Interleukin-21 engineering enhances NK cell activity against glioblastoma via CEBPD.

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM.

The RNA binding protein Arid5a drives IL-17-dependent autoantibody-induced glomerulonephritis.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPß, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

CEBPD aggravates apoptosis and oxidative stress of neuron after ischemic stroke by Nrf2/HO-1 pathway.

CCAAT enhancer binding protein delta (CEBPD) is a transcription factor and plays an important role in apoptosis and oxidative stress, which are the main pathogenesis of ischemic stroke. However, whether CEBPD regulates ischemic stroke through targeting apoptosis and oxidative stress is unclear. Therefore, to answer this question, rat middle cerebral artery occlusion (MCAO) reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) primary cortical neuron were established to mimic ischemic reperfusion injury. We found that CEBPD was upregulated and accompanied with increased neurological deficit scores and infarct size, and decreased neuron in MCAO rats. The siRNA targeted CEBPD inhibited CEBPD expression in rats, and meanwhile lentivirus system was used to blocked CEBPD expression in primary neuron. CEBPD degeneration decreased neurological deficit scores, infarct size and brain water content of MCAO rats. Knockdown of CEBPD enhanced cell viability and reduced apoptosis as well as oxidative stress in vivo and in vitro. CEBPD silencing promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the expression of heme oxygenase 1 (HO-1). Newly, CEBPD facilitated the transcription of cullin 3 (CUL3), which intensified ischemic stroke through Nrf2/HO-1 pathway that was proposed by our team in the past. In conclusion, targeting CEBPD-CUL3-Nrf2/HO-1 axis may be contributed to cerebral ischemia therapy.

The Janus kinase 1 is critical for pancreatic cancer initiation and progression.

Interleukin-6 (IL-6)-class inflammatory cytokines signal through the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and promote the development of pancreatic ductal adenocarcinoma (PDAC); however, the functions of specific intracellular signaling mediators in this process are less well defined. Using a ligand-controlled and pancreas-specific knockout in adult mice, we demonstrate in this study that JAK1 deficiency prevents the formation of KRASG12D-induced pancreatic tumors, and we establish that JAK1 is essential for the constitutive activation of STAT3, whose activation is a prominent characteristic of PDAC. We identify CCAAT/enhancer binding protein δ (C/EBPδ) as a biologically relevant downstream target of JAK1 signaling, which is upregulated in human PDAC. Reinstating the expression of C/EBPδ was sufficient to restore the growth of JAK1-deficient cancer cells as tumorspheres and in xenografted mice. Collectively, the findings of this study suggest that JAK1 executes important functions of inflammatory cytokines through C/EBPδ and may serve as a molecular target for PDAC prevention and treatment.

Inhibition of MMP8 effectively alleviates manic-like behavior and reduces neuroinflammation by modulating astrocytic CEBPD.

There is an intrinsic relationship between psychiatric disorders and neuroinflammation, including bipolar disorder. Ouabain, an inhibitor of Na+/K+-ATPase, has been implicated in the mouse model with manic-like behavior. However, the molecular mechanisms linking neuroinflammation and manic-like behavior require further investigation. CCAAT/Enhancer-Binding Protein Delta (CEBPD) is an inflammatory transcription factor that contributes to neurological disease progression. In this study, we demonstrated that the expression of CEBPD in astrocytes was increased in ouabain-treated mice. Furthermore, we observed an increase in the expression and transcript levels of CEBPD in human primary astrocytes following ouabain treatment. Transcriptome analysis revealed high MMP8 expression in human primary astrocytes following CEBPD overexpression and ouabain treatment. We confirmed that MMP8 is a CEBPD-regulated gene that mediates ouabain-induced neuroinflammation. In our animal model, treatment of ouabain-injected mice with M8I (an inhibitor of MMP8) resulted in the inhibition of manic-like behavior compared to ouabain-injected mice that were not treated with M8I. Additionally, the reduction in the activation of astrocytes and microglia was observed, particularly in the hippocampal CA1 region. Excessive reactive oxygen species formation was observed in ouabain-injected mice, and treating these mice with M8I resulted in the reduction of oxidative stress, as indicated by nitrotyrosine staining. These findings suggest that MMP8 inhibitors may serve as therapeutic agents in mitigating manic symptoms in bipolar disorder.

CEBPD REGULATES OXIDATIVE STRESS AND INFLAMMATORY RESPONSES IN HYPERTENSIVE CARDIAC REMODELING.

ABSTRACT: Hypertension seems to inevitably cause cardiac remodeling, increasing the mortality of patients. This study aimed to explore the molecular mechanism of CCAAT/enhancer-binding protein delta (CEBPD)-mediated oxidative stress and inflammation in hypertensive cardiac remodeling. The hypertensive murine model was established through angiotensin-II injection, and hypertensive mice underwent overexpressed CEBPD vector injection, cardiac function evaluation, and observation of histological changes. The cell model was established by angiotensin-II treatment and transfected with overexpressed CEBPD vector. Cell viability and surface area and oxidative stress (reactive oxygen species/superoxide dismutase/lactate dehydrogenase/malondialdehyde) were assessed, and inflammatory factors (TNF-α/IL-1ß/IL-6/IL-10) were determined both in vivo and in vitro . The levels of CEBPD, miR-96-5p, inositol 1,4,5-trisphosphate receptor 1 (IP3R), natriuretic peptide B, and natriuretic peptide A, collagen I, and collagen III in tissues and cells were determined. The binding relationships of CEBPD/miR-96-5p/IP3R 3' untranslated region were validated. CEBPD was reduced in cardiac tissue of hypertensive mice, and CEBPD upregulation improved cardiac function and attenuated fibrosis and hypertrophy, along with reductions of reactive oxygen species/lactate dehydrogenase/malondialdehyde/TNF-α/IL-1ß/IL-6 and increases in superoxide dismutase/IL-10. CEBPD enriched on the miR-96-5p promoter to promote miR-96-5p expression, whereas CEBPD and miR-96-5p negatively regulated IP3R. miR-96-5p silencing/IP3R overexpression reversed the alleviative role of CEBPD overexpression in hypertensive mice. In summary, CEBPD promoted miR-96-5p to negatively regulate IP3R expression to inhibit oxidative stress and inflammation, thereby alleviating hypertensive cardiac remodeling.

The dual role of C/EBPδ in cancer.

CCAAT/Enhancer-Binding Protein delta (C/EBPδ) is a transcription factor involved in differentiation and inflammation. While sparsely expressed in adult tissues, aberrant expression of C/EBPδ has been associated with different cancers. Initially, re-expression of C/EBPδ in cell cultures limited tumor cell proliferation, assigning it a tumor suppressor role. However, opposing observations were made in pre-clinical models and patients, suggesting that C/EBPδ not only mediates cell proliferation but dictates a broader spectrum of tumorigenesis-related effects. It is now widely accepted that C/EBPδ contributes to an inflammatory, tumor-promoting microenvironment, aids hypoxia adaption and contributes to the recruitment of blood vessels for improved nutrient supply to tumor cells and facilitated extravasation. This review summarizes the work published on this transcription factor in the field of cancer over the past decade. It points out areas in which a consensus on C/EBPδ's role appears to emerge and seek to explain seemingly contradictory results.

CEBPD is a master transcriptional factor for hypoxia regulated proteins in glioblastoma and augments hypoxia induced invasion through extracellular matrix-integrin mediated EGFR/PI3K pathway.

Hypoxia contributes to the initiation and progression of glioblastoma by regulating a cohort of genes called hypoxia-regulated genes (HRGs) which form a complex molecular interacting network (HRG-MINW). Transcription factors (TFs) often play central roles for MINW. The key TFs for hypoxia induced reactions were explored using proteomic analysis to identify a set of hypoxia-regulated proteins (HRPs) in GBM cells. Next, systematic TF analysis identified CEBPD as a top TF that regulates the greatest number of HRPs and HRGs. Clinical sample and public database analysis revealed that CEBPD is significantly up-regulated in GBM, high levels of CEBPD predict poor prognosis. In addition, CEBPD is highly expressed in hypoxic condition both in GBM tissue and cell lines. For molecular mechanisms, HIF1α and HIF2α can activate the CEBPD promotor. In vitro and in vivo experiments demonstrated that CEBPD knockdown impaired the invasion and growth capacity of GBM cells, especially in hypoxia condition. Next, proteomic analysis identified that CEBPD target proteins are mainly involved in the EGFR/PI3K pathway and extracellular matrix (ECM) functions. WB assays revealed that CEBPD significantly positively regulated EGFR/PI3K pathway. Chromatin immunoprecipitation (ChIP) qPCR/Seq analysis and Luciferase reporter assay demonstrated that CEBPD binds and activates the promotor of a key ECM protein FN1 (fibronectin). In addition, the interactions of FN1 and its integrin receptors are necessary for CEBPD-induced EGFR/PI3K activation by promoting EGFR phosphorylation. Furthermore, GBM sample analysis in the database corroborated that CEBPD is positively correlated with the pathway activities of EGFR/PI3K and HIF1α, especially in highly hypoxic samples. At last, HRPs are also enriched in ECM proteins, indicating that ECM activities are important components of hypoxia induced responses in GBM. In conclusion, CEPBD plays important regulatory roles in the GBM HRG-MINW as a key TF, which activates the EGFR/PI3K pathway through ECM, especially FN1, mediated EGFR phosphorylation.

C/EBP-Family Redundancy Determines Patient Survival and Lymph Node Involvement in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPß or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan-Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPß and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPß as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPß and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBß and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

C/EBPδ Suppresses Motility-Associated Gene Signatures and Reduces PDAC Cell Migration.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive human cancers and occurs globally at an increasing incidence. Metastases are the primary cause of cancer-related death and, in the majority of cases, PDAC is accompanied by metastatic disease at the time of diagnosis, making it a particularly lethal cancer. Regrettably, to date, no curative treatment has been developed for patients with metastatic disease, resulting in a 5-year survival rate of only 11%. We previously found that the protein expression of the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) negatively correlates with lymph node involvement in PDAC patients. To better comprehend the etiology of metastatic PDAC, we explored the role of C/EBPδ at different steps of the metastatic cascade, using established in vitro models. We found that C/EBPδ has a major impact on cell motility, an important prerequisite for tumor cells to leave the primary tumor and to reach distant sites. Our data suggest that C/EBPδ induces downstream pathways that modulate actin cytoskeleton dynamics to reduce cell migration and to induce a more epithelial-like cellular phenotype. Understanding the mechanisms dictating epithelial and mesenchymal features holds great promise for improving the treatment of PDAC.

Cortisol Stimulates Local Progesterone Withdrawal Through Induction of AKR1C1 in Human Amnion Fibroblasts at Parturition.

Fetal membrane activation is seen as being one of the crucial triggering components of human parturition. Increased prostaglandin E2 (PGE2) production, a common mediator of labor onset in virtually all species, is recognized as one of the landmark events of membrane activation. Fetal membranes are also equipped with a high capacity of cortisol regeneration by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), and the cortisol regenerated potently induces PGE2 synthesis, an effect normally suppressed by progesterone during gestation. There is no precipitous decline of progesterone synthesis in human parturition. It is intriguing how this suppression is lifted in parturition. Here, we investigated this issue by using human amnion tissue and primary amnion fibroblasts which synthesize the most PGE2 in the fetal membranes. Results showed that the expression of 11ß-HSD1 and aldo-keto reductase family 1 member C1 (AKR1C1), a progesterone-inactivating enzyme, increased in parallel in human amnion tissue with gestational age toward the end of gestation and at parturition. Cortisol induced AKR1C1 expression via the transcription factor CCAAT enhancer binding protein δ (C/EBPδ) in amnion fibroblasts. Inhibition of AKR1C1 not only blocked progesterone catabolism induced by cortisol, but also enhanced the suppression of cortisol-induced cyclooxygenase-2 (COX-2) expression by progesterone in amnion fibroblasts. In conclusion, our results indicate that cortisol regenerated in the fetal membranes triggers local progesterone withdrawal through enhancement of AKR1C1-mediated progesterone catabolism in amnion fibroblasts, so that the suppression of progesterone on the induction of COX-2 expression and PGE2 synthesis by cortisol can be lifted for parturition.

CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids.

BACKGROUND: CCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function. METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures). RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM. CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

FOXO1 cooperates with C/EBPδ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting.

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin-proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO-CCAAT/enhancer-binding protein δ (C/EBPδ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4-/- (loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPδ. During starvation, C/EBPδ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPδ prevented the induction of the ubiquitin-proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPδ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPδ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.

CCAAT/enhancer binding protein (C/EBP) delta promotes the expression of PTX3 and macrophage phagocytosis during A. fumigatus infection.

Given the increasing incidence of pulmonary aspergillosis, it is important to understand the natural defense mechanisms by which the body can kill Aspergillus fumigatus conidia. Pentraxin 3 (PTX3) plays a nonredundant role in resistance to A. fumigatus. Here, we found that the key predicted PTX3 transcription factor, CCAAT/enhancer-binding protein δ (CEBPD), was up-regulated during A. fumigatus conidia infection. Functionally, CEBPD significantly promoted the expression of PTX3 and the phagocytic ability of macrophages. Mechanistically, CEBPD activated the PTX3 by directly binding to the promoter region of the PTX3 gene. We also showed that the RNA-binding protein human antigen R promoted CEBPD expression. These findings provide new insights into the crucial role of CEBPD in the phagocytosis of A. fumigatus conidia by macrophages and highlight this protein as a potential therapeutic target for invasive pulmonary aspergillosis.

PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

CEBPD Potentiates the Macrophage Inflammatory Response but CEBPD Knock-Out Macrophages Fail to Identify CEBPD-Dependent Pro-Inflammatory Transcriptional Programs.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target.

Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.

Glycyrrhizic acid suppresses early stage of adipogenesis through repression of MEK/ERK-mediated C/EBPß and C/EBPδ expression in 3T3-L1 cells.

Glycyrrhizic acid (GA), a major constituent of the root of licorice (Glycyrrhiza glabra), and has various biological activities, including anti-obesity property. However, the molecular mechanism of anti-adipogenic effect of GA is still unclear. In this study, we investigated the anti-adipogenic effects of GA in mouse adipocytic 3T3-L1 cells and elucidated its underlying molecular mechanism. GA decreased the intracellular triglyceride level. The expression levels of the adipogenic and lipogenic genes were lowered by treatment with GA in a concertation-dependent manner. In contrast, GA did not affect the lipolytic gene expression and the released glycerol level. GA suppressed the early stage of adipogenesis when it was added for 0-3 h after initiation of adipogenesis. Moreover, GA reduced the mRNA levels of CCAAT/enhancer binding protein (C/EBP) ß and C/EBPδ, both of which activate the early stage of adipogenesis. Furthermore, GA decreased phosphorylation of extracellular signal-regulated kinase [ERK: p44/42 mitogen-activated protein kinase (MAPK)] in the early stage of adipogenesis. In addition, a MAPK kinase (MEK) inhibitor, PD98059 reduced the C/EBPß and C/EBPδ gene expression. These results indicate that GA suppressed the early stage of adipogenesis through repressing the MEK/ERK-mediated C/EBPß and C/EBPδ expression in 3T3-L1 cells. Thus, GA has an anti-adipogenic ability and a possible agent for treatment of obesity.

MCL1 participates in leptin-promoted mitochondrial fusion and contributes to drug resistance in gallbladder cancer.

Obesity is a risk factor for gallbladder cancer (GBC) development, and it correlates with shorter overall survival. Leptin, derived from adipocytes, has been suggested to contribute to the growth of cancer cells; however, the detailed mechanism of leptin in GBC drug resistance remains uninvestigated. In this study, our finding that patients with GBC with a higher BMI were associated with increased GBC risks, including shortened survival, is clinically relevant. Moreover, obese NOD/SCID mice exhibited a higher circulating concentration of leptin, which is associated with GBC growth and attenuated gemcitabine efficacy. We further revealed that leptin can inhibit gemcitabine-induced GBC cell death through myeloid cell leukemia 1 (MCL1) activation. The transcription factor C/EBP δ (CEBPD) is responsive to activated STAT3 (pSTAT3) and contributes to MCL1 transcriptional activation upon leptin treatment. In addition, MCL1 mediates leptin-induced mitochondrial fusion and is associated with GBC cell survival. The findings in this study suggest the involvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrial fusion and survival and provide a potentially new therapeutic target to improve the efficacy of gemcitabine in patients with GBC.

The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.