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Estrogen receptor-positive and progesterone receptor-negative (ER+/PR-) breast cancers account for 15% to 25% of all human breast cancers and display more aggressive malignant characteristics than ER+/PR+ cancers. However, the molecular mechanism underlying development of ER+/PR- breast cancers still remains elusive. We show here that Tip30 deletion dramatically accelerated the onset of mammary tumors in the MMTV-Neu mouse model of breast cancer. The mammary tumors arising in Tip30(-/-)/MMTV-Neu mice were exclusively ER+/PR-. The growth of these ER+/PR- tumors depends not only on estrogen but also on progesterone despite the absence of detectable PR. Tip30 is predominantly expressed in ER+ mammary epithelial cells, and its deletion leads to an increase in the number of phospho-ERα-positive cells in mammary glands and accelerated activation of Akt in MMTV-Neu mice. Moreover, we found that Tip30 regulates the EGFR pathway through controlling endocytic downregulation of EGFR protein level and signaling. Together, these findings suggest a novel mechanism in which loss of Tip30 cooperates with Neu activation to enhance the activation of Akt signaling, leading to the development of ER+/PR- mammary tumors., (©2010 AACR.)

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.

Ovarian steroid hormones play a critical role in regulating mammary gland growth and development. The mammary gland sequentially acquires and cyclically exhibits proliferative responses to estrogen and/or progesterone from birth to postmenopause. The focus of this review is to present our current understanding of estrogen and progesterone receptor distribution in epithelial and stromal cells and their functions in relation to mammary gland development. Insights gained from the study of the normal mammary gland are relevant to our understanding of the conditions which may predispose women to the development of breast cancer as well as to alterations in hormonal regulation that occur in breast cancer.

Mammary stromal cells can modulate steroid hormone responsiveness both in vivo and in vitro. One of the mechanisms by which stromal cells can influence epithelial cell behavior is by modifying the composition of the extracellular matrix (ECM). In this report, we have investigated the effects of five ECM molecules on control of epithelial cell proliferation by estrogen (E2) and progestin (R5020) under serum-free culture conditions. To assess the contribution of mammary gland differentiation in determining epithelial cell interactions with ECM, the behavior of mammary epithelial cells derived from nulliparous and pregnant mice was compared. We report the novel finding that the proliferative responses of mammary epithelial cells to progestin is influenced by specific ECM molecules. However, the primary determinant of hormonal responsiveness is the developmental state of the gland from which the epithelial cells were derived. Nulliparous-derived epithelial cells, proliferated in response to R5020 only on fibronectin (FN) and collagen IV (Col IV). The more highly differentiated, pregnancy-derived epithelial cells were not responsive to E2 or R5020 on any ECM. To determine if steroid hormone receptors were targets of ECM-mediated effects, ER and PR levels were analyzed. In both nulliparous and pregnancy-derived cultures, PR binding levels were maintained at similar levels on all ECMs. However, ER levels were not maintained in nulliparous-derived cultures, and this may have contributed to the lack of a significant response to E2. Alternatively or in addition, E2-induced responses may require additional signals or growth factors that are provided by stromal cells in vivo or by serum supplementation in vitro. These results demonstrate the ECM molecules, fibronectin and collagen IV, can modulate responsiveness of mammary epithelial cells to R5020 in vitro, and may be the mediators of stromal influences on hormone responsiveness in vivo. However, the specific effects of ECM and hormones are also determined by the developmental state of the mammary gland from which the cells are derived. Thus, mammary gland differentiation, ovarian hormones, and ECM composition may act in concert to determine the outcome of hormone treatment on cell proliferation.

The appearance, epithelial and stromal cell distribution of estrogen receptors (ER) in normal mouse mammary gland were determined between 1 and 10 weeks of age using immunohistochemistry. The effect of ovariectomy and estrogen (E)-treatment on the distribution and concentration of ER-positive cells at various ages was also analyzed. These studies demonstrate that ER are present in both mammary epithelial and stromal cells before the mammary gland exhibits a proliferative response or increase in progesterone receptor concentration as a result of E-treatment. Furthermore, an analysis of E-treatment suggests that although ER are present at an early age, there may be additional factors that determine the nature and extent of E-responsiveness.

The present studies were undertaken to determine the relative distribution of estrogen and progesterone receptors (PgR) in the epithelial, adipose, and connective tissues of normal mouse mammary gland and to determine in which tissue(s) the modulations in PgR concentration and mammary gland sensitivity to estrogenic stimulation of PgR occur. Mammary epithelium was separated from adipose and connective tissues surgically in vivo according to the cleared fat pad technique. Binding studies using [3H]R5020 or [3H]estradiol were carried out on cytoplasmic extracts of intact mammary gland or cleared fat pads which were devoid of epithelium and contained only mammary adipose and connective tissues. We found that mammary adipose and connective tissues contain separate, high affinity, steroid-specific, macromolecular binding sites for estrogen and progesterone, and the majority of PgR are present only in the glandular epithelium. Furthermore, the modulation in PgR concentration relative to mammary gland development as well as the estrogenic regulation of PgR concentration appear to be restricted to the epithelial component of mammary gland. Since the major mammary gland developmental changes that occur during pregnancy and lactation also occur in the epithelium, it is proposed that the receptor modulations may serve a regulatory function in mammary gland.

Cytoplasmic estrogen receptor (CER) activation and nuclear estrogen receptor (NER) forms have been examined in normal virgin and lactating mammary gland by 1) measuring [3H]estradiol-receptor dissociation kinetics, and by 2) sucrose density gradient analysis of sedimentation behavior on high salt gradients. The data show two forms of CER in both virgin and lactating mammary gland: 1) a fast dissociating-nonactivated form which sediments at 4 S, and 2) a slow dissociating-activated form which sediments at 5 S. The 5 S slow dissociating estrogen receptor (ER) is the predominant form found in the nucleus. CER are activated by exposure to elevated temperature, high salt concentration, dilution, or ammonium sulfate fractionation. This is demonstrated by an increase in the proportion of the slow dissociating, 5 S ER form. Sodium molybdate inhibits activation and prevents heat- or ammonium sulfate-induced increase in the amount of the slow-dissociating CER as well as the 4 S to 5 S increase in sedimentation coefficient. We also found that cytoplasmic and nuclear 5 S ER forms were susceptible to 1) cold-induced dissociation, and to 2) degradation by nuclear protease activity. Thus, whereas mammary gland CER activation and NER forms described herein are very similar to those reported for other estrogen target tissues, the experimental conditions required to demonstrate the activated-nuclear form of ER differ substantially from those reported for uterine tissue. These findings may be important for assessing differences in ER forms in certain disease states such as mammary neoplasia. Previously we have shown that lactating mammary gland, in contrast to virgin mammary gland, is not responsive to estrogen. Having compared CER and NER forms and receptor activation in virgin vs. lactating mammary gland, we find no differences between ER in these two tissue states that can explain the absence of responsiveness to estrogen during lactation.