Oncogene (2002) 21, 1009 ± 1016 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Expression of the PAX2 oncogene in human breast cancer and its role in progesterone-dependent mammary growth Gary B Silberstein*,1, Gregory R Dressler2 and Katharine Van Horn1 1Department of Molecular, Cell and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California, CA 95064, USA; 2Department of Pathology, University of Michigan, Ann Arbor, Michigan, MI 48109, USA In this study, we ®rst describe expression of the paired known to regulate several mammotrophic growth factor domain transcription factor PAX2 in the normal and pathways, including insulin-like growth factor, epider- cancerous human breast, then demonstrate in a murine mal growth factor, and transforming growth factor beta model a novel function for PAX2 in the regulation of (Dey et al., 1994; Drummond et al., 1992; Vicanek et al., progesterone stimulation of secondary ductal growth. In 1997; Werner et al., 1993). WT1 is also expressed in the human mammary tissue, PAX2 expression was coin- kidney, where it is positively regulated by the paired- cident with sub-populations of mammary ductal cells, domain transcription factor PAX2 and is required for some of which possessed an undierentiated histiotype, the metanephric mesenchyme to respond to signals and was also found in 450% of the human breast inducing cell proliferation (Dehbi et al., 1996; Kreidberg tumors surveyed (n=38). In the mouse, mammary et al., 1993). During embryonic kidney development parenchyma with a targeted deletion of PAX2 developed PAX2 expression is transient whereas persistent expres- normal ductal systems when grafted into wild-type host sion of the gene in Wilms' kidney tumors (Dressler and mammary fat pads, but failed to undergo higher order Douglass, 1992), renal cell carcinoma (Gnarra and side-branching and lobular development in response to Dressler, 1995), and polycystic kidneys (Ostrom et al., progesterone. A previously unsuspected PAX2/WT1 2000) suggests multiple roles in maintaining the dedier- (Wilms' tumor suppressor gene) regulatory axis in the entiated or proliferative state and preventing apoptosis mammary gland was also indicated. Using RT ± PCR, a of the renal epithelium. Deregulated expression of PAX2 signi®cant reduction in WT1 mRNA expression was can transform cells in vitro and promote tumor detected in the PAX2 mutant glands compared to wild- formation in nude mice, indicating a direct role for type counterparts and double-antibody immunohisto- PAX2 in tumorigenesis (Maulbecker and Gruss, 1993; chemistry detected the co-localization of PAX2 and Stuart and Gruss, 1996). PAX2 expression in the WT1 in the nuclei of normal and cancerous breast cells. mammary gland and its potential relationship with These data indicate a role for PAX2 (and possibly WT1) WT1 in that tissue has not been previously investigated, in the regulation of the progesterone response of the in part due to the death of PAX2 mutant mice within the mature mammary gland. The potential contribution of ®rst hours after birth. Here we report the unexpected PAX2 to breast tumor pathogenesis is discussed. expression of PAX2 in normal human breast tissue, as Oncogene (2002) 21, 1009 ± 1016. DOI: 10.1038/sj/onc/ well as in a high percentage of breast tumors. To begin to 1205172 understand a functional role, we have rescued the mammary anlagen from PAX2-null mouse embryos to Keywords: breast cancer; mammary; PAX2; progester- wild-type host mammary glands and now report that one; Wilms' tumor PAX2 is necessary for progesterone-dependent growth of lateral ductal branches. Introduction Results Previously, we demonstrated that the WT1 Wilms' tumor suppressor gene was expressed in normal human Expression of PAX2 in the normal and cancerous human breast tissue as well as in a high percentage of breast breast tumors (Silberstein et al., 1997). The WT1 protein may act as an activator or suppressor of transcription and is The expression of PAX2 in the human breast was investigated by immunohistochemistry and in situ hybridization. Using a polyclonal anti-PAX2 antibody (Dressler and Douglass, 1992), nuclear staining was *Correspondence: GB Silberstein, Sinsheimer Laboratories, detected in cells from the two primary developmental University of California, Santa Cruz, CA 95064, USA; lineages comprising myoepithelial and luminal cells in E-mail: [email protected] Received 11 October 2001; revised 5 November 2001; accepted 7 alveolar (Figure 1a) as well as ductal (Figure 1b) November 2001 elements of the tissue. These cells are arranged in two PAX2 mammary expression and function GB Silberstein et al 1010 Figure 1 PAX2 expression in normal and cancerous human mammary tissue. The nuclei of immuno-positive cells stained brown; counter stain: methyl green. No counter stain was used for the in situ hybridization, (a) Lobule cluster (cross- and grazing sections). PAX2 positive cells with oval as well as polygonal nuclear morphology (arrows within the lumens, L) lined the lobule lumens and were often adjacent to unstained cells. Immuno-positive myoepithelial cells (arrows on periphery of lobules) and immuno-negative counterparts (arrowheads) were also detected (Bar=20 mm). (b) Duct (longitudinal section of one side of a duct; high magni®cation). The arrangement of PAX2 immuno-positive and -negative cells mimicked pattern seen in lobules with strongly staining luminal cells often adjacent to non-staining cells (boxes). Myoepithelial cells (outside dotted line) stained lighter than ductal cells (small arrow) or were negative for PAX2 (arrowhead). (Bar=5 mm). (c) In situ hybridization localization of PAX2 mRNA in normal mammary duct (small arrow) and side-branch (large arrow). Control hybridization with sense-strand probe (inset). (Bar=15 mm). (d) Ductal carcinoma in situ. PAX2 positive cells (arrows); PAX2 negative cells (arrowheads). (Bar=20 mm). Inset: PAX2 mRNA in situ hybridization. Cells expressing PAX2 mRNA (arrows) surround PAX2 non-expressing cells (arrowheads). (Bar=25 mm). (e) In®ltrating ductal carcinoma. In®ltrating tumor cells had large, anaplastic nuclei (large arrows) or small nuclei with compact chromatin (small arrows); both populations were negative for PAX2 protein. Inset: PAX2 positive cells (arrows) in vicinity of main tumor (Bar=20 mm) Oncogene PAX2 mammary expression and function GB Silberstein et al 1011 circular layers and form a tube-within-a-tube. The Table 1 Survey of PAX2 protein expression in normal and innermost cells line the lumen, forming the ductal or cancerous breast tissue acinar tube proper, and give rise to secretory structures Tissue PAX2 positive a PAX2 negative b PAX2-neg/pos c at pregnancy, while the myoepithelium forms the Tumor 40% (15/38) 47% (18/38) 13% (5/38) epithelial boundary with the stroma and is comprised Vicinal d 20 1 - of contractile cells that facilitate milk extrusion (Silberstein, 2001). aTumors with 80% or more of cells immunopositive for PAX2, b The histiotype of certain PAX2 positive cells with estimated by inspection. PAX2 immunostaining was absent in greater than 90% of the tumor cells. cTumors with an approximately large, oval nuclei and diuse chromatin (Figure 1b) equal mixture of PAX2 negative and positive cells. dTissue that was was consistent with that of putative mammary stem excised from the vicinity of a tumor, contained normal-appearing cells (Smith and Medina, 1988; Stingl et al., 1998; structures and cells that exhibited ductal and lobular PAX2 staining Toker, 1967; Williams and Daniel, 1983). PAX2 patterns as in Figure 1a,b. Tumors studied: n = 38; classes represented: 27 in®ltrating ductal carcinomas, seven in®ltrating positive luminal cells with this nuclear morphology lobular carcinomas, medullary, colloid and adenocarcinoma, one coexisted with cells not ®tting this histiotype (Figure each, and one unclassi®ed 1a). The not-infrequent side-by-side arrangement of PAX2 positive and negative cells (Figure 1b, boxes) may have functional signi®cance as paracrine signaling grade and steroid receptor positive tumors were is known to be important in progesterone-dependent roughly equally divided between PAX2 positive and lateral branching (Brisken et al., 1998, 2000). Myoe- negative tumors. High grade and receptor negative pithelial cells also exhibited a mixed, PAX2 positive tumors were more likely to be PAX2 negative; 86% of and negative staining pattern (Figure 1a,b), with some Grade 3 tumors and 75% of receptor negative tumors myoepithelial cells having less immunoreactivity than were PAX2 negative. WT1 and PAX2 expression were adjacent luminal cells (Figure 1b). The localization of strongly correlated; 72% of PAX2 positive tumors PAX2 mRNA in mammary tissue was detected by in were WT1 positive, while 85% of WT1 negative situ hybridization and re¯ected PAX2 immuno-stain- tumors were also PAX2 negative. This study added ing. In the pictured section of a branched duct PAX2, 18 more cases to the patient population originally for example, PAX2 mRNA was found throughout the screened for WT1 expression (Silberstein et al., 1997). structure, with possibly higher amounts in the tips of All 18 cases showed some degree of WT1 staining, developing branches (Figure 1c). No signal was seen supporting recent ®ndings that a much higher with a sense probe (Figure 1c, inset). percentage of breast tumors are WT1-positive than Once we established the expression of PAX2 in the was originally reported (Loeb