Romaine I. Fernando

The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells

Metastatic disease is responsible for the majority of human cancer deaths. Understanding the molecular mechanisms of metastasis is a major step in designing effective cancer therapeutics. Here we show that the T-box transcription factor Brachyury induces in tumor cells epithelial-mesenchymal transition (EMT), an important step in the progression of primary tumors toward metastasis. Overexpression of Brachyury in human carcinoma cells induced changes characteristic of EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and an increase in cell migration and invasion. Brachyury overexpression also repressed E-cadherin transcription, an effect partially mediated by Slug. Conversely, inhibition of Brachyury resulted in downregulation of mesenchymal markers and loss of cell migration and invasion and diminished the ability of human tumor cells to form lung metastases in a xenograft model. Furthermore, we found Brachyury to be overexpressed in various human tumor tissues and tumor cell lines compared with normal tissues. We also determined that the percentage of human lung tumor tissues positive for Brachyury expression increased with the stage of the tumor, indicating a potential association between Brachyury and tumor progression. The selective expression of Brachyury in tumor cells and its role in EMT and cancer progression suggest that Brachyury may be an attractive target for antitumor therapies.

Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway.

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17β-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 → Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1–452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.

Influence of IL-8 on the epithelial–mesenchymal transition and the tumor microenvironment

The phenomenon of epithelial-mesenchymal transition (EMT) has gained attention in the field of cancer biology for its potential contribution to the progression of carcinomas. Tumor EMT is a phenotypic switch that promotes the acquisition of a fibroblastoid-like morphology by epithelial tumor cells, resulting in enhanced tumor cell motility and invasiveness, increased metastatic propensity and resistance to chemotherapy, radiation and certain small-molecule-targeted therapies. Tumor cells undergoing EMT are also known to increase the secretion of specific factors, including cytokines, chemokines and growth factors, which could play an important role in tumor progression. This review summarizes the current knowledge on the secretory properties of epithelial tumor cells that have undergone an EMT, with an emphasis on the potential role of the IL-8-IL-8 receptor axis on the induction and/or maintenance of tumor EMT and its ability to remodel the tumor microenvironment.

Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

WEE1 Inhibition Alleviates Resistance to Immune Attack of Tumor Cells Undergoing Epithelial–Mesenchymal Transition

Aberrant expression of the T-box transcription factor brachyury in human carcinomas drives the phenomenon of epithelial-mesenchymal transition (EMT), a phenotypic modulation that facilitates tumor dissemination and resistance to conventional therapies, including chemotherapy and radiotherapy. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrate that high levels of brachyury also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated attack was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon with loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. In support of a causal connection, pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggested that reconstituting CDK1 activity to threshold levels may be sufficient to restore immunosurveillance of mesenchymal-like cancer cells that have escaped previous immune detection or eradication.

An autocrine loop between TGF-β1 and the transcription factor Brachyury controls the transition of human carcinoma cells into a mesenchymal phenotype

The epithelial–mesenchymal transition (EMT) is a process associated with the metastasis of solid tumors as well as with the acquisition of resistance to standard anticancer modalities. A major initiator of EMT in carcinoma cells is TGF-β, which has been shown to induce the expression of several transcription factors ultimately responsible for initiating and maintaining the EMT program. We have previously identified Brachyury, a T-box transcription factor, as an inducer of mesenchymal features in human carcinoma cells. In this study, a potential link between Brachyury and TGF-β signaling has been investigated. The results show for the first time that Brachyury expression is enhanced during TGF-β1–induced EMT in various human cancer cell lines, and that a positive feedback loop is established between Brachyury and TGF-β1 in mesenchymal-like tumor cells. In this context, Brachyury overexpression is shown to promote upregulation of TGF-β1 at the mRNA and protein levels, an effect mediated by activation of the TGF-β1 promoter in the presence of high levels of Brachyury. Furthermore, inhibition of TGF-β1 signaling by a small-molecule inhibitor of TGF-β receptor type I decreases Brachyury expression, induces a mesenchymal-to-epithelial transition, and renders cancer cells more susceptible to chemotherapy. This study thus has implications for the future development of clinical trials using TGF-β inhibitors in combination with other anticancer agents. Mol Cancer Ther; 12(9); 1805–15. ©2013 AACR.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Breast Cancer Cell Proliferation Is Inhibited by BAD REGULATION OF CYCLIN D1

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G1 to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G1 transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser75 and Ser99. Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

Paclitaxel induced apoptosis in breast cancer cells requires cell cycle transit but not Cdc2 activity

PurposePaclitaxel (PTX) is a widely used chemotherapy agent and may cause cell death by apoptosis subsequent to microtubule (MT) disruption. In this paper, we have investigated whether cell cycle transit and or Cdc2 (Cdk1) activity is required for the apoptosis induced by PTX.MethodsCell cycle was analyzed by flow cytometry, Cdc2 was assayed bio chemically. Cdc2 activity was decreased by siRNA and dominant negative (dn) Cdc2 expression. Cells were arrested by chemical or biological inhibitors in a G1or S phase. Apoptosis was measured by DNA fragmentation and examination of nuclei by microscopy. JNK and AKT activations were assessed as well.Results Cell cycle inhibition was highly effective in decreasing PTX induced apoptosis. MT morphology was not altered by these inhibitors. PTX induced JNK activity or AKT mediated BAD phosphorylation was unaffected by cell cycle inhibitors. Abrogation of Cdc 2 activity was without effect on PTX induced apoptosis.ConclusionsWhile cell cycle transit is required for PTX induced apoptosis; Cdc2 activity is not required.

An immunotherapeutic intervention against tumor progression: Targeting a driver of the epithelial-to-mesenchymal transition.

Targeting the epithelial-to-mesenchymal transition (EMT) is emerging as a novel intervention against tumor progression and metastatic dissemination, as well as the resistance to chemo- and radiotherapy displayed by multiple carcinomas. We have recently developed an immunotherapeutic approach to target a major driver of EMT, the T-box transcription factor T (also known as brachyury). This therapeutic paradigm is currently being tested in patients with advanced carcinomas in the context of a Phase I clinical trial.