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

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.

Figure 1. Energy stress induces NBR2 expression through the LKB1-AMPK pathway

(a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 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). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d). (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) 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 real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) 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 real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 2. NBR2 regulates AMPK-mTORC1 signaling under energy stress

(a) Bar graph showing NBR2 shRNA-mediated knockdown efficiency by real-time PCR analysis in 786-O and MDA-MB-231 cells (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (b, c) 786-O or MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in medium with different concentrations of glucose for 24 hours. Cell lysates were then analyzed by Western blotting. (d) 786-O or MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in 0 or 5 mM 2DG-containing medium for 12 (for MDA-MB-231 cells) or 16 (for 786-O cells) hours. Cell lysates were then analyzed by Western blotting. (e) MDA-MB-231 cells infected with either control shRNA or NBR2 shRNA were cultured in 0 or 100 µM A769662-containing medium for 12 hours. Cell lysates were then analyzed by Western blotting. Source data for a can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 3. NBR2 regulates cell proliferation, apoptosis, and autophagy in response to energy stress

(a) Bar graph showing the percentages of S phase (Brdu positive) cells in control shRNA or NBR2 shRNA-infected MDA-MB-231 cells which were cultured in 25 or 0 mM glucose-containing medium for 24 hours (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (b, c) Bar graph showing the –Glucose/+Glucose ratio of S phase percentages in control shRNA or NBR2 shRNA-infected 786-O cells (b) or MDA-MB-231 cells (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (d–f) Control shRNA or NBR2 shRNA-infected 786O cells or MDA-MB-231 cells were cultured in medium with different concentrations of glucose for 24 hours, then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells (d for 786O cells, e for MDA-MB-231 cells, Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test), or to Western blotting analysis to measure Caspase-3 cleavage (f). (g–h) Bar graph showing the percentages of cells with LC3-GFP punctate localization in control shRNA or NBR2 shRNA-infected 786-O cells (g) or MDA-MB-231 cells (h), which were transfected with GFP-LC3 and then cultured in 25 or 0 mM glucose-containing medium for 12 (for MDA-MB-231 cells) or 18 (for 786-O cells) hours (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test). (i, j) 786-O (i) or MDA-MB-231 (j) cells infected with either control shRNA or NBR2 shRNA were cultured in glucose free 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 a, b, c, d, e, i, j can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 4. NBR2 inhibits tumor development

(a, b) 786-O (a) or MDA-MB-231 cells (b) infected with either control shRNA or NBR2 shRNA were seeded in soft agar containing high or low concentrations of glucose as indicated. Bar graph showing the mean colony numbers from the soft agar assay (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test). (c) Relative tumor volumes of MDA-MB-231 xenograft tumors infected with either control shRNA or NBR2 shRNA at different weeks (Mean ± s.e.m., n = 5 xenograft tumors, *: P < 0.05; **: P < 0.01 two-tailed paired Student’s t-test). (d) Protein lysates obtained from xenograft tumors infected with either control shRNA or NBR2 shRNA at the end point were subjected to Western blotting analysis as indicated. (e, f) The box plot showing the expression pattern of NBR2 for each pair of tumor and normal samples in BRCA (e, n=104 matched pairs, Sutdent's t-test and Wilcoxon test) and KIRC (f, n=65 matched pairs, Sutdent's t-test and Wilcoxon test). The boxes show the median ±1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile range from the box boundaries. (g) Kaplan Meier plots of breast cancer patients stratified by the expression levels of NBR2 (n_high = 1767, n_low = 1787, Log-Rank Test). Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 5. Energy stress induces NBR2 interaction with AMPK

(a, b) Nuclear and cytoplasmic fractions of 786O cells were subjected to either real-time PCR (a, Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) or Western blotting analysis (b). (c) In vitro-synthesized biotinylated sense (S) or antisense (AS) NBR2 #1 were incubated with protein lysates from HEK293T cells transfected with various vectors as indicated. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (d, f) In vitro-synthesized biotinylated sense (S) NBR2 or antisense (AS) NBR2 with different splicing isoforms were incubated with protein lysates from 786-O cells which had been cultured in 25 or 0 mM glucose-containing medium for 24 hours. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (e) In vitro-synthesized biotinylated sense (S) or antisense (AS) NBR2 #1 were incubated with purified human AMPK α protein. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (g) 786-O cells were cultured in 0 or 25 mM glucose-containing medium for 24 hours. Protein lysates were prepared and immunoprecipitated with AMPK α antibody or IgG. The RNA levels of NBR2 in immunoprecipitates or cell lysates (input) were measured by real-time PCR (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (h) In vitro-synthesized biotinylated NBR2 #1 were incubated with protein lysates from HEK293T cells transfected with various vectors and subjected to glucose starvation. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. Source data for a, g can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 6. NBR2 promotes AMPK kinase activity

(a, b) Protein lysates were prepared from HEK293T (a) or UMRC2 cells (b) with overexpression of EV or NBR2 expression vectors, and analyzed by Western blotting. (c) UMRC2 cells stably expressing EV or NBR2 expression vectors were cultured in 25 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). (d) Left panel: Schematic diagram showing different truncation mutants of NBR2 #1 and the summary of their binding capabilities to AMPK α. Right panel: In vitro-synthesized biotinylated sense (S), antisense (AS), or different truncation (T) mutants of NBR2 #1 were incubated with protein lysates from 786-O cells which had been cultured in glucose free medium for 24 hours. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (e) Protein lysates were prepared from HEK293T or UMRC2 cells with overexpression of EV, NBR2 #1 full length (FL), or T1 mutant expression vectors, and analyzed by Western blotting. (f) UMRC2 cells stably expressing EV, NBR2 #1 FL, or T1 mutant expression vectors were cultured in 25 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). (g) AMPK complex precipitated from HEK293T cells was subjected to the kinase assay in the presence of ATP, in vitro synthesized RNAs and GST-ACC 1–130 aa fusion proteins as indicated. The kinase activity of AMPK was measured by phosphorylation of ACC at S79 site. (h) In vitro purified active human AMPK complex was subjected to in vitro kinase assays in the presence of ATP, SAMS peptide and in vitro synthesized biotinylated sense (S)/antisense (AS)/T1 mutant (T1) NBR2 #1 or several chemical compounds (Compound C, A769662, AMP) as indicated (see Materials & Methods for details). The Kinase activity was measured by the luminescence with a plate-reading illuminometer (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for c, f, h can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.

Figure 7. The functional effects of NBR2 are partially mediated by AMPK

(A and B) UMRC2 cells stably expressing EV or NBR2 expression vectors were transfected with AMPK siRNA (AMPK si1 or si2) or control siRNA (Ctrl si). Protein lysates were prepared and analyzed by Western blotting (a), or cells were cultured in 25 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (b) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c–g) MDA-MB-231 cells with stable expression of control shRNA (Ctrl sh) or NBR2 shRNA (NBR2 sh) were infected with empty vector (EV) or constitutively active AMPK (AMPK CA). These cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and protein lysates were prepared and analyzed by Western blotting (c); The cells were cultured in 0 mM glucose-containing medium for different days as indicated, and then subjected to crystal violet staining to measure cell number (d) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test); The cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells cells (e) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test), or to Western blotting analysis to measure PARP cleavage (f); The cells were seeded in soft agar containing high or low concentrations of glucose as indicated. Bar graph showing the mean colony numbers from the soft agar assay (g) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test). (h) Relative tumor volumes of MDA-MB-231 xenograft tumors of different genotypes at different weeks (Mean ± s.d., n = 5 xenograft tumors, *: P < 0.05, **: P < 0.01, two-tailed paired Student’s t-test). Source data for b, d, e can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.