure 6: NBR2 promotes AMPK kinase activity.
Figure 7: The functional effects of NBR2 are partially mediated by AMPK.

Similar content being viewed by others

References

  1. Ulitsky, I. & Bartel, D. P. lincRNAs: genomics, evolution, and mechanisms. Cell 154, 26–46 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. EP Consortium An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

    Article  Google Scholar 

  3. Batista, P. J. & Chang, H. Y. Long noncoding RNAs: cellular address codes in development and disease. Cell 152, 1298–1307 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gupta, R. A. et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Huarte, M. et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142, 409–419 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Prensner, J. R. et al. Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression. Nat. Biotechnol. 29, 742–749 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Engreitz, J. M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Rinn, J. L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang, K. C. et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120–124 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Guttman, M. & Rinn, J. L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. van Heesch, S. et al. Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes. Genome Biol. 15, R6 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hawley, S. A. et al. Complexes between the LKB1 tumor suppressor, STRAD α/β and MO25 α/β are upstream kinases in the AMP-activated protein kinase cascade. J. Biol. 2, 28 (2003).

    PubMed  PubMed Central  Google Scholar 

  14. Woods, A. et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13, 2004–2008 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Shaw, R. J. et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl Acad. Sci. USA 101, 3329–3335 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hardie, D. G., Schaffer, B. E. & Brunet, A. AMPK: an energy-sensing pathway with multiple inputs and outputs. Trends Cell Biol. 26, 190–201 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gwinn, D. M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Inoki, K., Zhu, T. & Guan, K. L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shackelford, D. B. & Shaw, R. J. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 9, 563–575 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Huang, J. & Manning, B. D. The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem. J. 412, 179–190 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Hezel, A. F. & Bardeesy, N. LKB1; linking cell structure and tumor suppression. Oncogene 27, 6908–6919 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Alessi, D. R., Sakamoto, K. & Bayascas, J. R. LKB1-dependent signaling pathways. Annu. Rev. Biochem. 75, 137–163 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Faubert, B. et al. AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo. Cell Metab. 17, 113–124 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Xu, C. F. et al. Isolation and characterisation of the NBR2 gene which lies head to head with the human BRCA1 gene. Hum. Mol. Genet. 6, 1057–1062 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Sim, A. T. & Hardie, D. G. The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase. FEBS Lett. 233, 294–298 (1988).

    Article  CAS  PubMed  Google Scholar 

  29. Bungard, D. et al. Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science 329, 1201–1205 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Corradetti, M. N., Inoki, K., Bardeesy, N., DePinho, R. A. & Guan, K. L. Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome. Genes Dev. 18, 1533–1538 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hardie, D. G. AMPK-sensing energy while talking to other signaling pathways. Cell Metab. 20, 939–952 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gad, S. et al. Characterisation of a 161 kb deletion extending from the NBR1 to the BRCA1 genes in a French breast-ovarian cancer family. Hum. Mutat. 21, 654 (2003).

    Article  PubMed  Google Scholar 

  33. Jin, H. et al. Structural evolution of the BRCA1 genomic region in primates. Genomics 84, 1071–1082 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Pandey, R. R. et al. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell 32, 232–246 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Feng, J. et al. The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev. 20, 1470–1484 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lee, S. W. et al. Skp2-dependent ubiquitination and activation of LKB1 is essential for cancer cell survival under energy stress. Mol. Cell 57, 1022–1033 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lin, A. et al. The FoxO-BNIP3 axis exerts a unique regulation of mTORC1 and cell survival under energy stress. Oncogene 33, 3183–3194 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Gan, B. et al. Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells. Nature 468, 701–704 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gan, B. et al. mTORC1-dependent and -independent regulation of stem cell renewal, differentiation, and mobilization. Proc. Natl Acad. Sci. USA 105, 19384–19389 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gan, B. et al. FoxOs enforce a progression checkpoint to constrain mTORC1-activated renal tumorigenesis. Cancer Cell 18, 472–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lin, A. et al. FoxO transcription factors promote AKT Ser473 phosphorylation and renal tumor growth in response to pharmacological inhibition of the PI3K-AKT pathway. Cancer Res. 74, 1682–1693 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gyorffy, B., Surowiak, P., Budczies, J. & Lanczky, A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS ONE 8, e82241 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Gyorffy, B. et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res. Treat. 123, 725–731 (2010).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all members of the Gan laboratory for their advice and technical assistance. This research has been supported by grants from MD Anderson Cancer Center, US Department of Defense (TS093049), Cancer Prevention & Research Institute of Texas (RP130020), National Institutes of Health (CA181196 and CA190370), Ellison Medical Foundation (AG-NS-0973-13), and Gabrielle’s Angel Foundation for Cancer Research (to B.G.). B.G. is a Kimmel Scholar and an Ellison Medical Foundation New Scholar. H.Liang is supported by the National Institutes of Health (CA143883, CA175486); the R. Lee Clark Fellow Award from The Jeanne F. Shelby Scholarship Fund; a grant from the Cancer Prevention and Research Institute of Texas (RP140462); and the Mary K. Chapman Foundation and the Lorraine Dell Program in Bioinformatics for Personalization of Cancer Medicine. L.H. is supported by Cancer Prevention & Research Institute of Texas (RR150085). H.-K.L. is supported by the National Institutes of Health (CA182424 and CA193813). B.G., J.C., J.W. and H.Liang are members of the MD Anderson Cancer Center, and are supported by the National Institutes of Health Core Grant CA016672.

Author information

Author notes

  1. Xiaowen Liu and Zhen-Dong Xiao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA

    Xiaowen Liu, Zhen-Dong Xiao, Wenqi Wang, Hyemin Lee, Li Zhuang, Junjie Chen & Boyi Gan

  2. Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, USA

    Leng Han

  3. Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA

    Jiexin Zhang, Jing Wang & Han Liang

  4. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA

    Szu-Wei Lee, Hui-Kuan Lin & Boyi Gan

  5. Program of Genes and Development, and Program of Cancer Biology, The University of Texas Graduate School of Biomedical Sciences, 1515 Holcombe Boulevard, Houston, Texas 77030, USA

    Szu-Wei Lee, Junjie Chen, Hui-Kuan Lin & Boyi Gan

  6. Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, USA

    Hui-Kuan Lin

  7. Graduate Institute of Basic Medical Science, China Medical University, Taichung 404, Taiwan

    Hui-Kuan Lin

Authors
  1. Xiaowen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhen-Dong Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leng Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jiexin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Szu-Wei Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenqi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hyemin Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Li Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Junjie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hui-Kuan Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Han Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Boyi Gan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.-D.X. initiated the project and identified NBR2 as an energy-stress-induced lncRNA. X.L. performed most of the experiments with assistance from Z.-D.X., H.Lee, and L.Z. J.Z. and J.W. analysed RNA-seq data set. L.H. and H.Liang conducted computational analysis on NBR2 expression and status in human cancers. W.W., J.C., S.-W.L. and H.-K.L. provided reagents. B.G. supervised the study. X.L., Z.-D.X. and B.G. designed the experiments and wrote the manuscript. All authors commented on the manuscript.

Corresponding author

Correspondence to Boyi Gan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The schematic diagram of the genomic region of human BRCA1 and NBR2 genes with different splicing isoforms.

Arrows represent the direction of transcription.

Supplementary Figure 2 AMPK inactivation by compound C or AMPKα siRNA treatment.

(a) MDA-MB-231 cells were treated with 20 μM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to Western blotting analysis to measure AMPK activation. (b) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to Western blotting analysis to measure AMPK activation. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Supplementary Figure 3 NBR2 knockdown affects autophagy and cell proliferation in response to energy stress.

(a) The effect of NBR2 deficiency on GFP-LC3 puncta formation. 786-O cells infected with either control shRNA or NBR2 shRNA were transfected with GFP-LC3 plasmid, and then cultured in 25 or 0 mM glucose-containing medium for 18 hours. GFP-LC3 punctate foci were then detected using fluorescence microscopy. (Scale bars, 20 μm) (b) The effect of NBR2 deficiency on ULK1 phosphorylation and p62 degradation in response to glucose starvation. MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in 25 or 0 mM glucose-containing medium for 12 h. Cell lysates were then analyzed by Western blotting. (c,d) Cells infected with either control shRNA or NBR2 shRNA were cultured in 1 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (Mean ± s.d., n = 3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for c,d can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Supplementary Figure 4 Mechanistic studies of NBR2 regulation of AMPK.

(a) Protein lysates were prepared from HEK293T cells transfected with empty vector (EV), NBR2 full length (FL), T4, or T5 fragment expression vectors, and analyzed by Western blotting as indicated. (b) Protein lysates prepared from UMRC2 cells stably expressing EV or NBR2 #1 expression vectors were immunoprecipitated by IgG, LKB1 or Folliculin antibodies, and then were analyzed by Western blotting as indicated. Aliquots of the protein lysates (input) were also analyzed directly. (c) 786-O cells infected with either control shRNA or NBR2 shRNA were cultured in medium containing 0 or 25 mM glucose for 12 hours. Protein lysates were prepared and immunoprecipitated by IgG or LKB1 antibodies, and then were analyzed by Western blotting as indicated. Aliquots of the protein lysates (input) were also analyzed directly. (d) Empty vector (EV) or LKB1-infected Hela cells were transfected with EV or NBR2 #1 expression vectors. Protein lysates were prepared and analyzed by Western blotting as indicated. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Supplementary Figure 5 The working model of the reciprocal regulation between NBR2 and AMPK under energy stress, and its relevance to cancer development.

See discussion for detailed description.

Supplementary Figure 6 NBR2 deficiency affects AMPK activation under long periods of energy stress.

(a) MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in 0 mM glucose-containing medium for different hours, and protein lysates were prepared and analyzed by Western blotting. (b) MDA-MB-231 cells were cultured in 0 mM glucose-containing medium for different hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n = 3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for b can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Supplementary Figure 7 NBR2 deficiency does not affect BRCA1 expression.

(a) MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure BRCA1 expression (Mean ± s.d., n = 3 biologically independent extracts, two-tailed paired Student’s t-test). (b) Cell lysates were also analyzed by Western blotting as indicated. Source data for a can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1221 kb)

Supplementary Table 1

Supplementary Information (XLSX 25 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, X., Xiao, ZD., Han, L. et al. LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress. Nat Cell Biol 18, 431–442 (2016). https://doi.org/10.1038/ncb3328

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3328

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer