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Type 2 diabetes (T2D) is known as an inflammatory disease. NRF2 (Nuclear Factor Erythroid 2 Like2) encodes a transcription factor that binds to antioxidant response elements (AREs) and regulates the expression of genes involved in many antioxidant responses. This study aimed to gain insight into individual anti-inflammatory activity to prevent T2D development in humans. We performed a genome-wide association study (GWAS) to identify genetic variants influencing NRF2 expression in LCLs (lymphoblastoid cell lines) generated from 74 different individuals. Association analyses between T2D or its related traits and genetic risk score (GRS) calculated by combining genetic variants detected from GWAS for cellular NRF2 expression were performed using data from 8715 subjects. The T2D prediction model using GRS was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristics (ROC) curve. Our GWAS identified six genetic variants (SNP) showing suggestive evidence of associations with cellular NRF2 expression (P

BACKGROUND Relaxation of prostate smooth muscle tone is a key strategy for the medical treatment of lower urinary tract symptoms (LUTS) in men. However, potassium channel's physiological role inhuman prostatic smooth muscle (HPrSM) has yet to be determined. OBJECTIVES In this study, we examined the molecular characteristics and physiological roles of Kv7 channels in HPrSM. MATERIALS AND METHODS The expressions of KCNQ1-5 (Kv7 channel pore-forming α-subunits) and KCNE1-5 (β-regulatory subunits) isoforms in HPrSM were examined using real-time PCR. The relaxation effect of ML213 was investigated by cumulatively adding ML213 to the prostate strips. Kv7 currents were recorded using an amphotericin-B perforated patch-clamp technique. RESULTS In HPrSM cells, KCNQ4, KCNQ5, and KCNE4 isoforms were predominantly expressed, while KCNQ1, KCNQ5, and KCNE3 isoforms were the most abundantly expressed in human prostatic tissues. Western blot analysis revealed the protein expression of the Kv7.1, 7.4, and 7.5 channel subtypes in human prostate tissues (n = 3). ML213 (an activator of Kv7.2/7.4/7.5) induced the concentration-dependent relaxation of HPrSM strips (n = 15, p = 0.001), and this effect was attenuated by pretreatment with XE991 (a Kv7.1-7.5 blocker). In electrophysiology studies, ML213 significantly increased the amplitude of whole-cell Kv7 currents in HPrSM cells. ML213-induced outward currents were greater than retigabine (a Kv7.2-7.5 channel activator). The subsequent addition of XE991 completely inhibited the ML213-induced currents (n = 9, p

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory–secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory–secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory–secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory–secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory–secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory–secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma.

Copper(II) complexes bearing nonsteroidal anti-inflammatory drugs (NSAIDs) are known to potently kill cancer stem cells (CSCs), a subpopulation of tumour cells with high metastatic and relapse fidelity. One of the major disadvantages associated to these copper(II) complexes is their instability in the presence of strong cellular reductants (such as ascorbic acid). Here we present a biologically stable copper(II)-NSAID complex containing a bathocuproinedisulfonic acid disodium ligand and two indomethacin moieties, Cu(bathocuproinedisulfonic acid disodium)(indomethacin)2, 2. The copper(II) complex, 2 kills bulk breast cancer cells and breast CSC equally (in the sub-micromolar range) and displays very low toxicity against non-tumorigenic breast and kidney cells (IC50 value > 100 µM). Three-dimensional cell culture studies show that 2 can significantly reduce the number and size of breast CSC mammospheres formed (from single suspensions) to a similar level as salinomycin (an established anti-breast CSC agent). The copper(II) complex, 2 is taken up reasonably by breast CSCs and localises largely in the cytoplasm (>90%). Cytotoxicity studies in the presence of specific inhibitors suggest that 2 induces CSC death via a reactive oxygen species (ROS) and cyclooxygenase isoenzyme-2 (COX-2) dependent apoptosis pathway.

Epithelial to mesenchymal transition (EMT) is a critical step in the metastasis of epithelial cancer cells. Butyrate, which is produced from dietary fiber by colonic bacterial fermentation, has been reported to influence EMT. However, some studies have reported that butyrate promotes EMT, while others have reported an inhibitory effect. To clarify these controversial results, it is necessary to elucidate the mechanism by which butyrate can influence EMT. In this study, we examined the potential role of annexin A1 (ANXA1), which was previously reported to promote EMT in breast cancer cells, as a mediator of EMT regulation by butyrate. We found that ANXA1 mRNA and protein were expressed in highly invasive melanoma cell lines (A2058 and A375), but not in SK-MEL-5 cells, which are less invasive. We also showed that butyrate induced ANXA1 mRNA and protein expression and promoted EMT-related cell invasion in SK-MEL-5 cells. Downregulation of ANXA1 expression using specific small interfering RNAs in butyrate-treated SK-MEL-5 cells resulted in increased expression of the epithelial marker E-cadherin and decreased cell invasion. Moreover, overexpressing ANXA1 decreased the expression of the E-cadherin. Collectively, these results indicate that butyrate induces the expression of ANXA1 in human melanoma cells, which then promotes invasion through activating the EMT signaling pathway.

Traditional Chinese medicine (TCM) originated in ancient China has been practiced over thousands of years for treating various symptoms and diseases. However, the molecular mechanisms of TCM in treating these diseases remain unknown. In this study, we employ a systems pharmacology-based approach for connecting GWAS diseases with TCM for potential drug repurposing and repositioning. We studied 102 TCM components and their target genes by analyzing microarray gene expression experiments. We constructed disease-gene networks from 2558 GWAS studies. We applied a systems pharmacology approach to prioritize disease-target genes. Using this bioinformatics approach, we analyzed 14,713 GWAS disease-TCM-target gene pairs and identified 115 disease-gene pairs with q value

Motivation: Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. Results: We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. Availability and implementation: KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. Contact: aikchoon.tan@ucdenver.edu Supplementary information: Supplementary data are available at Bioinformatics online.

In response to oncogenic activation, cells initially undergo proliferation followed by an irreversible growth arrest called oncogene-induced senescence (OIS), an endogenous defense mechanism against tumorigenesis. Oncogenic activation of ERK1/2 is essential for both the initial phase of cellular proliferation as well as subsequent premature senescence, but little is known about the specific contribution of ERK1 versus 2 to OIS. Here we show that depletion of ERK2 but not ERK1 by shRNA knockdown in MEFs leads to continuous proliferation bypassing senescence even in the presence of oncogenic HRAS(V12). Upon depletion of ERK2, induction of both p19(Arf) and p16(Ink4a) was significantly compromised after oncogenic HRAS(V12) expression, attenuating activation of the key tumor suppressors p53 and pRb. Here we demonstrate that ERK2 but not ERK1 indirectly regulates p19(Arf) and p16(Ink4a) both at the transcriptional and translational level. Oncogenic Ras expression after ERK2 knockdown downregulates Fra-1 and c-Jun, components of the activator protein-1 (AP-1) heterodimer essential for transactivation of p19(Arf). Similarly we show a significant decrease in the activation of p38 MAPK and ETS family members which are involved in the induction of p16(Ink4a). The role of ERK2 in translational regulation is observed by the lack of tuberin (TSC2) and p70 ribosomal S6 kinase 1 (p70S6K1) phosphorylation, components of the mTOR pathway, which enhances p19(Arf) mRNA translation during oncogenic Ras-induced senescence. These observations suggest that ERK2 but not ERK1 contributes to upregulation of p19(Arf) and p16(Ink4a) in a transcription- and translation-dependent manner during oncogenic Ras-induced senescence. Taken together, our data indicate that ERK2 is the key ERK isoform mediating the senescence signaling pathway downstream of oncogenic Ras.

Individuals with Down syndrome exhibit remarkably reduced incidence of most solid tumors including pancreatic cancer. Multiple mechanisms arising from the genetic complexity underlying Down syndrome has been suggested to contribute to such a broad cancer protection. In this study, utilizing a genetically engineered mouse model of pancreatic cancer, we demonstrate that trisomy of the Down syndrome critical region-1 (Dscr1), an endogenous calcineurin inhibitor localized on chromosome 21, suppresses the progression of pancreatic intraepithelial neoplasia-1A (PanIN-1A) to PanIN-1B lesions without affecting the initiation of PanIN lesions mediated by oncogenic Kras(G12D). In addition, we show that Dscr1 trisomy attenuates nuclear localization of nuclear factor of activated T-cells (NFAT) accompanied by upregulation of the p15(Ink4b) tumor suppressor and reduction of cell proliferation in early PanIN lesions. Our data suggest that attenuation of calcineurin-NFAT signaling in neoplastic pancreatic ductal epithelium by a single extra copy of Dscr1 is sufficient to inhibit the progression of early PanIN lesions driven by oncogenic Kras, and thus may be a potential mechanism underlying reduced incidence of pancreatic cancer in Down syndrome individuals.

This study aimed to identify functional associations of cis-regulatory regions with gout susceptibility using data resulted from a genome-wide association study (GWAS), and to show a genetic architecture for gout with interaction effects among genes within each of the identified functions. The GWAS was conducted with 8314 control subjects and 520 patients with gout in the Korea Association REsource cohort. However, genetic associations with any individual nucleotide variants were not discovered by Bonferroni multiple testing in the GWAS (P>1.42 × 10(-7)). Genomic regions enrichment analysis was employed to identify functional associations of cis-regulatory regions. This analysis revealed several biological processes associated with gout susceptibility, and they were quite different from those with serum uric acid level. Epistasis for susceptibility to gout was estimated using entropy decomposition with selected genes within each biological process identified by the genomic regions enrichment analysis. Some epistases among nucleotide sequence variants for gout susceptibility were found to be larger than their individual effects. This study provided the first evidence that genetic factors for gout susceptibility greatly differed from those for serum uric acid level, which may suggest that research endeavors for identifying genetic factors for gout susceptibility should not be heavily dependent on pathogenesis of uric acid. Interaction effects between genes should be examined to explain a large portion of phenotypic variability for gout susceptibility.

Summary: We report the creation of Drug Signatures Database (DSigDB), a new gene set resource that relates drugs/compounds and their target genes, for gene set enrichment analysis (GSEA). DSigDB currently holds 22 527 gene sets, consists of 17 389 unique compounds covering 19 531 genes. We also developed an online DSigDB resource that allows users to search, view and download drugs/compounds and gene sets. DSigDB gene sets provide seamless integration to GSEA software for linking gene expressions with drugs/compounds for drug repurposing and translational research. Availability and implementation: DSigDB is freely available for non-commercial use at http://tanlab.ucdenver.edu/DSigDB. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: aikchoon.tan@ucdenver.edu

Population stratification can produce spurious genetic associations in genome-wide association studies (GWASs). Mixed model methodology has been regarded useful for correcting population stratification. This study explored statistical power and false discovery rate (FDR) with the data simulated for dichotomous traits. Empirical FDRs and powers were estimated using fixed models with and without genomic control and using mixed models with and without reflecting loci linked to the candidate marker in genetic relationships. Population stratification with admixture degree ranged from 1% to 10% resulted in inflated FDRs from the fixed model analysis without genomic control and decreased power from the fixed model analysis with genomic control (P0.05). Meanwhile, population stratification could not change FDR and power estimates from the mixed model analyses (P0.05). We suggest that the mixed model methodology was useful to reduce spurious genetic associations produced by population stratification in GWAS, even with a high degree of admixture (10%).

Use of mixed models is in the spotlight as an emerging method for genome-wide association studies (GWASs). This study investigated the statistical power for identifying nucleotide variants associated with quantitative traits using the mixed model methodology. Quantitative traits were simulated through design of heritability, the number of causal variants (NCV), the number of polygenic variants, and genetic variance ratio of causal to polygenic variants (VRCTP). Statistical power estimates were influenced not only by individual factors of heritability, NCV, and VRCTP, but also by their interactions (P0.05). As the genetic variance ratio decreased, the difference in power between heritabilities of 0.3 and 0.5 increased with the use of 20 causal variants, but decreased when there were 100 causal variants (P0.05). The power empirically estimated from the simulation study would be applicable to the design of GWAS for quantitative traits with known genetic parameters by predicting the degree of false negative associations.

The incidence of most solid tumors is remarkably reduced in individuals with Down syndrome. Using mouse models of Down syndrome, we have previously shown that this decrease in tumor incidence is due, in part, to suppression of tumor angiogenesis as a consequence of attenuated calcineurin signaling in endothelial cells. Our prior studies utilized xenografted tumors in a transgenic mouse model with three copies of the Down syndrome critical region-1 (Dscr1) gene, a chromosome 21-encoded endogenous calcineurin inhibitor. These data indicate that upregulated Dscr1 contributes to broad cancer protection by suppressing tumor angiogenesis through inhibiting the calcineurin pathway in the vascular endothelium. However, it still remains to be confirmed whether a single extra copy of Dscr1 is also sufficient to suppress tumor angiogenesis in slow growing spontaneous tumors that more accurately recapitulate molecular features of human malignancies. In this study, utilizing LSL-Kras(G12D) mice, an inducible and autochthonous model of human lung adenocarcinoma, on a Dscr1 transgenic mouse background, we show that a single extra transgenic copy of Dscr1 provides a survival advantage in these mice developing spontaneous lung tumors driven by oncogenic Kras(G12D) without affecting either initiation or progression of spontaneous lung tumors. Furthermore, we show that Dscr1 trisomy significantly reduces microvessel density in lung tumors and thus limits the growth of lung tumors through decreased proliferation and increased apoptosis of lung tumor cells. These data provide evidence that a single extra copy of Dscr1 is sufficient to suppress tumor angiogenesis during spontaneous lung tumorigenesis and further support our hypothesis that suppression of tumor angiogenesis by an additional copy of Dscr1 contributes to the reduced cancer incidence in individuals with Down syndrome and the calcineurin pathway in the tumor vasculature is a potential target for cancer treatment.

Background Triple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis. Results We integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. Wevalidated our predictions using published and new experimental data. Conclusions In summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that betulinic acid (BA), a triterpene from the bark of white birch, had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells. METHODOLOGY/PRINCIPAL FINDINGS: BA inhibited the protein expression and the transcriptional activities of hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. Consistently, BA blocked hypoxia-induced phosphorylation, DNA binding activity and nuclear accumulation of STAT3. In addition, BA significantly reduced cellular and secreted levels of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Furthermore, BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxic PC-3 cells, implying anti-angiogenic activity of BA under hypoxic condition. Of note, chromatin immunoprecipitation (ChiP) assay revealed that BA inhibited binding of HIF-1α and STAT3 to VEGF promoter. Furthermore, silencing STAT3 using siRNA transfection effectively enhanced the reduced VEGF production induced by BA treatment under hypoxia. CONCLUSIONS/SIGNIFICANCE: Taken together, our results suggest that BA has anti-angiogenic activity by disturbing the binding of HIF-1α and STAT3 to the VEGF promoter in hypoxic PC-3 cells.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively overexpressed in several human cancer cells and related to various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined whether betulinic acid (BA), a triterpene from the bark of white birch, could inhibit hypoxia-mediated activation of STAT3 and HIF-1 alpha in androgen independent human prostate cancer PC-3 cells. Our results show that BA inhibited the transcriptional activities and nuclear accumulation of both HIF-1 alpha and STAT3 under hypoxia. In addition, BA significantly reduced cellular and secreted levels of hypoxia-induced vascular endothelial growth factor (VEGF), a critical angiogenic factor, in PC-3 cells. BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced PC-3 cells. Moreover, silencing HIF 1 alpha and STAT3 by siRNA transfection augmented the suppression of VEGF expression by BA under hypoxia. Mechanistically, BA inhibited the recruitment of HIF-1 alpha and STAT3 on the VEGF promoter. Taken together, these findings suggest that BA may exert angio-prevention via inactivation of hypoxia-induced STAT3 and HIF-1 alpha in PC-3 cells. The in vivo anti-angiogenic and anti-PCa efficacy are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2011-2047