Deborah A. Sanders

The transcription factor FOXM1 is a cellular target of the natural product thiostrepton

Transcription factors are proteins that bind specifically to defined DNA sequences to promote gene expression. Targeting transcription factors with small molecules to modulate the expression of certain genes has been notoriously difficult to achieve. The natural product thiostrepton is known to reduce the transcriptional activity of FOXM1, a transcription factor involved in tumorigenesis and cancer progression. Herein we demonstrate that thiostrepton interacts directly with FOXM1 protein in the human breast cancer cells MCF-7. Biophysical analyses of the thiostrepton-FOXM1 interaction provide additional insights on the molecular mode of action of thiostrepton. In cellular experiments, we show that thiostrepton can inhibit the binding of FOXM1 to genomic target sites. These findings illustrate the potential druggability of transcription factors and provide a molecular basis for targeting the FOXM1 family with small molecules.

Human hair growth in vitro: a model for the study of hair follicle biology

The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. We have developed an in vitro human hair growth model that enables us to maintain isolated human hair follicles for up to 10 days, during which time they continue to grow at an in vivo rate producing a keratinised hair fibre. We have shown that epidermal growth factor (EGF) in our system mimics the in vivo depilatory action of EGF in sheep, and suggest that this occurs as a result of EGF stimulating outer root sheath (ORS) cell proliferation which results in the disruption of normal mechanisms of cell-cell interaction in the hair follicle. We identify transforming growth factor-beta (TGF-beta) as a possible negative regulator of hair follicle growth and show that physiological levels of insulin-like growth factor-I (IGF-I) can support the same rates of hair follicle growth as supraphysiological levels of insulin. Furthermore, in the absence of insulin hair follicles show premature entry into a catagen-like state. This is prevented by physiological levels of IGF-I. Finally we demonstrate that the hair follicle is an aerobic glycolytic, glutaminolytic tissue and discuss the possible implications of this metabolism.

G-Quadruplex-Binding Benzo[a]phenoxazines Down-Regulate c-KIT Expression in Human Gastric Carcinoma Cells

There is considerable interest in the structure and function of G-quadruplex nucleic acid secondary structures, their cellular functions, and their potential as therapeutic targets. G-Quadruplex sequence motifs are prevalent in gene promoter regions and it has been hypothesized that G-quadruplex structure formation is associated with the transcriptional status of the downstream gene. Using a functional cell-based assay, we have identified two novel G-quadruplex ligands that reduce the transcription of a luciferase reporter driven from the G-quadruplex-containing c-KIT promoter. We have further shown that endogenous c-KIT expression in a human gastric carcinoma cell line is also reduced on treatment with these molecules. Biophysical analysis using surface plasmon resonance has shown that these molecules preferentially bind with high affinity to one of the two G-quadruplex sequences in the c-KIT promoter over double-stranded DNA. This work highlights the utility of cell-based reporter assays to identify new G-quadruplex binding molecules that modulate transcription and identifies benzo[a]phenoxazine derivatives as potential antitumor agents.

Soluble FLT1 sensitizes endothelial cells to inflammatory cytokines by antagonizing VEGF receptor-mediated signalling

AIMS Pre-eclampsia affects 5-7% of pregnancies, and is a major cause of maternal and foetal death. Elevated serum levels of placentally derived splice variants of the vascular endothelial growth factor (VEGF) receptor, soluble fms-like tyrosine kinase-1 (sFLT1), are strongly implicated in the pathogenesis but, as yet, no underlying mechanism has been described. An excessive inflammatory-like response is thought to contribute to the maternal endothelial cell dysfunction that characterizes pre-eclampsia. We hypothesized that sFLT1 antagonizes autocrine VEGF-A signalling, rendering endothelial cells more sensitive to pro-inflammatory factors also released by the placenta. We tested this by manipulating VEGF receptor signalling and treating endothelial cells with low doses of tumour necrosis factor-α (TNF-α). METHODS AND RESULTS Application of recombinant sFLT1 alone did not activate human umbilical vein endothelial cells (HUVECs). However, antagonizing the autocrine actions of endothelial VEGF-A and/or placenta growth factor (PlGF) by pre-incubation with recombinant sFLT1, anti-FLT1, anti-VEGF receptor 2 (KDR), anti-VEGF-A, VEGF receptor tyrosine kinase inhibitor SU5614, or knocking-down FLT1 or KDR transcripts rendered cells more sensitive to low doses of TNF-α. Each treatment increased activation, as measured by increases in endothelial intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), endothelin 1 (ET-1), von Willebrand factor (vWF), and leucocyte adhesion, and led to reduction in AKT Ser⁴⁷³ and endothelial nitric oxide synthase (eNOS) Ser¹¹⁷⁷ phosphorylation. CONCLUSIONS Our data describe a mechanism by which sFLT1 sensitizes endothelial cells to pro-inflammatory factors, providing an explanation for how placental stress may precipitate the pre-eclamptic syndrome.

Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells

BackgroundThe forkhead transcription factor FOXM1 is a key regulator of the cell cycle. It is frequently over-expressed in cancer and is emerging as an important therapeutic target. In breast cancer FOXM1 expression is linked with estrogen receptor (ERα) activity and resistance to endocrine therapies, with high levels correlated with poor prognosis. However, the precise role of FOXM1 in ER positive breast cancer is not yet fully understood.ResultsThe study utilizes chromatin immunoprecipitation followed by high-throughput sequencing to map FOXM1 binding in both ERα-positive and -negative breast cancer cell lines. The comparison between binding site distributions in the two cell lines uncovered a previously undescribed relationship between binding of FOXM1 and ERα. Further molecular analyses demonstrated that these two factors can bind simultaneously at genomic sites and furthermore that FOXM1 regulates the transcriptional activity of ERα via interaction with the coactivator CARM1. Inhibition of FOXM1 activity using the natural product thiostrepton revealed down-regulation of a set of FOXM1-regulated genes that are correlated with patient outcome in clinical breast cancer samples.ConclusionsThese findings reveal a novel role for FOXM1 in ERα transcriptional activity in breast cancer and uncover a FOXM1-regulated gene signature associated with ER-positive breast cancer patient prognosis.

Pyridostatin analogues promote telomere dysfunction and long-term growth inhibition in human cancer cells

The synthesis, biophysical and biological evaluation of a series of G-quadruplex interacting small molecules based on a N,N′-bis(quinolinyl)pyridine-2,6-dicarboxamide scaffold is described.

Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition

The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA binding domain (DBD), and activates proliferation- and differentiation-associated genes. Aberrant overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here — from a high-throughput screen applied to a library of 54,211 small molecules — we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds: FDI-6 (NCGC00099374) is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional down-regulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically down regulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-seq. This small molecule mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

Herein, we demonstrate the design, synthesis, biophysical properties, and preliminary biological evaluation of 6-substituted indenoisoquinolines as a new class of G-quadruplex stabilizing small molecule ligands. We have synthesized 6-substituted indenoisoquinolines 1a-e in two steps from commercially available starting materials with excellent yields. The G-quadruplex stabilization potential of indenoisoquinolines 1a-e was evaluated by fluorescence resonance energy transfer-melting analysis, which showed that indenoisoquinolines show a high level of stabilization of various G-quadruplex DNA structures. Indenoisoquinolines demonstrated potent inhibition of cell growth in the GIST882 patient-derived gastrointestinal stromal tumor cell line, accompanied by inhibition of both c-Kit transcription and KIT oncoprotein levels.

Analysis of PPARα-dependent and PPARα-independent transcript regulation following fenofibrate treatment of human endothelial cells

Fenofibrate is a synthetic ligand for the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha and has been widely used in the treatment of metabolic disorders, especially hyperlipemia, due to its lipid-lowering effect. The molecular mechanism of lipid-lowering is relatively well defined: an activated PPARα forms a PPAR–RXR heterodimer and this regulates the transcription of genes involved in energy metabolism by binding to PPAR response elements in their promoter regions, so-called “trans-activation”. In addition, fenofibrate also has anti-inflammatory and anti-athrogenic effects in vascular endothelial and smooth muscle cells. We have limited information about the anti-inflammatory mechanism of fenofibrate; however, “trans-repression” which suppresses production of inflammatory cytokines and adhesion molecules probably contributes to this mechanism. Furthermore, there are reports that fenofibrate affects endothelial cells in a PPARα-independent manner. In order to identify PPARα-dependently and PPARα-independently regulated transcripts, we generated microarray data from human endothelial cells treated with fenofibrate, and with and without siRNA-mediated knock-down of PPARα. We also constructed dynamic Bayesian transcriptome networks to reveal PPARα-dependent and -independent pathways. Our transcriptome network analysis identified growth differentiation factor 15 (GDF15) as a hub gene having PPARα-independently regulated transcripts as its direct downstream children. This result suggests that GDF15 may be PPARα-independent master-regulator of fenofibrate action in human endothelial cells.