Author Correction: LSD1 activation promotes inducible EMT programs and modulates the tumour microenvironment in breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LSD1 activation promotes inducible EMT programs and modulates the tumour microenvironment in breast cancer.

Complex regulatory networks control epithelial-to-mesenchymal transition (EMT) but the underlying epigenetic control is poorly understood. Lysine-specific demethylase 1 (LSD1) is a key histone demethylase that alters the epigenetic landscape. Here we explored the role of LSD1 in global epigenetic regulation of EMT, cancer stem cells (CSCs), the tumour microenvironment, and therapeutic resistance in breast cancer. LSD1 induced pan-genomic gene expression in networks implicated in EMT and selectively elicits gene expression programs in CSCs whilst repressing non-CSC programs. LSD1 phosphorylation at serine-111 (LSD1-s111p) by chromatin anchored protein kinase C-theta (PKC-θ), is critical for its demethylase and EMT promoting activity and LSD1-s111p is enriched in chemoresistant cells in vivo. LSD1 couples to PKC-θ on the mesenchymal gene epigenetic template promotes LSD1-mediated gene induction. In vivo, chemotherapy reduced tumour volume, and when combined with an LSD1 inhibitor, abrogated the mesenchymal signature and promoted an innate, M1 macrophage-like tumouricidal immune response. Circulating tumour cells (CTCs) from metastatic breast cancer (MBC) patients were enriched with LSD1 and pharmacological blockade of LSD1 suppressed the mesenchymal and stem-like signature in these patient-derived CTCs. Overall, LSD1 inhibition may serve as a promising epigenetic adjuvant therapy to subvert its pleiotropic roles in breast cancer progression and treatment resistance.

MiR-200 can repress breast cancer metastasis through ZEB1-independent but moesin-dependent pathways.

The microRNA-200 (miR-200) family has a critical role in regulating epithelial-mesenchymal transition and cancer cell invasion through inhibition of the E-cadherin transcriptional repressors ZEB1 and ZEB2. Recent studies have indicated that the miR-200 family may exert their effects at distinct stages in the metastatic process, with an overall effect of enhancing metastasis in a syngeneic mouse breast cancer model. We find in a xenograft orthotopic model of breast cancer metastasis that ectopic expression of members of the miR-200b/200c/429, but not the miR-141/200a, functional groups limits tumour cell invasion and metastasis. Despite modulation of the ZEB1-E-cadherin axis, restoration of ZEB1 in miR-200b-expressing cells was not able to alter metastatic potential suggesting that other targets contribute to this process. Instead, we found that miR-200b repressed several actin-associated genes, with the knockdown of the ezrin-radixin-moesin family member moesin alone phenocopying the repression of cell invasion by miR-200b. Moesin was verified to be directly targeted by miR-200b, and restoration of moesin in miR-200b-expressing cells was sufficient to alleviate metastatic repression. In breast cancer cell lines and patient samples, the expression of moesin significantly inversely correlated with miR-200 expression, and high levels of moesin were associated with poor relapse-free survival. These findings highlight the context-dependent effects of miR-200 in breast cancer metastasis and demonstrate the existence of a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis, through which miR-200 can regulate tumour cell plasticity and metastasis.

Hoxb8 regulates expression of microRNAs to control cell death and differentiation.

Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17∼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17∼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17∼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17∼92 expression through c-Myc, a known transcriptional regulator of the miR-17∼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.

Circulating microRNAs predict biochemical recurrence in prostate cancer patients.

BACKGROUND: Circulating microRNAs (miRNAs) are emerging as promising biomarkers for prostate cancer. Here, we investigated the potential of these molecules to assist in prognosis and treatment decision-making. METHODS: MicroRNAs in the serum of patients who had experienced rapid biochemical recurrence (BCR) (n=8) or no recurrence (n=8) following radical prostatectomy (RP) were profiled using high-throughput qRT-PCR. Recurrence-associated miRNAs were subsequently quantitated by qRT-PCR in a validation cohort comprised of 70 patients with Gleason 7 cancers treated by RP, 31 of whom had undergone disease progression following surgery. The expression of recurrence-associated miRNAs was also examined in tumour tissue cohorts. RESULTS: Three miRNAs - miR-141, miR-146b-3p and miR-194 - were elevated in patients who subsequently experienced BCR in the screening study. MiR-146b-3p and miR-194 were also associated with disease progression in the validation cohort, as determined by log-rank tests and Cox proportional hazards regression. Multivariate analysis revealed that miR-146b-3p possessed prognostic information beyond standard clinicopathological parameters. Analysis of tissue cohorts revealed that miR-194 was robustly expressed in the prostate, elevated in metastases, and its expression in primary tumours was associated with a poor prognosis. CONCLUSION: Our study suggests that circulating miRNAs, measured at the time of RP, could be combined with current prognostic tools to predict future disease progression in men with intermediate risk prostate cancers.

Sensing solutions for collecting spatio-temporal data for wildlife monitoring applications: a review.

Movement ecology is a field which places movement as a basis for understanding animal behavior. To realize this concept, ecologists rely on data collection technologies providing spatio-temporal data in order to analyze movement. Recently, wireless sensor networks have offered new opportunities for data collection from remote places through multi-hop communication and collaborative capability of the nodes. Several technologies can be used in such networks for sensing purposes and for collecting spatio-temporal data from animals. In this paper, we investigate and review technological solutions which can be used for collecting data for wildlife monitoring. Our aim is to provide an overview of different sensing technologies used for wildlife monitoring and to review their capabilities in terms of data they provide for modeling movement behavior of animals.

Cytokeratin 17 and p63 are markers of the HPV target cell, the cervical stem cell.

BACKGROUND: Basic research on HPV has focused on identifying the genetic changes that lead to cervical carcinoma. However, while focusing on the molecular biology of the cancer, understanding of its cellular biology has lagged: the target cell of the HPV infection is unknown. MATERIALS AND METHODS: In this study we identified the stem cell population of the cervical epithelium by monoclonal antibodies against p63, a homologue of the tumor suppressor gene p53 and cytokeratin 17 (CK17). RESULTS: We noted p63 expression consistently in the nuclei of reserve cells, hyperplasia of the reserve cells and the basal layer of the ectocervical epithelium, while CK17 only stained endocervical reserve cells and reserve cell hyperplasia. CONCLUSION: We conclude that both p63 and CK17 are suitable markers for cervical stem cell identification. Both markers, therefore, qualify for the identification of the HPV target cell.

Imposex and organotin concentrations in Buccinum undatum and Neptunea antiqua from the North Sea: relationship to shipping density and hydrographical conditions.

The presence and the development of imposex were investigated in the common whelk (Buccinum undatum) and the red whelk (Neptunea antiqua) from the open North Sea and the Skagerrak. Imposex development was related to levels of organotins in snails and in the fine fractions (< 63 microm) of the sediments they inhabit. The sampling locations were classified according to three levels of traffic densities of ships of > or = 100 gt per day passing within 15 Nautical miles of the sampling station, shipping levels being: high (> 10 ships day(-1)), intermediate (5--10 ships day(-1)), and low (< 5 ships day(-1)). Sampling stations were also classified according to presence or absence of a vertically stratified water column. In the snails the body levels of the butyltin metabolites MBT and DBT and the parent phenyltin compound TPT, were higher than those of TBT and PT metabolites. In the sediment, the parent compounds and the mono-substituted metabolites MBT and MPT were present in the highest concentrations. The highest body levels of all organotin compounds and the highest imposex indices for the common whelk were found at those locations in the Southern Bight and the German Bight that had a high shipping density as well as a homogeneously mixed water column during the whole year. At these locations sediment levels of organotins were also higher than at other sites. In contrast, the body levels of organotins were low and imposex was sometimes even completely absent in snails from stratified deep-water stations in the Skagerrak, despite a very high shipping density in the entrance area of the Baltic. In sediments from stratified locations with low or intermediate shipping densities, organotin compounds were below or close to their respective limits of detection. These stations were located in areas with a stratified water column during the whole year. The results can be explained by postulating a much higher resistance for dissolved organotins to migrate through a pycnocline. Organotins could only transgress through a pycnocline when adsorbed to settling particles that manage to transgress the boundary between layers. N. antiqua could only be obtained in sufficient numbers from deeper water stations, which almost all had a stratified water column. At stations where both snail species were obtained and imposex was present, the imposex index was higher in the red whelk. Hence N. antiqua seems to be the more sensitive species of the two. In the red whelk, imposex development increased with shipping density too, though in the smaller samples the trend was not significant. Average biota-sediment accumulation factors (BSAFs; normalised for lipid content in snails and TOC content in the fraction < 63 microm in sediments) for Buccinum ranged from 0.4 to 1.0 for butyltins and were similar to literature values reported for TBT in other marine species. Higher average BSAF values were found for phenyltins 1.5 (MPT) to 17 (TPT). The high values for TPT match the ranges expected from equilibrium partitioning concepts of persistent hydrophobic compounds. The ratio of live snails to the total number of live snails plus empty shells ranged between 2.5 and 93%. This parameter might be a useful indicator to compare past and present densities of populations of both species in different areas of the North Sea.

A NF-kappa B/Sp1 region is essential for chromatin remodeling and correct transcription of a human granulocyte-macrophage colony-stimulating factor transgene.

The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb transgene containing the entire human GM-CSF gene has also been shown to display inducible, position-independent, copy number-dependent transcription in mouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter and activity assessed following the generation of transgenic mice. Of four mutations introduced into the transgene promoter, only one, in an NF-kappaB/Sp1 region, led to decreased induction of the transgene in splenocytes or bone marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, but not when transiently transfected into the same cell lines. The mutation alters the NF-kappaB family members that bind to the NF-kappaB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersensitive site that is normally generated at the promoter following T cell activation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-kappaB/Sp1 region plays a critical role in chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.

Reconstitution of T cell-specific transcription directed by composite NFAT/Oct elements.

The complex nature of most promoters and enhancers makes it difficult to identify key determinants of tissue-specific gene expression. Furthermore, most tissue-specific genes are regulated by transcription factors that have expression profiles more widespread than the genes they control. NFAT is an example of a widely expressed transcription factor that contributes to several distinct patterns of cytokine gene expression within the immune system and where its role in directing specificity remains undefined. To investigate distinct combinatorial mechanisms employed by NFAT to regulate tissue-specific transcription, we examined a composite NFAT/AP-1 element from the widely active GM-CSF enhancer and a composite NFAT/Oct element from the T cell-specific IL-3 enhancer. The NFAT/AP-1 element was active in the numerous cell types that express NFAT, but NFAT/Oct enhancer activity was T cell specific even though Oct-1 is ubiquitous. Conversion of the single Oct site in the IL-3 enhancer to an AP-1 enabled activation outside of the T cell lineage. By reconstituting the activities of both the IL-3 enhancer and its NFAT/Oct element in a variety of cell types, we demonstrated that their T cell-specific activation required the lymphoid cofactors NIP45 and OCA-B in addition to NFAT and Oct family proteins. Furthermore, the Oct family protein Brn-2, which cannot recruit OCA-B, repressed NFAT/Oct enhancer activity. Significantly, the two patterns of combinatorial regulation identified in this study mirror the cell-type specificities of the cytokine genes that they govern. We have thus established that simple composite transcription factor binding sites can indeed establish highly specific patterns of gene expression.

Generation of an improved luciferase reporter gene plasmid that employs a novel mechanism for high-copy replication.

We have engineered the reporter gene plasmid pXPG by incorporating a novel high-copy origin of replication and a modified luciferase gene into a pXP1-derived vector that efficiently blocks read-through transcription in eukaryotic cells. pXPG contains the Luc+ luciferase gene derived from pGL3 that lacks a peroxisomal targeting sequence, thereby allowing accumulation of luciferase protein in the cytoplasm rather than subcellular organelles of transfected eukaryotic cells. pXPG has distinct advantages over pGL3, because it contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation signal present in pGL3. pXPG contains a novel mutation near the origin of replication that increases plasmid copy number in Escherichia coli. This mutation alters the -10 sequence in the RNA II promoter of the ColE1 origin of replication from TAATCT to TAATAT. As this sequence is a closer match to the consensus -10 element, we suggest that the mutation increases copy number by increasing the rate of transcription of the RNA II replication primer. This novel mechanism for increasing copy number may have more widespread applications than the commonly used pUC high-copy origin of replication mutation. Unlike pUC, which reverts to low copy number at 30 degrees C, the pXPG mutation supports a higher copy number at both 37 and 30 degrees C.

The human granulocyte-macrophage colony-stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% +/- 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% +/- 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway.

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.

A T cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site.

Interleukin-3 (IL-3) is a cytokine that is expressed primarily in activated T cells. Here we identified an inducible T cell-specific enhancer 14 kb upstream of the IL-3 gene that responded to activation of T cell receptor signaling pathways. The IL-3 enhancer spanned an inducible cyclosporin A-sensitive DNase I-hypersensitive site found only in T cells. Four NFAT-like elements exist within the enhancer. The two most active NFAT-like elements were located at the center of the DNase I-hypersensitive site. One of these NFAT-like elements encompassed overlapping Oct- and NFATp/c-binding sites, which functioned in a highly synergistic manner. We suggest that the T cell-specific expression of the IL-3 gene is partly controlled through the enhancer by cooperation between Oct and NFAT family proteins.

Regulation of GM-CSF gene transcription by core-binding factor.

GM-CSF gene activation in T cells is known to involve the transcription factors nuclear factor-kappa B, AP-1, NFAT, and Sp1. Here we demonstrate that the human GM-CSF promoter and enhancer also encompass binding sites for core-binding factor (CBF). Significantly, the CBF sites are in each case contained within the minimum essential core regions required for inducible activation of transcription. Furthermore, these core regions of the enhancer and promoter each encompass closely linked binding sites for CBF, AP-1, and NFATp. The GM-CSF promoter CBF site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site. A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment. Stepwise deletions into the proximal promoter also revealed that the CBF site, but not the YY-1 site, was required for efficient induction of transcriptional activation. The AML1 and CBF beta genes that encode CBF each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia. This study adds GM-CSF to a growing list of cytokines and receptors that are regulated by CBF and which control the growth, differentiation, and activation of hemopoietic cells. The GM-CSF locus may represent one of several target genes that are dysregulated in acute myeloid leukemia.

Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT.

Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.

Human granulocyte-macrophage colony-stimulating factor enhancer function is associated with cooperative interactions between AP-1 and NFATp/c.

The promoter of the human granulocyte-macrophage colony-stimulating factor gene is regulated by an inducible upstream enhancer. The enhancer encompasses three previously defined binding sites for the transcription factor NFAT (GM170, GM330, and GM550) and a novel NFAT site defined here as the GM420 element. While there was considerable redundancy within the enhancer, the GM330, GM420, and GM550 motifs each functioned efficiently in isolation as enhancer elements and bound NFATp and AP-1 in a highly cooperative fashion. These three NFAT sites closely resembled the distal interleukin-2 NFAT site, and methylation interference assays further defined GGA(N)9TCA as a minimum consensus sequence for this family of NFAT sites. By contrast, the GM170 site, which also had conserved GGA and TCA motifs but in which these motifs were separated by 15 bases, supported strong independent but no cooperative binding of AP-1 and NFATp, and this site functioned poorly as an enhancer element. While both the GM330 and GM420 elements were closely associated with the inducible DNase I-hypersensitive site within the enhancer, the GM420 element was the only NFAT site located within a 160-bp HincII-BalI fragment defined by deletion analysis as the essential core of the enhancer. The GM420 element was unusual, however, in containing a high-affinity NFATp/c-binding sequence (TGGAAAGA) immediately upstream of the sequence TGACATCA which more closely resembled a cyclic AMP response-like element than an AP-1 site. We suggest that the cooperative binding of NFATp/c and AP-1 requires a particular spacing of sites and that their cooperativity and induction via independent pathways ensure very tight regulation of the granulocyte-macrophage colony-stimulating factor enhancer.

The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-cell line to identify additional regulatory elements. We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an AP1-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.

Effects of xylitol and fluoride on the response to glucose pulses of Streptococcus mutans T8 growing in continuous culture.

Streptococcus mutans T8 was grown glucose-limited at pH 7.0 in a chemostat and pulsed, under pH free-fall conditions, with glucose, xylitol or a mixture of the two. Experiments were conducted in the absence or continual presence of low levels (1 mmol.l-1) of fluoride. Culture filtrates of samples taken at frequent intervals were assayed for carbohydrate and fermentation end-products. Fluoride had little effect on the organism's response to glucose until the culture pH fell to ca. 5.0, at which point the rate of lactate production was reduced some 3-fold. Xylitol affected the response to glucose but its effect was most marked in the presence of fluoride. Under these conditions, the rate of lactate production was reduced at least 3-fold, the pH did not fall to 5.0 and only about 50% of the added glucose was consumed. This suggests that xylitol can augment the metabolic effects on S. mutans of low levels of fluoride.