The product of the WT1 Wilms tumor suppressor gene controls the expression of genes encoding components of the insulin-like growth factor and transforming growth factor ␤ signaling systems. The role of these growth factors in breast tumor growth led us to investigate possible WT1 gene expression in normal and cancerous breast tissue. WT1 was detected by immunohistochemistry in the normal mammary duct and lobule, and the patterns of expression were consistent with developmental regulation. In a survey of 21 infiltrating tumors, 40% lacked immunodetectable WT1 altogether and an additional 28% were primarily WT1-negative. Cytoplasmic, but not nuclear, localization of WT1 was noted in some tumor cells and WT1 was detected, sometimes at high levels, in more-advanced estrogenreceptor-negative tumors. In this highly malignant subset, the tumor suppressor protein p53, which can physically interact with WT1, was also sometimes detected. WT1 mRNA was detected in normal and tumor tissue by reverse transcription-coupled PCR. Alternative splicing of the WT1 mRNA may regulate gene targeting of the WT1 protein through changes either in its regulatory or zinc-finger domains. The relative proportions of WT1 mRNA splice variants were altered in a random sample of breast tumors, providing evidence that different tumors may share a common WT1-related defect resulting in altered regulation of target genes.

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In this study, we ®rst describe expression of the paired domain transcription factor PAX2 in the normal and cancerous human breast, then demonstrate in a murine model a novel function for PAX2 in the regulation of progesterone stimulation of secondary ductal growth. In human mammary tissue, PAX2 expression was coincident with sub-populations of mammary ductal cells, some of which possessed an undi erentiated histiotype, and was also found in 450% of the human breast tumors surveyed (n=38). In the mouse, mammary parenchyma with a targeted deletion of PAX2 developed normal ductal systems when grafted into wild-type host mammary fat pads, but failed to undergo higher order side-branching and lobular development in response to progesterone. A previously unsuspected PAX2/WT1 (Wilms' tumor suppressor gene) regulatory axis in the mammary gland was also indicated. Using RT ± PCR, a signi®cant reduction in WT1 mRNA expression was detected in the PAX2 mutant glands compared to wildtype counterparts and double-antibody immunohistochemistry detected the co-localization of PAX2 and WT1 in the nuclei of normal and cancerous breast cells. These data indicate a role for PAX2 (and possibly WT1) in the regulation of the progesterone response of the mature mammary gland. The potential contribution of PAX2 to breast tumor pathogenesis is discussed.

It is well established that 85-90% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female Sprague-Dawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/ kg body wt. When at least one mammary tumor had grown to 1.0-1.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.0-1.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a regrowth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17β-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like Abbreviations: α-lac, α-lactalbumin; E2, 17β-estradoil; EGF, epidermal growth factor; EGFR, EGF receptor; ERα (-β), estrogen receptor-α (-β); ERK-1 (-2), extracellular signal-regulated kinase-1 (-2); GH, growth hormone; HDT, hormone-dependent tumor; HIT, hormone-independent tumor; IGF-I (-II), insulin-like growth factor-I (-II); IGF-IR, IGF-I receptor; IRS-1 (-2), insulin receptor substrate-1 (-2); MAP kinase, mitogen-activated protein kinase; MNU, N-methyl-N-nitrosourea; OVX, ovariectomized; P4, progesterone; P450arom, P450 aromatase; PR, progesterone receptor; PRL, prolactin; RIA, radioimmunoassay; RPA, ribonuclease protection assay; TGF-α, transforming growth factor-α.© Oxford University Press 2039 growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. Ho...