The Differentiation and Neoplasia Section studies inductive signaling in tissue development/morphogenesis and, in parallel, its dysregulation in tumorigenesis with emphasis on the ligands that mediate normal tissue interactions and the pathways and targets that are activated in response to signaling. Our focus has been on development of the urogenital tract, which features reciprocal interactions between two distinct mesodermal progenitors, highly coordinated tissue movements, mesenchymal-epithelial transition, integration of structures from different lineages, reiterative cycles of development, and a tumor that caricatures nephrogenesis. More specifically we are interested in the signaling mechanisms that direct metanephric mesenchyme (MM) to convert to the epithelia of the nephron. Wilms tumor (WT) is characterized by an expanded blastemal/progenitor population with a restricted capacity for epithelial conversion. It is our long-term goal to identify targets on which WT cells depend for survival or dysregulated signaling that can be reprogrammed to allow cells to differentiate to a more benign phenotype. We and others have implicated the cytokine leukemia inhibitory factor (LIF) in the induction of nephronic epithelia from MM. LIF stimulates tyrosine and serine phosphorylation of STAT3 and STAT1 in MM progenitors. Despite the enormous literature on STAT signaling, the vast majority of studies have focused on its role in immune function or the inflammatory process. The involvement of STATs during development, with the exception of studies in Drosophila or ES cells, has largely gone unexplored. In efforts to better understand the role of these factors in differentiation, we have examined the expression of each STAT family member and, where possible, their phosphorylation during nephrogenesis. STATs 1, 3, 5, and 6 are all highly expressed throughout metanephric development and in the adult kidney, and STATs 1, 3, and 5 are phosphorylated only during development. These findings have led us to investigate the biological function of these molecules using conditional loss-of-function (LOF) mouse models. Although non-renal phenotypes have been reported for individual Stat knockouts, the ability of a single cytokine to activate more than one STAT family member is well established and suggests that functional redundancy is likely. A preliminary assessment of a LOF mutant for Stat3 has revealed extensive defects in the skeletal system but no obvious alterations in the kidney other than size differences. We are therefore generating conditional double mutants for Stats 1 and 3 and Stats 1 and 5 in order to circumvent the complications of redundancy. WNT4 has been shown to mediate mesenchymal-epithelial transition (MET) in MM in vivo, and LIF or GSK3 inhibitors similarly induce MET in explant culture. In efforts to understand the signaling mechanism(s) responsible for this signature event in nephron formation,we have examined the ability of each of the three molecules to activate calcium-dependent NFAT signaling, having demonstrated that the MET process occurs independently of beta-catenin/canonical Wnt signaling. We have now determined that NFAT is activated by each inductive factor and that constitutive activation of NFAT is sufficient to induce epithelial markers in MM. Moreover, inhibition of NFAT signaling blocks the expression of these markers. These studies were facilitated by 1. development of cell culture conditions that promote the expansion of nephrogenic progenitors for biochemical analyses without compromising their capacity to form tubules and 2. the design, synthesis, and analysis of a potent peptide inhibitor for beta-catenin that targets canonical Wnt signaling. This inhibitor was approved for patenting by NCI. Having implicated LIF signaling in MET of MM, we also asked whether STAT activation occurs in WTs. Constitutive phosphorylation of a STAT family member has been observed in many types of solid and hematologic cancers. An evaluation of WTs, revealed that most contain phosphorylated S727 in STAT1. When tumor cells expressed a mutant form (S727A) of STAT1, which cannot be phosphorylated, they lost their ability for anchorage-independent growth. Furthermore, downregulation of STAT1 induced apoptosis in these cells. Microarray analysis identified Mcl1, Hsp27, and Cux1 as downstream targets of STAT1 signaling, and these same genes were found to be highly expressed in WTs. Consistent with these observations, we reported that activation of STAT1 in normal MM with interferon-gamma (IFN-gamma) stimulated cell proliferation, inhibited tubule formation, and also induced the expression of MCL1 and CUX1, factors that promote cell survival and proliferation. Studies of WTs have been hampered by the absence of good animal models or tumor cell lines. We have taken three independent approaches to address this problem. Firstly, we have generated a mouse line carrying an activatable Stat1-S727E transgene that should mimic the phosphorylated form of the molecule. Additionally,we have conditionally expressed an Ifn-gamma transgene in mouse MM in order to constitutively activate STAT1 and confirm its role in WT pathogenesis. A similar approach yielded medulloblastomas in neural tissues. Secondly, we are mapping a genetic locus linked to nephroblastoma susceptibility/resistance in offspring of susceptible Nb strain rats versus resistant F344 strain rats, since inheritance was transmitted as an autosomal incomplete dominant trait. Thirdly, we are developing new WT cell lines for biochemical and molecular studies using conditions optimized for the culture of MM. These conditions promote the outgrowth and passage of cells from histologically distinct Wilms tumors and are currently examining their ability to form tumors in soft agar and in xenotransplantation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005093-33
Application #
8348866
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2011
Total Cost
$1,246,888
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Yun, Kangsun; Perantoni, Alan O (2017) Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection. Differentiation 94:1-7
Hall, Michael D; Murray, Caroline A; Valdez, Michael J et al. (2017) Mesoderm-specific Stat3 deletion affects expression of Sox9 yielding Sox9-dependent phenotypes. PLoS Genet 13:e1006610
Tanigawa, Shunsuke; Taguchi, Atsuhiro; Sharma, Nirmala et al. (2016) Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells. Cell Rep :
Tanigawa, Shunsuke; Perantoni, Alan O (2016) Modeling renal progenitors - defining the niche. Differentiation 91:152-8
Tanigawa, Shunsuke; Sharma, Nirmala; Hall, Michael D et al. (2015) Preferential Propagation of Competent SIX2+ Nephronic Progenitors by LIF/ROCKi Treatment of the Metanephric Mesenchyme. Stem Cell Reports 5:435-47
Plisov, Sergey; Wang, Honghe; Tarasova, Nadya et al. (2014) Protein/peptide transduction in metanephric explant culture. Methods Mol Biol 1092:255-67
Yun, Kangsun; Ajima, Rieko; Sharma, Nirmala et al. (2014) Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension. Hum Mol Genet 23:6807-14
Murphy, Andrew J; Pierce, Janene; de Caestecker, Christian et al. (2014) CITED1 confers stemness to Wilms tumor and enhances tumorigenic responses when enriched in the nucleus. Oncotarget 5:386-402
Murphy, Andrew J; Pierce, Janene; de Caestecker, Christian et al. (2014) Aberrant activation, nuclear localization, and phosphorylation of Yes-associated protein-1 in the embryonic kidney and Wilms tumor. Pediatr Blood Cancer 61:198-205
Kanda, Shoichiro; Tanigawa, Shunsuke; Ohmori, Tomoko et al. (2014) Sall1 maintains nephron progenitors and nascent nephrons by acting as both an activator and a repressor. J Am Soc Nephrol 25:2584-95

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