ZIA BC 005093 (ZIA) | |||
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Title | Growth-Differentiation Factors in Organogenesis | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Perantoni, Alan | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $748,932 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Chemotherapy (5.0%) Childhood Cancers (50.0%) |
Kidney Cancer (90.0%) Kidney Disease (90.0%) Urinary System (90.0%) Wilm's Tumor (45.0%) |
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Research Type | |||
Endogenous Factors in the Origin and Cause of Cancer Development and Characterization of Model Systems |
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Abstract | |||
The Renal 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 (MET), 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 (MET). 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 tumor cells to differentiate to a more benign phenotype. We identified several growth factors and small molecule pathway agonists that preserve and propagate long-term the nephronic progenitor. We have determined that the cytokine leukemia inhibitory factor (LIF) in combination with Rho kinase inhibitor (ROCKi) maintains and selectively expands the Six2+ nephronic stem cell population in culture. Moreover, these propagated stem cells retain their capacity to convert to all segments of the nephron, demonstrating that they are multipotent progenitors. LIF principally acts through the JAK/STAT pathway and up regulates the expression of several renal stem cell markers, e.g., Six2 and Pax2. Mechanistically, LIF stimulates JNK activation, which induces MM proliferation and enhances cell competence to differentiate. The Rho kinase inhibitor (ROCKi) attenuates the LIF-induced JNK activation thus inhibiting the differentiation of the progenitor. An investigation into the mechanism(s) mediated by LIF/ROCKi in these cells revealed that our conditions facilitate the nuclear localization of Yes-associated protein (YAP), a transcriptional co-activator and component of the Hippo signaling pathway. Furthermore, silencing Yap gene expression by siRNA knockdown in MM cells decreased the expression of progenitor markers and increased levels of MET markers, suggesting that YAP maintains MM cells in an undifferentiated state. Our conditions have also proven successful in promoting the growth of mouse and human Six2+ nephronic progenitors. However, this required further culture optimization with a Wnt agonist and Bmp family member in addition to LIF/ROCKi. In this case, Six2+ cells could be maintained for several passages and induced to form all segments of the nephronic epithelia. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation and raises the possibility that they may be applied to models of tissue repair/regeneration. We are now collaborating with another lab that studies renal damage in mice in efforts to determine if our cultured progenitors can facilitate kidney repair following injury. We have now also applied progenitor culture conditions to the propagation of human Wilms tumor (WT) cells from several different patients and have determined that these same factors selectively expand the Six2+ progenitor from tumor tissues. These cells also retain the expression of several other ""stemness"" and WT-associated genes, such as NCAM and PAX2. These cells are stable for several passages and retain a tumorigenic phenotype based upon their ability for anchorage-independent growth. Cultured cells from these tumors have now been xenografted into NSG mice to further assess tumorigenic behavior. Notably, WT cells readily form organoids when placed in nonadherent culture dishes. |