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...

The mammogenic actions of estrogen, although undisputed, lack definition due to uncertainties concerning the relative importance of systemic vs. local actions of the hormone. In addition to its well known, indirect effects on mammary tissue through pituitary intermediaries such as PRL and GH, recent evidence points to, but does not prove, direct estrogen action on mammary targets. The ability of exogenous estrogen to directly and locally stimulate mammary growth in vivo was previously shown in endocrine-ablated animals using small plastic pellets containing estradiol. The more important question of whether the direct action of endogenous estrogen is required for normal mammary growth and morphogenesis in the endocrine-intact animal is now investigated using direct-acting, slow-release plastic implants containing pure antiestrogens (antiestrogens with no estrogenic properties) inserted into the growth region of mammary glands. Local growth inhibition only in the immediate vicinity of the implants and not in other glands in the same mouse demonstrated the requirement of mammary tissues for endogenous, locally acting estrogen. Local actions of antiestrogens on ducts mimicked the ovariectomy-induced loss of systemic estrogen with respect to time course and morphology, with complete inhibition of ductal growth in 14 days. A second effect, in which locally acting antiestrogens simplified the pattern of ductal branching, was observed in both immature and mature animals. Two distinct mitogenic pathways, one governing ductal elongation and the other ductal maintenance, were thus affected. The inhibitory effects of antiestrogen treatment were fully reversible and not accompanied by obvious cytotoxicity. We conclude from these studies of localized estrogen receptor blockade that with respect to ductal mammogenesis, the action of estrogen is direct (acting at the level of the gland itself) and not primarily through the stimulation of pituitary mammogens.

During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERa, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovariectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERa, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERa, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERa, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERa, PR, and Pax-2. We hypothesize that the delays reflect a previously unrecognized timing system, which is present in all ovarian target tissues.

Parity in humans and rats provides significant protection against mammary tumor development. This study was carried out to investigate whether treatment of parous rats with mammotropic hormones would affect methyl-nitrosourea (MNU)-induced mammary carcinogenesis. Parous rats were treated with 17beta-estradiol (E2), progesterone (P4) and thyroxine (T4) alone or in combination. E2 (20 microg/60 days) and P4 (20 mg/60 days) were administered by silastic tubing and T4 in the drinking water (3 microg T4/ml). Hormonal treatments commenced 7 days before MNU injection and continued for 33 weeks. Animals were palpated weekly for tumor detection. The effects of the hormonal treatments on the circulating concentrations of E2, P4, growth hormone (GH), prolactin (PRL), T4 and insulin-like growth factor-I (IGF-I) after 7 days of treatment, the time of MNU injection, was assessed. Animals treated with E2 had significantly elevated circulation concentrations of GH, PRL and P4, and serum levels of E2 were more consistent in this group than in the other animal groups. P4 treatment caused elevation in P4 concentration in serum but did not affect the circulating levels of other hormones. The proliferation of the mammary gland at the time of MNU injection was elevated in animal groups treated with E2 either alone or with P4 and T4 and in animals treated with P4 alone, but the mammary gland was most differentiated in untreated parous rats and least in animals treated with E2 either alone or with P4 and T4. Mammary tumor incidence was 10% in parous rats that did not receive any hormonal treatment. Treatments with E2 or P4 alone significantly increased the susceptibility of parous animals to 67 and 50.0%, respectively; a tumor incidence similar to that of untreated AMV rats (64%). Parous rats treated with E2 plus P4 had tumor incidence higher than 90%. T4 administered did not affect mammary carcinogenesis.