The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity

Abstract

WTX encodes a tumor suppressor gene inactivated in Wilms tumor and recently implicated in WNT signaling through enhancement of cytoplasmic ␤-catenin (CTNNB1) degradation. Here, we report that WTX translocates to the nucleus, a property that is modified by an endogenous splicing variant and is modulated by a nuclear export inhibitor. WTX is present in distinct subnuclear structures and co-localizes with the paraspeckle marker p54NRB/NONO, suggesting a role in transcriptional regulation. Notably, WTX binds WT1, another Wilms tumor suppressor and stem cell marker that encodes a zinc-finger transcription factor, and enhances WT1-mediated transcription of Amphiregulin, an endogenous target gene. Together, these observations suggest a role for WTX in nuclear pathways implicated in the transcriptional regulation of cellular differentiation programs.cancer biology ͉ subcellular localization ͉ tumor suppression W ilms tumor, the most common pediatric kidney cancer, is a pluripotent tumor that arises from a kidney-specific stem cell population (1). Genetic studies of Wilms tumor have consistently implicated genes that are critical mediators of differentiation, underscoring the link between normal kidney development and tumorigenesis. Loss of function mutations in WT1, a transcription factor with multiple roles in the developing kidney, are present in 5%-10% of cases (2-4), often in association with activating mutations in ␤-catenin (CTNNB1) (5, 6), a key regulator of WNT signaling. Loss of imprinting leading to increased expression of insulin-like growth factor 2 (IGF2) has also been implicated in approximately one-third of cases (7,8). In addition to these well-defined abnormalities, several other loci have been implicated by loss of heterozygosity studies, suggesting significant genetic heterogeneity in this disease (9).We have recently identified an X chromosome gene, WTX, which is targeted by deletions and truncations in up to 30% of Wilms tumors (10). All WTX mutations were somatic, targeting the single X chromosome in males or the active X chromosome in females and thus leading to complete inactivation in both sexes. WTX belongs to a gene family comprised of 3 genes with no significant homology to known functional domains. Biochemical studies of WTX have identified regions that mediate binding to APC and ␤-catenin and an N-terminal domain that mediates localization to the plasma membrane (11,12). Functional studies performed with a WTX ORF (ORF) predicted by automated database annotation (WTX-PRED, 804 aa from a transcript composed of 3 exons) indicated that WTX encodes a component of the ␤-catenin destruction complex that inhibits WNT signaling by increasing the degradation of ␤-catenin in the cytoplasm (12). However, our characterization of the endogenous 7.5-kb WTX mRNA transcript in diverse tissues does not confirm expression of the WTX-PRED third exon, revealing instead a larger 1135-aa ORF (WTX-WT) encoded by a longer second exon with an additional 350 aa in the C terminus compared to WTX-PRED (Fig. 1B) (10).H...

Functional characterization of Wilms tumor-suppressor WTX and tumor-associated mutants

“…In contrast, transient overexpression of WT1 did not cause death of these cells. Immunoblot analysis showed the presence of two protein species in lysates of cells transfected with WTX-FL, WTX565 and WTX830 consistent with a previously demonstrated internal splicing event that deletes amino acids 51-326 of WTX (Jenkins et al, 2009;Rivera et al, 2009) (Figure 1b; Supplementary data 2). Although knockdown of WTX was shown to increase b-catenin levels (Major et al, 2007), overexpression of EGFP-WTX protein did not affect the steady state level of total and active b-catenin in HEK293 and other cell types (Figure 1b; Supplementary data 2).…”

“…Deletion constructs WTX358 and WTX565 were designed based on Wilms tumor-associated truncation mutants (Rivera et al, 2009). WTX overexpression was previously reported to cause profound cell death on transfection.…”

“…Previously, WTX was shown to stimulate WT1-mediated activation of Amphiregulin (Rivera et al, 2009). To gain insight into the growth suppressive properties of WTX, we compared alterations in gene expression modulated by WTX-FL, WTX358, WTX565 and WTX831-1135 with gene expression in HEK293-EGFP cells in the presence of doxycycline, when WTX expression and biological activity in the cell cycle was maximum.…”

“…WTX is likely to have a role during kidney development, as WTX is expressed in an overlapping pattern with WT1 expression during kidney development and modulates WT1 activity acting as a transcriptional cofactor (Rivera et al, 2009). WTX may have other functions as well.…”

Adenoma development in familial adenomatous polyposis and MUTYH‐associated polyposis: somatic landscape and driver genes

“…We did not observe somatic bi‐allelic mutations in adenomas from females, suggesting that Lyonization may be responsible for loss of WTX function. WTX was originally identified as a gene involved in the development of Wilms' tumour of the kidney 28 and has reported roles in the regulation of the WNT pathway, TP53 and cell fate, and in the localization of the tumour suppressor protein WT1 29, 30, 31. Germline truncating mutations in WTX have also been linked to a sclerosing skeletal dysplasia (OSCS; MIM300373) and are not considered to be associated with an increased risk of tumours, although relatively early‐onset colorectal cancer occurred in one of 25 adult patients in the original report 32.…”