lncRNA NBR2 modulates cancer cell sensitivity to phenformin through GLUT1

Abstract

Biguanides, including metformin (widely used in diabetes treatment) and phenformin, are AMP-activated protein kinase (AMPK) activators and potential drugs for cancer treatment. A more in-depth understanding of how cancer cells adapt to biguanide treatment may provide important therapeutic implications to achieve more effective and rational cancer therapies. NBR2 is a glucose starvation-induced long non-coding RNA (lncRNA) that interacts with AMPK and regulates AMPK activity upon glucose starvation. Here we show that phenformin treatment induces NBR2 expression, and NBR2 deficiency sensitizes cancer cells to phenformin-induced cell death. Surprisingly, unlike glucose starvation, phenformin does not induce NBR2 interaction with AMPK, and correspondingly, NBR2 deficiency does not affect phenformin-induced AMPK activation. We further reveal that NBR2 depletion attenuates phenformin-induced glucose transporter GLUT1 expression and glucose uptake. GLUT1 deficiency sensitizes cancer cells to phenformin-induced cell death, whereas GLUT1 restoration in NBR2 deficient cells rescues the increased cell death upon phenformin treatment. Together, the results of our study reveal that NBR2-GLUT1 axis may serve as an adaptive response in cancer cells to survive in response to phenformin treatment, and identify a novel mechanism coupling lncRNA to biguanide-mediated biology.

Keywords: AMPK; GLUT1; NBR2; biguanide; long non-coding RNA; phenformin.

Figure 1.

Phenformin induces NBR2 expression and NBR2 deficiency renders cancer cells more sensitive to phenformin-induced cell death. (A) Various cell lines were treated with 0 or 2mM phenformin for 18–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression. Three independent experiments were performed and the values were expressed as the mean ± SD, *: P< 0.05, **: P < 0.01. (B and C) Bar graph showing NBR2 shRNA-mediated knockdown efficiency by real-time PCR analysis under basal and phenformin treatment in 786-O (B) and MDA-MB-231 cells (C). Three independent experiments were performed and the values were expressed as the mean ± SD, **: P < 0.01. (D and E) Control shRNA or NBR2 shRNA-infected 786O cells (D) or MDA-MB-231 cells (E) were treated with 0 or 2 mM phenformin for 18 hours, then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells. Three independent experiments were performed and the values were expressed as the mean ± SD, **: P < 0.01, ***: P < 0.001. (F and G) Control shRNA or NBR2 shRNA-infected 786O cells (F) or MDA-MB-231 cells (G) were treated with 0 or 2 mM phenformin for 18 hours. Cell lysates were then analyzed by Western blotting to measure PARP cleavage.

Figure 2.

NBR2 does not regulate phenformin-induced AMPK activation and mTORC1 inactivation. (A and B) Control shRNA or NBR2 shRNA-infected 786O cells (A) or MDA-MB-231 cells (B) were treated with 0 or 2mM phenformin for 12 hours. Cell lysates were then analyzed by Western blotting. (C) In vitro-synthesized biotinylated sense (S) NBR2 were incubated with protein lysates from 786-O cells which had been cultured in 25 or 0 mM glucose-containing medium for 24 hours, or treated with 2 mM phenformin for 18 hours. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (D) 786-O cells were cultured in 0 or 25 mM glucose-containing medium for 24 hours, or treated with 2 mM phenformin for 18 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. Three independent experiments were performed and the values were expressed as the mean ± SD. *: P < 0.05.

Figure 3.

NBR2 regulates GLUT1 expression and glucose uptake in response to phenformin treatment. (A and B) 786O cells (A) or MDA-MB-231 cells (B) were treated with 0 or 2 mM phenformin for 12 hours, and then subjected to analyses to measure glucose uptake. Three independent experiments were performed and the values were expressed as the mean ± SD. ***: P< 0.001. (C and D) 786O cells (C) or MDA-MB-231 cells (D) were treated with 0 or 2 mM phenformin for 12 hours, and then subjected to analyses to measure lactate production. Three independent experiments were performed and the values were expressed as the mean ± SD. *: P < 0.05. (E and F) 786O cells (E) or MDA-MB-231 cells (F) were treated with 0 or 2 mM phenformin for 12 hours, and then subjected to real-time PCR analysis to measure the expression of indicated glucose transporters. Three independent experiments were performed and the values were expressed as the mean ± SD. ***: P < 0.001. (G and H) 786O cells (G) or MDA-MB-231 cells (H) were treated with 0 or 2 mM phenformin for 18 hours. Cell lysates were then analyzed by Western blotting.

Figure 4.

NBR2 regulates phenformin-induced cell death at least partly through GLUT1. (A) 786O cells (upper panel) or MDA-MB-231 cells (lower panel) were transfected with control or 2 independent GLUT1 siRNAs. Protein lysates were prepared and analyzed by Western blotting as indicated. (B) 786O cells (upper panel) or MDA-MB-231 cells (lower panel) transfected with control or GLUT1 siRNAs were treated with 0 or 2 mM phenformin for 18 hours, and then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PtdIns negative cells. Three independent experiments were performed and the values were expressed as the mean ± SD. ***: P< 0.001. (C) 786O cells (upper panel) or MDA-MB-231 cells (lower panel) transfected with control or GLUT1 siRNAs were treated with 0 or 2 mM phenformin for 18 hours, protein lysates were prepared and analyzed by Western blotting to detect PARP cleavage. (D and E) 786O cells (D) or MDA-MB-231 cells (E) with stable expression of control shRNA or NBR2 shRNA were infected with empty vector (EV) or GLUT1. Protein lysates were prepared and analyzed by Western blotting. (F and G) 786O cells (F) or MDA-MB-231 cells (G) with indicated genotypes were treated with 2 mM phenformin for 12 hours, and then subjected to analyses to measure glucose uptake. Three independent experiments were performed and the values were expressed as the mean ± SD. *: P < 0.05, ***: P < 0.001. (H and I) 786O cells (H) or MDA-MB-231 cells (I) with indicated genotypes were treated with 0 or 2 mM phenformin for 18 hours, and then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells. Three independent experiments were performed and the values were expressed as the mean ± SD. *: P < 0.05, **: P < 0.01.

Figure 5.

The subcellular localization of NBR2 under phenformin treatment. (A) Western blotting analysis for Vinculin (cytoplasmic marker) and Lamin A/C (nuclear marker) in total, cytoplasmic (Cyt) and nuclear (Nul) fractions of 786-O cells. (B) Real-time PCR showing the relative expression levels for GAPDH, U1 and NBR2 in cytoplasmic and nuclear fractions of 786-O cells. Three independent experiments were performed and the values were expressed as the mean ± SD. **: P < 0.01.