Puberty-specific promotion of mammary tumorigenesis by a high animal fat diet.

INTRODUCTION: Increased animal fat consumption is associated with increased premenopausal breast cancer risk in normal weight, but not overweight, women. This agrees with our previous findings in obesity-resistant BALB/c mice, in which exposure to a high saturated animal fat diet (HFD) from peripuberty through adulthood promoted mammary tumorigenesis. Epidemiologic and animal studies support the importance of puberty as a life stage when diet and environmental exposures affect adult breast cancer risk. In this study, we identified the effects of peripubertal exposure to HFD and investigated its mechanism of enhancing tumorigenesis. METHODS: Three-week-old BALB/c mice fed a low-fat diet (LFD) or HFD were subjected to 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. At 9 weeks of age, half the mice on LFD were switched to HFD (LFD-HFD group) and half the mice on HFD were switched to LFD (HFD-LFD group). Tumor gene expression was evaluated in association with diet and tumor latency. RESULTS: The peripubertal HFD reduced the latency of DMBA-induced mammary tumors and was associated with tumor characteristics similar to those in mice fed a continuous HFD. Notably, short-latency tumors in both groups shared gene expression characteristics and were more likely to have adenosquamous histology. Both HFD-LFD and continuous HFD tumors showed similar gene expression patterns and early latency. Adult switch from HFD to LFD did not reverse peripubertal HFD tumor promotion. Increased proliferation, hyperplasia, and macrophages were present in mammary glands before tumor development, implicating these as possible effectors of tumor promotion. Despite a significant interaction between pubertal diet and carcinogens in tumor promotion, peripubertal HFD by itself produced persistent macrophage recruitment to mammary glands. CONCLUSIONS: In obesity-resistant mice, peripubertal HFD is sufficient to irreversibly promote carcinogen-induced tumorigenesis. Increased macrophage recruitment is likely a contributing factor. These results underscore the importance of early life exposures to increased adult cancer risk and are consistent with findings that an HFD in normal weight premenopausal women leads to increased breast cancer risk. Notably, short-latency tumors occurring after peripubertal HFD had characteristics similar to human basal-like breast cancers that predominantly develop in younger women.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty.

INTRODUCTION: Puberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined. METHODS: Pubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL). RESULTS: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression. CONCLUSIONS: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Pubertal high fat diet: effects on mammary cancer development.

INTRODUCTION: Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD. METHODS: Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis. RESULTS: HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages. CONCLUSIONS: Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.

Toward a digital analysis of environmental impacts on rodent mammary gland density during critical developmental windows.

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies.

Benzophenone-3 promotion of mammary tumorigenesis is diet-dependent.

Benzophenone-3 is a putative endocrine disrupting chemical and common ingredient in sunscreens. The potential of endocrine disrupting chemicals to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis, demands evaluation of its potential in promoting breast cancer. This study identifies the effects of BP-3 on mammary tumorigenesis with high-fat diet during puberty versus adulthood in Trp53-null transplant BALB/c mice. Benzophenone-3 exposure yielded levels in urine similar to humans subjected to heavy topical sunscreen exposure. Benzophenone-3 was protective for epithelial tumorigenesis in mice fed lifelong low-fat diet, while promotional for epithelial tumorigenesis in mice fed adult high-fat diet. Benzophenone-3 increased tumor cell proliferation, decreased tumor cell apoptosis, and increased tumor vascularity dependent on specific dietary regimen and tumor histopathology. Even in instances of an ostensibly protective effect, other parameters suggest greater risk. Although benzophenone-3 seemed protective on low-fat diet, spindle cell tumors arising in these mice showed increased proliferation and decreased apoptosis. This points to a need for further studies of benzophenone-3 in both animal models and humans as a potential breast cancer risk factor, as well as a more general need to evaluate endocrine disrupting chemicals in varying dietary contexts.

Estrogen and progesterone are critical regulators of Stat5a expression in the mouse mammary gland.

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Progestin-regulated luminal cell and myoepithelial cell-specific responses in mammary organoid culture.

Normal mammary gland development requires the coordinated proliferation and morphogenesis of both mammary luminal epithelial cells (LECs) and myoepithelial cells (MECs). Cell proliferation in cultured mammary organoids containing both LECs and MECs is not increased by progestin (R5020) or 17beta-estradiol (E2) alone or R5020+E2 but is increased by E2-regulated, mammary stroma-derived Hepatocyte growth factor (HGF) and further increased by HGF+R5020. We investigated the effects of HGF and/or R5020 on morphology and LEC- and MEC-specific in vitro proliferation in organoids. HGF-induced tubulogenesis was initiated and carried out by LECs starting with cellular extensions, followed by the formation of chains and cords, and culminating in tubule formation. MECs did not appear to have an active role in this process. Whereas HGF by itself caused maximal proliferation of LECs, HGF+R5020 produced a synergistic and specific increase in MEC proliferation. Because only LECs expressed progesterone receptors (PRs), we investigated the role of receptor activator of nuclear factor-kappaB ligand (RANKL), a progestin-induced paracrine factor, in mediating increased MEC proliferation. Quantitative RT-PCR showed that RANKL mRNA was induced by R5020 or HGF+R5020 and RANKL protein colocalized with PRs in LECs. The increased proliferation of MECs in response to HGF+R5020 could be blocked by neutralizing antibody to RANKL and reproduced by treatment with HGF plus exogenous RANKL in place of R5020. Neither R5020, nor exogenously administered RANKL increased proliferation of LECs. These results led us to conclude that RANKL, induced by progestin in PR-positive cells, is secreted and interacts with HGF to specifically increase proliferation of PR-negative MECs.

Progesterone receptor isoform functions in normal breast development and breast cancer.

Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat, and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is recent advances in understanding the role of the PR isoform-specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies and their relevance to our understanding of the role of progestins in breast cancer etiology.

The Proliferative Response to p27 Down-Regulation in Estrogen Plus Progestin Hormonal Therapy is Lost in Breast Tumors.

Increased proliferation and breast cancer risk has been observed in postmenopausal women receiving estrogen (E) + progestin hormone replacement therapy (HRT). Progestin action is mediated through two progesterone receptor (PR) isoforms, PRA and PRB, with unique transcriptional activity and function. The current study examines hormonal regulation of PR isoforms in the normal postmenopausal human breast and the mechanism by which progestins increase proliferation and breast cancer risk. Archival benign breast biopsies from postmenopausal and premenopausal women, and luminal breast tumor biopsies from postmenopausal women, were analyzed for regulation of PRA and PRB expression by E and E+medroxyprogesterone acetate (MPA). In the postmenopausal breast without HRT, PRA and PRB expression was decreased compared to the premenopausal breast. Both E (n = 12) and E+MPA (n = 13) HRT in the postmenopausal breast were associated with increased PRA and PRB expression, increased nuclear cyclin E expression, and decreased nuclear p27 expression compared to no HRT (n = 16). With E+MPA HRT, there was a further decrease in nuclear p27 and increased Receptor Activator of NF-kappa B Ligand (RANKL) expression compared to E-alone HRT. In luminal breast cancers, E+MPA HRT (n = 6) was also associated with decreased nuclear expression of the cell cycle inhibitor p27 compared to E HRT (n = 6), but was not associated with increased proliferation. These results suggest that p27 mediates progestin-induced proliferation in the normal human breast and that regulation of this proliferative response by E+MPA is lost in breast tumors.

C/EBPß LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

CCAAT/enhancer binding protein ß (C/EBPß) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPß regulation of PR expression. All three C/EBPß isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPß and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPß isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPß and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPß and PRB largely co-localized. We suggest a critical role for C/EBPß, particularly LIP, in PRB expression.

Differential hormonal regulation and function of progesterone receptor isoforms in normal adult mouse mammary gland.

In normal mouse mammary gland, the mitogenic action of progesterone (P) is mediated by two P receptor (PR) isoforms, PRA and PRB. PRA is predominantly expressed in the adult virgin, and PRB is predominantly expressed during pregnancy. To investigate hormonal regulation of PR isoform expression and isoform-specific functions in vivo, adult ovariectomized BALB/c mice were treated for 3, 5, or 10 d with estrogen (E), P, or estrogen plus progesterone (E+P). Using an immunohistochemical approach with isoform-specific antibodies, we investigated hormonal regulation of PRA and PRB and their functional roles in proliferation and morphogenesis. Significant E-induced proliferation was only observed after 5 d at the distal tips of ducts; there was no sidebranching or alveologenesis. P induced proliferation that resulted in sidebranching and alveologenesis, but E+P treatment produced more proliferation sooner and more extensive sidebranching and alveologenesis. PRA levels were increased by E and decreased by P. Increased PRB levels were induced by treatment with P or E+P and coincided with the formation of alveoli. PRA was the predominant PR isoform expressed during sidebranching, and colocalization of PRA with 5-bromo-2'-deoxyuridine revealed that proliferation of PRA-positive and -negative cells was responsible for P-induced sidebranching. PRB was the predominant PR isoform expressed during alveologenesis, and colocalization of PRB with 5-bromo-2'-deoxyuridine showed that both PRB-positive and -negative cells proliferated during alveolar expansion. These results demonstrate different hormonal regulation of PRA and PRB levels in vivo and suggest that P can induce proliferation through either PRA or PRB via direct and paracrine mechanisms.

Progesterone receptor isoforms and proliferation in the rat mammary gland during development.

Progesterone (P), acting through progesterone receptor (PR) isoforms A and B, plays an important role in normal mammary gland development and is implicated in the etiology of breast cancer. Because of significant similarities between human and rat mammary gland development and hormonal responsiveness of mammary cancers, we investigated P action in the rat mammary gland. By immunohistochemical methods we determined PRA and PRB expression at puberty, sexual maturity, pregnancy, and lactation and after postlactational involution and their functional roles in the regulation of proliferation. PRA expression was restricted to luminal epithelial cells, whereas PRB was expressed in both luminal and myoepithelial cells, indicating a novel role of PRB in myoepithelial cell regulation. The majority of PRA-positive (PRA+) cells coexpressed PRB. In the pubertal and adult virgin mammary gland, PRA+PRB+ cells also expressed nuclear cyclin D1 but did not contain the proliferation marker bromodeoxyuridine. Based on a lack of phosphorylated retinoblastoma protein expression and the expression patterns of the cyclin-dependent kinase inhibitors p21 and p27 in these cells, we conclude that PRA+PRB+ cells appear to be cell cycle arrested and do not proliferate. PRA+ cells were decreased in the adult gland and during and after pregnancy. The percentage of PRB+ cells was relatively constant throughout development, and in a significant proportion of cells, only PRB was detected. During development, and especially during pregnancy, a high percentage of PRB+ cells were positive for bromodeoxyuridine. From this observation, we conclude that these cells proliferate and that P acting through PRB may directly stimulate proliferation.

Pubertally Initiated High-Fat Diet Promotes Mammary Tumorigenesis in Obesity-Prone FVB Mice Similarly to Obesity-Resistant BALB/c Mice.

Premenopausal breast cancer is associated with increased animal fat consumption among normal-weight but not overweight women. Our previous findings in obesity-resistant BALB/c mice showed that a diet high in saturated animal fat (HFD) promotes mammary tumorigenesis in both DMBA carcinogenesis and Trp53-null transplant models. Having made these observations in BALB/c mice, which have very modest HFD weight gain, we determined the effects of HFD in FVB mice, which gain significant weight on HFD. Three-week-old FVB mice fed a low-fat diet or HFD were subjected to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis. Like BALB/c mice, HFD promoted mammary tumorigenesis. Development of tumors largely occurred prior to mice becoming obese, indicating the role of animal-derived HFD rather than resulting obesity in tumor promotion. Also similar to BALB/c mice, early-occurring adenosquamous mammary tumors were abundant among HFD-fed FVB mice. Tumors from HFD mice also had increased intra-tumor M2 macrophages. Prior to tumor development, HFD accelerated normal mammary gland development and increased mammary M2 macrophages, similarly to BALB/c mice. The promotional effects of puberty-initiated HFD on carcinogen-induced mammary cancer are thus largely weight gain-independent. Like BALB/c mice, HFD promoted adenosquamous tumors, suggesting a role for early age HFD in promoting this subtype of triple negative mammary cancer. M2 macrophage recruitment was common to both mouse strains. We speculate that a similar effect of HFD on immune function may contribute to epidemiological findings of increased breast cancer risk in young, premenopausal, normal-weight women who consume a diet high in saturated animal fat.

Amphiregulin as a Novel Serum Marker of Puberty in Girls.

STUDY OBJECTIVE: Amphiregulin is a member of the epidermal growth factor family. In breast tissue, amphiregulin is a mediator of estrogen and progesterone signaling. The objectives were to examine the relationship of amphiregulin levels during peripuberty with estrogen levels. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: The participants in this analysis were a subset from a longitudinal study of pubertal maturation, the Breast Cancer and the Environment Research Program. They were recruited between ages 3 and 7 years. Blood specimens were selected for hormone analysis between 24 months before and 6 months after breast development. Serum amphiregulin levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: Amphiregulin levels were measured in 188 girls; 8.5% had a maternal history of breast cancer, and 30.9% of samples were below the limit of detection. Amphiregulin levels were greatest at 18 months before the onset of breast development (P < .006), and the rise in estrone levels between -24 and -18 months was correlated with the increase in amphiregulin levels in the same time period (P = .0002). After adjustment for time relative to breast development, amphiregulin levels were associated with maternal breast cancer (P = .024). Tracking of amphiregulin levels was highly significant (P < .0001) within a given individual. CONCLUSION: Amphiregulin levels peaked at 18 months before the onset of breast development, were temporally related to the rise in serum estrone, and were significantly associated with maternal history of breast cancer. Elevated amphiregulin levels at puberty might be a predictor of increased breast cancer risk.

Pubertal and adult windows of susceptibility to a high animal fat diet in Trp53-null mammary tumorigenesis.

Premenopausal breast cancer is associated with increased animal fat consumption among normal weight, but not overweight women (Farvid et al., 2014). Our previous findings in obesity-resistant BALB/c mice similarly showed promotion of carcinogen-induced mammary tumorigenesis by a diet high in saturated animal fat (HFD). This effect was specific to pubertal versus adult HFD. This study identifies the effects of HFD during puberty versus adulthood in Trp53-null transplant BALB/c mice and investigates its mechanism of enhancing tumorigenesis. Either pubertal or adult HFD is sufficient to increase incidence of Trp53-null mammary tumors. Puberty-restricted HFD exposure promoted tumor cell proliferation, increased angiogenesis, and increased recruitment of total and M2 macrophages in epithelial tumors. Adult-restricted exposure to HFD similarly increased proliferation, angiogenesis, recruitment of total and M2 macrophages, and additionally reduced apoptosis. Adult HFD also increased incidence of spindle cell carcinomas resembling claudin-low breast cancer, and thus adult HFD in the Trp53-null transplantation system may be a useful model for human claudin low breast cancer. Importantly, these results on Trp53-null and our prior studies on DMBA-induced mammary tumorigenesis demonstrate a pubertal window of susceptibility to the promotional effects of HFD, indicating the potential of early life dietary intervention to reduce breast cancer risk.

Progesterone receptor isoforms A and B: temporal and spatial differences in expression during murine mammary gland development.

Progesterone is a potent mitogen in the mammary gland. Based on studies using cells and animals engineered to express progesterone receptor (PR) isoforms A or B, PRA and PRB are believed to have different functions. Using an immunohistochemical approach with antibodies specific for PRA only or PRB only, we show that PRA and PRB expression in mammary epithelial cells is temporally and spatially separated during normal mammary gland development in the BALB/c mouse. In the virgin mammary gland when ductal development is active, the only PR protein isoform expressed was PRA. PRA levels were significantly lower during pregnancy, suggesting a minor role at this stage of development. PRB was abundantly expressed only during pregnancy, during alveologenesis. PRA and PRB colocalization occurred in only a small percentage of cells. During pregnancy there was extensive colocalization of PRB with 5-bromo-2'-deoxyuridine (BrdU) and cyclin D1; 95% of BrdU-positive cells and 83% of cyclin D1-positive cells expressed PRB. No colocalization of PRA with either BrdU or cyclin D1 was observed at pregnancy. In the virgin gland, PRA colocalization with BrdU or cyclin D1 was low; only 27% of BrdU-positive cells and 4% of cyclin D1-positive cells expressed PRA. The implication of these findings is that different actions of progesterone are mediated in PRB positive vs. PRA-positive cells in vivo. The spatial and temporal separation of PR isoform expression in mouse mammary gland provides a unique opportunity to determine the specific functions of PRA vs. PRB in vivo.

Experimental mouse model of hormonal therapy effects on the postmenopausal mammary gland.

Menopausal hormone therapy, in particular estrogen plus progestin therapy, has been associated with increased breast cancer risk. In order to understand the basis for increased breast cancer risk, more information is needed about the effects of menopausal hormone therapies on the breast. In this review we describe studies carried out in a mouse model of early vs. late postmenopausal states. We investigated the effects of 1) estrogen alone, 2) combined continuous estrogen + progestin, 3) systemically vs. locally administered estrogen and progestin, and 4) the effect of pregnancy on the response to hormonal therapies. We analysed the effects on mammary gland morphology and proliferation. Estrogen therapy started in late postmenopause caused a greater proliferative response than when started in early postmenopause. In parous, late postmenopausal mice the greater proliferative response to estrogen was not observed. Overall, the greatest proliferative response was observed with combined continuous estrogen + progestin hormone therapy and did not differ significantly in early vs. late nulliparous or parous postmenopausal mice. Both estrogen and progestin were found to act directly on the mammary gland rather than through systemically mediated effects. The possible implications of these findings for menopausal hormone therapy in women and breast cancer risk are discussed.

Progestins and breast cancer.

Progesterone (P) regulates proliferation and differentiation in the normal mammary gland in mouse, rat and human. Progesterone has also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is on recent advances in understanding the role of the progesterone receptor (PR) and functional significance of PR isoforms, PRA and PRB, in the normal mammary gland and in mammary cancer in mouse, rat and human.

Hepatocyte growth factor is required for progestin-induced epithelial cell proliferation and alveolar-like morphogenesis in serum-free culture of normal mammary epithelial cells.

The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.

Estrogen mediates mammary epithelial cell proliferation in serum-free culture indirectly via mammary stroma-derived hepatocyte growth factor.

Epithelial-stromal cell interactions are important for normal development and function of the mouse mammary gland. The steroid hormone estrogen is required for epithelial cell proliferation and ductal development in vivo. Recent studies of estrogen receptor alpha knockout mice indicate that estrogen-induced proliferation is dependent upon the presence of estrogen receptor in mammary stromal cells, but not in epithelial cells. The purpose of the present study was to identify the underlying mechanism of estrogen-dependent stroma-derived effects on mammary epithelium. We have developed a minimally supplemented serum-free medium, collagen gel primary mammary coculture system to address the issue of stroma-derived, estrogen-dependent effects on epithelial cell proliferation. Conditioned medium from mammary fibroblasts or coculture with mammary fibroblasts caused increased epithelial cell proliferation and produced tubular/ductal morphology. Hepatocyte growth factor (HGF) was identified as the mediator of this effect, as the proliferative activity in fibroblast-conditioned medium was completely abolished by neutralizing antibody to HGF, whereas neutralizing antibodies to either epidermal growth factor or IGF-I had no effect. Treatment of mammary fibroblasts with estrogen increased the production of HGF. From these results we conclude that estrogen may indirectly mediate mammary epithelial cell proliferation via the regulation of HGF in mammary stromal cells and that HGF plays a crucial role in estrogen-induced proliferation in vivo.