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

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. Oncogenes and tumour suppressors have been linked to the regulation of glucose metabolism, thereby connecting genetic alterations in cancers to their glucose metabolic phenotype. In particular, the MYC oncogene produces Myc protein that directly regulates glucose metabolic enzymes as well as genes involved in mitochondrial biogenesis. In this regard, we sought to determine the role of MYC in altering the mitochondrial proteome in order to understand further the regulation of tumour metabolism. We studied human P-493 B cells that bear a tetracycline-repressible MYC construct, such that tetracycline withdrawal results in rapid induction of Myc and mitochondrial biogenesis, followed by cell proliferation. By comparing the mitochondrial proteomes of tetracycline-treated and untreated cells with high Myc expression, we found eight mitochondrial proteins that are distinctly differentially expressed in response to Myc (Fig. 1a, b and Supplementary Table 1). Mitochondrial glutaminase expression (GLS, molecular mass of ,58 kDa) was increased ,10-fold in response to Myc. As such, we determined the response of glutaminase to Myc induction in a time-course study using anti-GLS antibody (Fig. 1c) and found that GLS levels diminish with decreased Myc expression and recover on Myc re-induction. However, the level of the mitochondrial protein TFAM remained virtually unaltered. GLS levels also correlate with Myc levels in another human B cell line (CB33) and one (CB33-Myc) with constitutive Myc expression. Because human prostate cancer is linked to Myc expression, we sought to determine whether reduction of Myc expression by short interfering RNA (siRNA) in the human PC3 prostate cancer cell line is also associated with reduction of GLS expression (Fig. 1d). Similar to the human lymphoid cells, the PC3 cells also displayed a correlation between Myc and GLS levels. We then sought to determine whether the marked alteration of GLS levels in response to Myc is functionally linked to Myc-induced cell proliferation. Although there are two major known tissue-specific GLS isoforms, GLS1 and GLS2 (refs 16, 17), our data show that only GLS1 is predominantly expressed in P493-6 or PC3 cells (Supplementary Fig. 1). We first determined whether gain of GLS1 function through overexpression in PC3 cells would rescue the diminished growth rate associated with siRNA-mediated reduction of Myc (Supplementary Fig. 2) and found that ectopic GLS1 expression alone is insufficient to stimulate growth. In light of the observation that no single gene could substitute for Myc and that Myc is a pleiotropic transcription factor, this outcome was not particularly surprising. As such, we reduced the expression of GLS1 (hereafter referred to as GLS) by RNA interference (GLS siRNA) and found that P-493-6 cell proliferation is markedly attenuated by GLS siRNA but not by control siRNA (Fig. 2a). Likewise, proliferation of the human PC3 prostate cancer cell line was diminished by GLS siRNA (Fig. 2a), indicating that GLS is necessary for cell proliferation. Because glutamine is converted by GLS to glutamate for further catabolism by the tricarboxylic acid (TCA) cycle, and previous studies indicate that overexpression of Myc sensitizes human cells to glutamine-withdrawal-induced apoptosis, we determined the metabolic responses of P493-6 or PC3 cells to glutamine deprivation (Fig. 2b). The growth of both cell lines was diminished significantly by glutamine withdrawal and moderately with glucose withdrawal. Glutamine withdrawal also resulted in a decrease in ATP levels (Fig. 2c) associated with a diminished cellular oxygen consumption rate (Supplementary Fig. 3a, b). Reduction of GLS by RNA interference (RNAi) also reduced ATP levels (Fig. 2d). Because glutamine is a precursor for glutathione, glutathione levels were measured by flow cytometry and were found to be diminished with glutamine withdrawal or RNAi-mediated reduction of GLS (Supplementary Fig. 4 and Supplementary Table 2) that is also associated with an increase in reactive oxygen species (ROS) levels (Supplementary Fig. 3c) and cell death in the P493-6 cells (Fig. 2e and Supplementary Fig. 5). Of note,