Stress-induced Transcription Factor Research Articles

Abstract 65: HIRA is a master regulator of the proliferation-to-dormancy switch in breast cancer

Abstract Most cancer-related deaths are caused by recurrent metastatic disease with latency periods that range from years to even decades. This halt in disease progression is driven by cancer dormancy, a stage in which residual disease is present but remains asymptomatic before reawakening and often causes lethal forms of the disease. Cell fate decisions, such as the ones that underlie dormancy and reawakening of cancer cells, often require extensive epigenetic remodeling, which in turn allows for the expression of specific genetic programs that trigger cell fate decisions. Therefore, epigenetic remodeling is likely essential for the switch between dormant and proliferative states throughout the metastatic process. Here we focus on epigenetic regulator, histone H3.3 chaperone HIRA and its role in the proliferation-to-dormancy switch in breast cancer cells. In this study, we demonstrate that HIRA suppression leads to cell cycle arrest and induces canonical dormancy pathways via phosphorylation of p38 and induction of p27 expression. In addition, HIRA suppression triggers induction of stress-inducible transcription factors NRF2, MITF and ATF3 to enable the activation of genetic programs that maintain homeostasis and promote survival in dormant cells. Our data also portray that HIRA is suppressed in the well-established isogenic models of dormancy when compared to their metastatic counterparts. Altogether we present for the first-time histone chaperone HIRA as a major regulatory point of cell fate for disseminated cancer cells by controlling their transition between dormant and proliferative states, and therefore suggest HIRA as a novel and tremendously needed therapeutic target for metastatic breast cancer. Citation Format: Stanislav Drapela, Devesh Raizada, Ananya Mukherjee, Joanne Tejero, Didem Ilter, Jose Javier Bravo-Cordero, Ana P Gomes. HIRA is a master regulator of the proliferation-to-dormancy switch in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 65.

Transcriptional profiling of the response to starvation and fattening reveals differential regulation of autophagy genes in mammals.

Nutrient deprivation (starvation) induced by fasting and hypercaloric regimens are stress factors that can influence cell and tissue homeostasis in mammals. One of the key cellular responses to changes in nutrient availability is the cell survival pathway autophagy. While there has been much research into the protein networks regulating autophagy, less is known about the gene expression networks involved in this fundamental process. Here, we applied a network algorithm designed to analyse omics datasets, to identify sub-networks that are enriched for induced genes in response to starvation. This enabled us to identify two prominent active modules, one composed of key stress-induced transcription factors, including members of the Jun, Fos and ATF families, and the other comprising autophagosome sub-network genes, including ULK1. The results were validated in the brain, liver and muscle of fasting mice. Moreover, differential expression analysis of autophagy genes in the brain, liver and muscle of high-fat diet-exposed mice showed significant suppression of GABARAPL1 in the liver. Finally, our data provide a resource that may facilitate the future identification of regulators of autophagy.

Activating Transcription Factor 3 Diminishes Ischemic Cerebral Infarct and Behavioral Deficit by Downregulating Carboxyl-Terminal Modulator Protein.

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor and a familiar neuronal marker for nerve injury. This factor has been shown to protect neurons from hypoxic insult in vitro by suppressing carboxyl-terminal modulator protein (CTMP) transcription, and indirectly activating the anti-apoptotic Akt/PKB cascade. Despite prior studies in vitro, whether this neuroprotective pathway also exists in the brain in vivo after ischemic insult remains to be determined. In the present study, we showed a rapid and marked induction of ATF3 mRNA throughout ischemia-reperfusion in a middle cerebral artery (MCA) occlusion model. Although the level of CTMP mRNA was quickly induced upon ischemia, its level showed only a mild increase after reperfusion. With the gain-of-function approach, both pre- and post-ischemic administration of Ad-ATF3 ameliorated brain infarct and neurological deficits. Whereas, with the loss-of-function approach, ATF3 knockout (KO) mice showed bigger infarct and worse functional outcome after ischemia. In addition, these congenital defects were rescued upon reintroducing ATF3 to the brain of KO mice. ATF3 overexpression led to a lower level of CTMP and a higher level of p-Akt(473) in the ischemic brain. On the contrary, ATF3 KO resulted in upregulation of CTMP and downregulation of p-Akt(473) instead. Furthermore, post-ischemic CTMP siRNA knockdown led to smaller infarct and better behaviors. CTMP siRNA knockdown increased the level of p-Akt(473), but did not alter the ATF3 level in the ischemic brain, upholding the ATF3→CTMP signal cascade. In summary, our proof-of-principle experiments support the existence of neuroprotective ATF3→CTMP signal cascade regulating the ischemic brain. Furthermore, these results suggest the therapeutic potential for both ATF3 overexpression and CTMP knockdown for stroke treatment.

Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

HIV Tat-Conjugated Histone H3 Peptides Induce Tumor Cell Death Via Cellular Stress Responses.

Histone H3 is a nucleosome scaffold protein that is involved in a variety of intracellular processes. Aberrant modification of H3 is important in carcinogenesis. In contrast, free histones in cells can act as stimuli to trigger cellular immune responses and cell death. In this study, we linked cell-penetrating peptide HIV Tat to a histone H3 fragment to achieve intracellular delivery in tumor cells. We found that Tat-conjugated histone polypeptides localized to nuclei of lung and breast cancer cells and caused cell death. A trans-configured Tat sequence displayed dramatically improved peptide half-life and cytotoxicity. Mechanistic studies demonstrated that treatment with the peptides significantly elevated mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species (ROS) production, as well as levels of stress-inducible transcription factor ATF3 (activating transcription factor 3) and AP-1 (activating protein-1). Cytotoxicity of the peptide was significantly reduced by inhibition of AP-1 activity and ROS production. These results suggest the potential of Tat-conjugated H3 peptides as antitumor agents to induce cell death via increased cellular stress response by activating p38-MAPK signaling and intracellular ROS production.

98 (PB088) - Discovery and validation of biomarkers to support clinical development of NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor

Background: Heat Shock Factor (HSF1) is a stress-inducible transcription factor that plays a key role in the activation of the eukaryotic heat shock response. HSF1 is hijacked by cancer cells to activate a set of genes that overlap with, but are not identical to, the classical heat shock response. Whereas the HSF1 pathway has been shown to play a key role in oncogenesis and multiple hallmark features of malignancy in experimental cancers, and its amplification/expression/activity correlates with poor clinical outcome, knockout of HSF1 does not impair viability in model organisms – suggesting a potential therapeutic window.

A hypothetical model of skewed DNA methylation balance in the enhancer regions containing differentially methylated cytosines associated with non-malignant complex diseases

Epigenome-wide association studies have been widely conducted over the past decade to identify the epigenetic determinants of human complex diseases. Numerous differentially methylated cytosines (DMCs) associated with diseases or environmental exposure have been identified till date. An important hallmark of DMCs in non-malignant complex diseases is that they are strongly enriched in CpG sites showing small changes to intermediate levels of methylation. This paradigm of subtle change in DMC methylation hampers our understanding of epigenetic regulation in non-malignant complex diseases and the application of DMCs as biomarkers in clinical practice. Herein, we propose a hypothetical model of skewed DNA methylation balance in the enhancer regions containing differentially methylated cytosines associated with non-malignant complex diseases. The DMCs are strongly enriched in enhancers, which are characterized by a highly dynamic balance of DNA methylation. The DNA methylation balance plays an important role in cellular adaptation to adverse environment; however, in the case of certain diseases, the balance becomes skewed toward one end of dynamic range. Binding stress-inducible transcription factors determines DNA methylation at the enhancer region, resulting in epigenetic heterogeneity. The dynamics of DNA methylation balance can be evaluated by analyzing the DNA co-methylation status of DMCs and their neighboring CpG sites. Targeted bisulfite resequencing method is suggested to analyze the change in DNA co-methylation in both case-control and longitudinal cohort studies. Cell models are necessary to study the molecular mechanisms of DNA methylation changes. We also suggest that complex obstetrical syndromes associated with placental disorders may be excellent models for investigating common features of DNA methylation in non-malignant complex diseases.

Sugarcane ScDREB2B-1 Confers Drought Stress Tolerance in Transgenic Nicotiana benthamiana by Regulating the ABA Signal, ROS Level and Stress-Related Gene Expression

The dehydration-responsive element-binding protein (DREB) is a subgroup member of the AP2/ERF family and actively participates in the response of plants to abiotic stress. Although DREB genes have been studied in a variety of plant species, there are few reports of DREB genes in sugarcane (Saccharum spp.). In this study, a novel full-length cDNA sequence of the ScDREB2B-1 gene was cloned from the Saccharum hybrid ROC22, whose encoding protein contained only one AP2-conserved domain and was clustered into the DREB (A-2) subgroup. The diverse promoter elements in the ScDREB2B-1 gene and the accumulated transcripts of its homologous gene (SsAP2/ERF-107) in S. spontaneum under drought stress suggest that the ScDREB2B-1 gene may play a role in drought response. In addition, reverse transcription quantitative PCR analysis showed that the expression level of the ScDREB2B-1 gene was upregulated in the root and leaf of ROC22 under polyethylene glycol, sodium chloride and abscisic acid (ABA) treatments. The yeast two-hybrid experiment demonstrated that ScDREB2B-1 had transcriptional self-activation activity. Compared with wild-type plants, the overexpression of the ScDREB2B-1 gene improved the drought tolerance of the transgenic Nicotiana benthamiana by activating the ABA pathway to enhance the expression of the ABA-responsive gene (NbNCED) and ABA content, regulate the intracellular reactive oxygen species (ROS) level (enhance the transcripts of ROS synthase-related gene NbRbohB and the activities of catalase, peroxidase and superoxide dismutase) and increase the relative water content, proline content and expression level of osmotic stress-related genes (NbERD and NbLEA). Collectively, our data indicate that ScDREB2B-1 is a stress-inducible and ABA-responsive transcription factor gene that responds to drought stress by regulating ABA signaling, ROS levels and stress-related gene expression. This study contributes to a better understanding of the biological function of ScDREB2B-1, which could serve as a foundation for future resistance breeding in sugarcane.

Transcriptomic and physiological responses of contrasting maize genotypes to drought stress.

Drought is a significant environmental stress factor that adversely affects maize productivity. However, many details regarding the molecular mechanisms of maize against drought are still unclear. In this study, leaf transcriptomics and physiological traits of two maize genotypes with differing drought resistance were analyzed. Transcriptome sequencing identified 8985 and 7305 differentially expressed genes (DEGs) in SD902 and SD609, respectively. Functional analysis suggested that numerous genes are highly involved in oxidative defense, protein modification, photosynthesis, phytohormone response, MAPK signaling, and transcription factors (TFs). Compared to SD902, SD609 had a higher expression of DEGs related to antioxidant enzymes, photosynthetic electron transport, heat shock proteins, and indole-3-acetic acid (IAA) signaling under drought conditions, which might contribute to its tolerance mechanisms to drought. Stress-induced TFs may play a crucial regulatory role in genotypic differences. Moreover, the physiological changes and gene expression abundance determined using quantitative reverse transcription polymerase chain reaction were consistent with the RNA sequencing data. The study results suggest that the higher drought tolerance of SD609 than SD902 can be attributed to stronger stress defense capabilities, IAA signal transduction, and more stable photosynthesis. Our findings provide new insights into the molecular mechanisms of maize against drought stress, and the candidate genes identified may be used in breeding drought-tolerant maize cultivars.

Abstract ND08: NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor

Abstract We report the discovery of NXP800 — a first-in-class orally active HSF1 pathway inhibitor identified in our academic laboratory at the Institute of Cancer Research, London — and its progression into Phase I clinical trials in collaboration with our partner Nuvectis Pharma. HSF1 (Heat Shock Factor 1) is an ancient stress-inducible transcription factor that plays a key role in the transcriptional activation of the eukaryotic heat shock response. This leads to expression of many heat shock proteins, including molecular chaperones, and through this HSF1 acts as a master transcriptional regulator of proteostasis. Importantly, HSF1 is hijacked by cancer cells to activate a set of genes that overlaps with, but is not identical to, the classical heat shock response. The HSF1 pathway has been shown to play a key role in oncogenesis and the hallmark features of malignancy and is important in the initiation and progression of many experimental cancer models. Importantly, while HSF1 has been demonstrated to be important in mouse models of oncogenesis, knockout of the homologue of HSF1 has only modest effects in mice and flies under non-stressed laboratory conditions – supporting the potential for a therapeutic window. In addition, gene amplification, expression and activation of HSF1 has been shown to predict clinical outcome in many human cancers. Despite strong scientific rationale and pre-clinical validation, pharmaceutical exploitation has been limited because analysis of the available molecular structures of HSF1 shows it to be very difficult to drug. Because of this, we set out to discover inhibitors of the HSF1 pathway using an initial phenotypic screen that reports on the activation of the HSF1-regulated gene HSP72. This identified the bisamide series that was followed up by a detailed understanding of structure-activity relationships and medicinal chemistry optimization, particularly to improve pharmaceutical properties, leading to the selection of NXP800 (CCT361814) as the development candidate. NXP800 has good pharmacokinetic properties in mice, including oral bioavailability; also, pharmacodynamic studies show modulation of biomarkers of the HSF1 pathway and stress response in human tumour xenograft models. NXP800 exhibits striking therapeutic activity in xenografts of human ovarian clear cell ovarian cancer (OCCC) and endometrioid ovarian cancer, two serious conditions of high unmet medical need with limited treatment options – including prolonged tumour growth inhibition and regression at doses that show good toleration. The efficacy and tolerability data indicate a clear therapeutic window, supported by selectivity and safety profiting in pharmacology and kinome panels. In addition, we identified deficiency in ARID1A, a component of the SWI/SNF chromatin remodelling complex, as indicative of greater therapeutic responsiveness. This was confirmed through broader evaluation in human cancer cell line panels that also indicates therapeutic potential for NXP800 in additional cancer types. Studies to support mechanism of action and identify patient selection biomarkers will be described. A Phase 1 trial of NXP800 is now open, incorporating validated predictive, pharmacokinetic and pharmacodynamic biomarkers that provide a robust Pharmacological Audit Trail and support the prediction of dose-to-human. Citation Format: Paul Workman. NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND08.

The multifaceted role of ATF4 in regulating glucose-stimulated insulin secretion

Stress-inducible transcription factor ATF4 is essential for survival and identity of β-cell during stress conditions. However, the physiological role of ATF4 in β-cell function is not yet completely understood. To understand the role of ATF4 in glucose-stimulated insulin secretion (GSIS), β-cell-specific Atf4 knockout (βAtf4KO) mice were phenotypically characterized. Insulin secretion and mechanistic analyses were performed using islets from control Atf4f/f and βAtf4KO mice to assess key regulators for triggering and amplifying signals for GSIS. βAtf4KO mice displayed glucose intolerance due to reduced insulin secretion. Moreover, βAtf4KO islets exhibited a decrease in both the insulin content and first-phase insulin secretion. The analysis of βAtf4KO islets showed that ATF4 is required for insulin production and glucose-stimulated ATP and cAMP production. The results demonstrate that ATF4 contributes to the multifaceted regulatory process in GSIS even under stress-free conditions.

Targeting the Interaction of GABAB Receptors With CHOP After an Ischemic Insult Restores Receptor Expression and Inhibits Progressive Neuronal Death.

GABAB receptors control neuronal excitability via slow and prolonged inhibition in the central nervous system. One important function of GABAB receptors under physiological condition is to prevent neurons from shifting into an overexcitation state which can lead to excitotoxic death. However, under ischemic conditions, GABAB receptors are downregulated, fostering over-excitation and excitotoxicity. One mechanism downregulating GABAB receptors is mediated via the interaction with the endoplasmic reticulum (ER) stress-induced transcription factor CHOP. In this study, we investigated the hypothesis that preventing the interaction of CHOP with GABAB receptors after an ischemic insult restores normal expression of GABAB receptors and reduces neuronal death. For this, we designed an interfering peptide (R2-Pep) that restored the CHOP-induced downregulation of cell surface GABAB receptors in cultured cortical neurons subjected to oxygen and glucose deprivation (OGD). Administration of R2-Pep after OGD restored normal cell surface expression of GABAB receptors as well as GABAB receptor-mediated inhibition. As a result, R2-Pep reduced enhanced neuronal activity and inhibited progressive neuronal death in OGD stressed cultures. Thus, targeting diseases relevant protein-protein interactions might be a promising strategy for developing highly specific novel therapeutics.

Phytomelatonin as a signaling molecule for protein quality control via chaperone, autophagy, and ubiquitin–proteasome systems in plants

Physiological effects mediated by melatonin are attributable to its potent antioxidant activity as well as its role as a signaling molecule in inducing a vast array of melatonin-mediated genes. Here, we propose melatonin as a signaling molecule essential for protein quality control (PQC) in plants. PQC occurs by the coordinated activities of three systems: the chaperone network, autophagy, and the ubiquitin-proteasome system. With regard to the melatonin-mediated chaperone pathway, melatonin increases thermotolerance by induction of heat shock proteins and confers endoplasmic reticulum stress tolerance by increasing endoplasmic reticulum chaperone proteins. In chloroplasts, melatonin-induced chaperones, including Clps and CpHSP70s, play key roles in the PQC of chloroplast-localized proteins, such as Lhcb1, Lhcb4, and RBCL, during growth. Melatonin regulates PQC by autophagy processes, in which melatonin induces many autophagy (ATG) genes and autophagosome formation under stress conditions. Finally, melatonin-mediated plant stress tolerance is associated with up-regulation of stress-induced transcription factors, which are regulated by the ubiquitin-proteasome system. In this review, we propose that melatonin plays a pivotal role in PQC and consequently functions as a pleiotropic molecule under non-stress and adverse conditions in plants.

Nickel oxide nanoparticles induce apoptosis and ferroptosis in airway epithelial cells via ATF3.

Exposure to nickel oxide nanoparticles (NiONPs), which have been widely produced and applied in industry, leads to adverse pulmonary and systemic effects. The aim of this study is to investigate the involvement of apoptosis and ferroptosis in NiONPs-induced acute lung injury (ALI). Intratracheal instillation of NiONPs into mice elevated the levels of pro-inflammatory cytokines, neutrophils, and proteins in the bronchoalveolar lavage fluid, and triggered apoptosis and ferroptosis in the lung tissues. Consistently, NiONPs-induced apoptosis and ferroptosis were observed in in vitro experiments using human lung epithelial cells. Activating transcription factor 3 (ATF3), a stress-inducible transcription factor, was upregulated by NiONPs exposure in both murine lung tissues and human lung epithelial cells. Moreover, human lung epithelial cells with ATF3 deficiency exhibited a lower level of apoptosis and ferroptosis when exposed to NiONPs. Collectively, our findings demonstrated that ATF3 was responsive to NiONPs exposure, and promoted NiONPs-induced apoptosis and ferroptosis in lung epithelial cells, indicating that ATF3 is a potential biomarker and therapeutic target for NiONPs-associated ALI.

Hif1α and Wnt are required for posterior gene expression during Xenopus tropicalis tail regeneration

Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1α, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1α is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1α and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1α is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate that Hif1α has regulatory roles in Wnt target gene expression across multiple tissue contexts.

Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.

A novel NRF2/ARE inhibitor gossypol induces cytotoxicity and sensitizes chemotherapy responses in chemo-refractory cancer cells.

NRF2/ARE signaling pathway is a principal regulator of cellular redox homoeostasis. The stress-induced transcription factor, NRF2, can shield cells from the oxidative damages via binding to the consensus antioxidant-responsive element (ARE) and driving several cyto-protective genes expression. Increasing evidence indicated that aberrant activation of NRF2 in malignant cells may support their survival through various pathways to detoxify chemotherapy drugs, attenuate drug-induced oxidative stress, or induce drug efflux, all of which are crucial in developing drug resistance. Accordingly, NRF2 is a potential drug target for improving the effectiveness of chemotherapy and to reverse drug resistance in cancer cells. A stable ARE-driven reporter human head and neck squamous cell carcinoma (HNSCC) cell line, HSC3-ARE9, was established and utilized to screen novel NRF2 inhibitors from a compound library. The cotton plant derived phenolic aldehyde-gossypol was selected for further analyses. The effects of gossypol in cancer cells were determined by western blotting, RT-qPCR, clonogenic assay, and cell viability assays. The gossypol-responsive gene expression levels were assessed in the Oncomine database. The effects of gossypol on conferring chemo-sensitization were evaluated in etoposide-resistant and cisplatin-resistant cancer cells. Our study is the first to identify that gossypol is effective to reduce both basal and NRF2 activator tert-butylhydroquinone (t-BHQ)-induced ARE-luciferase activity. Gossypol diminishes NRF2 protein stability and thereby leads to the suppression of NRF2/ARE pathway, which resulted in decreasing the expression levels of NRF2 downstream genes in both time- and dose-dependent manners. Inhibition of NRF2 by gossypol significantly decreases cell viabilities in human cancer cells. In addition, we find that gossypol re-sensitizes topoisomerase II poison treatment in etoposide-resistant cancer cells via suppression of NRF2/ABCC1 axis. Moreover, gossypol suppresses NRF2-mediated G6PD expression thereby leads to induce synthetic lethality with cisplatin not only in parental cancer cells but also in cisplatin-resistant cancer cells. These findings suggest that gossypol is a novel NRF2/ARE inhibitor, and can be a potential adjuvant chemotherapeutic agent for treatment of chemo-refractory tumor.

Integrative analysis of the metabolome and transcriptome reveal the phosphate deficiency response pathways of alfalfa

Understanding the mechanisms underlying the responses to inorganic phosphate (Pi) deficiency in alfalfa will help enhance Pi acquisition efficiency and the sustainable use of phosphorous resources. Integrated global metabolomic and transcriptomic analyses of mid-vegetative alfalfa seedlings under 12-day Pi deficiency were conducted. Limited seedling growth were found, including 13.24%, 16.85% and 33.36% decreases in height, root length and photosynthesis, and a 24.10% increase in root-to-shoot ratio on day 12. A total of 322 and 448 differentially abundant metabolites and 1199 and 1061 differentially expressed genes were identified in roots and shoots. Increased (>3.68-fold) inorganic phosphate transporter 1;4 and SPX proteins levels in the roots (>2.15-fold) and shoots (>2.50-fold) were related to Pi absorption and translocation. The levels of phospholipids and Pi-binding carbohydrates and nucleosides were decreased, while those of phosphatases and pyrophosphatases in whole seedlings were induced under reduced Pi. In addition, nitrogen assimilation was affected by inhibiting high-affinity nitrate transporters (NRT2.1 and NRT3.1), and nitrate reductase. Increased delphinidin-3-glucoside might contribute to the gray-green leaves induced by Pi limitation. Stress-induced MYB, WRKY and ERF transcription factors were identified. The responses of alfalfa to Pi deficiency were summarized as local systemic signaling pathways, including root growth, stress-related responses consisting of enzymatic and nonenzymatic systems, and hormone signaling and systemic signaling pathways including Pi recycling and Pi sensing in the whole plant, as well as Pi recovery, and nitrate and metal absorption in the roots. This study provides important information on the molecular mechanism of the response to Pi deficiency in alfalfa.

Stress Induced Transcription Factors Transactivate the Herpes Simplex Virus 1 Infected Cell Protein 27 (ICP27) Transcriptional Enhancer.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons, including sensory neurons within trigeminal ganglia. During latency, lytic cycle viral gene expression is silenced. However, stressful stimuli can trigger reactivation from latency. The viral tegument protein, VP-16, transactivates all immediate early (IE) promoters during productive infection. Conversely, cellular factors are expected to trigger viral gene expression during early stages of reactivation from latency and in non-neuronal cells that do not support high levels of productive infection. The glucocorticoid receptor (GR), synthetic corticosteroid dexamethasone, and certain stress-induced transcription factors cooperatively transactivate infected cell protein 0 (ICP0) and ICP4 promoters. Since ICP27 protein expression is required for productive infection, we hypothesized that the ICP27 promoter is transactivated by stress-induced transcription factors. New studies have demonstrated that ICP27 enhancer sequences were transactivated by GR and Krüppel-like factor 15 (KLF15). Mutation of a consensus Sp1 binding site within ICP27 enhancer sequences impaired transactivation by GR and KLF15. Chromatin immunoprecipitation studies have demonstrated that GR and KLF15 occupy ICP27 promoter sequences during productive infection. Cells transfected with an ICP27 enhancer fragment revealed the GR and KLF15 occupancy of ICP27 enhancer sequences required the intact Sp1 binding site. Notably, GR and KLF15 form a feed-forward transcription loop in response to stress, suggesting these cellular factors promote viral replication following stressful stimuli.

Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress

The emergence of new primate-specific genes is an essential factor in human and primate brain development and functioning. POU2F1/Oct-1 is a transcription regulator in higher eukaryotes which is involved in the regulation of development, differentiation, stress response, and other processes. We have demonstrated that the Tigger2 transposon insertion into the POU2F1 gene which occurred in the primate lineage led to the formation of an additional exon (designated the Z-exon). Z-exon-containing primate-specific Oct-1Z transcript includes a short upstream ORF (uORF) located at its 5’-end and the main ORF encoding the Oct-1Z protein isoform (Pou2F1 isoform 3, P14859-3), which differs from other Oct-1 isoforms by its N-terminal peptide. The Oct-1Z-encoding transcript is expressed mainly in human brain cortex. Under normal conditions, the translation of the ORF coding for the Oct-1Z isoform is repressed by uORF. Under various stress conditions, uORF enables a strong increase in the translation of the Oct-1Z-encoding ORF. Increased Oct-1Z expression levels in differentiating human neuroblasts activate genes controlling stress response, neural cell differentiation, brain formation, and organogenesis. We have shown that the Oct-1Z isoform of the POU2F1/Oct-1 transcription factor is an example of a primate-specific genomic element contributing to brain development and cellular stress defense.