c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism

Abstract

Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.

Figure 1

1a. The expanded insets of 2-dimensional gels reveal the induction of glutaminase (GLS; highlighted by white circles) by Myc in P493-6 B cells. For each condition, 350μg of mitochondrial protein lysate was resolved on 18 cm immobilized pH gradient (IPG) strips as the first dimension followed by 10% Bis-Tris SDS-PAGE as the second dimension, which is marked by molecular mass markers. Protein spots were visualized by silver staining. Six independent biological experiments were performed for each condition. Table S1 summarizes the identity of the spots with the same numbering system as depicted in the figure. 1b. Immunoblot with anti-GLS antibody of a 1-dimensional SDS-PAGE gel of mitochondrial proteins (20 μg/lane) validates the induction of GLS by Myc discovered in Figure 1a. TFAM represents a control mitochondrial protein. 1c. P493-6 cells were treated with tetracycline (Tet) for different lengths of time to inhibit Myc expression or were treated first with tetracycline for 48h and then washed (Wash) to remove tetracycline with the times after wash out indicated. Cells were then harvested for immunoblot assay for GLS or c-Myc. Anti-tubulin antibody and anti-TFAM were used for loading controls. 1d. Human CB33 lymphoblastoid cells, CB33-Myc cells, and PC3 cells transfected with siRNA against c-Myc (siMYC) or control siRNA (siCont) were used for immunoblot assays. Experiments were replicated with similar results.

Figure 2

2a. Top panel, immunoblots document that GLS protein level was diminished by transfecting the cells with siRNA for GLS1 (siGLS) as compared to non transfection (No tx) or control siRNA (siCont); Lower panel, Growth inhibition of P493 and PC3 cells by siGLS. The results shown are mean ± SD, n=3. 2b. Growth inhibition of P493 and PC3 cells cultured under control, glucose- or glutamine- deprived conditions. The results shown are mean ± SD, n=3. 2c. Cells were cultured with normal medium or medium without glucose ((−)gluc)or glutamine ((−)Q) for 48h and harvested for ATP assay as described in Methods section. The results shown (mean ± SD, n=2) were relative ATP levels per microgram total protein normalized to the control (Cont) normal medium group. 2d. ATP levels in control cells (Cont) or cells transfected with siRNA for GLS (siGLS) or control siRNA (siCont). 72h after transfection, cells were harvested for ATP assay. The results shown (mean ± SD, n=2) were relative ATP levels per microgram total protein normalized to the non transfected control group (Cont). 2e. Cells were transfected with siRNA for GLS (siGLS) or control siRNA (siCont) and cultured with 10mM N-acetylcysteine (NAC), or 5mM oxaloacetate (OAA), or no addition control. Left panel shows cell counts (mean ± SD, n=5) of different groups at 72h after transfection; complete cell growth curve is available in Figure S5. Right panel shows percentage cell death at 72h after transfection. Percentage Cell death indicates Annexin positive plus Annexin V and 7-AAD positive cells. Primary data are shown in Figure S4. All experiments in Figure 2 were repeated at least twice. All experiments with P493 cells were in the absence of tetracycline. (*) denotes mean (± SD) that is significantly different (P < 0.05 by t test).

Figure 3

3a. P493-6 cells were treated with tetracycline (Tet) for different lengths of time to inhibit Myc expression or were treated first with tetracycline for 48h and then washed (Wash) to remove tetracycline with the times after wash out indicated. RNA was then harvested for real-time PCR for GLS1 as described in Methods section. Data are shown as mean ± SD, n = 3 PCR reactions. 3b. Northern analysis of miR-23a and miR-23b expression in P493 cells treated with or without tetracycline for 24h and then transfected with anti-sense miR-23a and miR-23b LNAs or Scrambled control LNA. 48h after transfection, cells were harvested and northern blot assays were performed as described in the Methods section. U6 snRNA probe was used as the loading control. 3c. Chromatin immunoprecipitation (ChIP) assay with P493 cells documents Myc binding to the promoter region of C9orf3, whose transcript is processed to miR-23b. The positions of the amplicons are depicted in the cartoon of the C9orf3 gene below the bar graphs (mean ± SD, n=3) demonstrating the binding of Myc in the amplicon 2 region in a tetracycline dependent manner. Anti-HGF serves as a non-specific antibody control. 3d. Inhibition of GLS-3′-UTR luciferase reporter by miR-23. Upper panel. Glutaminase reporter (wild-type GLS-3′UTR or mutant Mut-GLS-3′-UTR) or control (PGL3) luciferase constructs were transfected into MCF-7 cells with the following oligonucleotides: scrambled control LNA (cont-LNA) nucleotide or antisense (miR-23-LNA). The ratio of normalized reporter to control luciferase activity is shown. Cells were co-transfected either with 100ng reporter vectors and 4ng pSV-Renilla, and further co-transfected with 10nM LNA antisense for miR-23 or control LNA. After 24h, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Data shown are luciferase activity (RLU = relative light unit) normalized to control group (mean ± SD, n=4). (*) denotes mean (± SD) that is significantly different (P < 0.05 by t test). Lower panel illustrates miR-23a, miR-23b, GLS-3′UTR and MutGLS-3′UTR sequences. 3e. Analysis of GLS protein levels in P493 and PC3 cells treated with control (Cont-LNA) or antisense miR-23 LNAs (miR-23-LNA). Left panel, P493 cells were treated with or without tetracycline for 24h and then transfected with antisense miR-23a and miR-23b LNAs or Scramble control probe. After 72h, cells were harvested for immunoblot assay with anti-Myc and anti-GLS antibodies. Tubulin serves as a loading control. Right panel, PC3 cells were transfected with siRNA for MYC (siMyc) or control siRNA (siCont). After 24h, cells were transfected with LNA knockdown probes for miR-23a and miR-23b or Scramble control probe. Cells were cultured for 72h and then were harvested for immunoblot assay. Each experiment was repeated twice with a representative experiment shown.