Susan A. Krum

Positive Cross-Regulatory Loop Ties GATA-3 to Estrogen Receptor α Expression in Breast Cancer

The transcription factor GATA-3 is required for normal mammary gland development, and its expression is highly correlated with estrogen receptor α (ERα) in human breast tumors. However, the functional role of GATA-3 in ERα-positive breast cancers is yet to be established. Here, we show that GATA-3 is required for estradiol stimulation of cell cycle progression in breast cancer cells. The role of GATA-3 in estradiol signaling requires the direct positive regulation of the expression of the ERα gene itself by GATA-3. GATA-3 binds to two cis-regulatory elements located within the ERα gene, and this is required for RNA polymerase II recruitment to ERα promoters. Reciprocally, ERα directly stimulates the transcription of the GATA-3 gene, indicating that these two factors are involved in a positive cross-regulatory loop. Moreover, GATA-3 and ERα regulate their own expression in breast cancer cells. Hence, this transcriptional coregulatory mechanism accounts for the robust coexpression of GATA-3 and ERα in human breast cancers. In addition, these results highlight the crucial role of GATA-3 for the response of ERα-positive breast cancers to estradiol. Moreover, they identify GATA-3 as a critical component of the master cell-type–specific transcriptional network including ERα and FoxA1 that dictates the phenotype of hormone-dependent breast cancer. [Cancer Res 2007;67(13):6477–83]

Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival

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.

Unique ERα Cistromes Control Cell Type-Specific Gene Regulation

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-ERalpha by expression microarrays. We find that fewer than 10% of the 17beta-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-ERalpha cells, we compared the ERalpha 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 ERalpha 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 ERalpha cistrome in MCF7 cells, it is not expressed in U2OS-ERalpha cells, and forkhead motifs are not enriched in the ERalpha cistrome in these cells. Finally, the ERalpha 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 ERalpha occupancy of epigenetically marked cis-regulatory regions of target genes.

WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer

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.

Novel functions for NFκB: inhibition of bone formation

NFκB is a family of transcription factors involved in immunity and the normal functioning of many tissues. It has been well studied in osteoclasts, and new data indicate an important role for NFκB in the negative regulation of bone formation. In this article, we discuss how NFκB activation affects osteoblast function and bone formation. In particular, we describe how reduced NFκB activity in osteoblasts results in an increase in bone formation via enhanced c-Jun N-terminal kinase (JNK) activity, which regulates FOSL1 (also known as Fra1) expression. Furthermore, we discuss how estrogen and NFκB crosstalk in osteoblasts acts to oppositely regulate bone formation. Future NFκB-targeting treatments for osteoporosis, rheumatoid arthritis and other inflammatory bone diseases could lead to increased bone formation concurrent with decreased bone resorption.

Coactivator Function Defines the Active Estrogen Receptor Alpha Cistrome

ABSTRACT 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.

A functional link between Wnt signaling and SKP2-independent p27 turnover in mammary tumors

Loss of the CDK inhibitor p27(KIP1) is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27(KIP1) were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27(KIP1). Interestingly we found that Wnt-induced turnover of p27(KIP1) was independent from classical SCF(SKP2)-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27(KIP1) in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27(KIP1) degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27(KIP1) levels and cell cycle progression in mammalian cells.

BRCA1 associates with processive RNA polymerase II

The human BRCA1 tumor suppressor interacts with transcriptional machinery, including RNA polymerase II (RNA pol II). We demonstrated that interaction with RNA pol II is a conserved feature of BRCA1 proteins from several species. We found that full-length BRCA1 proteins universally fail to activate transcription in classic GAL4-UAS one-hybrid assays and that the activity associated with the human BRCA1 C terminus was poorly conserved in closely related homologs of the gene. Fractionation studies demonstrated that BRCA1 proteins from all species tested interacted specifically with hyperphosphorylated pol II (IIO), in preference to hypophosphorylated RNA pol II (IIA) expected at promoters. BRCA1-RNA pol II complexes showed evidence of a multiply phosphorylated heptad repeat domain in the catalytic subunit (p220) of RNA pol II, and the complex was highly functional in transcriptional run-off assays. Interestingly, endogenous BRCA1 associated with a large fraction of the processive RNA pol II activity present in undamaged cells, and the interaction was disrupted by DNA-damaging agents. Preferential interaction with processive RNA pol II in undamaged cells places BRCA1 in position to link late events in transcription with repair processes in eukaryotic cells.

Direct transcriptional targets of sex steroid hormones in bone

The sex steroid hormones, androgens and estrogens, via their respective nuclear receptors, regulate bone mineral density in humans and mice. Very little is known about the direct targets of the androgen and estrogen receptors in bone cells. First, models of hormone and receptor deficiency in mouse and human bone are discussed. This review then focuses on the direct targets of the receptors in osteoblasts and osteoclasts. A direct target of a NR is defined here as a gene that is regulated by NR binding to the DNA (either through DNA binding or association with a DNA binding protein) at an enhancer or promoter of that gene. The experimental evidence that illustrates androgen and estrogen gene regulation in osteoblasts and osteoclasts will be summarized and compared with the phenotype of the hormones in vivo. J. Cell. Biochem. 112: 401–408, 2011.

Unraveling estrogen action in osteoporosis

A decrease in estrogen levels at menopause leads to a rapid loss of bone mineral density and an increase in fracture risk. For over ten years it has been known that the beneficial effects of estrogen are due in part to the ability of estrogen to suppress osteoclastogenic cytokine production in T-cells and osteoblasts. In addition to suppressing these cytokines, estrogen has been shown to induce the apoptotic death of osteoclasts. A variety of different mechanisms have been suggested to explain the estrogen regulation of osteoclast survival. One hypothesis is that estrogen, via rapid non-genomic signaling, induces apoptosis without the need for direct binding of estrogen receptor α (ERα) to DNA. A second hypothesis proposes that estrogen-stimulation of ERα in osteoclasts induces the expression Fas Ligand which in turn leads to cell death via an autocrine mechanism. In contrast, recent work from our lab has led to a genomic model of estrogen action in which estrogen acts to induce ERα binding to transcriptional enhancers in the Fas Ligand gene leading to its up-regulation in osteoblasts which through a paracrine mechanism induces apoptosis in osteoclasts. Here we will focus on these differing models of the mechanism of estrogen-mediated osteoclast apoptosis.