Furthermore, SIX2 and -catenin have been shown to share gene regulatory networks in the embryonic kidney, providing additional evidence that these two transcriptional regulators tightly coordinate the mesenchymal-to-epithelial transition of the CM . WNT pathway signaling, and gene expression of specific WNT pathway participants were evaluated. Relative to controls, WiT49 cells overexpressing SIX2 showed significantly enhanced anchorage-independent growth and early-passage proliferation representing surrogates of cell survival. Interestingly, overexpression of SIX2 generally repressed TCF/LEF-dependent canonical WNT signaling, which activates and coordinates both differentiation and stem pathways, but significantly heightened canonical WNT signaling through the survivin promoter, a mechanism that exclusively maintains the stem state. In summary, when overexpressed in Sch-42495 racemate a human WT cell line, SIX2 enhances cell survival and appears to shift the balance in WNT/-catenin signaling away from a differentiation path and toward a stem cell survival path. Introduction Wilms tumor (WT), the most common childhood kidney cancer, retains gene expression profiles and histologic elements characteristic of the embryonic kidney and so is classified among embryonal tumors , , . Typically, WTs show a triphasic pattern of cellular features, comprised principally of 1 1) blastema, its putative cancer stem cell and the malignant analogue of nephron progenitors, 2) epithelia, which appears more differentiated as primitive tubules and glomeruli but lacks physiologic function and tissue architecture, and 3) stroma, which consists mostly of connective tissue fibroblasts but can show smooth or skeletal muscle, and even cartilaginous, differentiation , . A predominant pattern of blastema, particularly if persisting after neoadjuvant therapy, represents a histologic marker of treatment resistance and has been shown to portend a worse prognosis , . The 2013 Childrens Oncology Group blueprint for renal tumors therefore challenges investigators to identify the mechanisms that maintain blastema interminably and confer treatment resistance to WT as targets for more efficacious drugs, answers to which likely rest in the mysteries of cancer stem cell self-renewal and evasion of standard therapies . (cells are spawned, a state of interminable self-perpetuation is created. CBP/-cateninCdependent symmetric cell division, which sets up a perpetual loop, has been proposed as a self-maintenance mechanism of the cancer stem cell . How SIX2 and -catenin interact in the highly regulated coordination of CM asymmetric cell division is incompletely understood; moreover, how this balance shifts to symmetric cell division in the cancer stem cell remains more elusive yet represents a candidate Sch-42495 racemate target of new therapies , , . To coordinate the critical balance between maintaining a sufficient nephron progenitor pool while simultaneously spawning committed epithelial cells within the CM of the murine embryonic kidney, SIX2 and -catenin have been shown to share regulatory gene networks, and a tight interplay has been observed between SIX2 and Wnt9b , . Curiously, both SIX2 and -catenin are broadly activated in WT, which represents an ideal paradigm to study self-renewal of an embryonal cancer stem cell and its potential for epithelial conversion, given the typical appearance of WT blastema immediately adjacent to or surrounding a variety of epithelial structures?, Ngfr , . Although much has been revealed regarding the function of SIX2 and canonical WNT signaling in the coordinated process of nephron development, insufficient evidence has been uncovered regarding these transcriptional regulators in blastema self-renewal and maintenance of the Sch-42495 racemate WT cancer stem cell. Analogous to its functional significance in the CM, we hypothesized therefore that exuberant SIX2 expression confers a survival mechanism to WT and preferentially drives -catenin toward the CBP-dependent arm of the canonical WNT pathway to maintain the stem state. These studies were designed to test SIX2 as a survival mechanism and a modifier of the canonical WNT pathway in the WT context. Methods SIX2 Cellular Distribution in WT WT clinical specimens To evaluate SIX2 as a marker of the putative WT stem cell, or blastema, we conducted a comprehensive immunohistochemical (IHC) analysis of its cellular distribution in relation to neural cell adhesion molecule (NCAM, a cell surface marker of the WT stem cell) , . Because SIX2 provides self-renewal to the CM, we also examined SIX2 as a marker of proliferation by evaluating its association with proliferating cell nuclear antigen (PCNA, a marker of proliferating cells) expression across a large sampling of WT specimens. Briefly, using formalin-fixed paraffin-embedded renal tumor and adjacent kidney specimens collected prospectively and archived in our Institutional Review Board (IRB)-approved laboratory embryonal tumor repository, we created three tissue microarrays (TMA) comprised of 223 total punches (~?1 mm in diameter each). Two hundred and fourteen of these punches were derived from 43 consecutive childhood renal tumors (41 WTs, 1 clear.