Jennifer K. Richer

Differential Gene Regulation by the Two Progesterone Receptor Isoforms in Human Breast Cancer Cells

The PR-A and PR-B isoforms of progesterone receptors (PR) have different physiological functions, and their ratio varies widely in breast cancers. To determine whether the two PR regulate different genes, we used human breast cancer cell lines engineered to express one or the other isoform. Cells were treated with progesterone in triplicate, time-separated experiments, allowing statistical analyses of microarray gene expression data. Of 94 progesterone-regulated genes, 65 are uniquely regulated by PR-B, 4 uniquely by PR-A, and only 25 by both. Almost half the genes encode proteins that are membrane-bound or involved in membrane-initiated signaling. We also find an important set of progesterone-regulated genes involved in mammary gland development and/or implicated in breast cancer. This first, large scale study of PR gene regulation has important implications for the measurement of PR in breast cancers and for the many clinical uses of synthetic progestins. It suggests that it is important to distinguish between the two isoforms in breast cancers and that isoform-specific genes can be used to screen for ligands that selectively modulate the activity of PR-A or PR-B. Additionally, use of natural target genes, rather than “consensus” response elements, for transcription studies should improve our understanding of steroid hormone action.

MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents.

The transcription factor ZEB1 is normally not expressed in epithelial cells. When inappropriately expressed in carcinomas, ZEB1 initiates epithelial to mesenchymal transition due to its ability to repress E-cadherin and other genes involved in polarity. Recently, ZEB1 and ZEB2 have been identified as direct targets of the microRNA-200c family. We find that miR-200c levels are high in well-differentiated endometrial, breast, and ovarian cancer cell lines, but extremely low in poorly differentiated cancer cells. Low or absent miR-200c results in aberrant expression of ZEB1 and consequent repression of E-cadherin. Reinstatement of miR-200c to such cells restores E-cadherin and dramatically reduces migration and invasion. Microarray profiling reveals that in addition to ZEB1 and ZEB2, other mesenchymal genes (such as FN1, NTRK2, and QKI), which are also predicted direct targets of miR-200c, are indeed inhibited by addition of exogenous miR-200c. One such gene, class III β-tubulin (TUBB3), which encodes a tubulin isotype normally found only in neuronal cells, is a direct target of miR-200c. This finding is of particular significance because we show that restoration of miR-200c increases sensitivity to microtubule-targeting agents by 85%. Because expression of TUBB3 is a common mechanism of resistance to microtubule-binding chemotherapeutic agents in many types of solid tumors, the ability of miR-200c to restore chemosensitivity to such agents may be explained by its ability to reduce TUBB3. Because miR-200c is crucial for maintenance of epithelial identity, behavior, and sensitivity to chemotherapy, we propose that it warrants further investigation as a therapeutic strategy for aggressive, drug-resistant cancers. [Mol Cancer Ther 2009;8(5):OF1–12]

The Transcription Factor ZEB1 Is Aberrantly Expressed in Aggressive Uterine Cancers

The transcription factor ZEB1 (deltaEF1 in mice) has been implicated in cellular processes during development and tumor progression including epithelial to mesenchymal transition. deltaEF1 null mice die at birth, but heterozygotes expressing a LacZ reporter inserted into the deltaEF1 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.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors

BackgroundTumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive.ResultsHere we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators.ConclusionsOur findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

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.

New Human Breast Cancer Cells to Study Progesterone Receptor Isoform Ratio Effects and Ligand-independent Gene Regulation

All known progesterone target cells coexpress two functionally different progesterone receptor (PR) isoforms: 120-kDa B-receptors (PR-B) and N-terminally truncated, 94-kDa A-receptors (PR-A). Their ratio varies in normal and malignant tissues. In human breast cancer cells, homodimers of progesterone-occupied PR-A or PR-B regulate different gene subsets. To study PR homo- and heterodimers, we constructed breast cancer cell lines in which isoform expression is controlled by an inducible system. PR-negative cells or cells that stably express one or the other isoform were used to construct five sets of cells: (i) PR-negative control cells (Y iNull), (ii) inducible PR-A cells (Y iA), (iii) inducible PR-B cells (Y iB), (iv) stable PR-B plus inducible PR-A cells (B iA), and (v) stable PR-A plus inducible PR-B cells (A iB). Expression levels of each isoform and/or the PR-A/PR-B ratios could be tightly controlled by the dose of inducer as demonstrated by immunoblotting and transcription studies. Induced PRs underwent normal progestin-dependent phosphorylation and down-regulation and regulated exogenous promoters as well as endogenous gene expression. Transcription of exogenous promoters was dependent on the PR-A/PR-B ratio, whereas transcription of endogenous genes was more complex. Finally, we have described several genes that are regulated by induced PR-A even in the absence of ligand.

MicroRNAs Link Estrogen Receptor Alpha Status and Dicer Levels in Breast Cancer

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1− cells (∼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3′ UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1− cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3′ UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα− cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1− cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα− breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.

Nuclear receptor conformation, coregulators, and tamoxifen-resistant breast cancer

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.

Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring

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.

Expression profiling of human breast cancers and gene regulation by progesterone receptors.

Even the first expression profiling studies of breast cancers have generated new insights. They suggest for example, that information about tumor aggressiveness, prognosis, metastatic potential, or treatment outcome is encoded in, and can be deduced from, the primary tumor. On the other hand no clinical genomic array data have yet been published that deal with hormonal aspects of breast tumorigenesis, tumor progression, or therapeutics. Rather, studies have focused on experimental model systems. We review below the currently published data on array profiling in clinical breast cancer, then describe our studies in breast cancer cell lines and xenograft models dealing with progesterone receptors (PRs) and the role of progesterone. We demonstrate that the two PR isoforms, PR-A and PR-B, have mostly nonoverlapping molecular signatures when liganded by progesterone, with PR-B the more active form. Additionally, we document the surprising finding that unliganded PRs can regulate gene transcription, with PR-A the more active form. In ovariectomized mice supplemented with estradiol but lacking measurable progesterone, PR-B-expressing tumors grow to twice the size of PR-A-expressing ones. We conclude that in breast cancers, PRs are more than simple markers of estrogen receptor function. Rather, presence of PRs and the ratio of the two isoforms directly influence tumor phenotype, even in the absence of ligand.