Project description:Progesterone receptors (PRs) are critical context-dependent transcription factors required for normal uterine (PR-A) and mammary gland (PR-B) development. Progesterone is proliferative in the breast, where PR-target genes include paracrine factors that mediate mammary stem cell self-renewal. In the context of altered signal transduction that typifies breast tumorigenesis, dysregulated (i.e. hyper-phosphorylated) PRs likely contribute to tumor progression by promoting cancer cell pro-survival and proliferation. Notably, in breast cancer cells, progestin-bound PRs induce rapid MAPK activation leading to selective regulation of growth-promoting genes by phosphorylated PR species. Functional domains within PR that interact with c-Src and estrogen receptors (ER) have been identified as indirect routes to MAPK activation. Herein, we describe a common docking (CD) domain located within the PR-B N-terminus, a motif first described in MAPKs that facilitates direct interactions between MAPKs and MEK1 or MAPK-phosphatases (MKPs). Mutation of negatively-charged amino acids, previously determined to be critical for CD domain function in MAPKs, within PR-B (mCD PR) did not alter MEK-binding or progestin-induced rapid signaling (i.e. MAPK activation) and PR transcriptional activity as measured by PRE-luciferase (reporter) assays. Microarray gene-expression analysis revealed that endogenous genes regulated by wt PR, but not mCD PR, are involved in critical cellular pathways regulating growth, proliferation, survival, and cancer. mCD PR failed to undergo ligand-induced phosphorylation on Ser81, a ck2-dependent site required for progestin-regulation of select growth-promoting genes (BIRC3, HSD11β2, HbEGF). Progestin-induced PR Ser81 phosphorylation mapped to CD domain-dependent binding of PR-B to MKP3, but did not require phosphatase activity. Receptors containing either mutant CD domains (mCD PR) or point mutations of Ser81 (S79/81A PR) failed to upregulate STAT5 and Wnt1, key PR-target gene products that act as critical mediators of mammary stem cell expansion. Inhibition of JAK/STAT signaling blocked progestin-induced STAT5 and Wnt1 expression. ChIP assays demonstrated that wt, but not phospho-mutant (S79/81A), PR-B was co-recruited to a PRE-containing enhancer region of the Wnt1 gene along with MKP3, ck2 and STAT5. Our studies reveal a novel scaffolding action of MKP3 mediated by interaction with the PR CD domain and required for ck2-dependent PR Ser81 phosphorylation. Co-regulation of select target genes by phospho-Ser81 PR and phospho-STAT5 is likely a global mechanism required for the activation of growth promoting programs active during normal mammary gland development and relevant to mechanisms of breast cancer progression. The study contains 6 different sample groups measured in triplicate, for a total of 18 individual samples (18 arrays). From parental T47D-Y human breast cancer cell lines, we created three stable clones expressing (1) an empty vector (pSG5), (2) the wild type progesterone receptor isoform B (pSG5-PR-B), or (3) a mutant mutant CD domain PR-B. These cell lines were treated with either (1) vehicle control (ethanol) or (2) R5020 10e-8 M for 6 hours before total RNA harvest. Thus, the experiment contains three cell lines, and two treatments (6 sample groups) treated and analyzed in triplicate (18 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.

Project description:Background Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. Methods To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. Results Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. Conclusions We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Project description:Progesterone receptors (PRs) are critical context-dependent transcription factors required for normal uterine (PR-A) and mammary gland (PR-B) development. Progesterone is proliferative in the breast, where PR-target genes include paracrine factors that mediate mammary stem cell self-renewal. In the context of altered signal transduction that typifies breast tumorigenesis, dysregulated (i.e. hyper-phosphorylated) PRs likely contribute to tumor progression by promoting cancer cell pro-survival and proliferation. Notably, in breast cancer cells, progestin-bound PRs induce rapid MAPK activation leading to selective regulation of growth-promoting genes by phosphorylated PR species. Functional domains within PR that interact with c-Src and estrogen receptors (ER) have been identified as indirect routes to MAPK activation. Herein, we describe a common docking (CD) domain located within the PR-B N-terminus, a motif first described in MAPKs that facilitates direct interactions between MAPKs and MEK1 or MAPK-phosphatases (MKPs). Mutation of negatively-charged amino acids, previously determined to be critical for CD domain function in MAPKs, within PR-B (mCD PR) did not alter MEK-binding or progestin-induced rapid signaling (i.e. MAPK activation) and PR transcriptional activity as measured by PRE-luciferase (reporter) assays. Microarray gene-expression analysis revealed that endogenous genes regulated by wt PR, but not mCD PR, are involved in critical cellular pathways regulating growth, proliferation, survival, and cancer. mCD PR failed to undergo ligand-induced phosphorylation on Ser81, a ck2-dependent site required for progestin-regulation of select growth-promoting genes (BIRC3, HSD11β2, HbEGF). Progestin-induced PR Ser81 phosphorylation mapped to CD domain-dependent binding of PR-B to MKP3, but did not require phosphatase activity. Receptors containing either mutant CD domains (mCD PR) or point mutations of Ser81 (S79/81A PR) failed to upregulate STAT5 and Wnt1, key PR-target gene products that act as critical mediators of mammary stem cell expansion. Inhibition of JAK/STAT signaling blocked progestin-induced STAT5 and Wnt1 expression. ChIP assays demonstrated that wt, but not phospho-mutant (S79/81A), PR-B was co-recruited to a PRE-containing enhancer region of the Wnt1 gene along with MKP3, ck2 and STAT5. Our studies reveal a novel scaffolding action of MKP3 mediated by interaction with the PR CD domain and required for ck2-dependent PR Ser81 phosphorylation. Co-regulation of select target genes by phospho-Ser81 PR and phospho-STAT5 is likely a global mechanism required for the activation of growth promoting programs active during normal mammary gland development and relevant to mechanisms of breast cancer progression.

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Progesterone Receptor-Cyclin D1 Complexes Induce Cell Cycle-Dependent Transcriptional Programs In Breast Cancer Cells

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