Euan W. Baxter

FGFR1-Induced Epithelial to Mesenchymal Transition through MAPK/PLCγ/COX-2-Mediated Mechanisms

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγand MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E2 levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.

A Modular Enhancer Is Differentially Regulated by GATA and NFAT Elements That Direct Different Tissue-Specific Patterns of Nucleosome Positioning and Inducible Chromatin Remodeling

ABSTRACT We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer.

RUNX1 transformation of primary embryonic fibroblasts is revealed in the absence of p53

The mammalian Runx gene family (Runx1–3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19ARF-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently prooncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFβ indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

A conserved insulator that recruits CTCF and cohesin exists between the closely related but divergently regulated interleukin-3 and granulocyte-macrophage colony-stimulating factor genes.

ABSTRACT The human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating-factor (GM-CSF, or CSF2) gene cluster arose by duplication of an ancestral gene. Although just 10 kb apart and responsive to the same signals, the IL-3 and GM-CSF genes are nevertheless regulated independently by separate, tissue-specific enhancers. To understand the differential regulation of the IL-3 and GM-CSF genes we have investigated a cluster of three ubiquitous DNase I-hypersensitive sites (DHSs) located between the two genes. We found that each site contains a conserved CTCF consensus sequence, binds CTCF, and recruits the cohesin subunit Rad21 in vivo. The positioning of these sites relative to the IL-3 and GM-CSF genes and their respective enhancers is conserved between human and mouse, suggesting a functional role in the organization of the locus. We found that these sites effectively block functional interactions between the GM-CSF enhancer and either the IL-3 or the GM-CSF promoter in reporter gene assays. These data argue that the regulation of the IL-3 and the GM-CSF promoters depends on the positions of their enhancers relative to the conserved CTCF/cohesin-binding sites. We suggest that one important role of these sites is to enable the independent regulation of the IL-3 and GM-CSF genes.

The Human IL-3/Granulocyte-Macrophage Colony-Stimulating Factor Locus Is Epigenetically Silent in Immature Thymocytes and Is Progressively Activated during T Cell Development

The closely linked IL-3 and GM-CSF genes are located within a cluster of cytokine genes co-expressed in activated T cells. Their activation in response to TCR signaling pathways is controlled by specific, inducible upstream enhancers. To study the developmental regulation of this locus in T lineage cells, we created a transgenic mouse model encompassing the human IL-3 and GM-CSF genes plus the known enhancers. We demonstrated that the IL-3/GM-CSF locus undergoes progressive stages of activation, with stepwise increases in active modifications and the proportion of cytokine-expressing cells, throughout the course of T cell differentiation. Looking first at immature cells, we found that the IL-3/GM-CSF locus was epigenetically silent in CD4/CD8 double positive thymocytes, thereby minimizing the potential for inappropriate activation during the course of TCR selection. Furthermore, we demonstrated that the locus did not reach its maximal transcriptional potential until after T cells had undergone blast cell transformation to become fully activated proliferating T cells. Inducible locus activation in mature T cells was accompanied by noncoding transcription initiating within the enhancer elements. Significantly, we also found that memory CD4 positive T cells, but not naive T cells, maintain a remodeled chromatin structure resembling that seen in T blast cells.

Selection for Loss of p53 Function in T-Cell Lymphomagenesis Is Alleviated by Moloney Murine Leukemia Virus Infection in myc Transgenic Mice

ABSTRACT Thymic lymphomas induced by Moloney murine leukemia virus (MMLV) have provided many examples of oncogene activation, but the role of tumor suppressor pathways in these tumors is less clear. These tumors display little evidence of loss of heterozygosity, and MMLV is only weakly synergistic with the Trp53 null genotype, suggesting that viral lymphomagenesis involves mechanisms which do not require mutational loss of Trp53function. To explore this relationship in greater depth, we infected CD2-myc transgenic mice with MMLV and examined the role of Trp53 in the genesis of these tumors. Most (19 of 27) of the tumors from MMLV-infected, CD2-myc Trp53+/− mice retained the wild-typeTrp53 allele in vivo while tumors of uninfected CD2-myc Trp53 +/− mice invariably showed allele loss from a significant fraction of primary tumor cells. The functional integrity of the Trp53gene in these tumors was indicated by ongoing allele loss or selection for mutational stabilization during in vitro propagation and by the radiosensitivity of selected Trp53 +/−tumor cell lines. An inverse correlation was noted between retention of the wild-type Trp53 allele and expression of p19ARF, providing further evidence of negative-feedback control of the latter by p53. However, expression of p19ARFdoes not appear to be counterselected in the absence of p53, and its integrity in Trp53 +/− tumors was indicated by its transcriptional upregulation on Trp53 wild-type allele loss in vitro in selected tumor cell lines. The role of MMLV was investigated further by analysis of proviral insertion sites in tumors of CD2-myc transgenic mice sorted forTrp53 genotype. A proportion of tumors showed insertions at Runx2, an oncogene which has been shown to collaborate independently with CD2-myc and with theTrp53 null genotype, and at a novel common integration site (ptl-1) on chromosome 8. Genotypic analysis of the panel of tumors suggested that neither of these integrations is functionally redundant with loss of p53, but it appears that the combination of the MMLV oncogenic program with the CD2-myc oncogene relegates p53 loss to a late step in tumor progression or in vitro culture. While the means by which these tumors preempt the p53 tumor suppressor response remains to be established, this study provides further evidence that irreversible inactivation of this pathway is not a prerequisite for tumor development in vivo.

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR–92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer.

The Inducible Tissue-Specific Expression of the Human IL-3/GM-CSF Locus Is Controlled by a Complex Array of Developmentally Regulated Enhancers

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I-hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the −34- to −40-kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible cyclosporin A-sensitive enhancer at −37 kb that increased IL-3 promoter activity 40-fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells, and myeloid progenitor cells.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action

Significance Autoimmune disease pathogenesis is driven by inflammation, induced partly by IgG autoantibody-containing immune complexes binding to Fc gamma receptors (FcγRs). These receptors are valid therapeutic targets in the treatment of autoimmunity. FcγRIIIa is one of a family of highly homologous receptors for IgG antibodies; previous attempts at therapeutic blockade have resulted in off-target effects involving cells that express the almost identical protein FcγRIIIb. Here we report the identification of functionally specific protein-based inhibitors (Affimer proteins) of FcγRIIIa and the structural/functional basis of their selectivity. As molecular research tools FcγRIIIa-specific Affimer proteins provide the ability to block IgG interaction with a single receptor. Our findings suggest that highly selective protein-based blocking agents that may have therapeutic applications can be readily produced. Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Abstract B127: Loss of miR-92 expression in breast epithelial cells is associated with cancer progression

MicroRNAs (miR) are a class of short non-coding RNAs that regulate gene expression and are commonly dysregulated in cancers, including those of the breast. MiR-92 is thought to function as an oncogene, promoting cell proliferation and reducing apoptosis. We have previously shown that expression of ERβ1 is negatively regulated by miR-92 in unselected non-microdissected breast cancers, providing a mechanism for down-regulation of this putative tumour suppressor gene. Here, we aimed to confirm the function of ERβ1 in breast cancer cells and examined expression of miR-92, specifically in the breast epithelium, during breast cancer progression. We also investigated the role of miR-92 in fibroblasts and examined whether differential expression in these cells might modify the behaviour of breast cancer epithelial cells. Stable ERβ1 overexpression was achieved using a MoMLV-based vector (pFBneo) or silenced by siRNA and effects were examined using various functional assays. We used laser microdissection (LMD) to isolate matched normal, co-incident DCIS and invasive tissue from 10 breast cancer patients. RNA was extracted, cDNA synthesised and preamplified using MegaPlex PreAmp Primers. MiR-92 expression was analysed by real-time PCR. Expression of ERβ1 protein was concurrently analysed for each patient by immunohistochemistry. LMD was also used to isolate matched normal and cancer-associated fibroblasts from 21 breast cancer patients and miR-92 expression was examined as above. Loss and gain of miR-92 in fibroblasts was achieved using anti-miR-92 miRNA and miRNASelect pEP-hsa-mir-92a-1 expression vector, respectively. The invasive potential of these fibroblasts was examined using a 2D modified Boyden chamber assay. ERβ1 overexpression decreased growth and induced cell death in T47D and BT-20 breast cancer cells. Surprisingly, we found that whilst absolute miR-92 expression levels varied across patients the expression pattern of miR-92 was consistent in all cases; miR-92 levels decreased during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p In summary, whilst confirming a potential tumour suppressor role for ERβ1 we have shown that expression is not inversely correlated with its negative regulator miR-92 in breast epithelium, suggesting other methods of regulation. Expression of miR-92 was lost, rather than gained, in breast epithelial cells during breast cancer progression correlating with a shift in ERβ1 staining from nuclei to the cytoplasm. While miR-92 expression levels remained unchanged in stromal fibroblasts, our data support a functional role in fibroblasts and show that differential miR-92 expression in these cells can influence the invasive capacity of breast cancer epithelial cells. Finally, our study highlights the importance of isolating specific cell types prior to expression analyses since changes in the epithelium may be masked by expression from the stroma and vice versa. Citation Format: Laura Smith, Euan Baxter, Andrew Hanby, Thomas Hughes, Rebecca Millican-Slater, Eldo Verghese, Valerie Speirs. Loss of miR-92 expression in breast epithelial cells is associated with cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B127.