Therapy-Induced Senescence: Novel Approaches for Markers Identification.

Therapy-induced senescence (TIS) represents a major cellular response to anticancer treatments. Both malignant and non-malignant cells in the tumor microenvironment undergo TIS and may be harmful for cancer patients since TIS cells develop a senescence-associated secretory phenotype (SASP) that can sustain tumor growth. The SASP also modulates anti-tumor immunity, although the immune populations involved and the final results appear to be context-dependent. In addition, senescent cancer cells are able to evade senescence growth arrest and to resume proliferation, likely contributing to relapse. So, research data suggest that TIS induction negatively affects therapy outcomes in cancer patients. In line with this, new interventions aimed at the removal of senescent cells or the reprogramming of their SASP, called senotherapy, have become attractive therapeutic options. To date, the lack of reliable, cost-effective, and easy-to-use TIS biomarkers hinders the application of recent anti-senescence therapeutic approaches in the clinic. Hence, the identification of biomarkers for the detection of TIS tumor cells and TIS non-neoplastic cells is a high priority in cancer research. In this review article, we describe the current knowledge about TIS, outline critical gaps in our knowledge, and address recent advances and novel approaches for the discovery of TIS biomarkers.

Glutamine Metabolism in Cancer Stem Cells: A Complex Liaison in the Tumor Microenvironment.

In this review we focus on the role of glutamine in control of cancer stem cell (CSC) fate. We first provide an overview of glutamine metabolism, and then summarize relevant studies investigating how glutamine metabolism modulates the CSC compartment, concentrating on solid tumors. We schematically describe how glutamine in CSC contributes to several metabolic pathways, such as redox metabolic pathways, ATP production, non-essential aminoacids and nucleotides biosynthesis, and ammonia production. Furthermore, we show that glutamine metabolism is a key regulator of epigenetic modifications in CSC. Finally, we briefly discuss how cancer-associated fibroblasts, adipocytes, and senescent cells in the tumor microenvironment may indirectly influence CSC fate by modulating glutamine availability. We aim to highlight the complexity of glutamine's role in CSC, which supports our knowledge about metabolic heterogeneity within the CSC population.

NGAL as a Potential Target in Tumor Microenvironment.

The signaling network between cancer and stromal cells plays a crucial role in tumor microenvironment. The fate of tumor progression mainly depends on the huge amount of information that these cell populations exchange from the onset of neoplastic transformation. Interfering with such signaling has been producing exciting results in cancer therapy: just think of anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies that, acting as immune checkpoint inhibitors, interrupt the inhibitory signaling exerted by cancer cells on immune cells or the CAR-T technology that fosters the reactivation of anti-tumoral immunity in a restricted group of leukemias and lymphomas. Nevertheless, many types of cancers, in particular solid tumors, are still refractory to these treatments, so the identification of novel molecular targets in tumor secretome would benefit from implementation of current anti-cancer therapeutical strategies. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a secreted protein abundantly expressed in the secretome of various human tumors. It represents a promising target for the multiple roles that are played inside cancer and stromal cells, and also overall in their cross-talk. The review focuses on the different roles of NGAL in tumor microenvironment and in cancer senescence-associated secretory phenotype (SASP), highlighting the most crucial functions that could be eventually targetable in cancer therapy.

CIKS/DDX3X interaction controls the stability of the Zc3h12a mRNA induced by IL-17.

IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.

Fructose induces prothrombotic phenotype in human endothelial cells : A new role for "added sugar" in cardio-metabolic risk.

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5 mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.

Down-regulation of wild-type p53-induced phosphatase 1 (Wip1) plays a critical role in regulating several p53-dependent functions in premature senescent tumor cells.

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser(15) and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser(15) phosphorylation is associated with enhanced phosphorylation at Ser(46), increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.

Iron Metabolism in Cancer and Senescence: A Cellular Perspective.

Iron participates in a number of biological processes and plays a crucial role in cellular homeostasis. Alterations in iron metabolism are considered hallmarks of cancer and drivers of aggressive behaviors, such as uncontrolled proliferation, resistance to apoptosis, enhanced metastatic ability, increased cell plasticity and stemness. Furthermore, a dysregulated iron metabolism has been associated with the development of an adverse tumor microenvironment. Alterations in iron metabolism have been described in cellular senescence and in aging. For instance, iron has been shown to accumulate in aged tissues and in age-related diseases. Furthermore, in vitro studies demonstrate increases in iron content in both replicative and stress-induced senescent cells. However, the role, the mechanisms of regulation and dysregulation and the effects of iron metabolism on senescence remain significantly less characterized. In this review, we first provide an overview of iron metabolism and iron regulatory proteins. Then, we summarize alterations in iron homeostasis in cancer and senescence from a cellular point of view.

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Trabectedin suppresses escape from therapy-induced senescence in tumor cells by interfering with glutamine metabolism.

Conventional and targeted cancer therapies may induce a cellular senescence program termed therapy-induced senescence. However, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, driving tumor recurrence after treatments. Cells that escape from therapy-induced senescence are characterized by a plastic, cancer stem cell-like phenotype, and recent studies are beginning to define their unique metabolic features, such as glutamine dependence. Here, we show that the antineoplastic drug trabectedin suppresses escape from therapy-induced senescence in all cell lines studied, and reduces breast cancer stem-like cells, at concentrations that do not affect the viability of senescent tumor cells. We demonstrate that trabectedin downregulates both the glutamine transporter SLC1A5 and glutamine synthetase, thereby interfering with glutamine metabolism. On the whole, our results indicate that trabectedin targets a glutamine-dependent cancer stem-like cell population involved in evasion from therapy-induced senescence and suggest a therapeutic potential for trabectedin combined with pro-senescence chemotherapy in tumor treatment.

Thyroid hormone and androgen signals mutually interplay and enhance inflammation and tumorigenic activation of tumor microenvironment in prostate cancer.

Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the fifth leading cause of death worldwide. The majority of PCas are androgen-sensitive, with a significant up-regulation of Androgen Receptor (AR) that causes a stimulatory effect on growth and progression of cancer cells. For this reason, the first-line therapy for PCa is androgen ablation, even if it ultimately fails due to the onset of hormone-refractory state, in which the malignant cells do not sense the androgen signal anymore. Besides androgens, a growing number of evidence suggests that Thyroid Hormones (THs) mediate tumor-promoting effects in a variety of human cancers, as Epithelial-to-Mesenchymal Transition (EMT), invasion and metastasis and also stimulation of angiogenesis and tumor metabolism. Moreover, epidemiological studies demonstrated an increased risk for PCa in patients with lower levels of Thyreotropin (TSH). Here, we investigated if intracellular TH metabolism affects Benign Prostatic Hyperplasia (BPH) and PCa formation and progression. We found that the intracellular TH metabolism is a crucial determinant of PCa behavior. We observed that a dynamic stage-specific expression of the THs modulating enzymes, the deiodinases, is required for the progression of BPH to PCa malignancy. By acting simultaneously on epithelial cancer cells and fibroblasts, THs exert a proliferative and pro-inflammatory effect cooperating with androgens. These findings suggest that androgens and THs may interplay and mediate a coordinate effect on human PCa formation and progression. In light of our results, future perspective could be to explore the potential benefits of THs intracellular modulators aimed to counteract PCa progression.

The neutrophil gelatinase-associated lipocalin (NGAL), a NF-kappaB-regulated gene, is a survival factor for thyroid neoplastic cells.

NF-kappaB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-kappaB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-kappaB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-kappaB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-kappaB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-kappaB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

Analysis of the Contribution of NF-κB in the Regulation of Chemotherapy-Induced Cell Senescence by Establishing a Tetracycline-Regulated Cell System.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Glutamine promotes escape from therapy-induced senescence in tumor cells.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

NGAL promotes recruitment of tumor infiltrating leukocytes.

We have previously shown that Neutrophil Gelatinase-Associated Lipocalin (NGAL) is strongly expressed in thyroid carcinomas, especially of anaplastic type, where it protects neoplastic cells from serum deprivation-induced apoptosis and enhances tumor invasivity by regulating MMP-9 activity. Here we demonstrate that NGAL-containing conditioned medium from human anaplastic thyroid carcinoma (ATC) cells is able to induce monocyte migration via up-regulation of a number of different chemokines. The enhanced chemokines transcription is due to the NGAL-mediated intracellular iron uptake. Very importantly, mice tumor allografts raised from subcutaneous injection of syngeneic colon carcinoma cell lines, expressing high levels of NGAL, show a dense leukocyte infiltrate which strongly decreases in tumor allografts from NGAL-depleted cell injected mice. Our results indicate that the NGAL promotes leukocytes recruitment in tumor microenvironment through iron-mediated chemokines production.

RbAp48 is a target of nuclear factor-kappaB activity in thyroid cancer.

CONTEXT: We have recently shown that nuclear factor (NF)-kappaB activity is constitutively elevated in anaplastic human thyroid carcinomas. The inhibition of NF-kappaB in the anaplastic thyroid carcinoma cell line (FRO) leads to increased susceptibility to apoptosis induced by chemotherapeutic drugs and to the block of oncogenic activity. OBJECTIVES: To understand better the molecular mechanisms played by NF-kappaB in thyroid oncogenesis, we performed a differential proteomic analysis between FRO transfected with a superrepressor form of inhibitor of kappaBalpha (IkappaBalphaM) and the parental counterpart (FRO Neo cells). RESULTS: Differential proteomic analysis revealed that the retinoblastoma-associated protein 48 (RbAp48) is down-regulated in the absence of functional NF-kappaB. Immunohistochemical analysis of normal and pathological human thyroid specimens confirmed that RbAp48 is strongly overexpressed in primary human carcinomas. Reduction of RbAp48 expression using small interfering RNA determined the suppression of tumorigenicity, very likely due to the decrease of their growth rate rather than to an increased susceptibility to apoptosis. In addition, we showed that NF-kappaB, at least in part, transcriptionally controls RbAp 48. A functional NF-kappaB consensus sequence was located within the promoter region of RbAp48 human gene, and embryonic fibroblasts isolated from the p65 knockout mouse (murine embryonic fibroblasts p65-/-) showed decreased expression of RbAp48. CONCLUSION: Our results show that RbAp48 is a NF-kappaB-regulated gene playing an important role in thyroid cancer cell autonomous proliferation.

The chemokine scavenging receptor D6/ACKR2 is a target of miR-146a in thyroid cancer.

We have previously shown that miR-146a, a NF-κB-regulated microRNA, is strongly expressed in human specimens and cell lines derived from anaplastic thyroid carcinomas (ATC) where it mediates some of the NF-κB pro-tumorigenic functions. By using a bioinformatic analysis, we identified the chemokine scavenger receptor D6/ ACKR2 as a target of miR146a in human ATC. We found that the expression of D6/ ACKR2 was up-regulated in miR-146a-null ATC cell lines and that the 3' UTR of D6/ ACKR2 mRNA was able to inhibit its expression in parental, but not in miR-146a-null ATC cells. Since human specimens from primary ATC showed a low expression of D6/ ACKR2 compared to normal thyroid tissues, we analyzed the effects of D6/ACKR2 over-expression in ATC cells. Different chemokines added to the conditioned medium of D6/ACKR2 over-expressing ATC cells partially failed to drive in vitro monocyte migration, and tumors derived from the injection of the same cells in nude mice showed a decreased number of infiltrating macrophages. Taken together, these results indicate that ATC cells down-regulate D6/ACKR2 expression through miR-146a activity to sustain leukocyte trafficking inside tumor microenvironment and shed light on a novel mechanism by which NF-κB indirectly inhibits the expression and the function of anti-tumorigenic gene in thyroid cancer.

NF-kappaB in solid tumors.

Cancer is a multistep process during which cells acquire genetic alterations that drive the progressive transformation of normal cells into highly malignant cells. Self-sufficiency in growth, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, are signatures of transformed cells. NF-kappaB is a key actor in tumorigenesis given its ability to control the expression and the function of a number of genes involved in these processes. Indeed, constitutive activation of NF-kappaB is a common feature of many human tumors, while its sustained activation during inflammation predisposes normal cells to neoplastic transformation. Since suppression of NF-kappaB has been shown to inhibit oncogenic potential of transformed cells, targeting it should be effective in the prevention and treatment of cancer.

Expression of functional tissue factor in activated T-lymphocytes in vitro and in vivo: A possible contribution of immunity to thrombosis?

OBJECTIVE: T-lymphocyte activation plays an important role in the pathophysiology of acute coronary syndromes (ACS). Plaques from ACS patients show a selective oligoclonal expansion of T-cells, indicating a specific, antigen-driven recruitment of T-lymphocytes within the unstable lesions. At present, however, it is not known whether T-cells may contribute directly to thrombosis by expressing functional tissue factor (TF). Accordingly, the aim of the present study was to investigate whether T-cells are able to express functional TF in their activated status. METHODS: In vitro, CD3(+)-cells, isolated from buffy coats, were stimulated with anti-CD3/CD28 beads, IL-6, TNF-α, IL-17, INF-γ or PMA/ionomycin. Following stimulation, TF expression on cell-surface, at gene and protein levels, as well as its procoagulant activity in whole cells and microparticles was measured. In vivo, TF expression was evaluated in CD3(+)-cells isolated from the aorta and the coronary sinus of ACS-NSTEMI and stable coronary artery disease (SCAD) patients. The presence of CD3(+)-TF(+)cells was also evaluated by immunohistochemistry in thrombi aspirated from ACS-STEMI patients. RESULTS: PMA/ionomycin and IL-17 plus INF-γ stimulation resulted in a significant TF increase at gene and protein levels as well as at cell-surface expression. This was accompanied by a parallel increase in FXa generation, both in whole cells and in microparticles, indicating that the induced membrane-bound TF was active. Furthermore, transcardiac TF gradient was significantly higher in CD3(+)-cells obtained from ACS-patients compared to SCAD-patients. Interestingly, thrombi from ACS-STEMI patients resulted enriched in CD3(+)-cells, most of them expressing TF. CONCLUSIONS: Our data demonstrate that activated T-lymphocytes in vitro express functional TF on their membranes, suggesting a direct pathophysiological role of these cells in the thrombotic process; this hypothesis is further supported by the observations in vivo that CD3(+)-cells from coronary circulation of ACS-NSTEMI patients show increased TF levels and that coronary thrombi from ACS-STEMI patients are enriched in CD3(+)-cells expressing TF.

The adipokine apelin-13 induces expression of prothrombotic tissue factor.

Adipocytes are cells able to produce and secrete several active substances (adipokines) with direct effects on vascular cells. Apelin, one of the most recently identified adipokines has been studied in cardiovascular system physiology in regard to vessel vasodilation and myocardial contraction, but it has not yet completely characterised for its pathophysiological role in cardiovascular disease and especially in acute coronary syndromes (ACS). Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of apelin 12 and apelin 13 on TF in human umbilical endothelial cells (HUVECs) and monocytes. Cells were stimulated with increasing concentrations of apelin 12 or apelin 13 and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and pro-coagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that apelin 13 but not apelin 12 induces transcription of mRNA for TF. In addition, we show that this adipokine promotes surface expression of TF that is functionally active. Apelin 13 effects on TF appear modulated by the activation of the G-protein-transcription factor nuclear factor (NF)-κB axis since G-protein inhibitors suppressed NF-κB mediated TF expression. Data of the present study, although in vitro, indicate that apelin-13, induces a procoagulant phenotype in HUVECs and monocytes by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

Endoplasmic reticulum stress causes thyroglobulin retention in this organelle and triggers activation of nuclear factor-kappa B via tumor necrosis factor receptor-associated factor 2.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.