Nuclear accumulation of beta-catenin protein indicates activation of wnt signaling in chemically induced rat nephroblastomas

Abstract

Aberrant wnt signaling caused by mutations in CTNNB1 occurs in about 15% of Wilms tumors, and these mutations appear to be dependent on the concomitant mutational inactivation of the zinc-finger protein WT1. Nuclear beta-catenin protein, a substitute marker of active wnt signaling, has been detected in an even higher proportion (>50%) of Wilms tumors, suggesting alternative genetic pathways leading to beta-catenin activation. Thus, targeting wnt signaling may become an important future therapeutic strategy in Wilms tumor patients. Currently, chemically induced rat nephroblastomas provide the only available rodent model for this tumor. To determine the contribution of active wnt signaling in this model, we investigated 24 chemically induced rat nephroblastomas for beta-catenin protein expression and for Ctnnb1 and WT1 mutations. Immunohistochemistry showed focal strong nuclear accumulation of beta-catenin protein in 18 of 24 tumors, although in a heterogenous pattern. Blastemal and mesenchymal compartments displayed nuclear-positive cells more frequently than areas of epithelial differentiation. Interestingly, we found no mutation of exon 3 of Ctnnb1 and no mutation within the zinc-finger region of WT1 in any of the 24 tumors analyzed. In conclusion, our findings suggest activation of wnt signaling in the majority (63%) of chemically induced rat nephroblastomas. Nuclear expression of beta-catenin in the absence of Ctnnb1 mutations implies, however, alternate mutational targets in rat nephroblastomas.

Figure 1. Immunohistological expression of β-catenin in carcinogen induced rat nephroblastomas

Normal rat kidney shows focal membranous expression on the lateral site of the tubular epithelial cells, distal tubules show stronger expression than proximal tubules, no nuclear expression can be found (a), tumor tissue (b) presented on the same slide often shows much more cytosolic β-Catenin expression; epithelial tumor cells without (c) and with nuclear expression of β-Catenin (d); blastemal component of a rat nephroblastoma with strong membranous and cytosolic expression (e), some blastemal components also reveal nuclear β-Catenin expression (f); mesenchymal differentiated cells with (h) and without (g) nuclear β-Catenin expression.

Figure 2. TOPflash/FOPflash ratio

Expression ratio of the N-ethylnitrosourea generated ENU-T1 cell line after transfection with reporter plasmids pTOPflash and pFOPflash to examine the activation grade of wnt-signalling pathway in these cells. HCT116 cell line with activated wnt-signalling shows a nearly nine fold higher TOPflash tah FOPflash activity (TOP/FOP ratio 8,86; σ = 2,46). 293 T cells are presenting themselves with low activated wnt signalling pathway (TOP/FOP ratio 1,79; σ = 0,25). ENU-T1 cells are showing low, but not significant activation (TOP/FOP ratio 1,38; σ = 0,47).