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Adenocarcinoma cells from the pleural effusion of a patient with breast cancer were injected into the mammary glands of nude mice and grown into solid tumors. A cell line derived from these tumors expressed α-smooth muscle actin but not human cytokeratin 7, indicating “activated” stroma of mouse origin. Cells in mitosis exhibited mainly polyploid mouse karyotypes, but 30% had mixed mouse and human chromosomes, among which 8% carried mouse/human translocations. Nuclei of interphase cells were 64% hybrid. Hybrid mouse/human nuclei were also detected in the primary xenograft. Thus, synkaryons formed in the solid tumor by spontaneous fusion between the malignant human epithelium and the surrounding normal host mouse stroma. The transformed stroma-derived cells are tumorigenic with histopathologic features of malignancy, suggesting a new mechanism for tumor progression. (Cancer Res 2006; 66(16): 8274-9)

Supplementary Figure 1 from Estrogen Insensitivity in a Model of Estrogen Receptor–Positive Breast Cancer Lymph Node Metastasis

The lymphatic system is a common avenue for the spread of breast cancer cells and dissemination through it occurs at least as frequently as hematogenous metastasis. Approximately 75% of primary breast cancers are estrogen receptor (ER) positive and the majority of these maintain receptor expression as lymph node (LN) metastases. However, it is unknown if ER function is equivalent in cancer cells growing in the breast and in the LNs. We have developed a model to assess estrogen responsiveness in ER+ breast tumors and LN metastases. Fluorescent ER+ MCF-7 tumors were grown in ovariectomized nude mice supplemented with estradiol. Once axillary LN metastasis arose, estradiol was withdrawn (EWD), for 1 or 4 weeks, or continued, to assess estradiol responsiveness. On EWD, proliferation rates fell similarly in tumors and LN metastases. However, estradiol-dependent ER down-regulation and progesterone receptor induction were deficient in LN metastases, indicating that ER-dependent transcriptional function was altered in the LN. Cancer cells from estradiol-treated and EWD primary tumors and matched LN metastases were isolated by laser capture microdissection. Global gene expression profiling identified transcripts that were regulated by the tissue microenvironment, by hormones, or by both. Interestingly, numerous genes that were estradiol regulated in tumors lost estradiol sensitivity or were regulated in the opposite direction by estradiol in LN metastases. We propose that the LN microenvironment alters estradiol signaling and may contribute to local antiestrogen resistance. [Cancer Res 2007;67(21):10582–91]

Supplementary Figures 1-3 from Tissue-Specific Pathways for Estrogen Regulation of Ovarian Cancer Growth and Metastasis

Supplementary Video from Spontaneous Fusion with, and Transformation of Mouse Stroma by, Malignant Human Breast Cancer Epithelium

Supplementary Tables 1-4 from Tissue-Specific Pathways for Estrogen Regulation of Ovarian Cancer Growth and Metastasis

Progesterone and progesterone receptors (PR) have a storied albeit controversial history in breast cancers. As endocrine therapies for breast cancer progressed through the twentieth century from oophorectomy to antiestrogens, it was recognized in the 1970s that the presence of estrogen receptors (ER) alone could not efficiently predict treatment responses. PR, an estrogen regulated protein, became the first prognostic and predictive marker of response to endocrine therapies. It remains today as the gold standard for predicting the existence of functional, targetable ER in breast malignancies. PRs were subsequently identified as highly structured transcription factors that regulate diverse physiological processes in breast cancer cells. In the early 2000s, the somewhat surprising finding that prolonged use of synthetic progestin-containing menopausal hormone therapies was associated with increased breast cancer incidence raised new questions about the role of PR in ‘tumorigenesis’. Most recently, PR have been linked to expansion of cancer stem cells that are postulated to be the principal cells reactivated in occult or dormant disease. Other studies establish PR as dominant modulators of ER activity. Together, these findings mark PR as bona fide targets for progestin or antiprogestin therapies, yet their diverse actions have confounded that use. Here we summarize the early history of PR in breast cancer; debunk the theory that progesterone causes cancer; discuss recent discoveries that PR regulate cell heterogeneity; attempt to unify theories describing PR as either good or bad actors in tumors; and discuss emerging areas of research that may help explain this enigmatic hormone and receptor.

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient — that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146(+)) or lack (CD146(–)) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146(–) fibroblasts promoted increased metastasis compared with CD146(+) fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146(+) CAFs produced an environment rich in basement membrane proteins, while CD146(–) CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146(–) CAFs predicted for likelihood of lymph node involvement even in small primary tumors (

Luminal breast cancers express estrogen (ER) and progesterone (PR) receptors, and respond to endocrine therapies. However, some ER+PR+ tumors display intrinsic or acquired resistance, possibly related to PR. Two PR isoforms, PR-A and PR-B, regulate distinct gene subsets that may differentially influence tumor fate. A high PR-A:PR-B ratio is associated with poor prognosis and tamoxifen resistance. We speculate that excessive PR-A marks tumors that will relapse early. Here we address mechanisms by which PR-A regulate transcription, focusing on SUMOylation. We use receptor mutants and synthetic promoter/reporters to show that SUMOylation deficiency or the deSUMOylase SENP1 enhance transcription by PR-A, independent of the receptors' dimerization interface or DNA binding domain. De-SUMOylation exposes the agonist properties of the antiprogestin RU486. Thus, on synthetic promoters, SUMOylation functions as an independent brake on transcription by PR-A. What about PR-A SUMOylation of endogenous human breast cancer genes? To study these, we used gene expression profiling. Surprisingly, PR-A SUMOylation influences progestin target genes differentially, with some upregulated, others down-regulated, and others unaffected. Hormone-independent gene regulation is also PR-A SUMOylation dependent. Several SUMOylated genes were analyzed in clinical breast cancer database. In sum, we show that SUMOylation does not simply repress PR-A. Rather it regulates PR-A activity in a target selective manner including genes associated with poor prognosis, shortened survival, and metastasis.

Luminal breast cancers represent approximately 75% of cases. Explanations into the causes of endocrine resistance are complex and are generally ascribed to genomic mechanisms. Recently, attention has been drawn to the role of epigenetic modifications in hormone resistance. We review these here. Epigenetic modifications are reversible, heritable and include changes in DNA methylation patterns, modification of histones and altered microRNA expression levels that target the receptors or their signaling pathways. Large-scale analyses indicate distinct epigenomic profiles that distinguish breast cancers from normal and benign tissues. Taking advantage of the reversibility of epigenetic modifications, drugs that target epigenetic modifiers, given in combination with chemotherapies or endocrine therapies, may represent promising approaches to restoration of therapy responsiveness in these cases.

Progesterone plays a key role in the development, differentiation and maintenance of female reproductive tissues and has multiple non-reproductive neural functions. Depending on the cell and tissue, the hormonal environment, growth conditions and the developmental stage, progesterone can either stimulate cell growth or inhibit it while promoting differentiation. Progesterone receptors (PRs) belong to the steroid hormone receptor superfamily of ligand-dependent transcription factors. PR proteins are subject to extensive post-translational modifications that include phosphorylation, acetylation, ubiquitination and SUMOylation. The interplay among these modifications is complex with alteration of the receptors by one factor influencing the impact of another. Control over these modifications is species-, tissue- and cell-specific. They in turn regulate multiple functions including PR stability, their subcellular localization, protein-protein interactions and transcriptional activity. These complexities may explain how tissue- and gene-specific differences in regulation are achieved in the same organism, by the same receptor protein and hormone. Here we review current knowledge of PR post-translational modifications and discuss how these may influence receptor function focusing on human breast cancer cells. There is much left to be learned. However, our understanding of this may help to identify therapeutic agents that target PR activity in tissue-specific, even gene-specific ways.

All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.

Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like “triple negative” cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that more than half of primary ER + PR + breast cancers contain an ER − PR − CK5 + “luminobasal” subpopulation exceeding 1% of cells. Starting from ER + PR + luminal cell lines, we generated lines with varying luminal to luminobasal cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone-resistant cells matches that of clinical triple negative basal-like and claudin-low disease. Luminobasal cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by γ-secretase inhibitors. Our data establish a previously unrecognized plasticity of ER + PR + luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition.

Patients with estrogen receptor (ER)+ breast cancer are at chronic and prolonged risk of metastatic recurrences, which are usually fatal. The most frequent sites of ER+ breast cancer metastasis are bone, liver, lung, and brain, with individual tumors preferring one or more sites. The underlying mechanisms of site-specific metastasis are poorly understood and are critical for prevention and treatment of metastases to more lethal (brain, liver, lung) vs. static (bone) sites. The purpose of this study is to use single-cell RNA sequencing (sc RNA-seq) of ER+ breast cancer cells dispersed to breast tropic sites to define unique molecular determinants of organ-specific metastasis. Our group has developed ER+ patient-derived xenografts (PDX) at the University of Colorado Denver (UCD) labeled with constitutive luciferase-GFP. Our ER+ PDX fall into two general categories-pure “luminal” tumors with high ER plus/minus progesterone receptor (PR) (>50% of cells) and an absence of cytokeratin 5 (CK5)+ cells, and “luminobasal” tumors with a low percent of ER+PR+ cells (5-10%) and high CK5+ cells (15-90%). Using intracardiac (ic) dispersal as a measure of late-stage metastasis, we have defined the organ homing preference of several of these lines. Luminal PDX frequently colonize bone, with occasional secondary lesions to brain and liver. Luminobasal PDX ubiquitously colonize liver, with secondary lesions mostly to lung. We performed sc RNA-seq of luminal UCD65 brain metastatic and luminobasal UCD46 liver metastatic cells compared to their cognate primary tumors grown in the mammary fat pad. UCD65 brain metastatic vs. primary tumor cells have elevated expression of PR and carbonic anhydrase 2 (CA2), a cellular guard against intracellular pH changes, and a prospective PR-regulated gene. UCD46 liver metastatic cells have activated TGFbeta signaling, and increased cancer stem cell markers. We conclude that ER+ breast cancer PDX retain innate preference for specific breast-tropic organs, that this may relate to their ER+ subtype (luminal vs. luminobasal), and that sc RNA-seq may identify key molecular signatures of ER+ breast cancer metastases. We are profiling additional tumors and sites to make further comparisons and generate testable hypotheses. Furthermore, our models allow for functional studies to test innovative interventions and therapeutics that target site-specific metastases. This work was supported by grants from the Breast Cancer Research Foundation (16-072, CAS, KBH, PK), the NIH (CA140985, CAS) and the UCD RNA Biosciences Initiative (AEG, CAS, PK). Citation Format: Nuria Padilla Just, Jessica Finlay-Schultz, Austin E. Gillen, Diana M. Cittelly, Elizabeth A. Wellberg, Kathryn B. Horwitz, Peter Kabos, Carol A. Sartorius. Single-cell RNA sequencing defines regulatory networks in ER+ breast cancer organ-specific metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3027.

Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.

We have conducted an in silico analysis of progesterone response elements (PRE) in progesterone receptor (PR) up-regulated promoters. Imperfect inverted repeats, direct repeats, and half-site PRE are widespread, not only in PR-regulated, but also in non-PR-regulated and random promoters. Few resemble the commonly used palindromic PRE with three nucleotide (nt) spacers. We speculated that PRE may be necessary but insufficient to control endogenous PR-dependent transcription. A search for PRE partners identified a highly conserved 234-nt sequence invariably located within 1-2 kb of transcription start sites. It resembles ALU repeats and contains binding sites for 11 transcription factors. The 234-nt sequence of the PR-regulated 8-oxoguanine DNA glycosylase promoter was cloned in the forward or reverse orientation in front of zero, one, or two inverted repeat PRE, and one or tandem PRE half-sites, driving luciferase. Under these conditions the 234-nt sequence functions as a co-response element (coRE). From the PRE or tandem half-sites, the reverse coRE is a strong activator of PR and glucocorticoid receptor-dependent transcription. The forward coRE is a powerful repressor. The prevalence of PRE half-sites in natural promoters suggested that PR monomers regulate transcription. Indeed, dimerization-domain mutant PR monomers were stronger transactivators than wild-type PR on PRE or tandem half-sites. This was repressed by the forward coRE. We propose that in natural promoters the coRE functions as a composite response element with imperfect PRE and half-sites to present variable, orientation-dependent transcription factors for interaction with nearby PR.

This transcript is based on my The Year in Basic Science lecture at ENDO 2008. I reviewed current data surrounding hormone replacement therapy and the relationship between systemic estrogen plus progestin (E+P) treatment and increased breast cancer risk, and I explored the hypothesis that women who develop breast cancer while on E+P had occult, undiagnosed disease before they started therapy. Beginning with recent hormone replacement therapy data focusing on E+P and its association with breast cancer to set the stage, the lecture then reviewed our newly published data that progestins expand breast cancer stem cells. Finally, the issues of occult or undiagnosed breast cancer in presumably healthy women, and of tumor dormancy in breast cancer survivors, were brought to bear on the discussion. Taken together, these apparently disparate themes allowed me to suggest the idea that systemic progestins have the ability to reawaken cancers that were presumed to be either nonexistent or cured. To avoid this potentially devastating outcome while retaining the benefits of E+P, I advocated the use of local P delivery methods, rather than the currently popular systemic routes.

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.

There are two major subtypes of human breast cancers: the luminal, estrogen, and progesterone receptor-positive, cytokeratin 18-positive (ER + PR + CK18 + ) subtype, and the basal ER − PR − CK18 − CK5 + subtype. Tumor-initiating cells (CD44 + ) have been described for human breast cancers; whether these are common to the two subtypes is unknown. We have identified a rare population of cells that are both CD44 + and ER − PR − CK5 + in luminal-like ER + PR + T47D human breast tumor xenografts. The tumor-isolated CD44 + cell fraction was highly enriched for clonogenic ( in vitro culture) and tumorigenic ( in vivo reimplantation) cells compared with the CD44 − cell fraction. Rare ER − PR − CK5 + cells were present within CD44 + -derived colonies. Tumor-isolated cells placed in minimal media also contained rare ER − PR − CK5 + cells at early time points (+ PR + CK5 − cells, conversely, increased linearly with colony growth. Similary, tumors originating in vivo from CD44 + cells contained a rare static ER − PR − CK5 + population, an intermediate ER − PR − CK5 − population, and an expanding ER + PR + CK5 − population. Putative ER + PR + CK5 + transitional cells could be seen only in colonies or tumors treated with a progestin. We propose that luminal ER + PR + breast tumors contain a minor ER − PR − CK5 + population that has the capacity to generate the majority of ER + PR + CK18 + CK5 − cells. Luminal breast cancers are treated with endocrine therapies that target ER. The rare ER − PR − CK5 + progenitor cells would escape such treatments and survive to repopulate the tumor.

Approximately 30% of patients with estrogen receptor (ER) positive breast cancers exhibit de novo or intrinsic resistance to endocrine therapies. The purpose of this study was to define genes that distinguish ER+ resistant from ER+ responsive tumors, prior to the start of hormone therapies. Previously untreated post-menopausal patients with ER+ breast cancers were treated for 4 months in a neoadjuvant setting with the aromatase inhibitor exemestane alone, or in combination with the antiestrogen tamoxifen. Matched pre- and post-treatment tumor samples from the same patient, were analyzed by gene expression profiling and were correlated with response to treatment. Genes associated with tumor shrinkage achieved by estrogen blockade therapy were identified, as were genes associated with resistance to treatment. Prediction Analysis of Microarrays (PAM) identified 50 genes that can predict response or intrinsic resistance to neoadjuvant endocrine therapy of ER+ tumors, 8 of which have been previously implicated as useful biomarkers in breast cancer. In summary, we identify genes associated with response to endocrine therapy that may distinguish ER+, hormone responsive breast cancers, from ER+ tumors that exhibit intrinsic or de novo resistance. We suggest that the estrogen signaling pathway is aberrant in ER+ tumors with intrinsic resistance. Lastly, the studies show upregulation of a "lipogenic pathway" in non-responsive ER+ tumors that may serve as a marker of intrinsic resistance. This pathway may represent an alternative target for therapeutic intervention.

The two, nearly identical, isoforms of human progesterone receptors (PR), PR-B and -A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear receptors. Other PR-B functions—N-/C-terminal interactions, steroid receptor coactivator-1 coactivation, ligand-dependent down-regulation—also require an intact BUS. All three are autonomous in PR-A, and map to N-terminal regions common to both PR. This suggests that the N-terminal structure adopted by the two PR is different, and that for PR-B, this is controlled by BUS. Indeed, gene expression profiling of breast cancer cells stably expressing PR-B, PR-BdL140, or PR-A shows that mutation of AF3 destroys PR-B-dependent gene transcription without converting PR-B into PR-A. In sum, AF3 in BUS plays a critical modulatory role in PR-B, and in doing so, defines a mechanism for PR-B function that is fundamentally distinct from that of PR-A.

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of E1 (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Down-regulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.

Activation of the type I insulin-like growth factor receptor (IGF-IR) regulates several aspects of the malignant phenotype, including cancer cell proliferation and metastasis. Phosphorylation of adaptor proteins downstream of IGF-IR may couple IGF action to specific cancer phenotypes. In this study, we sought to determine if insulin receptor substrate-1 and -2 (IRS-1 and -2) mediate distinct biological effects in breast cancer cells. Insulin receptor substrate-1 and IRS-2 were expressed in T47D-YA breast cancer cells, which lack IRS-1 and -2 expression, yet retain functional IGF-IR. In the absence of IRS-1 and -2 expression, IGF-IR activation was unable to stimulate proliferation or motility in T47D-YA cells. Expression of IRS-1 resulted in IGF-I-stimulated proliferation, but did not affect motility. In contrast, expression of IRS-2 enhanced IGF-I-stimulated motility, but did not stimulate proliferation. The alphaIR-3, an inhibitor of the IGF-IR, was unable to affect these IGF-stimulated phenotypes unless IRS-1 or -2 was expressed. Thus, IGF-IR alone is unable to regulate important breast cancer cell phenotypes. In these cells, IRS proteins are required for and mediate distinct aspects of IGF-IR-stimulated behaviour. As multiple agents targeting the IGF-IR are currently in early clinical trials, IRS expression should be considered as a potential biomarker for IGF-IR responsiveness.

The transcription factor ZEB1 (δEF1 in mice) has been implicated in cellular processes during development and tumor progression including epithelial to mesenchymal transition. δEF1 null mice die at birth, but heterozygotes expressing a LacZ reporter inserted into the δEF1 gene live and reproduce. Using these mice, we observed ZEB1 promoter activity in the virgin myometrium, and stroma and myometrium of the pregnant uterus. ZEB1 protein is up-regulated in the myometrium and endometrial stroma after progesterone or estrogen treatment of ovariectomized mice. In the normal human uterus, ZEB1 protein is increased in the myometrium and stroma during the secretory stage of the menstrual cycle. ZEB1 is not expressed in the normal endometrial epithelium. In malignancies of the uterus, we find that ZEB1 (a) is overexpressed in malignant tumors derived from the myometrium (leiomyosarcomas), (b) is overexpressed in tumor-associated stroma of low-grade endometrioid adenocarcinomas, and (c) is aberrantly expressed in the tumor epithelial cells of aggressive endometrial cancers. Specifically, in grade 3 endometrioid adenocarcinomas and uterine papillary serous carcinomas, ZEB1 could be expressed in the epithelial-derived carcinoma cells as well as in the stroma. In malignant mixed Müllerian tumors, the sarcomatous component always expresses ZEB1, and the carcinomatous component can also be positive. In summary, ZEB1 is normally regulated by both estrogen and progesterone receptors, but in uterine cancers, it is likely no longer under control of steroid hormone receptors and becomes aberrantly expressed in epithelial-derived tumor cells, supporting a role for ZEB1 in epithelial to mesenchymal transitions associated with aggressive tumors. (Cancer Res 2006; 66(7): 3893-902)

In breast cancers, estrogen receptor (ER) levels are highly correlated with response to endocrine therapies. We sought to define mechanisms of estrogen (E) signaling in a solid breast tumor model using gene expression profiling. ER+ T47D-Y human breast cancer cells were grown as xenografts in ovariectomized nude mice under four conditions: 1) 17β-estradiol for 8 wk (E); 2) without E for 8 wk (control); 3) E for 7 wk followed by 1 wk of E withdrawal (Ewd); or 4) E for 8 wk plus tamoxifen for the last week. E-regulated genes were defined as those that differed significantly between control and E and/or between E and Ewd or control and Ewd. These protocols generated 188 in vivo E-regulated genes that showed two major patterns of regulation. Approximately 46% returned to basal states after Ewd (class I genes); 53% did not (class II genes). In addition, more than 70% of class II-regulated genes also failed to reverse in response to tamoxifen. These genes may be interesting for the study of hormone-resistance issues. A subset of in vivo E-regulated genes appears on lists of clinical ER discriminator genes. These may be useful therapeutic targets or markers of E activity. Comparison of in vivo E-regulated genes with those regulated in identical cells in vitro after 6 and 24 h of E treatment demonstrate only 11% overlap. This indicates the extent to which gene expression profiles are uniquely dependent on hormone-treatment times and the cellular microenvironment.

Introduction Medroxyprogesterone acetate (MPA), the major progestin used for oral contraception and hormone replacement therapy, has been implicated in increased breast cancer risk. Is this risk due to its progestational or androgenic properties? To address this, we assessed the transcriptional effects of MPA as compared with those of progesterone and dihydrotestosterone (DHT) in human breast cancer cells. Method A new progesterone receptor-negative, androgen receptor-positive human breast cancer cell line, designated Y-AR, was engineered and characterized. Transcription assays using a synthetic promoter/reporter construct, as well as endogenous gene expression profiling comparing progesterone, MPA and DHT, were performed in cells either lacking or containing progesterone receptor and/or androgen receptor. Results In progesterone receptor-positive cells, MPA was found to be an effective progestin through both progesterone receptor isoforms in transient transcription assays. Interestingly, DHT signaled through progesterone receptor type B. Expression profiling of endogenous progesterone receptor-regulated genes comparing progesterone and MPA suggested that although MPA may be a somewhat more potent progestin than progesterone, it is qualitatively similar to progesterone. To address effects of MPA through androgen receptor, expression profiling was performed comparing progesterone, MPA and DHT using Y-AR cells. These studies showed extensive gene regulatory overlap between DHT and MPA through androgen receptor and none with progesterone. Interestingly, there was no difference between pharmacological MPA and physiological MPA, suggesting that high-dose therapeutic MPA may be superfluous. Conclusion Our comparison of the gene regulatory profiles of MPA and progesterone suggests that, for physiologic hormone replacement therapy, the actions of MPA do not mimic those of endogenous progesterone alone. Clinically, the complex pharmacology of MPA not only influences its side-effect profile; but it is also possible that the increased breast cancer risk and/or the therapeutic efficacy of MPA in cancer treatment is in part mediated by androgen receptor.

Progesterone in hormone replacement therapy (HRT) preparations increases, while hysterectomy greatly reduces, the incidence of breast cancer. Cross-talk between the progesterone and growth factor signaling pathways occurs at multiple levels and this maybe a key factor in breast cancer survival and progression. To test this hypothesis, we characterized the effect of progesterone pre-treatment on the sensitization of the epidermal growth factor (EGF) signaling pathway to EGF in the breast cancer cell line ZR-75. For the first time in ZR-75 cells and in agreement with previous work using synthetic progestins, we demonstrate that pre-treatment with the natural ligand progesterone increases EGF receptor (EGFR) levels and subsequent ligand-dependent phosphorylation. Downstream we demonstrate that progesterone alone increases erk-1 + 2 phosphorylation, potentiates EGF-phosphorylated erk-1 + 2 and maintains these levels elevated for 24 h; over 20 h longer than in vehicle treated cells. Additionally, progesterone increased the levels of STAT5, another component of the EGF signaling cascade. Progesterone increased EGF mediated transcription of a c-fos promoter reporter and the nuclear localization of the native c-fos protein. Furthermore, progesterone and EGF both alone and in combination, significantly increase cell proliferation. Several results presented herein demonstrate the conformity between the action of the natural ligand progesterone with that of synthetic progestins such as MPA and R5020 and allows the postulation that the progestin/progesterone-dependent increase of EGF signaling provides a survival advantage to burgeoning cancer cells and may contribute to the breast cancer risk associated with endogenous progesterone and with progestin-containing HRT.

The progesterone receptor (PR) is a ligand-dependent transcription factor that promotes progestin-stimulated expression of target genes. Two functional PR isoforms, PRA and PRB, are expressed in progestin-responsive cells. PRA and PRB have distinct roles in gene expression and in mammary gland development. One role of PRs in T47-D cells is regulating expression of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor. This study explores the isoform specificity of this PR function using parental T47-Dco cells that express both PRA and PRB and clonal derivatives that express either PRA (YA cells) or PRB (YB cells) or lack PR (Y cells). Treatment with progesterone induces VEGF mRNA and protein ∼2-fold in T47-Dco and YA cells and 3–7-fold in YB cells, suggesting that PRA inhibits PRB-dependent induction of VEGF. This is consistent with the observation that clinically relevant progestins induce a much higher level of VEGF in YB cells than in YA cells. Another novel finding in this report is that estradiol (10−8 m) induces VEGF production from YB cells. However, this induction is not blocked by 100-fold excess tamoxifen or ICI-182,780. Moreover, both tamoxifen (10−6 m) and ICI-182,780 (10−6 m) function as agonists for VEGF in YB cells. Small interfering RNA against PR or estrogen receptor abrogated estradiol and tamoxifen induction, indicating that the agonist-like response of these compounds in YB cells is estrogen receptor and PR dependent. Estradiol, tamoxifen, and ICI-182780 also induce VEGF in BT-474 cells when their PRB levels were elevated by transfecting an expression plasmid for PRB, but not when the cells were transfected with vector alone. These results indicate that (a) PRB preferentially regulates VEGF expression in breast cancer cells and (b) PRB-enriched tumor cells may produce more VEGF, have a better developed vasculature, and potentially are more resistant to tamoxifen and ICI-182,780 than cells that express an equivalent or higher level of PRA than PRB. These results imply that PRB-enriched breast tumors may respond well to anticancer therapies that include inhibitors of angiogenesis.

Progesterone receptors (PR) are present in two isoforms, PR-A and PR-B. The B-upstream segment (BUS) of PR-B is a 164 amino acid N-terminal extension that is missing in PR-A and is responsible for the functional differences reported between the two isoforms. BUS contains an activation function (AF3) which is defined by a core domain between residues 54-154 whose activity is dependent upon a single Trp residue and two LXXLL motifs. We have also identified sites both within and outside of BUS that repress the strong synergism between AF3 and AF1 in the N-terminal region and AF2 in the hormone binding domain. One of these repressor sites is a consensus binding motif for the small ubiquitin-like modifier protein, SUMO-1 (387IKEE). The DNA binding domain (DBD) structure is also important for function. When BUS is linked to the glucocorticoid receptor DBD, AF3 activity is substantially attenuated, suggesting that binding to a DNA response element results in allosteric communication between the DBD and N-terminal functional regions. Lastly, biochemical and biophysical analyses of highly purified PR-B and PR-A N-terminal regions reveal that they are unstructured unless the DBD is present. Thus, the DBD stabilizes N-terminal structure. We propose a model in which the DBD through DNA binding, and BUS through protein-protein interactions, stabilize active receptor conformers within an ensemble distribution of active and inactive conformational states. This would explain why PR-B are stronger transactivators than PR-A.

Background Luminal, estrogen receptor-positive breast cancers represent more than 70% of cases. Despite initial good prognoses one third of Luminal cancers eventually recur locally or at distant sites and exhibit hormone resistance. Here we demonstrate that factors elaborated by malignant stromal cells can induce Luminal tumor cells proliferation and promote angiogenesis and hormone independence. We recently isolated a malignant mouse mammary gland stromal cell line named BJ3Z that increases proliferation and angiogenesis in estrogen-free xenografted Luminal MCF-7 breast cancer cells. Methods BJ3Z and Normal mouse mammary Fibroblasts (NMFs) were expression profiled using microarray assays. Messenger RNA levels were confirmed by RT-PCR and by immunohistochemistry (IHC). Breast cancer MCF-7, BT-474, BT-20 and MDA-MB-231cell lines and stromal BJ3Z and NMFs were grown for in vitro assays: breast cancer cell lines were treated with stromal cells conditioned media, for three-dimensional (3D) mono and co-cultures in Matrigel, proliferation was measured by Bromo-deoxyuridine (BrdU) incorporation using IHC. Tubule formation in vitro, a proxy for angiogenesis, was assessed using 3D cultured Human Umbilical cord Vascular Endothelial Cells (HUVEC). Results We show that under estrogen-free conditions, BJ3Z cells but not NMFs increase proliferation of co-cultured Luminal but not basal-like human breast cancer cells in 2D or as 3D Matrigel colonies. Gene expression profiling, RT-PCR analysis and IHC of colony-derived BJ3Z cells and NMFs shows that Platelet Derived Growth Factor ligands (PDGF-A and -B) are elaborated by BJ3Z cells but not NMFs; while PDGF receptors are present on NMFs but not BJ3Z cells. As a result, in colony co-culture assays, BJ3Z cells but not NMFs increase MCF-7 cell proliferation. This can be mimicked by direct addition of PDGF-BB, and blocked by the PDGF receptor inhibitor Imatinib Mesylate. Both normal and malignant stromal cells enhance angiogenesis in an in vitro model. This effect is also due to PDGF and is suppressed by Imatinib. Conclusions We provide evidence that Luminal breast cancer cells can be targeted by the PDGF signaling pathway leading to estrogen-independent proliferation and angiogenesis. We speculate that stroma-directed therapies, including anti-PDGFR agents like Imatinib, may be useful in combination with other therapies for treatment of luminal cancers. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-735) contains supplementary material, which is available to authorized users.

Introduction Luminal, estrogen receptor-positive (ER+) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models. Methods Immunocompromised, ovariectomized (ovx’d) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER+PR+ cytokeratin 5-negative (CK5−) E3 and MCF-7 cells, basal ER−PR−CK5+ estrogen withdrawn-line 8 (EWD8) cells, and basal ER−PR−CK5− MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx’d mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers. Results ER−PR− cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx’d mice injected with ER+PR+ cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER+PR+CK5− cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER−PR−CK5+ cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx’d control mice injected with ER+PR+ cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER+ but PR−. Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration. Conclusions We conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER−PR− subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx’d mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0489-4) contains supplementary material, which is available to authorized users.

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the “B-upstream segment” (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54–90 (R1) and 120–154 (R2). R1 contains a consensus 55LXXLL59 motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at115LXXLL119 and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

To understand the basis for functional differences between the two human progesterone receptors (PR), we have carried out a detailed biochemical and biophysical analysis of the N-terminal region of each isoform. Extending our previous work on the A-isoform (Bain, D. L, Franden, M. A., McManaman, J. L., Takimoto, G. S., and Horwitz, K. B. (2000) J. Biol. Chem. 275, 7313-7320), here we present studies on the N-terminal region of the B-isoform (NT-B) and compare its properties to its A-receptor counterpart (NT-A). As seen previously with NT-A, NT-B is quantitatively monomeric in solution, yet undergoes N-terminal-mediated assembly upon DNA binding. Limited proteolysis, microsequencing, and sedimentation analyses indicate that the B-isoform exists in a non-globular, extended conformation very similar to that of NT-A. Additionally, the 164 amino acids unique to the B-isoform (BUS) appear to be in a more extended conformation relative to sequences common to both receptors and do not exist as an independent structural domain. However, sedimentation studies of NT-A and NT-B show differences in the ensemble distribution of their conformational states. We hypothesize that isoform-specific functional differences are not due to structural differences, per se. Rather, the transcriptional element BUS, or possibly other transcription factors, causes a redistribution of the conformational ensemble by stabilizing a more functionally active set of conformations in NT-B.

The antiestrogen tamoxifen is an effective treatment for estrogen receptor positive breast cancers, slowing tumor growth and preventing disease recurrence, with relatively few side effects. However, many patients who initially respond to treatment, later become resistant to treatment. Tamoxifen has both agonist and antagonist activities, which are manifested in a tissue-specific pattern. Development of tamoxifen resistance can be characterized by an increase in the partial agonist properties of the antiestrogen in the breast, resulting in loss of growth inhibition and even inappropriate tumor stimulation. Nuclear receptor function is modulated by transcriptional coregulators, which either enhance or repress receptor activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies N-CoR suppressed the agonist properties of tamoxifen and RU486, while L7/SPA increased agonist effects. We speculated that the relative level of these coactivators and corepressors might determine the balance of agonist and antagonist properties of mixed antagonists such as tamoxifen. Using quantitative RT-PCR we therefore measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

The development of tamoxifen resistance and consequent disease progression are common occurrences in breast cancers, often despite the continuing expression of estrogen receptors (ER). Tamoxifen is a mixed antagonist, having both agonist and antagonist properties. We have suggested that the development of tamoxifen resistance is associated with an increase in its agonist-like properties, resulting in loss of antagonist effects or even inappropriate tumor stimulation. Nuclear receptor function is influenced by a family of transcriptional coregulators, that either enhance or suppress transcriptional activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies, N-CoR suppressed the agonist properties of tamoxifen and RU486, and L7/SPA increased agonist effects. We speculated that the relative levels of these coactivators and corepressors may determine the balance of agonist and antagonist properties of mixed antagonists, such as tamoxifen. Using quantitative RT-PCR, we, therefore, measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen-resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.

The role of the N-terminal region in nuclear receptor function was addressed by a biochemical and biophysical analysis of the progesterone receptor A-isoform lacking only the hormone binding domain (NT-A). Sedimentation studies demonstrate that NT-A is quantitatively monomeric, with a highly asymmetric shape. Contrary to dogma, the N-terminal region is structured as demonstrated by limited proteolysis. However, N-terminal structure is strongly stabilized by the DNA binding domain, possibly explaining the lack of structure seen in isolated activation domains. Upon DNA binding, NT-A undergoes N-terminal mediated assembly, suggestive of DNA-induced allostery, and consistent with changes in protease accessibility of sites outside the DNA binding domain. Microsequencing reveals that protease-accessible regions are limited to previously identified phosphorylation motifs and to functional domain boundaries.

Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.

STATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like progesterone receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a beta-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21(WAF1) and c-fos promoters. These data demonstrate the convergence of progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland.

During late stages of breast cancer progression, tumors frequently acquire steroid hormone resistance with concurrent amplification of growth factor receptors; this alteration predicts a poor prognosis. We show here that following treatment with the progestin, R5020, breast cancer cells undergo a "biochemical shift" in the regulation of epidermal growth factor (EGF)-stimulated signaling pathways: R5020 potentiates the effects of EGF by up-regulating EGFR, c-ErbB2 and c-ErbB3 receptors, and by enhancing EGF-stimulated tyrosine phosphorylation of signaling molecules known to associate with activated type I receptors. Independently of EGF, R5020 increases Stat5 protein levels, association of Stat5 with phosphotyrosine-containing proteins, and tyrosine phosphorylation of JAK2 and Shc. Furthermore, progestins "prime" breast cancer cells for growth signals by potentiating EGF-stimulated p42/p44 mitogen-activated protein kinase (MAPK), p38 MAP kinase, and JNK activities. Although the levels of cyclin D1, cyclin E, and p21(WAF1), are up-regulated by R5020 alone, they are synergistically up-regulated by EGF in the presence of R5020. Up-regulation of cell cycle proteins by EGF is blocked by inhibition of p42/p44 MAPK only in the presence of R5020, supporting a shift in the regulation of these cell cycle mediators from MAPK-independent to MAPK-dependent pathways. In summary, progesterone selectively increases the sensitivity of key kinase cascades to growth factors, thereby priming cells for stimulation by latent growth signals. These data support a model in which breast cancer cell growth switches from steroid hormone to growth factor dependence.

Progesterone has biphasic effects on proliferation of breast cancer cells; it stimulates growth in the first cell cycle, then arrests cells at G1/S of the second cycle accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21. We now show that progesterone regulates transcription of the p21 promoter by an unusual mechanism. This promoter lacks a canonical progesterone response element. Instead, progesterone receptors (PRs) interact with the promoter through the transcription factor Sp1 at the third and fourth of six Sp1 binding sites located downstream of nucleotide 154. Mutation of Sp1 site 3 eliminates basal transcription, and mutation of sites 3 and 4 eliminates transcriptional up-regulation by progesterone. Progesterone-mediated transcription is further prevented by overexpression of E1A, suggesting that CBP/p300 is required. Indeed, in HeLa cells, Sp1 and CBP/p300 associate with stably integrated flag-tagged PRs in a multiprotein complex. Since many signals converge on p21, cross-talk between PRs and other factors co-localized on the p21 promoter, may explain how progesterone can be either proliferative or differentiative in different target cells.

The B-isoform of human progesterone receptors (PR) contains three activation functions (AF3, AF1, and AF2), two of which (AF1 and AF2) are shared with the A-isoform. AF3 is in the B-upstream segment (BUS), the far N-terminal 164 amino acids of B-receptors; AF1 is in the 392-amino acid N-terminal region common to both receptors; and AF2 is in the C-terminal hormone binding domain. B-receptors are usually stronger transactivators than A-receptors due to transcriptional synergism between AF3 and one of the two downstream AFs. We now show that the N terminus of PR common to both isoforms contains an inhibitory function (IF) located in a 292-amino acid segment lying upstream of AF1. IF represses the activity of A-receptors but is not inhibitory in the context of B-receptors due to constraints imparted by BUS. As a result, IF inhibits AF1 or AF2 but not AF3, regardless of the position of IF relative to BUS. IF is functionally independent and strongly represses transcription when it is fused upstream of estrogen receptors. These data demonstrate the existence of a novel, transferable inhibitory function, mapping to the PR N terminus, which begins to assign specific roles to this large undefined region.

Pregnancy-associated breast cancers (PABC) generally present at advanced stages and have a poor prognosis. The reasons are unclear but we hypothesized that the continuous high levels of estrogens and progesterone were involved. We have now carried out a detailed analysis of PABC compared to tumors of age-matched nonpregnant (non-PABC) women. Malignant epithelia and tumor-associated stroma of PABC and non-PABC were isolated by laser capture microdissection and gene expression profiled. Additionally, normal breast epithelia and stroma adjacent to the two tumor types were analyzed. Lastly, subsets of previously identified E- and P-regulated genes were defined in all tissues. We find that PABC signatures cluster with established breast cancer subtypes. Major hormone-regulated genes whose expression correlated with epithelia of PABC dealt with regulation of cell proliferation, metabolism, and tumor aggressiveness, including genes used to predict tumor recurrence. Compared to normal epithelia, a significant number of genes associated with cell cycle processes were enriched in PABC, many of which are hormone regulated. Thus, compared to normal epithelia, many of the genes that were differentially expressed in epithelia of PABC were distinct from those differentially expressed in non-PABC. With regard to the tumor microenvironment, immune-related genes were enriched in tumor-associated stroma of PABC. Compared to normal stroma, PABC-associated stroma overexpressed immune response genes, while genes involved in angiogenesis and extracellular matrix deposition were more commonly downregulated. This suggests that the heightened aggressiveness of PABC may involve a predisposition to metastasis through extracellular matrix degradation, plus angiogenesis independence. Moreover, genes encoding cell proliferative factors, signaling, immunomodulators and cell death, were hormone regulated in stroma. In sum, these analyses demonstrate complex patterns of enrichment and hormonal regulation of genes in PABC and suggest that it may have a distinct biological nature.

Mucinous breast cancer (MBC) is mainly a disease of postmenopausal women. Pure MBC is rare and augurs a good prognosis. In contrast, MBC mixed with other histological subtypes of invasive disease loses the more favorable prognosis. Because of the relative rarity of pure MBC, little is known about its cell and tumor biology and relationship to invasive disease of other subtypes. We have now developed a human breast cancer cell line called BCK4, in which we can control the behavior of MBC. BCK4 cells were derived from a patient whose poorly differentiated primary tumor was treated with chemotherapy, radiation and tamoxifen. Malignant cells from a recurrent pleural effusion were xenografted in mammary glands of a nude mouse. Cells from the solid tumor xenograft were propagated in culture to generate the BCK4 cell line. Multiple marker and chromosome analyses demonstrate that BCK4 cells are human, near diploid and luminal, expressing functional estrogen, androgen, and progesterone receptors. When xenografted back into immunocompromised cycling mice, BCK4 cells grow into small pure MBC. However, if mice are supplemented with continuous estradiol, tumors switch to invasive lobular carcinoma (ILC) with mucinous features (mixed MBC), and growth is markedly accelerated. Tamoxifen prevents the expansion of this more invasive component. The unexpected ability of estrogens to convert pure MBC into mixed MBC with ILC may explain the rarity of the pure disease in premenopausal women. These studies show that MBC can be derived from lobular precursors and that BCK4 cells are new, unique models to study the phenotypic plasticity, hormonal regulation, optimal therapeutic interventions, and metastatic patterns of MBC.

The nuclear receptors belong to a superfamily of proteins, many of which are ligand-regulated, that bind to specific DNA sequences and control specific gene transcription. Recent data show that, in addition to contacting the basal transcription machinery directly, nuclear receptors inhibit or enhance transcription by recruiting an array of coactivator or corepressor proteins to the transcription complex. In this review we define the properties of these putative coregulatory factors; we describe the basal and coregulatory factors that are currently known to interact with nuclear receptors; we suggest various mechanisms by which coactivators and corepressors act; we discuss issues that are raised by the presence of multiple, perhaps competing, coregulatory factors; and we speculate how these additional regulatory layers may explain the heterogeneity of hormone responses that are observed in normal and malignant tissues.

To study the function of human progesterone receptor (hPR) phosphorylation, we have tested four sets of serine to alanine substitution mutants: 10 serine clusters, located in regions common to both hPR isoforms (the M-series mutants) were mutated in A-receptors and B-receptors; 6 serine clusters located in the B-upstream segment (BUS; the B-series mutants) were mutated individually and collectively and cloned into B-receptors and into BUS-DBD-NLS, a constitutive transactivator, in which the AF3 function of BUS is fused to the DNA binding domain (DBD) and nuclear localization signal (NLS) of hPR. Transcription by most of the M-series mutants resembles that of wild-type A- or B-receptors. Mutation of 3 sites, Ser190 at the N terminus of A-receptors, a cluster of serines just upstream of the DBD, or Ser676 in the hinge region, inhibits transcription by 20-50% depending on cell or promoter context. These sites lie outside the AF1 activation function. M-series mutants are substrates for a hormone-dependent phosphorylation step, and they all bind well to DNA. Progressive mutation of the B-series clusters leads to the gradual dephosphorylation of BUS, but only the 6-site mutant, involving 10 serine residues, is completely dephosphorylated. These data suggest that in BUS alternate serines are phosphorylated or dephosphorylated at any time. However, even when BUS is completely dephosphorylated, both BUS-DBD-NLS and full-length B-receptors remain strong transactivators. Mutant B-receptors also do not acquire the dominant negative properties of A-receptors, and they retain the ability to activate transcription in synergy with 8-Br-cAMP and antiprogestins. We conclude that phosphorylation has subtle effects on the complex transcriptional repertoire that distinguishes the two hPR isoforms and does not influence transactivation mediated by AF1 or AF3, but subserves other functions.

This year represents the 20th anniversary of our first demonstration that human breast cancers contain progesterone receptors (PR), and are markers of hormone dependence. These receptors are now routinely measured in tumours not only as markers of hormone dependence, but of disease prognosis. Theoretically, their central function in breast cancers is not as markers, but as effectors of the proliferative signals of endogenous progesterone in premenopausal women, and as targets for progestins and antiprogestins. At present, PR are rarely measured for these functional purposes. The actions of PR are complex, and responsiveness to progestin agonists or antagonists will depend on the gene whose activity is being measured, the peculiarities of the cell and tissue under study, and most importantly, the PR isoform that predominates in a tissue or tumour. The differential expression of PR isoforms, serves, we believe, to fine-tune responsiveness to this important reproductive hormone. Knowledge, not just of the PR content of a tissue, but of the expression of B- vs Areceptors in that tissue, will be vital to understanding the effects of progestins therein. Recent studies with PR have forced us to revise the standard model of steroid receptor action. The conventional model, which depicts receptors as ligand-activated proteins that bind to specific DNA sequences at ‘consensus’ hormone response elements and activate transcription, is not incorrect. It is, however, oversimplified, as studies with PR demonstrate. These demands include requirements for both positive and negative transcriptional regulation; for tissue specificity of action; and for regulation of composite and simple gene promoters. Multiple functional domains control intramolecular contacts, intermolecular protein-protein interactions, and DNA binding. As steroid antagonists are synthetic rather than natural hormones, their binding produces structural alterations in the receptors that unveil additional novel interactive capabilities. While antiprogestins competitively inhibit agonists by forming non-productive receptor-DNA complexes, this is not their sole mechanism of action. Antiprogestin effects may also be mediated by receptor interactions with coactivators whose function is in turn controlled by non-steroidal signals. When two different signalling pathways are activated simultaneously they can cooperate to produce unintended effects. Additionally, it seems clear that antagonist-occupied receptors can act without binding to canonical PREs, or without binding to DNA at all, relying perhaps on tethering proteins. This may be a consequence of the unusual allosteric structure imparted on the receptors by synthetic ligands. For some of these unusual actions, the receptors may even be monomeric rather than dimeric. Investigators should not assume when studying antiprogestins that a specific mechanism is operating. These novel actions begin to explain two properties of steroid antagonists that have puzzled investigators. One is the common observation that antagonists are agonists in some normal tissues. The other, an extension of the first, is that in malignant cells, antagonists can acquire agonist-like properties as tumours progress, leading to treatment failure. Although such tumours are called ‘resistant’, they may in fact be responding quite well to the antagonist! With respect to receptor protein structure, we are only beginning to appreciate its complexity. For example, it appeared initially that the structural independence of functional domains permitted analysis of receptor fragments by fusing them to heterologous proteins. However, we now know that important functional domains can overlap; that other functional domains may be discontinuous; and that one domain can modulate the activity of another. This means that analysis of receptor fragments in chimeras is an incomplete test of domain function, and that we need innovative experimental strategies to understand this intramolecular cross-talk.

Endocrine therapy used either prophylactically or therapeutically for the treatment of locally advanced or metastatic breast cancers offers many advantages to patients whose tumors contain functional estrogen (ER) and progesterone (PR) receptors. The range of treatments defined as endocrine include surgical ablation of endocrine glands, administration of pharmacologic doses of steroid hormones, chemical blockade of steroid hormone biosynthesis, and inhibition of endogenous steroid hormone action at the tumor with synthetic antagonists. The last of these approaches is the most widely used, making the antiestrogen tamoxifen the preferred first-line therapeutic agent for treatment of hormone-dependent metastatic breast cancer. The wide-spread use of tamoxifen reflects its efficacy and low toxicity, and the fact that it makes good physiological sense to block the local proliferative effects of estrogens directly at the breast. But are estrogens the only hormones with a proliferative impact on the breast and on breast cancers? This chapter focuses on evidence that progesterone also has proliferative actions in the breast; on preliminary data showing that progesterone antagonists may be new tools for the management of metastatic breast cancer; and on recent data suggesting that antiprogestin-occupied PR have novel mechanisms of action that bear on tissue specificity and development of hormone resistance.