Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

SUMMARY Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from non-irradiated hosts. Molecular and genetic approaches show that TGFβ mediated tumor acceleration; molecular signatures implicated TGFβ and genetically reducing TGFβ abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGFβ independent and linked to mammary stem cell activity. Thus the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms.

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.

Estrogen receptor alpha (ER␣), 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 ER␣ 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 tamoxifeninducible activity of ER␣ and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ER␣-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 ER␣.E strogen receptor alpha (ER␣), a member of the nuclear receptor superfamily of transcription factors, mediates the actions of estrogen in normal physiology and disease (17). ER␣ is expressed in the normal mammary gland and in 70% of human breast cancers and is a key driver of breast cell proliferation (16,26,83). Directed overexpression of ER␣ in the mammary gland is sufficient to induce hyperplasia, and blockade of ER␣ activity by hormonal therapies (aromatase inhibitors, tamoxifen, and fulvestrant) reduces recurrence and improves clinical outcomes of ER␣-positive breast cancer patients (19,22). Two activation functions mediate the transcriptional activity of ER␣, a C-terminal liganddependent AF2 and an N-terminal ligand-independent AF1 (89). Regulation of ER␣ activity via the C-terminal AF2 has been wellcharacterized through biochemical and crystallographic studies and forms the basis for our understanding of hormonal therapy for breast cancer (10,34,84). In the canonical activation pathway, ligand binding initiates C-terminal structural rearrangements that facilitate downstream events, including dimerization, DNA binding, and coregulator interactions, ultimately engaging the basal transcriptional machinery to regulate gene expression. However, ER␣ can also be activated by growth factors and kinases, which phosphorylate the receptor N terminus and other domains to regulate transcription in the absence of direct ligand engagement (for reviews, see references 27, 47, 75, 94, and 95). In contrast to the C-terminal AF2 domain, biochemical and structural mechanisms that control N-terminal AF1 remain poorly understood (48,91).Multiple challenges have hindered molecular dissection of AF1 regulation. First, AF1 resid...

IntroductionEpidemiological 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.MethodsPubertal 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.ResultsHFD 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.ConclusionsTaken 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.

208BrdU = bromodeoxyuridine; Col 1 = collagen type 1; ECM = extracellular matrix; EGF = epidermal growth factor; ER = estrogen receptor; FCM = fibroblast-conditioned medium; FN = fibronectin; HGF = hepatocyte growth factor; IGF-1 = insulin-like growth factor I; LM = laminin; PBS = phosphate-buffered saline; PR = progesterone receptor; R5020 = promegestone. Breast Cancer Research Vol 5 No 4 Haslam and Woodward IntroductionMammary gland growth and development are mediated through the complex interactions of steroid hormones, polypeptide hormones, growth stimulatory factors and growth inhibitory factors. Normal development and function of the mammary gland are also dependent upon complex interactions between epithelial cells and stromal cells [1,2]. Stromal cells can regulate the epithelium by the production of soluble growth stimulatory and/or inhibitory factors; and components of the extracellular matrix such as collagens, fibronectin and laminin can also act as signaling molecules for epithelial cells, via specific integrins on epithelial cells. Epithelial cells also secrete factors that influence proliferation and function of adjacent epithelial and stromal cells (Fig. 1).Although there have been numerous studies of signalling mediated by the extracellular matrix and integrin in normal mammary gland and breast cancer cell lines, none has addressed the role of stroma in mediating and modulating steroid hormone action. There is increasing evidence that a number of responses to estrogen and/or progesterone in the mammary gland may be mediated indirectly through paracrine effects. This review focuses on recent studies from our laboratory addressing interactions between epithelial cells and stromal cells and between steroid hormones and growth factors in the normal murine mammary gland and in human breast cancer cells. Steroid hormones and mammary gland developmentEstrogen and progesterone are required for proliferation and morphogenesis of the normal mammary gland. Estrogen drives ductal development during puberty, whereas estrogen + progesterone mediate the proliferative and morphological changes of ductal side-branching and alveologenesis that occur at sexual maturity and during pregnancy [1,2]. Progesterone is also mitogenic in the premenopausal and postmenopausal human breast [3]. The greater risk of breast cancer in postmenopausal women receiving combined estrogen plus progestin hormone replacement therapy than in those receiving AbstractMammary epithelial cells comprise the functional component of the normal gland and are the major target for carcinogenesis in mammary cancer. However, the stromal compartment of the normal gland and of tumors plays an important role in directing proliferative and functional changes in the epithelium. In vivo and in vitro studies of the murine mammary gland have provided insights into novel stromadependent mechanisms by which estrogen and progesterone action in the epithelium can be modulated by hepatocyte growth factor (HGF) and the extracellular matrix proteins, collagen typ...

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.

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