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Piperlongumine downregulates Sp1, Sp3, Sp4 and Sp-regulated genes: effects of Tiron.

PDF file - 75KB, Supplemental Figure S1. Inactivation of NR4A1 induces ER stress and autophagy in vitro and in vivo.

PDF file, 204K, Effects of proteasome and ROS inhibitors.

Supplemental Table 1, NMR Results, Supplemental Table 2, Supplemental Figures 1 and 2

PDF file - 36KB, Supplemental Table S2. Proteins showing differential expression in Panc1 cells by transfection of siNR4A1.

PDF file - 33KB, Supplemental Figure S2. Inactivation of NR4A1 induces DNA fragmentation and ER Ca2+ release in Panc-1 cells.

PDF file - 50KB, Supplemental Figure S5. Effect of siNR4A2 on induction of apoptosis and ER stress in pancreatic cancer cells.

The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that bound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental toxicants; however, there is increasing evidence that the AHR is an important new drug target for treating multiple diseases including breast cancer. Treatment of estrogen receptor (ER)-negative MDA-MB-231 and BT474 breast cancer cells with TCDD or the selective AHR modulator 6-methyl-1,3,-trichlorodibenzofuran (MCDF) inhibited breast cancer cell invasion in a Boyden chamber assay. These results were similar to those previously reported for the antimetastic microRNA-335 (miR-335). Both TCDD and MCDF induced miR-335 in MDA-MB-231 and BT474 cells and this was accompanied by downregulation of SOX4, a miR-335-regulated (inhibited) gene. The effects of TCDD and MCDF on miR-335 and SOX4 expression and interactions of miR-335 with the 3′-UTR target sequence in the SOX4 gene were all inhibited in cells transfected with an oligonucleotide (iAHR) that knocks down the AHR, thus confirming AHR-miR-335 interactions. MCDF (40 mg/kg/d) also inhibited lung metastasis of MDA-MB-231 cells in a tail vein injection model, showing that the AHR is a potential new target for treating patients with ER-negative breast cancer, a disease where treatment options and their effectiveness are limited. Mol Cancer Ther; 11(1); 108–18. ©2011 AACR.

PDF file, 220K, Role of E2F in modulating miR expression.

PDF file - 20KB, Supplemental Table S1. List of real-time PCR primer sequences.

Supplementary Figures 1-5 from Aryl Hydrocarbon Receptor Agonists Induce MicroRNA-335 Expression and Inhibit Lung Metastasis of Estrogen Receptor Negative Breast Cancer Cells

PDF file - 53KB, Supplemental Figure S4. DIM-C-pPhOH induces ER stress through inactivation of nuclear NR4A1.

PDF file - 125KB, Supplemental Table S3. mRNAs showing differential expression in Panc1 cells by transfection of siNR4A1.

PDF file - 37KB, Supplemental Figure S3. Top 10 significant enriched canonical pathways altered by transfection of Panc-1 cells with siNR4A1.

PDF file, 42K, Data mining for ZBTB4 expression.

Androgen-insensitive DU145 and PC3 human prostate cancer cells express high levels of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and treatment of cells with methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) inhibited cell growth and downregulated Sp1, Sp3, and Sp4 expression. CDODA-Me (15 mg/kg/d) was a potent inhibitor of tumor growth in a mouse xenograft model (PC3 cells) and also decreased expression of Sp transcription factors in tumors. CDODA-Me–mediated downregulation of Sp1, Sp3, and Sp4 was due to induction of the transcriptional repressor ZBTB4, which competitively binds and displaces Sp transcription factors from GC-rich sites in Sp1-, Sp3-, Sp4-, and Sp-regulated gene promoters. ZBTB4 levels are relatively low in DU145 and PC3 cells due to suppression by miR paralogs that are members of the miR-17-92 (miR-20a/17-5p) and miR-106b-25 (miR-106b/93) clusters. Examination of publically available prostate cancer patient array data showed an inverse relationship between ZBTB4 and miRs-20a/17-5p/106b/93 expression, and increased ZBTB4 in patients with prostate cancer was a prognostic factor for increased survival. CDODA-Me induces ZBTB4 in prostate cancer cells through disruption of miR–ZBTB4 interactions, and this results in downregulation of pro-oncogenic Sp transcription factors and Sp-regulated genes. Mol Cancer Ther; 11(9); 1852–62. ©2012 AACR.

NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNA interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum stress, including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and endoplasmic reticulum stress by these agents was attenuated after cotreatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and endoplasmic reticulum stress; thus, demonstrating that NR4A1 regulates levels of endoplasmic reticulum stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of the ROS/endoplasmic reticulum stress and proapoptotic pathways.Implications: The NR4A1 receptor is pro-oncogenic, regulates the ROS/endoplasmic reticulum stress pathways, and inactivation of the receptor represents a novel pathway for inducing cell death in pancreatic cancer. Mol Cancer Res; 12(4); 527–38. ©2014 AACR.

PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells, PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal germinal center (GC)-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole–derived NR4A1 antagonists including 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoter-dependent luciferase activity, and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells, Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in PD-L1–expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy.Significance:These findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis.

Supplemental Figure S4. NR4A1 antagonist inhibit 4T1-luc cell growth and invasion, and induce apoptosis. 4T1-Luc cells were treated with different concentrations of CDIM8 or Cl-OCH3 and effects on cell proliferation (A), invasion (B) and induction of Annexin V staining (C) were determined as outlined in the Materials and Methods. Results are expressed as means {plus minus} SD for at least 3 replicate determinations per treatment group and significant (p

Supplemental Table S1. Summary of reagents, antibodies, immune staining sources, oligonucleotides and primers

Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2′-dihydroxy substituents and this included 2,2′-dihydroxy-, 2,2′,4′-trihydroxy-, and 2,2′,5′-trihydroxychalcones. In contrast, 2′,4,5′-, 2′3′,4′-, 2′,4,4′-trihydroxy, and 2′,3-, 2′,4-, 2′,4′-, and 2′,5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2′-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.

Unsubstituted flavone induced CYP1A1, CYP1B1 and UGT1A1 gene expression in Caco2 cells and was characterized as an aryl hydrocarbon receptor (AhR) agonist. The structure-activity relationships among 15 mono- and dihydroxyflavones showed that addition of one or two hydroxyl groups resulted in active (e.g.: 5- and 6- mono- and 5,6-dihydroxyflavones) and inactive (e.g.: 7-mono, 7,4′ and 6,4′-dihydroxyflavones) AhR ligands. Ligand docking studies of flavone, mono- and dihydroxyflavones to the human AhR resulted in similar docking scores that varied from −3.48 to −4.58 kcal/mol and these values did not distinguish between AhR-active and AhR-inactive mono- and dihydroxyflavones. The AhR-inactive flavones were subsequently investigated as AhR antagonists by determining their activities as inhibitors of TCDD-induced expression of CYP1A1, CYP1AA2 and UGT 1A1 gene expression in Caco2 cells. Initial studies with 7,4′-dihydroxyflavone showed that this compound was an AhR antagonist in Caco2 cells and resembled the activity of the classical AhR antagonist CH223191. With few exceptions most of the remaining AhR-inactive compounds in terms of inducing AhR responsive genes were also AhR antagonists. Thus, based on modeling studies, mono- and dihydroxyflavones bind with similar affinities to the AhR and exhibit AhR agonist or antagonist activities, however, the structural requirements (substitution patterns) for predicting these opposing activities were not apparent and could only be determined using bioassays.

INTRODUCTION: The orphan nuclear receptor 4A2 (NR4A2) has been extensively characterized in subcellular regions of the brain and is necessary for the function of dopaminergic neurons. The NR4A2 ligand, 1,1-bis (3(1)-indoly1)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) inhibits markers of neuroinflammation and degeneration in mouse models and in this study we investigated expression and function of NR4A2 in glioblastoma (GBM). METHODS: Established and patient-derived cell lines were used as models and the expression and functions of NR4A2 were determined by western blots and NR4A2 gene silencing by antisense oligonucleotides respectively. Effects of NR4A2 knockdown and DIM-C-pPhCl on cell growth, induction of apoptosis (Annexin V Staining) and migration/invasion (Boyden chamber and spheroid invasion assay) and transactivation of NR4A2-regulated reporter genes were determined. Tumor growth was investigated in athymic nude mice bearing U87-MG cells as xenografts. RESULTS: NR4A2 knockdown and DIM-C-pPhCl inhibited GBM cell and tumor growth, induced apoptosis and inhibited migration and invasion of GBM cells. DIM-C-pPhCl and related analogs also inhibited NR4A2-regulated transactivation (luciferase activity) confirming that DIM-C-pPhCl acts as an NR4A2 antagonist and blocks NR4A2-dependent pro-oncogenic responses in GBM. CONCLUSION: We demonstrate for the first time that NR4A2 is pro-oncogenic in GBM and thus a potential druggable target for patients with tumors expressing this receptor. Moreover, our bis-indole-derived NR4A2 antagonists represent a novel class of anti-cancer agents with potential future clinical applications for treating GBM.

Many of the protective responses observed for flavonoids in the gastrointestinal track resemble aryl hydrocarbon receptor (AhR)-mediated effects. Therefore, we examined the structure–activity relationships of isoflavones and isomeric flavone and flavanones as AhR ligands on the basis of their induction of CYP1A1, CYP1B1, and UGT1A1 gene expression in colon cancer Caco2 cells and young adult mouse colonocyte (YAMC) cells. Caco2 cells were significantly more Ah-responsive than YAMC cells, and this was due, in part, to flavonoid-induced cytotoxicity in the latter cell lines. The structure–activity relationships for the flavonoids were complex and both response and cell context specific; however, there was significant variability in the AhR activities of the isomeric substituted isoflavones and flavones. For example, 4′,5,7-trihydroxyisoflavone (genistein) was AhR-inactive whereas 4′,5,7-trihydroxyflavone (apigenin) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells. In contrast, both 5,7-dihydroxy-4-methoxy substituted isoflavone (biochanin A) and flavone (acacetin) induced all three AhR-responsive genes; 4′,5,7-trimethoxyisoflavone was a potent AhR agonist, and the isomeric flavone was AhR-inactive. These results coupled with simulation studies modeling flavonoid interaction within the AhR binding pocket demonstrate that the orientation of the substituted phenyl ring at C-2 (flavones) or C-3 (isoflavones) on the common 4-H-chromen-4-one ring strongly influences the activities of isoflavones and flavones as AhR agonists.

Tryptophan metabolites exhibit aryl hydrocarbon receptor (AhR) agonist activity and recent studies show that the phenylalanine metabolites serotonin and carbidopa, a drug used in treating Parkinson's disease, activated the AhR. In this study, we identified the neuroactive hormone dopamine as an inducer of drug-metabolizing enzymes CYP1A1, CYP1B1, and UGT1A1 in colon and glioblastoma cells and similar results were observed for carbidopa. In contrast, carbidopa but not dopamine exhibited AhR activity in BxPC3 pancreatic cancer cells whereas minimal activity was observed for both compounds in Panc1 pancreatic cancer cells. In contrast with a previous report, the induction responses and cytotoxicity of carbidopa was observed only at high concentrations (100 µM) in BxPC3 cells. Our results show that similar to serotonin and several tryptophan metabolites, dopamine is also an AhR-active compound.

The aryl hydrocarbon receptor (AhR) was first identified as the intracellular protein that bound and mediated the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and dioxin-like compounds (DLCs). Subsequent studies show that the AhR plays an important role in maintaining cellular homeostasis and in pathophysiology, and there is increasing evidence that the AhR is an important drug target. The AhR binds structurally diverse compounds, including pharmaceuticals, phytochemicals and endogenous biochemicals, some of which may serve as endogenous ligands. Classification of DLCs and non-DLCs based on their persistence (metabolism), toxicities, binding to wild-type/mutant AhR and structural similarities have been reported. This review provides data suggesting that ligands for the AhR are selective AhR modulators (SAhRMs) that exhibit tissue/cell-specific AhR agonist and antagonist activities, and that their functional diversity is similar to selective receptor modulators that target steroid hormone and other nuclear receptors.

The aryl hydrocarbon receptor (AhR), a ligand‐activated transcription factor that senses xenobiotics, diet, and gut microbial‐derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5(+) stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal‐specific AhR knockout increases basal stem cell and crypt injury‐induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis‐associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR‐FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.

Background: The aryl hydrocarbon receptor (AhR) is expressed in gliomas and the highest staining is observed in glioblastomas. A recent study showed that the AhR exhibited tumor suppressor-like activity in established and patient-derived glioblastoma cells and genomic analysis showed that this was due, in part, to suppression of CXCL12, CXCR4 and MMP9. Methods: Selective AhR modulators (SAhRMs) including AhR-active pharmaceuticals were screened for their inhibition of invasion using a spheroid invasion assay in patient-derived AhR-expressing 15-037 glioblastoma cells and in AhR-silenced 15-037 cells. Invasion, migration and cell proliferation were determined using spheroid invasion, Boyden chambers and scratch assay, and XTT metabolic assays for cell growth. Changes in gene and gene product expression were determined by real-time PCR and Western blot assays, respectively. In vivo antitumorigenic activity of omeprazole was determined in SCID mice bearing subcutaneous patient-derived 15-037 cells. Results: Results of a screening assay using patient-derived 15-037 cells (wild-type and AhR knockout) identified the AhR-active proton pump inhibitor omeprazole as an inhibitor of glioblastoma cell invasion and migration only AhR-expressing cells but not in cells where the AhR was downregulated. Omeprazole also enhanced AhR-dependent repression of the pro-invasion CXCL12, CXCR4 and MMP9 genes, and interactions and effectiveness of omeprazole plus temozolomide were response-dependent. Omeprazole (100 mg/kg/injection) inhibited and delayed tumors in SCID mice bearing patient-derived 15-037 cells injected subcutaneously. Conclusion: Our results demonstrate that omeprazole enhances AhR-dependent inhibition of glioblastoma invasion and highlights a potential new avenue for development of a novel therapeutic mechanism-based approach for treating glioblastoma.

Scope This study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)-induced intestinal barrier damage in mice. Methods and results Aqueous coffee extracts induced Ah receptor (AhR) -responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon-derived Caco2 and YAMC cells. Tissue-specific AhR knockout (AhRf/f x Lgr5-GFP-CreERT2 x Villin-Cre), wild-type (Lgr5-CreERT2 x Villin-Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR-active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS-induced damage to intestinal barrier function and DSS-induced mucosal inflammatory genes such as IL-6 and TGF-β1 in wild-type (AhR+/+ ) but not AhR-/- mice. In contrast, coffee does not exhibit protective effects in intestinal-specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR-active microbial metabolites. Conclusions In colon-derived cells and in the mouse intestine, coffee induced several AhR-dependent responses including gene expression, inhibition of intestinal stem cell-enriched organoid growth, and inhibition of DSS-induced intestinal barrier damage. We conclude that the anti-inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

The aryl hydrocarbon receptor (AhR) is overexpressed in some patients with different tumor types, and the receptor can be a negative or positive prognostic factor. There is also evidence from both in vivo and in vitro cell culture models that the AhR can exhibit tumor-specific pro-oncogenic and tumor suppressor-like functions and therefore can be treated with AhR antagonists or agonists, respectively. Successful clinical applications of AhR ligands will require the synthesis and development of selective AhR modulators (SAhRMs) with tumor-specific AhR agonist or antagonist activity, and some currently available compounds such as indole-3-carbinol and diindolylmethane-(DIM) and synthetic AhR antagonists are potential drug candidates. There is also evidence that some AhR-active pharmaceuticals, including tranilast, flutamide, hydroxytamoxifen and omeprazole or their derivatives, may be effective AhR-dependent anticancer agents for single or combination cancer chemotherapies for treatment of breast and pancreatic cancers.

PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells, PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal germinal center (GC)-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole–derived NR4A1 antagonists including 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoter-dependent luciferase activity, and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells, Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in PD-L1–expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy. Significance: These findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis.

The aryl hydrocarbon receptor (AhR) plays an important role in maintaining cellular homeostasis and also in pathophysiology. For example, the interplay between the gut microbiome and microbially derived AhR ligands protects against inflammation along the gut–brain axis. The AhR and its ligands also inhibit colon carcinogenesis, but it has been reported that the AhR and its ligand kynurenine enhance glioblastoma (GBM). In this study, using both established and patient-derived GBM cells, we re-examined the role of kynurenine and the AhR in GBM, observing that kynurenine does not modulate AhR-mediated gene expression and does not affect invasion of GBM cells. Therefore, using an array of approaches, including ChIP, quantitative real-time PCR, and cell migration assays, we primarily focused on investigating the role of the AhR in GBM at the functional molecular and genomic levels. The results of transient and stable CRISPR/Cas9-mediated AhR knockdown in GBM cells indicated that loss of AhR enhances GBM tumor growth in a mouse xenograft model, increases GBM cell invasion, and up-regulates expression of pro-invasion/pro-migration genes, as determined by ingenuity pathway analysis of RNA-Seq data. We conclude that the AhR is a tumor suppressor–like gene in GBM; future studies are required to investigate whether the AhR could be a potential drug target for treating patients with GBM who express this receptor.

The present work involves an extensive analytical and toxicological description of a recently identified mixed halogenated carbazole found in sediment samples, 1,8-dibromo-3,6-dichloro-9H-carbazole (BCCZ). Concentrations and the relative effect potency (REP) were calculated for the target BCCZ in a set of stream sediments collected in 2008 in Ontario, Canada. The levels calculated for BCCZ as compared to those previously assessed for legacy persistent organic pollutants (POPs) in the same samples revealed a significant contribution of BCCZ to the total organic chemical contamination (

In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25-100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5'UTRs and 5'flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.

Specificity protein (Sp) transcription factor (TF) Sp1 is overexpressed in multiple tumors and is a negative prognostic factor for patient survival. Sp1 and also Sp3 and Sp4 are highly expressed in cancer cells and in this study, we have used results of RNA interference (RNAi) to show that the three TFs individually play a role in the growth, survival and migration/invasion of breast, kidney, pancreatic, lung and colon cancer cell lines. Moreover, tumor growth in athymic nude mice bearing L3.6pL pancreatic cancer cells as xenografts were significantly decreased in cells depleted for Sp1, Sp3 and Sp4 (combined) or Sp1 alone. Ingenuity Pathway Analysis (IPA) of changes in gene expression in Panc1 pancreatic cancer cells after individual knockdown of Sp1, Sp3 and Sp4 demonstrates that these TFs regulate genes and pathways that correlated with the functional responses observed after knockdown but also some genes and pathways that inversely correlated with the functional responses. However, causal IPA analysis which integrates all pathway-dependent changes in all genes strongly predicted that Sp1-, Sp3- and Sp4-regulated genes were associated with the pro-oncogenic activity. These functional and genomic results coupled with overexpression of Sp transcription factors in tumor vs. non-tumor tissues and decreased Sp1 expression with age indicate that Sp1, Sp3 and Sp4 are non-oncogene addiction (NOA) genes and are attractive drug targets for individual and combined cancer chemotherapies.

Several aryl hydrocarbon receptor (AhR)-active pharmaceuticals were screened as inhibitors of pancreatic cancer cell invasion and identified two compounds, omeprazole, that inhibited invasion. Inhibition of highly invasive Panc1 cell invasion by omeprazole involves an AhR-dependent non-genomic pathway, and omeprazole-mediated inhibition of Panc1 cell invasion was dependent on Jun-N-terminal kinase (JNK) and mitogen-activated kinase kinase 7 (MKK7). The failure of omeprazole to induce nuclear translocation of the AhR was not due to overexpression of cytosolic AhR partner proteins Hsp90 or XAP2, and results of DNA sequencing show that the AhR expressed in Panc1 cells was not mutated. Results of RNAseq studies indicate that omeprazole induced an AhR-dependent downregulation of several pro-invasion factors including activated leukocyte cell adhesion molecule (ALCAM), long chain fatty acid CoA-synthase (CSL4), stathmin 3 (STMN3) and neuropillin 2 (NRP2), and the specific functions of these genes are currently being investigated.

Environmental estrogens including trace contaminants and phytoestrogens have been classified as endocrine disruptors (EDs), and it is hypothesized that ED compounds may be etiologic agents in male reproductive tract problems and breast cancer in women. It is clear from experimental studies that industrial estrogenic compounds (xenoestrogens) such as bisphenol A and phytoestrogens are selective estrogen receptor modulators (SERMs) that may exhibit structure-dependent and tissue-specific estrogenic or antiestrogenic activities. However, to date there is minimal evidence showing that ED compounds are causal factors in breast cancer, male reproductive tract problems such as decreased sperm counts and fertility, or prostate and testicular cancers. Moreover, many of the so-called global male reproductive problems, such as decreased sperm counts, are currently not recognized as global problems and may be due to unidentified regional problems including obesity.

Specificity protein 1 (Sp1) transcription factor (TF) regulates expression of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) cells. RNA interference (RNAi) studies showed that among several lncRNAs expressed in HepG2, SNU-449 and SK-Hep-1 cells, highly upregulated in liver cancer (HULC) was regulated not only by Sp1 but also Sp3 and Sp4 in the three cell lines. Knockdown of Sp transcription factors and HULC by RNAi showed that they play important roles in HCC cell proliferation, survival and migration. The relative contribution of Sp1, Sp3, Sp4 and HULC on these responses in HepG2, SNU-449 and SK-Hep-1 cells were cell context- and response-dependent. In the poorly differentiated SK-Hep-1 cells, knockdown of Sp1 or HULC resulted in genomic and morphological changes, indicating that Sp1 and Sp1-regulated HULC are important for maintaining the mesenchymal phenotype in this cell line. Genomic analysis showed an inverse correlation between expression of genes after knockdown of HULC and expression of those genes in liver tumors from patients. The antidiabetic drug metformin down-regulates Sp proteins in pancreatic cancer, and similar results including decreased HULC expression were observed in HepG2, SNU-449 and SK-Hep-1 cells treated with metformin, indicating that metformin and other antineoplastic agents that target Sp proteins may have clinical applications for HCC chemotherapy.

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.

Piperlongumine is a natural product found in the plant species Piper longum, and this compound exhibits potent anticancer activity in multiple tumor types and has been characterized as an inducer of reactive oxygen species (ROS). Treatment of Panc1 and L3.6pL pancreatic, A549 lung, 786-O kidney, and SKBR3 breast cancer cell lines with 5 to 15 μmol/L piperlongumine inhibited cell proliferation and induced apoptosis and ROS, and these responses were attenuated after cotreatment with the antioxidant glutathione. Piperlongumine also downregulated expression of Sp1, Sp3, Sp4, and several pro-oncogenic Sp-regulated genes, including cyclin D1, survivin, cMyc, EGFR and hepatocyte growth factor receptor (cMet), and these responses were also attenuated after cotreatment with glutathione. Mechanistic studies in Panc1 cells showed that piperlongumine-induced ROS decreased expression of cMyc via an epigenetic pathway, and this resulted in downregulation of cMyc-regulated miRNAs miR-27a, miR-20a, and miR-17 and induction of the transcriptional repressors ZBTB10 and ZBTB4. These repressors target GC-rich Sp-binding sites to decrease transactivation. This pathway observed for piperlongumine in Panc1 cells has previously been reported for other ROS-inducing anticancer agents and shows that an important underlying mechanism of action of piperlongumine is due to downregulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes. Cancer Prev Res; 10(8); 467–77. ©2017 AACR.