Investigating the therapeutic potential of cellular FLICE-like inhibitory protein and TRAIL in preclinical models of breast cancer

Apoptosis is an important process in normal mammary gland physiology and evasion of apoptosis has also been identified as a hallmark of cancer. In breast cancer cells apoptotic resistance is an acquired feature that can promote tumour growth and progression. Induction of apoptosis by the extrinsic death ligand TRAIL has been shown to be a promising clinical therapy targeting a number of different cancer cells whilst sparing normal cells. Unfortunately most breast cancers are inherently resistant to TRAIL treatment. Herein it is shown that by reducing the expression of the downstream TRAIL inhibitor c-FLIP, a range of different breast cancer subtypes can be sensitised to TRAIL treatment resulting in significant cancer cell death. Significantly, suppression of c-FLIP in combination with TRAIL (FLIPi/TRAIL) ablated the tumour-initiating breast cancer stem cell (bCSC) subset, as defined by mammosphere formation assay, within cell lines. This selective killing of bCSCs translated to reduced tumour initiation and metastasis in animal transplant models. However, continued culture of FLIPi/TRAIL treated cell lines in adherent conditions resulted in bCSC re-acquisition suggesting a phenotypic plasticity of non-bCSC cells. Re-acquired bCSCs also demonstrated sensitivity to repeated FLIPi/TRAIL treatment and maintaining reduced c-FLIP expression prevented bCSC re-acquisition. These results substantiate the importance of resistance to apoptosis in tumour initiation and metastasis and identify the targeting of c-FLIP proteins as a promising anti-cancer therapeutic approach. Acquired resistance to existing mainstay therapies such as antiestrogens (AEs) (tamoxifen and Faslodex) and aromatase inhibitors (AIs) is an ongoing obstacle in treatment of a large number of breast cancer patients. AE-resistant models of breast cancer and a multiple endocrine-resistant patient sample demonstrated hypersensitivity to TRAIL. This sensitivity was observed in both in vitro and in vivo models of AE resistance and cell death was prevalent in both bulk tumour cells and bCSCs. Sensitisation was not attributed to combination AE/TRAIL treatment suggesting cellular changes during the acquisition of AE resistance are responsible for TRAIL sensitivity in these models. Further investigation suggested that the mechanism of AE-resistant cell sensitivity to TRAIL was not dependant on functional estrogen receptor signalling and is most likely dependant on the AE agent that the cancer cells have acquired resistance to. Interestingly tamoxifen-resistant MCF-7 cells were shown to have reduced c-FLIP protein expression compared to parental cells, further supporting c-FLIP’s potential in cancer therapy. Recent success in the use non-MHC-restricted γδ T cells as a targeted immunotherapy in clinical trials has identified this therapeutic methodology as desirable. Here it is shown that TRAIL is readily expressed by this subset of T cells that also demonstrate cytotoxicity to breast cancer cell lines. Neither the secretion of TRAIL or surface expression of TRAIL appeared to contribute significantly towards γδ T cell cytotoxicity and the majority of breast cancer cell death induced by γδ T cells would seem to be perforin-mediated. The suppression of c-FLIP in target cells increased γδ T cell cytotoxicity but again not via TRAIL. Preliminary results also indicated that the bCSCs of some cell lines were exquisitely sensitive to γδ T cell treatment. In summary these results indicate that targeting c-FLIP and TRAIL can be therapeutically beneficial in a range of different breast cancer subtypes by certain therapeutic strategies

Efficient intravenous tumor targeting using the αvβ6 integrin-selective precision virotherapy Ad5NULL-A20

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvβ6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvβ6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvβ6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvβ6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvβ6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvβ6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvβ6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes

Antibody drug conjugates against the receptor for advanced glycation end products (RAGE), a novel therapeutic target in endometrial cancer

Background The treatment of endometrial cancer (EC), the most common gynecological cancer, is currently hampered by the toxicity of current cytotoxic agents, meaning novel therapeutic approaches are urgently required. Methods A cohort of 161 patients was evaluated for the expression of the receptor for advanced glycation end products (RAGE) in endometrial tissues. The present study also incorporates a variety of in vitro methodologies within multiple cell lines to evaluate RAGE expression and antibody-drug conjugate efficacy, internalisation and intercellular trafficking. Additionally, we undertook in vivo bio-distribution and toxicity evaluation to determine the suitability of our chosen therapeutic approach, together with efficacy studies in a mouse xenograft model of disease. Results We have identified an association between over-expression of the receptor for advanced glycation end products (RAGE) and EC (H-score = Healthy: 0.46, SD 0.26; Type I EC: 2.67, SD 1.39; Type II EC: 2.20, SD 1.34; ANOVA, p < 0.0001). Furthermore, increased expression was negatively correlated with patient survival (Spearman’s Rank Order Correlation: ρ = − 0.3914, p < 0.05). To exploit this association, we developed novel RAGE-targeting antibody drug conjugates (ADC) and demonstrated the efficacy of this approach. RAGE-targeting ADCs were up to 100-fold more efficacious in EC cells compared to non-malignant cells and up to 200-fold more cytotoxic than drug treatment alone. Additionally, RAGE-targeting ADCs were not toxic in an in vivo pre-clinical mouse model, and significantly reduced tumour growth in a xenograft mouse model of disease. Conclusions These data, together with important design considerations implied by the present study, suggest RAGE-ADCs could be translated to novel therapeutics for EC patients

The contribution of macroalgae-associated fishes to small-scale tropical reef fisheries

Macroalgae-dominated reefs are a prominent habitat in tropical seascapes that support a diversity of fishes, including fishery target species. To what extent, then, do macroalgal habitats contribute to small-scale tropical reef fisheries? To address this question we: (1) Quantified the macroalgae-associated fish component in catches from 133 small-scale fisheries, (2) Compared life-history traits relevant to fishing (e.g. growth, longevity) in macroalgal and coral-associated fishes, (3) Examined how macroalgae-associated species can influence catch diversity, trophic level and vulnerability and (4) Explored how tropical fisheries change with the expansion of macroalgal habitats using a case study of fishery-independent data for Seychelles. Fish that utilised macroalgal habitats comprise 24% of the catch, but very few fished species relied entirely on macroalgal or coral habitats post-settlement. Macroalgal and coral-associated fishes had similar life-history traits, although vulnerability to fishing declined with increasing contribution of macroalgae association to the catch, whilst mean trophic level and diversity peaked when macroalgal-associated fish accounted for 20%-30% of catches. The Seychelles case study revealed similar total fish biomass on macroalgal and coral reefs, although the biomass of primary target species increased as macroalgae cover expanded. Our findings reinforce that multiple habitat types are needed to support tropical fishery stability and sustainability. Whilst coral habitats have been the focus of tropical fisheries management, we show the potential for macroalgae-associated fish to support catch size and diversity in ways that reduce vulnerability to overfishing. This is pertinent to seascapes where repeated disturbances are facilitating the replacement of coral reef with macroalgal habitats

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL

Towards a simple global-standard bioassay for a key ecosystem process: organic-matter decomposition using cotton strips

Cotton-strip bioassays are increasingly used to assess ecosystem integrity because they provide a standardized measure of organic-matter decomposition – a fundamental ecosystem process. However, several different cotton- strip assays are routinely used, complicating the interpretation of results across studies, and hindering broader synthesis. Here, we compare the decay rates and assemblages of bacteria and fungi colonizing the three most commonly used cotton materials: Artist’s canvas, Calico cloth, and Empa fabric. Cotton strips from each material type were incubated in 10 streams that span a wide range of physicochemical properties across five ecoregions. Additionally, to evaluate responses to environmental stress without potentially confounding biogeographical effects, we deployed identical bioassays in five streams across an acidification gradient within a single ecoregion. Across all streams decomposition rates (as tensile strength loss [TSL]) differed among the three cotton ma- terials; Calico cloth decomposed fastest (time to 50% TSL [T50]=16.7d), followed by the Empa fabric (T50 = 18.3 d) and then Artist’s canvas (T50 = 21.4 d). Despite these differences, rates of TSL of the three cotton materials responded consistently to variation in environmental conditions; TSL of each fabric increased with stream temperature, dissolved-nutrient concentrations and acid-neutralizing capacity, although Artist’s canvas and Calico cloth were more sensitive than Empa fabric. Microbial communities were similar among the mate- rials, and values of community structure (e.g., phylotype richness and diversity) were comparable to those reported for decaying leaves in streams from the same region, the major natural basal carbon resource in forested-stream ecosystems. We present linear calibrations among pairs of assays so that past and future studies can be expressed in a “common currency” (e.g., Artist’s-fabric equivalents) ‘past and future studies’ repeated two times in the sentence. Lastly, given its relatively low within-site variability, and the large number of streams where it has been used (> 700 across the globe), we recommend Artist’s fabric for future work. These results show that cotton provides an effective and realistic standardized substrate for studying heterotrophic microbial assemblages, and acts as a reasonable proxy for more chemically complex forms of detritus. These findings add to growing evidence that cotton-strip bioassays are simple, effective and easily standardized indicators of het- erotrophic microbial activity and the ecosystem processes that result

Survey and Excavation at the Henges of the Wharfe Valley, North Yorkshire, 2013-15

YesGeophysical survey at the three major henge monuments in the Wharfe Valley has provided details of survival and internal features. Excavation at Yarnbury has confirmed its Bronze Age date and has recovered material matching that from previous unrecorded excavations. The excavation has provided environmental data for the construction of the henge. The sites are placed in their regional context.British Academ

Breast cancer stem cells: obstacles to therapy

Only a fraction of the cells in a breast tumour are able to seed new tumour growth. These so-called breast cancer stem cells (bCSCs) are characterised by a number of discrete functional properties, some of which impact on therapeutic strategies aimed at eliminating these cells from tumours. Here we discuss how recent experimental evidence indicates that phenotypic plasticity is a central feature of tumour cell heterogeneity and drug resistance, traits that must be overcome in order to efficiently target bCSCs as a therapy for breast cancer. We propose that a better understanding of this fundamental property of breast cancer stem cells, over and above their identification in tumours, is a priority for improvement of patient survival