The role of cFLIP in breast cancer stem cells

Breast tumours have intrinsic heterogeneity. The cancer stem cell hypothesis is currently challenging the notion that all cancer cells are equally malignant, suggesting that it is important to evaluate the efficacy of potential anti-cancer drugs by their ability to target the stem-like population. TRAIL is a cytotoxic agent the efficacy of which has been limited by a lack of patient stratification in clinical trials (Lemke 2014). In pre-clinical studies TRAIL has shown specificity towards mesenchymal-like breast cancer cell lines (Rahman et al. 2009) We show here that TRAIL is able to target the tumoursphere-forming population of four out of six breast cancer cell lines, including two epithelial-like lines, the bulk population of which is TRAIL-resistant. Furthermore, TRAIL also reduced the tumour-initiating capacity of the MCF-7 line. In addition, we have also investigated a paracrine mechanism of sensitising breast cancer cell lines to TRAIL. We have shown that a soluble factor produced by MDA-MB-231 cells, fibroblasts, and cancer-associated fibroblasts (CAFs) can sensitise both MCF-7 cells and SKBR3 tumoursphere-forming cells to TRAIL. Our data shows that cytoplasmic levels of Cellular FLICE-Like Inhibitory Protein (cFLIP) – a naturally occurring inhibitor of TRAIL’s cell toxicity effects- are lower in TRAIL-sensitive cells and suggest that tumoursphere populations are TRAIL-sensitive due to the re-localisation of cFLIP to the nucleus. We believe cFLIP is nuclear in stem-like cells due to a role as a promoter of the Wnt pathway. We have shown that inhibition of cFLIP by siRNA resulted in a reduction in both beta-catenin protein levels and Wnt-target gene transcription in both the MCF-7 and MDA-MB-231 breast cancer cell lines. We have also demonstrated a novel role for cFLIP as a promoter of bCSC maintenance. We have found that inhibition of cFLIP by shRNA decreased the self-renewal of tumoursphere-forming cells and also reduced colony formation. As TRAIL alone does not completely eradicate tumoursphere-forming or tumour-initiating cells in any breast cancer cell line, we believe our data are evidence of bCSC heterogeneity existing in terms of susceptibility to TRAIL. We propose a model of phenotypic heterogeneity within breast cancer cell lines and bCSCs whereby there exist two populations of cells which can be distinguished based on TRAIL susceptibility correlating with the known distinction of epithelial-like or mesenchymal-like status and our novel observation of cFLIP localisation. While these findings are currently restricted to cell lines, if confirmed in primary breast cancer cells, the clinical implication of our model is that although TRAIL alone is a potential therapy, a much more effective therapeutic strategy would be to also inhibit cFLIP, the consequences of which would not just be a sensitisation to TRAIL but also a reduction in Wnt signalling, and potentially a reduction in bCSC self-renewal and proliferation

Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells

Purpose: Nuclear factor-kappa B (NF-κB) signalling has been shown to regulate properties of breast cancer stem cells. However, the specific contribution of the non-canonical NF-κB pathway, components of which are elevated in aggressive breast cancer has not been addressed. Methods: Through shRNA silencing of the Nfkb2 gene, the role of p100/p52 in 4T1 and N202.1A cell lines were assessed by NF-κB reporter, invasion, tumoursphere and orthotopic transplantation assays. The processing of p100 into p52 was also inhibited with a p97 ATPase inhibitor, NMS-873, and its effects on tumoursphere formation was assessed. Results: Knockdown of Nfkb2 led to opposing changes in NF-κB-dependent transcription. NF-κB activity was elevated in 4T1 cells and this resulted in increased motility, cancer stem cell (CSC) activity and tumourigenicity in vivo. Conversely, depletion of Nfkb2 in N202.1a cells decreased NF-κB activity, CSC properties and tumourigenicity in vivo. By selectively overexpressing the p52 subunit in Nfkb2 depleted cells, we found that the increased malignancy in 4T1 cells could not be reverted in the presence of p52, whereas the decreased tumourigenicity of N202.1a cells could be rescued by p52. These results indicate that p100 and its subunit p52 have opposing effects on breast CSC activity. Accordingly, inhibition of an upstream regulator of p100 processing was effective in reducing tumoursphere formation of N202.1A and SKBR3 (ErbB2 HIGH) cells without aggravating that of 4T1 and MDA-MB-231 (ErbB2LOW) cells. Conclusion: These findings indicate that inhibiting the processing of p100 may be a potential therapeutic strategy to suppress CSC activity in a subset of breast tumours. © 2017, Springer Science+Business Media New York

Inside the stemness engine: mechanistic links between deregulated transcription factors and stemness in cancer

Cell identity is largely determined by its transcriptional profile. In tumour, deregulation of transcription factor expression and/or activity enables cancer cell to acquire a stem-like state characterised by capacity to self-renew, differentiate and form tumours in vivo. These stem-like cancer cells are highly metastatic and therapy resistant, thus warranting a more complete understanding of the molecular mechanisms downstream of the transcription factors that mediate the establishment of stemness state. Here, we review recent research findings that provide a mechanistic link between the commonly deregulated transcription factors and stemness in cancer. In particular, we describe the role of master transcription factors (SOX, OCT4, NANOG, KLF, BRACHYURY, SALL, HOX, FOX and RUNX), signalling-regulated transcription factors (SMAD, β-catenin, YAP, TAZ, AP-1, NOTCH, STAT, GLI, ETS and NF-κB) and unclassified transcription factors (c-MYC, HIF, EMT transcription factors and P53) across diverse tumour types, thereby yielding a comprehensive overview identifying shared downstream targets, highlighting unique mechanisms and discussing complexities

Breast Cancer Risk Assessment and Primary Prevention Advice in Primary Care: A Systematic Review of Provider Attitudes and Routine Behaviours

Implementing risk-stratified breast cancer screening is being considered internationally. It has been suggested that primary care will need to take a role in delivering this service, including risk assessment and provision of primary prevention advice. This systematic review aimed to assess the acceptability of these tasks to primary care providers. Five databases were searched up to July–August 2020, yielding 29 eligible studies, of which 27 were narratively synthesised. The review was pre-registered (PROSPERO: CRD42020197676). Primary care providers report frequently collecting breast cancer family history information, but rarely using quantitative tools integrating additional risk factors. Primary care providers reported high levels of discomfort and low confidence with respect to risk-reducing medications although very few reported doubts about the evidence base underpinning their use. Insufficient education/training and perceived discomfort conducting both tasks were notable barriers. Primary care providers are more likely to accept an increased role in breast cancer risk assessment than advising on risk-reducing medications. To realise the benefits of risk-based screening and prevention at a population level, primary care will need to proactively assess breast cancer risk and advise on risk-reducing medications. To facilitate this, adaptations to infrastructure such as integrated tools are necessary in addition to provision of education

The theoretical basis of a nationally implemented type 2 diabetes prevention programme: how is the programme expected to produce changes in behaviour?

From Springer Nature via Jisc Publications RouterHistory: received 2020-11-25, registration 2021-05-04, accepted 2021-05-04, online 2021-05-13, pub-electronic 2021-05-13, collection 2021-12Publication status: PublishedFunder: Health Services and Delivery Research Programme; doi: http://dx.doi.org/10.13039/501100002001; Grant(s): 16/48/07Abstract: Background: It is considered best practice to provide clear theoretical descriptions of how behaviour change interventions should produce changes in behaviour. Commissioners of the National Health Service Diabetes Prevention Programme (NHS-DPP) specified that the four independent provider organisations must explicitly describe the behaviour change theory underpinning their interventions. The nationally implemented programme, launched in 2016, aims to prevent progression to Type 2 diabetes in high-risk adults through changing diet and physical activity behaviours. This study aimed to: (a) develop a logic model describing how the NHS-DPP is expected to work, and (b) document the behaviour change theories underpinning providers’ NHS-DPP interventions. Methods: A logic model detailing how the programme should work in changing diet and activity behaviours was extracted from information in three specification documents underpinning the NHS-DPP. To establish how each of the four providers expected their interventions to produce behavioural changes, information was extracted from their programme plans, staff training materials, and audio-recorded observations of mandatory staff training courses attended in 2018. All materials were coded using Michie and Prestwich’s Theory Coding Scheme. Results: The NHS-DPP logic model included information provision to lead to behaviour change intentions, followed by a self-regulatory cycle including action planning and monitoring behaviour. None of the providers described an explicit logic model of how their programme will produce behavioural changes. Two providers stated their programmes were informed by the COM-B (Capability Opportunity Motivation – Behaviour) framework, the other two described targeting factors from multiple theories such as Self-Regulation Theory and Self-Determination Theory. All providers cited examples of proposed links between some theoretical constructs and behaviour change techniques (BCTs), but none linked all BCTs to specified constructs. Some discrepancies were noted between the theory described in providers’ programme plans and theory described in staff training. Conclusions: A variety of behaviour change theories were used by each provider. This may explain the variation between providers in BCTs selected in intervention design, and the mismatch between theory described in providers’ programme plans and staff training. Without a logic model describing how they expect their interventions to work, justification for intervention contents in providers’ programmes is not clear

Does the design of the NHS Diabetes Prevention Programme intervention have fidelity to the programme specification? A document analysis

Aims To assess fidelity of the Healthier You: NHS Diabetes Prevention Programme (NHS‐DPP), a behavioural intervention for people in England at high risk of developing type 2 diabetes, to the specified programme features. Methods Document analysis of the NHS‐DPP programme specification, including National Institute for Health and Care Excellence (NICE) PH38 diabetes prevention guidance. This was compared with the intervention design (framework response documents and programme manuals) from all four independent providers delivering the NHS‐DPP. Documents were coded using the Template for Intervention Description and Replication framework (describing service parameters) and the Behaviour Change Technique Taxonomy v1. Results Providers demonstrated good fidelity to service parameters of the NHS‐DPP. The NHS‐DPP specification indicated 19 unique behaviour change techniques. Framework responses for the four providers contained between 24 and 32 distinct behaviour change techniques, and programme manuals contained between 23 and 45 distinct behaviour change techniques, indicating variation in behaviour change content between providers’ intervention documents. Thus, each provider planned to deliver 74% of the unique behaviour change techniques specified, and a large amount of behaviour change content not mandated. Conclusions There is good fidelity to the specified service parameters of the NHS‐DPP; however, the four providers planned to deliver approximately three‐quarters of behaviour change techniques specified by the NHS‐DPP. Given that behaviour change techniques are the ‘active ingredients’ of interventions, and some of these techniques in the programme manuals may be missed in practice, this highlights possible limitations with fidelity to the NHS‐DPP programme specification at the intervention design stage

Is the NHS Diabetes Prevention Programme Intervention Delivered as Planned? An Observational Study of Fidelity of Intervention Delivery

Background The NHS Diabetes Prevention Programme (NHS-DPP) has been delivered by four commercial organizations across England, to prevent people with impaired glucose tolerance developing Type 2 diabetes. Evidence reviews underpinning the NHS-DPP design specification identified 19 Behavior Change Techniques (BCTs) that are the intervention “active ingredients.” It is important to understand the discrepancies between BCTs specified in design and BCTs actually delivered. Purpose To compare observed fidelity of delivery of BCTs that were delivered to (a) the NHS-DPP design specification, and (b) the programme manuals of four provider organizations. Methods Audio-recordings were made of complete delivery of NHS-DPP courses at eight diverse sites (two courses per provider organization). The eight courses consisted of 111 group sessions, with 409 patients and 35 facilitators. BCT Taxonomy v1 was used to reliably code the contents of NHS-DPP design specification documents, programme manuals for each provider organization, and observed NHS-DPP group sessions. Results The NHS-DPP design specification indicated 19 BCTs that should be delivered, whereas only seven (37%) were delivered during the programme in all eight courses. By contrast, between 70% and 89% of BCTs specified in programme manuals were delivered. There was substantial under-delivery of BCTs that were designed to improve self-regulation of behavior, for example, those involving problem solving and self-monitoring of behavior. Conclusions A lack of fidelity in delivery to the underlying evidence base was apparent, due to poor translation of design specification to programme manuals. By contrast, the fidelity of delivery to the programme manuals was relatively good. Future commissioning should focus on ensuring the evidence base is more accurately translated into the programme manual contents

Cytoplasmic levels of cFLIP determine a broad susceptibility of breast cancer stem/progenitor-like cells to TRAIL

Background The clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged. Methods We used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively. Results TRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling. Conclusion These data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution

Preventing type 2 diabetes:A research agenda for behavioural science

Aims The aim of this narrative review was to identify important knowledge gaps in behavioural science relating to type 2 diabetes prevention, to inform future research in the field. Methods Seven researchers who have published behaviour science research applied to type 2 diabetes prevention independently identified several important gaps in knowledge. They met to discuss these and to generate recommendations to advance research in behavioural science of type 2 diabetes prevention. Results A total of 21 overlapping recommendations for a research agenda were identified. These covered issues within the following broad categories: (a) evidencing the impact of whole population approaches to type 2 diabetes prevention, (b) understanding the utility of disease-specific approaches to type 2 diabetes prevention such as Diabetes Prevention Programmes (DPPs) compared to generic weight loss programmes, (c) identifying how best to increase reach and engagement of DPPs, whilst avoiding exacerbating inequalities, (d) the need to understand mechanism of DPPs, (e) the need to understand how to increase maintenance of changes as part of or following DPPs, (f) the need to assess the feasibility and effectiveness of alternative approaches to the typical self-regulation approaches that are most commonly used, and (g) the need to address emotional aspects of DPPs, to promote effectiveness and avoid harms. Conclusions There is a clear role for behavioural science in informing interventions to prevent people from developing type 2 diabetes, based on strong evidence of reach, effectiveness and cost-effectiveness. This review identifies key priorities for research needed to improve existing interventions