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.

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.

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.

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.

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.

Epidermal growth factor receptor (EGFR) signaling is a key mediator of hormone-induced leukocyte infiltration in the pubertal female mammary gland.

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.

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.

Progesterone stimulates proliferation and promotes cytoplasmic localization of the cell cycle inhibitor p27 in steroid receptor positive breast cancers.

Progestins are reported to increase the risk of more aggressive estrogen receptor positive, progesterone receptor positive (ER+ PR+) breast cancers in postmenopausal women. Using an in vivo rat model of ER+ PR + mammary cancer, we show that tumors arising in the presence of estrogen and progesterone exhibit increased proliferation and decreased nuclear expression of the cell cycle inhibitor p27 compared with tumors growing in the presence of estrogen alone. In human T47D breast cancer cells, progestin increased proliferation and decreased nuclear p27 expression. The decrease of nuclear p27 protein was dependent on activation of Src and PI3K by progesterone receptor isoforms PRA or PRB. Importantly, increased proliferation and decreased nuclear p27 expression were observed in invasive breast carcinoma compared with carcinoma in situ. These results suggest that progesterone specifically regulates intracellular localization of p27 protein and proliferation. Therefore, progesterone-activated pathways can provide useful therapeutic targets for treatment of more aggressive ER+ PR+ breast cancers.

Progesterone decreases levels of the adhesion protein E-cadherin and promotes invasiveness of steroid receptor positive breast cancers.

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.

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.

A potential role of progestin-induced laminin-5/α6-integrin signaling in the formation of side branches in the mammary gland.

Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and α6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-κB ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

Perfluorooctanoic acid effects on ovaries mediate its inhibition of peripubertal mammary gland development in Balb/c and C57Bl/6 mice.

Exposure to perfluorooctanoic acid (PFOA), a synthetic perfluorinated compound and an agonist of peroxisome proliferator-activated receptor α (PPARα), causes stunted mouse mammary gland development in various developmental stages. However, the underlying mechanisms remain poorly understood. We found that peripubertal PFOA exposure significantly inhibited mammary gland growth in both Balb/c and C57Bl/6 wild type mice, but not in C57Bl/6 PPARα knockout mice, and Balb/c mice were more sensitive to PFOA inhibition. PFOA caused (1) delayed or absence of vaginal opening and lack of estrous cycling during the experimental period; (2) decreases in ovarian steroid hormonal synthetic enzyme levels; and (3) reduced expression of estrogen- or progesterone-induced mammary growth factors. Supplementation with exogenous estrogen and/or progesterone reversed the PFOA inhibitory effect on mammary gland. These results indicate that PFOA effects on ovaries mediate its inhibition of mammary gland development in Balb/c and C57Bl/6 mice and that PPARα expression is a contributing factor.

Regulation of estrogen receptor α N-terminus conformation and function by peptidyl prolyl isomerase Pin1.

Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors.

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.

Signal transducer and activator of transcription 5a mediates mammary ductal branching and proliferation in the nulliparous mouse.

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a-/- mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (E+P), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-kappaB ligand (RANKL). Stat5a-/- mammary glands were defective in inducing RANKL in response to E+P treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), was altered in Stat5a-/- mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in E+P-treated mammary epithelial cells.

Perfluorooctanoic acid effects on steroid hormone and growth factor levels mediate stimulation of peripubertal mammary gland development in C57BL/6 mice.

Perfluorooctanoic acid (PFOA) is a synthetic, widely used perfluorinated carboxylic acid and a persistent environmental pollutant. It is an agonist of peroxisome proliferator-activated receptor alpha (PPARalpha). Studies have shown that PFOA causes hepatocellular hypertrophy, tumorigenesis, and developmental toxicity in rodents, and some of its toxicity depends on the expression of PPARalpha. Our recent study revealed a stimulatory effect of peripubertal PFOA treatment (5 mg/kg) on mammary gland development in C57Bl/6 mice. The present study was designed to examine the underlying mechanism(s). It was found that mammary gland stimulation by PFOA was similarly observed in PPARalpha knockout and wild-type C57Bl/6 mice. The presence of ovaries was required for PFOA treatment (5 mg/kg) to stimulate mammary gland development with significant increases in the levels of enzymes involved in steroid hormone synthesis in both PFOA-treated wild-type and PPARalpha knockout mouse ovaries. PFOA treatment significantly increased serum progesterone (P) levels in ovary-intact mice and also enhanced mouse mammary gland responses to exogenous estradiol (E), P, and E + P. In addition, PFOA treatment resulted in elevated mammary gland levels of epidermal growth factor receptor (EGFR), estrogen receptor alpha, amphiregulin (Areg, a ligand of EGFR), hepatocyte growth factor, cyclin D1, and proliferating cell nuclear antigen (PCNA) in both wild-type and PPARalpha knockout mouse mammary glands. These results indicate that PFOA stimulates mammary gland development in C57Bl/6 mice by promoting steroid hormone production in ovaries and increasing the levels of a number of growth factors in mammary glands, which is independent of the expression of PPARalpha.