Carol A. Lange

Roles of the EZH2 histone methyltransferase in cancer epigenetics

EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. This methylated H3-K27 chromatin mark is commonly associated with silencing of differentiation genes in organisms ranging from plants to flies to humans. Studies on human tumors show that EZH2 is frequently over-expressed in a wide variety of cancerous tissue types, including prostate and breast. Although the mechanistic contributions of EZH2 to cancer progression are not yet determined, functional links between EZH2-mediated histone methylation and DNA methylation suggest partnership with the gene silencing machinery implicated in tumor suppressor loss. Here we review the basic molecular biology of EZH2 and the findings that implicate EZH2 in different cancers. We also discuss EZH2 connections to other silencing enzymes, such as DNA methyltransferases and histone deacetylases, and we consider progress on deciphering mechanistic consequences of EZH2 overabundance and its potential roles in tumorigenesis. Finally, we review recent findings that link EZH2 roles in stem cells and cancer, and we consider prospects for integrating EZH2 blockade into strategies for developing epigenetic therapies.

Transcriptional hyperactivity of human progesterone receptors is coupled to their ligand-dependent down-regulation by mitogen-activated protein kinase-dependent phosphorylation of serine 294

ABSTRACT Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in part by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiated signaling pathways, we overexpressed mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase) kinase kinase 1 (MEKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEKK1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased progestin-mediated transcription 8- to 10-fold above normal PR-driven transcription levels. This was dependent on the presence of a progesterone response element and functional PR. PR protein levels were unchanged by MEKK1 alone but were extensively down-regulated by MEKK1 plus the progestin R5020. MEKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consensus site known to mediate ligand-dependent PR degradation. MEK inhibitors blocked phosphorylation of Ser294 and attenuated PR transcriptional hyperactivity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing mutant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-induced transcriptional synergy but respond to progestins alone. Similarly, c-myc protein levels are synergistically increased by epidermal growth factor and R5020 in cells expressing wild-type PR, but not S294A PR. Thus, highly stable mutant PR are functional in response to progestins but are incapable of cross talk with MAPK-driven pathways. These studies demonstrate a paradoxical coupling between steroid receptor down-regulation and transcriptional hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone control of breast cancer cell growth.

Convergence of Progesterone and Epidermal Growth Factor Signaling in Breast Cancer POTENTIATION OF MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS

During late stages of breast cancer progression, tumors frequently acquire steroid hormone resistance with concurrent amplification of growth factor receptors; this alteration predicts a poor prognosis. We show here that following treatment with the progestin, R5020, breast cancer cells undergo a “biochemical shift” in the regulation of epidermal growth factor (EGF)-stimulated signaling pathways: R5020 potentiates the effects of EGF by up-regulating EGFR, c-ErbB2 and c-ErbB3 receptors, and by enhancing EGF-stimulated tyrosine phosphorylation of signaling molecules known to associate with activated type I receptors. Independently of EGF, R5020 increases Stat5 protein levels, association of Stat5 with phosphotyrosine-containing proteins, and tyrosine phosphorylation of JAK2 and Shc. Furthermore, progestins “prime” breast cancer cells for growth signals by potentiating EGF-stimulated p42/p44 mitogen-activated protein kinase (MAPK), p38 MAP kinase, and JNK activities. Although the levels of cyclin D1, cyclin E, and p21WAF1, are up-regulated by R5020 alone, they are synergistically up-regulated by EGF in the presence of R5020. Up-regulation of cell cycle proteins by EGF is blocked by inhibition of p42/p44 MAPK only in the presence of R5020, supporting a shift in the regulation of these cell cycle mediators from MAPK-independent to MAPK-dependent pathways. In summary, progesterone selectively increases the sensitivity of key kinase cascades to growth factors, thereby priming cells for stimulation by latent growth signals. These data support a model in which breast cancer cell growth switches from steroid hormone to growth factor dependence.

Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity.

STATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like progesterone receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a β-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21WAF1 and c-fospromoters. These data demonstrate the convergence of progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland.

Progesterone Receptors Upregulate Wnt-1 To Induce Epidermal Growth Factor Receptor Transactivation and c-Src-Dependent Sustained Activation of Erk1/2 Mitogen-Activated Protein Kinase in Breast Cancer Cells

ABSTRACT Progesterone receptor (PR) ligand binding induces rapid and transient (5- to 10-min) activation of cytosolic c-Src-Ras-Erk1/2 mitogen-activated protein kinase (MAPK) signaling that is independent of PR functioning as transcription factors. Here, we have explored the integration of PR-dependent transcription and rapid signaling events in breast cancer cells. PR-B, but not PR-A, induced robust and sustained (6- to 72-h) Erk1/2 activation that was required for elevated cyclin D1 protein but not mRNA levels. Sustained Erk1/2 activation in response to progestins occurred via a novel mechanism distinct from rapid signaling initiated by PR/c-Src interactions and required the PR-B DNA-binding domain (DBD). PR/progestin upregulated epidermal growth factor receptor (EGFR) and Wnt-1. In response to PR-induced Wnt-1 signaling, matrix metalloprotease (MMP)-mediated membrane-proximal shedding of EGFR ligands transactivated EGFR and induced persistent downstream c-Src and Erk1/2 activities. T47D cell anchorage-independent growth was stimulated by progestins and blocked by inhibition of Erk1/2, c-Src, EGFR, or RNA interference of Wnt-1. Similarly, cell growth in soft agar required the PR DBD but was sensitive to disruption of PR/c-Src interactions, suggesting that both PR-B-induced rapid signaling events and nuclear actions contribute to this response. Our discovery that progestins are capable of robust autocrine activation of EGFR and sustained Erk1/2 signaling provides further support for the physiological linkage of growth factor and steroid hormone signaling. PR-B-induced sustained MAPK signaling may provide prosurvival or proliferative advantages to early breast cancer lesions.

Breast Tumor Kinase (Protein Tyrosine Kinase 6) Regulates Heregulin-Induced Activation of ERK5 and p38 MAP Kinases in Breast Cancer Cells

Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

Progesterone Receptor Rapid Signaling Mediates Serine 345 Phosphorylation and Tethering to Specificity Protein 1 Transcription Factors

Human progesterone receptors (PR) rapidly activate cytosolic signaling pathways, in addition to their classical function as ligand-activated transcription factors. Using ER+/PR-B+ T47D breast cancer cells, we probed the role of progestin-stimulated rapid PR signaling in the transcriptional regulation of target genes involved in breast cancer cell proliferation. Epidermal growth factor receptor (EGFR) was rapidly activated after a 10-min treatment with R5020. Progestin induced EGFR-, c-Src-, and MAPK-dependent phosphorylation of PR-B on the MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with specificity protein 1 (Sp1) transcription factors to regulate PR cell cycle (p21) and growth-promoting (EGFR) target genes whose promoters lack canonical progesterone response element sequences. Inhibitors of EGFR, c-Src, or MAPK activities blocked PR tethering to Sp1 and progestin-stimulated S-phase entry. Mutant PR-B receptors defective for c-Src binding (mPro) were not phosphorylated on Ser345 in response to progestin and failed to interact with Sp1. Hormone-induced complexes containing Sp1 and wild-type PR-B, but not S345A or mPro PR-B, were recruited to Sp1 sites within the endogenous p21 promoter. Progestin-induced S-phase entry was attenuated in T47D cells containing wild-type PR-B and treated with EGFR, c-Src, or MAPK kinase inhibitors or in T47D cells stably expressing mPro or mutant DNA-binding domain PR-B. In sum, rapid progestin-activated PR signaling leads to PR Ser345 phosphorylation and tethering to Sp1. These events are critical for progestin-stimulated regulation of Sp1 target genes and breast cancer cell proliferation. Our data demonstrate the therapeutic potential for PR-targeted breast cancer treatment by exploiting multiple nodes along the PR signaling pathway, including PR-B, EGFR, c-Src, MAPK, or Sp1.

Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2.

ABSTRACT Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27−/− mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.

Insulin receptor substrates mediate distinct biological responses to insulin-like growth factor receptor activation in breast cancer cells

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

Membrane progesterone receptor expression in mammalian tissues: A review of regulation and physiological implications

The recent discovery of a novel, membrane localized progestin receptor (mPR) unrelated to the classical progesterone receptor (PR) in fishes and its subsequent identification in mammals suggests a potential mediator of non-traditional progestin actions, particularly in tissues where PR is absent. While early studies on mPR focused on final oocyte maturation in fishes, more current studies have examined mPRs in multiple mammalian systems in both reproductive and non-reproductive tissues as well as in diseased tissues. Here we review the current data on mPR in mammalian systems including male and female reproductive tracts, liver, neuroendocrine tissues, the immune system and breast and ovarian cancer. We also provide new data demonstrating mPR expression in the RAW 264.7 immune cell line and bone marrow-derived macrophages as well as mPR expression and downstream gene regulation in ovarian cancer cells.