Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC

Abstract

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ(1)-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using (13)C,(15)N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies.

Fig. 1.

MYC robustly suppresses the expression of POX/PRODH protein. (A) Scheme of proline and glutamine interconversion. GLS, glutaminase; GS, glutamine synthetase; GSA, glutamic-γ-semialdehyde; α-KG, α-ketoglutarate; P5C, Δ1-pyyroline-5-carboxylate; P5CDH, P5C dehydrogenase; P5CS, P5C synthase; POX/PRODH, proline oxidase/dehydrogenase; PYCR1, P5C reductase 1; TCA cycle, tricarboxylic acid cycle. (B and D Upper) Western blots for POX/PRODH and MYC in P493 cells with or without tetracycline (T) treatment, using GAPDH as a loading control. (B and D Lower) Densitometry analysis shows the band density ratios of MYC and POX/PRODH to loading control. Data shown represent one of three independent experiments. (C and E) POX/PRODH mRNA levels were measured by real-time RT-PCR using GAPDH as an internal control. The relative folds were calculated to the group without tetracycline treatment. The results shown are mean ± SEM, n = 3. P values were obtained by one-way analysis of variance. *P < 0.05 and **P < 0.001 compared with 0 h control.

Fig. 2.

POX/PRODH suppression is necessary for MYC-mediated cancer cell proliferation and survival in P493 cells. P493 cells were firstly transfected with siRNA against POX/PRODH (designated as siPOX) or negative control siRNA (siNeg) for 24 h, then treated with tetracycline (Tet) for 2 or 4 d. (A) The knockdown of POX/PRODH was confirmed by Western blot. (B) ROS production was performed by dichlorofluorescein (DCF) assay. (C) After 4 d of tetracycline treatment, apoptosis in the cells was monitored by Annexin V-FITC and propidium iodide (PI) staining. The results are representative of two separate experiments in triplicate. The number in the bottom right corner shows the percentage of Annexin V-positive cells, expressed as mean ± SEM. (D) The relative living cell number was determined by trypan blue exclusion assay. The results are shown as mean ± SEM (n = 3). All P values were obtained by the Student t test.

Fig. 3.

MYC indirectly suppresses POX/PRODH expression at the transcriptional level. (A) PC3 cells were cotransfected with luciferase reporter PRODH-Luc containing PRODH promoter and siMYC or siNeg, using pRL-null renilla luciferase reporter as an internal control for normalizing transfection efficiency. PRODH promoter luciferase activity was determined by using the dual luciferase assay kit. Data shown are mean ± SEM (n = 3). P values were obtained by the Student t test. (B) Chromatin immunoprecipitation assay of the PRODH promoter in P493 cells treated with or without tetracycline. Soluble chromatin was immunoprecipitated by using anti-MYC antibody or no antibody as control. A portion of the sonicated chromatin was used as DNA input control. A known target gene of MYC, cyclin-dependent kinase 4 (CDK4), was used as a positive control. Immunoprecipitates were analyzed by PCR with specific primers for the PRODH and CDK4 gene areas containing canonical or noncanonical MYC binding sequence (E-box).

Fig. 4.

MYC suppresses POX/PRODH protein expression primarily through increasing miR-23b*. (A and B) The expression of miR-23b* was monitored by real-time RT-PCR in P493 cells treated with or without tetracycline (T). U6 was used as an internal control. Results were determined in triplicate and repeated in two independent experiments. Data are shown as mean ± SEM. P values were obtained by one-way analysis of variance. **P < 0.001 compared with 0 h control in A. (C) P493 cells were transfected with miR-23b* antagomir (Anti-23b*) to inhibit the expression of miR-23b*, and scrambled RNA was used as negative control (Neg RNA). (D) P493 cells were transfected with mimic miR-23b* to enhance the expression of miR-23b* when tetracycline was added to the medium to inhibit MYC expression. (C and D Upper) POX/PRODH protein was detected by Western blot. (C and D Lower) The normalization of POX/PRODH protein relative to loading control by densitometry analysis. Data shown represent one of three independent experiments.

Fig. 5.

MYC markedly increases the biosynthesis of proline from glutamine (A) Western blots of the enzymes in proline and glutamine catabolism pathway in P493 cells treated with tetracycline for different lengths of time. (B) PC3 cells were transfected with siRNA against MYC (siMYC) or negative control siRNA (siNeg). The enzymes in the proline and glutamine metabolic pathways were analyzed by Western blots. Experiments were replicated with similar results. (C) Intracellular proline levels in MYC-On and MYC-Off cells. (D) The expected labeling patterns from [U-¹³C,¹⁵N]-glutamine (Gln) and preexisting unlabeled Gln to different isotopologues of proline (Pro) and the intermediates of the TCA cycle via glutamate (Glu). [¹³C₅,¹⁵N₂]-Gln can be catabolized via glutaminase (GLS) to produce ¹³C₅,¹⁵N-glutamate (Glu) (m+6), which can be converted directly to ¹³C₅,¹⁵N-Pro (m+6) via the pathway depicted in Fig. 1A. Alternatively, ¹³C₅,¹⁵N-Glu can be converted to α-ketoglutarate (α-KG) and metabolized via the TCA cycle. The reverse reaction of α-KG to Glu enables ¹⁵N reincorporation into Glu (e.g., production of ¹⁵N₁-Glu) via transaminases (TA) and/or Glu dehydrogenase (GDH) activity. The carbon tracings shown illustrate the ¹³C fate after three turns of the TCA cycle. ●, ¹²C or ¹⁴N; red circle, ¹³C in the first turn; green circle, ¹³C in the second turn; pink circle, ¹³C in the third turn; blue circle and N, ¹⁵N; AA, amino acids; single and double-headed solid arrows: single irreversible and reversible reactions, respectively; dashed arrows, multistep reactions; AA, amino acids; CS, citrate synthase; PDH, pyruvate dehydrogenase. (E and F) The GC-MS analysis of [¹³C,¹⁵N]-Gln contribution to citrate and proline synthesis in P493 cells with MYC On and Off. All GC-MS data were corrected for natural abundance isotopic contribution and normalized to cell pellet wet weight. Each value is an average of duplicate samples. The entire experiment was repeated three times.