Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone‐forming osteoblasts and bone‐resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen‐induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre‐osteoclasts. We have characterized a cell‐type‐specific hormone‐inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor‐alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre‐osteoclasts by the same osteoblast‐dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre‐osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

Wnt/β‐catenin signalling has been suggested to be active in basal‐like breast cancer. However, in highly aggressive metastatic triple‐negative breast cancers (TNBC) the role of β‐catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active β‐catenin in human TNBC and predicts survival‐outcome of patients with both TNBC and basal‐like tumours. We provide evidence that transgenic murine Wnt10b ‐driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical β‐catenin signalling leading to up‐regulation of HMGA2. Treatment of mouse and human triple‐negative tumour cells with two Wnt/β‐catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse‐free‐survival and metastasis in TNBC patients.

Estrogens play an important role in normal physiology and in a variety of pathological states involving diverse tissues including breast and bone. The mechanism by which estrogens exert cell type- and disease-specific effects, however, remains to be explained. We have compared the gene expression profile of the MCF7 breast cancer cell line with that of the osteoblast-like cell line U2OS-ERα by expression microarrays. We find that fewer than 10% of the 17β-estradiol (E2)-regulated genes are common to both cell types. We have validated this in primary calvarial osteoblasts. To dissect the mechanism underlying the cell type-specific E2 regulation of gene expression in MCF7 and U2OS-ERα cells, we compared the ERα binding sites on DNA in the two cell types by performing chromatin immunoprecipitation (ChIP) on genomic tiling arrays (ChIP-on-chip). Consistent with the distinct patterns of E2-regulated gene expression in these two cell lines, we find that the vast majority of ERα binding sites are also cell type specific and correlate both in position and number with cell type-specific gene regulation. Interestingly, although the forkhead factor FoxA1 plays a critical role in defining the ERα cistrome in MCF7 cells, it is not expressed in U2OS-ERα cells, and forkhead motifs are not enriched in the ERα cistrome in these cells. Finally, the ERα cistromes are correlated with cell type-specific epigenetic histone modifications. These results support a model for the cell type-specific action of E2 being driven primarily through specific ERα occupancy of epigenetically marked cis-regulatory regions of target genes.

Proper activation of transcriptional networks in complex organisms is central to the response to stimuli. We demonstrate that the selective activation of a subset of the estrogen receptor alpha (ERα) cistrome in MCF7 breast cancer cells provides specificity to the estradiol (E2) response. ERα-specific enhancers that are subject to E2-induced coactivator-associated arginine methyltransferase 1 (CARM1) action are critical to E2-stimulated gene expression. This is true for both FoxA1-dependent and independent enhancers. In contrast, a subset of E2-suppressed genes are controlled by FoxA1-independent ERα binding sites. Nonetheless, these are sites of E2-induced CARM1 activity. In addition, the MCF7 RNA polymerase II cistrome reveals preferential occupancy of E2-regulated promoters prior to stimulation. Interestingly, E2-suppressed genes tend to lie in otherwise silent genomic regions. Together, our results suggest that the transcriptional response to E2 in breast cancer cells is dependent on the interplay between polymerase II pre-occupied promoters and the subset of the ERα cistrome associated with coactivation.