7490

WT1

Wilms tumor 1;WT1;Wilms tumor 1

Wilms tumor protein|amino-terminal domain of EWS|last three zinc fingers of the DNA-binding domain of WT1

11p13

protein-coding

PURPOSE: Although metastasis is the primary determinant of poor survival of patients with osteogenic sarcoma, some patients live much longer than others, indicating metastatic heterogeneity underlying survival outcome. The purpose of the investigation was to identify genes underlying survival outcome of patients with osteogenic sarcoma metastasis. EXPERIMENTAL DESIGN: We have used microarray to first compare mRNA expression between normal bone and osteogenic sarcoma specimens, identified genes overexpressed in osteogenic sarcoma, and compared expression of the selected gene between a poorly metastatic (SAOS) and two highly metastatic cell lines (LM8 and 143B). Finally, expression of the selected gene was assessed by immunostaining of osteogenic sarcoma samples with known survival outcome. RESULTS: Microarray analysis revealed 5.3-fold more expression of WT1 mRNA in osteogenic sarcoma compared with normal bone and >2-fold overexpression in 143B and LM8 cells compared with SAOS. Furthermore, WT1 mRNA was absent in normal bone (10 of 10) by reverse transcription-PCR but present in osteogenic sarcoma-derived cell lines (5 of 8). One hundred percent (42 of 42) of low-grade osteogenic sarcoma specimens expressed no WT1 as determined by immunostaining; however, 24% (12 of 49) of the high-grade specimens showed intense staining. Mean survival of patients with high-grade metastatic osteogenic sarcoma but low WT1 staining (27 of 37) was 96.5 +/- 129.3 months, whereas mean survival of patients with high-grade metastatic osteogenic sarcoma having intense staining (10 of 37) was 18.3 +/- 12.3 months (P > 0.0143). All splice variants of WT1 mRNA, including a hitherto unknown variant (lacking exons 4 and 5), were found to be expressed in osteogenic sarcoma. CONCLUSION: WT1 seems to be associated with very poor survival of patients with osteogenic sarcoma metastasis.

BACKGROUND: Although metastatic disease is the primary cause of death from epithelial ovarian carcinoma, to the authors knowledge the cellular mechanisms that regulate intraperitoneal metastasis are largely unknown. Metastasizing ovarian carcinoma cells encounter a collagen-rich microenvironment because the submesothelial matrix is comprised mainly of interstitial collagens Types I and III. METHODS: Immunohistochemistry using primary and metastatic ovarian carcinoma samples was employed to detect expression of Wilms tumor gene protein 1 (WT1). Three-dimensional (3D) collagen culture, real-time reverse transcriptase-polymerase chain reaction, and immunofluorescent staining were used to evaluate changes in WT1 RNA and protein expression in response to 3D collagen culture. Boyden chamber invasion assay, scratch-wound motility assay, and Western blot analysis were used to establish the function of WT1 in ovarian carcinoma cells. RESULTS: To model intraperitoneal invasion in vitro, ovarian cancer cells were cultured in a 3D collagen microenvironment. 3D collagen culture resulted in robust induction of WT1 at the mRNA and protein levels. WT1 expression was prevalent in primary ovarian tumors and was retained in paired peritoneal metastases. Functional studies supported a role for WT1 in intraperitoneal invasion, because siRNA knockdown of WT1 expression reduced the ability of ovarian cancer cells to invade 3D collagen gels. CONCLUSIONS: The data from the current study identify a novel regulatory mechanism for the control of WT1 expression and provide evidence for a functional role of WT1 protein in the control of cellular invasive activity.

BACKGROUND: One key step in the development of prostate cancer (PCa) metastasis is the loss of E-cadherin expression associated with increased cellular motility and tumor invasion. This loss of E-cadherin expression is also required during normal embryogenesis and similar transcriptional repressors have been identified in both processes. We have previously reported the presence of one such transcription factor, WT1 in high Gleason grade prostate tumor tissues, and its absence in non-neoplastic or benign prostatic hyperplasia tissues. RESULTS: To better understand the effect of WT1 on E-cadherin expression and migration of PCa cells we quantified WT1 and E-cadherin mRNA levels in normal prostate epithelial and PCa cell lines with varying migratory potential. In WT1 transfected cells E-cadherin transcript levels were decreased, while they were increased in siWT1-RNA transfected PCa cells, suggesting that elevated WT1 expression was sufficient to dampen E-cadherin levels and potentially enhance migratory ability. To delineate the mechanism of WT1-mediated repression of E-cadherin, potential WT1 binding sites were tested in vitro and in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells was assessed by Chromatin Immunoprecipitation. The effect of WT1 binding was measured in reporter assays; in PC3 and DU145 cells WT1 decreased the activity of the proximal E-cadherin promoter. Using site-directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for this repression by WT1. Transwell migration and wound healing assays revealed that in LNCaP cells with low migratory potential, over-expression of WT1 was sufficient to enhance migration, conversely, in the highly migratory PC3 cells silencing of WT1 decreased migration. CONCLUSIONS: These findings suggested that WT1 expression in high grade prostate cancer may contribute to migration and metastasis. Thus, in prostate cancer WT1 may function as a novel oncogene facilitating development of the lethal metastatic phenotype.