Un-Ho Jin

Role of the Aryl Hydrocarbon Receptor in Carcinogenesis and Potential as a Drug Target

The aryl hydrocarbon receptor (AHR) is highly expressed in multiple organs and tissues, and there is increasing evidence that the AHR plays an important role in cellular homeostasis and disease. The AHR is expressed in multiple tumor types, in cancer cell lines, and in tumors from animal models, and the function of the AHR has been determined by RNA interference, overexpression, and inhibition studies. With few exceptions, knockdown of the AHR resulted in decreased proliferation and/or invasion and migration of cancer cell lines, and in vivo studies in mice overexpressing the constitutively active AHR exhibited enhanced stomach and liver cancers, suggesting a pro-oncogenic role for the AHR. In contrast, loss of the AHR in transgenic mice that spontaneously develop colonic tumors and in carcinogen-induced liver tumors resulted in increased carcinogenesis, suggesting that the receptor may exhibit antitumorigenic activity prior to tumor formation. AHR ligands also either enhanced or inhibited tumorigenesis, and these effects were highly tumor specific, demonstrating that selective AHR modulators that exhibit agonist or antagonist activities represent an important new class of anticancer agents that can be directed against multiple tumors.

MICROBIOME-DERIVED TRYPTOPHAN METABOLITES AND THEIR ARYL HYDROCARBON RECEPTOR-DEPENDENT AGONIST AND ANTAGONIST ACTIVITIES

The tryptophan metabolites indole, indole-3-acetate, and tryptamine were identified in mouse cecal extracts and fecal pellets by mass spectrometry. The aryl hydrocarbon receptor (AHR) agonist and antagonist activities of these microbiota-derived compounds were investigated in CaCo-2 intestinal cells as a model for understanding their interactions with colonic tissue, which is highly aryl hydrocarbon (Ah)–responsive. Activation of Ah-responsive genes demonstrated that tryptamine and indole 3-acetate were AHR agonists, whereas indole was an AHR antagonist that inhibited TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)–induced CYP1A1 expression. In contrast, the tryptophan metabolites exhibited minimal anti-inflammatory activities, whereas TCDD decreased phorbol ester-induced CXCR4 [chemokine (C-X-C motif) receptor 4] gene expression, and this response was AHR dependent. These results demonstrate that the tryptophan metabolites indole, tryptamine, and indole-3-acetate modulate AHR-mediated responses in CaCo-2 cells, and concentrations of indole that exhibit AHR antagonist activity (100–250 μM) are detected in the intestinal microbiome.

Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents

ABSTRACT Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents.

Minireview: Role Of Orphan Nuclear Receptors in Cancer and Potential as Drug Targets

The nuclear orphan receptors for which endogenous ligands have not been identified include nuclear receptor (NR)0B1 (adrenal hypoplasia congenita critical region on chromosome X gene), NR0B2 (small heterodimer partner), NR1D1/2 (Rev-Erbα/β), NR2C1 (testicular receptor 2), NR2C2 (testicular receptor 4), NR2E1 (tailless), NR2E3 (photoreceptor-specific NR [PNR]), NR2F1 chicken ovalbumin upstream promoter transcription factor 1 (COUP-TFI), NR2F2 (COUP-TFII), NR2F6 (v-erbA-related protein), NR4A1 (Nur77), NR4A2 (Nurr1), NR4A3 (Nor1), and NR6A1 (GCNF). These receptors play essential roles in development, cellular homeostasis, and disease including cancer where over- or underexpression of some receptors has prognostic significance for patient survival. Results of receptor knockdown or overexpression in vivo and in cancer cell lines demonstrate that orphan receptors exhibit tumor-specific pro-oncogenic or tumor suppressor-like activity. For example, COUP-TFII expression is both a positive (ovarian) and negative (prostate and breast) prognostic factor for cancer patients; in contrast, the prognostic activity of adrenal hypoplasia congenita critical region on chromosome X gene for the same tumors is the inverse of COUP-TFII. Functional studies show that Nur77 is tumor suppressor like in acute leukemia, whereas silencing Nur77 in pancreatic, colon, lung, lymphoma, melanoma, cervical, ovarian, gastric, and some breast cancer cell lines induces one or more of several responses including growth inhibition and decreased survival, migration, and invasion. Although endogenous ligands for the orphan receptors have not been identified, there is increasing evidence that different structural classes of compounds activate, inactivate, and directly bind several orphan receptors. Thus, the screening and development of selective orphan receptor modulators will have important clinical applications as novel mechanism-based agents for treating cancer patients overexpressing one or more orphan receptors and also for combined drug therapies.

The Nuclear Receptor TR3 Regulates mTORC1 Signaling in Lung Cancer Cells Expressing Wild-type p53

The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small-cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3, which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.

Aryl Hydrocarbon Receptor Agonists Induce MicroRNA-335 Expression and Inhibit Lung Metastasis of Estrogen Receptor Negative Breast 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.

The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis

BackgroundPatients with ER-negative breast tumors are among the most difficult to treat and exhibit low survival rates due, in part, to metastasis from the breast to various distal sites. Aryl hydrocarbon receptor (AHR) ligands show promise as antimetastatic drugs for estrogen receptor (ER)-negative breast cancer.MethodsTriple negative MDA-MB-231 breast cancer cells were treated with eight AHR-active pharmaceuticals including 4-hydroxtamoxifen, flutamide leflunomide, mexiletine, nimodipine, omeprazole, sulindac and tranilast, and the effects of these compounds on cell proliferation (MTT assay) and cell migration (Boyden chamber assay) were examined. The role of the AHR in mediating inhibition of MDA-MB-231 cell invasion was investigated by RNA interference (RNAi) and knockdown of AHR or cotreatment with AHR agonists. Lung metastasis of MDA-MB-231 cells was evaluated in mice administered cells by tail vein injection and prometastatic gene expression was examined by immunohistochemistry.ResultsWe showed that only the proton pump inhibitor omeprazole decreased MDA-MB-231 breast cancer cell invasion in vitro. Omeprazole also significantly decreased MDA-MB-231 cancer cell metastasis to the lung in a mouse model (tail vein injection), and in vitro studies showed that omeprazole decreased expression of at least two prometastatic genes, namely matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4). Results of RNA interference studies confirmed that omeprazole-mediated downregulation of CXCR4 (but not MMP-9) was AHR-dependent. Chromatin immunoprecipitation assays demonstrated that omeprazole recruited the AHR to regions in the CXCR4 promoter that contain dioxin response elements (DREs) and this was accompanied by the loss of pol II on the promoter and decreased expression of CXCR4.ConclusionsAHR-active pharmaceuticals such as omeprazole that decrease breast cancer cell invasion and metastasis may have important clinical applications for late stage breast cancer chemotherapy.

Diindolylmethane Analogs Bind NR4A1 and Are NR4A1 Antagonists in Colon Cancer Cells

1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds exhibit antineoplastic activity in multiple cancer cell lines and the p-hydroxyphenyl analog (DIM-C-pPhOH) inactivates nuclear receptor 4A1 (NR4A1) in lung and pancreatic cancer cell lines. Using a series of 14 different p-substituted phenyl C-DIMs, we show that several compounds including DIM-C-pPhOH directly interacted with the ligand binding domain of NR4A1. Computational-based molecular modeling studies showed high-affinity interactions of DIM-C-pPhOH and related compounds within the ligand binding pocket of NR4A1, and these same compounds decreased NR4A1-dependent transactivation in colon cancer cells transfected with a construct containing 3 tandem Nur77 binding response elements linked to a luciferase reporter gene. Moreover, we also show that knockdown of NR4A1 by RNA interference (small interfering NR4A1) or treatment with DIM-C-pPhOH and related compounds decreased colon cancer cell growth, induced apoptosis, decreased expression of survivin and other Sp-regulated genes, and inhibited mammalian target of rapamycin signaling. Thus, C-DIMs such as DIM-C-pPhOH directly bind NR4A1 and are NR4A1 antagonists in colon cancer cells, and their antineoplastic activity is due, in part, to their interactions with nuclear NR4A1.

Omeprazole Inhibits Pancreatic Cancer Cell Invasion through a Nongenomic Aryl Hydrocarbon Receptor Pathway.

Omeprazole and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are aryl hydrocarbon receptor (AhR) agonists that inhibit the invasion of breast cancer cells through inhibition of CXCR4 transcription. Treatment of highly invasive Panc1 pancreatic cancer cells with TCDD, omeprazole, and seven other AhR-active pharmaceuticals showed that only omeprazole and tranilast, but not TCDD, inhibited invasion in a Boyden chamber assay. Similar results were observed in MiaPaCa2 cells, another quasimensenchymal pancreatic ductal adenocarcinoma (QM-PDA) pancreatic cancer cell line, whereas invasion was not observed with BxPC3 or L3.6pL cells, which are classified as classical (less invasive) pancreatic cancer cells. It was also observed in QM-PDA cells that TCDD, omeprazole, and tranilast did not induce CYP1A1 or CXCR4 and that treatment with these compounds did not result in nuclear uptake of AhR. In contrast, treatment of BxPC3 and L3.6pL cells with these AhR ligands resulted in induction of CYP1A1 (by TCDD) and nuclear uptake of AhR, which was similar to that observed for Ah-responsive MDA-MB-468 breast and HepG2 liver cancer cell lines. Results of AhR and AhR nuclear translocator (Arnt) knockdown experiments in Panc1 and MiaPaCa2 cells demonstrated that omeprazole- and tranilast-mediated inhibition of invasion was AhR-dependent but Arnt-independent. These results demonstrate that in the most highly invasive subtype of pancreatic cancer cells (QM-PDA) the selective AhR modulators omeprazole and tranilast inhibit invasion through a nongenomic AhR pathway.

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells.

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.