90 YEARS OF PROGESTERONE: Progesterone and progesterone receptors in breast cancer: past, present, future.

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.

Fibroblast subtypes define a metastatic matrisome in breast cancer.

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 (<5 cm). Clearly small tumors enriched for CD146- CAFs require aggressive treatments.

SUMOylation Regulates Transcription by the Progesterone Receptor A Isoform in a Target Gene Selective Manner.

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.

Role of epigenetic modifications in luminal breast cancer.

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.

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells.

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.

Malignant stroma increases luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth factor signaling.

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.

Modeling luminal breast cancer heterogeneity: combination therapy to suppress a hormone receptor-negative, cytokeratin 5-positive subpopulation in luminal disease.

INTRODUCTION: Many Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call "Luminobasal" is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment. METHODS: To address the relationships between ER+PR+CK5- and ER-PR-CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models. RESULTS: We show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy. CONCLUSIONS: We propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.

Post-translational modifications of the progesterone receptors.

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.

Genomic signatures of pregnancy-associated breast cancer epithelia and stroma and their regulation by estrogens and progesterone.

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.

ER, PR, HER2, Ki-67 and CK5 in Early and Late Relapsing Breast Cancer-Reduced CK5 Expression in Metastases.

Breast cancer can recur even decades after the primary therapy. Markers are needed to predict cancer progression and the risk of late recurrence. The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), proliferation marker Ki-67, and cytokeratin CK5 were studied to find out whether their expression or occurrence in subgroups of breast cancers correlated with the time of recurrence. The expression of HER2, ER, PR, Ki-67, and CK5 was studied by IHC in 72 primary breast cancers and their corresponding recurrent/metastatic lesions. The patients were divided into three groups according to the time of the recurrence/metastasis: before two years, after 5 years, and after 10 years. Based on their IHC profiles, the tumors were divided into surrogates of the genetically defined subgroups of breast cancers and the subtype definitions were as follows: luminal A (ER or PR+HER2-), luminal B (ER or PR+HER2+), HER2 overexpressing (ER-PR-HER2+), triple-negative (ER-PR-HER2-), basal-like (ER-PR-HER2-CK5+), non-classified (ER-PR-HER2-CK5-) and luminobasal (ER or PR+CK5+). In multivariate analysis, tumor size and HER2 positivity were a significant risk of early cancer relapse. The metastases showed a significantly lower CK5 expression. CK5 positivity distinguished triple negative tumors into rapidly and slowly recurring cancers. The IHC subtype ER or PR+HER2- luminal A presented a significantly lower risk of early tumor recurrence. Ki-67 expression denoted early-relapsing tumors and correlated linearly with tumor progression, since Ki-67 positivity declined gradually from early-relapsing toward late-recurring cancers.

Estrogen switches pure mucinous breast cancer to invasive lobular carcinoma with mucinous features.

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.

Control of progesterone receptor transcriptional synergy by SUMOylation and deSUMOylation.

BACKGROUND: Covalent modification of nuclear receptors by the Small Ubiquitin-like Modifier (SUMO) is dynamically regulated by competing conjugation/deconjugation steps that modulate their overall transcriptional activity. SUMO conjugation of progesterone receptors (PRs) at the N-terminal lysine (K) 388 residue of PR-B is hormone-dependent and suppresses PR-dependent transcription. Mutation of the SUMOylation motif promotes transcriptional synergy. RESULTS: The present studies address mechanisms underlying this transcriptional synergy by using SUMOylation deficient PR mutants and PR specifically deSUMOylated by Sentrin-specific proteases (SENPs). We show that deSUMOylation of a small pool of receptors by catalytically competent SENPs globally modulates the cooperativity-driven transcriptional synergy between PR observed on exogenous promoters containing at least two progesterone-response elements (PRE2). This occurs in part by raising PR sensitivity to ligands. The C-terminal ligand binding domain of PR is required for the transcriptional stimulatory effects of N-terminal deSUMOylation, but neither a functional PR dimerization interface, nor a DNA binding domain exhibiting PR specificity, are required. CONCLUSION: We conclude that direct and reversible SUMOylation of a minor PR protein subpopulation tightly controls the overall transcriptional activity of the receptors at complex synthetic promoters. Transcriptional synergism controlled by SENP-dependent PR deSUMOylation is dissociable from MAPK-catalyzed receptor phosphorylation, from SRC-1 coactivation and from recruitment of histone deacetylases to promoters. This will provide more information for targeting PR as a part of hormonal therapy of breast cancer. Taken together, these data demonstrate that the SUMOylation/deSUMOylation pathway is an interesting target for therapeutic treatment of breast cancer.

Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch.

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.

Progesterone receptors, their isoforms and progesterone regulated transcription.

This review discusses mechanisms by which progesterone receptors (PR) regulate transcription. We examine available data in different species and tissues regarding: (1) regulation of PR levels; and (2) expression profiling of progestin-regulated genes by total PRs, or their PRA and PRB isoforms. (3) We address current views about the composition of progesterone response elements, and postulate that PR monomers acting through "half-site" elements are common, entailing cooperativity with neighboring DNA-bound transcription factors. (4) We summarize transcription data for multiple progestin-regulated promoters as directed by total PR, or PRA vs. PRB. We conclude that current models and methods used to study PR function are problematical, and recommend that future work employ cells and receptors appropriate to the species, focusing on analyses of the effects of endogenous receptors targeting endogenous genes in native chromatin.

Unliganded progesterone receptors attenuate taxane-induced breast cancer cell death by modulating the spindle assembly checkpoint.

Whether the presence of steroid receptors in luminal breast cancers renders them resistant to taxanes remains uncertain. Here we assess the role of progesterone receptors (PR) on taxane-induced cell death. We previously showed that estrogen receptor (ER)-positive human breast cancer cells that inducibly express PR-A or PR-B isoforms were protected from taxane-stimulated apoptosis when compared to the identical cells lacking PR. Surprisingly, PR-dependent protection occurred in the absence of progesterone, demonstrating that the unliganded receptors were biologically active. The present studies demonstrate that unliganded PR, focused on PR-A, protect breast cancer cells from taxane-stimulated apoptosis. The studies identify genes regulated by taxanes in isogenic ER-positive cells that either lack or express PR-A. We show that unliganded PR-A alters the gene expression pattern controlled by taxanes, especially multiple genes involved in the spindle assembly checkpoint, a group of proteins that insure proper attachment of microtubules to kinetochores during mitosis. Importantly, taxanes and unliganded PR regulate many of these genes in opposite directions. As a result, mitotic slippage is exacerbated by the presence of PR, leading to an increase in the number of multinucleated cells both in vitro and in xenograft tumors. We describe a simple new assay for assessing multinucleation in paraffin sections. We speculate that rather than inducing cell death, unliganded PR exploits multinucleation to promote cell survival from taxane therapy. This can be prevented with antiprogestin.

Cytokeratin 5 positive cells represent a steroid receptor negative and therapy resistant subpopulation in luminal breast cancers.

A majority of breast cancers are estrogen receptor (ER) positive and have a luminal epithelial phenotype. However, these ER⁺ tumors often contain heterogeneous subpopulations of ER⁻ tumor cells. We previously identified a population of cytokeratin 5 (CK5) positive cells within ER⁺ and progesterone receptor positive (PR⁺) tumors that is both ER⁻PR⁻ and CD44⁺, a marker of breast tumor-initiating cells (TICs). These CK5⁺ cells have properties of TICs in luminal tumor xenografts, and we speculated that they are more resistant to chemo- and anti-ER-targeted therapies than their ER⁺ neighbors. To test this, we used ER⁺PR⁺ T47D and MCF7 breast cancer cells. CK5⁺ cells had lower proliferative indices than CK5⁻ cells, were less sensitive to 5-fluorouracil and docetaxel, and cultures became enriched for CK5⁺ cells after treatments. CK5⁺ cells were less prone to drug-induced apoptosis than CK5⁻ cells. In cells treated with 17ß-estradiol (E) plus anti-estrogens tamoxifen or fulvestrant, ER protein levels decreased, and CK5 protein levels increased, compared to controls treated with E alone. In ER⁺ tumors from patients treated with neoadjuvant endocrine therapies ER gene expression decreased, and CK5 gene expression increased in post compared to pre-treatment tumors. The number of CK5⁺ cells in tumors also increased in post- compared to pre-treatment tumors. We conclude that an ER⁻PR⁻CK5⁺ subpopulation found in many luminal tumors is resistant to standard endocrine and chemotherapies, relative to the majority ER⁺PR⁺CK5⁻ cells. Compounds that effectively target these cells are needed to improve outcome in luminal breast cancers.

An immunohistochemical method to study breast cancer cell subpopulations and their growth regulation by hormones in three-dimensional cultures.

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions.

Tissue-specific pathways for estrogen regulation of ovarian cancer growth and metastasis.

Menopausal estrogen (E2) replacement therapy increases the risk of estrogen receptor (ER)-positive epithelial ovarian cancers (EOC). Whether E2 is tumorigenic or promotes expansion of undiagnosed preexisting disease is unknown. To determine E2 effects on tumor promotion, we developed an intraperitoneal mouse xenograft model using ZsGreen fluorescent ER(-) 2008 and ER(+) PEO4 human EOC cells. Tumor growth was quantified by in vivo fluorescent imaging. In ER(+) tumors, E2 significantly increased size, induced progesterone receptors, and promoted lymph node metastasis, confirming that ERs are functional and foster aggressiveness. Laser-captured human EOC cells from ER(-) and ER(+) xenografted tumors were profiled for expression of E2-regulated genes. Three classes of E2-regulated EOC genes were defined, but <10% were shared with E2-regulated breast cancer genes. Because breast cancer selective ER modulators (SERM) are therapeutically ineffective in EOC, we suggest that our EOC-specific E2-regulated genes can assist pharmacologic discovery of ovarian-targeted SERM.

Vascular endothelial growth factor secreted by activated stroma enhances angiogenesis and hormone-independent growth of estrogen receptor-positive breast cancer.

"Reactive" or activated stroma characterizes many malignancies including breast cancers. Recently, we isolated a reactive mouse mammary gland stromal cell line called BJ3Z. These cells express alpha-smooth muscle actin and stromal cell-derived factor 1 (SDF-1) and are tumorigenic when injected into mice. Here we show that, in vivo, BJ3Z cells influence the angiogenesis and proliferation of xenografted estrogen receptor (ER)-positive MCF-7 human breast cancer cell-derived solid tumors. The growth-promoting effects of BJ3Z cells are equivalent to those of estradiol (E(2)). BJ3Z cells also increase the proliferation of normal mouse mammary luminal cells adjacent to tumors. In vitro, BJ3Z cells reorganize and increase the proliferation of cocultured malignant MCF-7 and normal human breast MCF10A cells grown as organoids in three-dimensional culture. The effects of BJ3Z cells on MCF-7 cells are equivalent to those of E(2). In contrast, BJ3Z cells do not alter the growth of highly aggressive ER-negative MDA-MB-231 human breast cancer cells. We show that BJ3Z cells secrete vascular endothelial growth factor (VEGF). The growth of MCF-7 organoids induced by BJ3Z can be inhibited by antagonists of VEGF and SDF-1. Conversely, recombinant VEGF stimulates the proliferation of MCF-7, but not MDA-MB-231, organoids. We conclude that, in addition to angiogenesis, VEGF released by activated stroma increases the growth of ER-positive malignant epithelial cells and of adjacent normal epithelium. Because activated stroma can substitute for E(2) and fosters hormone-independent growth of ER-positive tumors, we suggest that breast cancers exhibiting intrinsic hormone resistance may respond to antiangiogenic therapies.