Abstract 1126: Gene expression profiling reveals that WT1 and WTX control cell growth through similar gene networks but different specific genes

The WTX Wilms tumor suppressor is present on the X chromosome, and hence a single WTX suppression may be sufficient to promote carcinogenesis in a subset of Wilms tumors. Thought WTX mutations were only occasionally found in other tumors, the study of this protein may give new insights into growth suppressive pathways. Unlike the WT1 tumor suppressor gene, WTX lacks known functional protein domains. Previous biochemical studies suggested that WTX inhibits WNT signaling by degrading beta-catenin and similarly, WT1 inhibits WNT signaling. The study of WTX function was hampered by the fact that transient expression of WTX but not WT1 caused profound cell death. Hence we constructed inducible cell lines expressing WTX and tumor associated WTX mutants in HEK293 cells. Similar to our results with WT1, induction of WTX resulted in a 40% increase in G1/G0 cells and a concomitant decrease of cells in S phase. A short, tumor-associated truncation mutant of WTX (Arg358ter) led to a more modest 15-20% increase in G1 while a longer truncation mutant (Leu561ter) led to cell cycle arrest to a similar extent as wild type WTX. Wild-type WTX decreased cell growth by nearly 50% while Arg358ter led to only a 7% decrease in growth and Leu561 led to a 22% decrease. WT1 and WTX both slow growth and cause cell cycle arrest, but gene expression profiling showed that only a few specific target genes were regulated in common by the proteins. Nevertheless, the two tumor suppressors converged on similar pathways as Ingenuity Pathway Analysis showed that gene networks implicated in the cell cycle, cell death, cancer, cellular growth, and cardiovascular system development were both affected by both WT1 and WTX. Gene networks involved in gastrointestinal disease, nucleic acid metabolism, and reproductive system disease were uniquely enriched in WTX targets, while lipid metabolism, molecular transport, endocrine system disorders, cellular assembly and organization, and gene expression functions were enriched among WT1 target genes. When gene expression changes mediated by wild-type WTX and the Leu561Ter were compared, there was a 50% overlap of WTX up-regulated genes and 30% overlap in down-regulated genes. By contrast, the Arg358Ter regulated few genes in common with wild-type WTX, implying that a domain containing amino acid residues from 358 and 561 may be critical for tumor suppressor function of WTX. Collectively these data indicate that although both associated with tumor development, Arg 358 is a near complete loss of function mutant while Arg 561 is a hypomorphic form of WTX. WTX regulates similar sets of genes as WT1 to suppress cell growth through cell cycle arrest and induction of apoptosis. Whether patients harboring the shorter truncation mutations of WTX have a distinct phenotype remains to be determined. Further study will be necessary to determine how these genes are directly or indirectly regulated by WTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1126.