Transcription Factor Response Elements Research Articles

Hepa-ToxMOA: a pathway-screening method for evaluating cellular stress and hepatic metabolic-dependent toxicity of natural products.

In the field of drug discovery, natural products have emerged as therapeutic agents for diseases such as cancer. However, their potential toxicity poses significant obstacles in the developing effective drug candidates. To overcome this limitation, we propose a pathway-screening method based on imaging analysis to evaluate cellular stress caused by natural products. We have established a cellular stress sensing system, named Hepa-ToxMOA, which utilizes HepG2 cells expressing green fluorescent protein (GFP) fluorescence under the control of transcription factor response elements (TREs) for transcription factors (AP1, P53, Nrf2, and NF-κB). Additionally, to augment the drug metabolic activity of the HepG2 cell line, we evaluated the cytotoxicity of 40 natural products with and without S9 fraction-based metabolic activity. Our finding revealed different activities of Hepa-ToxMOA depending on metabolic or non-metabolic activity, highlighting the involvement of specific cellular stress pathways. Our results suggest that developing a Hepa-ToxMOA system based on activity of drug metabolizing enzyme provides crucial insights into the molecular mechanisms initiating cellular stress during liver toxicity screening for natural products. The pathway-screening method addresses challenges related to the potential toxicity of natural products, advancing their translation into viable therapeutic agents.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Osteoprotegerin Gene Polymorphisms Are Associated with Subclinical Atherosclerosis in the Mexican Mestizo Population.

Subclinical atherosclerosis (SA) is the presence of coronary calcification in the absence of cardiovascular symptoms, and it usually progresses to atherosclerotic disease. Studies have shown an association of osteoprotegerin gene (OPG) variants with calcification process in cardiovascular diseases; however, to this day there are no studies that evaluate individuals in the asymptomatic stage of atherosclerotic disease. Therefore, the purpose of this study was to analyze the association of four genetic variants and haplotypes of the OPG gene with the development of SA, through TaqMan genotyping assays. We also aimed to identify potential response elements for transcription factors in these genetic variants. The study included 1413 asymptomatic participants (1041 were controls and 372 were individuals with SA). The rs3102735 polymorphism appeared as a protective marker (OR = 0.693; 95% CI = 0.493–0.974; pheterozygote = 0.035; OR = 0.699; 95% CI = 0.496–0.985; pcodominant 1 = 0.040) and two haplotypes were associated with SA, one as a decreased risk: GACC (OR = 0.641, 95% CI = 0.414–0.990, p = 0.045) and another as an increased risk: GACT (OR = 1.208, 95% CI = 1.020–1.431, p = 0.029). Our data suggest a lower risk of SA in rs3102735 C carriers in a representative sample of Mexican mestizo population.

Proteome-Wide Profiling of Readers for DNA Modification.

DNA modifications, represented by 5‐methylcytosine (5mC), 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxylcytosine (5caC), play important roles in epigenetic regulation of biological processes. The specific recognition of DNA modifications by the transcriptional protein machinery is thought to be a potential mechanism for epigenetic‐driven gene regulation, and many modified DNA‐specific binding proteins have been uncovered. However, the panoramic view of the roles of DNA modification readers at the proteome level remains largely unclear. Here, a recently developed concatenated tandem array of consensus transcription factor (TF) response elements (catTFREs) approach is employed to profile the binding activity of TFs at DNA modifications. Modified DNA‐binding activity is quantified for 1039 TFs, representing 70% of the TFs in the human genome. Additionally, the modified DNA‐binding activity of 600 TFs is monitored during the mouse brain development from the embryo to the adult stages. Readers of these DNA modifications are predicted, and the hierarchical networks between the transcriptional protein machinery and modified DNA are described. It is further demonstrated that ZNF24 and ZSCAN21 are potential readers of 5fC‐modified DNA. This study provides a landscape of TF–DNA modification interactions that can be used to elucidate the epigenetic‐related transcriptional regulation mechanisms under physiological conditions.

Establishment of a New-generation High-throughput Proteomic Profiling of Transcription Factor in Human Atrial Tissue

To explore for the establishment of an experimental technique for profiling transcription factors, namely transcription factor response elements (TFRE), with high throughput and efficiency using human atrial tissue. Postoperative right atrial tissues from 2 patients, one with preoperative atrial fibrillation and the one with no preoperative atrial fibrillation, were included in the study. The nucleus protein was extracted from the human atrial tissue, and the protein concentration was then measured. A solution with a complex formed through combining magnetic beads with concatenated tandem array of the consensus transcription factor response element DNA sequence (beads-catTFRE) was prepared, and the beads-catTFREs were then used to enrich transcription factors in the nucleoprotein extraction. SDS-PAGE electrophoresis was performed after dissociating beads-catTFRE from nucleoprotein with high temperature and high salt. The gel was then cut and faded before enzymolysis by trypsin in the gels was performed. Acetonitrile was used to extract the peptides from the gels, and the peptide solution was then dried. After that, we dissolved the peptides and performed mass spectrum tests, and the data were analyzed and processed with Firmiana one-stop proteomic analysis platform. In this study, 220 and 181 transcription factors were identified in the normal right atrial tissue and the right atrial tissue with atrial fibrillation, respectively. A total of 241 transcription factors were identified in the two groups. Among the 241 transcription factors, 12 were in the top 10% of those transcription factors that were above the median expression level of the normal right atrial tissue, and 12 transcription factors were in the top 10% of those above the median expression level of the right atrial tissue with atrial fibrillation. The high-throughput profiling method established in this study has high coverage, and the data collected can be used to support further validation studies.

Systematical Identification of the Protective Effect of Danhong Injection and BuChang NaoXinTong Capsules on Transcription Factors in Cerebral Ischemia Mice Brain.

Cerebral ischemia has led to a high rate of both disability and mortality with massive healthcare costs. Although transcriptional regulation is typically mediated by different combinations of TFs, a combined regulatory unit to synergistically activate transcription has remained unclear in cerebral ischemia, especially in different drug treatments. In this study, TFs alterations after 6 h cerebral ischemic injury and repair were performed by a concatenated tandem array of consensus transcription factor response elements (catTFREs), and vital TFs were obtained by TFs-target imbalanced network. Drug intervention used Danhong injection (DHI) and BNC (BuChang NaoXinTong Capsules), which has been widely prescribed in Chinese herb medicine for the treatment of cerebrovascular and cardiovascular diseases. There were 198 TFs identified after 6 h MCAO operation, and six TFs (Sox2, Smad3, FoxO1, Creb1, Egr,1 and Smad4) were considered as critical TFs in response to cerebral ischemia. Moreover, Smad3 was identified as a hub TF among six vital TFs, and the transcription activity of Smad3 was further verified. These 6 TFs were all reversed by DHI or BNC, indicating different medications may regulate different transcription factors through TF synergy. Moreover, validation results indicated that Smad3 was a putative target TF for DHI and BNC-mediated protection against cerebral ischemia. The observations of the present study provide a fresh understanding of biomolecules and possible new avenues for therapeutic interventions, in addition to the new intervention pattern for different treatments for ischemia stroke.

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Docosahexaenoic acid (DHA, 22:6n-3) and oleic acid (18:1n-9) can alter the DNA methylation of individual CpG loci in vivo and in vitro, although the targeting mechanism is unknown. We tested the hypothesis that the targeting of altered methylation is associated with putative transcription factor response elements (pTREs) proximal to modified loci. Jurkat cells were treated with 22:6n-3 or 18:1n-9 (both 15 μM) for eight days and DNA methylation measured using the MethylationEPIC 850K array. 1596 CpG loci were altered significantly (508 hypermethylated) by 22:6n-3 and 563 CpG loci (294 hypermethylated) by 18:1n-9. 78 loci were modified by both fatty acids. Induced differential methylation was not modified by the PPARα antagonist GW6471. DNA sequences proximal to differentially methylated CpG loci were enriched in zinc-finger pTREs. These findings suggest that zinc-finger-containing transcription factors may be involved in targeting altered DNA methylation modifying processes induced by fatty acids to individual CpG loci.

Docosahexaenoic acid and oleic acid induce altered DNA methylation of individual CpG loci in Jurkat T cells

Docosahexaenoic acid (DHA, 22:6n-3) and oleic acid (18:1n-9) can alter the DNA methylation of individual CpG loci in vivo and in vitro, although the targeting mechanism is unknown. We tested the hypothesis that the targeting of altered methylation is associated with putative transcription factor response elements (pTREs) proximal to modified loci. Jurkat cells were treated with 22:6n-3 or 18:1n-9 (both 15 μM) for eight days and DNA methylation measured using the MethylationEPIC 850K array. 1596 CpG loci were altered significantly (508 hypermethylated) by 22:6n-3 and 563 CpG loci (294 hypermethylated) by 18:1n-9. 78 loci were modified by both fatty acids. Induced differential methylation was not modified by the PPARα antagonist GW6471. DNA sequences proximal to differentially methylated CpG loci were enriched in zinc-finger pTREs. These findings suggest that zinc-finger-containing transcription factors may be involved in targeting altered DNA methylation modifying processes induced by fatty acids to individual CpG loci.

Tokoronin Contained in Dioscorea tokoro Makino ex Miyabe Suppressed α-MSH-Induced Melanogenesis in B16 Cells via Suppression of Classical MAPK Pathway Activation.

In this study, melanogenesis inhibition in B16 cells by eight compounds, namely, tokorogenin, tokoronin, yononin, gracillin, proto-yonogenin, proto-tokoronin, proto-yononin, and proto-gracillin, isolated from Dioscorea tokoro Makino ex Miyabe were evaluated. The results of the cytotoxicity and α-MSH-induced melanogenesis inhibition effects of the eight compounds revealed that tokoronin was the most effective in terms of low-cytotoxicity and melanogenesis inhibition. Tokoronin downregulated α-MSH-induced melanogenesis via suppression of the expression of the three types of melanogenesis-related enzymes [tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2] by the inhibition of phospho-microphthalmia-associated transcription factor (p-MITF) and cAMP response element binding protein (CREB) levels. p-MITF and CREB are regulated by various kinases [Akt, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK)]. As the results of measurement of the combined effects of tokoronin with inhibitors or promoters of these kinases, no change in the biological activity of tokoronin by Akt inhibitor (wortmannin) or p38 MAPK inhibitor (SB202190) was observed, however, the effect of tokoronin was reduced by the MEK/ERK inhibitor (U0126) and promoted by the MEK/ERK activator (FGF2). Therefore, it was deduced that tokoronin first inactivated ERK; then, it suppressed p-MITF and CREB levels; and finally, α-MSH-induced melanogenesis was suppressed.

Daily interpersonal stress, sleep duration, and gene regulation during late adolescence

BackgroundPsychological stress and poor sleep are associated with a wide range of negative health outcomes, which are thought to be mediated in part by alterations in immune processes. However, the molecular bases of links among stress, sleep, and immune processes are not completely understood, particularly during adolescence when sensitivity to stress and problems with sleep tend to increase. In the current study, we investigated whether various stressors (daily stress, major life events, perceived stress), sleep indices (duration, efficiency), and their interactions (e.g., moderating effects) are associated with expression of genes bearing response elements for transcription factors that regulate inflammatory and anti-viral processes. MethodEighty-seven late adolescents completed daily checklists of their social experiences across a 15-day period and reported on their major life events during the previous year. They also completed actigraphy-based assessments of sleep quality and duration during 8 consecutive nights. An average of 5.5 months later, participants reported on their global perceptions of stress during the previous month and provided blood samples for genome-wide expression profiling of mRNA from peripheral blood mononuclear cells (PBMCs). ResultsHigher levels of daily interpersonal stress and shorter sleep duration were associated with upregulation of inflammation-related genes bearing response elements for proinflammatory transcription factor nuclear factor kappa B (NF-κB). Shorter sleep duration was also linked to downregulation of antiviral-related genes bearing response elements for interferon response factors (IRFs). Lastly, there was a significant interaction between daily stress and shorter sleep duration, such that the association between daily stress and inflammation-related gene expression was exacerbated in the context of shorter sleep duration. Results were independent of sex, ethnicity, parent education, body mass index, and smoking and alcohol history. ConclusionEveryday interpersonal stress and shortened sleep can be consequential for upstream NF-κB signaling pathways relevant to inflammatory processes during late adolescence. Notably, the occurrence of both may lead to even greater activation of NF-κB signaling.

De novo construction of mammalian synthetic inhibitory transcription factor and promoter pairs

Transcriptional regulation is crucial for regulated gene expression. Due to the complexity, it has been difficult to engineer eukaryotic transcription factor (TF) and promoter pairs. The few availabilities of eukaryotic TF and promotor pairs limit their practical use for clinical or industrial applications. Here, we report a de novo construction of synthetic inhibitory transcription factor and promoter pairs for mammalian transcriptional regulation. The design of synthetic TF was based on the fusion of DNA binding domain and Kruppel associated box transcription regulating domain (KRAB). The synthetic promoter was constructed by inserting the corresponding TF response element after SV40 promoter. We constructed and tested five synthetic inhibitory transcription factor and promoter pairs in cultured mammalian cells. The inhibition capability and orthogonality were verified by flow cytometry. In summary, we demonstrate the feasibility of constructing mammalian inhibitory TF and promoter pairs, which could be standardized for advanced gene-circuit design and various applications in the mammalian synthetic biology.

Association of high 5-hydroxymethylcytosine levels with Ten Eleven Translocation 2 overexpression and inflammation in Sjögren's syndrome patients

Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögren's syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögren's syndrome etiopathogenesis.

Analysis of transcriptional factors expression profile during dedifferentiation of liver cell cultured in vitro

Primary hepatocytes are widely used in drug metabolism and toxicity assessment. As the culture of primary hepatocytes in vitro is a process of dedifferentiation, hepatocytes lose normal metabolic detoxification function gradually. The mechanism of hepatocyte dedifferentiation has been not clear so far. TFs play an important role in the dedifferentiation and non-parenchymal cells can maintain the function of hepatocytes in vitro. However, the current methods cannot be used in effective identification and quantitative analysis of a large number of TFs. In this paper, the mo-culture system (only primary hepatocytes) and co-culture system (primary hepatocytes and non-parenchymal cells) were established. The cells were cultured for 24 h, 48 h, 72 h as monolayer. The changes of TFs during the culture were obtained by TOT (Transcription factor response elements on tip) transcription factor enrichment method and mass spectrometry. A total of 219 TFs were identified in three individual replicates. The result revealed that up-regulated TFs were enriched in cell proliferation, death and immune response pathways, and down-regulated TFs were involved in metabolism pathway. The establishment of such culture-TFs identification system is of great significance to reveal the mechanism of primary hepatocyte dedifferentiation and crosstalk between hepatocytes and non-parenchymal cells.

Parkinson's disease genetic risk in a midbrain neuronal cell line

In genome-wide association studies of complex diseases, many risk polymorphisms are found to lie in non-coding DNA and likely confer risk through allele-dependent differences in gene regulatory elements. However, because distal regulatory elements can alter gene expression at various distances on linear DNA, the identity of relevant genes is unknown for most risk loci. In Parkinson's disease, at least some genetic risk is likely intrinsic to a neuronal subpopulation of cells in the brain regions affected. In order to compare neuron-relevant methods of pairing risk polymorphisms to target genes as well as to further characterize a single-cell model of a neurodegenerative disease, we used the portionally-dopaminergic, neuronal, mesencephalic-derived cell line LUHMES to dissect differentiation-specific mechanisms of gene expression. We compared genome-wide gene expression in undifferentiated and differentiated cells with genome-wide histone H3K27ac and CTCF-bound regions. Whereas promoters and CTCF binding were largely consistent between differentiated and undifferentiated cells, enhancers were mostly unique. We matched the differentiation-specific appearance or disappearance of enhancers with changes in gene expression and identified 22,057 enhancers paired with 6388 differentially expressed genes by proximity. These enhancers are enriched with at least 13 transcription factor response elements, driving a cluster of genes involved in neurogenesis. We show that differentiated LUHMES cells, but not undifferentiated cells, show enrichment for PD-risk SNPs. Candidate genes for these loci are largely unrelated, though a subset is linked to synaptic vesicle cycling and transport, implying that PD-related disruption of these pathways is intrinsic to dopaminergic neurons.

An integrated approach to identify critical transcription factors in the protection against hydrogen peroxide-induced oxidative stress by Danhong injection

Oxidative stress plays a vital role in many pathological processes of the cardiovascular diseases. However, the underlying mechanism remains unclear, especially on a transcription factor (TF) level. In this study, a new method, concatenated tandem array of consensus transcription factor response elements (catTFREs), and an Illumina-based RNA-seq technology were integrated to systematically investigate the role of TFs in hydrogen peroxide (H2O2)-induced oxidative stress in cardiomyocytes; the damage was then rescued by Danhong injection (DHI), a Chinese standardized product approved for cardiovascular diseases treatment. The overall gene expression revealed cell apoptosis and DNA repair were vital for cardiomyocytes in resisting oxidative stress. By comprehensively integrating the transcription activity of TFs and their downstream target genes, an important TFs-target network were constructed and 13 TFs were identified as critical TFs in DHI-mediated protection in H2O2-induced oxidative stress. By using the integrated approach, seven TFs of these 13 TFs were also identified in melatonin-mediated protection in H2O2-induced damage. Furthermore, the transcription activity of DNA-(apurinic or apyrimidinic site) lyase (Apex1), Myocyte-specific enhancer factor 2D (Mef2d) and Pre B-cell leukemia transcription factor 3 (Pbx3) was further verified in pluripotent stem cell-derived cardiomyocytes. This research offers a new understanding of cardiomyocytes in response to H2O2-induced oxidative stress and reveals additional potential therapeutic targets. The combination of two parallel omics datasets (corresponding to the transcriptome and proteome) can reduce the noise in high-throughput data and reveal the fundamental changes of the biological process, making it suitable and reliable for investigation of critical targets in many other complicated pathological processes.

Family environments and leukocyte transcriptome indicators of a proinflammatory phenotype in children and parents.

High conflict and low warmth in families may contribute to immune cells developing a tendency to respond to threats with exaggerated inflammation that is insensitive to inhibitory signaling. We tested associations between family environments and expression of genes bearing response elements for transcription factors that regulate inflammation: nuclear factor kappa B (NF-κB) and glucocorticoid receptor. The overall sample (47 families) completed interviews, questionnaires, and 8-week daily diary assessments of conflict and warmth, which were used to create composite family conflict and warmth scores. The diaries assessed upper respiratory infection (URI) symptoms, and URI episodes were clinically verified. Leukocyte RNA was extracted from whole blood samples provided by a subsample of 42 children (8-13 years of age) and 73 parents. In children, higher conflict and lower warmth were related to greater expression of genes bearing response elements for the proinflammatory transcription factor NF-κB, and more severe URI symptoms. In parents, higher conflict and lower warmth were also related to greater NF-κB-associated gene expression. Monocytes and dendritic cells were implicated as primary cellular sources of differential gene expression in the sample. Consistent with existing conceptual frameworks, stressful family environments were related to a proinflammatory phenotype at the level of the circulating leukocyte transcriptome.

Polymorphisms in CARS are associated with gastric cancer risk: a two-stage case-control study in the Chinese population.

The cysteinyl transfer RNA synthetase gene (CARS) is located on chromosome band 11p15.5, which is an important tumor-suppressor gene region. Mutations in CARS have been identified in many kinds of cancers; however, evidence for a relationship between genetic variants in CARS and gastric cancer at the population level is still lacking. Thus, we explored the association of variants in CARS with gastric cancer using a two-stage case-control strategy in Chinese. We undertook a two-stage case-control study to investigate the association between polymorphisms in CARS and risk of gastric cancer with use of an Illumina Infinium® BeadChip and an ABI 7900 system. Four single nucleotide polymorphisms (SNPs) were significantly associated with gastric cancer risk in both the discovery stage and the validation stage after adjustment for age and sex. In addition, the combined results of the two stages showed these SNPs were related to gastric cancer risk (P falsediscoveryrate≤0.001 for rs384,490, rs729662, rs2071101, and rs7394702). In silico analyses revealed that rs384490 and rs7394702 could affect transcription factor response elements or DNA methylation of CARS, and rs729662 was associated with the prognosis of gastric cancer. Additionally, expression quantitative trait loci analysis showed rs384490 and rs729662 might alter expression of CARS-related genes. The potential functional SNPs in CARS might influence the biological functions of CARS or CARS-related genes and ultimately modify the occurrence and development of gastric cancer in Chinese. Further large-scale population-based studies or biological functional assays are warranted to validate our findings.

Transcription Factor Response Elements on Tip: A Sensitive Approach for Large-Scale Endogenous Transcription Factor Quantitative Identification.

The ability to map endogenous transcription factors (TFs) DNA binding activity at the proteome scale will greatly enhance our understanding of various biological processes. Here we report a highly sensitive, rapid, and high-throughput approach, transcription factor response elements on tip-mass spectrometry (TOT-MS), that allows for quantitative measurement of endogenous TFs. A total of 150 TFs from 1 μg of nuclear extracts can be quantified with single shot mass spectrometry detection in 1 h of machine time. Up to 755 TFs, which is comparable to the depth of RNA-seq, were identified by TOT coupled with on-tip small size reverse-phase liquid chromatography. We further demonstrated the capability of TOT-MS by interrogating the dynamic change of TFs in the epidermal growth factor (EGF) signaling pathway. This approach should find broad applications in elucidating the TF landscape from limited amounts of biological materials.

Systematic investigation of transcription factors critical in the protection against cerebral ischemia by Danhong injection.

Systematic investigations of complex pathological cascades during ischemic brain injury help to elucidate novel therapeutic targets against cerebral ischemia. Although some transcription factors (TFs) involved in cerebral ischemia, systematic surveys of their changes during ischemic brain injury have not been reported. Moreover, some multi-target agents effectively protected against ischemic stroke, but their mechanisms, especially the targets of TFs, are still unclear. Therefore, a comprehensive approach by integrating network pharmacology strategy and a new concatenated tandem array of consensus transcription factor response elements method to systematically investigate the target TFs critical in the protection against cerebral ischemia by a medication was first reported, and then applied to a multi-target drug, Danhong injection (DHI). High-throughput nature and depth of coverage, as well as high quantitative accuracy of the developed approach, make it more suitable for analyzing such multi-target agents. Results indicated that pre-B-cell leukemia transcription factor 1 and cyclic AMP-dependent transcription factor 1, along with six other TFs, are putative target TFs for DHI-mediated protection against cerebral ischemia. This study provides, for the first time, a systematic investigation of the target TFs critical to DHI-mediated protection against cerebral ischemia, as well as reveals more potential therapeutic targets for ischemic stroke.

Abstract 81: RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines

Abstract We previously observed aberrant activation of NF-kB in head and neck squamous cell carcinoma (HNSCC), however, the signaling constitutively activating NF-kB in solid cancers has not been well defined. Recently, The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-kB, and death pathways. These genetic alterations include amplification of FADD and BIRC2/3, mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatics analyses and protein structural and interactive tools, we identified ∼60 proteins that potentially interact with these genetically altered molecules. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, of which FADD amplification and/or overexpression ranked first with 37%. We searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for these alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed RNA-seq and exome DNA-seq in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. Among the top 30 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns for ∼57% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-kB transcription factor response elements upstream of the α-lactamase reporter gene (Gene BLAzer). Using these NF-kB reporter cell lines, genome-wide RNAi screening assays have been performed and the regulatory and signaling molecules involved in NF-κB and death pathways in responding to TNF-α have been identified. We further validated the screening results by knockdown of 35 genes individually using two siRNAs for each genes, and found that knockdown of 16 genes significantly modulated TNF-α and Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival. We observed that TNF-α and LTβ cross-activate classical and alternative NF-κB pathways and regulate key molecules that serve differential roles in proliferation, survival and migration in HNSCC. Our data help define key molecules modulating aberrant classical and alternative NF-kB activation, as candidate molecular biomarkers for diagnosis and prognosis and targets for further preclinical and clinical investigation in HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, 74; and NCI/NIH, under contract number HHSN261200800001E). Citation Format: Hui Cheng, Xinping Yang, Anthony Saleh, Shaleeka Cornelius, Carter Van Waes, Zhong Chen, Emine Guven-Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov. RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 81.