Published OnlineFirst January 18, 2016; DOI: 10.1158/1078-0432.CCR-15-1511 Biology of Human Tumors Clinical Cancer Research Role of NADH Dehydrogenase (Ubiquinone) 1 Alpha Subcomplex 4-Like 2 in Clear Cell Renal Cell Carcinoma Denise R. Minton1,2,7, Leiping Fu1, Nigel P. Mongan3, Maria M. Shevchuk4,5, David M. Nanus5,6, and Lorraine J. Gudas1,5 Abstract Purpose: We delineated the functions of the hypoxia-inducible analyzed the proliferation, clonogenicity, metabolite levels, cell factor-1a (HIF1a) target NADH dehydrogenase (ubiquinone) 1 structure, and autophagy in ccRCC cell lines in culture. alpha subcomplex 4-like 2 (NDUFA4L2) in clear cell renal cell Results: We found that NDUFA4L2 mRNA and protein are carcinoma (ccRCC) and characterized NDUFA4L2 as a novel highly expressed in ccRCC samples but undetectable in normal molecular target for ccRCC treatment. kidney tissue samples, and that NDUFA4L2 mRNA expression Experimental Design: We evaluated normal kidney and ccRCC correlates with tumor stage and lower overall survival. In addition, patient microarray and RNAseq data from Oncomine and The we demonstrated that NDUFA4L2 is an HIF1a target in ccRCC Cancer Genome Atlas for NDUFA4L2 mRNA levels and the and that NDUFA4L2 knockdown has a profound antiproliferative clinical implications of high NDUFA4L2 expression. In addition, effect, alters metabolic pathways, and causes major stress in we examined normal kidney and ccRCC patient tissue samples, cultured RCC cells. human ccRCC cell lines, and murine models of ccRCC for NDU- Conclusions: Collectively, our data show that NDUFA4L2 is a FA4L2 mRNA and protein expression. Utilizing short hairpin novel molecular target for ccRCC treatment. Clin Cancer Res; 1–11. RNA, we performed NDUFA4L2 knockdown experiments and Ó2016 AACR. Introduction The majority of ccRCCs possess an inactivating mutation in the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to Clear cell renal cell carcinoma (ccRCC) is the most common the stabilization of hypoxia-inducible factors (HIF; refs. 6, 7). form of kidney cancer, representing more than 80% of all diag- HIF1a and HIF2a are transcription factors that influence expres- noses (1). ccRCC is a disease of altered cell metabolism (2), and sion of many genes involved in altered metabolic pathways in many genes known to contribute to the development and/or cancer, including glucose uptake, enhanced glycolytic metabo- progression of ccRCC are involved in sensing oxygen, iron, lism, and decreased mitochondrial respiration (8). We previously nutrients, and energy (3). Altered metabolism is now a widely demonstrated that expression of a constitutively active HIF1a accepted hallmark of cancer (4), and metabolic alterations greatly mutant protein exclusively in kidney proximal tubule cells is influence tumor development and progression (5). Thus, target- sufficient to drive a program of early tumorigenesis in a murine ing metabolic abnormalities in ccRCC may provide novel oppor- model of ccRCC called the TRACK (TRAnsgenic model of Cancer tunities for developing more effective treatments (2). of the Kidney) model (9). TRACK mice exhibit increased expres- sion of HIF1a target genes that are associated with a shift in 1Department of Pharmacology,Weill Cornell Medical College (WCMC) metabolism from mitochondrial oxidative phosphorylation to of Cornell University, New York, New York. 2Weill Cornell Graduate increased aerobic glycolysis and lactate production, similar to School of Medical Sciences–Pharmacology Program, WCMC, New what is observed in human ccRCC (10). In addition, our meta- York, New York. 3Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, The University of Nottingham, Sut- bolomics data show that TRACK kidneys and human ccRCC ton Bonington Campus, Loughborough, United Kingdom. 4Depart- samples have increased levels of glycolytic intermediates and ment of Pathology and Laboratory Medicine, WCMC of Cornell Uni- 5 lactate, and decreased levels of metabolites of the tricarboxylic versity, New York, New York. Meyer Cancer Center, WCMC of Cornell fi University, New York, New York. 6Division of Hematology and Medical acid (TCA) cycle (10). Similar to our ndings in the TRACK Oncology of the Department of Medicine,WCMC of Cornell University, kidneys, Arreola and colleagues found that stable expression of New York, New York. 7Department of Pathology, New York University HIF1a in primary kidney epithelial cells increased expression of (NYU) School of Medicine, New York, New York. key glycolytic enzymes and favored pyruvate conversion into Note: Supplementary data for this article are available at Clinical Cancer lactate under nutrient poor conditions (11). In contrast, stable Research Online (http://clincancerres.aacrjournals.org/). expression of HIF2a in primary kidney epithelial cells did not Corresponding Author: Lorraine J. Gudas, Department of Pharmacology, Weill appear to mediate glycolysis or inhibit oxidative phosphorylation Cornell Medical College (WCMC) of Cornell University, 1300 York Avenue, New (11). Indeed, HIF2a had minimal influence on glycolysis and was York, NY 10065. Phone: 212-746-6250; Fax: 212-746-8858; found to support oxidative phosphorylation in a nutrient rich E-mail:[email protected] environment (11). doi: 10.1158/1078-0432.CCR-15-1511 In the current study, we evaluated the levels and role of the gene Ó2016 American Association for Cancer Research. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 18, 2016; DOI: 10.1158/1078-0432.CCR-15-1511 Minton et al. formed to assess HIF1a binding in human umbilical vein endo- Translational Relevance thelial cells in hypoxic and normoxic conditions. In this study, we show that NADH dehydrogenase (ubiqui- none) 1 alpha subcomplex 4-like 2 (NDUFA4L2) is a novel Tissue samples molecular target for clear cell renal cell carcinoma (ccRCC) Human tissue samples were obtained from patients who treatment. We demonstrate that NDUFA4L2 is highly underwent radical or partial nephrectomy at Weill Cornell Med- expressed at the mRNA and protein levels in human ccRCC, ical College (WCMC). The pathologist selected samples of tumor yet is not expressed in normal, healthy kidney tissue. We also and normal kidney tissue to be used for research. The Institutional show that NDUFA4L2 mRNA expression is high across all Review Board at WCMC approved this study. stages of ccRCC compared with normal kidney, with an even Male C57BL/6 wild-type mice were obtained from Jackson greater expression in higher-stage ccRCC. In addition, patients Laboratories and TRACK mice were generated as described pre- with the highest expression of NDUFA4L2 in their tumors viously (9). All procedures involving the use of mice were exhibit a lower overall survival. Knockdown of NDUFA4L2 in approved by the WCMC Institutional Animal Use and Care cell culture models of ccRCC greatly impairs proliferation, Committee. perturbs metabolic pathways, and causes major stress on RCC cells. Cell proliferation and colony formation sssays RCC4 (ATCC) and SKRC48 (Memorial Sloan Kettering Cancer Center, New York, NY; cell lines were not authenticated) cell lines were cultured in DMEM (MP Biomedicals; 1033122) containing 10% FBS (Gibco, Life Technologies; 10439-016), Caki1 cells (NDUFA4L2, Gene ID: 56901). NDUFA4L2 is one of the most (ATCC) were grown in McCoy 5a medium modified (Gibco; highly expressed genes in almost all ccRCC samples compared 16600-082) containing 10% FBS, and 786-O and HK2 cells with healthy kidney tissue, according to several sets of microarray (ATCC) were grown in RPMI1640 medium (Gibco; 11875- data from the Oncomine database (12–15) and RNA sequencing 093) containing 10% FBS. Cells were cultured in a humidified (RNAseq) data from The Cancer Genome Atlas (TCGA; refs. 16, tissue culture incubator at 37 C in a 10% CO2 atmosphere. 17), and NDUFA4L2 mRNA expression is similarly increased by 4 For proliferation assays, cells were plated at 2.5 Â 10 per well >50-fold in TRACK kidneys (10, 16). Hypoxia induces NDU- in multiwell culture plates. Cell numbers were counted in trip- FA4L2 expression via a mechanism involving HIF1a, but not licate using an electron particle counter (Coulter Z; Beckman HIF2a (18). Blocking HIF1a by RNA interference abrogated Coulter) after 1, 3, 5, and 7 days of culture. For colony formation NDUFA4L2 expression in a VHL-negative ccRCC cell line, whereas assays, cells were plated at 500 per 6-well plate. After approxi- blocking HIF2a expression by RNA interference did not affect the mately 14 days, cells were stained with crystal violet. The number expression of NDUFA4L2 (18). Under hypoxic conditions, NDU- of colonies was quantified using ImageJ software (NIH, Bethesda, FA4L2 is involved in lowering mitochondrial oxygen consump- MD). tion and complex I mitochondrial activity, which in turn causes a shift from mitochondrial respiration to anaerobic glycolysis and Knockdown of NDUFA4L2 using shRNA reduces reactive oxygen species production (18). Thus, NDU- We produced short hairpin RNA (shRNA) containing lentiviral FA4L2 plays a role in altering cellular metabolism when HIF1a particles as described previously (23). We used two pLKO shNDU- is stabilized and active within a cell. In the current study, we FA4L2 vectors directed specifically toward human NDUFA4L2 analyzed the functions of NDUFA4L2 in