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Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation Through E2F1-Mediated Increase of Cyclins S

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Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation Through E2F1-Mediated Increase of Cyclins S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/12/21/mol.116.106757.DC1 1521-0111/91/3/189–196$25.00 http://dx.doi.org/10.1124/mol.116.106757 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:189–196, March 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation through E2F1-Mediated Increase of Cyclins s Jonathan Choiniere, Jianguo Wu, and Li Wang Department of Physiology and Neurobiology, Institute for Systems Genomics, University of Connecticut, Storrs, Connecticut (J.C., J.W., L.W.); Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut (L.W.); Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut (L.W.); and School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China (L.W.) Downloaded from Received August 31, 2016; accepted December 9, 2016 ABSTRACT Hepatocellular carcinoma (HCC) is a common form of cancer PDK4-knockdown HCC cells also progressed faster through with prevalence worldwide. There are many factors that lead to the cell cycle, which concurrently expressed high levels of molpharm.aspetjournals.org the development and progression of HCC. This study aimed to cyclins and E2F1 as seen in the Pdk42/2 mice. Interestingly, identify potential new tumor suppressors, examine their function the induced cyclin E1 and cyclin A2 caused by Pdk4 deficiency as cell cycle modulators, and investigate their impact on the was repressed by arsenic treatment in mouse liver and in HCC cyclin family of proteins and cyclin-dependent kinases (CDKs). cells. E2f1 deficiency in E2f12/2 mouse liver or knockdown E2F1 In this study, the pyruvate dehydrogenase kinase (PDK)4 gene using small hairpin RNAs in HCC cells significantly decreased was shown to have potential tumor suppressor characteristics. cyclin E1, cyclin A2, and E2F1 proteins. In contrast, inhibition of PDK4 expression was significantly downregulated in human PDK4 activity in HCC cells increased cyclin E1, cyclin A2, and HCC. Pdk42/2 mouse liver exhibited a consistent increase in E2F1 proteins. These findings demonstrate that PDK4 is a critical cell cycle regulator proteins, including cyclin D1, cyclin E1, cyclin regulator of hepatocyte proliferation via E2F1-mediated regula- A2, some associated CDKs, and transcription factor E2F1. tion of cyclins. at ASPET Journals on September 23, 2021 Introduction 2003). Identification of cyclin modulators will aid in the under- standing of HCC development and progression. Liver cancer is the second leading cause of cancer-related Pyruvate dehydrogenase kinase PDK4 is a mitochondrial death worldwide, with an estimated 745,500 deaths yearly. protein with a histidine kinase domain that inhibits the The majority of liver cancer deaths are identified as hepato- pyruvate dehydrogenase complex (PDC). Inhibition of the cellular carcinoma (HCC). The mechanisms behind HCC PDC results in reduced conversion of pyruvate to acetyl- progression are continually studied. Many factors that in- CoA. Acetyl-CoA is used in the citric acid cycle to carry out crease the risk of HCC have been identified, including cellular respiration (Sugden and Holness, 2003). PDK4 is a environmental factors, viral infection, alcohol consumption, major factor in cellular respiration, since it works to inhibit and smoking (Kanda et al., 2015). Cyclins are vital cell cycle the progression from glycolysis to the citric acid cycle. Cellular regulators that normally function to ensure the control of respiration is a major physiologic factor that was shown to be cellular proliferation. The cyclin family of proteins and the altered in cancer cells (Scatena, 2012); therefore, PDK4 is a associated cyclin-dependent kinases (CDKs) have been shown prime molecular suspect in crosstalk between cellular respi- to be significantly elevated in HCC tissues (Masaki et al., ration and cell cycle progression. Because liver cells are highly aerobic and metabolically active, liver tissue and associated cells serve as excellent test subjects. This research was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01-ES025909], the Arsenic is identified as a group 1 carcinogen and is the 20th National Institutes of Health National Institute of Diabetes and Digestive most common element in the earth’s crust (International and Kidney Diseases [R01-DK080440, R01-DK104656, and P30-DK34989 (to Agency for Research on Cancer, 2012). Over 100 million people Yale Liver Center)], the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants R21-AA022482 and R21-AA024935], worldwide are exposed to arsenic-contaminated drinking and the U.S. Department of Veterans Affairs [Merit Award 1I01BX002634]. water, making it a global health concern (Polya and Charlet, dx.doi.org/10.1124/mol.116.106757. s This article has supplemental material available at molpharm. 2009). Arsenic has been reported to induce epigenetic alter- aspetjournals.org. ations related to HCC progression (Liu et al., 2014) and to ABBREVIATIONS: Aza, 59-aza-29-deoxycytidine; CDK, cyclin-dependent kinase; DNMT, DNA methyltransferase; HCC, hepatocellular carcinoma; MeDIP, methylated DNA immunoprecipitation on chromatin immunoprecipitation; PCNA, proliferating cell nuclear antigen; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; SHP, small heterodimer partner; shRNA, small hairpin RNA; WT, wild type. 189 190 Choiniere et al. silence hepatic PDK4 expression through activation of histone Liver Specimens. The coded human liver specimens were H3K9 methyltransferase G9a (Zhang et al., 2016). obtained through the Liver Tissue Procurement and Distribution Nuclear receptors are a class of proteins that mediate the System (Minneapolis, MN) and were described previously (He et al., activity of hormones and have been implicated in various 2008). Because we did not ascertain individual identities associated diseases, including HCC (Rudraiah et al., 2016). The small with the samples, the Institutional Review Board for Human Research at the University of Connecticut determined that the project was not heterodimer partner (SHP) is a unique transcriptional re- research involving human subjects. pressor (Zhou et al., 2010) and has been implicated as a critical Cell Culture Experiments, Fluorescence-Activated Cell inhibitor of HCC progression (Zhang et al., 2011b) by regulat- Sorting Analysis, and Luciferase Promoter Assay. Huh7 ing multiple pathways involved in tumor growth, including (Zhang and Wang, 2011) and 293T (Yang and Wang, 2012) cells cell proliferation (Zhang et al., 2008), apoptosis (Farhana were described previously. Cultured cells were maintained in et al., 2007; Zhang et al., 2010), and migration and invasion Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with (Yang et al., 2016). Intriguingly, SHP represses DNA meth- 10% fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin ylation via suppressing DNA methyltransferase (DNMT) (Mediatech, Manassas, VA). High- and low-efficiency (shPDK4 H expression (Zhang and Wang, 2011; Zhang et al., 2012), and shPDK4 L, respectively) PDK4 knockdown cell lines were suggesting that modulating SHP function by its agonist may generated by a lentiviral vector containing the small hairpin RNA (shRNA) along with packing vector and envelope vector. These be useful to develop epigenetic-based therapeutic treatment of components were cotransfected into 293T cells using X-tremeGENE Downloaded from HCC. HP DNA transfection reagent (Sigma-Aldrich) according to the Using an unbiased approach that included a combination manufacturer’s protocol. The supernatant containing virus particles of methylated DNA immunoprecipitation on chromatin was collected and concentrated. Suspended virus was applied to immunoprecipitation (MeDIP) and RNA sequencing (RNA- target cells with 6 mg/ml polybrene. The shPDK4H cell line was seq) methods in Shp2/2 mice, this study identified PDK4 as generated using shRNA (TRCN0000006264, clone ID NM_002612.2- a potential tumor suppressor gene. The expression of PDK4 2954s1c) from Sigma-Aldrich, and the shPDK4 L was generated was markedly reduced in human HCC specimens and in using shRNA (TRCN0000194917, clone ID NM_002612.2-1297s1c1) molpharm.aspetjournals.org 2 2 m mouse liver tumors. The Pdk4 / liver had a significant also from Sigma-Aldrich. shPDK4L cells were treated with 15 M induction in multiple cyclin proteins, with the most striking arsenic for 24 hours. E2F1 shRNAs were from Sigma-Aldrich (shE2F1 1: TRCN0000039659, clone ID NM_005225.1-502s1c1; and shE2F1 2: elevation of cyclin E1, cyclin A2, and E2F1, whereas Pdk4- TRCN0000010328, clone ID NM_005225.x-1171s1c1). The PDK4 knockdown HCC cells exhibited a similar activation of cyclin inhibitor diisopropylamine dichloroacetate was from Fisher Scien- proteins and progressed faster through the G2/M phase of the tific (cat. no. AAH6134103, Hampton, NH). cell cycle compared with control cells. Interestingly, arsenic Western Blotting, Quantitative Polymerase Chain Reaction, decreased the levels of cyclin proteins in both mouse livers and Transient Transfection and Luciferase Assay. Western and cultured HCC cells that were induced by Pdk4 de- blotting and quantitative polymerase chain reaction
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