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Anti-apoptotic Bcl-2 family proteins are often highly expressed in chemotherapy-resistant cancers and impair mitochondrial outer membrane permeabilisation (MOMP), a key requirement for caspase activation via the intrinsic apoptosis pathway. Interestingly, while Bcl-2 overexpression in HeLa cervical cancer cells abrogated caspase processing in response to intrinsic apoptosis induction by staurosporine, tunicamycin or etoposide, residual caspase processing was observed following proteasome inhibition by bortezomib, epoxomicin or MG-132. Similar responses were found in Bcl-2 overexpressing H460 NSCLC cells and Bax/Bak-deficient mouse embyronic fibroblasts. Mild caspase processing resulted in low DEVDase activities, which were MOMP independent and persisted for long periods without evoking immediate cell death. Surprisingly, depletion of caspase-3 and experiments in caspase-7-depleted MCF-7-Bcl-2 cells indicated that the DEVDase activity did not originate from effector caspases. Instead, FADD-dependent caspase-8 activation was the major contributor to the slow, incomplete substrate cleavage. Casapse-8 activation was independent of death ligands but required the induction of autophagy and the presence of Atg5. Depletion of XIAP or addition of XIAP-antagonizing peptides resulted in a switch towards efficient apoptosis execution, suggesting that the requirement for MOMP was bypassed by activating the caspase-8/caspase-3 axis. Combination treatments of proteasome inhibitors and XIAP antagonists therefore represent a promising strategy to eliminate highly resistant cancer cells which overexpress anti-apoptotic Bcl-2 family members.

Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 ± 1.5% at MOMP onset. This was increased to 11.2±3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation. Apoptosis is an important physiological cell death process during development, and a key cell death pathway for the elimination of damaged or superfluous cells in the adult. In the 'mitochondrial' or 'intrinsic' apoptosis pathway, the permeabilisation of the mitochondrial outer membrane permeabilisation (MOMP) represents the key decisive process. 1 MOMP enables the release of mitochondrial intermembrane proteins into the cytosol which then activate a family of cytosolic cysteine proteases, the caspases. Bax and Bak are multidomain proapoptotic Bcl-2 family proteins required for the process of MOMP. 2 Although Bak is localised to mitochondria in nonapoptotic cells, large quantities of Bax are found in the cytosol and only translocate to mitochondria during apoptosis. 3 To activate MOMP, Bax and Bak undergo specific conformational changes that enable both proteins to anchor in the mitochondrial outer membrane and to oligomerise. [4][5][6] Although the mechanisms of Bax and Bak activation during apoptosis is still an area of intensive research, evidence is growing that specific proapoptotic proteins of the Bcl-2 protein super-family, in particular the Bcl-2 homology-domain 3 (BH3)-only proteins tBid and Bim, may be able to directly activate Bax and Bak. [5][6][7] Bax and Bak homo-tetramers and higher oligomers are believed to physically form the release channels in the mitochondrial outer membrane large enough to allow for release of intermembrane proteins. 7,8

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-to-use tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them.

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein-protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future.

MiRNAs have the potential to support AD diagnosis and patient care.

3Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, 4we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a 5 stand-alone signature and as a constituent of further refined prognostic stratification tools. 6Experimental Design: Apoptosis competency of primary tumor samples from n=120 stage III 7 colorectal cancer patients was calculated by APOPTO-CELL from measured protein concentrations of 35Among all clinicopathological, demographic, and molecular predictors analyzed, enriched-apoptosis 36 systems modeling delivered the highest ranking independent prognostic biomarker. Furthermore, 37 apoptosis modelling can be combined with molecular tumor subtyping to further refine risk 38 predictions. 39We report the clinical validation of diagnostic tools for stage III CRC patients that could deliver 40 superior quality of care by personalizing cancer treatment. 42Word count: 119.

Our understanding of the mitochondrial or intrinsic apoptosis pathway and its role in chemotherapy resistance has increased significantly in recent years by a combination of experimental studies and mathematical modelling. This combined approach enhanced the quantitative and kinetic understanding of apoptosis signal transduction, but also provided new insights that systems-emanating functions (i.e., functions that cannot be attributed to individual network components but that are instead established by multi-component interplay) are crucial determinants of cell fate decisions. Among these features are molecular thresholds, cooperative protein functions, feedback loops and functional redundancies that provide systems robustness, and signalling topologies that allow ultrasensitivity or switch-like responses. The successful development of kinetic systems models that recapitulate biological signal transduction observed in living cells have now led to the first translational studies, which have exploited and validated such models in a clinical context. Bottom-up strategies that use pathway models in combination with higher-level modelling at the tissue, organ and whole body-level therefore carry great potential to eventually deliver a new generation of systems-based diagnostic tools that may contribute to the development of personalised and predictive medicine approaches. Here we review major achievements in the systems biology of intrinsic apoptosis signalling, discuss challenges for further model development, perspectives for higher-level integration of apoptosis models and finally discuss requirements for the development of systems medical solutions in the coming years.