In previous studies, we reported the constitutive activation of STAT1 in Wilms tumors. Activation of a STAT family member occurs in a variety of human neoplasms but generally involves Y705 phosphorylation of STAT3. While investigating the possible dysregulation of STATs in Wilms tumors, we discovered the constitutive phosphorylation of S727 in STAT1 and demonstrated its role in Wilms tumorigenesis. Since this neoplasm is a prototype for arrested cellular differentiation in cancer, we hypothesized that STAT1 signaling might also play an important role in the developing kidney. An analysis of early rat metanephric development revealed both tyrosine and serine phosphorylation of STAT1 and STAT3 from the beginning of rudiment formation. For STAT3, pY705 was detected throughout the nephrogenic zone including the cap, which is populated with renal progenitors. Levels were maximal at the initiation of metanephric development (E13.5). For STAT1, pY701 was also observed in the nephrogenic zone but was additionally detected in newly formed nephronic epithelia. Levels were maximal at E15.5 and diminished postnataly with maturation. STAT5 pY694 was localized primarily to epithelial structures but was maximal at E18.5. These results suggest that these transcription factors function at different stages of metanephric development. To better understand the relationship among the various STATs during nephrogenesis, we utilized explant cultures of metanephric mesenchyme (MM), the progenitor of nephronic epithelia and putative origin of Wilms tumor. Interferon-gamma (IFNg), unlike leukemia inhibitory factor (LIF), caused STAT1, but not STAT3, activation in MM. In functional opposition to LIF, which induces tubulogenesis in MM, IFNg stimulated proliferation in MMs and inhibited nephron formation following induction. Furthermore, a peptide inhibitor designed by our NCI-Frederick collaborator Nadya Tarasova to target the STAT1 N-domain specifically disrupted STAT1-dependent transcription and induced tubule formation in explanted MMs. These findings indicate that STAT1 activation provides a critical regulatory signal in MM specification, consistent with its role in Wilms tumorigenesis, and further suggest that disruption of STAT1 activation may provide a novel target for Wilms tumor therapy. We are currently evaluating the role of individual STATs during metanephric development by conditional loss-of-function alleles, targeting the loss to the mesoderm, the metanephric mesenchyme, or the ureteric bud. We are also examining conditional gain-of-function alleles for biological activity. The ectopic expression of interferon-gamma in MM resulted in renal hypoplasia. The kidneys exhibited one of two phenotypes: either a 50% size reduction or complete loss of the kidney. Mutants with reduced kidney size had comparable rates of proliferation and apoptosis with normal kidneys. However, the mutants also display a looped-tail phenotype typical of a defect in the planar cell polarity (PCP) pathway. We are now assessing this possibility in the kidney. Additionally, we have generated constructs with mutant forms of STAT1 that conditionally target S727 activity (floxed stop codons upstream of the mutant Stat form). These include a S727-to-E727 mutation, which is constitutively active, and a S727-to-A727, which is inactive for phosphorylation at this site. Mouse lines have now been produced bearing these constructs. In addition to our work on STATs, we are actively collaborating with CDBL PI Terry Yamaguchi on a characterization of the renal phenotype of Wnt5a conditional loss-of-function mutants. Wnt5a has previously been associated with noncanonical Wnt mechanisms, notably the PCP pathway. It is expressed normally in the ureteric bud and surrounding interstitial stroma. Inactivation of Wnt5a in mesoderm with TCre yields mutants with duplexed kidneys, shortened ureters, and aberrant branching morphogenesis in the metanephros. The presence of hypercellular thickened collecting ducts suggests PCP involvement, so we are exploring this possibility. Another feature of Wilms tumor is the frequent activation of the Wnt pathway, as demonstrated in our 2010 publication on rat nephroblastomas, which show nuclear localization of beta-catenin in most tumors. This pathway is also critical to nephronic development;however, the exact mechanism involved is not clear. Studies have shown an essential role for Wnt/beta-catenin in mesenchymal-epithelial transition (MET) during nephron formation. Intriguingly, analysis of transgenic mice expressing a beta-catenin-transactivated lacZ reporter revealed that the reporter is not expressed in metanephric mesenchyme (MM) or nephronic tubules, raising the possibility that beta-catenin acts through a TCF/LEF-independent mechanism to promote MET and tubule formation. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of beta-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of beta-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of beta-catenins helix C, which is required for transcriptional activation. One inhibitor blocked TCF-dependent transcription and induced degradation of beta-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca2+-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca2+ and caused phosphorylation of CaMKII. Moreover, treatment with Ionomycin, a Ca2+-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney. Furthermore, they demonstrate that canonical Wnt activation can be chemically dissected in cells without interfering with mechanisms of morphogenesis or differentiation. Thus it may be possible to selectively block canonical Wnt activation in tumors that depend upon signaling by this mechanism for survival without affecting their ability to differentiate. We are currently testing the efficacy of these peptides on tumors with constitutively active beta-catenin. Finally, we also continue to investigate the susceptibility of rat to develop nephroblastomas. The Noble rat is exquisitely sensitive to chemical induction of these tumors, while the F344 rat is insensitive. When crossed, animals develop a tumor incidence suggestive of the involvement of a single autosomal locus with incomplete dominance. Since the etiology of Wilms tumor remains largely undefined and since we have ruled out known genetic lesions, it is possible that we are targeting an important novel event in this model. Accordingly, we are currently mapping that locus. Thus far, we have completed the animal portion of the study, generating F1s which were then backcrossed with susceptible Noble rats. The F2s were exposed in utero to a single dose of the direct alkylating agent ethylnitrosourea, which induces a high incidence of tumors in susceptible rats. We have also completed the histology and are currently concluding the SNP analyses for this genome-wide association study.
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