Oncogene (2007) 26, 3423–3430 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Isoforms of Wilms’ tumor suppressor gene (WT1) have distinct effects on mammary epithelial cells EA Burwell, GP McCarty, LA Simpson, KA Thompson and DM Loeb Division of Pediatric Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA The role of WT1 (Wilm’s tumor suppressor gene) in breast tumors, and they observed a correlation between loss of cancer is controversial, with evidence for both tumor- WT1 expression and expression of the estrogen receptor promoting andtumor-suppressing activities. In orderto (ER). They, therefore, concluded that WT1 plays a address this question, we expressed different WT1 tumor suppressor role in breast cancer. Consistent with isoforms in the mammary epithelial cell line H16N-2, this was a report by Zhang et al. (2003) that WT1 which does not express endogenous WT1. Cells were suppresses the clonal growth of MDA-MB-231 cells in stably transfectedwith either WT1 ( ÀEx5/ÀKTS) or soft agar. In contrast, our group reported overexpres- WT1 ( þ Ex5/ þ KTS) under the control of the inducible sion of WT1 in breast cancer (Loeb et al., 2001). Using metallothionein promoter. Induction of WT1 (ÀEx5/ reverse transcription–polymerase chain reaction (RT– ÀKTS) upregulatedp21, causing a slowing of prolifera- PCR), we were only able to detect WT1 expression in tion anda G2-phase cell cycle arrest. In artificial one of 20 samples of mammary epithelium, but we basement membrane, the WT1 (ÀEx5/ÀKTS) isoform detected WT1 expression in 27 of 31 primary breast promotedthe appearance of highly organizedacinar carcinoma specimens. Subsequently, Miyoshi et al. cellular aggregates. In contrast, WT1 ( þ Ex5/ þ KTS) (2002) reported a correlation between increased WT1 hadno effect on p21 or proliferation, but rather causedan expression and poor long-term survival in women with epithelial–mesenchymal transition anda redistribution of breast cancer, and Zapata-Benavides et al. (2002) E-cadherin from the cell membrane to the cytoplasm. This reported a correlation between WT1 expression levels isoform also causes the cellular aggregates growing in and the proliferative rates of breast cancer cell lines. artificial basement membrane to appear significantly less Taken together, these data suggest that WT1 might organizedthan control cells. Thus, different WT1 iso- function as a tumor-promoting gene in breast cancer. forms have distinct effects in this cell line, suggesting that Complex transcriptional and translational regulation depending on the ratio of WT1 isoform expression in of the WT1 gene results in the expression of several mammary epithelial cells, WT1 couldfunction to either protein isoforms. The most abundant of these isoforms promote or suppress a transformedphenotype. result from two independent alternative splicing events Oncogene (2007) 26, 3423–3430. doi:10.1038/sj.onc.1210127; (Haber et al., 1991). One of these alternative splices published online 11 December 2006 includes or excludes exon 5 from the mature mRNA, resulting in proteins that contain or lack a 17 amino acid Keywords: breast cancer; oncogene; epithelial–mesenchymal segment in the N-terminal transcriptional regulatory transition;cellcycle;alternativesplicing domain. The other alternative splice involves the use of one of the two distinct splice acceptor sites at the N-terminus of exon 10, resulting in the inclusion of three additional amino acids (KTS) in the larger protein Introduction isoform, altering the spacing of the zinc-fingers that compose the DNA-binding domain. The presence or The WT1 (Wilm’s tumor suppressor gene) gene encodes absence of this so-called KTS insert has profound a zinc finger transcription factor that has been functional consequences for the mature protein, affect- implicated in the pathogenesis of breast cancer. Silber- ing both DNA-binding and subcellular localization, but stein et al. (1997) reported that WT1 is expressed in the functional importance of exon 5 remains unclear mammary epithelial cells, with higher levels of expres- (Larsson et al., 1995; Davies et al., 1998; Natoli et al., sion in myoepithelium than in luminal epithelium. They 2002; Reynolds et al., 2003). also reported a loss of WT1 expression in most breast Variable expression of WT1 isoforms in breast tumors has been reported (Silberstein et al., 1997; Loeb et al., Correspondence: Dr DM Loeb, Division of Pediatric Oncology, 2001). We, therefore, hypothesized that different WT1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins isoforms might have different effects on mammary University, Bunting-Blaustein Cancer Research Building, 1650 Orleans epithelial cells, thus accounting for the conflicting St., Baltimore, MD 21231, USA. E-mail: [email protected] reports of the role of this gene in the development of Received 16 December 2005; revised 13 September 2006; accepted 4 October breast cancer. To test this hypothesis, we expressed two 2006; published online 11 December 2006 distinct WT1 isoforms in the H16N-2 cell line. This Effects of WT1 isoforms on breast epithelial cells EA Burwell et al 3424 immortalized, but not transformed, mammary epithelial cell line does not express endogenous WT1 (Loeb et al., 2001). Because they are the most structurally divergent isoforms, we chose to investigate the role of the WT1 isoform containing both exon 5 and the KTS insert (designated WT1 ( þ Ex5/ þ KTS)) and the isoform lacking both these alternatively spliced sequences (designated WT1 (ÀEx5/ÀKTS)). We found that WT1 (ÀEx5/ÀKTS) inhibited proliferation and promoted an Figure 1 WT1 expression in stably transfected cell lines. Cells were treated overnight with ( þ ) or without (À) 100 mM ZnCl2 to acinar growth pattern in cells surrounded by extra- induce WT1 expression, and cell lysates were subjected to Western cellular matrix, characteristics of a tumor suppressor blotting with anti-WT1.(a) Cell lines expressing WT1 (ÀEx5/ gene, whereas WT1 ( þ Ex5/ þ KTS) caused a morpho- ÀKTS) and (b) Cell lines expressing WT1 ( þ Ex5/ þ KTS). Equal logical transition from an epithelial to a more mesench- protein loading was confirmed by Western blotting with antibody ymal phenotype, accompanied by alterations in the against tubulin. expression of E-cadherin and vimentin, consistent with a tumor-promoting function. Gene expression profiling hypothesis, we performed proliferation assays using cell revealed that these WT1 isoforms induced distinct gene lines stably transfected with the WT1 isoforms under the expression patterns consistent with the observed pheno- control of the metallothionein promoter. Cells were typic differences. These data support the hypothesis plated with or without ZnCl2 to induce WT1 expression, that different WT1 isoforms have distinct effects on and relative cell number was determined after 48 h. mammary epithelial cells. Induction of WT1 (ÀEx5/ÀKTS) resulted in a signifi- cant slowing of proliferation, induction of WT1 ( þ Ex5/ þ KTS) had no effect, and the addition of ZnCl2 to Results control cells did not affect their proliferation (Figure 2a). Cell cycle analysis revealed that WT1 Establishment of stable transfectants (ÀEx5/ÀKTS) causes an accumulation of cells in the H16N-2 cells were transfected with one of four different G2 phase of the cell cycle, but the addition of ZnCl2 to plasmids, and stable transfectants were isolated by the other cell lines had no effect on cell cycle distribution selection with G418. The plasmids designated (Figure 2b). Thus, WT1 (ÀEx5/ÀKTS) slows the pCB6WT(À/À) and pCB6WT( þ / þ ) direct the expres- proliferation of H16N-2 cells through induction of a sion of WT1 isoforms ((ÀEx5/ÀKTS) and ( þ Ex5/ G2-cell cycle arrest, whereas WT1 ( þ Ex5/ þ KTS) does þ KTS), respectively) under the control of the consti- not affect proliferation. tutively active cytomegalovirus (CMV) immediate-early WT1 (ÀEx5/ÀKTS) upregulates p21 expression promoter. Plasmids designated pMTWT(À/À) and in Saos2 cells (Englert et al., 1997), but not in 32D cl3 pMTWT( þ / þ ) direct the expression of the same cells (Loeb et al., 2002). Because p21 causes a cell cycle WT1 isoforms under the control of the zinc-inducible arrest and is a WT1 target gene in some cell lines, metallothionein promoter. Although cell lines expres- we investigated whether upregulation of p21 accompa- sing WT1 (ÀEx5/ÀKTS) under the control of the nied the WT1 (ÀEx5/ÀKTS)-induced cell cycle arrest metallothionein promoter were readily obtained, only in H16N-2 cells. As determined by western blot- one stably transfected clone that constitutively expressed ting, induction of WT1 (ÀEx5/ÀKTS), but not WT1 this isoform was obtained, despite numerous attempts. ( þ Ex5/ þ KTS), upregulated p21 expression In contrast, WT1 ( þ Ex5/ þ KTS) was easily expressed (Figure 2c). These data strongly suggest that WT1 both constitutively and in an inducible form. Cell lines (ÀEx5/ÀKTS) induces a G2-phase cell cycle arrest with inducible expression of WT1 (ÀEx5/ÀKTS) were through upregulation of p21. designated H16NMTWTA1, A2, and A3 and cell lines with inducible expression of WT1 ( þ Ex5/ þ KTS) were Effect of WT1 isoforms on morphology designated H16NMTWTD1, D2 and D3. Control cell It was difficult to generate clones constitutively expres- lines were designated H16NMT1, H16NMT2 and sing WT1 (ÀEx5/ÀKTS), and we also noticed that the H16NMT3. Western blotting was performed to verify cells expressing WT1 ( þ Ex5/ þ KTS) had a different expression of transfected WT1 proteins (Figure 1). morphology from control cells and from cells expressing Because there was only one clone constitutively expres- WT1 (ÀEx5/ÀKTS). Control cells grow with an sing WT1 ( Ex5/ KTS), further work was limited to À À epithelial morphology, tending to cluster together and the inducible expression system. Data obtained with form a regular, cobblestone pattern (Figure 3a). Cells H16NMTWTA1 and H16NMTWTD1 cells are shown, stably transfected with inducible WT1 (ÀEx5/ÀKTS) but all results were confirmed in multiple subclones. grow in a similar pattern (Figure 3b).