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

Abstract

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