AP-1 Transcription Factor Subunit Research Articles

Abstract P5-11-04: Anti-proliferative effect of oral metronomic vinorelbine (mVNB) in PAM50 Luminal/HER2-negative early breast cancer (SOLTI-1501 VENTANA): A randomized, three-arm, window-of-opportunity study

Abstract BACKGROUND: The anti-proliferative effect of mVNB alone or in combination with endocrine therapy in patients with hormone receptor-positive/HER2- breast cancer (BC) has been scarcely addressed. METHODS: Postmenopausal women with untreated stage I-III BC were randomized (1:1:1) to receive 3 weeks of letrozole (LTZ) 2.5mg/day, oral mVNB 50mg 3 days/week or the combination. The 1ary objective was to evaluate, within PAM50 Luminal A/B disease, if the anti-proliferative effect of mVNB+LTZ was superior to monotherapy. An anti-proliferative effect was defined as the mean relative decrease of the PAM50 11-gene Proliferation Score in each arm. 2ary objectives included safety and the comparison of the anti-proliferative effect between arms. An unplanned analysis of stromal tumor infiltrating lymphocytes (sTILs) was performed. PAM50 analyses were performed using the nCounter®-based Breast Cancer 360TM panel. Changes in the expression of 790 genes/signatures tracking multiple biological processes from tumor cells and the microenvironment were evaluated within each arm using paired (surgery vs. baseline) univariate analyses. P-values were corrected for multiple comparisons using false discovery rate (FDR). RESULTS: A total of 61 patients were randomized and 54 paired samples (89%) were analyzed. Main patient characteristics were mean age 67, mean tumor size 1.7 cm, stage I (55.7%) and grade 1-2 (90%). Grade 3 toxicities occurred in 3.3% of cases. Baseline samples were Luminal A (72.3%) or B (27.7%). The anti-proliferative effect of mVNB+LTZ (-73.2%) was superior to both monotherapy arms combined (-49.9%; p=0.001) and mVNB (-19.1%; p<0.001). The anti-proliferative effect of mVNB+LTZ (-73.2%) was higher compared to LTZ (-65.7%) but did not reach statistical significance (p=0.328). Across the mVNB+LTZ, LTZ and VNB arms, 413 (52.3%), 403 (51.0%) and 21 (2.6%) genes/signatures were found differentially expressed (FDR<5%) between baseline and surgery samples. Compared to mVNB+LTZ baseline samples, surgical samples showed higher expression of AP-1 transcription factor subunits FOS and JUN, inflammatory chemokines (e.g. CCL4 and IL6), stromal-related genes (e.g. CAV1 and stroma signature) and immune infiltration (e.g. CD8 T-cell signature) and lower expression of proliferation-related genes (e.g. MKI67 and UBE2C), estrogen receptor-signaling and Risk of Recurrence. Of the 413 genes found differentially expressed in surgical samples compared to baseline samples in the mVNB+LTZ arm, 108 (26.2%) were not found in the LTZ arm. Among them, high expression of LAG3, CD24, CD84 and CCR5. Under the microscope, sTILs (≥10% at week 3) were observed in 6.6% (mVNB), 15% (LTZ) and 26% (mVNB+LTZ) of the cases. In tumors with ≤10% TILs at baseline, an increase in TILs was observed following LTZ (p=0.049) and mVNB+LTZ (p=0.012). CONCLUSIONS: mVNB is well-tolerated and presents antiproliferative activity alone and in combination with LTZ. The increase of activated CD8 T-cells or TILs observed with LTZ+mVNB opens the possibility of studying combinations with immunotherapy. Further investigation comparing these biological results with other metronomic schedules or combinations is warranted. Citation Format: Prat A, Adamo B, Pascual T, Perez Fidalgo JA, Blanch S, Martínez N, Gomez Pardo P, Lopez Gonzalez A, Murillo Jaso L, Amillano K, Vidal M, Paré L, Canes J, Galvan P, Gonzalez Farre B, Ortega Cebrián V, Gonzalez X, Bellet Ezquerra M, Villagrasa P, Ciruelos E. Anti-proliferative effect of oral metronomic vinorelbine (mVNB) in PAM50 Luminal/HER2-negative early breast cancer (SOLTI-1501 VENTANA): A randomized, three-arm, window-of-opportunity study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-04.

Comprehensive bioinformatics analysis of critical lncRNAs, mRNAs and miRNAs in non‑alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common fatty liver disease in developed countries, in which fat accumulation in the liver is induced by non-alcoholic factors. The present study was conducted to identify NAFLD-associated long non-coding RNAs (lncRNAs), mRNAs and microRNAs (miRNAs). The microarray dataset GSE72756, which included 5 NAFLD liver tissues and 5 controls, was acquired from the Gene Expression Omnibus database. Differentially expressed lncRNAs (DE-lncRNAs) and mRNAs (DE-mRNAs) were detected using the pheatmap package. Using the clusterProfiler package and Cytoscape software, enrichment and protein-protein interaction (PPI) network analyses were conducted to evaluate the DE-mRNAs. Next, the miRNA-lncRNA-mRNA interaction network was visualized using Cytoscape software. Additionally, RP11-279F6.1 and AC004540.4 expression levels were analyzed by reverse transcription quantitative polymerase chain reaction. There were 318 DE-lncRNAs and 609 DE-mRNAs identified in the NAFLD tissues compared with the normal tissues. Jun proto-oncogene, AP-1 transcription factor subunit (JUN), which is regulated by AC004540.4 and RP11-279F6.1, exhibited higher degree compared with other nodes in the PPI network. Furthermore, miR-409-3p and miR-139 (targeting JUN) were predicted as PPI network nodes. In the miRNA-lncRNA-mRNA network, miR-20a and B-cell lymphoma 2-like 11 (BCL2L11) were among the top 10 nodes. Additionally, BCL2L11, AC004540.4 and RP11-279F6.1 were targeted by miR-20a, miR-409-3p and miR-139 in the miRNA-lncRNA-mRNA network, respectively. RP11-279F6.1 and AC004540.4 expression was markedly enhanced in NAFLD liver tissues. These key RNAs may be involved in the pathogenic mechanisms of NAFLD.

Novel compound VB-037 inhibits Aβ aggregation and promotes neurite outgrowth through enhancement of HSP27 and reduction of P38 and JNK-mediated inflammation in cell models for Alzheimer's disease

The pathogenesis of Alzheimer's disease (AD) is involved in the aggregation of misfolded amyloid β (Aβ), which upregulates the activity of acetylcholinesterase (AChE), increases the production of reactive oxygen species (ROS), enhances neuroinflammation, and eventually leads to neuronal death. Therefore, compounds targeting these mechanisms may be candidates for multitarget drugs in AD treatment. We found that two quinoline derivatives, VB-030 and VB-037, markedly reduced Aβ aggregation and ROS levels in the thioflavin T biochemical assay and Tet-On Aβ-green fluorescent protein (GFP) 293 AD cell model. These compounds further improved neurite outgrowth, reduced AChE activity and upregulated the molecular chaperone heat shock protein family B [small] member 1 (HSP27), whereas knockdown of HSP27 counteracted the compounds' neuroprotective effects on the Tet-On Aβ-GFP SH-SY5Y AD neuronal model. Furthermore, VB-037 attenuated lipopolysaccharide (LPS)/interferon (IFN)-γ-induced activation of BV-2 microglial cells. In addition, VB-037 demonstrated its potential to diminish LPS/IFN-γ-induced upregulation of caspase 1 activity, expression of interleukin (IL)-1β, and active phosphorylation of mitogen-activated protein kinase 14 (P38), mitogen-activated protein kinase 8 (JNK), and Jun proto-oncogene, AP-1 transcription factor subunit (JUN) signalings, as well as improve cell viability in the Tet-On Aβ-GFP SH-SY5Y AD neuronal model. Our findings strongly indicate the potential of VB-037 for modifying AD progression by targeting multiple mechanisms, thereby offering a new drug development avenue for AD treatment.

Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, although the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G protein-coupled receptor (GPCR) homolog US28 is required for successful latent infection. We now show that US28 protein (pUS28) provided in trans complements the US28Δ lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 h. However, this repression is only maintained in the presence of continual pUS28 expression provided in trans Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this we find that US28Δ infection results in an increase in AP-1 binding to the MIEP, compared with WT latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28Δ infection to levels similar to those we observe during WT latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.

Comparative transcriptome analysis reveals regulators mediating breast muscle growth and development in three chicken breeds

Objective: The goal of this study was to investigate the mechanisms of muscle growth and development of three chicken breeds.Participants: Eighteen chickens, including three different breeds with different growth speeds (White Broiler, Daheng, and Commercial Layers of Roman), were used.Methods: Total RNA from breast muscle of these chickens was subjected to a gene expression microarray. Differentially expressed genes (DEGs) were screened and functional enrichment analysis was performed using DAVID. Seven DEGs were confirmed by quantitative reverse transcription PCR.Results: Overall, 8,398 DEGs were found among the different lines. The DEGs between each two lines that were unique for a developmental stage were greater than those that were common during all stages. Functional analysis revealed that DEGs across the entire developmental process were primarily involved in positive cell proliferation, growth, cell differentiation, and developmental processes. Genes involved in muscle regulation, muscle construction, and muscle cell differentiation were upregulated in the faster-growing breed compared to the slower-growing breed. DEGs including myosin heavy chain 15 (MYH15), myozenin 2 (MYOZ2), myosin-binding protein C (MYBPC3), insulin-like growth factor 2 (IGF2), apoptosis regulator (BCL-2), AP-1 transcription factor subunit (JUN), and AP-1 transcription factor subunit (FOS) directly regulated muscle growth or were in the center of the protein–protein interaction network. Pathways, including the extracellular matrix (ECM)-receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and focal adhesion, were the most enriched DEGs between lines or within lines under different developmental stages.Conclusions: Genes involved in muscle construction and cell differentiation were differentially expressed among the three breeds.

ITGB4 is a novel prognostic factor in colon cancer.

Integrin β4 (ITGB4) has been reported to be involved in carcinomas. Currently, ITGB4 has been characterized in colon cancer, however, its clinical significance is not very clear. In the present study, we utilized the large public datasets from NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases and collected clinical samples in our center to investigate the transcriptional expressions of ITGB4 in colon cancer, and then explored the associations of ITGB4 with clinicopathological features and overall survival. The statistical analyses suggested that ITGB4 mRNA expressions were up-regulated significantly in colon cancer. High ITGB4 expression was observed to be associated with elder onset age, proximal tumor location, and high microsatellite instability (MSH) status. Further, Kaplan-Meier curves and univariate analysis demonstrated high ITGB4 expression was significantly associated with unfavorable overall survival in colon cancer (HR=1.292, 95%CI=1.084-1.540, P=0.004). And significant association was also found after adjusting the confounding factors including age, gender, and stage (adjusted HR=1.254, 95%CI=1.050-1.497, P=0.012). The annotation of ITGB4 co-expressed genes suggested the pathways including cell growth, positive regulation of cell migration, and apoptotic signaling might be involved in the potential mechanisms of ITGB4 in colon cancer development. The molecular regulation mechanism of ITGB4 ectopic expression in colon cancer was also explored and the results indicated that ITGB4 might be up-regulated by the transcription factor FOSL1 (FOS like 1, AP-1 Transcription Factor Subunit) and its promoter hypomethylation. Our results revealed that ITGB4 might be a therapeutic target and prognosis marker for individual therapy of colon cancer.

Bioinformatics analysis to identify key genes and pathways influencing synovial inflammation in osteoarthritis.

Osteoarthritis (OA) is a chronic arthropathy that occurs in the middle-aged and elderly population. The present study aimed to identify gene signature differences between synovial cells from OA synovial membrane with and without inflammation, and to explain the potential mechanisms involved. The differentially expressed genes (DEGs) between 12 synovial membrane with inflammation and 12 synovial membrane without inflammation from the dataset GSE46750 were identified using the Gene Expression Omnibus 2R. The DEGs were subjected to enrichment analysis, protein-protein interaction (PPI) analysis and module analysis. The analysis results were compared with text-mining results. A total of 174 DEGs were identified. Gene Ontology enrichment results demonstrated that functional molecules encoded by the DEGs primarily had extracellular location, molecular functions predominantly involving ‘chemokine activity’ and ‘cytokine activity’, and were associated with biological processes, including ‘inflammatory response’ and ‘immune response’. The Kyoto Encyclopedia of Genes and Genomes results demonstrated that DEGS may function through pathways associated with ‘rheumatoid arthritis’, ‘chemokine signaling pathway’, ‘complement and coagulation cascades’, ‘TNF signaling pathway’, ‘intestinal immune networks for IgA production’, ‘cytokine-cytokine receptor interaction’, ‘allograft rejection’, ‘Toll-like receptor signaling pathway’ and ‘antigen processing and presentation’. The top 10 hub genes [interleukin (IL)6, IL8, matrix metallopeptidase (MMP)9, colony stimulating factor 1 receptor, FOS proto-oncogene, AP1 transcription factor subunit, insulin-like growth factor 1, TYRO protein tyrosine kinase binding protein, MMP3, cluster of differentiation (CD)14 and CD163] and four gene modules were identified from the PPI network using Cytoscape. In addition, text-mining was used to identify the commonly used drugs and their targets for the treatment of OA. It was initially verified whether the results of the present study were useful for the study of OA treatment targets and pathways. The present study provided insight for the molecular mechanisms of OA synovitis. The hub genes and associated pathways derived from analysis may be targets for OA treatment. IL8 and MMP9, which were validated by text-mining, may be used as molecular targets for the OA treatment, while other hub genes require further validation.

Comparison of gene expression profiles between human erythroid cells derived from fetal liver and adult peripheral blood.

BackgroundA key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease and β-thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain.MethodsWe obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+ cells were cultured in vitro in a fetal bovine serum-based culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset.ResultsFL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by meta-analysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. The in vivo relevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogen-activated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways.DiscussionThe genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression.

Vγ9Vδ2 T cells inhibit immature dendritic cell transdifferentiation into osteoclasts through downregulation of RANK, c‑Fos and ATP6V0D2.

Osteoimmunological studies have revealed that T cells exert a powerful impact on the formation and activity of osteoclasts and bone remodeling. Evidence demonstrates that immature dendritic cells (iDCs) are more efficient transdifferentiating into osteoclasts (OCs) than monocytes. However, whether Vγ9Vδ2 T (γδ T) cells stimulate or inhibit iDC transdifferentiation into OCs has never been reported. The aim of the present study was to investigate the effects of γδ T cells on this transdifferentiation process. γδ T cells and iDCs were isolated from the peripheral blood of healthy volunteers separately and were co-cultured with Transwelll inserts, with γδ T cells in the upper chamber and iDCs in the lower chamber. IDCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB (RANK) ligand. Tartrate resistant acid phosphatase (TRAP) assay and dentine resorption assay were performed to detect OC formation and their resorption capacity, respectively. The mRNA expression of OCs was examined using a micro-array and real time-quantitative polymerase chain reaction to trace the changes during iDC transdifferentiation into OCs. The results demonstrated that γδ T cells significantly inhibited the generation of the TRAP-positive OCs from iDCs and their resorption capacity. The microarray analysis identified decreased expression level of Fos proto-oncogene AP-1 transcription factor subunit (c-Fos), ATPase H+ transporting V0 subunit d (ATP6V0D2) and cathepsin K when iDCs were co-cultured with γδ T cells. These genes are associated with OC differentiation, indicating that γδ T cells suppressed iDCs osteoclastogenesis by downregulation of the RANK/c-Fos/ATP6V0D2 signaling pathway. The present findings provide novel insights into the interactions between human γδ T cells and iDCs, and demonstrate that γδ T cells are capable of inhibiting OC formation and their activity via downregulation of genes associated with OC differentiation.

Titanium particle‑mediated osteoclastogenesis may be attenuated via bidirectional ephrin‑B2/eph‑B4 signaling invitro.

The present study investigated the role of bidirectional ephrin-B2/erythropoietin-producing human hepatocellular receptor 4 (ephB4) signaling in the regulation of wear particle-mediated osteoclastogenesis in vitro. Mouse bone marrow macrophages (BMMs) were induced into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL, 50 ng/ml). EphB4-Fc, an osteoblast membrane surface receptor (4 µg/ml), was used to stimulate the ephrin-B2 ligand of osteoclasts in the presence and absence of titanium (Ti). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the number of osteoclasts, and phalloidin staining was used to examine the cytoskeletons of the osteoclasts. A bone pit absorption experiment was used to measure osteoclast function. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine osteoclastogenesis. ELISAs were used to detect the production of inflammatory factors. The data demonstrated that Ti significantly promoted the differentiation of BMMs into mature osteoclasts in the presence of RANKL and significantly promoted expression of the ephrin-B2, nuclear factor of activated T-cells 1 (NFATc1), TRAP, Fos proto-oncogene, AP-1 transcription factor subunit (C-FOS), and matrix metalloproteinase 9 (MMP9) genes. Phalloidin and TRAP staining revealed that following the addition of ephB4-Fc, the number, size and cytoskeletal elements of Key words: osteoclasts, osteoblasts, remodeling, ephrin-B2, osteoclastogenesis osteoclasts were significantly decreased compared with those in the titanium particle group without ephB4-Fc. Compared with the titanium particle group, the bone pit absorption experiment revealed significantly decreased absorption pit areas in the titanium particle+ephB4-Fc group. The expression of the NFATc1, TRAP, C-FOS and MMP9 genes was markedly decreased in the ephB4-Fc group; however, the expression of the ephrin-B2 gene was increased compared with the Ti particle group without ephB4-Fc after 5 days. Production of inflammatory cytokines was inhibited by Ti particles through bidirectional signals. Addition of ephB4-Fc inhibited the osteoclast-mediated formation of Ti particles via bidirectional ephrin-B2/ephB4 signaling. Activation of this bidirectional signaling pathway may be a potential clinical treatment for osteolysis surrounding prostheses.

Identification of potential markers of fatal hypothermia by a body temperature-dependent gene expression assay.

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice and even if findings due to cold exposure are evident, cold exposure is not necessarily a direct cause of death. Identification of useful molecular markers for the diagnosis of fatal hypothermia has not been successful. In this study, to identify novel molecular markers that inform the diagnosis of fatal hypothermia, we focused on skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We made rat models of mild, moderate, and severe hypothermia and performed body temperature-dependent gene expression analysis in the iliopsoas muscle using next-generation sequencing (NGS). NGS showed that after severe hypothermia, the expression levels of 91 mRNAs were more than double those in mild and moderate hypothermia and control animals. Gene ontology (GO) analysis indicated that these mRNAs are involved in a number of biological processes, including response to stress and lipids, and cellular response to hypoxia. The expression of four genes [connective tissue growth factor (Ctgf), JunB proto-oncogene, AP-1 transcription factor subunit (Junb), nuclear receptor subfamily 4, group A, member 1 (Nr4a1), and Syndecan 4 (Sdc4)] and the level of one protein (CTGF) were induced only by severe hypothermia. These genes and protein are involved in muscle regeneration, tissue repair, and lipid metabolism. These results indicate that heat production to maintain body temperature in a process leading to fatal hypothermia might be performed by the iliopsoas muscle, and that Ctgf, Junb, Nr4a1, and Sdc4 genes are potential diagnostic markers for fatal hypothermia.

MiR-144-3p Targets FosB Proto-oncogene, AP-1 Transcription Factor Subunit (FOSB) to Suppress Proliferation, Migration, and Invasion of PANC-1 Pancreatic Cancer Cells.

This study aimed to investigate the role of miR-144-3p in pancreatic cancer (PC) carcinogenesis and to explore the mechanism of its function in PC. miR-144-3p was downregulated in PC tissues and cells. miR-144-3p overexpression significantly inhibited PC cell proliferation, migration, and invasion. FosB proto-oncogene, AP-1 transcription factor subunit (FOSB) was a target gene of miR-144-3p. miR-144-3p could repress PC cell proliferation, migration, and invasion by inhibiting the expression of FOSB. In conclusion, miR-144-3p plays an important role in PC cell proliferation, migration, and invasion by targeting FOSB. miR-144-3p may provide a new target for the development of therapeutic agents against PC.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n= 15) and performed immunohistochemical analyses. Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre miceexposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

Endogenous authentic OCT4A proteins directly regulate FOS/AP-1 transcription in somatic cancer cells

OCT4A is well established as a master transcription factor for pluripotent stem cell (PSC) self-renewal and a pioneer factor for initiating somatic cell reprogramming, yet its presence and functionality in somatic cancer cells remain controversial and obscure. By combining the CRISPR-Cas9-based gene editing with highly specific PCR assays, highly sensitive immunoassays, and mass spectrometry, we provide unequivocal evidence here that full-length authentic OCT4A transcripts and proteins were both present in somatic cancer cells, and OCT4A proteins were heterogeneously expressed in the whole cell population and when expressed, they are predominantly localized in cell nucleus. Despite their extremely low abundance (approximately three orders of magnitude lower than in PSCs), OCT4A proteins bound to the promoter/enhancer regions of the AP-1 transcription factor subunit c-FOS gene and critically regulated its transcription. Knocking out OCT4A in somatic cancer cells led to dramatic reduction of the c-FOS protein level, aberrant AP-1 signaling, dampened self-renewal capacity, deficient cell migration that were associated with cell growth retardation in vitro and in vivo, and their enhanced sensitivity to anticancer drugs. Taken together, we resolve the long-standing controversy and uncertainty in the field, and reveal a fundamental role of OCT4A protein in regulating FOS/AP-1 signaling-centered genes that mediate the adhesion, migration, and propagation of somatic cancer cells.

A gene interaction network‑based method to measure the common and heterogeneous mechanisms of gynecological cancer.

Gynecological malignancies are a leading cause of mortality in the female population. The present study intended to identify the association between three severe types of gynecological cancer, specifically ovarian cancer, cervical cancer and endometrial cancer, and to identify the connective driver genes, microRNAs (miRNAs) and biological processes associated with these types of gynecological cancer. In the present study, individual driver genes for each type of cancer were identified using integrated analysis of multiple microarray data. Gene Ontology (GO) has been used widely in functional annotation and enrichment analysis. In the present study, GO enrichment analysis revealed a number of common biological processes involved in gynecological cancer, including ‘cell cycle’ and ‘regulation of macromolecule metabolism’. Kyoto Encyclopedia of Genes and Genomes pathway analysis is a resource for understanding the high-level functions and utilities of a biological system from molecular-level information. In the present study, the most common pathway was ‘cell cycle’. A protein-protein interaction network was constructed to identify a hub of connective genes, including minichromosome maintenance complex component 2 (MCM2), matrix metalloproteinase 2 (MMP2), collagen type I α1 chain (COL1A1) and Jun proto-oncogene AP-1 transcription factor subunit (JUN). Survival analysis revealed that the expression of MCM2, MMP2, COL1A1 and JUN was associated with the prognosis of the aforementioned gynecological cancer types. By constructing an miRNA-driver gene network, let-7 targeted the majority of the driver genes. In conclusion, the present study demonstrated a connection model across three types of gynecological cancer, which was useful in identifying potential diagnostic markers and novel therapeutic targets, in addition to in aiding the prediction of the development of cancer as it progresses.

Dysregulated genes targeted by microRNAs and metabolic pathways in bladder cancer revealed by bioinformatics methods.

The present study aimed to identify bladder cancer-associated microRNAs (miRNAs) and target genes, and further analyze the potential molecular mechanisms involved in bladder cancer. The mRNA and miRNA expression profiling dataset GSE40355 was downloaded from the Gene Expression Omnibus database. The Limma package in R was used to identify differential expression levels. The Human microRNA Disease Database was used to identify bladder cancer-associated miRNAs and Target prediction programs were used to screen for miRNA target genes. Enrichment analysis was performed to identify biological functions. The Database for Annotation, Visualization and Integration Discovery was used to perform OMIM_DISEASE analysis, and then protein-protein interaction (PPI) analysis was performed to identify hubs with biological essentiality. ClusterONE plugins in cytoscape were used to screen modules and the InterPro database was used to perform protein domain enrichment analysis. A group of 573 disease dysregulated genes were identified in the present study. Enrichment analysis indicated that the muscle organ development and vascular smooth muscle contraction pathways were significantly enriched in terms of disease dysregulated genes. miRNAs targets (frizzled class receptor 8, EYA transcriptional coactivator and phosphatase 4, sacsin molecular chaperone, calcium voltage-gated channel auxiliary subunit β2, peptidase inhibitor 15 and catenin α2) were mostly associated with bladder cancer. PPI analysis revealed that calmodulin 1 (CALM1), Jun proto-oncogene, AP-1 transcription factor subunit (JUN) and insulin like growth factor 1 (IGF1) were the important hub nodes. Additionally, protein domain enrichment analysis indicated that the serine/threonine protein kinase active site was enriched in module 1 extracted from the PPI network. Overall, the results suggested that the IGF signaling pathway and RAS/MEK/extracellular signal-regulated kinase transduction signaling may exert vital molecular mechanisms in bladder cancer, and that CALM1, JUN and IGF1 may be used as novel potential therapeutic targets.

NOS1 mediates AP1 nuclear translocation and inflammatory response

A hallmark of the AP1 functioning is its nuclear translocation, which induces proinflammatory cytokine expression and hence the inflammatory response. After endotoxin shock AP1 transcription factor, which comprises Jun, ATF2, and Fos family of proteins, translocates into the nucleus and induces proinflammatory cytokine expression. In the current study, we found, NOS1 inhibition prevents nuclear translocation of the AP1 transcription factor subunits. Pharmacological inhibition of NOS1 impedes translocation of subunits into the nucleus, suppressing the transcription of inflammatory genes causing a diminished inflammatory response. In conclusion, the study shows the novel mechanism of NOS1- mediated AP1 nuclear translocation, which needs to be further explored.

MicroRNA hsa-let-7e-5p as a potential prognosis marker for rectal carcinoma with liver metastases.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that target mRNAs for translational repression or cleavage. The present study was conducted to identify differentially expressed miRNAs in primary tumor tissues of rectal carcinoma (RC) that may be associated with heterochrony hepatic metastasis (HHM). Samples were collected exclusively from patients with RC but not colon cancer (CC); Next-generation high-throughput sequencing technology and bioinformatics tools were used to profile and analyze small RNAs and their corresponding targets in primary tumor tissues with HHM (n=2) or without metastases (non-metastatic, NM; n=2). A total of 24 known miRNAs were identified to be differentially expressed (P<0.01; absolute value of log2-fold change ≥1). Hsa-let-7e-5p exhibited the most significant elevation in tissues with HHM (log2-fold change=2.62). By combining online informatics resources and previous mRNA sequencing data, it was identified that 54 validated target genes of let-7e were downregulated in primary tumor tissues with HHM. A number of these target genes have been demonstrated to be directly involved in tumor metastasis (including MYC proto-oncogene, bHLH transcription factor, high-mobility group AT-Hook 2, peptidase inhibitor 3, KIT proto-oncogene receptor tyrosine kinase, Jun proto-oncogene, AP-1 transcription factor subunit and ribonuclease T2), or have physiological associations to immunity (including C-C motif chemokine receptor 4 and cluster of differentiation 40 ligand) and cellular metabolism (including peroxisome proliferator-activated receptor γ, coactivator 1 α). Next, 14 target genes were selected for reverse transcription-quantitative polymerase chain reaction analysis in non-sequenced samples, and the downregulation of 10 target genes in RC samples with HHM was confirmed. In addition, it was demonstrated that hsa-let-7e-5p stimulated colorectal cancer cell migration in vitro. The miRNA hsa-let-7e-5p may serve as a potential biomarker for rectal carcinoma-associated HHM, facilitating the identification of patients with RC who are at risk of developing HHM.

Heterodimers of the transcriptional factors NFATc3 and FosB mediate tissue factor expression for 15(S)-hydroxyeicosatetraenoic acid–induced monocyte trafficking

Tissue factor (TF) is expressed in vascular and nonvascular tissues and functions in several pathways, including embryonic development, inflammation, and cell migration. Many risk factors for atherosclerosis, including hypertension, diabetes, obesity, and smoking, increase TF expression. To better understand the TF-related mechanisms in atherosclerosis, here we investigated the role of 12/15-lipoxygenase (12/15-LOX) in TF expression. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major product of human 15-LOXs 1 and 2, induced TF expression and activity in a time-dependent manner in the human monocytic cell line THP1. Moreover, TF suppression with neutralizing antibodies blocked 15(S)-HETE-induced monocyte migration. We also found that NADPH- and xanthine oxidase-dependent reactive oxygen species (ROS) production, calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation, and interactions between nuclear factor of activated T cells 3 (NFATc3) and FosB proto-oncogene, AP-1 transcription factor subunit (FosB) are involved in 15(S)-HETE-induced TF expression. Interestingly, NFATc3 first induced the expression of its interaction partner FosB before forming the heterodimeric NFATc3-FosB transcription factor complex, which bound the proximal AP-1 site in the TF gene promoter and activated TF expression. We also observed that macrophages from 12/15-LOX-/- mice exhibit diminished migratory response to monocyte chemotactic protein 1 (MCP-1) and lipopolysaccharide compared with WT mouse macrophages. Similarly, compared with WT macrophages, monocytes from 12/15-LOX-/- mice displayed diminished trafficking, which was rescued by prior treatment with 12(S)-HETE, in a peritonitis model. These observations indicate that 15(S)-HETE-induced monocyte/macrophage migration and trafficking require ROS-mediated CaMKIV activation leading to formation of NFATc3 and FosB heterodimer, which binds and activates the TF promoter.

Low expression of microRNA-1908 predicts a poor prognosis for patients with ovarian cancer.

MicroRNAs (miRs) serve important roles in cancer genesis and progression. The expression of miR-1908 has been reported in a number of types of cancer; however, the clinical significance of miR-1908 in human ovarian cancer (OC) remains unclear. A total of 491 patients with OC from The Cancer Genome Atlas project cohort were selected and divided into two groups according to the median expression level of miR-1908. Univariate and multivariate analyses, using the Kaplan-Meier method and Cox regression, were performed to identify the characteristics that predict OC prognosis. Bioinformatics tools were used to identify potential targets of miR-1908. It was identified that the low expression of miR-1908 is associated with a poor prognosis for OC (P<0.05). The potential target genes of miR-1908 included podocan-like 1, JunB AP-1 transcription factor subunit, homeobox B8, SET binding factor 1 and sirtuin 2; high expression of these five genes additionally predicted a poor prognosis. These results suggest that miR-1908 may be a suitable target for the development of novel approaches in OC diagnosis and therapy in the future.