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Activated ERBB2/HER2 Licenses Sensitivity to Apoptosis upon Endoplasmic Reticulum Stress through a PERK-Dependent Pathway
Abstract
HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately
20% of human breast tumors. Truncated or mutant isoforms which show increased
oncogenicity compared to 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 (ER) stress, altering the unfolded protein
response (UPR) of these cells. Deregulation of the ERK, AKT and mTOR activities
elicited by mutant ERBB were involved in mediating this differential UPR response,
elevating the response to ER stress and apoptotic cell death. Mechanistic investigations
revealed that the increased sensitivity of mutant ERBB2-expressing cells to ER 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 ER stress against mutant ERBB2-
expressing breast tumor cells.
... Several years ago, it was reported that DRs and the Caspase-8/ Bid pathway were involved in executing apoptosis upon ER stress in cancer cells [4,5,26]. Subsequently, the contribution and requirement of DRs in ER stress was more widely studied and has met with some controversy, as their role is possibly cell-type dependent [4-6, 10, 27]. ...
... Chemicals or physiological stimuli that perturb ER homeostasis are no exception [34], and a role for BIM as an universal mediator of cell death by ER stressors was proposed by studies in knock-out mice [9]. However, it was also reported that DRs, mainly DR5, are involved in ER stress-mediated cell death [4,5,26]. This has led to controversy and to the more recent realization that the role of DRs in ER-stress-mediated cell death could well be cell-type dependent [10,29]. ...
Impairments in protein folding in the endoplasmic reticulum (ER) lead to a condition called ER stress, which can trigger apoptosis via the mitochondrial or the death receptor (extrinsic) pathway. There is controversy concerning involvement of the death receptor (DR)4 and DR5-Caspase-8 –Bid pathway in ER stress-mediated cell death, and this axis has not been fully studied in B-cell malignancies. Using three B-cell lines from Mantle Cell Lymphoma, Waldenström’s macroglobulinemia and Multiple Myeloma origins, we engineered a set of CRISPR KOs of key components of these cell death pathways to address this controversy. We demonstrate that DR4 and/or DR5 are essential for killing via TRAIL, however, they were dispensable for ER-stress induced-cell death, by Thapsigargin, Brefeldin A or Bortezomib, as were Caspase-8 and Bid. In contrast, the deficiency of Bax and Bak fully protected from ER stressors. Caspase-8 and Bid were cleaved upon ER-stress stimulation, but this was DR4/5 independent and rather a result of mitochondrial-induced feedback loop subsequent to Bax/Bak activation. Finally, combined activation of the ER-stress and TRAIL cell-death pathways was synergistic with putative clinical relevance for B-cell malignancies.
... Initiator caspase-8 is an essential component of the extrinsic pathway of apoptosis signaling induced upon death receptor activation by their cognate ligands [3,4,45] and cellular stress [46][47][48][49]. Furthermore, a growing body of evidences indicates that the activation of the mitochondria-operated pathway is a key event in the feedback amplification of apical apoptotic caspases by effector caspases [50]. ...
Different studies have reported that inhibiting the mevalonate pathway with statins may increase the sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the signaling mechanism leading to this sensitization remains largely unknown. We investigated the role of the YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex in the metabolic control of TRAIL sensitivity by the mevalonate pathway. We show that depleting nuclear YAP/TAZ in tumor cells, either via treatment with statins or by silencing YAP/TAZ expression with siRNAs, facilitates the activation of apoptosis by TRAIL. Furthermore, the blockage of TEAD transcriptional activity either pharmacologically or through the ectopic expression of a disruptor of the YAP/TAZ interaction with TEAD transcription factors, overcomes the resistance of tumor cells to the induction of apoptosis by TRAIL. Our results show that the mevalonate pathway controls cellular the FLICE-inhibitory protein (cFLIP) expression in tumor cells. Importantly, inhibiting the YAP/TAZ-TEAD signaling pathway induces cFLIP down-regulation, leading to a marked sensitization of tumor cells to apoptosis induction by TRAIL. Our data suggest that a combined strategy of targeting TEAD activity and selectively activating apoptosis signaling by agonists of apoptotic TRAIL receptors could be explored as a potential therapeutic approach in cancer treatment.
... The PERK-ATF4 signaling pathway is one of the indispensable ERS signaling pathways in addition to the IRE1-XBP1 and ATF6 pathways, all of which can lead to the transcription of CHOP, a pivotal mediator in the development of sepsis [46]. Emerging evidence suggests that the PERK-ATF4-CHOP pathway is related to the activation of cytoprotective autophagy upon ERS in different cellular models [47][48][49]. Our previous studies demonstrated that the PERK-ATF4-CHOP signaling pathway was critical for SESN2 alleviating the apoptosis, pyroptosis and ferroptosis of dendritic cells in sepsis [45,46,50]. ...
Background:
Ribophagy is a selective autophagic process that specifically degrades dysfunctional or superfluous ribosomes to maintain cellular homeostasis. Whether ribophagy can ameliorate the immunosuppression in sepsis similar to endoplasmic reticulum autophagy (ERphagy) and mitophagy remains unclear. This study was conducted to investigate the activity and regulation of ribophagy in sepsis and to further explore the potential mechanism underlying the involvement of ribophagy in T-lymphocyte apoptosis.
Methods:
The activity and regulation of nuclear fragile X mental retardation-interacting protein 1 (NUFIP1)-mediated ribophagy in T lymphocytes during sepsis were first investigated by western blotting, laser confocal microscopy and transmission electron microscopy. Then, we constructed lentivirally transfected cells and gene-defective mouse models to observe the impact of NUFIP1 deletion on T-lymphocyte apoptosis and finally explored the signaling pathway associated with T-cell mediated immune response following septic challenge.
Results:
Both cecal ligation and perforation-induced sepsis and lipopolysaccharide stimulation significantly induced the occurrence of ribophagy, which peaked at 24 h. When NUFIP1 was knocked down, T-lymphocyte apoptosis was noticeably increased. Conversely, the overexpression of NUFIP1 exerted a significant protective impact on T-lymphocyte apoptosis. Consistently, the apoptosis and immunosuppression of T lymphocytes and 1-week mortality rate in NUFIP1 gene-deficient mice were significantly increased compared with those in wild-type mice. In addition, the protective effect of NUFIP1-mediated ribophagy on T lymphocytes was identified to be closely related to the endoplasmic reticulum stress apoptosis pathway, and PERK-ATF4-CHOP signaling was obviously involved in downregulating T-lymphocyte apoptosis in the setting of sepsis.
Conclusions:
NUFIP1-mediated ribophagy can be significantly activated to alleviate T lymphocyte apoptosis through the PERK-ATF4-CHOP pathway in the context of sepsis. Thus, targeting NUFIP1-mediated ribophagy might be of importance in reversing the immunosuppression associated with septic complications.
... glands from SjD patients (Barrera et al., 2016) and is also known to be linked to apoptosis via caspase activation (Egger et al., 2007;Martín-Pérez et al., 2014). To test the effect of GPR78 overexpression on ER stress, mRNA expression level of two ER stress markers, ATF4 and CHOP, were analyzed by using qRT-PCR. ...
Objective:
Sjögren's disease (SjD) has a strong sex bias, suggesting an association with sex hormones. Male SjD represents a distinct subset of the disease, but the pathogenic mechanisms of male SjD is poorly characterized. The aim of this study is to identify initiating events related to the development of gland hypofunction and autoimmunity in male SjD patients.
Materials and methods:
Human minor salivary glands were transcriptomically analyzed with microarrays to detect differentially expressed genes in male SjD patients. Identified genes were tested on their involvement in the disease using conditional transgenic mice and gene-overexpressing cells.
Results:
GPR78, an orphan G protein-coupled receptor, was overexpressed in salivary glands of male SjD patients compared with male healthy controls and female SjD patients. Male GPR78 transgenic mice developed salivary gland hypofunction with increased epithelial apoptosis, which were not seen in control or female transgenic mice. In cell culture, GPR78 overexpression decreased lysosomal integrity, leading to caspase-dependent apoptotic cell death. GPR78-induced cell death in vitro was inhibited by treatment with estradiol.
Conclusion:
GPR78 overexpression can induce apoptosis and salivary gland hypofunction in male mice through lysosomal dysfunction and increased caspase-dependent apoptosis in salivary gland epithelium, which may drive disease in humans.
... This mechanism is activated under conditions of EnR stress owing to the accumulation of ROS and misfolded proteins. Activated UPR promotes the reduction of misfolded proteins, and if this does not occur, tumor cell apoptosis is induced through the activation of caspases [137]. ...
Breast cancer is the most frequent malignant neoplasm in females. While conventional treatments such as chemotherapy and radiotherapy are available, they are highly invasive and toxic to oncological patients. Melatonin is a promising molecule for the treatment of breast cancer with antitumor effects on tumorigenesis and tumor progression. The aim of this systematic review was to synthesize knowledge about the antitumor effect of melatonin on breast cancer in experimental models and propose the main mechanisms of action already described in relation to the processes regulated by melatonin. PubMed, Web of Science, and Embase databases were used. The inclusion criteria were in vitro and in vivo experimental studies that used different formulations of melatonin as a treatment for breast cancer, without year or language restrictions. Risk of bias for studies was assessed using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tool. Data from selected articles were presented as narrative descriptions and tables. Seventy-five articles on different breast cancer cell lines and experimental models treated with melatonin alone, or in combination with other compounds were included. Melatonin showed antitumor effects on proliferative pathways related to the cell cycle and tumorigenesis, tumor death, angiogenesis, and tumor metastasis, as well as on oxidative stress and immune regulatory pathways. These effects were either dependent or independent of melatonin receptors. Herein, we clarify the antitumor action of melatonin on different tumorigenic processes in breast cancer in experimental models.
Systematic review registration: PROSPERO database (CRD42022309822/https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022309822).
Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.
Traumatic brain injury (TBI) is a common trauma with high mortality and disability rates worldwide. However, the current management of this disease is still unsatisfactory. Therefore, it is necessary to investigate the pathophysiological mechanisms of TBI in depth to improve the treatment options. In recent decades, abundant evidence has highlighted the significance of endoplasmic reticulum stress (ERS) in advancing central nervous system (CNS) disorders, including TBI. ERS following TBI leads to the accumulation of unfolded proteins, initiating the unfolded protein response (UPR). Protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1 (IRE1), and activating transcription factor 6 (ATF6) are the three major pathways of UPR initiation that determine whether a cell survives or dies. This review focuses on the dual effects of ERS on TBI and discusses the underlying mechanisms. It is suggested that ERS may crosstalk with a series of molecular cascade responses, such as mitochondrial dysfunction, oxidative stress, neuroinflammation, autophagy, and cell death, and is thus involved in the progression of secondary injury after TBI. Hence, ERS is a promising candidate for the management of TBI.
Zinc oxide nanoparticles (ZnO NPs) have recently been used as food preservatives and additives because of their good antibacterial and nutritional functions. This study performed RNA-seq analyses to evaluate the potential toxicity of ZnO NPs on goat mammary epithelial cells (GMECs) in vitro. Our results suggested that the ZnO NP treatment significantly reduced GMEC viability in a time- and dose-dependent manner. Transcriptomic analysis showed that ZnO NP exposure changed the expression levels of more than 500 genes in GMECs, including various biological pathways. We observed that decreased mitochondrial membrane potential caused mitochondrial dysfunction. Further study indicated that the treatment of cells with ZnO NPs resulted in the accumulation of reactive oxygen species (ROS), which led to oxidative stress. Meanwhile, the expression of genes (TNFα, TNFR1, FADD, Caspase 8 and Caspase 6) associated with the death receptor pathway was upregulated, which indicated the death receptor-mediated extrinsic apoptosis pathway was activated. Moreover, the expression levels of Bax, Cytc, Caspase 3 and Caspase 9 were upregulated, while the expression levels of Bcl2 were downregulated, which indicated mitochondria-mediated intrinsic apoptosis pathway was activated. More notably, ZnO NP exposure increased the expression levels of ER stress-related genes (PERK, ATF4, eIF2α and CHOP) and proteins (p-PERK, p-eIF2α, PERK and CHOP). Furthermore, gene ontology (GO) terms and genes related to autophagy were altered, suggesting that exposure to ZnO NPs might activate autophagy in GMECs. In summary, our findings showed that ZnO NPs could exert significant toxic effects on GMECs through multiple mechanisms. These pathways are related to each other and influence each other to participate in ZnO NPs-induced the damage of GMECs. Thus, their safe use in the feed and food industry should be considered. Meanwhile, RNA-seq might represent a new method of assessing the toxicity mechanisms of nanomaterials.
Cancer cells adapt multiple mechanisms to counter intense stress on their way to growth. Tumor microenvironment stress leads to canonical and noncanonical endoplasmic stress (ER) responses, which mediate autophagy and are engaged during proteotoxic challenges to clear unfolded or misfolded proteins and damaged organelles to mitigate stress. In these conditions, autophagy functions as a cytoprotective mechanism in which malignant tumor cells reuse degraded materials to generate energy under adverse growing conditions. However, cellular protection by autophagy is thought to be complicated, contentious, and context-dependent; the stress response to autophagy is suggested to support tumorigenesis and drug resistance, which must be adequately addressed. This review describes significant findings that suggest accelerated autophagy in cancer, a novel obstacle for anticancer therapy, and discusses the UPR components that have been suggested to be untreatable. Thus, addressing the UPR or noncanonical ER stress components is the most effective approach to suppressing cytoprotective autophagy for better and more effective cancer treatment.
Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1–JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1–JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE–JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1–JNK pathway.
A conserved domain in the extracellular region of the 60- and 80-kilodalton tumor necrosis factor receptors (TNFRs) was identified
that mediates specific ligand-independent assembly of receptor trimers. This pre–ligand-binding assembly domain (PLAD) is
physically distinct from the domain that forms the major contacts with ligand, but is necessary and sufficient for the assembly
of TNFR complexes that bind TNF-α and mediate signaling. Other members of the TNFR superfamily, including TRAIL receptor 1
and CD40, show similar homotypic association. Thus, TNFRs and related receptors appear to function as preformed complexes
rather than as individual receptor subunits that oligomerize after ligand binding.
Unlabelled:
Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment.
Significance:
We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.
A genome-wide characterization of active translation of messenger RNA
following inhibition of mTOR will transform our view of this signalling
protein's regulatory role in cancer. See Article p.55 & Letter p.109
The HER-2/neu oncogene is a member of the crbB-like oncogene family, and is related to, but distinct from, the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 189 primary human breast cancers were investigated. HER-2/ neu was found to be amplified from 2- to greater than 20-fold in 30% of the tumors. Correlation of gene amplification with several disease parameters was evaluated. Amplification of die HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. It retained its significance even when adjustments were made for other known prognostic factors. Moreover, HER-2/neu amplification had greater prognostic value than most currently used prognostic factors, including hormonal-receptor status, in lymph node-positive disease. These data indicate that this gene may play a role in the biologic behavior and/or padiogenesis of human breast cancer.
Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2α (eIF2α) in response to a stress signal from the endoplasmic reticulum (ER). Activation of this process leads to reduced rates of initiation of protein translation during ER stress. Here we describe the cloning of perk, a gene encoding a type I transmembrane ER- resident protein. PERK has a lumenal domain that is similar to the ER- stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2α kinases, PKR and HRI. ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2α on serine residue 51, inhibiting translation of messenger RNA into protein. These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress.
Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However,
the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo
fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated
by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent
and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we
have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through
the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP
homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of
NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis
is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7.
Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced
apoptotic pathway, and both contribute to ER stress-induced apoptosis.
Many cellular processes including apoptosis, autophagy, translation, energy metabolism, and inflammation are controlled by the mammalian target of rapamycin (mTOR) kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR). Although both of these signaling nodes have attracted wide attention in fundamental cell biology and drug discovery, crosstalk between the two pathways has emerged only very recently. mTOR complex 1 (mTORC1) operates both upstream and downstream of ER stress signals, which can either enhance or antagonize the anabolic output of mTORC1. Upon prolonged ER stress, mTORC1 contributes to apoptotic signaling by suppressing the survival kinase Akt through feedback inhibition. Likewise, chronic ER stress obstructs activation of Akt by mTOR complex 2. This review surveys our knowledge of mTOR-ER stress intersections and highlights potential therapeutic implications.
The vast majority of proteins that a cell secretes or displays on its surface first enter the endoplasmic reticulum (ER),
where they fold and assemble. Only properly assembled proteins advance from the ER to the cell surface. To ascertain fidelity
in protein folding, cells regulate the protein-folding capacity in the ER according to need. The ER responds to the burden
of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways, collectively termed
the unfolded protein response (UPR). Together, at least three mechanistically distinct branches of the UPR regulate the expression
of numerous genes that maintain homeostasis in the ER or induce apoptosis if ER stress remains unmitigated. Recent advances
shed light on mechanistic complexities and on the role of the UPR in numerous diseases.