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Succinate Dehydrogenase Inhibition Leads to Epithelial-Mesenchymal Transition and Reprogrammed Carbon Metabolism." Cancer & Metabolism

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Succinate Dehydrogenase Inhibition Leads to Epithelial-Mesenchymal Transition and Reprogrammed Carbon Metabolism. Succinate dehydrogenase inhibition leads to epithelial- mesenchymal transition and reprogrammed carbon metabolism The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Aspuria, Paul-Joseph P., Sophia Y. Lunt, Leif Varemo, Laurent Vergnes et al. "Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism." Cancer & Metabolism. (2014 Dec 15); 2 (1):21. As Published http://dx.doi.org/10.1186/2049-3002-2-21 Publisher BioMed Central Ltd. Version Final published version Citable link http://hdl.handle.net/1721.1/92499 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0 Aspuria et al. Cancer & Metabolism 2014, 2:21 http://www.cancerandmetabolism.com/content/2/1/21 Cancer & Metabolism RESEARCH Open Access Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism Paul-Joseph P Aspuria1*, Sophia Y Lunt2,3†, Leif Väremo4†, Laurent Vergnes5, Maricel Gozo1,6, Jessica A Beach1,6, Brenda Salumbides1,KarenReue5, W Ruprecht Wiedemeyer1,7,JensNielsen4, Beth Y Karlan1,7 and Sandra Orsulic1,7 Abstract Background: Succinate dehydrogenase (SDH) is a mitochondrial metabolic enzyme complex involved in both the electron transport chain and the citric acid cycle. SDH mutations resulting in enzymatic dysfunction have been found to be a predisposing factor in various hereditary cancers. Therefore, SDH has been implicated as a tumor suppressor. Results: We identified that dysregulation of SDH components also occurs in serous ovarian cancer, particularly the SDH subunit SDHB. Targeted knockdown of Sdhb in mouse ovarian cancer cells resulted in enhanced proliferation and an epithelial-to-mesenchymal transition (EMT). Bioinformatics analysis revealed that decreased SDHB expression leads to a transcriptional upregulation of genes involved in metabolic networks affecting histone methylation. We confirmed that Sdhb knockdown leads to a hypermethylated epigenome that is sufficient to promote EMT. Metabolically, the loss of Sdhb resulted in reprogrammed carbon source utilization and mitochondrial dysfunction. This altered metabolic state of Sdhb knockdown cells rendered them hypersensitive to energy stress. Conclusions: These data illustrate how SDH dysfunction alters the epigenetic and metabolic landscape in ovarian cancer. By analyzing the involvement of this enzyme in transcriptional and metabolic networks, we find a metabolic Achilles’ heel that can be exploited therapeutically. Analyses of this type provide an understanding how specific perturbations in cancer metabolism may lead to novel anticancer strategies. Keywords: Succinate dehydrogenase, SDH, Ovarian cancer, EMT, Carbon metabolism, Epigenetics Background several molecular subtypes with different clinical out- Ovarian cancer is the most lethal gynecological malig- comes [4,5]. One of the poor prognosis subtypes contains nancy in the United States, resulting in approximately an epithelial-mesenchymal transition (EMT) signature 22,000 new diagnoses and 14,000 deaths in 2013 [1]. [5]. Therefore, understanding and characterizing EMT in The prognosis of ovarian cancer is poor and has seen ovarian cancer is of great importance. limited improvement in the past few decades with a 5-year EMT is a biological process that converts epithelial survival rate of roughly 44% [2]. High-grade serous ovar- cells to motile, mesenchymal cells [6]. It is largely defined ian cancer is the most common and most lethal subtype by the loss of plasma membrane E-cadherin and conversion of ovarian cancer, contributing to >70% of deaths from from a cobblestone-like morphology to a more spindle- ovarian cancer [3]. Integrative large-scale analyses of pa- shaped and less adherent state. It was initially characterized tient tumors with serous histology have revealed that the as a fundamental event in morphogenesis during embry- disease is highly heterogeneous but can be clustered into onic tissue and organ development [7]. However, emerging evidence suggests that cancer cells utilize the EMT pro- * Correspondence: [email protected] † gram to enhance invasiveness, increase migratory ability, Equal contributors and acquire stem-like characteristics such as chemore- 1Women’s Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA sistance. EMT reprogramming involves several layers of Full list of author information is available at the end of the article © 2014 Aspuria et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aspuria et al. Cancer & Metabolism 2014, 2:21 Page 2 of 15 http://www.cancerandmetabolism.com/content/2/1/21 regulation and has been the subject of intense research, in EMT. Sdhb knockdown cells were found to have a especially in the context of cancer [6]. One area that has hypermethylated epigenome whereby increased H3K27 not been extensively studied is the role of metabolic methylation was sufficient to induce EMT. Using a meta- enzymes in the regulation and maintenance of EMT. bolomic approach to investigate the metabolic effects of Alterations in metabolism have been implicated in decreased SDH activity, we found that Sdhb knockdown cancer, with the main focus on the Warburg effect, a leads to altered glucose and glutamine utilization through phenomenon in which cancer cells upregulate glycolysis central carbon metabolism as well as mitochondrial and lactate production while decreasing glucose contribu- dysfunction. These data demonstrate the metabolic and tion to the citric acid (TCA) cycle in the mitochondria, epigenetic consequences of decreased activity of a mito- even in the presence of sufficient oxygen [8-10]. However, chondrial metabolic enzyme in ovarian cancer. mitochondrial metabolism is also important for cancer proliferation. For example, mutations in the mitochondrial Methods enzyme succinate dehydrogenase (SDH) have been repor- Plasmids and reagents ted in a wide variety of cancers [11,12]. Also known as pLKO.1-based short hairpin constructs specific for mouse mitochondrial respiratory Complex II, SDH is the only Sdhb and human SDHB, as well as scrambled control enzyme to participate in both the TCA cycle and electron sequences, were obtained from Sigma-Aldrich (St. Louis, transport chain (ETC.). In the TCA cycle, SDH catalyzes MO, USA). shRNA sequences are listed in Additional the oxidation reaction where succinate is converted to file 1: Table S1. GSK-J1 and GSK-J4 were obtained fumarate, which is coupled to the reduction of ubiquinone from Tocris Bioscience (Bristol, Avon, UK). Metformin, to ubiquinol in the ETC. [12]. SDH is a holoenzyme con- p-iodonitrotetrazolium, oligomycin, carbonyl cyanide 4- sisting of four essential subunits: SDHA (a flavoprotein), (trifluoromethoxy)phenylhydrazone chloride (FCCP), SDHB (iron-sulfur protein), and two membrane anchor and rotenone-myxothiazol were purchased from Sigma- units SDHC and SDHD. SDH assembly requires two fac- Aldrich. tors, SDHAF1 and SDHAF2 [13], while its function is influenced by the deacetylase activity of SIRT3 [14]. SDHx Cell lines loss-of-function mutations have been found to predispose C1 mouse ovarian cancer cells were generated as pre- individuals to hereditary pheochromocytoma (PCC), para- viously described [19-21]. HEY cells were obtained from ganglioma (PGL), gastrointestinal stromal tumor (GIST), American Type Culture Collection (ATCC). Both cell and renal cell carcinoma [12]. Interestingly, it has been lines were grown in Dulbecco’s modified Eagle’s medium shown that SDHx mutations are associated with hallmarks (DMEM) supplemented with 10% FBS unless otherwise of EMT in PCC and PGL as a result of alterations to the indicated. epigenome [15,16]. Furthermore, the SDH regulators SDHAF1 and SIRT3 have been identified as tumor sup- Lentiviral-mediated gene knockdown pressor genes in lung and breast cancer, respectively pLKO.1-sh1-Sdhb, pLKO.1-sh2-Sdhb,pLKO.1-sh1-SDHB, [14,17]. This has led to the definition of SDH as a mito- pLKO.1-sh2-SDHB, and pLKO.1 scrambled shRNA plas- chondrial tumor suppressor and a key player in cancer cell mids were co-transfected with the delta 8.9 packaging differentiation. plasmid and VSVG plasmid into Lenti-X™ 293 T cells Ovarian cancer is characterized by a remarkable degree using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, of genomic disarray but extremely low frequency of re- USA). After overnight incubation, the cell culture medium current genomic alterations [4]. The dearth of common was changed to DMEM supplemented with 30% FBS. The therapeutic targets in ovarian cancer samples highlights medium was harvested 48 h later and filtered through a the need for individualized therapy or different approaches 0.45-μM filter. The viral supernatant was used for infec- to identifying patient groups with common character- tion of C1 and HEY cells with polybrene (4 μg/ml). istics that can be therapeutically exploited. Interest- Cells were infected overnight,
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