The Role of Endocrine Disruption Chemical-Regulated Aryl Hydrocarbon Receptor Activity in the Pathogenesis of Pancreatic Diseases and Cancer.

The aryl hydrocarbon receptor (AHR) serves as a ligand-activated transcription factor crucial for regulating fundamental cellular and molecular processes, such as xenobiotic metabolism, immune responses, and cancer development. Notably, a spectrum of endocrine-disrupting chemicals (EDCs) act as agonists or antagonists of AHR, leading to the dysregulation of pivotal cellular and molecular processes and endocrine system disruption. Accumulating evidence suggests a correlation between EDC exposure and the onset of diverse pancreatic diseases, including diabetes, pancreatitis, and pancreatic cancer. Despite this association, the mechanistic role of AHR as a linchpin molecule in EDC exposure-related pathogenesis of pancreatic diseases and cancer remains unexplored. This review comprehensively examines the involvement of AHR in EDC exposure-mediated regulation of pancreatic pathogenesis, emphasizing AHR as a potential therapeutic target for the pathogenesis of pancreatic diseases and cancer.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Regulation of a long noncoding RNA MALAT1 by aryl hydrocarbon receptor in pancreatic cancer cells and tissues.

Environmental toxicants such as dioxins and polycyclic aromatic carbons are risk factors for pancreatitis and pancreatic cancer. These toxicants activate aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, of which activation regulates many downstream biological events, including xenobiotic metabolism, inflammation, and cancer cell growth and transformation. Here, we identified that environmental toxicant-activated AHR increased expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in pancreatic cancer cells and pancreatic tissues. The MALAT1 is a long noncoding (lnc) RNA which interacts with Enhancer of Zeste 2 (EZH2), a histone methyltransferase with epigenetic silencer activity, and the MALAT1-EZH2 interaction increased its epigenetic silencing activity. In contrast, AHR antagonist, CH223191 or resveratrol, counteracted the AHR-mediated MALAT1 induction and MALAT1-enahnced EZH2 activity. Collectively, these results revealed a novel pathway of how environmental exposure leads to epigenetic alteration via activation of AHR-MALAT1-EZH2 signaling axis under pancreatic tissue- and cancer cell-context.

NR2E3 is a key component in p53 activation by regulating a long noncoding RNA DINO in acute liver injuries.

Damage-induced long noncoding RNA (DINO) is a long noncoding RNA that directly interacts with p53 and thereby enhances p53 stability and activity in response to various cellular stresses. Here, we demonstrate that nuclear receptor subfamily 2 group E member 3 (NR2E3) plays a crucial role in maintaining active DINO epigenetic status for its proper induction and subsequent p53 activation. In acetaminophen (APAP)- or carbon tetrachloride-induced acute liver injuries, NR2E3 knockout (KO) mice exhibited far more severe liver injuries due to impaired DINO induction and p53 activation. Mechanistically, NR2E3 loss both in vivo and in vitro induced epigenetic DINO repression accompanied by reduced DINO chromatin accessibility. Furthermore, compared with the efficient reversal by a typical antidote N-acetylcysteine (NAC) treatment of APAP-induced liver injury in wild-type mice, the liver injury of NR2E3 KO mice was not effectively reversed, indicating that an intact NR2E3-DINO-p53-signaling axis is essential for NAC-mediated recovery against APAP-induced hepatotoxicity. These findings establish that NR2E3 is a critical component in p53 activation and a novel susceptibility factor to drug- or toxicant-induced acute liver injuries.-Khanal, T., Leung, Y.-K., Jiang, W., Timchenko, N., Ho, S.-M., Kim, K. NR2E3 is a key component in p53 activation by regulating a long noncoding RNA DINO in acute liver injuries.

Loss of NR2E3 represses AHR by LSD1 reprogramming, is associated with poor prognosis in liver cancer.

The aryl hydrocarbon receptor (AHR) plays crucial roles in inflammation, metabolic disorder, and cancer. However, the molecular mechanisms regulating AHR expression remain unknown. Here, we found that an orphan nuclear NR2E3 maintains AHR expression, and forms an active transcriptional complex with transcription factor Sp1 and coactivator GRIP1 in MCF-7 human breast and HepG2 liver cancer cell lines. NR2E3 loss promotes the recruitment of LSD1, a histone demethylase of histone 3 lysine 4 di-methylation (H3K4me2), to the AHR gene promoter region, resulting in repression of AHR expression. AHR expression and responsiveness along with H3K4me2 were significantly reduced in the livers of Nr2e3rd7 (Rd7) mice that express low NR2E3 relative to the livers of wild-type mice. SP2509, an LSD1 inhibitor, fully restored AHR expression and H3K4me2 levels in Rd7 mice. Lastly, we demonstrated that both AHR and NR2E3 are significantly associated with good clinical outcomes in liver cancer. Together, our results reveal a novel link between NR2E3, AHR, and liver cancer via LSD1-mediated H3K4me2 histone modification in liver cancer development.

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.

Specificity protein (Sp) transcription factors and metformin regulate expression of the long non-coding RNA HULC.

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.

Deregulation of NR2E3, an orphan nuclear receptor, by benzo(a)pyrene-induced oxidative stress is associated with histone modification status change of the estrogen receptor gene promoter.

We previously reported that NR2E3, an orphan nuclear receptor, plays an important role in maintaining the basal expression of estrogen receptor α (ER) and that the NR2E3 level is highly correlated with the relapse-free survival of breast cancer patients. Here, we investigated the role of NR2E3 in benzo(a)pyrene (BaP)-mediated cell injury. BaP treatment reduced NR2E3 homo-dimer formation and expression and subsequently decreased ER expression. The chromatin immunoprecipitation assay results showed that the treatment of MCF-7 breast cancer cells and the mouse liver with BaP released NR2E3 from the ER promoter to transform the transcriptionally active histone modification status into a repressive state. NR2E3 depletion in MCF-7 cells also induced a similar inactive epigenetic status in the ER promoter region, indicating that NR2E3 is an essential epigenetic player that maintains basal ER expression. Interestingly, these negative effects of BaP on the expression levels of NR2E3 and ER were rescued by antioxidant treatment. Collectively, our study provides novel evidence to show that BaP-induced oxidative stress decreases ER expression, in part by regulating NR2E3 function, which modulates the epigenetic status of the ER promoter. NR2E3 is likely an essential epigenetic player that maintains basal ER expression to protect cells from BaP-induced oxidative injury.

The transcriptional repressor ZBTB4 regulates EZH2 through a MicroRNA-ZBTB4-specificity protein signaling axis.

ZBTB4 is a transcriptional repressor and examination of publically-available microarray data sets demonstrated an inverse relationship in the prognostic value and expression of ZBTB4 and the histone methyltransferase EZH2 in tumors from breast cancer patients. The possibility of functional interactions between EZH2 and ZBTB4 was investigated in breast cancer cells and the results showed that EZH2 is directly suppressed by ZBTB4 which in turn is regulated (suppressed) by miR-106b and other paralogues from the miR-17-92, miR-106b-25 and miR-106a-363 clusters that are highly expressed in breast and other tumors. ZBTB4 also acts a suppressor of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and RNA interference studies show that Sp proteins are required for EZH2 expression. The prediction analysis results from breast cancer patient array data sets confirm an association of Sp1-dependent EZH2 gene signature with decreased survival of breast cancer patients. Disruption of oncogenic miR-ZBTB4 signaling axis by anticancer agent such as betulinic acid that induce down-regulation of Sp proteins in breast cancer cells resulted in inhibition of tumor growth and colonization of breast cancer cells in a mouse model. Thus, EZH2 is reciprocally regulated by a novel signaling network consisting of Sp proteins, oncogenic miRs and ZBTB4, and modulation of this gene network is a novel therapeutic approach for treatment of breast cancer and possibly other cancers.

MicroRNA-26b represses colon cancer cell proliferation by inhibiting lymphoid enhancer factor 1 expression.

microRNAs (miR) can act as oncogenes and tumor suppressors and several miRs are associated with cancer development and progression through the modulation of multiple cellular processes. miR26b is downregulated in several cancers and tumors and miR26b directly targets the lymphoid enhancer factor 1 (Lef1)3'UTR and inhibits endogenous Lef1 expression. We report that miR26b expression is associated with human colon cancer through the regulation of LEF1 expression in colon cancer cells. Analyses of multiple colon cancer cell lines revealed an inverse correlation between miR26b and LEF1 expression. Normal human colon cells express low levels of LEF1 and high levels of miR26b; however, human colon cancer cells have decreased miR26b expression and increased LEF1 expression. We demonstrate that miR26b expression is a potent inhibitor of colon cancer cell proliferation and significantly decreases LEF1 expression. The LEF1-regulated genes cyclin D1 and c-Myc were indirectly repressed by miR26b and this was consistent with decreased proliferation. miR26b overexpression in SW480 colon cancer cells also inhibited tumor growth in nude mice and this was due to decreased tumor growth and not apoptosis. Analyses of human colon cancer databases also demonstrated a link between miR26b and LEF1 expression. c-Myc expression is associated with multiple cancers and we propose that miR26b may act as a potential therapeutic agent in reducing cancer cell proliferation through repressing LEF1 activation of c-Myc and cyclin D1 expression.

Mechanism of action of phenethylisothiocyanate and other reactive oxygen species-inducing anticancer agents.

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.

Inhibition of rhabdomyosarcoma cell and tumor growth by targeting specificity protein (Sp) transcription factors.

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c-MET, insulin-like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp-regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients.

Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs.

BACKGROUND: Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. METHODS: The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. RESULTS: The IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 µM for curcumin to 0.7 µM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors. CONCLUSIONS: These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.

Induction of the transcriptional repressor ZBTB4 in prostate cancer cells by drug-induced targeting of microRNA-17-92/106b-25 clusters.

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-18ß-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.

FOXM1 mediates Dox resistance in breast cancer by enhancing DNA repair.

Transcription factors are direct effectors of altered signaling pathways in cancer and frequently determine clinical outcomes in cancer patients. To uncover new transcription factors that would determine clinical outcomes in breast cancer, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that Forkhead box protein M1 (FOXM1) is the top-ranked survival-associated transcription factor in patients with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NFκB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in breast cancer cells in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes, and our findings provide mechanistic insights into how FOXM1 mediates resistance to Dox and evidence that FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox.

Betulinic acid targets YY1 and ErbB2 through cannabinoid receptor-dependent disruption of microRNA-27a:ZBTB10 in breast cancer.

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 µmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid-dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor-dependent, thus identifying a new cellular target for this anticancer agent.

Reconstruction of nuclear receptor network reveals that NR2E3 is a novel upstream regulator of ESR1 in breast cancer.

ESR1 is one of the most important transcription factors and therapeutic targets in breast cancer. By applying systems-level re-analysis of publicly available gene expression data, we uncovered a potential regulator of ESR1. We demonstrated that orphan nuclear receptor NR2E3 regulates ESR1 via direct binding to the ESR1 promoter with concomitant recruitment of PIAS3 to the promoter in breast cancer cells, and is essential for physiological cellular activity of ESR1 in estrogen receptor (ER)-positive breast cancer cells. Moreover, expression of NR2E3 was significantly associated with recurrence-free survival and a favourable response to tamoxifen treatment in women with ER-positive breast cancer. Our results provide mechanistic insights on the regulation of ESR1 by NR2E3 and the clinical relevance of NR2E3 in breast cancer.

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.