Biology subjects

Longley, D. B.

Publications and source records attributed to Longley, D. B..

14 recordsLinked to original sources

bioRxiv2026

FLIP is essential for oncogenic KRAS-driven lung cancer

Mutations in KRAS represent the most common oncogenic event in human cancer and occur in approximately 30% of lung adenocarcinomas. The mechanisms by which lung tumours evade apoptosis induced by oncogenic KRAS-driven stress remain incompletely understood. Here, we identify the anti-apoptotic regulator FLIP (CFLAR) as a critical dependency in KRAS-mutant lung cancers. We demonstrate that KRAS-mutant human lung cancer cell lines exhibit elevated FLIP expression and enhanced dependence on FLIP for survival compared to KRAS wild-type counterparts. Subsequently, using genetically engineered mouse models (GEMMs), we show that FLIP is essential for Kras-driven lung tumour development in vivo. In vitro, FLIP-deficient lung cancer cells display spontaneous, caspase-8- dependent apoptosis and hyper-sensitivity to the immune/inflammatory cytokines TNF and TRAIL. Strikingly, FLIP-null lung cancer cells fail to engraft even in highly immunodeficient orthotopic models that lack TRAIL-expressing immune cells but retain TNF-expressing monocytes. Moreover, silencing of TNFR1 or TNF but not TRAIL-R2 rescued constitutive caspase-8-dependent apoptosis in FLIP null lung cancer cells, implicating TNF/TNFR1 in mediating this apoptotic response. Mechanistically, we find that mutant KRAS sustains FLIP expression via ERK1/2 signalling, thereby protecting cells from caspase-8 activation. Notably, KRAS inhibition downregulates FLIP, sensitising cells to TNF- and TRAIL-induced apoptosis. These findings uncover a novel KRAS-ERK-FLIP axis that protects tumour cells from caspase-8-mediated apoptosis and reveal FLIP as a key survival factor co-opted by KRAS-mutant lung cancers. Beyond identifying FLIP as a promising therapeutic target in KRAS mutant lung cancer, our work also provides mechanistic insight into the pro-apoptotic effects of KRAS inhibitors and suggests that FLIP expression may serve as a predictive biomarker to enhance patient stratification and the therapeutic efficacy of these agents in lung cancer.

cancer biology↗

bioRxiv2026

A neural network model delivers a highly prognostic protein signature in cancer stem cells that identifies relapse in stage III colorectal cancer patients.

BackgroundStage III colorectal cancer poses a significant threat of metastasis development, as tumour resection and adjuvant chemotherapy do not guarantee prolonged disease-free survival. ObjectiveThe spatial, quantitative, and qualitative characteristics of various cell types within tumour tissues could be key to developing accurate prognostic AI models. DesignTissue microarrays created from primary tumour tissues collected during surgical resection from a cohort of 493 stage III colorectal cancer (CRC) patients were analysed for 61 protein markers at the single-cell level using multiplexed immunofluorescence imaging via the Cell DIVE platform. Subsequent cell-type classification enabled quantitative cell-type analyses, co-localisation neighbourhood assessments, and cell-type-specific protein signature discoveries that distinguish between early and late/non-recurring patient samples. ResultsThis study identifies a stem cell protein profile that drives tumour relapse. A deep neural network (DNN) model, based on a stem cell protein signature composed of BAX, MLKL, FLIP, GLUT1, and CDX2, provided accurate prognosis for stage III CRC patients in both discovery and validation cohorts and in an independent validation cohort. Nodal count-based metric further increased prognosis accuracy. Our study also revealed distinct spatial arrangements of immune, endothelial, and stem cells that were linked to early tumour recurrence. ConclusionOur findings propose a clinically promising prognostic tool based on a five-protein stem cell signature. These markers not only predict chemotherapy resistance in cancer stem cells but also suggest potential therapeutic strategies such as combinatorial treatments incorporating small molecule inhibitors targeting FLIP and GLUT1. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIMore than 20% of stage III colorectal cancer patients will experience early tumour recurrence within the first 3 years post treatment that includes surgery and adjuvant 5-FU based chemotherapy treatment. C_LIO_LISeveral studies pointed towards involvements of number of cell type specific spatial neighbourhoods in tumour progression where some immune tumour microenvironment promoting angiogenesis and intravasation events, some may provide immunosuppression. C_LIO_LICancer stem cells could be responsible for metastatic tumour spread, early recurrence and chemoresistance. C_LI What this study addsO_LISpatial single cell quantitative multiplex profiling of 45 cancer hallmark proteins and 15 cell identity markers in 493 stage III CRC patients tissue samples demonstrated significant differences in cellular proximity neighbourhoods, cell type specific abundance and expression between the early and late recurrence samples. C_LIO_LIWe discover that macrophages show spatial association with the blood vessels in early recurrence samples. Moreover, we observed conglomeration of B cells and macrophages with Tregulatory, Thelper and Tcytotoxic cells in association with early recurrences. C_LIO_LIWe showed that stromal abundance of Tregulatory, Thelper, Tcytotoxic cells and monocytes are significantly in late, and no recurrence samples compared to early recurrence samples. C_LIO_LIThe most differential expression profile that differentiates late and no recurrence samples from the early recurrence samples is related to the stem cell population. Particularly, we found overexpression of GLUT1, FLIP and downregulation of BAX, BAK, MLKL and CDX2 proteins in the cancer stem cell of early recurrence samples. C_LIO_LIWe built a neural network based on the cancer stem cell protein signature (BAX, MLKL, FLIP, GLUT1 and CDX2 proteins) that delivers a high-performance prognostic classifier. C_LI How this study might affect research, practice or policyO_LIOur results propose a clinically promising prognostic tool based on a five-protein stem cell signature that outperforms existing clinical and proposed transcriptomic based signatures for separation between risk groups. C_LIO_LIMoreover, our five-protein signature markers not only predict stem cell chemotherapy resistance and therefore tumour recurrence but also suggest potential therapeutic strategies. For instance, this approach could guide combinatorial treatments at high risk of chemoresistance, such as incorporating small molecule inhibitors targeting FLIP (currently in discovery phase) and GLUT1 (already under preclinical trial evaluation). C_LI

cancer biology↗

bioRxiv2025

Multiplex analysis of colorectal cancer tissue describes the composition, cell biology and spatial effects of cell-in-cell events and identifies a T cell-dependent prognostic signature

Cell competition is an emerging mechanism in which mammalian tissues maintain homeostasis by eliminating less fit (loser) cells through direct interactions with fitter (winner) neighbouring cells. In cancer, these competitive interactions may drive tumour evolution; however, spatial organisation and clinical relevance of these events remain poorly understood. One mechanism by which winner cells eliminate loser cells is engulfment, resulting in cell-in-cell (CIC) formation. Although CICs have been observed in many tumour types for over a century, their cellular composition, spatial context, interactions with the tumour microenvironment, and biological significance in human cancers remain unclear. Here, we systematically characterised the cellular identity and functional states of CICs in situ, examined their spatial interactions within the tumour microenvironment, and assessed their clinical relevance using spatially resolved single-cell data from a large cohort of colorectal cancer patients. We demonstrate that CICs occur predominantly between cancer cells but also involve cancer stem cell (CSC)-like populations and cytotoxic T cells. Engulfed (inner) cancer and CSC-like cells display molecular features consistent with a loser-cell phenotype, including increased apoptosis and reduced proliferation, whereas outer cancer cells exhibit winner-cell features such as upregulated glycolysis. Live-cell time-lapse experiments demonstrate that glucose accumulates in inner cells during lysosomal degradation following cell engulfment. Spatial analysis further revealed distinct CIC neighbourhoods, which we defined based on proximity to engulfment events. Cells within these regions, particularly CSC-like cells and cytotoxic T cells, exhibit increased metabolic stress, suggesting local competition for nutrients. Importantly, the presence of cytotoxic T cells within CIC neighbourhoods and spatial co-occurrence patterns between cancer cells and CSC-like populations are associated with improved patient outcomes. Together, our findings demonstrate that cell engulfment defines spatially organised competitive niches and may reflect cell competition within complex tumour microenvironments.

cancer biology↗

bioRxiv2025

High-Resolution Spatial Proteomics Characterises Colorectal Cancer Consensus Molecular Subtypes

BackgroundIdentification of the consensus molecular subtypes (CMS) opened significant potential for understanding the tumor biology and intertumoral heterogeneity of colorectal cancer (CRC). However, molecular subtyping in CRC traditionally relies on bulk transcriptomics, therefore, lacks spatial and single-cell level aspect. MethodsWe constructed tissue microarrays using tumor cores from 222 CRC patients. Arrays were stained and imaged using 54 cell identity and cancer hallmark markers, delivering spatially resolved protein profiles of >2 million cells. RNA sequencing data and CMS classification were also available for these patients. After segmentation of cancer, stromal and immune cells, we investigated intratumoral heterogeneity within CMS subtypes using spatially resolved single-cell protein profiling (>2 million cells). We compared cell types, their spatial organization and their expression of cancer hallmark-related proteins in CMS 1-4 subtypes. ResultsWe revealed tissue atlases illustrating the cell types/states, spatial heterogeneity, cellular neighborhoods, cellular network, and single-cell protein profiles of CMS tumors. CMS1 tumors had more CD3+, CD8+, and PD1+ immune cells that were found in the epithelial layer frequently. CMS1 was also associated with higher levels of metabolic reprogramming markers such as upregulated glycolysis. CMS2 showed immune segregation, reactive stroma patterns and higher levels of apoptotic and proliferative signaling proteins. CMS3 exhibited clustered cancer cells with high RIP3 levels, suggesting a pro-inflammatory microenvironment. CMS4 displayed stromal-centric and immune-evasive tumors characterized by decreased HLA-1 levels and regulatory T-cell exclusion from epithelium. ConclusionWe present a spatial protein atlas of CRC at single-cell resolution and demonstrate novel aspects of CMS tumour structures.

cancer biology↗

bioRxiv2024

Mitochondrial metabolism is a key determinant of chemotherapy sensitivity in Colorectal Cancer

Therapy resistance is attributed to over 80% of cancer deaths per year emphasizing the urgent need to overcome this challenge for improved patient outcomes. Despite its widespread use in colorectal cancer (CRC) treatment, resistance to 5-fluorouracil (5FU) remains poorly understood. Here, we investigate the transcriptional responses of CRC cells to 5FU treatment, revealing significant metabolic reprogramming towards heightened mitochondrial activity. Utilizing CRC models, we demonstrate sustained enhancement of mitochondrial biogenesis and function following 5FU treatment, leading to resistance in both in vitro and in vivo settings. Furthermore, we show that targeting mitochondrial metabolism, specifically by inhibiting Complex I (CI), sensitizes CRC cells to 5FU, resulting in delayed tumour growth and prolonged survival in preclinical models. Additionally, our analysis of patient data suggests that oxidative metabolism signatures may predict responses to 5FU-based chemotherapy. These findings shed light on mechanisms underlying 5FU resistance and propose a rational strategy for combination therapy in CRC, emphasizing the potential clinical benefit of targeting mitochondrial metabolism to overcome resistance and enhance patient outcomes.

cancer biology↗

bioRxiv2024

Spatial Effects of Infiltrating T cells on Neighbouring Cancer Cells and Prognosis in Stage III CRC patients

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks, as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil-based chemotherapy. Images underwent segmentation for tumour, stroma and immune cells, and cancer cell state protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell - T cell interactions at single-cell level. In our discovery cohort (MSK), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (HV) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (Discovery cohort: p = 0.07, Validation cohort: p = 0.19). We next utilized our region-based nearest neighbourhood approach to determine the spatial relationships between cytotoxic T cells, helper T cells and cancer cell clusters. In the both cohorts, we found that lower distance between cytotoxic T cells, T helper cells and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (Discovery cohort: p = 0.01, Validation cohort: p = 0.003).

cancer biology↗

bioRxiv2023

The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

The activation of apoptosis signalling by TRAIL (TNF-related apoptosis-inducing ligand) through receptor binding is a fundamental mechanism of cell death induction and is often perturbed in cancer cells to enhance their cell survival and treatment resistance. Ubiquitination plays an important role in the regulation of TRAIL-mediated apoptosis, and here we investigate the role of the E3 ubiquitin ligase Itch in TRAIL-mediated apoptosis in oesophageal cancer cells. Knockdown of Itch expression resulted in resistance to TRAIL-induced apoptosis, caspase-8 activation, Bid cleavage and also promoted cisplatin resistance. Whilst the assembly of the death-inducing signalling complex (DISC) at the plasma membrane was not perturbed relative to the control, the TRAIL-R2 receptor was mis-localised in the Itch-knockdown cells. Further, we observed significant mitochondrial widening with an increased cholesterol content. An inhibitor of cholesterol trafficking, U18666A, was able to replicate some of the effects of Itch knockdown, including protection from TRAIL-induced apoptosis, reduced caspase-8 activation, Bid cleavage and Cisplatin resistance. This study highlights the importance of Itch in regulating the crosstalk between mitochondrial cholesterol and TRAIL-induced apoptosis.

cell biology↗

bioRxiv2021

Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC)

ObjectiveTranscriptomic-based subtyping, Consensus Molecular Subtyping (CMS) and CRC Intrinsic Subtyping (CRIS), identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence of Fusobacterium nucleatum (Fn) and Fusobacteriales, CMS/CRIS subtyping, cell type composition, immune infiltrates and host contexture to refine patients stratification and identify druggable context-specific vulnerabilities. DesignWe coupled cell culture experiments with characterization of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from 2 independent CRC patient cohorts (Taxonomy: n=140; TCGA-COAD-READ: n=605). ResultsIn vitro, Fn infection induced inflammation via NF{kappa}B/TNF in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, MSI tumours, with infiltration of macrophages M1, reduced macrophages M2, and high IL6/IL8/IL1{beta} signaling. Analysis of the Taxonomy cohort suggested that Fn was prognostic for CMS4/CRIS-B patients, despite having lower Fn load than CMS1 patients. In the TCGA-COAD-READ cohort, we likewise identified a differential association between Fusobacteriales relative abundance and outcome when stratifying patients in mesenchymal (either CMS4 and/or CRIS-B) vs. non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately 2-fold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the 3-way association between Fusobacteriales prevalence, molecular subtyping, and host contexture with logistic models with an interaction term disentangled the pathogen/host-signaling relationship and identified aberrations (including EMT/WNT/NOTCH) as candidate targets. ConclusionThis study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology. Significance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIFusobacterium nucleatum (Fn), a commensal Gram-negative anaerobe from the Fusobacteriales order, is an onco-bacterium in CRC as a causal relationship between Fn prevalence and CRC pathogenesis, progression and treatment response has been reported in vivo. C_LIO_LIBroad spectrum antibiotics has proven moderately successful in reducing tumour growth in preclinical models. However, the use of antibiotics to treat bacterium-positive cases in the clinic is not a viable option as it may further alter the already dysbiotic gut microbiome of CRC patients and may also have limited efficacy against Fn which penetrates and embeds deeply within the tumour. C_LIO_LIThe highly heterogenous CRC patient population can be classified into distinct molecular subtypes (CMS and CRIS) based on gene expression profiles mirroring the underlying transcriptional programs. Patients classified as CMS4 and CRIS-B exhibit a mesenchymal phenotype and have poorer outcome. C_LI What are the new findings?O_LIFn/Fusobacteriales prevalence is associated with immune involvement (decrease in macrophages M1 and increase in macrophages M2) and activation of specific signalling programs (inflammation, DNA damage, WNT, metastasis, proliferation, cell cycle) in the host tumours. C_LIO_LIThe prevalence of bacteria from the Fusobacteriales order, largely driven by Fn species, play an active or opportunistic role depending on the underlying host tumour biology and microenvironment. C_LIO_LIFn and other species of the Fusobacteriales order are enriched in CMS1 (immuno, microsatellite unstable) patients compared to CMS2-4 cases. C_LIO_LIFn/Fusobacteriales prevalence is associated with worse clinical outcome in patients with mesenchymal-rich CMS4/CRIS-B tumours, but not in patients with other molecular subtypes. C_LI How might it impact on clinical practice in the foreseeable future?O_LIFn/Fusobacteriales screening and transcriptomic-based molecular subtyping should be considered to identify patients with mesenchymal-rich tumours and high bacterium prevalence and to inform disease management. C_LIO_LIFn/Fusobacteriales prevalence may need to be addressed exclusively in patients with mesenchymal-rich high-stromal infiltrating tumours rather than a blanket-approach to treat all pathogen-positive patients. C_LIO_LIClinical management of the disease for this subpopulation of high-risk patients with unfavourable clinical outcome could be attained by administering compounds currently in clinical trials that target aberrations in the host signaling pathways (NOTCH, WNT, EMT) and tumour microenviroment (inflammasome, activated T cells, complement system, and macrophage chemotactism and activation). C_LI

cancer biology↗

bioRxiv2021

Stratification Of Chemotherapy-Treated Stage III Colorectal Cancer Patients Using Multiplexed Imaging And Single Cell Analysis Of T Cell Populations

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Predictive biomarkers for identification of patients with increased risk for disease recurrence are currently lacking, with progress in biomarker discovery hindered by the diseases inherent heterogeneity. The immune profile of colorectal tumors has previously been found to have prognostic value. The aims of this study were to evaluate immune signatures in the tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single cell analysis technology (Cell DIVE). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin chemotherapy. Single sections underwent multilplexed immunofluorescence with Cy3- and Cy5-conjugated antibodies for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase and S6). We applied a probabilistic multi-class, multi-label classification algorithm based on multi-parametric models to build statistical models of protein expression to classify immune cells. Expert annotations of immune cell markers were made on a range of images, and Support Vector Machines (SVM) were used to derive a statistical model for cell classification. Images were also manually scored independently by a Pathologist as high, moderate or low, for stromal and total immune cell content. Excellent agreement was found between manual and total automated scores (p<0.0001). Higher levels of multi-marker classified regulatory T cells (CD3+CD4+FOXP3+PD1-) were significantly associated with disease-free survival (DFS) and overall-survival (OS) (p=0.049 and 0.032), compared to FOXP3 alone. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cells with greater potential for predicting patient outcomes.

cancer biology↗

bioRxiv2020

Clinical positioning of the IAP antagonist tolinapant (ASTX660) in colorectal cancer

Cancer cells frequently express elevated levels of Inhibitor of Apoptosis Proteins (IAPs): cIAPI, cIAP2 and XIAP. Elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in microsatellite stable (MSS) stage-III colorectal cancer (CRC) patients treated with adjuvant chemotherapy, suggesting their involvement in promoting resistance. Preclinical analysis of the IAP inhibitor tolinapant in CRC cell lines demonstrated robust on-target effects and caspase-8-dependent apoptosis that was inhibited by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinipant-induced apoptosis was augmented by standard-of-care chemotherapy (FOLFOX) in CRC disease models, due (at least in part) to FOLFOX-induced downregulation of Class-I histone deacetylases, leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, this effect could be phenocopied using a Class-I HDAC inhibitor. Further analyses revealed that caspase-8-knockout RIPK3-positive CRC models were sensitive to tolinostat-induced necroptosis, an effect that could be exploited with the FDA-approved caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX chemotherapy in poor prognosis MSS CRC with elevated cIAP1/2 expression.

cancer biology↗

bioRxiv2020

TRAIL signalling promotes entosis in colorectal cancer

Entosis is a form of non-phagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumours show evidence of entosis, however factors controlling entosis remain to be elucidated. Here we find that the death receptor ligand TRAIL is a potent activator of entosis in colon cancer cells. CLEM/3D confocal microscopy analysis revealed ultrastructural features of entosis and subsequent entotic cell death of inner cells upon TRAIL treatment. Induction of entosis and apoptosis by TRAIL were mutually exclusive events but both required the presence of caspase-8. Bax/Bak double knock-out or caspase inhibition altered the fate of inner cells from entotic cell death to survival and escape. Analysis of colorectal cancer tumours showed a significant association between expression levels of TRAIL and CICs. Notably, the presence of CICs in the invasive front regions of colorectal tumours was significantly correlated with adverse patient prognosis.

cancer biology↗

bioRxiv2019

FLIP(L) determines p53 induced life or death

How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Here, we demonstrate that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes a critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a novel ligand-independent TRAIL-R2/caspase-8 complex, which, by activating BID, induces mitochondrial-mediated apoptosis. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Thus, we report two previously undescribed, novel FLIP(L)-dependent mechanisms that determine cell fate following p53 activation.

cancer biology↗

bioRxiv2019

Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

Depending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)s DISC-processed p43-form. Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCFSkp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIPs association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled.

molecular biology↗

bioRxiv2017

Bcl-xL as a poor prognostic biomarker and predictor of response to adjuvant chemotherapy specifically in BRAF-mutant stage II and III colon cancer

BackgroundBRAF mutation occurs in 8-15% of colon cancers (CC), and is associated with poor prognosis in metastatic disease. Compared to wild-type BRAF (BRAFWT) disease, stage II/III CC patients with BRAF mutant (BRAFMT) tumors have shorter overall survival after relapse; however, time-to-relapse is not significantly different. The aim of this investigation was to identify, and validate, novel predictors of relapse of stage II/III BRAFMT CC.\n\nPatients and methodsWe used gene expression data from a cohort of 460 patients (GSE39582) to perform a supervised classification analysis based on risk-of-relapse within BRAFMT stage II/III CC, to identify transcriptomic biomarkers associated with prognosis within this genotype. These findings were validated using immunohistochemistry in an independent population-based cohort of Stage II/III CC (n=691), applying Cox proportional hazards analysis to determine associations with survival.\n\nResultsHigh gene expression levels of Bcl-xL, a key regulator of apoptosis, were associated with increased risk of relapse, specifically in BRAFMT tumors (HR=8.3, 95% CI 1.7-41.7), but not KRASMT/BRAFWT or KRASWT/BRAFWT tumors. High Bcl-xL protein expression in BRAFMT, untreated, stage II/III CC was confirmed to be associated with an increased risk of death in an independent cohort (HR=12.13, 95% CI 2.49-59.13). Additionally, BRAFMT tumors with high levels of Bcl-xL protein expression appeared to benefit from adjuvant chemotherapy (P for interaction =0.006), indicating the potential predictive value of Bcl-xL expression in this setting.\n\nConclusionsThese findings provide evidence that Bcl-xL gene and/or protein expression identifies a poor prognostic subgroup of BRAFMT stage II/III CC patients, who may benefit from adjuvant chemotherapy.\n\nKey MessageO_LIUsing a combination of computational biology discovery and immunohistochemistry validation in independent patient cohorts, we show that high expression of the apoptosis regulator Bcl-xL is associated with disease relapse specifically within BRAF mutant stage II/III colon cancer.\nC_LIO_LIThis data could enable tailored disease management to reduce relapse rates in the most aggressive subtype.\nC_LI

cancer biology↗