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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.

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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.
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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.

Mentions: We next sought to determine the extent to which the functional effects of NBR2 are mediated by NBR2 regulation of AMPK activation. We first examined whether overexpression of NBR2 still exerted its functional effects in AMPKα knockdown cells. Such analyses revealed that, while overexpression of NBR2 increased ACC phosphorylation, decreased S6 phosphorylation, and suppressed cell proliferation in control siRNA (Ctrl si) transfected cells, such effects were attenuated in AMPKα knockdown (AMPK si) cells (Fig. 7a, b). As a complementary approach, we also examined whether restoration of constitutively active (CA) AMPK (1–312 a.a. of AMPK α1) would rescue any of the defects observed in NBR2 deficient cells. Our data revealed that overexpression of AMPK CA in NBR2 knockdown cells restored ACC or S6 phosphorylation under glucose starvation condition as expected (Fig. 7c), and correspondingly, significantly rescued cell proliferation, apoptosis, and anchorage independence growth under glucose starvation conditions in NBR2 deficient cells (Fig. 7d–g). Importantly, restoration of AMPK CA in NBR2 deficient background significantly attenuated the enhanced xenograft tumor development caused by NBR2 deficiency (Fig. 7h). Taken together, our data strongly suggested that the functional effects of NBR2 are at least partially dependent on AMPK.


lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress
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.
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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.
Mentions: We next sought to determine the extent to which the functional effects of NBR2 are mediated by NBR2 regulation of AMPK activation. We first examined whether overexpression of NBR2 still exerted its functional effects in AMPKα knockdown cells. Such analyses revealed that, while overexpression of NBR2 increased ACC phosphorylation, decreased S6 phosphorylation, and suppressed cell proliferation in control siRNA (Ctrl si) transfected cells, such effects were attenuated in AMPKα knockdown (AMPK si) cells (Fig. 7a, b). As a complementary approach, we also examined whether restoration of constitutively active (CA) AMPK (1–312 a.a. of AMPK α1) would rescue any of the defects observed in NBR2 deficient cells. Our data revealed that overexpression of AMPK CA in NBR2 knockdown cells restored ACC or S6 phosphorylation under glucose starvation condition as expected (Fig. 7c), and correspondingly, significantly rescued cell proliferation, apoptosis, and anchorage independence growth under glucose starvation conditions in NBR2 deficient cells (Fig. 7d–g). Importantly, restoration of AMPK CA in NBR2 deficient background significantly attenuated the enhanced xenograft tumor development caused by NBR2 deficiency (Fig. 7h). Taken together, our data strongly suggested that the functional effects of NBR2 are at least partially dependent on AMPK.

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