Progesterone induces adult mammary stem cell expansion.

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer.

Progesterone receptor A predominance is a discriminator of benefit from endocrine therapy in the ATAC trial.

Progesterone receptor (PR) function, while essential in normal human breast, is also implicated in breast cancer risk. The two progesterone receptors, PRA and PRB, are co-expressed at equivalent levels in normal breast, but early in carcinogenesis normal levels of PRA:PRB are frequently disrupted, and predominance of one isoform, usually PRA, results. In model systems, PRA and PRB have different activities, and altering the PRA:PRB ratio in cell lines alters PR signaling. The purpose of this study was to determine whether hormonal or reproductive factors contribute to imbalanced PRA:PRB expression in breast tumors and the impact of PRA:PRB imbalance on disease outcome. The relative expression of PRA and PRB proteins was determined by dual immunofluorescence histochemistry in archival breast tumors and associations with clinical and reproductive history assessed. PRA:PRB expression was not influenced by reproductive factors, whereas exogenous hormone use (menopausal hormone treatment, MHT) favored PRB expression (p < 0.035). The PRA:PRB ratio may be a discriminator of response to endocrine therapy in the TransATAC sample collection, with high PRA:PRB ratio predicting earlier relapse for women on tamoxifen, but not anastrozole (mean lnPRA:PRB ratio; HR (95 % CI) tamoxifen 2.45 (1.20-4.99); p value 0.02; anastrozole 0.80 (0.36-1.78); p value 0.60). The results of this study show that PRA:PRB imbalance in breast cancers is not associated with lifetime endogenous endocrine and reproductive factors, but is associated with MHT use, and that PRA predominance can discriminate those women who will relapse earlier on tamoxifen treatment. These data support a role for imbalanced PRA:PRB expression in breast cancer progression and relative benefit from endocrine treatment.

Hormone-responsive model of primary human breast epithelium.

Retention of hormone responsiveness in primary culture models of human breast is essential for studies aimed at understanding the mechanisms of action of the ovarian hormones in the human breast. In this chapter we describe the development of a culture model of primary human breast that retains critical features of the tissue in vivo. We find that primary normal human breast tissue in embedded culture recapitulates the morphology, cell lineages, functional gene expression characteristics and estrogen and progesterone receptor responsiveness of the breast in vivo. The ratio of luminal to myoepithelial cells after culture recapitulates that observed in the uncultured tissue, highlighting the fact that progenitor cells capable of giving rise to both epithelial cell lineages are retained in this model system. By contrast, primary cells placed into monolayer culture, even for a single passage, lose bipotent progenitors, and the myoepithelial lineage predominates, demonstrating the rapidity with which phenotypic changes and selection occur in normal breast cells, unless cultured under conditions that prevent this outcome. Primary matrix-embedded culture of normal human breast cells provides researchers with a new opportunity to understand ovarian hormone action in the human breast.

Poor-prognosis estrogen receptor-positive breast cancer identified by histopathologic subclassification.

PURPOSE: Identification of biologically and clinically distinct breast cancer subtypes could improve prognostic assessment of primary tumors. The characteristics of "molecular" breast cancer subtypes suggest that routinely assessed histopathologic features in combination with limited biomarkers may provide an informative classification for routine use. EXPERIMENTAL DESIGN: Hierarchical cluster analysis based on components of histopathologic grade (tubule formation, nuclear pleomorphism, and mitotic score), expression of ER, cytokeratin 5/6, and HER2 amplification identified four breast cancer subgroups in a cohort of 270 cases. Cluster subgroup membership was compared with observed and Adjuvant! Online predicted 10-year survival. Survival characteristics were confirmed in an independent cohort of 300 cases assigned to cluster subgroups using a decision tree model. RESULTS: Four distinct breast cancer cluster subgroups (A-D) were identified that were analogous to molecular tumor types and showed a significant association with survival in both the original and validation cohorts (P < 0.001). There was a striking difference between survival for patients in cluster subgroups A and B with ER(+) breast cancer (P < 0.001). Outcome for all tumor types was well estimated by Adjuvant! Online, with the exception of cluster B ER(+) cancers where Adjuvant! Online was too optimistic. CONCLUSIONS: Breast cancer subclassification based on readily accessible pathologic features could improve prognostic assessment of ER(+) breast cancer.

Focal subnuclear distribution of progesterone receptor is ligand dependent and associated with transcriptional activity.

The progesterone receptor (PR) is a critical mediator of progesterone action in the female reproductive system. Expressed in the human as two proteins, PRA and PRB, the receptor is a ligand-activated nuclear transcription factor that regulates transcription by interaction with protein cofactors and binding to specific response elements in target genes. We previously reported that PR was located in discrete subnuclear foci in human endometrium. In this study, we investigated the role of ligand in the formation of PR foci and their association with transcriptional activity. PR foci were detected in mouse uterus and normal human breast tissues and were more abundant when circulating progesterone was high. In human malignant tissues, PR foci were aberrant: foci were larger in endometrial cancers than in normal endometrium, and in breast cancers hormone-dependence was decreased. Chromatin disruption also increased foci size and decreased ligand dependence, suggesting that altered nuclear architecture may contribute to the aberrant PR foci observed in endometrial and breast cancers. In breast cancer cells, movement of PR into foci required exposure to ligand and was blocked by transcriptional inhibitors and by prolonged inhibition of proteasomal degradation. Foci contained PR dimers, and fluorescence resonance energy transfer demonstrated that PR foci contained the highest concentration of receptor dimers in the nucleus. PR in foci colocalized with transcription factors and nascent RNA transcripts only in the presence of ligand, and inhibition of coactivator recruitment inhibited PR foci formation. The demonstration that focal distribution of PR within the nucleus is associated with transcription suggests a link between the subnuclear distribution of PR and its transcriptional activity that is likely to be important for normal cellular function of PR.

Overlapping and distinct expression of progesterone receptors A and B in mouse uterus and mammary gland during the estrous cycle.

In rodents, progesterone receptors (PRs) A and B have different and often nonoverlapping roles, and this study asked whether different activities of the PR proteins in mouse are related to differences in their expression in reproductive tissues. The individual expression of PRA and PRB was determined immunohistochemically in mammary gland and uterus during the estrous cycle or in response to endocrine manipulation. In the mammary gland, PRA and PRB were colocated in PR+ epithelial cells, with little change during the estrous cycle. In the uterus, PRA was not detected in luminal epithelium at any stage of the cycle, and PR+ luminal cells expressed only PRB. In the stroma and myometrium, PRA and PRB levels fluctuated with cyclical systemic hormone exposure. Observation of functional end points suggested that augmented stromal and/or myometrial PRA in proestrus inhibited estrogen receptor expression and epithelial proliferation. Colocation of PRA and PRB was hormonally regulated, and ovariectomy did not reproduce the expression of PRA and PRB in the uterus during the estrous cycle. Whereas PRB was the only PR in the luminal epithelium in cycling mice, ovariectomy restored PRA expression, resulting in PRA-PRB colocation. In stroma and myometrium, PRA and PRB colocated in PR+ cells, but ovariectomy reduced PRA levels more than PRB, resulting in PRB-only-expressing cells. This study has shown that nonoverlapping PRA and PRB expression in the uterus, in particular the lack of PRA, and expression of PRB only in the luminal epithelium throughout the estrous cycle, is likely to contribute to the distinct roles of PRA and PRB in the adult mouse.

Subnuclear distribution of progesterone receptors A and B in normal and malignant endometrium.

The nuclear progesterone receptor (PR), which is expressed as two proteins with different functions (PRA and PRB), is expressed in the normal endometrium and endometrial cancer. Our previous work has shown that there is a disruption to the relative PR isoform expression in progression to malignancy in endometrial cancer in which expression of a single isoform is common. A consistent feature in these studies was discrete punctate distribution of PRA and PRB in the nuclei of endometrial cancer cells. In this study PRA and PRB distribution within the nucleus was examined in vivo in the normal endometrium during the menstrual cycle, and in endometrial cancer, by dual immunofluorescence and confocal microscopy using cohorts in which PRA and PRB expression levels have previously been characterized. In the normal endometrium, PR was distributed evenly within the nucleus and also localized in discrete subnuclear foci. In the proliferative phase, even PR distribution was predominant and both PR isoforms were colocated and distributed evenly. In the secretory phase, there was a marked increase in the proportion of nuclei containing PR distributed into discrete foci, and PRB was the predominant isoform in nuclear foci. There was an inverse relationship between even and focal PR distribution in the menstrual cycle, suggesting that hormonal fluctuations were involved in movement of PR into focal nuclear locations. In endometrial cancers colocalization of PRA and PRB was infrequent, and there was no relationship between even and focal PR isoform distribution, unlike the normal endometrium. PRA was predominantly evenly distributed in endometrial cancers, whereas PRB was focal. Even PRB distribution in endometrial cancer was not often noted. Multivariate analysis showed that PRA expression was highly predictive of even nuclear distribution in endometrial cancers and PRB expression of distribution into foci, and these associations were independent of total PRA and PRB levels. Nuclear distribution of PR isoforms was associated with clinical grade, where tumors of high grade had significantly fewer nuclei containing even PRA distribution and focal PRB distribution, compared with tumors of low grade. In the normal endometrium, localization of PR into nuclear foci coincides with high progesterone levels, suggesting that altered intranuclear PR distribution is hormonally regulated. Nuclear distribution may be an important component of gene regulation in target tissues, and disruptions in PR distribution in endometrial cancer could affect the function of PR and contribute to aberrant hormonal responses.

Progesterone action in human tissues: regulation by progesterone receptor (PR) isoform expression, nuclear positioning and coregulator expression.

Progesterone is a critical regulator of normal female reproductive function, with diverse tissue-specific effects in the human. The effects of progesterone are mediated by its nuclear receptor (PR) that is expressed as two isoforms, PRA and PRB, which are virtually identical except that PRA lacks 164 amino acids that are present at the N-terminus of PRB. Considerable in vitro evidence suggests that the two PRs are functionally distinct and in animals, tissue-specific distribution patterns of PRA and PRB may account for some of the diversity of progesterone effects. In the human, PRA and PRB are equivalently expressed in most target cells, suggesting that alternative mechanisms control the diversity of progesterone actions. PR mediates the effects of progesterone by association with a range of coregulatory proteins and binding to specific target sequences in progesterone-regulated gene promoters. Ligand activation of PR results in redistribution into discrete subnuclear foci that are detectable by immunofluorescence, probably representing aggregates of multiple transcriptionally active PR-coregulator complexes. PR foci are aberrant in cancers, suggesting that the coregulator composition and number of complexes is altered. A large family of coregulators is now described and the range of proteins known to bind PR exceeds the complement required for transcriptional activation, suggesting that in the human, tissue-specific coregulator expression may modulate progesterone response. In this review, we examine the role of nuclear localization of PR, coregulator association and tissue-specific expression in modulating progesterone action in the human.

DNA replication licensing and progenitor numbers are increased by progesterone in normal human breast.

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.

Trafficking of circulating pro-NK cells to the decidualizing uterus: regulatory mechanisms in the mouse and human.

Natural Killer (NK) lymphocytes, strongly expressing CD56, become abundant in the human uterus three to five days after the mid-menstrual cycle surge in pituitary-derived luteinizing hormone (LH). The primary functions of LH are to initiate final oocyte maturation/ovulation and to contribute to decidualization of the uterine stroma. Decidualization is the transformation of estrogen-primed uterine stromal fibroblasts into large hormone-producing cells under the influence of progesterone (P4). Decidual CD56bright (dNK) cells are a distinct, transient, tissue-specific NK cell subset that undergoes proliferation, terminal differentiation, and then death prior to menses. If pregnancy occurs, dNK cells increase during first trimester, then decline and are virtually absent in late pregnancy. In mouse models, pregnancy-associated uterine NK (uNK) cells appear coincident with onset of decidualization during embryonic implantation. Murine uNK cells traffic from the circulation to the antimesometrial side of the uterus and migrate to the mesometrial side of each implantation site. Here they proliferate and are implicated in regulation of midgestation structural changes to major arteries supplying the placenta, before dying in late gestation. Emerging data indicate that interactions between lymphocytes and endothelial cells within the uterine microenvironment are mediated by classical molecules associated with lymphocyte trafficking in immune surveillance and in response to inflammation. Here, we review factors influencing NK cell trafficking to decidualizing murine and human uteri and the differentiation and functions of these cells within the uterus.

Expression of progesterone receptor A and B isoforms in low-grade endometrial stromal sarcoma.

The progesterone receptor (PR) exists as two isoforms, PRA and PRB. In vitro studies have shown that these proteins are functionally distinct, suggesting that their relative expression can influence progesterone response. Low-grade endometrial stromal sarcoma (LGESS) is an uncommon tumor that usually expresses PR. In normal endometrial stroma, both PR isoforms are present with PRA predominant throughout the menstrual cycle. The relative expression of PRA and PRB in LGESS has not been previously reported. All nine cases of primary LGESS (seven uterine, two extrauterine) expressed PRB. Eight tumors also contained PRA and it was the predominant isoform in seven cases. These tumors had similar histopathologic appearances, whereas a case with approximately equal PR isoform expression showed features of sex cord or smooth muscle differentiation. An extrauterine tumor expressing only PRB had myxoid stroma. Recurrent tumor in two cases, which expressed predominantly PRA in the primary, contained reduced levels of PR consisting predominantly or entirely of PRB after prolonged interval progestin therapy. Most primary LGESSs showed PR isoform expression similar to normal endometrial stroma, consistent with the highly differentiated phenotype of this tumor. Variant differentiation or disease recurrence was accompanied by an altered PR isoform profile that could impact on hormone response.

Germ-line mutations in BRCA1 or BRCA2 in the normal breast are associated with altered expression of estrogen-responsive proteins and the predominance of progesterone receptor A.

The breast cancer susceptibility genes BRCA1 and BRCA2 are responsible for a large proportion of familial breast and ovarian cancer, yet little is known of how disruptions in the functions of the proteins these genes encode increased cancer risk preferentially in hormone-dependent tissue. There is no information on whether a germ-line mutation in BRCA1 or BRCA2 causes disruptions in hormone-signaling pathways in the normal breast. In this study markers of hormone responsiveness were measured in prophylactically removed normal breast tissue (n = 31) in women bearing a germ-line pathogenic mutation in one of the BRCA genes. The estrogen receptor (ER) and proteins associated with ER action in hormone-sensitive tissues, namely, PS2 and the progesterone receptor (PR), were detected immunohistochemically. ER expression was not different in BRCA mutation carriers than in noncarriers, but there was a reduction in PS2 expression. PR expression was also reduced, and there was a striking lack of expression of the PRB isoform, which resulted in cases with PRA-only expression in BRCA1 and BRCA2 mutation carriers. The alterations in PS2 and PR expression were similar in the BRCA1 and BRCA2 carriers, demonstrating that although these proteins are structurally and functionally distinct, there is overlap in their interaction with hormone-signaling pathways. This study provides evidence for altered cell function arising from loss of function of one BRCA allele in the normal breast, leading to PS2 loss, preferential PRB loss, and expression of PRA alone. In breast cancer development, PRA overexpression becomes evident in premalignant lesions and is associated with features of poor prognosis in invasive disease and altered cell function in vitro. The results of this study suggest that heterozygosity for a germ-line mutation in BRCA1 or BRCA2 results in development of PRA predominance. This is likely to lead to changes in progesterone signaling in hormone-dependent tissues, which may be a factor in the increased risk of cancer in these tissues in women with germ-line BRCA1 or BRCA2 mutations.