A Murine Model of K-RAS and β-Catenin Induced Renal Tumors Expresses High Levels of E2F1 and Resembles Human Wilms Tumor

Abstract

Purpose: Wilms tumor is the most common renal neoplasm of childhood. We previously found that restricted activation of the WNT/β-catenin pathway in renal epithelium late in kidney development is sufficient to induce small primitive neoplasms with features of epithelial Wilms tumor. Metastatic disease progression required simultaneous addition of an activating mutation of the oncogene K-RAS. We sought to define the molecular pathways activated in this process and their relationship to human renal malignancies.

Materials and methods: Affymetrix® expression microarray data from murine kidneys with activation of K-ras and/or Ctnnb1 (β-catenin) restricted to renal epithelium were analyzed and compared to publicly available expression data on normal and neoplastic human renal tissue. Target genes were verified by immunoblot and immunohistochemistry.

Results: Mouse kidney tumors with activation of K-ras and Ctnnb1, and human renal malignancies had similar mRNA expression signatures and were associated with activation of networks centered on β-catenin and TP53. Up-regulation of WNT/β-catenin targets (MYC, Survivin, FOXA2, Axin2 and Cyclin D1) was confirmed by immunoblot. K-RAS/β-catenin murine kidney tumors were more similar to human Wilms tumor than to other renal malignancies and demonstrated activation of a TP53 dependent network of genes, including the transcription factor E2F1. Up-regulation of E2F1 was confirmed in murine and human Wilms tumor samples.

Conclusions: Simultaneous activation of K-RAS and β-catenin in embryonic renal epithelium leads to neoplasms similar to human Wilms tumor and associated with activation of TP53 and up-regulation of E2F1. Further studies are warranted to evaluate the role of TP53 and E2F1 in human Wilms tumor.

Keywords: Wilms tumor; animal; beta catenin; kidney; models; oncogenes.

Figure 1. Gene expression from tumor bearing murine Kras^G12D/Catnb^Δex3 kidneys is significantly different than that of Kras^G12D, Catnb^Δex3, or control kidneys

The microarray gene-expression data were analyzed among the four biological groups using Principal component analysis (A) and unsupervised hierarchical clustering analysis (B) with the open-source R scripts, version 2.10.1 (www.r-project.org). The Kras^G12D/Catnb^Δex3 kidneys are indicated by the red circle for principal component analysis and are at the far right two columns in the hierarchical clustering analysis. In both analyses they are significantly different than the other three genotypes, which are similar to each other.

Figure 2. Canonical Wnt/β-catenin target genes are up-regulated in Kras^G12D/Catnb^Δex3 kidneys

Immunoblot demonstrating higher levels of the Wnt targets FoxA2, survivin, Axin2, and c-Myc in Kras^G12D/Catnb^Δex3 kidneys compared to the other three groups.

Figure 3. The gene signature that distinguishes murine Kras^G12D/Catnb^Δex3 kidneys and human renal malignancies from normal/control tissues are centered on CTNNB1 and TP53

Using the comparative analysis tool EXALT the gene expression of murine Kras^G12D/Catnb^Δex3 kidneys and human renal malignancies were shown to be similar to each other and significantly different from normal tissues/controls. The concordant gene signature that defined this difference was generated and Ingenuity pathway analysis (IPA) of this signature shows gene networks that are centered on CTNNB1 (A) and TP53 (B).

Figure 4. The gene signatures that distinguish murine Kras^G12D/Catnb^Δex3 kidneys and human WT from other renal malignancies or normal/control tissues are centered on TP53

Using the comparative analysis tool EXALT the gene expression of murine Kras^G12D/Catnb^Δex3 and human WT were found to be similar to each other and significantly different from normal tissues/controls. The concordant gene signature defining this association was generated and IPA of this signature shows a gene network centered TP53 (A). The same approach showed the gene expression of murine Kras^G12D/Catnb^Δex3 kidneys and human WT were more similar to each other that other renal neoplasms. IPA of the corresponding concordant gene signature again showed a gene network centered TP53 (B).

Figure 5. E2F1 is up regulated in murine Kras^G12D/Catnb^Δex3 kidneys

Mice were bred and sacrificed at 15–20 weeks of age. Kidneys were snap frozen and RNA extracted and RT-PCR performed for E2F1 (A). Results are shown compared to kidneys from control mice and analyzed across groups using the 2^−ΔΔC_T method with * indicating p value <0.05. Whole protein lysates were extracted and tested by immunoblot (B) and FFPE kidneys were tested for E2F1 expression by IHC (C–F).

Figure 6. E2F1 is expressed at high levels in the majority of human WT’s

IHC was performed for E2F1 on a TMA of 32 human WT’s. Strong nuclear staining was seen in 30/32 (94%) WT’s and seen in blastemal elements (A, low power, B high power), stromal elements (* in B), and epithelial elements (C, low power, D high power).