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

Wei Liua,1, Anne Leb, Chad Hancocka, Andrew N. Lanec,d, Chi V. Dange, Teresa W.-M. Fanf,d,1, and James M. Phanga,1

aMetabolism and Cancer Susceptibility Section, Basic Research Laboratory, National Cancer Institute, Frederick, MD 21702; bDepartment of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; Departments of cMedicine and fChemistry, Center for Regulatory Environmental Analytical Metabolomics and dJames Graham Brown Cancer Center, University of Louisville, KY 40202; and eAbramson Cancer Center, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104

Edited by Matt Vander Heiden, Massachusetts Institute of Technology, Cambridge, MA, and accepted by the Editorial Board April 23, 2012 (received for review February 24, 2012)

In addition to glycolysis, the oncogenic transcription factor c-MYC because of its ability to generate reactive oxygen species (ROS) (MYC) stimulates glutamine catabolism to fuel growth and pro- (6, 7). In addition to inducing apoptotic cell death, POX/ liferation of cancer cells through up-regulating (GLS). PRODH negatively regulates the growth of various cancer cells, Glutamine is converted to glutamate by GLS, entering the tri- causes cell cycle arrest at the G2-M checkpoint, and inhibits carboxylic acid cycle as an important energy source. Less well- tumor formation in a mouse xenograft model (7, 9). Sub- recognized, glutamate can also be converted to proline through sequently, the absence or reduction of POX/PRODH was ob- 1 Δ -pyrroline-5-carboxylate (P5C) and vice versa. This study sug- served in a variety of human tumor tissues compared with their gests that some MYC-induced cellular effects are due to MYC reg- normal tissue counterparts (9, 11), and miR-23b* was found to ulation of proline metabolism. Proline oxidase, also known as be one of the mechanisms mediating POX/PRODH down-reg- fi proline (POX/PRODH), the rst in proline ulation in renal tumors (11). catabolism, is a mitochondrial tumor suppressor that inhibits pro- The MYC oncogene is frequently dysregulated in human can- liferation and induces apoptosis. MiR-23b* mediates POX/PRODH cers. It encodes a transcription factor, c-MYC (herein termed CELL BIOLOGY down-regulation in human kidney tumors. MiR-23b* is processed MYC), which links altered cellular metabolism to carcinogenesis. from the same transcript as miR-23b; the latter inhibits the trans- In addition to its known function in regulating glucose metabolism lation of GLS. Using MYC-inducible human Burkitt lymphoma (12), MYC recently has been documented to induce the expres- model P493 and PC3 human prostate cancer cells, we showed that sion of mitochondrial glutaminase (GLS) to stimulate glutamine MYC suppressed POX/PRODH expression primarily through up- catabolism (13, 14). The importance of glutamine catabolism in regulating miR-23b*. The growth inhibition in the absence of fi MYC was partially reversed by POX/PRODH knockdown, indicating cancer cell metabolism was reemphasized by these ndings. In the importance of suppression of POX/PRODH in MYC-mediated addition to its use for synthesis of proteins, nucleotides, and lipids cellular effects. Interestingly, MYC not only inhibited POX/PRODH, (15, 16), glutamine is converted to glutamate by GLS. Glutamate but also markedly increased the of proline biosynthesis not only is an essential component of , but also an from glutamine, including P5C synthase and P5C reductase 1. MYC- important energy source via anaplerotic input into the tri- induced proline biosynthesis from glutamine was directly con- carboxylic acid (TCA) cycle after conversion to α-ketoglutarate firmed using 13C,15N-glutamine as a tracer. The metabolic link be- (α-KG). Less well-recognized, glutamate can also be converted to tween glutamine and proline afforded by MYC emphasizes the proline through Δ1-pyrroline-5-carboxylate (P5C) catalyzed se- complexity of tumor metabolism. Further studies of the relation- quentially by P5C synthase (P5CS) and P5C reductase (PYCR) ship between glutamine and proline metabolism should provide (Fig. 1A). Conversely, proline can be converted to glutamate a deeper understanding of tumor metabolism while enabling the through proline catabolism sequentially catalyzed by POX/ development of novel therapeutic strategies. PRODH and P5C dehydrogenase (P5CDH). The proline-derived glutamate can be further converted to α-KG, or used to generate amino acids | redox signaling | reactive oxygen species | miRNA | glutamine through (GS). Thus, we won- metabolic tumor suppressor dered whether oncogenic MYC could affect proline metabolism and, specifically, modulate proline catabolism by the tumor sup- rowing tumors alter their metabolic profiles to meet the pressor POX/PRODH. In this study, we investigated the effects Gbioenergetic and biosynthetic demands of increased cell of MYC on proline catabolism and proline biosynthesis from growth and proliferation (1–3). Many oncogenes and tumor glutamine, especially its effect on the expression of POX/PRODH suppressors have been linked to tumor metabolic regulation and its regulatory mechanisms. The contribution of POX/PRODH (4, 5). Proline oxidase, also known as to MYC-mediated tumor cell behavior was further established. (POX/PRODH), as a mitochondrial inner membrane enzyme is involved in the first step of proline catabolism and has been identified as one of a few mitochondrial tumor suppressors (6– PRODH Author contributions: W.L., A.L., C.H., A.N.L., C.V.D., T.W.-M.F., and J.M.P. designed re- 10). The encoding POX/PRODH also known as search; W.L., A.L., C.H., A.N.L., and T.W.-M.F. performed research; W.L., C.H., A.N.L., C.V.D., was initially identified as a p53-induced gene in a screening study T.W.-M.F., and J.M.P. analyzed data; and W.L., A.N.L., T.W.-M.F., and J.M.P. wrote the paper. (8). Here, we will refer to this enzyme as POX/PRODH and to The authors declare no conflict of interest. PRODH. the gene as Intensive investigation has led to the rec- This article is a PNAS Direct Submission. M.V.H. is a guest editor invited by the Editorial ognition of important functions of proline metabolism in human Board. tumors. Earlier work in our laboratory has demonstrated the Freely available online through the PNAS open access option. important roles of POX/PRODH in the apoptosis induced by 1To whom correspondence may be addressed. E-mail: [email protected], liuwei7997@ cytotoxic agents and by peroxisome proliferator activated re- gmail.com, or [email protected]. γ γ ceptor (PPAR ) and its ligands (6, 10). Hyperexpression of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. POX/PRODH in cancer cells is sufficient to initiate apoptosis 1073/pnas.1203244109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203244109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A

B POX/PRODH D POX/PRODH MYC MYC GAPDH GAPDH Fig. 1. MYC robustly suppresses the expres- sion of POX/PRODH protein. (A) Scheme of 0 24 48 72 96 120 Time (h) 72 72 72 48 72 120 120 48 72 Time (h) proline and glutamine interconversion. GLS, -T -T +T Wash -T +T Wash +T glutaminase; GS, glutamine synthetase; GSA, glutamic-γ-semialdehyde; α-KG, α-ketogluta- 0.8 MYC 1.0 MYC POX/PRODH POX/PRODH rate; P5C, Δ1-pyyroline-5-carboxylate; P5CDH, 0.6 0.8 P5C dehydrogenase; P5CS, P5C synthase; POX/PRODH, proline oxidase/dehydrogenase; 0.6 0.4 PYCR1, P5C reductase 1; TCA cycle, tricar- 0.4 boxylic acid cycle. (B and D Upper) Western 0.2 blots for POX/PRODH and MYC in P493 cells 0.2 with or without tetracycline (T) treatment, Protien expression Protien 0.0 expression Protein using GAPDH as a loading control. (B and D (Relative optical density) optical (Relative 0 24 48 72 96 120 Time (h) 0.0 (Relative optical density) optical (Relative 72 72 48 72 120 120 48 72 Time (h) Lower) Densitometry analysis shows the band +T -T +T Wash -T +T Wash density ratios of MYC and POX/PRODH to

C 7 E p<0.05 loading control. Data shown represent one of p<0.05 6 4 three independent experiments. (C and E) ** ** p<0.001 5 POX/PRODH mRNA levels were measured by 3 4 real-time RT-PCR using GAPDH as an internal p<0.05 3 control. The relative folds were calculated to 2 p<0.05 2 the group without tetracycline treatment. The * ± 1 1 results shown are mean SEM, n =3.P values POX/PRODH mRNA relative expression relative were obtained by one-way analysis of vari- POX/PRODH mRNA 0 expression relative 0 24 48 72 96 120 Time (h) 0 < < 72 72 48 72 120 120 48 72 Time (h) ance. *P 0.05 and **P 0.001 compared +T -T +T Wash -T +T Wash with 0 h control.

Results MYC is functionally linked to MYC-induced proliferation and MYC Robustly Suppresses the Expression of POX/PRODH Protein. To cell survival became an interesting question. We knocked down investigate whether MYC plays a role in the regulation of POX/ the expression of POX/PRODH by siRNA in P493 cells with PRODH expression in human cancer, we used human Burkitt MYC suppressed by tetracycline. Western blot confirmed the lymphoma model P493 cells that bear a tetracycline-repressible knockdown of POX/PRODH (Fig. 2A). In Fig. 2B, at different MYC construct. In the absence of tetracycline, ectopic MYC is time points of tetracycline treatment, POX/PRODH siRNA induced in a tumorigenic state that resembles human Burkitt consistently reduced the production of ROS, although the sup- lymphoma. We analyzed the changes of POX/PRODH protein pression of MYC by tetracycline decreased the accumulation of and mRNA expression in response to MYC. As shown in Fig. 1B, ROS at day 4 (-Tet + siNeg vs. +Tet + siNeg as shown in Fig. POX/PRODH protein increased in a time-dependent fashion and 2B), which may reflect the different effects of various MYC reached approximately 3.8-fold and 7.5- fold when P493 cells were regulated on ROS production at various stages (17–19). treated with tetracycline for 24 h and 120 h, respectively. When Correspondingly, the apoptosis assay by flow cytometry showed MYC expression was recovered by tetracycline removal, the POX/ that knockdown of POX/PRODH by siRNA decreased the PRODH protein diminished markedly (Fig. 1D). However, POX/ percentage of apoptotic and dead cells occurring with MYC PRODH mRNA did not show any obvious increase until 72 h after suppression (Fig. 2C). In contrast, the number of living cells tetracycline treatment (∼1.7-fold), and even at 120 h, it increased suggested that POX/PRODH siRNA could significantly rescue only ∼4.7-fold (Fig. 1 C and E). Thus, the changes of POX/ 30∼40% of the diminished growth rates, which were 46% and PRODH mRNA levels were delayed and plateaued at a lower 82% at day 2 and day 4, respectively, resulting from MYC sup- level compared with changes in POX/PRODH protein levels. pression by tetracycline (Fig. 2D). These results indicated that Because overexpression of MYC was also found in human POX/PRODH suppression participates in MYC-mediated can- prostate cancer, we tested whether MYC had the same effects on cer cell proliferation and survival. the expression of POX/PRODH in the PC3 human prostate To extend the above conclusion from the tetracycline-con- cancer cells. MYC knockdown in PC3 cells by short interfering trolled cells to cancer cells overexpressing MYC, we performed RNA (siRNA) resulted in the inhibition of POX/PRODH ex- the same assays in PC3 prostate cancer cells. We first confirmed pression with a pattern similar to the P493 cells (Fig. S1A). When the decreased expression of MYC and POX/PRODH by their MYC was knocked down 85%, POX/PRODH protein expression respective siRNAs using Western blots (Fig. S2A). As shown in increased ∼4.2-fold, whereas POX/PRODH mRNA levels only Fig. S2B, knockdown of MYC by siRNA first resulted in the increased ∼1.6-fold (Fig. S1 A and B). decrease of ROS production at 3 d followed by a small increase at 6 d. When POX/PRODH siRNA was used to reduce the POX/PRODH Suppression Is Essential for MYC-Mediated Cancer Cell increased expression of POX/PRODH resulting from MYC Proliferation and Survival. As mentioned above, POX/PRODH siRNA, the production of ROS was decreased at both time has been identified as a tumor suppressor, whose expression is points. The reduction of MYC by MYC siRNA reduced cell suppressed in a variety of tumors; it induces apoptosis through growth 53.0% and 68.6% at 3 and 6 d, respectively, which could the generation of ROS and inhibits tumor growth in a xenograft be recovered 19.5% and 71.6% by POX/PRODH siRNA (Fig. mouse model. Whether the suppression of POX/PRODH by S2C). Apoptosis assay showed that the percentage of apoptotic

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203244109 Liu et al. Downloaded by guest on September 28, 2021 siNeg + PRODH-Luc -T+siNeg A B 2.0 A PRODH -T+siPOX p<0.05 siMyc + -Luc +T+siNeg +T+siPOX p<0.001 p<0.01 1.5 p<0.01 2.0 p<0.05 POX 1.0

ROS 1.5 MYC 0.5 GAPDH 1.0 0.0 (Relative Fluorescence) (Relative 2ds 4ds activity C -T+siNeg -T+siPOX 0.5 Relative luciferase Relative 0.0 25 p<0.001 p<0.01 72 h 96 h

20

15 B

10

+T+siNeg +T+siPOX 5 0 % Annexin V + cells Annexin %

siNeg siNeg T+ Positive control: - -T+siPOX +T+ +T+siPOX CDK4 (110bp)

D 6 -T+siNeg PRODH promoter -T+siPOX -2858 to -2783 +T+siNeg 4 +T+siPOX (76bp)

2 PRODH promoter p<0.001 p<0.05 -1040 to -812

(229bp) CELL BIOLOGY Relative cell number 0 0d 2ds 4ds

Fig. 2. POX/PRODH suppression is necessary for MYC-mediated cancer cell -T +T proliferation and survival in P493 cells. P493 cells were firstly transfected Fig. 3. MYC indirectly suppresses POX/PRODH expression at the transcrip- with siRNA against POX/PRODH (designated as siPOX) or negative control tional level. (A) PC3 cells were cotransfected with luciferase reporter PRODH- siRNA (siNeg) for 24 h, then treated with tetracycline (Tet) for 2 or 4 d. (A) Luc containing PRODH promoter and siMYC or siNeg, using pRL-null renilla The knockdown of POX/PRODH was confirmed by Western blot. (B) ROS luciferase reporter as an internal control for normalizing transfection effi- production was performed by dichlorofluorescein (DCF) assay. (C) After 4 d ciency. PRODH promoter luciferase activity was determined by using the of tetracycline treatment, apoptosis in the cells was monitored by Annexin V- dual luciferase assay kit. Data shown are mean ± SEM (n =3).P values were FITC and propidium iodide (PI) staining. The results are representative of obtained by the Student t test. (B) Chromatin immunoprecipitation assay of two separate experiments in triplicate. The number in the bottom right the PRODH promoter in P493 cells treated with or without tetracycline. corner shows the percentage of Annexin V-positive cells, expressed as mean ± Soluble chromatin was immunoprecipitated by using anti-MYC antibody or SEM. (D) The relative living cell number was determined by trypan blue no antibody as control. A portion of the sonicated chromatin was used as exclusion assay. The results are shown as mean ± SEM (n =3).AllP values DNA input control. A known target gene of MYC, cyclin-dependent kinase 4 were obtained by the Student t test. (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). cells increased threefold with MYC siRNA, which was decreased ∼40% by POX/PRODH knockdown (Fig. S2D). The mechanism about ROS production by POX/PRODH directly to the PRODH gene, we performed the chromatin im- through complex III of the electron transport chain, and the munoprecipitation (ChIP) assay in P493 cells, using cyclin- contribution of ROS generated by POX/PRODH to reduced dependent kinase 4 (CDK4), which has been reported to be growth resulting from MYC suppression are described in SI up-regulated by MYC (20), as a positive control. None of the Results and Discussion and shown in Fig. S3. PRODH promoter regions containing either canonical or non- canonical MYC binding sites showed significant PCR amplifi- MYC Indirectly Suppresses POX/PRODH Expression at the Transcriptional cation (Fig. 3B), demonstrating that MYC does not directly Level. As described above, MYC decreased POX/PRODH interact with the PRODH gene, and the decreased POX/ mRNA expression although it was not comparable with its in- PRODH mRNA expression may be mediated through other hibition of POX/PRODH protein levels. To further confirm its transcription factors. This result is consistent with the delayed regulation of POX/PRODH transcription, we tested the effect of changes of POX/PRODH mRNA levels regulated by MYC as MYC on PRODH promoter activity in PC3 prostate cancer cells showed in Fig. 1. by transfecting the PRODH promoter/luciferase reporter con- struct containing PRODH promoter region (10). As shown in MYC Suppresses POX/PRODH Protein Expression Primarily Through Fig. 3A, knockdown of MYC resulted in the increase of PRODH Increasing miR-23b*. The above data showed that POX/PRODH promoter activity, indicating MYC regulates POX/PRODH at protein, but not mRNA, robustly responds to the alteration of the transcriptional level. However, it is unknown whether MYC MYC levels in P493 and PC3 cells. Thus, we sought regulatory acts as a transcription factor by binding directly to the PRODH effects of MYC on POX/PRODH at the posttranscriptional promoter area. Analysis of PRODH promoter nucleotide se- level. Earlier study showed that MYC suppresses the expression quence revealed one canonical MYC binding site 5′-CACGTG- of miR-23b in P493 and PC3 cells (14). Our previously published 3′ (E-box) and one noncanonical binding site (5′-ACGGTG-3′) study indicated that miR-23b* mediates the loss of POX/PRODH at −2808 to −2813 bp and −637 to −642 bp of the PRODH pro- protein in renal cancers (11). Because miR-23b and miR-23b* moter region, respectively. To investigate whether MYC binds originate from the same transcript, we first determined the effects

Liu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 of MYC on miR-23b* and then tested whether the suppression miRNA, such as the indirect regulation at the transcriptional of POX/PRODH by MYC was related to its effects on miR-23b* level as shown above. in P493 cells. In Fig. 4 A and B, real-time PCR assays showed We further confirmed the suppression of POX/PRODH by that miR-23b* levels decreased with diminished MYC expres- MYC through miR-23b* in PC3 cells by transfecting the lucif- sion and then increased on MYC reinduction in a manner erase reporter with the POX/PRODH mRNA 3′ UTR sequence compatible with the POX/PRODH protein levels observed in containing the miR-23b* binding site, designated as pPOX 3′ Fig. 1 B and D. PC3 prostate cancer cells also showed the same UTR (11). pMIR-Report, the original reporter without the relationship between MYC and the levels of miR-23b*, i.e., MYC POX/PRODH mRNA 3′ UTR was used as a control. Negative knockdown by siRNA resulted in the decrease of miR-23b* ex- siRNAs (siNeg) or MYC siRNAs (siMYC) were cotransfected pression (Fig. S4A). These results were distinct from the reported into PC3 cells. As shown in Fig. S4B, MYC knockdown by siR- decrease of miR-23b by MYC. The nonparallel expression of the NAs increased luciferase activity of pPOX 3′ UTR significantly, sibling miRNAs regulated by MYC implied that MYC may dif- indicating the decrease of miR-23b* by siMYC. As expected, ferentially affect the processing of miRNA, including their sta- without MYC knockdown, the luciferase activity of pPOX 3′ bilization and degradation. SI Results and Discussion and Fig. S5 UTR was much lower than that of the original pMIR-Report provide the preliminary data that showed that MYC regulated because of high levels of miR-23b* binding to POX/PRODH the differential expression of miR-23b* and miR-23b partially mRNA 3′ UTR, thereby suppressing luciferase expression. through up-regulating Agonaute 2 protein (Ago 2), a key player in miRNAs stability and degradation. MYC Markedly Increases the Biosynthesis of Proline from Glutamine. To assess whether the suppression of POX/PRODH by MYC MYC has been shown to enhance GLS expression and glutamine relates to the increase of miR-23b*, we transfected P493 cells catabolism (13, 14). Because glutamine and proline are in- with antagomirs against miR-23b* to inhibit the high expression terconvertible (Fig. 1A), we investigated the MYC regulation of of miR-23b* induced by MYC overexpression in the absence of glutamine and proline metabolism by studying the enzymes of tetracycline. As shown in Fig. 4C, POX/PRODH protein level glutamine-proline metabolic pathway in P493 cells. As shown in increased 1.5-fold after miR-23b* was inhibited by antagomirs. Fig. 5A, MYC robustly increased the expression of GLS, P5CS, Additionally, we transfected P493 cells with mimic miR-23b* and PYCR1 in the pathway from glutamine to proline and de- under MYC inhibition by tetracycline. Ectopic miR-23b* ex- creased the expression of POX/PRODH, P5CDH, and GS in the pression at high levels was confirmed by real-time PCR after the pathway from proline to glutamine. PC3 human prostate cancer transfection. As expected, mimic miR-23b* resulted in a marked cells displayed the same correlation between MYC and gluta- decrease in POX/PRODH protein (Fig. 4D). However, the de- mine and proline metabolism. The reduction of MYC by MYC crease of POX/PRODH still was not comparable with that siRNA resulted in the increase of POX/PRODH, P5CDH, and without tetracycline treatment, indicating that MYC could sup- GS and decrease of GLS, P5CS, and PYCR1 (Fig. 5B). We then press POX/PRODH expression through pathways other than measured the intracellular levels of proline in P493 MYC-On and MYC-Off cells. As expected, MYC dramatically increased the intracellular levels of proline (Fig. 5C). To confirm directly the production of proline from glutamine p<0.001 A B 1.5 induced by MYC, we traced the conversion of glutamine to p<0.001 1.5 p<0.001 proline by gas chromatography–mass spectrometry (GC-MS), 1.0 1.0 Fourier transform-ion cyclotron resonance MS (FT-ICR-MS), and NMR using [U-13C,15N]-glutamine (Gln) as a tracer in P493 0.5

0.5 expression cells. Glutamine was labeled at all five carbon and both nitrogen expression

** miR-23b* relative miR-23b* relative ** ** ** atoms, which were incorporated into newly synthesized proline. ** 0.0 0.0 13 15 0 24 48 72 96 120 Time (h) 72 72 48 72 Time (h) Fig. 5D shows the expected labeling patterns from [U- C, N]- +T -T +T Wash Gln, including the intermediates of the TCA cycle and proline - + + + T (Pro) via glutamate (Glu). As described in detail (21), MYC C D -- + -Mimic 23b* -- -+ Neg RNA induced glutamine oxidation via the TCA cycle such that all of POX/PRODH the intermediates of the TCA cycle derived from glutamine were POX/PRODH α MYC increased by MYC, including -KG, succinate, fumarate, malate, MYC fi GAPDH and citrate. To con rm the previous results, the GC-MS analysis GAPDH 13 15 0.8 of C, N-Gln contribution to citrate in P493 cells with MYC- 2.0 -T E 13 0.6 On and MYC-Off is shown in Fig. 5 . The production of C5- 1.5 citrate (m+5) from Gln tracer can be explained by the reductive 0.4 1.0 carboxylation of α-KG, which entails a reversal of the citrate to 0.2 α-KG reaction catalyzed by aconitase and isocitrate de- 0.5

POX/PRODH protein POX/PRODH – 0.0 hydrogenase (IDH) as recently reported in other cells (22 25). (Relative optical density) optical (Relative

POX/PRODH protein POX/PRODH 0.0 -T +T

(Relative optical density) However, this process is unlikely to be the major pathway in P493 -T+Neg RNA -T+Anti-23b* ic 23b* +Neg RNA fi +T+Mim +T cells under normoxia, as discussed (21). This nding is also consistent with the recent reports that showed the glutamine- Fig. 4. MYC suppresses POX/PRODH protein expression primarily through dependent reductive carboxylation is most prevalent either under increasing miR-23b*. (A and B) The expression of miR-23b* was monitored hypoxia or in cells with a TCA cycle deficiency (23–25). by real-time RT-PCR in P493 cells treated with or without tetracycline (T). U6 Fig. 5F shows the GC-MS analysis of 13C,15N-Gln contribution was used as an internal control. Results were determined in triplicate and to proline synthesis with or without tetracycline treatment. m+1 repeated in two independent experiments. Data are shown as mean ± SEM. < to m+6 represent incremental increases of neutron masses P values were obtained by one-way analysis of variance. **P 0.001 com- resulting from one to five 13C and zero or one 15N incorporation pared with 0 h control in A.(C) P493 cells were transfected with miR-23b* 13 15 into proline. Thus, the m+6 labeled species of proline ( C5, N- antagomir (Anti-23b*) to inhibit the expression of miR-23b*, and scrambled 13 15 RNA was used as negative control (Neg RNA). (D) P493 cells were transfected Pro) is the direct product from C5, N2-Gln catabolism, with mimic miR-23b* to enhance the expression of miR-23b* when tetra- whereas the rest of the labeled species can be derived from the cycline was added to the medium to inhibit MYC expression. (C and D Upper) parent glutamine tracer via metabolic scrambling through the POX/PRODH protein was detected by Western blot. (C and D Lower)The TCA cycle, transamination (TA), and/or normalization of POX/PRODH protein relative to loading control by (GDH) activity (Fig. 5D). The increased expression of MYC with densitometry analysis. Data shown represent one of three independent tetracycline withdrawal markedly and consistently increased the experiments. levels of m+1 to m+6 isotopologues of proline, although the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203244109 Liu et al. Downloaded by guest on September 28, 2021 Fig. 5. MYC markedly increases the bio- ABC synthesis of proline from glutamine (A) Western 1 d 3ds 4ds - + - + - + Tet blots of the enzymes in proline and glutamine POX/PRODH POX/PRODH catabolism pathway in P493 cells treated with 1.5 tetracycline for different lengths of time. (B) P5CDH P5CDH p=0.001 PC3 cells were transfected with siRNA against GS GS 1.0 MYC (siMYC) or negative control siRNA (siNeg). PYCR1 PYCR1 0.5 The enzymes in the proline and glutamine P5CS P5CS metabolic pathways were analyzed by Western GLS GLS Myc-On) to (Relative 0.0 blots. Experiments were replicated with similar Intracellular proline level MYC ON MYC OFF MYC MYC results. (C) Intracellular proline levels in MYC- GAPDH On and MYC-Off cells. (D) The expected labeling GAPDH patterns from [U-13C,15N]-glutamine (Gln) and preexisting unlabeled Gln to different iso- D E topologues of proline (Pro) and the inter- mediates of the TCA cycle via glutamate (Glu). 13 15 [ C5, N2]-Gln can be catabolized via gluta- 13 15 minase (GLS) to produce C5, N-glutamate (Glu) (m+6), which can be converted directly to 13 15 C5, N-Pro (m+6) via the pathway depicted in 13 15 Fig. 1A. Alternatively, C5, N-Glu can be con- verted to α-ketoglutarate (α-KG) and metabo- lized via the TCA cycle. The reverse reaction of F α-KG to Glu enables 15N reincorporation into 15 Glu (e.g., production of N1-Glu) via trans- aminases (TA) and/or Glu dehydrogenase (GDH) activity. The carbon tracings shown illustrate the 13C fate after three turns of the TCA cycle. ●, 12Cor14N; red circle, 13Cinthefirst turn; green circle, 13C in the second turn; pink circle,

13C in the third turn; blue circle and N, 15N; AA, CELL BIOLOGY 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.

ratios of concentrations for the various labeled isotopologues of Discussion proline were not constant. The nonconstant ratios of proline As the only proteinogenic secondary , proline is me- isotopologues between MYC On and MYC Off are likely due to tabolized by its own family of enzymes that respond to various the following reasons. As stated above, the various labeled spe- stresses and participate in redox regulation and metabolic sig- cies of proline except for m+6 can be derived from the various naling. Recent studies defining the regulation of this system carbon and nitrogen scrambling processes through the TCA cy- suggest that proline is a “stress substrate” and proline metabo- fi cle, transamination, and/or GDH activity, as shown in Fig. 5D lism may be a potential antitumor target. POX/PRODH, the rst enzyme in proline catabolism, is induced by genotoxic (p53) (8), after three turns. Scrambling of carbon detected in proline via fl γ the TCA cycle is consistent with the production of labeled citrate in ammatory (PPAR and its ligands) (10), and nutrient stress with different scrambled labeling patterns (e.g., m+2 and m+4 (glucose deprivation) (26). Proline catabolism catalyzed by POX/ E PRODH generates electrons to produce ROS and initiates isotopologues in Fig. 5 ). The m+2 and m+4 isotopologues of a variety of downstream effects, including blockage of the cell citrate are the respective products of three and one turn of the 13 cycle and initiation of apoptosis. In this sense, POX/PRODH Krebs cycling (Fig. 5D), whereas the m+5 ( C5-citrate) and m+6 13 functions as a metabolic tumor suppressor, which is supported by ( C6-citrate) can be contributed from pyruvate carboxylation its low expression or loss in tumors and the inhibition of tumor and TCA cycle-independent ATP citrate lyase plus malic enzyme formation in a mouse xenograft tumor model by its ectopic ex- reaction sequence as shown in Le et al. (21). MYC suppression pression. In this work, we showed that oncogenic transcription may have differential effects on these processes. Moreover, the factor MYC inhibits POX/PRODH expression and, thereby, abundant m+5 isotopologue of Pro implies the replacement of inhibits its tumor suppressor function. When MYC is suppressed, 15Nby14N as all five carbons should be labeled because of the the increase of POX/PRODH induces ROS generation and ap- 13 high abundance of its precursor C5-Glu (21). The FT-ICR-MS optosis, leading to decreased cell proliferation and growth. analysis in Fig. S6 showed the fractional distribution of Glu, These results suggest that MYC-induced suppression of POX/ aspartate (Asp) and proline isotopologues. Consistent with that PRODH contributes to MYC-mediated changes of cell behavior of proline, 13Cand15N isotopologue distributions of Glu and including proliferation and metabolic reprogramming that, in turn, contributes to tumorigenesis and tumor progression. Asp were differentially modulated by MYC expression. Transformed cells from different origins typically up-regulate In addition, the NMR spectral analysis in Fig. S7 showed the both glucose and glutamine consumption as sources of metabolic extensive 13C enrichment in proline from glutamine induced by fi fi energy and as precursors for biosynthesis of macromolecules (13, MYC, which further con rmed the GC-MS data. These ndings 27). The MYC oncogene, which plays a critical role in many are consistent with the marked increases in the enzymes medi- human cancers, is considered a master regulator of cell metab- ating proline synthesis from glutamate. Thus, MYC not only olism and proliferation. It not only promotes glucose uptake and stimulates the conversion of glutamine to glutamate, but also induces aerobic glycolysis, but also enhances glutamine uptake markedly enhances subsequent conversion of glutamate to pro- and stimulates glutamine catabolism. As mentioned above, glu- line via proline biosynthetic enzymes. tamine metabolism is linked to biosynthesis of protein, nucleotide

Liu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 and lipids, redox homeostasis, and energy metabolism. However, attractive model for understanding the up-regulation of the the report from Wise et al. suggests that little of the glutamine proline synthetic pathway by MYC (34–36). uptake stimulated by MYC is used for macromolecular synthesis In summary, we showed the suppression of POX/PRODH by (13). MYC-induced glutamine catabolism is the reprogramming MYC, and demonstrated the effect of this reprogramming on cell of mitochondrial metabolism to sustain cellular viability and proliferation and survival, which makes POX/PRODH a poten- TCA cycle anaplerosis (13). The most recent findings reported tial target for cancer therapeutics. The regulatory mechanism for by Le et al. (21) and Wang et al. (28) have emphasized the the decreased expression of tumor suppressor POX/PRODH in metabolic reprogramming controlled by MYC in transformed human cancer is in part mediated posttranscriptionally by MYC cells and activated T cells. The latter showed that MYC-driven via miRNA. The metabolic link between glutamine and proline glutamine catabolism couples with multiple biosynthetic path- afforded by MYC emphasizes the complexity of tumor metabo- ways, especially ornithine and polyamine biosynthesis (28). lism, an area deserving additional studies. Interestingly, our current studies show that MYC markedly increased glutamine-derived proline biosynthesis. However, Materials and Methods how does this biosynthetic pathway fit into the MYC-driven Cells and Cell Culture. MYC-inducible human Burkitt lymphoma model P493 metabolic reprogramming? cells, PC3 human prostate cancer cells were maintained in RPMI medium 1640 The physiological relationship between glutamine and proline with 10% (vol/vol) FBS. metabolism was observed during the 1970s by Windmueller and Spaeth (29). In rat small intestine, a rapidly proliferating tissue, Additional Methods. Detailed descriptions of methods for real-time RT- glutamine was an important substrate, with a utilization rate PCR analysis, small RNAs transfection, Western blot, luciferase assay, mea- nearly two-thirds of that for glucose. Interestingly, proline was surement of ROS, apoptosis assay, chromatin immunoprecipitation assay, a quantitatively important product from glutamine. Similarly, in measurement of intracellular proline levels, measurement of glutamate and GSH, and GC-MS, FT-ICR-MS and NMR studies of [13C,15N]-Gln contribution to cultured L-M cells, Stoner and Merchant showed a net increase proline biosynthesis are available in supporting information. of free proline accompanying the utilization of glutamine (30). In the tumor microenvironment, a metabolic commensalism between ACKNOWLEDGMENTS. We thank Dr. Ziqiang Zhu for insightful comments areas under the varying influence of MYC and HIF-1 or between and Dr. Ziqiang Zhu, Julie Tan, and Radhika Burra for excellent technical tumor and stromal cells has been proposed (3, 31–33). The assistance. This research was supported by the Intramural Research Program metabolic advantage afforded by the increased conversion of of the National Institutes of Health (NIH); the National Cancer Institute; glutamine to proline remains unclear, but the conversion of one the Center for Cancer Research; the National Science Foundation, the Experimental Program to Stimulate Competitive Research (EPSCoR) Grant nonessential amino acid to another for protein synthesis seems EPS-0447479; NIH Grants P20RR018733 from the National Center for Re- unlikely. The previously described metabolic interlock between search Resources 1R01CA118434-01A2 (to T.W.-M.F.), 3R01CA118434-02S1 proline synthesis and pentose phosphate pathway offers an (to T.W.-M.F.), and R21CA133688 (to A.N.L.); and the Brown Foundation.

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