Metabolism in cells adapts quickly to changes in nutrient availability and cellular differentiation status, including growth conditions in cell culture settings. The last decade saw a vast increase in three-dimensional (3D) cell culture techniques, engendering spheroids and organoids. These methods were established to improve comparability to in vivo situations, differentiation processes and growth modalities. How far spheroids mimic in vivo metabolism, however, remains enigmatic. Here, to our knowledge, we compare for the first time metabolic fingerprints between cells grown as a single layer or as spheroids with freshly isolated in situ tissue. While conventionally grown cells express elevated levels of glycolysis intermediates, amino acids and lipids, these levels were significantly lower in spheroids and freshly isolated primary tissues. Furthermore, spheroids differentiate and start to produce metabolites typical for their tissue of origin. 3D grown cells bear many metabolic similarities to the original tissue, recommending animal testing to be replaced by 3D culture techniques.

The PIDDosome, a multiprotein complex constituted of the ‘p53-induced protein with a death domain (PIDD), ‘receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain' (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/β3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53−/− mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.