Christopher J. Ormandy

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women’s Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49fhi stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.

Cyclin D1, EMS1 and 11q13 amplification in breast cancer.

Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast where up to 15% carry this chromosomal abnormality. Originally 11q13 amplification was thought to involve a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. Although the region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich have made definitive identification of specific genes that contribute to the genesis and progression of breast cancer a difficult and continuing process. To date CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding the filamentous actin binding protein and c-Src substrate cortactin, are the favored candidates responsible for the emergence of two of the four amplification cores.

The Ets transcription factor Elf5 specifies mammary alveolar cell fate.

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development.

Osteoblasts Are a New Target for Prolactin: Analysis of Bone Formation in Prolactin Receptor Knockout Mice1

Bone development is a multistep process that includes patterning of skeletal elements, commitment of hematopoietic and/or mesenchymental cells to chondrogenic and osteogenic lineages, and further differentiation into three specialized cell types: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. Although PRL has a multitude of biological actions in addition to its role in the mammary gland, very little is known about its effect on bone. Mice carrying a germline null mutation for the PRL receptor gene have been produced in our laboratory and used to study the role of PRL in bone formation. In− /− embryos, we observed an alteration in bone development of calvaria. In adults, histomorphometric analysis showed that the absence of PRL receptors leads to a decrease in bone formation rate using double calcein labeling and a reduction of bone mineral density, measured by dual energy x-ray absorptiometry. In addition, serum estradiol, progesterone, testosterone, and PTH levels were analyzed. We al...

Mammary Gland Development in Prolactin Receptor Knockout Mice

A germ line null mutation of the prolactin receptor gene has been produced by replacing exon 5 with the Tk-NEO3 cassette. Heterozygous females showed almost complete failure of lactation following their first pregnancy when mated at 6–8 weeks of age. The severity of this phenotype was reduced when heterozygotes were mated at 20 weeks and was absent following a second pregnancy when assessed by pup survival, although a longer lag time prior to weight increase at normal rates was seen in these litters. Histological and whole mount analysis of virgin mammary glands showed that heterozygous glands were smaller in size due to a less developed ductal structure with fewer branch points. Virgin knockout glands were small, composed of sparse, large, poorly branched ducts. This work provides an ideal model to further study the role of the prolactin receptor and its ligands in mammary development and physiology.

Rescue of Preimplantatory Egg Development and Embryo Implantation in Prolactin Receptor-Deficient Mice after Progesterone Administration.

PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.

Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor‐bound (Grb)14, an adapter‐type signalling protein that associates with the insulin receptor (IR). Adult male Grb14−/− mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin‐induced 2‐deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue‐specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin‐induced tyrosine phosphorylation of insulin receptor substrate (IRS)‐1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS‐1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue‐specific modulator of insulin action, most likely via repression of IR‐mediated IRS‐1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

The role of prolactin and growth hormone in mammary gland development.

Development and differentiation of the mammary gland occur primarily during pregnancy. Females homozygous (-/-) for the null mutation of the PRL receptor (PRLR) gene are sterile due to a complete failure of blastocysts to implant. In progesterone-treated mice pregnancy is rescued but the mammary gland is severely underdeveloped. Interestingly, females hemizygous for the PRLR (+/-) in their first lactation show an almost complete failure to lactate. This phenotype disappears in the second and subsequent pregnancies in inbred 129/Sv mice but is maintained in inbred C57BL/6 mice. In GH receptor (GHR) KO mice litter size is markedly decreased, probably due to an ovarian defect. To assess the relevance of the GH and PRLRs in the mammary gland development, GHR and PRLR null epithelia were transplanted into cleared fat pads of wild-type mice. Such studies show that epithelial GHR is not required for functional mammary development. In contrast, epithelial PRLRs are required for mammary development and milk protein gene expression during pregnancy. Since ductal development is impaired in GHR -/- mice, it appears that GH signals through the stromal compartment. In summary, it is now established that GH and PRL activate Stat5 in separate compartments, reflecting their specific roles in ductal and alveolar development and differentiation.

Key stages in mammary gland development - The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium

Massive tissue remodelling occurs within the mammary gland during pregnancy, resulting in the formation of lobuloalveoli that are capable of milk secretion. Endocrine signals generated predominantly by prolactin and progesterone operate the alveolar switch to initiate these developmental events. Here we review the current understanding of the components of the alveolar switch and conclude with an examination of the role of the ets transcription factor Elf5. We propose that Elf5 is a key regulator of the alveolar switch.

Hedgehog Overexpression Is Associated with Stromal Interactions and Predicts for Poor Outcome in Breast Cancer

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.