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

View Article: PubMed Central - PubMed

ABSTRACT

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signaling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that lncRNA NBR2 (neighbor of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. Upon 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 tumor development in vivo. NBR2 is down-regulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, our study uncovers a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to further understand the regulation of kinase signaling by lncRNAs.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: AMPK functions as a critical metabolic checkpoint; defective AMPK signaling leads to increased cell proliferation yet decreased autophagy under conditions of energy stress, leading to apoptosis12, 20. The aforementioned data prompted us to examine the impact of NBR2 deficiency on cell proliferation, apoptosis, and autophagy in response to energy stress. Glucose starvation dramatically decreased S phase entry as measured by BrdU incorporation, and knockdown of NBR2 significantly attenuated the reduction of S phase entry upon glucose starvation (Fig. 3a–c). Thus, similar to cells with defective AMPK signaling18, NBR2 deficient cells continue cycling under energy stress.


lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress
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.
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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.
Mentions: AMPK functions as a critical metabolic checkpoint; defective AMPK signaling leads to increased cell proliferation yet decreased autophagy under conditions of energy stress, leading to apoptosis12, 20. The aforementioned data prompted us to examine the impact of NBR2 deficiency on cell proliferation, apoptosis, and autophagy in response to energy stress. Glucose starvation dramatically decreased S phase entry as measured by BrdU incorporation, and knockdown of NBR2 significantly attenuated the reduction of S phase entry upon glucose starvation (Fig. 3a–c). Thus, similar to cells with defective AMPK signaling18, NBR2 deficient cells continue cycling under energy stress.

View Article: PubMed Central - PubMed

ABSTRACT

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signaling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that lncRNA NBR2 (neighbor of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. Upon 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 tumor development in vivo. NBR2 is down-regulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, our study uncovers a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to further understand the regulation of kinase signaling by lncRNAs.

No MeSH data available.


Related in: MedlinePlus