Morphogenesis in the differentiating metanephros is regulated by reciprocal interactions between ureteric bud (UB) epithelia and the metanephric mesenchyme (MM). The UB invades the overlying MM and induces conversion of the mesenchyme into stromal and epithelial elements, which form the nephron. In turn, the MM stimulates the UB to grow and branch, forming the collecting duct system. The Differentiation and Neoplasia Section has focused on the elucidation of mechanisms of inductive signaling in metanephric development, seeking (1) the ligands responsible for nephronic differentiation, (2) other non-inductive regulatory factors of nephrogenesis, (3) the molecular targets of induction, and (4) alterations in inductive signaling during tumorigenesis. Thus far, we have identified three families of secreted soluble growth factors/cytokines produced by the UB that cooperate in inductive signaling to generate nephronic tubules. These include the fibroblast growth factors (notably FGF2 and 9), gp130-binding proteins (leukemia inhibitory factor; LIF), and transforming growth factor-beta (Tgf-beta2, activin A and B, and gdf11). In a recent collaborative effort, we have additionally identified, through gene targeting studies, Fgf8, a factor expressed by MM, as a critical player in nephrogenesis through upregulation of secreted patterning molecule Wnt4 and homeodomain transcription factor Lim1. We now plan to verify the roles of these factors in experiments designed to rescue kidney development through their replacement and overexpression. In the genetic studies, we have also discovered a role for Fgf8 in the development of male reproductive tract accessory tissues. To understand the targets of individual signaling pathways and their cooperative output, we have begun a systematic assessment of activated signaling in the MM. As part of this effort, we have generated an inducible immortalized cell line of MM that, under inductive conditions, differentiates to tubular epithelia and responds to the three described families of inductive factors. Furthermore, microarray analysis has provided a comprehensive characterization of transcriptional targets associated with induction. This line is now also being applied to the elucidation of proteomic changes that mediate or regulate differentiation of the MM progenitor. For this, we are specifically evaluating canonical and noncanonical pathways of Wnt signaling in order to clarify the mechanism invoked in tubule formation.Microarray analysis of a line we derived from the UB has identified high expression levels of multiple CXC chemokines. Primarily studied for their involvement in inflammatory responses, we find that they also function in normal kidney differentiation. CXCL7 is the principal ligand from this family expressed during development of the metanephros prior to induction, while CXCL1 is upregulated in induced MM. Furthermore, inhibition of signaling through their receptor CXCR2 dramatically inhibits development of the metanephros and promotes apoptosis of the MM, suggesting that it plays a critical role in the growth and maintenance of metanephric tissues. Adult tissues in general lack CXCR2, but a variety of human tumors express the receptors, suggesting that they function in tumorigenesis. Indeed, examination of several Wilms tumors reveals the expression of the CXCR2 receptor, and treatment of a Wilms tumor cell line with a CXCR2 inhibitor induces apoptosis in these cells. We are currently acquiring other chemical inhibitors described as more specific as well as evaluating the action of a soluble truncated CXCR2 receptor with possible inhibitory activity for CXCR2 signaling, which we have produced, that may target Wilms tumor cells by sequestering CXCR2 ligands and thus promote apoptosis in blastemal tumor cell populations. The ultimate goal of the DNS is to define subverted signaling pathways in tumorigenesis based upon normal growth and differentiation mechanisms required by tissue progenitors in development and tissue renewal. Accordingly, having identified several pathways responsible for normal cell maintenance and differentiation in the kidney, we have shifted efforts to the Wilms tumor, a caricature of metanephric development characterized by an accumulation of blastemal progenitors. Beginning with proteomic studies of Stat phosphorylation, the mediator of LIF induction, we have identified patterns of tyrosine and serine phosphorylation of Stats 1 and 3 that contribute to tumor behaviors. Thus far, we have evaluated several cell lines, including those from a Wilms tumor and the NCI-60 panel. We also expanded our findings to include 16 primary Wilms tumors and 4 normal tissues from tumor-bearing patients. In these studies, we have observed a consistent pattern of constitutive phosphorylation of Stat1 serine 727, but not tyrosine 701, in tumors - a pattern which does not occur in normal tissues. Furthermore, abrogation of altered Stat signaling, using a mutant form that prevents serine 727 phosphorylation, inhibits tumorigenesis as measured by Wilms tumor cell growth in soft agar and tumor cell invasion, and enhances cell survival under hypoxic conditions, suggesting that the altered patterns contribute to the neoplastic process. We have also implicated protein kinase CK2 in the aberrant phosphorylation and find that inhibition of CK2 also blocks tumorigenic behavior. Remarkably, gene expression profiles determined by microarray analyses of cells transfected with a mutant inactive or constitutively active Stat1 demonstrate regulation of many of the same genes profiled by others in Wilms tumor, suggesting that expression of this molecule is critical to the Wilms tumor phenotype. In addition, these profiles reveal the induction of invasive factors, e.g., fibulin 5 and MMP3, and downregulation of differentiation properties, e.g. K-cadherin and alpha8 integrin, with serine 727 phosphorylation. A test of a peptide inhibitor for Stat1 developed by the SBL proved unsuccessful, so they are currently designing another to target a different portion of the molecule. These studies provide compelling support for the principle that tumorigenesis is a disease of differentiation and that by dissecting inductive signaling, we may indeed gain insight into the mechanisms of tumor formation.
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