Published OnlineFirst January 12, 2016; DOI: 10.1158/0008-5472.CAN-15-1859 Cancer Tumor and Stem Cell Biology Research Formation of Renal Cysts and Tumors in Vhl/Trp53-Deficient Mice Requires HIF1a and HIF2a Desir ee Schonenberger€ 1, Sabine Harlander1,2, Michal Rajski1, Robert A. Jacobs1,2,3, Anne-Kristine Lundby1,2, Mojca Adlesic1, Tomas Hejhal1, Peter J. Wild4, Carsten Lundby1,2, and Ian J. Frew1,2 Abstract The von Hippel–Lindau (VHL) tumor suppressor gene is inac- chondrial abundance and oxidative capacity, glycogen accu- tivated in the majority of clear cell renal cell carcinomas (ccRCC), mulation, and acquisition of a clear cell phenotype in Vhl- but genetic ablation of Vhl alone in mouse models is insufficient deficient renal epithelial cells. HIF1a, but not HIF2a, induced to recapitulate human tumorigenesis. One function of pVHL is to Warburg-like metabolism characterized by increased glycoly- regulate the stability of the hypoxia-inducible factors (HIF), sis, decreased oxygen consumption, and decreased ATP pro- which become constitutively activated in the absence of pVHL. duction in mouse embryonic fibroblasts, providing insights In established ccRCC, HIF1a has been implicated as a renal tumor into the cellular changes potentially occurring in Vhl mutant suppressor, whereas HIF2a is considered an oncoprotein. In this renal cells before ccRCC formation. Importantly, deletion of study, we investigated the contributions of HIF1a and HIF2a to either Hif1a or Hif2a completely prevented the formation of ccRCC initiation in the context of Vhl deficiency. We found that renal cysts and tumors in Vhl/Trp53 mutant mice. These find- deleting Vhl plus Hif1a or Hif2a specifically in the renal ings argue that both HIF1a and HIF2a exert protumorigenic epithelium did not induce tumor formation. However, HIF1a functions during the earliest stages of cyst and tumor forma- and HIF2a differentially regulated cell proliferation, mito- tion in the kidney. Cancer Res; 76(7); 1–12. Ó2016 AACR. Introduction autochthonous mouse models that fully reproduce all of the characteristic morphologic and invasive properties of human Clear cell renal cell carcinoma (ccRCC) is the most frequent ccRCC; however, consistent with the hypothesis that multiple renal malignancy, and up to 92% of ccRCC tumors harbor cooperating mutations are required for ccRCC development, biallelic inactivation of the von Hippel–Lindau (VHL) tumor kidney epithelial cell–specific codeletion of Vhl with Pten (6), or suppressor gene (1). VHL mutations occur at the earliest stage with Kif3a to genetically ablate primary cilia (7), caused the of tumor formation (2). The absence of pVHL function is clearly formation of simple and atypical cystic lesions that are similar necessary for the growth of fully transformed ccRCC cell lines as to the ccRCC precursor cystic lesions found in the kidneys of xenografts (3). However, the fact that the frequency of VHL patients with inherited VHL disease. Deletion of Vhl together with mutant single epithelial cells vastly outweighs the number of homozygous loss of Trp53 (8) or with heterozygous loss of Bap1 ccRCC tumors in kidneys of familial VHL disease patients (4) and (9) gave rise to very similar phenotypes, including simple and the absence of tumors in a variety of renal epithelial cell–specific atypical cystic lesions as well as tumors containing cells that Vhl knockout mice (reviewed in ref. 5) argues that ccRCC forma- display cytoplasmic clearing and elevated mTORC1 activity, reca- tion requires mutations in addition to VHL. There are currently no pitulating many of the cellular and molecular changes that are characteristic of human ccRCC. pVHL controls many biological activities, including regulation 1Institute of Physiology, University of Zurich, Zurich, Switzerland. 2Zurich Center for Integrative Human Physiology, University of Zurich, of the stability of the hypoxia-inducible transcription factors a Zurich, Switzerland. 3Health and Physical Education, School of Teach- (HIF1a, HIF2a, and HIF3a, collectively HIFa; ref. 10), regulation ing and Learning, Western Carolina University, Cullowhee, North Car- of NF-kB activity (11), maintenance of the primary cilium (12), olina. 4Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland. activation of p53 (13), secretion of extracellular matrix compo- nents (14), promotion of DNA double-strand repair (15), regu- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). lation of the plane of cellular division (16, 17), and suppression of aneuploidy (16, 18). The combined loss of several or all of pVHL's D. Schonenberger,€ S. Harlander, and M. Rajski contributed equally to this article. functions could contribute to tumor initiation and progression. Corresponding Author: Ian J. Frew, University of Zurich, Winterthurerstrasse Loss of function mutations of VHL occur in ccRCC mutually 190, CH-8057 Zurich, Switzerland. Phone: 41-44-635-5004; Fax: 41-44-635- exclusively to rarer mutations in TCEB1, encoding Elongin C, 6814; E-mail: [email protected] and collectively these cause constitutive stabilization of HIFa in doi: 10.1158/0008-5472.CAN-15-1859 up to 95% of ccRCC tumors (1), implying that HIFa activation is a Ó2016 American Association for Cancer Research. major oncogenic driving force in ccRCC. HIF1a and HIF2a appear www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 12, 2016; DOI: 10.1158/0008-5472.CAN-15-1859 Schonenberger€ et al. to play opposite roles in determining the aggressiveness of estab- mice. Littermate mice that lacked the Cre transgene served as wild- lished ccRCCs; HIF1a inhibits, whereas HIF2a promotes tumor type controls. formation in ccRCC xenografts (19–21), copy loss of the chro- mosomal locus harboring the HIF1A gene predicts poor patient Immunohistochemistry a a outcome (22), and sporadic ccRCCs expressing HIF1 and HIF2 Immunohistochemistry and immunofluorescence of formalin- exhibit lower proliferation rates than ccRCCs expressing only fixed paraffin-embedded tissues were performed as previously a a HIF2 (23). ccRCC cell lines frequently express only HIF2 and described (6, 8) using antibodies listed in Supplementary Materi- a do not express functional HIF1 due to biallelic alterations of the als and Methods. HIF1A locus (24). Knockdown of HIF1A in ccRCC cell lines that express both HIF1a and HIF2a promotes xenograft tumor for- Respirometry mation (24). These studies argue that whereas HIF2a activity is Biopsies (1 mm3) of renal cortex and medulla were assayed for tumor promoting, there is a selection against HIF1a expression or oxygen consumption following the addition of different respira- activity during the progression of some cases of ccRCC. On the tory substrates and inhibitors using high-resolution respirometry other hand, because HIF1a is strongly expressed in single and (Oxygraph-2k, OROBOROS INSTRUMENTS Corp) according to multicellular clusters of VHL-null cells and in cystic lesions in VHL the protocol described in Supplementary Materials and Methods. patients (4), and because transgenic overexpression of HIF1a in mouse proximal tubular epithelial cells causes a clear cell appear- ance, increased proliferation, and a disorganized tubular mor- Microarrays phology (25), and because many human ccRCCs express HIF1a Primary adult mouse renal epithelial cells were cultured for 3 and HIF1a target genes (26), it may also be argued that HIF1a days and infected with Adeno-GFP (Vector Biolabs, 1060) or plays an important role in promoting ccRCC development. These Adeno-Cre (Vector Biolabs, 1700) as described (8). mRNA studies have largely focused on fully transformed, genetically was isolated 4 days after adenovirus infection, and Cy3- and complex ccRCC cell lines derived from advanced tumors, and it Cy5-labeled cDNA from GFP and Cre samples was competitively remains unclear if and how HIF1a and HIF2a contribute to the hybridized to Mouse GE 4 Â 44 K v2 Microarrays (Agilent). Dye earliest stages of initiation of ccRCC. swap experiments were used to control potential dye-specific An early event following VHL mutation is likely to be a hybridization effects. Analysis using R/Bioconductor package profound alteration of cellular metabolic pathways. Numerous limma was based on the average fold expression level changes HIF1a and/or HIF2a-dependent metabolic changes occur in (Cre/GFP) of three independent biologic experiments. Microarray ccRCC cell lines, including elevated glucose uptake and conver- data are accessible through GEO number GSE75745. sion to lactate with a concomitant reduction in mitochondrial oxidation of pyruvate, reduced mitochondrial biogenesis, Histological stains reduced mitochondrial complex I activity, altered cytochrome c Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) oxidase activity, increased pentose phosphate pathway flux, stains were conducted using standard protocols. For the visual- decreased oxidative glutaminolysis, and increased lipogenesis ization of lipids, frozen kidney sections were fixed with 10% through reductive glutamine metabolism (27–38). Because formalin for 15 minutes, washed in tap water, equilibrated in 60% ccRCC cell lines have complex genetic backgrounds that arose isopropanol, and stained with Oil Red O (3 mg/mL in 60% during tumor evolution, it is possible that some of these meta- isopropanol) overnight at 4C. After