Children heterozygous for a germline WT1 mutation are at a greatly elevated risk of developing WT, and tumors from these children have invariably mutated or lost the wild-type allele. In contrast, Wt1+/– mice do not develop tumors. We hypothesized that this was due to the inability of differentiating murine metanephric mesenchyme to accrue necessary mutations during the developmental stage at which it was at risk for tumorigenesis. We further hypothesized that this inability was due in large part to differences in genomic organization between humans and mice — specifically, the synteny of WT1 and IGF2 in humans that is not present in mice. Thus, we needed to engineer into murine developing kidney cells 2 genetic alterations, somatic Wt1 homozygous ablation and Igf2 upregulation, that can result from a single event in humans. The early and frequent occurrence of tumors observed in the engineered Wt1-Igf2 mouse strongly supports our hypotheses and serves as an example of the need to be cognizant of genomic architecture when developing mouse models for human diseases.
mouse also demonstrates that Wt1
ablation and Igf2
upregulation constitute 1 combination of rate-limiting events for tumorigenesis and provides important insights into the cellular effect of these 2 alterations. The role of WT1
ablation vis-a-vis LOH or LOI at 11p15 imprinted genes (sometimes referred to as WT2), a result of which is biallelic expression of IGF2, has been unclear. These 2 events are observed in approximately 20% and 70% of human WT, respectively, which has led to speculation that they represent distinct molecular etiologies for WT. However, as in the Wt1-Igf2
mouse tumors, both WT1
ablation and 11p15 LOH/LOI are present in some human tumors (14
). The Wt1-Igf2
mouse data suggest a model, discussed below, in which upregulation of Igf2
results in increased ERK1/2 phosphorylation, Wt1
ablation results in a block in mesenchyme differentiation, and the combined occurrence of these events leads to malignant tumors.
In addition to 11p15 LOH/LOI, a role for IGF2
in WT tumorigenesis is strongly suggested by the observation that patients with the somatic overgrowth syndrome Beckwith-Wiedemann syndrome who develop WT often carry germline mutations in an IGF2
imprinting control region (43
). IGF2 signals primarily through IGF-IR (39
), and the increased expression of pIRS, a substrate for activated IGF-IR, in mouse tumors confirms that this is also true for the Wt1-Igf2
tumors. However, the IGF-IR signal can be transduced via several alterative signaling pathways, and to our knowledge, the effector signaling pathway key for IGF2’s role in WT has not previously been known. The robust upregulation of pERK1/2 and lack of increased pAKT, pPDK1, pmTOR, and pSTAT3 in tumors indicates that in the Wt1-Igf2
mouse tumors, the IGF2 signal is transduced via the Ras/Raf/MEK/ERK pathway, which was not to our knowledge previously implicated in WT.
We determined that human WTs similarly display little evidence of activation of PDK1, STAT3, or AKT. In contrast, as in the mouse tumors, upregulation of pIRS1 and pERK1/2 was observed in many human WTs, confirming that the signaling pathway identified in the mouse model has indeed identified a pathway important in human tumors. That this pathway is not upregulated in all human tumors is likely in part a consequence of the known genetic heterogeneity of human WTs along with their more complex genetic background.
We detected no mutations in Ctnnb1, Wtx, and p53, genes mutated in 5%–20% of human tumors, in the mouse tumors (data not shown). Similarly, genome-wide comparative genome hybridization (CGH) analysis revealed no duplication and/or loss of large genomic regions (e.g., 16q, 12q, 1p) that are sometimes observed in human tumors (data not shown). Although we cannot rule out the presence of mutations at other, previously unimplicated, loci in the mouse tumors, these data suggest that Wt1 ablation and Igf2 upregulation are the critical genetic events in these tumors.
WT1 was originally identified by virtue of its role as a tumor suppressor gene; roughly 20% of WTs are homozygous for WT1 loss-of-function mutations. Interestingly, and seemingly contradictory to its role as a tumor suppressor gene, in mice, germline ablation of Wt1
leads to apoptosis of urogenital ridge cells at approximately E10.5, resulting in complete kidney agenesis. However, while we observed a slight increase in the number of TUNEL-positive cells in Wt1
-ablated kidney, there was no widespread apoptosis of metanephric mesenchyme following Wt1
ablation at approximately E13. This result indicates that loss of Wt1
function has different effects depending upon developmental stage: before invasion of the intermediate mesenchyme by the ureteric bud, Wt1
is essential for cell survival, but its loss in approximately E13 metanephric mesenchyme, following ureteric bud invasion, has only a weak apoptotic effect. The complete block in kidney development we observed histologically in vivo following genetic Wt1
ablation (which was previously described in vitro following Wt1
knockdown; ref. 24
) is consistent with this notion.
is expressed in the metanephric mesenchyme from which both the nephrons and the kidney stroma arise, its expression is increasingly restricted during nephrogenesis, such that its expression in adult kidney is primarily in podocytes, highly specialized epithelial cells in the glomerulus. Interestingly, and in contrast to a prior observation following in vitro knockdown of Wt1
in approximately E13 kidney (24
), we found no increase in cell proliferation following Wt1
ablation. However, our analysis of Wt1
-ablated E14.5 kidneys revealed that, while little histologic change was apparent compared with control kidneys at that time point, there was already significant alteration in the expression of genes (e.g., Wnt4
, and Lhx1
) upregulated during mesenchymal epithelialization and differentiation to form nephrons. The most statistically significant dysregulated gene, Wnt4
, is expressed in both ureteric bud and metanephric mesenchyme derivatives, and it is critical for the mesenchyme-to-epithelial transition that occurs during nephron development (32
). While the decrease in Wnt4
expression in Wt1
-ablated E14.5 kidneys was less than 2-fold, this change was nevertheless highly statistically significant. The somewhat modest change in Wnt4
expression is likely due to its normal expression in the ureteric bud derivatives; while Wt1
ablation affects Wnt4
expression in the mesenchyme, it cannot do so in ureteric bud derivatives that do not express Wt1
mutant kidneys display a developmental phenotype similar to that of Wt1
-ablated embryonic kidneys (45
), and in vitro studies have suggested that Wt1
indirectly upregulates Wnt4
). Our in vivo data strongly support this.
In contrast to Wnt4
, and Lhx1
, changes in the expression of Pbx1
, expressed in mesenchyme fated to become kidney stroma (26
), was not observed. Interestingly, there was also no change in expression of Six2
, a gene required for the maintenance of mesenchymal tubule progenitor cells (27
). These in vivo histology and gene expression data suggest a model whereby Wt1
ablation has little, if any, effect on stromagenic mesenchyme. Neither does it result in widespread loss of SIX2-positive nephron progenitors. Instead, it blocks — perhaps via loss of Wnt4
expression — the ability of induced mesenchyme to form renal vesicles. Mutant mesenchyme did not express K-cadherin, an early marker of epithelialization, but whether this is a primary consequence of Wt1
ablation or is secondary to the lack of renal vesicle initiation is not clear. The observation of epithelial differentiation in some components of both murine and human WTs implies that Wt1
-ablated, blocked mesenchyme can, in the context of other genetic alterations and/or cellular milieu, undergo epithelial differentiation, albeit aberrantly. Whether Igf2
upregulation plays a role in this regard is not known and requires further investigation.
From which mesenchymal cells did the Wt1-Igf2 tumors arise? The triphasic histology of stroma, blastema (similar to condensed mesenchyme), and epithelial tubules observed in both human and mouse tumors points to an origin in an early mesenchyme progenitor cell that then differentiates aberrantly. In this model, the expression in tumors of genes (Six1, Pax2, and Eya1) normally expressed in the nephrogenic mesenchyme could be a reflection of the impaired differentiation of the nephrogenic elements of the tumor. Alternatively, this nephrogenic gene expression profile could be a reflection of the tumor arising at this developmental stage, with epithelial and stromal components observed in the tumor arising as a result of aberrant differentiation and/or transdifferentiation. Additionally, even though Wt1 ablation did not alter the expression of markers for stromagenic mesenchyme or tubule progenitor cells, it is possible that Wt1 loss is tumorigenic in one or both of these cell types. Substitution of the ubiquitous Cre transgene with kidney compartment–specific Cre transgenes in the Wt1-Igf2 mouse model will be one robust approach to investigate these alternative models of tumorigenesis.
In summary, we have generated an endogenous mouse model for WT by engineering into mice 2 separate genetic alterations that commonly occur in humans as a result of a single genomic event. In doing so, we have defined one important combination of genetic alterations that act as the requisite rate-limiting 2 hits originally hypothesized for WT tumorigenesis (47
). Data from the Wt1-Igf2
mouse tumors and data from fetal kidneys that have sustained these 2 alterations individually suggest a model whereby loss of Wt1
function alters normal differentiation of the induced nephrogenic mesenchyme and Igf2
upregulation drives the proliferation of these abnormal cells through IGF-IR signaling transduced via pIRS1 and pERK1/2. A significant fraction of human WTs also displayed increased pIRS1 and pERK1/2. Thus, guided by the mouse tumors, we have identified a signaling pathway that likely plays an important role in the development of many human WTs. Because the Wt1-Igf2
mice carry the same alterations that occur in human tumors, they provide a highly relevant model for further investigating the underlying biology of tumor development, for understanding the functional significance of other gene mutations previously identified in subsets of human WTs, and for functionally identifying additional genes that have been implicated in WT by linkage, LOH, CGH, and other studies. Furthermore, the IGF pathway has become increasingly recognized as an attractive target for cancer therapy (25
), and the Wt1-Igf2
animals potentially provide a powerful model for assessing the short- and long-term efficacy of anti-IGF pathway molecules for cancer treatment and perhaps also prevention.