Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate.

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration.

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.

Development of a patient-derived explant model for prediction of drug responses in endometrial cancer.

OBJECTIVE: To undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting. METHODS: Fresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1-3 mm3 explants that were cultured at the air-liquid interface for 16-24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression. RESULTS: EC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment. CONCLUSIONS: Our PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.

Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.

Preclinical models that can accurately predict outcomes in the clinic are much sought after in the field of cancer drug discovery and development. Existing models such as organoids and patient-derived xenografts have many advantages, but they suffer from the drawback of not contextually preserving human tumour architecture. This is a particular problem for the preclinical testing of immunotherapies, as these agents require an intact tumour human-specific microenvironment for them to be effective. In this review, we explore the potential of patient-derived explants (PDEs) for fulfilling this need. PDEs involve the ex vivo culture of fragments of freshly resected human tumours that retain the histological features of original tumours. PDE methodology for anti-cancer drug testing has been in existence for many years, but the platform has not been widely adopted in translational research facilities, despite strong evidence for its clinical predictivity. By modifying PDE endpoint analysis to include the spatial profiling of key biomarkers by using multispectral imaging, we argue that PDEs offer many advantages, including the ability to correlate drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. As such, PDEs are a powerful model of choice for cancer drug and biomarker discovery programmes.

Exploration of Matrix Effects in Laser Ablation Inductively Coupled Plasma Mass Spectrometry Imaging of Cisplatin-Treated Tumors.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes.

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.

An optimised patient-derived explant platform for breast cancer reflects clinical responses to chemotherapy and antibody-directed therapy.

Breast Cancer is the most common cancer among women globally. Despite significant improvements in overall survival, many tumours are refractory to therapy and so novel approaches are required to improve patient outcomes. We have evaluated patient-derived explants (PDEs) as a novel preclinical platform for breast cancer (BC) and implemented cutting-edge digital pathology and multi-immunofluorescent approaches for investigating biomarker changes in both tumour and stromal areas at endpoint. Short-term culture of intact fragments of BCs as PDEs retained an intact immune microenvironment, and tumour architecture was augmented by the inclusion of autologous serum in the culture media. Cell death/proliferation responses to FET chemotherapy in BC-PDEs correlated significantly with BC patient progression-free survival (p = 0.012 and p = 0.0041, respectively) and cell death responses to the HER2 antibody therapy trastuzumab correlated significantly with HER2 status (p = 0.018). These studies show that the PDE platform combined with digital pathology is a robust preclinical approach for informing clinical responses to chemotherapy and antibody-directed therapies in breast cancer. Furthermore, since BC-PDEs retain an intact tumour architecture over the short-term, they facilitate the preclinical testing of anti-cancer agents targeting the tumour microenvironment.

The 'complexities' of life and death: death receptor signalling platforms.

Cell death is critical to the normal functioning of multi-cellular organisms, playing a central role in development, immunity, inflammation, and cancer progression. Two cell death mechanisms, apoptosis and necroptosis, are dependent on the formation of distinct multi-protein complexes including the DISC, Apoptosome, Piddosome and Necrosome following the induction of cell death by specific stimuli. The role of several of these key multi-protein signalling platforms, namely the DISC, TNFR1 complex I/II, the Necrosome and Ripoptosome, in mediating these pathways will be discussed, as well as the open questions and potential therapeutic benefits of understanding their underlying mechanisms.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

Compromised vitamin D receptor signalling in malignant melanoma is associated with tumour progression and mitogen-activated protein kinase activity.

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at the tumour base did not metastasize, a potentially important prognostic indicator. Decreased nuclear VDR correlated with increased cytoplasmic staining, suggesting the failure of nuclear entry as a primary cause of defective VDR signalling in MM. The histological subset analysis and MM cell line studies confirmed the inhibitory effect of MAPK activity on VDR signalling, but the pattern of VDR subcellular localization suggested failure of VDR nuclear entry as a primary effect of MAPK activity rather than direct inhibition of VDR-regulated transcription. Furthermore, high MAPK activity in tumours expressing cytoplasmic VDR was associated with worsened prognosis.

Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.

Modulation of the sensitivity of Jurkat T-cells to inhibition of protein synthesis by tumor necrosis factor α-related apoptosis-inducing ligand.

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in Jurkat T lymphoma cells. One of the characteristics of the phase preceding overt apoptosis is the marked downregulation of protein synthesis. We have investigated factors that can influence this response and have explored some of the signaling pathways involved. We show that interferon-α (IFNα) pretreatment desensitizes Jurkat cells to TRAIL-induced inhibition of protein synthesis, such that the concentration of TRAIL required for 50% inhibition is increased by 10-fold. The inhibition of translation is characterized by dephosphorylation of the eIF4E-binding protein 4E-BP1 and IFNα desensitizes Jurkat cells to this effect. IFNα also inhibits TRAIL-mediated dephosphorylation of the growth-promoting protein kinase B (Akt). Since Jurkat cells are defective for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and therefore have constitutive phosphoinositide 3-kinase (PI3K) activity, we investigated the consequences for protein synthesis of inhibiting PI3K using LY294002. Inhibition of PI3K partially inhibits translation, but also enhances the effect of a suboptimal concentration of TRAIL. However, LY294002 does not block the ability of IFNα to protect protein synthesis from TRAIL-induced inhibition. Data are presented suggesting that IFNα impairs the process of activation of caspase-8 within the TRAIL death-inducing signaling complex.