FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor, FLIP, is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were down-regulated by RNAi, IRinduced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the anti-tumor activity of IR in vivo. Therefore, FLIP down-regulation induced by HDAC inhibitors is a potential clinical strategy to radio-sensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The role of Ubiquitination in Apoptosis and Necroptosis

Cell death pathways have evolved to maintain tissue homoeostasis and eliminate potentially harmful cells from within an organism, such as cells with damaged DNA that could lead to cancer. Apoptosis, known to eliminate cells in a predominantly non-inflammatory manner, is controlled by two main branches, the intrinsic and extrinsic apoptotic pathways. While the intrinsic pathway is regulated by the Bcl-2 family members, the extrinsic pathway is controlled by the Death receptors, members of the tumour necrosis factor (TNF) receptor superfamily. Death receptors can also activate a pro-inflammatory type of cell death, necroptosis, when Caspase-8 is inhibited. Apoptotic pathways are known to be tightly regulated by post-translational modifications, especially by ubiquitination. This review discusses research on ubiquitination-mediated regulation of apoptotic signalling. Additionally, the emerging importance of ubiquitination in regulating necroptosis is discussed

Cytoplasmic FLIP(S) and nuclear FLIP(L) mediate resistance of castrate-resistant prostate cancer to apoptosis induced by IAP antagonists

Abstract Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC

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

OBJECTIVES: Transcriptomic-based subtyping, consensus molecular subtyping (CMS) and colorectal cancer 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 patient stratification and to identify druggable context-specific vulnerabilities. DESIGN: We coupled cell culture experiments with characterisation of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from two independent colorectal cancer patient cohorts (Taxonomy: n=140, colon and rectal cases of The Cancer Genome Atlas (TCGA-COAD-READ) cohort: n=605). RESULTS: In vitro, Fn infection induced inflammation via nuclear factor kappa-light-chain-enhancer of activated B cells/tumour necrosis factor alpha in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, microsatellite unstable () tumours, with infiltration of M1 macrophages, reduced M2 macrophages, and high interleukin (IL)-6/IL-8/IL-1β signalling. 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) versus non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately twofold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the three-way association between Fusobacteriales prevalence, molecular subtyping and host contexture with logistic models with an interaction term disentangled the pathogen–host signalling relationship and identified aberrations (including NOTCH, CSF1-3 and IL-6/IL-8) as candidate targets. CONCLUSION: This study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology