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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.


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.
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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.

Mentions: To study the potential function of NBR2 in mediating energy stress response, we generated 786-O cells (a kidney cancer cell line) and MDA-MB231 cells (a breast cancer cell line) with stable knockdown of NBR2 (Fig. 2a). We then analyzed whether knockdown of NBR2 affected any biochemical signaling surrogate induced by energy stress, including AMPK activation. As shown in Fig. 2b, glucose starvation potently induced phosphorylation of AMPK, or AMPK substrates acetyl CoA carboxylase (ACC) and Raptor18, 28. Notably, NBR2 knockdown significantly attenuated glucose starvation-induced phosphorylation of AMPK, ACC and Raptor. Accordingly, S6 and S6K de-phosphorylation induced by glucose deprivation was significantly compromised in NBR2 knockdown cells compared with control shRNA-infected cells (Fig. 2c). Finally, NBR2 knockdown also attenuated 2DG or A769662 treatment-induced AMPK activation and mTORC1 inactivation (Fig. 2d, e). Our results thus revealed that NBR2 depletion attenuates energy stress-induced AMPK activation and mTORC1 inactivation, and suggested a feed-forward mechanism on NBR2-AMPK regulation, in which AMPK initially promotes NBR2 expression in response to energy stress and NBR2 in turn regulates AMPK activation under energy stress (see Discussion).


lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress
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.
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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.
Mentions: To study the potential function of NBR2 in mediating energy stress response, we generated 786-O cells (a kidney cancer cell line) and MDA-MB231 cells (a breast cancer cell line) with stable knockdown of NBR2 (Fig. 2a). We then analyzed whether knockdown of NBR2 affected any biochemical signaling surrogate induced by energy stress, including AMPK activation. As shown in Fig. 2b, glucose starvation potently induced phosphorylation of AMPK, or AMPK substrates acetyl CoA carboxylase (ACC) and Raptor18, 28. Notably, NBR2 knockdown significantly attenuated glucose starvation-induced phosphorylation of AMPK, ACC and Raptor. Accordingly, S6 and S6K de-phosphorylation induced by glucose deprivation was significantly compromised in NBR2 knockdown cells compared with control shRNA-infected cells (Fig. 2c). Finally, NBR2 knockdown also attenuated 2DG or A769662 treatment-induced AMPK activation and mTORC1 inactivation (Fig. 2d, e). Our results thus revealed that NBR2 depletion attenuates energy stress-induced AMPK activation and mTORC1 inactivation, and suggested a feed-forward mechanism on NBR2-AMPK regulation, in which AMPK initially promotes NBR2 expression in response to energy stress and NBR2 in turn regulates AMPK activation under energy stress (see Discussion).

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.