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.

Functionally distinct roles for different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukaemia.

Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.

A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer.

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial-mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal-epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.

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.

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

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.

Mutant p53 drives invasion in breast tumors through up-regulation of miR-155.

Loss of p53 function is a critical event during tumorigenesis, with half of all cancers harboring mutations within the TP53 gene. Such events frequently result in the expression of a mutated p53 protein with gain-of-function properties that drive invasion and metastasis. Here, we show that the expression of miR-155 was up-regulated by mutant p53 to drive invasion. The miR-155 host gene was directly repressed by p63, providing the molecular basis for mutant p53 to drive miR-155 expression. Significant overlap was observed between miR-155 targets and the molecular profile of mutant p53-expressing breast tumors in vivo. A search for cancer-related target genes of miR-155 revealed ZNF652, a novel zinc-finger transcriptional repressor. ZNF652 directly repressed key drivers of invasion and metastasis, such as TGFB1, TGFB2, TGFBR2, EGFR, SMAD2 and VIM. Furthermore, silencing of ZNF652 in epithelial cancer cell lines promoted invasion into matrigel. Importantly, loss of ZNF652 expression in primary breast tumors was significantly correlated with increased local invasion and defined a population of breast cancer patients with metastatic tumors. Collectively, these findings suggest that miR-155 targeted therapies may provide an attractive approach to treat mutant p53-expressing tumors.

FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in breast cancer cells.

The transcription factor FOXP3 has been identified as a tumour suppressor in the breast and prostate epithelia, but little is known about its specific mechanism of action. We have identified a feed-forward regulatory loop in which FOXP3 suppresses the expression of the oncogene SATB1. In particular, we demonstrate that SATB1 is not only a direct target of FOXP3 repression, but that FOXP3 also induces two miRs, miR-7 and miR-155, which specifically target the 3'-UTR of SATB1 to further regulate its expression. We conclude that FOXP3-regulated miRs form part of the mechanism by which FOXP3 prevents the transformation of the healthy breast epithelium to a cancerous phenotype. Approaches aimed at restoring FOXP3 function and the miRs it regulates could help provide new approaches to target breast cancer.

Anti-CD3 monoclonal antibody treatment in newly diagnosed Type 1 diabetes patients: a hypothetical modelling analysis.

AIMS: Although limited clinical data exist for anti-CD3 monoclonal antibody therapies, it is believed that they may influence glycaemic control, endogenous insulin secretion and hypoglycaemic event rates in individuals newly diagnosed with Type 1 diabetes. In the absence of suitable empirical evidence, the objective of this study was to estimate the potential long-term clinical outcomes associated with treatment via a hypothetical modelling analysis. METHODS: Analyses were performed using a published and validated computer simulation model of diabetes in a hypothetical US cohort based on published literature and expert opinion. The efficacy of anti-CD3 monoclonal antibody treatment was estimated from clinical data and expert opinion and simulations were performed over a 60-year time horizon. The impact on quality of life associated with treatment was also captured via published utility values. RESULTS: Assuming that a treatment course of an anti-CD3 monoclonal antibody produced an initial reduction in glycated haemoglobin of -0.8%, and that the effects persisted for up to 5 years, treatment was projected to lead to an increase in undiscounted life expectancy of 0.43 years and an increase in quality-adjusted life expectancy of 0.36 quality-adjusted life years compared with conventional exogenous insulin. CONCLUSIONS: A course of a hypothetical anti-CD3 monoclonal antibody treatment associated with improved glycaemic control and, potentially, the preservation of pancreatic beta-cell function was estimated to lead to improved life expectancy and quality-adjusted life expectancy compared with conventional treatment in patients with newly diagnosed Type 1 diabetes.

Evaluation of exenatide vs. insulin glargine in type 2 diabetes: cost-effectiveness analysis in the German setting.

OBJECTIVES: The objective of this analysis was to determine the cost-effectiveness of exenatide vs. insulin glargine in patients with type 2 diabetes failing to achieve glycaemic control with oral antidiabetic agents, in the German setting, from a third-party payer perspective. METHODS: Data from a published randomized controlled trial were used in combination with a published, validated computer simulation model of type 2 diabetes to project clinical and cost outcomes over a time horizon of 10 years. Cost data were obtained from published literature and expert opinion. Clinical and cost outcomes were discounted at 5% per annum. Sensitivity analyses were performed to establish key drivers and parameters. RESULTS: Treatment with exenatide compared with insulin glargine was projected to be associated with improvements in life expectancy of 0.016 years and quality-adjusted life expectancy of 0.280 quality-adjusted life years (QALYs), increased lifetime direct medical costs of euro 3854 (euro 22 095 vs. euro 18 242) and an incremental cost-effectiveness ratio (ICER) of euro 13 746 per QALY. If quality of life was not taken into account, exenatide was associated with an ICER of euro 238 201 per life year gained vs. insulin glargine. Sensitivity analyses revealed that outcomes were most sensitive to changes in assumptions for (dis)utility values relating to weight change and the rate of self-monitored blood glucose testing. CONCLUSIONS: Exenatide was projected to be associated with similar clinical outcomes and increased costs compared with insulin glargine. Analysis of cost-effectiveness from a third-party perspective suggests that exenatide is likely to represent good value for money in the German setting.

Exenatide versus insulin glargine: a cost-effectiveness evaluation in patients with Type 2 diabetes in Switzerland.

OBJECTIVES: To investigate the long-term clinical and economic outcomes associated with exenatide versus insulin glargine as "add-on" treatments to oral therapy in individuals with Type 2 diabetes inadequately controlled with combination oral agents in the Swiss setting. METHODS: A computer simulation model of diabetes was used to project complications, life expectancy, quality-adjusted life expectancy and direct medical costs over a 35-year time horizon. Cohort characteristics and treatment effect data were derived from a 26-week randomized clinical trial comparing exenatide and insulin glargine. Modeled treatment effects included reductions in glycosylated hemoglobin (HbA1c) by -0.99% and -1.07% and in body mass index (BMI) by -0.80 and +0.55 kg/m2 with exenatide and insulin glargine respectively. Changes in systolic blood pressure and serum lipid levels were also captured. Simulations incorporated published quality of life utilities and Swiss costs from 2006. Extensive sensitivity analyses were conducted to assess the robustness of projected outcomes. Future clinical and economic outcomes were discounted at 2.5% per annum. RESULTS: In the base-case analysis exenatide was associated with comparable life expectancy (11,549 years versus 11,468 years) and an improvement in quality-adjusted life expectancy of 0.43 quality-adjusted life years (QALYs) versus insulin glargine over a 35-year time horizon. Exenatide was associated with a reduced cumulative incidence of most diabetes-related complications including an absolute reduction in myocardial infarction by 0.28%. Assuming an annual treatment cost of CHF 2,797.74 for exenatide, direct costs increased by CHF 8,378 per patient over the 35-year time horizon compared to insulin glargine. The resultant incremental cost-effectiveness ratio was CHF 19,450 per QALY gained for exenatide versus insulin glargine. CONCLUSIONS: Exenatide was associated with comparable life expectancy and an improvement in quality-adjusted life expectancy versus insulin glargine over a 35-year time horizon. Based on current standards exenatide would be a cost-effective treatment alternative to insulin glargine in Switzerland for Type 2 diabetes patients inadequately controlled on oral therapy.

Cost-effectiveness of pioglitazone in patients with type 2 diabetes and a history of macrovascular disease in a Swiss setting.

OBJECTIVES: To evaluate the cost-effectiveness of pioglitazone versus placebo, given in addition to existing treatment regimens, in patients with type 2 diabetes and evidence of macrovascular disease in Switzerland. METHODS: Event rates corresponding to macrovascular outcomes from the PROactive (Prospective Pioglitazone Clinical Trial in Macrovascular Events) trial of pioglitazone were used to project long-term clinical outcomes as part of a modified version of the previously validated CORE Diabetes Model. Direct medical costs associated with treatment regimens, complications and patient management were accounted in 2005 values based on Swiss-specific unit costs. Time horizon was set to lifetime (35 years). Future costs and clinical benefits were discounted at 2.5% annually in line with Swiss recommendations. One-way sensitivity analyses were performed. RESULTS: Addition of pioglitazone was associated with a reduced incidence of most diabetes-related complications, improved life expectancy (0.258 years) and improved quality-adjusted life expectancy (0.180 QALYs) compared with placebo. Pioglitazone treatment increased direct costs by CHF 10,914 per patient over a lifetime horizon. The incremental cost-effectiveness ratio (ICER) of pioglitazone versus placebo was CHF 42,274 per life-year gained and CHF 60,596 per QALY gained. ICERs were sensitive to variation in time horizon and duration of pioglitazone treatment effects. With a willingness to pay of CHF 80,000 per QALY in the Swiss setting, there was a 62.5% chance that pioglitazone would be cost-effective. CONCLUSIONS: Addition of pioglitazone to existing therapy was projected to reduce the long-term cumulative incidence of most diabetes complications and improve quality-adjusted life expectancy. Evaluation of incremental direct medical costs associated with these clinical benefits indicated that pioglitazone is likely to be a cost-effective treatment option in the Swiss setting over patient lifetimes.

The role of microRNAs in metastasis and epithelial-mesenchymal transition.

For a tumour cell to metastasize it must successfully negotiate a number of events, requiring a series of coordinated changes in the expression of many genes. MicroRNAs are small non-coding RNA molecules that post-transcriptionally control gene expression. As microRNAs are now recognised as master regulators of gene networks and play important roles in tumourigenesis, it is no surprise that microRNAs have recently been demonstrated to have central roles during metastasis. Recent work has also demonstrated critical roles for microRNAs in epithelial-mesenchymal transition, a phenotypic change underlain by altered gene expression patterns that is believed to mirror events in metastatic progression. These findings offer new potential for improved prognostics through expression profiling and may represent novel molecular treatment targets for future therapy. In this review, we summarise the multistep processes of metastasis and epithelial-mesenchymal transition and describe the recent discoveries of microRNAs that participate in controlling these processes.

Biphasic insulin aspart 70/30 vs. insulin glargine in insulin naïve type 2 diabetes patients: modelling the long-term health economic implications in a Swedish setting.

OBJECTIVES: To evaluate the long-term clinical and economic outcomes of biphasic insulin aspart 70/30 (BIAsp 70/30) treatment vs. insulin glargine in insulin naïve, type 2 diabetes patients failing oral antidiabetic drugs in a Swedish setting. METHODS: A published and validated computer simulation model (the CORE Diabetes Model) was used to project life expectancy, quality-adjusted life expectancy (QALE) and costs over patient lifetimes. Cohort characteristics [54.5% male, mean age 52.4 years, 9 years mean diabetes duration, mean glycosylated haemoglobin (HbA1c) 9.77%] and treatment effects were based on results from the Initiate Insulin by Aggressive Titration and Education (INITIATE) clinical trial. Direct medical costs were accounted in 2006 Swedish Kronor (SEK) and economic and clinical benefits were discounted at 3% per annum. RESULTS: Biphasic insulin aspart 70/30 treatment when compared with insulin glargine treatment was associated with improvements in discounted life expectancy of 0.21 years (13.10 vs. 12.89 years) and QALE of 0.21 quality-adjusted life years (QALYs) (9.16 vs. 8.96 QALYs). Reductions in the incidence of diabetes-related complications in the BIAsp 70/30 treatment arm led to reduced total costs of SEK 10,367 when compared with insulin glargine (SEK 396,475 vs. SEK 406,842) over patient lifetimes. BIAsp 70/30 treatment was projected to be dominant (cost and lifesaving) when compared with insulin glargine in the base case analysis. CONCLUSIONS: Biphasic insulin aspart 70/30 treatment was associated with improved clinical outcomes and reduced costs compared with insulin glargine treatment over patient lifetimes. These results were driven by improved HbA1c levels associated with BIAsp 70/30 compared with insulin glargine and the accompanying reduction in diabetes-related complications despite increases in body mass index.

Dopamine manipulations limited to preexposure are sufficient to modulate latent inhibition.

Four experiments are reported that demonstrated that dopamine (DA) transmission is involved in the acquisition of latent inhibition (LI) of a conditioned taste aversion. LI refers to weaker conditioning as a consequence of nonreinforced preexposure (PE) of the future conditioned stimulus. Although it is known to depend on DA transmission during the conditioning phase, it is usually thought that the cognitive processes involved in the establishment of LI (during the PE phase) are DA independent. Either amphetamine (AMPH; 0.5 or 1.0 mg/kg) or haloperidol (HAL; 0.1 mg/kg) were injected before 1 or all of the 3 PE sessions. AMPH blocked the acquisition of LI if it was injected before each or before only the last PE session and HAL potentiated LI.

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi.

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.