Role of NADH Dehydrogenase (Ubiquinone) 1 alpha subcomplex 4-like 2 in clear cell renal cell carcinoma

PURPOSE We delineated the functions of the HIF1α target NADH Dehydrogenase (Ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) in 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 (TCGA) for NDUFA4L2 mRNA levels and the clinical implications of high NDUFA4L2 expression. Additionally, 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 shRNA, 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. Additionally, we demonstrated that NDUFA4L2 is a HIF1α target in ccRCC and that NDUFA4L2 knockdown has a profound anti-proliferative 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

Degradation of p53 by Human Alphapapillomavirus E6 Proteins Shows a Stronger Correlation with Phylogeny than Oncogenicity

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.These data suggest that the alpha-HPV E6 proteins' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential

Abstract LB-486: Overexpression of a mutated, constitutively active HIF2α promotes the carcinogenesis of TRACK mouse

Abstract 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</jats:p

Supplementary Tables 1 - 2 from Activation of HIF2α in Kidney Proximal Tubule Cells Causes Abnormal Glycogen Deposition but not Tumorigenesis

&lt;p&gt;PDF file - 63K, Supplemental tables for GGT-HIF2αM3 transcript quantification&lt;/p&gt;</jats:p

Diversifying Selection in Human Papillomavirus Type 16 Lineages Based on Complete Genome Analyses

Human papillomavirus type 16 (HPV16) is the primary etiological agent of cervical cancer, the second most common cancer in women worldwide. Complete genomes of 12 isolates representing the major lineages of HPV16 were cloned and sequenced from cervicovaginal cells. The sequence variations within the open reading frames (ORFs) and noncoding regions were identified and compared with the HPV16R reference sequence (50). This whole-genome approach gives us unprecedented precision in detailing sequence-level changes that are under selection on a whole-viral-genome scale. Of 7,908 base pair nucleotide positions, 313 (4.0%) were variable. Within the 2,452 amino acids (aa) comprising 8 ORFs, 243 (9.9%) amino acid positions were variable. In order to investigate the molecular evolution of HPV16 variants, maximum likelihood models of codon substitution were used to identify lineages and amino acid sites under selective pressure. Five codon sites in the E5 (aa 48, 65) and E6 (aa 10, 14, 83) ORFs were demonstrated to be under diversifying selective pressure. The E5 ORF had the overall highest nonsynonymous/synonymous substitution rate (ω) ratio (M3 = 0.7965). The E2 gene had the next-highest ω ratio (M3 = 0.5611); however, no specific codons were under positive selection. These data indicate that the E6 and E5 ORFs are evolving under positive Darwinian selection and have done so in a relatively short time period. Whether response to selective pressure upon the E5 and E6 ORFs contributes to the biological success of HPV16, its specific biological niche, and/or its oncogenic potential remains to be established

Abstract 2857: Expression of mutated, constitutively active HIF1α in the mouse kidney results in early stage clear cell renal cell carcinoma

Abstract 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</jats:p

Supplemental Table 2 from Genome-Wide Profiling of TRACK Kidneys Shows Similarity to the Human ccRCC Transcriptome

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