Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iß by cytochrome c.

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iß interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iß to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iß. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iß, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iß's histone chaperone activity.

Opposing roles of TGF-ß and EGF in the regulation of TRAIL-induced apoptosis in human breast epithelial cells.

Transforming growth factor-beta (TGF-ß) induces the epithelial to mesenchymal transition (EMT) in breast epithelial cells and plays an important role in mammary morphogenesis and breast cancer. In non-transformed breast epithelial cells TGF-ß antagonizes epidermal growth factor (EGF) action and induces growth inhibition. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to participate in lumen formation during morphogenesis of human breast epithelial cells. Our previous work indicated that sensitivity of human breast epithelial cells to TRAIL can be modulated through the activation of the epidermal growth factor receptor-1 (EGFR). Here, we show that TGF-ß opposes EGF-mediated sensitization to TRAIL-induced caspase-8 activation and apoptosis in non-transformed breast epithelial cells. Death-inducing signalling complex (DISC) formation by TRAIL was significantly reduced in cells treated with TGF-ß. TGF-ß treatment activates cytoprotective autophagy and down-regulates TRAIL-R2 expression at the cell surface by promoting the intracellular accumulation of this receptor. Lastly, we demonstrate that EMT is not involved in the inhibitory effect of TGF-ß on apoptosis by TRAIL. Together, the data reveal a fine regulation by EGF and TGF-ß of sensitivity of human breast epithelial cells to TRAIL which may be relevant during morphogenesis.

Involvement of both caspase-8 and Noxa-activated pathways in endoplasmic reticulum stress-induced apoptosis in triple-negative breast tumor cells.

Recent evidences indicate that triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype show a basal activation of the unfolded protein response (UPR) that increases their sensitivity to endoplasmic reticulum (ER) stress although the underlying cell death mechanism remains largely unexplored. Here we show that both caspase-8-dependent and -independent apoptotic mechanisms are activated in TNBC cells undergoing sustained ER stress. Activation of the extrinsic apoptotic pathway by ER stress involves ATF4-dependent upregulation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5). In addition, accumulation of BH3-only protein Noxa at the mitochondria further contributes to apoptosis following ER stress in TNBC cells. Accordingly, simultaneous abrogation of both extrinsic and intrinsic apoptotic pathways is required to inhibit ER stress-induced apoptosis in these cells. Importantly, persistent FLICE-inhibitory protein (FLIP) expression plays an adaptive role to prevent early activation of the extrinsic pathway of apoptosis upon ER stress. Overall, our data show that ER stress induces cell death through a pleiotropic mechanism in TNBC cells and suggest that targeting FLIP expression may be an effective approach to sensitize these tumor cells to ER stress-inducing agents.

Oncogenic p95HER2/611CTF primes human breast epithelial cells for metabolic stress-induced down-regulation of FLIP and activation of TRAIL-R/Caspase-8-dependent apoptosis.

Oncogenic transformation triggers reprogramming of cell metabolism, as part of the tumorigenic process. However, metabolic reprogramming may also increase the sensitivity of transformed cells to microenvironmental stress, at the early stages of tumor development. Herein, we show that transformation of human breast epithelial cells by the p95HER2/611CTF oncogene markedly sensitizes these cells to metabolic stress induced by the simultaneous inhibition of glucose and glutamine metabolism. In p95HER2/611CTF-transformed cells, metabolic stress activates a TNF related apoptosis-inducing ligand (TRAIL)-R and caspase-8-dependent apoptotic process that requires prior down-regulation of cellular FLICE-like inhibitor protein (c-FLIP) levels. Importantly, sustained mTOR activation is involved in FLIP down-regulation and apoptosis induced by metabolic stress. In vivo experiments in immunodeficient mice demonstrate a requirement for caspase-8 in restraining primary tumor growth of xenografts with p95HER2/611CTF-transformed cells. Collectively, these data define a critical role of the extrinsic pathway of apoptosis in the control of tumor initiation by microenvironmental cues.

The long and winding road to cancer treatment: the TRAIL system.

Activation of cell surface death receptors of the tumor necrosis factor (TNF) receptor superfamily by the appropriate ligands represents an attractive therapeutic strategy to induce cell death by apoptosis in cancer cells. However, the toxic effects of TNF-alpha and CD95/Fas ligand (FasL) in normal tissues have significantly hampered the clinical application of these ligands in cancer treatment. TNF-related apoptosis-inducing ligand (TRAIL/APO-2L), another member of the TNF family, has been shown to induce apoptosis selectively in many tumor cell lines. Interestingly, TRAIL treatment also results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice and nonhuman primates. At the same time, recombinant TRAIL and agonistic TRAIL receptor antibodies show no significant cytotoxicity in these studies. Despite some adverse effects of certain TRAIL preparations, activation of proapoptotic TRAIL receptors represents a promising approach in cancer therapy. Herein we review what is known about proapoptotic TRAIL signaling, the role of intracellular survival pathways in the regulation of resistance to TRAIL and the activation of non-apoptotic signaling by TRAIL. We also discuss the role of the TRAIL system in tumorigenesis and the results of clinical trials with recombinant TRAIL and various TRAIL receptor agonistic antibodies, either as monotherapy or in combination with targeted or conventional chemotherapy.

Activated ERBB2/HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress through a PERK-dependent pathway.

HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately 20% of human breast tumors. Truncated or mutant isoforms that show increased oncogenicity compared with the wild-type receptor are found in many breast tumors. Here, we report that constitutively active ERBB2 sensitizes human breast epithelial cells to agents that induce endoplasmic reticulum stress, altering the unfolded protein response (UPR) of these cells. Deregulation of the ERK, AKT, and mTOR activities elicited by mutant ERBB2 was involved in mediating this differential UPR response, elevating the response to endoplasmic reticulum stress, and apoptotic cell death. Mechanistic investigations revealed that the increased sensitivity of mutant ERBB2-expressing cells to endoplasmic reticulum stress relied upon a UPR effector signaling involving the PERK-ATF4-CHOP pathway, upregulation of the proapoptotic cell surface receptor TRAIL-R2, and activation of proapoptotic caspase-8. Collectively, our results offer a rationale for the therapeutic exploration of treatments inducing endoplasmic reticulum stress against mutant ERBB2-expressing breast tumor cells.