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.

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.

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.

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.

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.

Rare steroid receptor-negative basal-like tumorigenic cells in luminal subtype human breast cancer xenografts.

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 (<10 cells); however, this population did not expand with increasing colony size. The number of ER(+)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.

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.

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

The Year in Basic Science: update of estrogen plus progestin therapy for menopausal hormone replacement implicating stem cells in the increased breast cancer risk.

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.

Progestins in hormone replacement therapies reactivate cancer stem cells in women with preexisting breast cancers: a hypothesis.

Why does hormone replacement therapy (HRT) with estrogens plus progestins increase the risk of breast cancer? First, experimental estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) human breast cancers contain a rare subpopulation of ER(-), PR(-) cancer stem cells. Especially in small, nascent ER+, PR+ tumor colonies, progestins, but not estrogens, reactivate cells with ER(-), PR(-) stem-like properties. Second, there is a reservoir of occult, undetected, preinvasive breast cancer in some women who are candidates for HRT. We propose that women who develop breast cancer while on estrogens plus progestins harbor undiagnosed nascent disease before the start of therapy. The progestin component, in a nonproliferative step, reactivates receptor-negative cancer stem cells within such germinal, perhaps even dormant tumors. After reacquiring receptors, these tumor cells are expanded by the mitogenic properties of estrogens. We argue that screening methods need to be improved to detect small, preexisting malignancies prior to the start of HRT. Women harboring such disease should be excluded from regimens that include systemic progestins.

Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases.

Genome-wide expression profiling has expedited our molecular understanding of the different subtypes of breast cancers, as well as defined the differences among genes expressed in primary tumors and their metastases. Laser-capture microdissection (LCM) coupled to gene expression analysis allows us to understand how specific cell types contribute to the total cancer gene expression signature. Expression profiling was used to define genes that contribute to breast cancer spread into and/or growth within draining lymph nodes (LN). Whole tumor xenografts and their matched whole LN metastases were compared to LCM captured cancer cells from the same tumors and matched LN metastases. One-thousand nine-hundred thirty genes were identified by the whole organ method alone, and 1,281 genes by the LCM method alone. However, less than 1% (30 genes) of genes that changed between tumors and LN metastases were common to both methods. Several of these genes have previously been implicated in cancer aggressiveness. Our data show that whole-organ and LCM based gene expression profiling yield distinctly different lists of metastasis-promoting genes. Contamination of the tumor cells, and cross reactivity of mouse RNA to human-specific chips may explain these differences, and suggests that LCM-derived data may be more accurate.

Estrogen regulated gene expression in response to neoadjuvant endocrine therapy of breast cancers: tamoxifen agonist effects dominate in the presence of an aromatase inhibitor.

Estrogens (E) and estrogen receptors (ER) are implicated in breast cancer growth and are targets of hormonal therapies. Such therapies commonly use aromatase inhibitors (AI) to block E production, or antiestrogens like tamoxifen (TAM), which targets ER. Here we compare genes in pre-and post-treatment tumor pairs of patients with ER+ tumors, that were treated preoperatively with the AI exemestane alone, or with exemestane plus TAM. The accompanying manuscript shows that tumors from patients treated with AI + TAM responded less well than tumors treated with AI alone. The present manuscript defines the E-signaling mechanisms underlying these differences, and describes genetic differences between hormone responsive versus intrinsically resistant ER+ tumors. Gene expression profiling was performed on paired tumor biopsies of individual patients before treatment, and after 4 months of treatment with AI or AI + TAM. Separately, E and TAM regulated genes were defined using a human breast cancer xenograft model. We demonstrate: (1) that AI alone alters global gene expression approximately 5 times more than AI + TAM, and is 11 times more effective in modifying expression of E regulated genes; (2) among E regulated genes, there is little overlap between AI and AI + TAM treatment groups. AI + TAM preferentially induce genes, like androgen receptors, expressing TAM "E-like" agonist activity, or genes uniquely regulated by TAM. (3) A pre-treatment 25 gene signature of ER+ tumors may predict response or intrinsic resistance to endocrine therapies. We conclude that in the presence of exemestane, the agonist properties of TAM are paradoxically exposed, diminishing the effectiveness of combination therapy.

Molecular signatures of neoadjuvant endocrine therapy for breast cancer: characteristics of response or intrinsic resistance.

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.

ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease.

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.

Progesterone receptor action: translating studies in breast cancer models to clinical insights.

Progesterone receptors (PR) are useful prognostic indicators of breast cancers likely to respond to anti-estrogen receptor (ER) therapies. However, the role of progesterone, therapeutic progestins, or unliganded or liganded PRin breast cancer development or progression remains controversial. PR are ligand-activated transcription factors that act in concert with intracellular signaling pathways as "sensors" of multiple growth factor inputs to hormonally regulated tissues, such as the breast. The recently defined induction of rapid signaling events upon progestin-binding to PR-B provides a means to ensure that receptors and coregulators are appropriately phosphorylated as part of optimal transcription complexes. PR-activated kinase cascades may provide additional avenues for progestin-regulated gene expression independent of PR nuclear action. Herein, we present an overview ofprogesterone/PR and signaling cross-talk in breast cancer models and discuss the potential significance ofprogestin/PR action in breast cancer biology using examples from both in vitro and in vivo models, as well as limited clinical data. Kinases are emerging as key mediators of PR action. Cross-talk between PR and membrane-initiated signaling events suggests a mechanism for coordinated regulation ofgene subsets by mitogenic stimuli in hormonally responsive normal tissues. Dysregulation of this cross-talk mechanism may contribute to breast cancer biology; further studies are needed to address the potential for targeting PR in addition to ER and selected protein kinases as part of more effective breast cancer therapies.

Estrogen receptor positive breast cancer metastasis: altered hormonal sensitivity and tumor aggressiveness in lymphatic vessels and lymph nodes.

Breast cancers commonly spread to lymph nodes (LNs). If the primary tumors are estrogen receptor (ER) and/or progesterone receptor (PR) positive, then the likelihood that LN metastases express receptors exceeds 80%. However, due to lack of ER+ models, little is known about the role of hormones in breast cancer spread or the effects of the LN microenvironment on hormone responsiveness. We have developed metastasis models using ZsGreen labeled MCF-7 and T47D human breast cancer cells. Tumors are tracked in living mice by whole-body imaging, and macrometastases or micrometastases are detected by intravital imaging or fluorescence microscopy. Tumor growth is estrogen dependent and required for intratumoral lymphangiogenesis. Seventy-five percent of all tumors and >95% of larger tumors generate LN metastases. Occasionally more distant metastases are also observed. "Triads" of primary tumors, tumor-filled draining lymphatic vessels, and tumor-filled LNs from the same mouse show that (a) proliferation, as measured by 5-bromo-2'-deoxyuridine uptake, is higher in the LN than in the primary tumor. (b) High ER levels are extensively down-regulated by estradiol in primary tumors. However, there is partial failure of ER down-regulation in LNs associated with (c) reduced PR expression. This suggests that ER are dysfunctional in the LN microenvironment and perhaps hormone resistant. (d) CD44 is sparsely expressed in primary tumor cells but homogeneously overexpressed in cells transiting the lymphatics and populating LNs. We hypothesize that CD44 expression targets tumor cells for transport to, and uptake in, LNs. If so, the CD44 pathway could be targeted therapeutically to slow or prevent LN metastases.