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Role and Regulation of Coordinately Expressed De Novo Purine Biosynthetic Enzymes PPAT and PAICS in Lung Cancer

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Role and Regulation of Coordinately Expressed De Novo Purine Biosynthetic Enzymes PPAT and PAICS in Lung Cancer Henry Ford Health System Henry Ford Health System Scholarly Commons Urology Articles Urology 9-15-2015 Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer. Moloy T. Goswami Guoan Chen Balabhadrapatruni Chakravarthi Satya Pathi Sharath Anand See next page for additional authors Follow this and additional works at: https://scholarlycommons.henryford.com/urology_articles Recommended Citation Goswami MT, Chen G, Chakravarthi BV, Pathi SS, Anand SK, Carskadon SL, Giordano TJ, Chinnaiyan AM, Thomas DG, Palanisamy N, Beer DG, and Varambally S. Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer. Oncotarget 2015; 6(27):23445-23461. This Article is brought to you for free and open access by the Urology at Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Urology Articles by an authorized administrator of Henry Ford Health System Scholarly Commons. Authors Moloy T. Goswami, Guoan Chen, Balabhadrapatruni Chakravarthi, Satya Pathi, Sharath Anand, Shannon Carskadon, Thomas Giordano, Arul Chinnaiyan, Dafydd Thomas, Nallasivam Palanisamy, David Beer, and Sooryanarayana Varambally This article is available at Henry Ford Health System Scholarly Commons: https://scholarlycommons.henryford.com/ urology_articles/201 www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 27 Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer Moloy T. Goswami1,2,*, Guoan Chen4,*, Balabhadrapatruni V.S.K. Chakravarthi1,2,8,*, Satya S. Pathi1,2,9, Sharath K. Anand2, Shannon L. Carskadon1,2, Thomas J. Giordano2,5, Arul M. Chinnaiyan1,2,3,5,6, Dafydd G. Thomas2,5, Nallasivam Palanisamy1,2,5,7, David G. Beer4,5, Sooryanarayana Varambally1,2,5,8 1Michigan Center for Translational Pathology, Ann Arbor, MI 48109, USA 2Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA 3Department of Urology, University of Michigan, Ann Arbor, MI 48109, USA 4Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA 5Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA 6Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI 48109, USA 7Department of Urology, Henry Ford Health System, Detroit, MI 48202, USA 8Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35233, USA 9Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA *These authors have contributed equally to this work Correspondence to: Sooryanarayana Varambally, e-mail: soorya@uab.edu David G. Beer, e-mail: dgbeer@umich.edu Keywords: lung adenocarcinoma, amplification, purine biosynthesis, glutamine, xenograft Received: May 04, 2015 Accepted: June 12, 2015 Published: June 23, 2015 ABSTRACT Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention. www.impactjournals.com/oncotarget 23445 Oncotarget INTRODUCTION involved in cancer and underscore the importance of meta- bolic dysregulation in cancer progression [22]. Lung cancer is the leading cause of cancer-related Purine nucleotides are synthesized through two deaths globally [1] with non-small cell lung cancer distinct pathways, either de novo utilizing simple (NSCLC) accounting for 80% of all lung cancers [2, 3]. In precursors like amino acids (glutamine, glycine and the United States, the overall 5-year survival rate of lung aspartate) and bicarbonate or through salvage of purine cancer patients is 16% and more than 50% of the cases bases released by the hydrolytic degradation of nucleic are diagnosed at advanced stages where curative treat- acids and nucleotides. De novo purine biosynthesis ment is not possible. Genomic and gene expression-based requires 5-phosphoribosyl-1-pyrophosphate (PRPP) characterizations have resulted in improved molecular sub- as a precursor and glutamine as co-substrate [23]. The typing of lung cancer and identification of driver mutations initial committed step in this pathway is catalyzed including epidermal growth factor receptor (EGFR- by PPAT leading to production of 5-phosphoribosyl- 10–30%), Kirsten rat sarcoma viral oncogene homolog 1-amine (5-PRA). PPAT is divergently transcribed from (KRAS-15–30%), and fibroblast growth factor receptor 1 a locus that also encodes PAICS, another enzyme in (FGFR1-20%), among others [4, 5]. Identification of the pathway that produces the intermediary metabolite EGFR activating mutations in its kinase domain led to N-succinyl-5-aminoimidazole-4-carboxamide-1-ribose- the development of EGFR tyrosine kinase inhibitors, erlo- 5’-phosphate (SAICAR), known to activate pyruvate tinib and gefitinib [5, 6]. Lung cancer patients harboring kinase isoform PKM2 under glucose-depleted condi- ALK gene fusions treated with crizotinib show similar tion [24]. After the initial committed step, in a series positive response [7, 8]. In recent studies, identification of enzymatic reactions, 5-PRA is converted to inosine of fusions of CD74-NRG1 that activate HER2:HER3 monophosphate (IMP), the precursor for nucleotides signaling offered therapeutic possibilities for intractable adenosine monophosphate (AMP) and guanosine mono- mucinous lung cancers [9, 10]. Thus, well-defined molecu- phosphate (GMP). lar stratification has become essential for the development In this study, we demonstrate that de novo purine of targeted treatment for specific molecular subtypes of biosynthetic pathway enzymes PPAT and PAICS show cancers and personalized therapy. increased expression in lung cancer and can serve Recent studies have identified various metabolic as prognostic markers for patient survival. Further, genes and associated metabolites that play a role in onco- our investigations indicate PPAT and PAICS genes genesis. For example, IDH1 and IDH2 mutations confer are necessary for lung tumorigenesis. We show that altered enzyme properties producing D-2-hydroxyglutarate PPAT and PAICS expression influences the pyruvate and triggering oncogenesis [11, 12]. Our earlier studies kinase activity. Finally, our results reveal glutamine revealed that the metabolite sarcosine and its biosynthetic mediated modulation govern PPAT and PAICS over- enzyme, glycine N-methyltransferase (GNMT) were expression. elevated in prostate cancer, while sarcosine dehydroge- nase (SARDH) and pipecolic acid oxidase (PIPOX) that RESULTS metabolize sarcosine, were reduced in prostate tumors [13, 14]. In addition, KRAS-mediated metabolic trans- De novo purine biosynthetic pathway genes are formation is known to activate the phosphate pentose overexpressed in lung cancers pathway and hexosamine biosynthesis in pancreatic can- cer [15]. Various oncogenes such as MYC have been The de novo purine biosynthetic pathway shown to induce purine and pyrimidine biosynthesis involves multiple enzymatic steps that convert genes including phosphoribosyl amidotransferase (PPAT), 5-phosphoribosyl-1-pyrophosphate (PRPP) to inosine phosphoribosylaminoimidazole carboxylase, phospho- monophosphate (IMP), a precursor for adenosine ribosylaminoimidazole succinocarboxamide synthetase monophosphate (AMP) and guanosine monophosphate (PAICS), adenylosuccinate Lyase (ADSL), dihydrooro- (GMP) production. Many of the bifunctional enzymes cat- tate dehydrogenase (DHODH) and others [16, 17]. In alyze more than one step in the pathway. Recent studies addition, a yeast screen identified the purine biosynthetic have suggested that N-succinyl-5- aminoimidazole-4- gene PAICS as an anti-apoptotic oncogene [18]. Aerobic carboxamide-1-ribose-5’-phosphate (SAICAR), a product glycolysis, also known as the Warburg effect, is a well- of the enzyme PAICS, is involved in activating pyruvate known metabolic aberration occurring during oncogenesis kinase PKM2 under glucose-depleting condition [24]. [19,
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