Myc promotes glutaminolysis in human neuroblastoma through direct activation of glutaminase 2 - PubMed (original) (raw)
Myc promotes glutaminolysis in human neuroblastoma through direct activation of glutaminase 2
Daibiao Xiao et al. Oncotarget. 2015.
Abstract
Deamidation of glutamine to glutamate by glutaminase 1 (GLS1, also called GLS) and GLS2 is an essential step in both glutaminolysis and glutathione (GSH) biosynthesis. However, mechanisms whereby cancer cells regulate glutamine catabolism remains largely unknown. We report here that N-Myc, an essential Myc family member, promotes conversion of glutamine to glutamate in MYCN-amplified neuroblastoma cells by directly activating GLS2, but not GLS1, transcription. Abrogation of GLS2 function profoundly inhibited glutaminolysis, which resulted in feedback inhibition of aerobic glycolysis likely due to thioredoxin-interacting protein (TXNIP) activation, dramatically decreasing cell proliferation and survival in vitro and in vivo. Moreover, elevated GLS2 expression is significantly elevated in MYCN-amplified neuroblastomas in comparison with non-amplified ones, correlating with unfavorable patient survival. In aggregate, these results reveal a novel mechanism deciphering context-dependent regulation of metabolic heterogeneities, uncovering a previously unsuspected link between Myc, GLS2 and tumor metabolism.
Keywords: N-Myc; cancer metabolism; glutaminase 2; glutamine; neuroblastoma.
Conflict of interest statement
CONFLICTS OF INTEREST
No conflict of interests were declared.
Figures
Figure 1. N-Myc induction promotes glutamine catabolism in association with GLS2 activation
A. and B. N-Myc activation promotes glutamine metabolism. SHEP MYCN-ER cells were treated with or without 100 nM 4-hydroxytamoxifen (4-OHT) for 24 hrs. Glutamine (A) and ammonium (B) levels in the media were analyzed using the Nova Flex and are presented as an average of triplicates. C–D. MYCN-ER induction activates GLS2 but not GLS1 expression. SHEP MYCN-ER cells were treated with 4-OHT for 0, 24 and 48 hrs. GLS1 and GLS2 levels were quantitated by real-time qPCR using ΔCT method (C) and immunoblot assays (D) NCL, which encodes nucleolin, was used as a positive control to monitor Myc transcriptional activities. Data shown are averages of representative triplicates from one cDNA sample. Actin was used as a loading control. Inset: nuclear fractions were isolated from SHEP MYCN-ER cells treated by 4-OHT as indicated, and N-Myc nuclear translocation was monitored by immunoblot. Histone H3 was used as a nuclear marker (also a loading control) and actin as a cytoplasmic marker (the last band in actin immunoblot was obtained from total cell extracts to confirm the efficacy of actin antibody). **p < 0.01; ***p < 0.005.
Figure 2. N-Myc is a novel GLS2 activator
A. and B. Effect of N-Myc depletion on GLS1 and GLS2 expression in Kelly and BE-2C cells. Relative MYCN, GLS1 and GLS2 mRNA levels were quantitated by real-time qPCR by the ΔCT quantitation method (A); Data shown are averages of representative triplicates from one cDNA sample. Relative protein levels were quantitated by western blot (B); actin was used as a loading control. C. Schematic representation of the Myc response element (Myc RE) within the first intron of GLS2 and its mutant (REmut). D. Binding of N-Myc to the GLS2, NCL and ACTIN promoters analyzed by ChIP assay in Kelly cells with a specific N-Myc antibody or isotype control IgG. Results are presented as averages of fold difference between N-Myc ChIP and IgG control (background) in triplicates. E. Luciferase assay performed using Myc RE and REmut constructs in the presence or absence of exogenous N-Myc expression. Data shown are averages of triplicates. **p < 0.01; ***p < 0.005.
Figure 3. GLS2 sustains proliferation and viability of _MYCN_-Amplified neuroblastoma cells
A. and B. Depletion of GLS2 expression by two specific shRNAs in Kelly and BE-2C cells. Relative GLS2 mRNA levels were quantitated by real-time qPCR (A); data shown are an average of triplicates from a single cDNA. GLS1 (GAC) and GLS2 protein levels were analyzed by western blot (B); actin was used as a loading control. C. Proliferation of Kelly and BE-2C cells cultured over 7 days, as measured by serial cell counts upon GLS2 inhibition. Data are shown as an average of triplicates. D. Relative clonogenic growth of Kelly and BE-2C cells expressing a control or specific GLS2 shRNA. E. Representative PI–Annexin V staining plots of Kelly cells treated with control or GLS2 siRNA or combination of GLS2 siRNA and dimethyl α-KG (4 mM). F. Depletion of GLS2 expression profoundly inhibited the xenograft tumor growth of Kelly cells (n = 6 tumors per group). G. Representative staining of active caspase3 in tumor sections with and without GLS2 inhibition. The scale bar represents 50 μm. **p < 0.01; ***p < 0.005.
Figure 4. GLS2 depletion inhibits conversion of glutamine to glutamate
A. Diagram depicting glutamine and glucose metabolism. See text for more details. B. and C. Effect of GLS2 inhibition on glutamine consumption (B) and glutamate production (C) D. and E. Effect of GLS1 depletion on glutamine consumption (D) and glutamate production (E) analyzed as in (B) and (C). F–H. Effect of N-Myc depletion (F) on glutamine consumption (G) and glutamate production (H) analyzed as in (B) and (C) Kelly or BE-2C cells were infected with indicated shRNAs and selected with puromycin for 24 hr, and then switched to fresh medium. After 24 hr, glutamine consumption and glutamate production was analyzed by respective assay kits, and normalized to the same cell number. Data were presented as percentages of control and are shown as averages of triplicates. *p < 0.05; **p < 0.01; ***p < 0.005.
Figure 5. Changes in α-KG contents, ATP production, GSH biosynthesis and ROS generation upon GLS2 depletion
A–D. Effects of GLS2 depleted on contents of α-KG (A), ATP (B) and GSH (C), as well as GSH/GSSG ratio (D). Kelly and BE-2C cells were infected with indicated GLS2 shRNAs and selected with puromycin for 24 hr, and then switched to fresh medium. After 24 hr, α-KG, ATP, and GSH contents were analyzed with respective assay kits and normalized to the same cell number. Data were presented as percentages of control and are shown as averages of triplicates. E–F. Effects of GLS2 depletion on ROS production in Kelly (E) and BE-2C (F) cells. Cells were transfected with indicated siRNAs for 40 hr. DCF staining was followed by FACS analysis. Fold changes in ROS generation were presented as an average of triplicates. *p < 0.05; **p < 0.01; ***p < 0.005.
Figure 6. GLS2 depletion inhibits aerobic glycolysis
Glucose consumption A. and lactate production B. upon GLS2 inhibition or glutamine deprivation. Kelly and BE-2C cells were transfected with indicated siRNAs for 24 hr and then switched to fresh medium. 24 hr later, glucose consumption and lactate production were analyzed with respective assay kits and normalized to the same cell number. Kelly and BE-2C cells that were glutamine-starved for 24 hr were used for comparison. Data were presented as percentages of control and are shown as averages of triplicates. C. GLS2 and TXNIP protein levels analyzed by western blot. Actin was used as a loading control. *p < 0.05; **p < 0.01.
Figure 7. Expression of GLS1 and GLS2 in primary neuroblastoma tumors
A. Relative expression of MYCN, GLS1 and GLS2 in 643 human neuroblastoma tumors. Non-Amp: MYCN non-amplified tumors (550); Amp: _MYCN_-amplified tumors (93). B. Relative GLS2 expression in _MYCN_-transgenic mouse neuroblastoma tumors. Control: mouse sympathetic ganglia; A, B, C and D represent tumor groups with an increasing malignancy, respectively. C. Representative N-Myc, c-Myc, GLS1 and GLS2 immunochemical staining in _MYCN_-amplified neuroblastoma tumors; sections from 20641 (a MYCN non-amplified, low-stage neuroblastoma tumor) were used as a negative control. The scale bar represents 50 μm. D. Kaplan–Meier survival curvesn of neuroblastoma patients based on GLS1 and GLS2 expression. Data were generated from Kocak dataset accessible at
.
References
- Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011;11:85–95. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Miscellaneous