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PHGDH Expression Is Required for Mitochondrial Redox Homeostasis

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PHGDH Expression Is Required for Mitochondrial Redox Homeostasis Published OnlineFirst June 8, 2016; DOI: 10.1158/0008-5472.CAN-16-0530 Cancer Molecular and Cellular Pathobiology Research PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Breast Cancer Stem Cell Maintenance, and Lung Metastasis Debangshu Samanta1,2, Youngrok Park1, Shaida A. Andrabi1,3, Laura M. Shelton4, Daniele M. Gilkes1,2,5, and Gregg L. Semenza1,2,5,6 Abstract Intratumoral hypoxia stimulates enrichment of breast cancer drial redox homeostasis, and increased apoptosis, which abrogat- stem cells (BCSC), which are critical for metastasis and patient ed BCSC enrichment under hypoxic conditions. PHGDH-deficient mortality. Here we report a metabolic adaptation that is required cells exhibited increased oxidant levels and apoptosis, as well as for hypoxia-induced BCSC enrichment and metastasis. Hypoxia- loss of BCSC enrichment, in response to treatment with carbopla- inducible factors coordinately regulate expression of genes encod- tin or doxorubicin. PHGDH-deficient cells were relatively weakly ing phosphoglycerate dehydrogenase (PHGDH) and five down- tumorigenic and tumors that did form were deficient in BCSCs, stream enzymes in theserinesynthesis pathway andmitochondrial abolishing metastatic capacity. Our findings highlight a role for one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH PHGDH in the formation of secondary (recurrent or metastatic) expression in both estrogen receptor–positive and negative breast tumors, with potential implications for therapeutic targeting of cancer cells led to decreased NADPH levels, disturbed mitochon- advanced cancers. Cancer Res; 76(15); 1–13. Ó2016 AACR. Introduction increased mitochondrial reactive oxygen species (ROS) produc- tion that occurs due to decreased electron transport chain effi- Breast cancer mortality occurs in patients whose cancer cells ciency under hypoxic conditions (16–23). metastasize to distant sites, such as the lungs, bones, and brain. Oncogenic transformation also activates pathways that generate Only a small percentage of the breast cancer cells in a primary ROS and place cancer cells at risk for apoptosis (24). Redox tumor have self-renewal capacity, which is necessary to form a homeostasis is dependent on a balance between levels of oxidants metastatic tumor, and are designated as breast cancer stem cells and antioxidants. The latter are dependent upon the generation of (BCSC) or tumor-initiating cells (1, 2). Compared with bulk NADPH, which is used to maintain glutathione, the principal breast cancer cells, BCSCs exhibit increased survival when treated cellular antioxidant, in a reduced form. Two glycolytic shunt path- with cytotoxic chemotherapy (3, 4), which actively induces the ways utilize glucose metabolites for NADPH generation: the pen- BCSC phenotype (5–7). Intratumoral hypoxia is common in tose phosphate pathway (PPP) diverts glucose-6-phosphate, advanced breast cancers (8) and induces the metastatic (9) and whereas the serine synthesis pathway (SSP) converts 3-phospho- BCSC (10) phenotypes through transcriptional activation of glycerate into serine via three reactions that are catalyzed by target genes by hypoxia-inducible factor 1 (HIF-1) and HIF-2 phosphoglycerate dehydrogenase (PHGDH), phosphoserine ami- (11–15). Adaptation of mammalian cells to chronic hypoxia notransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). involves a HIF-1–dependent switch from oxidative to glycolytic Serine is utilized as a substrate for one-carbon (folate cycle) metabolism, which is an adaptive response to, and ameliorates, metabolism (1CM), either in the cytosol or mitochondria. In the mitochondria (mito1CM), serine hydroxymethyl transferase 2 (SHMT2) catalyzes the reaction of serine and tetrahydrofolate 1 Institute for Cell Engineering, Johns Hopkins University School of (THF) to glycine and 5,10-methylene-THF (MTHF). MTHF dehy- Medicine, Baltimore, Maryland. 2McKusick-Nathans Institute of Genet- ic Medicine, Johns Hopkins University School of Medicine, Baltimore, drogenase 2 (MTHFD2) catalyzes the reaction of MTHF and þ Maryland. 3Department of Neurology, Johns Hopkins University NADP to generate formyl-MTHF and NADPH. Finally, MTHFD1L 4 School of Medicine, Baltimore, Maryland. Human Metabolome Tech- splits formyl-THF into THF and formate (Fig. 1A). The cytosolic nologies America, Inc., Boston, Massachusetts. 5Sidney Kimmel Com- prehensive Cancer Center, Johns Hopkins University School of Med- (cyto1CM) reactions are catalyzed by SHMT1 and MTHFD1 (which icine, Baltimore, Maryland. 6Departments of Pediatrics, Medicine, performs reactions catalyzed by both MTHFD2 and MTHFD1L). Radiation Oncology, and Biological Chemistry, Johns Hopkins Univer- PHGDH catalyzes the reaction that diverts 3-phosphoglycerate sity School of Medicine, Baltimore, Maryland. from the Embden–Meyerhof pathway (EMP) to the SSP. A short Note: Supplementary data for this article are available at Cancer Research hairpin RNA (shRNA) screen revealed that transformed breast Online (http://cancerres.aacrjournals.org/). cells required PHGDH expression for tumor xenograft formation Corresponding Author: Gregg L. Semenza, Johns Hopkins University School of (25). PHGDH gene amplification was found in 6% of breast Medicine, 733 N. Broadway, Suite 671, Baltimore, MD 21205. Fax: 443-287-5618; cancers and shRNA-mediated knockdown of PHGDH expression E-mail: [email protected] inhibited proliferation of breast cancer cells with PHGDH ampli- doi: 10.1158/0008-5472.CAN-16-0530 fication (25, 26). PHGDH overexpression was observed in 70% of À Ó2016 American Association for Cancer Research. estrogen receptor negative (ER ) breast cancers (25), indicating www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 8, 2016; DOI: 10.1158/0008-5472.CAN-16-0530 Samanta et al. A C Glucose 3-Phosphoglycerate Lactate NAD+ PHGDH NADH PHGDH MTHFD1L MTHFD2 PSPH 3-Phosphohydroxypyruvate SHMT2 MTHFD1 PSAT1 SHMT1 Glutamate PSAT1 α-Ketoglutarate MDA-231 Phosphoserine SUM-149 PSPH HCC-1954 Serine Glycine MCF-7 SHMT2 ZR-75.1 THF MTHF BT-474 GSH NADP+ MTHFD2 GSSG NADPH Hypoxic induction Formyl-THF No hypoxic induction Cytosol Mitochondrion MTHFD1L Figure 1. Hypoxia induces expression of mRNAs Formate encoding SSP and mito1CM enzymes. +THF A, enzymatic reactions. Glucose- derived 3-phosphoglycerate is metabolized to glycine and NADPþ is reduced to NADPH through the B P 20% O2 1% O2 No induction 1% O2 Induction ( < 0.001) activity of SSP (blue) and mito1CM (purple) enzymes. B, mRNA expression. Breast cancer cell lines 30 PHGDH 3.5 PSAT1 2 were exposed to 20% or 1% O for 300 2 3 24 hours and expression of mRNAs 250 1.5 2.5 encoding SSP and 1CM enzymes were 20 200 2 analyzed by RT-qPCR. The expression 1 150 1.5 of each mRNA was quantified relative 10 to 18S rRNA and then normalized to 100 1 0.5 the result obtained from MDA-MB-231 50 0.5 (MDA-231) cells at 20% O (mean Æ 0 0 2 0 0 SEM; n ¼ 3). C, summary of mRNA expression data (columns) in breast cancer cell lines (rows). Red, significantly increased expression 0.8 at 1% as compared to 20% O 12 2 3.5 7 (P < 0.001; Student t test); gray, no PSPH SHMT2 10 0.6 induction at 1% O2. 2.5 5 8 0.4 6 1.5 3 4 0.2 0.5 1 2 0 0 Relative mRNA expression mRNA Relative 60 2 1 12 MTHFD2 MTHFD1L 50 10 1.5 0.8 8 40 0.6 6 30 1 0.4 4 20 0.5 0.2 2 10 0 0 0 0 that a mechanism other than gene amplification must underlie 1CM enzymes, is required to maintain redox homeostasis in PHGDH overexpression in most breast cancers. We hypothesized hypoxic breast cancer cells, especially in BCSCs, which are par- that increased expression of PHGDH, as well as other SSP and ticularly sensitive to ROS (27). OF2 Cancer Res; 76(15) August 1, 2016 Cancer Research Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 8, 2016; DOI: 10.1158/0008-5472.CAN-16-0530 PHGDH Is Required for Breast Cancer Progression Materials and Methods Metabolite analysis Metabolites in culture media and cells were analyzed by cap- For details, see Supplementary Materials and Methods. illary electrophoresis and single or tandem mass spectrometry (MS/MS) relative to internal standards (Human Metabolome Cell culture Technologies) as described previously (29, 30). MCF-7, MDA-MB-231, HCC-1954, SUM-149, and SUM-159 cells were cultured as described previously (6). BT-474, ZR75.1, and T47D cells were cultured in RPMI1640 with 10% FBS. The cell Bioinformatics lines were obtained from Dr. Sara Sukumar (Johns Hopkins For the HIF signature, the The Cancer Genome Atlas Breast University, Baltimore, MD) in 2012. Cell authentication was Invasive Carcinoma Gene Expression Dataset of 1,215 patients performed by PCR analysis of short tandem repeats. was analyzed (31, 32). Tumor grade was analyzed using GOBO (33). Kaplan–Meier curves were generated using KM plotter (34). Lentivirus transduction Vectors encoding shRNA targeting HIF-1a and HIF-2a, and Results generation of MDA-MB-231 and MCF-7 subclones, were SSP and mito1CM enzyme expression is induced in hypoxic described previously (13, 28). pLKO.1-puro lentiviral vectors breast cancer cells þ encoding shRNA targeting PHGDH (Supplementary Table S1) Breast cancers are classified as ER , progesterone receptor þ were purchased from Sigma-Aldrich. Lentiviruses were packaged positive (PR ), human epidermal growth factor receptor 2 pos- þ À À À and transduced cells were selected as described previously (28). itive (HER2 ), or triple negative (ER /PR /HER2 ). We exposed six representative human breast cancer cell lines to 20% or 1% O2 þ þ þ þ Reverse transcription and quantitative real-time PCR for 24 hours: BT-474 (ER /PR /HER2 ), HCC-1954 (HER2 ), þ þ À À À Total RNA was extracted from cells and tumors using TRIzol MCF-7 (ER /PR ), MDA-MB-231 (ER /PR /HER2 ), SUM-149 À À À þ (Invitrogen) and treated with DNase I (Ambion). cDNA synthesis (ER /PR /HER2 ), and ZR-75.1 (ER ; ref.
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