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Supplemental Figure 1. Validation of some metabolic genes by semi-quantitative PCR.

Supplemental Table 2. All 98 GO biological process ontologies that are over-represented in the differentially expressed (DE) genes.

Supplemental Figure 2. Protein expression of NDUFA4L2, SLC16A3 and HK2 in clear cell proximal tubules.

Supplemental Table 1. Summary of number of reads for each sample.

Supplementary Table S1: shRNA sequences Supplementary Table S2: List of antibodies Supplementary Table S3: List of primer sequences Supplementary Figure S1: ChIP-seq analysis and hierarchical cluster analysis Supplementary Figure S2. Caki1, 786-O, and HK2 proliferation and Caki1 colony formation assays Supplementary Figure S3. Metabolomics analysis of RCC4 cells Supplementary Figure S4. TEM analysis of Caki1 cells Supplementary Figure S5. Western blot analysis of LC3 in Caki1, 786-O, and HK2 cells

Purpose: We delineated the functions of the hypoxia-inducible factor-1α (HIF1α) target NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) in clear cell renal cell carcinoma (ccRCC) and characterized NDUFA4L2 as a novel molecular target for ccRCC treatment.Experimental Design: We evaluated normal kidney and ccRCC patient microarray and RNAseq data from Oncomine and The Cancer Genome Atlas for NDUFA4L2 mRNA levels and the clinical implications of high NDUFA4L2 expression. In addition, we examined normal kidney and ccRCC patient tissue samples, human ccRCC cell lines, and murine models of ccRCC for NDUFA4L2 mRNA and protein expression. Utilizing short hairpin RNA, we performed NDUFA4L2 knockdown experiments and analyzed the proliferation, clonogenicity, metabolite levels, cell structure, and autophagy in ccRCC cell lines in culture.Results: We found that NDUFA4L2 mRNA and protein are highly expressed in ccRCC samples but undetectable in normal kidney tissue samples, and that NDUFA4L2 mRNA expression correlates with tumor stage and lower overall survival. In addition, we demonstrated that NDUFA4L2 is an HIF1α target in ccRCC and that NDUFA4L2 knockdown has a profound antiproliferative effect, alters metabolic pathways, and causes major stress in cultured RCC cells.Conclusions: Collectively, our data show that NDUFA4L2 is a novel molecular target for ccRCC treatment. Clin Cancer Res; 22(11); 2791–801. ©2016 AACR.

PDF file - 63K, Supplemental tables for GGT-HIF2αM3 transcript quantification

Renal cell carcinoma (RCC) is the most common primary cancer arising from the kidney in adults, with clear cell renal cell carcinoma (ccRCC) representing approximately 75% of all RCCs. Increased expression of the hypoxia-induced factors-1α (HIF1α) and HIF2α has been suggested as a pivotal step in ccRCC carcinogenesis, but this has not been thoroughly tested. Here, we report that expression of a constitutively activated form of HIF2α (P405A, P530A, and N851A, named as HIF2αM3) in the proximal tubules of mice is not sufficient to promote ccRCC by itself, nor does it enhance HIF1αM3 oncogenesis when coexpressed with constitutively active HIF1αM3. Neoplastic transformation in kidneys was not detected at up to 33 months of age, nor was increased expression of Ki67 (MKI67), γH2AX (H2AFX), or CD70 observed. Furthermore, the genome-wide transcriptome of the transgenic kidneys does not resemble human ccRCC. We conclude that a constitutively active HIF2α is not sufficient to cause neoplastic transformation of proximal tubules, arguing against the idea that HIF2α activation is critical for ccRCC tumorigenesis. Cancer Res; 73(9); 2916–25. ©2013 AACR.

PDF file - 75K, GGT-HIF2αM3 transcript quantification

Purpose: We delineated the functions of the hypoxia-inducible factor-1α (HIF1α) target NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) in clear cell renal cell carcinoma (ccRCC) and characterized NDUFA4L2 as a novel molecular target for ccRCC treatment. Experimental Design: We evaluated normal kidney and ccRCC patient microarray and RNAseq data from Oncomine and The Cancer Genome Atlas for NDUFA4L2 mRNA levels and the clinical implications of high NDUFA4L2 expression. In addition, we examined normal kidney and ccRCC patient tissue samples, human ccRCC cell lines, and murine models of ccRCC for NDUFA4L2 mRNA and protein expression. Utilizing short hairpin RNA, we performed NDUFA4L2 knockdown experiments and analyzed the proliferation, clonogenicity, metabolite levels, cell structure, and autophagy in ccRCC cell lines in culture. Results: We found that NDUFA4L2 mRNA and protein are highly expressed in ccRCC samples but undetectable in normal kidney tissue samples, and that NDUFA4L2 mRNA expression correlates with tumor stage and lower overall survival. In addition, we demonstrated that NDUFA4L2 is an HIF1α target in ccRCC and that NDUFA4L2 knockdown has a profound antiproliferative effect, alters metabolic pathways, and causes major stress in cultured RCC cells. Conclusions: Collectively, our data show that NDUFA4L2 is a novel molecular target for ccRCC treatment. Clin Cancer Res; 22(11); 2791–801. ©2016 AACR.

Renal cancers are highly aggressive and clinically challenging, but a transgenic mouse model to promote pathologic studies and therapeutic advances has yet to be established. Here, we report the generation of a transgenic mouse model of von Hippel–Lindau (VHL) renal cancer termed the TRACK model (transgenic model of cancer of the kidney). TRACK mice specifically express a mutated, constitutively active HIF1α in kidney proximal tubule (PT) cells. Kidney histologies displayed by TRACK mice are highly similar to histologies seen in patients with VHL disease, including areas of distorted tubular structure, cells with clear cytoplasm and increased glycogen and lipid deposition, multiple renal cysts, and early onset of clear cell renal cell carcinoma (ccRCC). Distorted tubules in TRACK mice exhibit higher levels of CA-IX, Glut1, and VEGF than tubules in nontransgenic control mice. Furthermore, these tubules exhibit increased numbers of endothelial cells, increased cell proliferation, and increased expression of the human ccRCC marker CD70(TNFSF7). Moreover, PT cells in kidney tubules from TRACK mice exhibit increased genomic instability, as monitored by elevated levels of γH2AX. Our findings establish that activated HIF1α in murine kidney PT cells is sufficient to promote cell proliferation, angiogenesis, genomic instability, and other phenotypic alterations characteristic of human VHL kidney disease, establishing the TRACK mouse as a valid preclinical model of human renal cell carcinoma. Cancer Res; 71(21); 6848–56. ©2011 AACR.

Human papillomavirus (HPV) type 97 was identified and the genome was cloned from cervicovaginal cells of a Costa Rican woman with a normal Pap smear. The HPV97 L1 open reading frame (ORF) was most closely related to HPV45 (84% identity) and HPV18 (79% identity), placing it into the high-risk α7 species. Ectopic expression of the HPV97 E6 and E7 proteins significantly decreased steady state p53 and pRb levels using an in vitro cotransfection assay, respectively. These data suggest that HPV97 shares a most recent common ancestor with HPV18 and HPV45 and should be evaluated in cancer specimens from different geographic populations. © 2007 Wiley-Liss, Inc.

We have cloned, sequenced, and characterized the complete genome of a novel human papillomavirus (HPV), candHPV62. During cloning, 2 candHPV62 viral isolates were recovered from a single cervical sample; 1 had all anticipated HPV open-reading frames (ORFs) intact, whereas the other exhibited an E1 frame-shift mutation. Further experiments indicated that the 2 strains were equivalent in abundance. It appears that an early mutation occurred within the E1 ORF, which was transcomplemented by an intact E1 protein. A search of the HPV database identified disruption of the E1 ORF in the cloned reference isolates of HPV16, HPV53, HPV56, and HPV72. These data suggest that disruption of the E1 ORF in genital HPVs is not uncommon.

Hypoxia inducible factor 1 alpha (HIF1α) is a transcription factor that is frequently stabilized and active in human clear cell renal cell carcinoma (ccRCC). We have found that constitutively active HIF1α is sufficient to cause neoplastic transformation in a murine model of ccRCC termed the TRACK model. RNA sequencing (RNAseq) and untargeted metabolomics analyses of samples from TRACK kidneys demonstrate that HIF1α activates the transcription of genes that cause increased glucose uptake, glycolysis, and lactate production, as well as a decrease in the flux of pyruvate entering the tricarboxylic acid (TCA) cycle and a decrease in oxidative phosphorylation; these changes are identical to those observed in human ccRCC samples. These studies show that a constitutively active HIF1α promotes tumorigenesis in TRACK mice by mediating a metabolic switch to aerobic glycolysis, i.e., the Warburg effect, and suggest that TRACK mice are a valid model to test novel therapies targeting metabolic changes to inhibit human ccRCC.

Renal cell carcinoma (RCC) is the most common cancer arising from the kidney in adults, with clear cell RCC (ccRCC) representing the majority of all RCCs. Expression of a human HIF1α triple-mutant (P402A, P564A, and N803A) construct in the proximal tubule cells of C57BL/6 mice [TRAnsgenic model of Cancer of the Kidney (TRACK); ref. 1] mimics the histologic changes found in early stage human ccRCC. To better understand the genomic landscape, a high-throughput sequence analysis was performed with cDNA libraries (RNAseq) derived from TRACK transgenic positive (TG+) kidney cortex along with human ccRCC transcripts from the Oncomine and The Cancer Genome Atlas databases. Importantly, the expression profiles of TRACK TG+ kidneys show significant similarities with those observed in human ccRCC, including increased expression of genes involved in glycolysis and the tricarboxylic acid cycle. Some of the transcripts overexpressed in both the TRACK mouse model and human ccRCC include ANKRD37, CA9, EGLN3, HK2, NDUFA4L2, and SLC16A3. These data suggest that constitutive activation of HIF1α in kidney proximal tubule cells transcriptionally reprograms the regulation of metabolic pathways in the kidney and that HIF1α is a major contributor to the altered metabolism observed in human ccRCC. Implications: TRACK (GGT-HIF1αM3) kidney mRNA profiles show similarities to human ccRCC transcriptome and phenotypes associated with the Warburg effect. Mol Cancer Res; 13(5); 870–8. ©2015 AACR.

The transcription factor HIF1α is implicated in the development of clear cell renal cell carcinoma (ccRCC). Although HIF1α was initially believed to be essential for ccRCC development, recent studies hypothesize an oncogenic role for HIF2α in ccRCC, but a tumor suppressor role for HIF1α, leading to uncertainty as to the precise roles of the different HIF transcription factors in this disease. Using evidence available from studies with human ccRCC cell lines, mouse xenografts, murine models of ccRCC, and human ccRCC specimens, we evaluate the roles of HIF1α and HIF2α in the pathogenesis of ccRCC. We present a convergence of clinical and mechanistic data supporting an important role for HIF1α in promoting tumorigenesis in a clinically important and large subset of ccRCC. This indicates that current understanding of the exact roles of HIF1α and HIF2α is incomplete and that further research is required to determine the diverse roles of HIF1α and HIF2α in ccRCC.The TRACK mouse ccRCC model with constitutively active HIF1α but not HIF2α expressed in proximal tubules develops RCC. HIF1α protein is expressed in the majority of human ccRCC specimens. Elevated HIF1α in ccRCC correlates with a worse prognosis. Many publications do not support a tumor suppressor role for HIF1α in ccRCC. HIF1α, but not HIF2α, is expressed in some types of cancer stem cells.

493 Background: Clear cell renal cell carcinoma (ccRCC) is characterized by altered metabolism, including a shift from oxidative phosphorylation to glycolysis and lactate production (the Warburg effect). Here we evaluate human ccRCC tissue samples and kidney samples from the TRACK transgenic mouse model of ccRCC (that express constitutively active HIF1α in proximal tubule cells) [1] to characterize the effect of HIF1α on the renal cancer metabolome. Methods: We performed untargeted metabolomics analyses of 12 primary human ccRCCs and 10 TRACK kidney samples. In addition, we analyzed human ccRCC mRNA expression (Oncomine database) and performed RNA sequencing (RNAseq) on six TRACK kidney samples for expression of HIF1α target genes that regulate metabolism. Results: Analysis of RNA expression of HIF1α target genes in human ccRCCs and TRACK kidneys identified significantly increased transcript levels of genes responsible for mediating glucose uptake and glycolysis and suppressing the activity of the TCA cycle and mitochondrial respiration, including glucose transporter 1 (SLC2A1), glycolytic enzyme phosphofructokinase (PFKP), TCA cycle inhibitor pyruvate dehydrogenase kinase (PDK1), oxidative phosphorylation inhibitor NADH dehydrogenase 1 alpha subcomplex, 4-like 2 (NDUFA4L2), and lactate transporter (SLC16A3). Metabolomics analysis similarly demonstrated a significant increase in glycolytic intermediates and lactate, in association with a decrease in metabolites of the TCA cycle. Conclusions: These studies show that in both human ccRCC and TRACK mice HIF1α mediates a metabolic switch to aerobic glycolysis, implicating HIF1α in RCC tumorigenesis. Additionally, these data suggest that targeting the metabolic pathway (or inhibition of HIF1α) is a novel approach to treat ccRCCs.

Papillomaviruses are a heterogeneous group of DNA viruses with closed circular double-stranded DNA genomes of about 8 kb in size that contain three general regions. An upstream regulatory region (URR) contains sequences that control transcription and replication, an early region contains genes (e.g., E6, E7, E1, E2, E4, and E5) involved primarily in enzymatic activities, and a structural region produces the L1 capsid protein and L2, which facilitates packaging of the viral DNA. Human papillomaviruses (HPVs) are classified by the sequence similarity of their genomes. A cloned HPV genome whose L1 open reading frame (ORF) displays less than 90% similarity to previously designated types is defined as a novel type. To date, more than 90 different genotypes of the HPV have been fully characterized (21). Intratypic variants and subtypes are defined as HPVs that vary by less than 10% in their L1 DNA sequences (5, 21). HPVs are causally involved in the etiology of cervical cancer and its precursor lesions (16, 17, 43, 49). Of the high-risk HPV types associated with cervical cancer, HPV16 is the most prevalent and is found in approximately half of all cancers (7, 49). Numerous variants of HPV16 have been identified in different geographic locations and ethnic groups (35, 42, 53, 68). Although all HPV16 isolates are closely related, previous studies inferred five distinct phylogenetic branches among HPV16 variants: European (E), Asian (As), Asian-American (As-Am), African-1 (Af-1), and African-2 (Af-2), corresponding to the geographic locations from which the samples were obtained (12, 34). Subsequent studies by sequence analyses of theHPV16 variants in other genomic regions (e.g., E6, L2, and L1) expanded and complemented this phylogenetic hypothesis (69). Although HPV16 variants are an important focus of phylogenetic studies and the molecular variants of E2, L2, L1, the URR, and especially the E6 region have been described in detail previously (28), covariation among different ORFs belonging to the same lineage or isolate have not been studied in great detail. HPV16 variants have demonstrable differences in biological properties in vitro which may be responsible, in part, for differences in pathogenicity, carcinogenic risk, and perhaps immunogenicity (28). Furthermore, HPV16 variants are associated with different cervical cancer risks (6, 32, 65, 67). Although the diversifying selection in the HPV16 E6 and E7 oncogenes has been described recently (20), the evolutionary basis of the entire genome, coevolutionary mechanisms among different HPV16 genes, and their underlying biological significance remain unknown. Obtaining whole-genome sequences representative of the major HPV16 variants allowed us to determine with certainty nucleotide and amino acid sequence changes that are of potential evolutionary importance. Comparison of synonymous (silent; dS) and nonsynonymous (amino acid-changing; dN) substitution rates in protein-coding genes provides an important means for investigating the forces of molecular evolution (72). The nonsynonymous/synonymous rate ratio (ω = dN /dS) measures selective pressure at the protein level. Nonsynonymous rates that are significantly higher than synonymous rates are accepted as evidence for molecular adaptation (71, 72). This criterion has been used to identify a number of genes under adaptive molecular evolution (45). In this report, the complete nucleotide sequences of and codon variations within HPV16 genomes representing the five major lineages were determined. By examining the ratio of dN to dS substitutions per site, diversifying selection acting on all of the eight protein-coding regions was evaluated. In addition, complete genome sequences for these variants allow us to reconstruct their genealogical relationships with unprecedented precision.

The distributions of human papillomavirus (HPV) types detected in cervical adenocarcinomas and squamous cell tumors differ. However, whether the distributions of intratypic HPV variants seen in these two histological forms of cervical disease differ is unknown. Our objective was to compare the distribution of HPV intratypic variants observed in squamous cell carcinomas (SCC) and cervical tumors of glandular origin (e.g., adenocarcinomas; AC) for two HPV types commonly observed in cervical tumors, HPV16 and HPV18. Participants in a multicenter case-control study of AC and SCC conducted in the eastern United States were studied. A total of 85 HPV16 and/or HPV18 positive individuals (31 diagnosed with AC, 43 diagnosed with SCC, and 11 population controls) were included. For HPV16-positive individuals, both the noncoding long control region and the E6 open reading frame were sequenced, and classified into phylogenetic-based lineage groups (European, Asian-American, African1, and African2). For HPV18-positive individuals, the long control region region only was sequenced and classified into known intratypic lineages (European, Asian-Amerindian, and African). The distribution of these different intratypic lineages among AC cases, SCC cases, and population controls was compared using standard methods. Non-European HPV16 and/or HPV18 intratypic variants were observed in 42% of ACs compared with 16% of SCCs and 18% of population controls (P = 0.04). Intratypic variants from the Asian-American lineage of HPV16 accounted for the differences seen between histological groups. The differences observed between AC and SCC cases were strongest for HPV16, and persisted in analysis restricted to Caucasian women, suggesting that the effect cannot be explained by differences in the ethnic make-up of AC versus SCC cases. Cervical AC and SCC differ not only with respect to the distribution of HPV types detected but also with respect to intratypic variants observed. Non-European HPV16 and/or HPV18 variants are commonly seen in AC. A possible hormonal mechanism is suggested to explain the observed findings.

Renal cell carcinoma (RCC) is the most common primary cancer arising from the kidney in adults, with clear cell renal cell carcinoma (ccRCC) representing approximately 75% of all RCCs. Increased expression of the hypoxia-induced factors-1α (HIF1α) and HIF2α has been suggested as a pivotal step in ccRCC carcinogenesis, but this has not been thoroughly tested. Here, we report that expression of a constitutively activated form of HIF2α (P405A, P530A, and N851A, named as HIF2αM3) in the proximal tubules of mice is not sufficient to promote ccRCC by itself, nor does it enhance HIF1αM3 oncogenesis when coexpressed with constitutively active HIF1αM3. Neoplastic transformation in kidneys was not detected at up to 33 months of age, nor was increased expression of Ki67 (MKI67), γH2AX (H2AFX), or CD70 observed. Furthermore, the genome-wide transcriptome of the transgenic kidneys does not resemble human ccRCC. We conclude that a constitutively active HIF2α is not sufficient to cause neoplastic transformation of proximal tubules, arguing against the idea that HIF2α activation is critical for ccRCC tumorigenesis. Cancer Res; 73(9); 2916–25. ©2013 AACR.

Background: VHL disease patients have an increased risk of developing clear cell renal cell carcinomas (ccRCC). Increased expression of HIF1α and/or HIF2α has been proposed as a key step in ccRCC carcinogenesis. We recently reported a mouse model of VHL renal cancer, the TRACK model (Fu et al, Cancer research. 2011;71(21):6848-56.). In the TRACK model, proximal tubule (PT) cell overexpression of a triple mutant (P402A, P564A and N803A) HIF1α in the mouse mimics human VHL kidney disease. Methods: Here we report a mouse model expressing a triple mutant (P405A, P531A and N847A) HIF2α in PT cells. Mutant HIF2α was expressed in both WT and HIF1α-expressing C57BL/6 mice. Results: Transgenic (TG) mice expressing this HIF2α construct specifically in PT cells exhibit vacuolization consistent with glycogen accumulation and hydropic degeneration. The activation of HIF2α was confirmed by increased expression of HIF2α target genes, e.g. CA9. Neoplastic transformation was not observed in the kidneys of HIF2α TG+ mice. In HIF1α/HIF2α-expressing mice, disorganized PT cells mimicking in situ carcinoma was observed in 5 mo old mouse kidneys, which was seen only at 14 mo in mice expressing only mutant HIF1α. Conclusions: These data suggest HIF2α accelerates carcinogenesis in the mutant HIF1α TRACK model. These mouse models provide excellent models to study VHL renal disease and carcinogenesis, and provide an opportunity for chemoprevention studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-486. doi:1538-7445.AM2012-LB-486

Introduction: Renal cell carcinoma (RCC) is the most common primary kidney cancer, with clear cell carcinoma (ccRCC) representing ∼75% of all RCCs. In patients with Von Hippel-Lindau (VHL) disease, inactivation of the VHL tumor suppressor gene results in increased expression of HIF1α, the development of numerous premalignant lesions as well as multifocal ccRCC. Previously, no mouse model that simulates human ccRCC has been established. We report the first transgenic mouse model that closely resembles the development of ccRCC. Methods: Three mutations (P402A, P564A and N803A) were introduced into human HIF1α cDNA by site-directed mutagenesis. Gamma-glutamyl transpeptidase (GGT or γ-GT) is specifically expressed in the proximal tubules of the kidney starting 3 weeks after birth. GGT promoter, triple mutant HIF1α ORF and a beta globin poly A were cloned to create the γ-HIF1α-M3 plasmid. A linearized fragment with the vector sequence removed was injected into the pronuclei of one-cell mouse embryos to create γ-HIF1α-M3 transgenic mice. Results: Five of 51 founder mice harbored the integrated target gene by Southern Blot analysis (1 lacked Germline transmission). Expression of the HIF1α transgene mRNA was confirmed by RT-PCR. Triple mutant HIF1α was expressed in the kidneys of transgenic positive (TG+) mice (lines 8, 25, 32 and 43) and not other organs tested (spleen, liver, heart, lung, intestine, skeletal muscle). TG+ mice and transgenic negative (TG-) littermates were sacrificed at 6-7 months. Histological review revealed clusters of “clear” cells with distorted, abnormal shapes in the outer cortex of TG+ kidneys. These “clear” cells contained large amounts of glycogen and lipid in their cytoplasm, as shown by Periodic Acid/Schiff stain and Oil Red O stain, respectively. Immunohistochemical analysis of HIF1α regulated genes revealed greatly increased expression of CA-IX, Glut-1, and VEGF in these “clear” cells, as well as an increased number of blood vessels (angiogenesis) as determined by Cd-31b. Although there was no clear evidence of neoplasia in 6-7 month old mice, pre-malignant “clear” cells exhibited increased proliferation (PCNA expression) and increased DNA double strand breaks (γH2AX expression). Analysis of the kidneys of 1.5 year-old TG+ mice revealed multiple renal cysts and disorganized proximal tubules that display an intratubular proliferation of atypical clear cells, suggesting a pre-invasive or in-situ tumor. Conclusions: Expression of mutated, constitutively active HIF1α protein in mouse kidney proximal tubule cells results in a transgenic phenotype similar to that observed in patients with VHL disease, including the development of premalignant lesions, multiple renal cysts and early ccRCC. This Transgenic Model of Cancer of the Kidney (TRACK) provides a model to study the genetic and molecular events that result in the development of ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2857. doi:10.1158/1538-7445.AM2011-2857