Kyeong-Ah Jung

Identification of aldo-keto reductases as NRF2-target marker genes in human cells.

Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphism in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammation, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expressions of AKRs were significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14(+) monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.

Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells.

The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma β-cell line, βTC-6. shRNA-mediated silencing of Nrf2 expression in βTC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ER stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in βTC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated-like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic β-cells by enhancing proteasome-mediated ERAD.

NRF2 inhibition represses ErbB2 signaling in ovarian carcinoma cells: Implications for tumor growth retardation and docetaxel sensitivity

NF-E2-related factor 2 (NRF2) is a transcription factor that regulates the expression of various antioxidant and detoxifying enzymes. Although the benefit of NRF2 in cancer prevention is well established, its role in cancer pathobiology was recently discovered. In this study, the role of NRF2 in tumor growth and docetaxel sensitivity was investigated in ErbB2-overexpressing ovarian carcinoma SKOV3 cells. Interfering RNA-mediated stable inhibition of NRF2 in SKOV3 cells repressed NRF2 signaling, resulting in cell growth arrest at G(0)/G(1) phase and tumor growth retardation in mouse xenografts. Microarray analysis revealed that ErbB2 expression is substantially reduced in NRF2-inhibited SKOV3 and this was further confirmed by RT-PCR and immunoblot analysis. Repression of ErbB2 led to a decrease in phospho-AKT and enhanced p27 protein, reinforcing the effect of NRF2 knockdown on SKOV3 growth. Furthermore, NRF2 inhibition-mediated ErbB2 repression increases the sensitivity of these cells to docetaxel cytotoxicity and apoptosis. The linkage between NRF2 and ErbB2 was confirmed in the ErbB2-positive breast cancer cell line BT-474: NRF2 knockdown suppressed ErbB2 expression and enhanced docetaxel sensitivity. Our results provide insight into the coordinated regulation of signaling molecules responding to environmental stress and suggest that NRF2 modulation might be a therapeutic strategy to limit tumor growth and enhance sensitivity to taxane-based chemotherapy.

The c-MET/PI3K Signaling is Associated with Cancer Resistance to Doxorubicin and Photodynamic Therapy by Elevating BCRP/ABCG2 Expression

Overexpression of BCRP/ABCG2, a xenobiotic efflux transporter, is associated with anticancer drug resistance in tumors. Proto-oncogene c-MET induces cancer cell proliferation, motility, and survival, and its aberrant activation was found to be a prognostic factor in advanced ovarian cancers. In the present study, we investigated the potential crossresistance of doxorubicin-resistant ovarian cancer cells to the pheophorbide a (Pba)–based photodynamic therapy (PDT), and suggest c-MET and BCRP/ABCG2 overexpression as an underlying molecular mechanism. The doxorubicin-resistant A2780 cell line (A2780DR), which was established by incubating A2780 with stepwise increasing concentrations of doxorubicin, showed low levels of cellular Pba accumulation and reactive oxygen species generation, and was more resistant to PDT cytotoxicity than A2780. In a microarray analysis, BCRP/ABCG2 was found to be the only drug transporter whose expression was upregulated in A2780DR; this increase was confirmed by Western blot and immunocytochemical analyses. As functional evidence, the treatment with a BCRP/ABCG2-specific inhibitor reversed A2780DR resistance to both doxorubicin and PDT. We identified that c-MET increase is related to BCRP/ABCG2 activation. The c-MET downstream phosphoinositide 3-kinase (PI3K)/AKT signaling was activated in A2780DR and the inhibition of PI3K/AKT or c-MET repressed resistance to doxorubicin and PDT. Finally, we showed that the pharmacological and genetic inhibition of c-MET diminished levels of BCRP/ABCG2 in A2780DR. Moreover, c-MET inhibition could repress BCRP/ABCG2 expression in breast carcinoma MDA-MB-231 and colon carcinoma HT29, resulting in sensitization to doxorubicin. Collectively, our results provide a novel link of c-MET overexpression to BCRP/ABCG2 activation, suggesting that this mechanism leads to crossresistance to both chemotherapy and PDT.

Enhanced 4-hydroxynonenal resistance in KEAP1 silenced human colon cancer cells.

Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.

Abstract 1502: The link between NRF2 and mitochondria through the regulation of miR-181c/mitochondria-encoded cytochrome c oxidase subunit-1

The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is involved in the environmental resistance of cancers by enhancing the antioxidant capacity. In the current study, we investigated the potential link between NRF2 and mitochondrial function in cancer cells. Global miRNA expression analysis of HT29 and HCT116 identified miR-181c as an NRF2-silencing-inducible miRNA, and miR-181c elevation was associated with the decrease in mitochondria-encoded cytochrome c oxidase subunit-1 (MT-CO1), a mitochondrial genome-encoded complex IV subunit of the electron transport chain. As a result of decreased complex IV activity, NRF2-silenced cancer cells exhibited reduced levels of mitochondrial membrane potential (MMP), oxygen consumption rate, and ATP production. Notably, these changes induced adaptive activation of AMP-activated protein kinase-α (AMPKα) and thereby, NRF2-silenced cells were more vulnerable to AMPK inhibitor-induced growth suppression. Similarly, mouse tumor xenografts derived from NRF2-silenced HT29 exhibited MT-CO1 reduction and AMPKα activation, thereby increasing responsiveness to the AMPK inhibitor treatment. Collectively, we showed a novel link of NRF2 to cancer mitochondria and AMPK signaling by elucidating miR-181c/MT-CO1 signaling as an underlying molecular mechanism. These results also suggest that it may an effective strategy to inhibit both NRF2 and AMPK to control adaptive tumor responses. Citation Format: Mi-Kyoung Kwak, Kyeong-Ah Jung, Donghyeok Kim, Sujin Lee. The link between NRF2 and mitochondria through the regulation of miR-181c/mitochondria-encoded cytochrome c oxidase subunit-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2017-1502

Abstract B27: Involvement of c-MET signaling in BCRP/ABCG2 activation and resistance to doxorubicin and photodynamic therapy

Overexpression of BCRP/ABCG2, a xenobiotic efflux transporter, is related to anticancer drug resistance in tumors. Proto-oncogene c-MET induces cancer cell proliferation, motility, and survival, and its aberrant activation was found to be a prognostic factor in advanced ovarian cancers. In this study, we demonstrate the potential cross-resistance of doxorubicin-resistant ovarian cancer cells to the pheophorbide a (Pba)-based photodynamic therapy (PDT) and suggest c-MET and BCRP/ABCG2 overexpression as an underlying molecular mechanism. The doxorubicin resistant A2780 cell line (A2780DR) showed enhanced resistance to PDT cytotoxicity with decreased level of reactive oxygen species generation and Pba accumulation than A2780. In microarray analysis, BCRP/ABCG2 is the sole drug transporter which was upregulated in A2780DR. The incubation with the BCRP/ABCG2 specific inhibitor reversed the both Pba-PDT and doxorubicin resistance in A2780DR. Importantly, we identified that the expression of c-Met, which is an oncogene activating PI3K signaling, was increased in A2780DR and the inhibition of PI3K/AKT or c-MET repressed resistance to doxorubicin and PDT. Finally, we showed that the pharmacological and genetic inhibition of c-MET diminished levels of BCRP/ABCG2 in A2780DR. Collectively, our results provide evidence that c-MET overexpression is linked to BCRP/ABCG2 activation and this mechanism leads to crossresistance to both chemotherapy and PDT. Citation Format: Bo-hyun Choi, Kyeong-Ah Jung, Mi-Kyoung Kwak. Involvement of c-MET signaling in BCRP/ABCG2 activation and resistance to doxorubicin and photodynamic therapy. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B27.

Abstract 2066: Identification of NRF2 target genes in human cells: Aldoketo reductases as a potential biomarker for NRF2 activity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The transcription factor NF-E2-related factor 2 (NRF2) plays a critical role in cellular defense system by up-regulating multiple antioxidant genes. Therefore, the identification of biomarker genes reflecting NRF2 activity would be important for the prediction of high-risk populations to environmental stresses. In the current study, we have investigated human genes of which expression is highly dependent on NRF2 to establish biomarker genes for NRF2 activity. For this purpose, NRF2-specific interfering RNA was stably introduced in normal human renal epithelial cells (HK-2) and human bronchial epithelial cells (NL-20), and changes in inducible genes were determined following treatment with 4 types of NRF2 activators: thiol-reacting sulforaphane, free-radical generating tert-butylhydroquinone, Michael acceptor cinnamic aldehyde and pro-oxidant hydrogen peroxide. These treatments showed relatively common alterations in gene expression, and among these, the expression of aldoketo reductase (AKR) 1C1 was highly inducible by all of these treatments in an NRF2-depedent manner. The levels for AKRs were found to be constitutively high in renal carcinoma A498 cells, of which NRF2 activity is elevated compared to normal renal epithelial HK-2 cells. In addition, the expression of AKRs was greatly enhanced by NRF2 activator treatments in human monocytes (U937), implying the potential utility of AKRs as a peripheral NRF2 marker. In conclusion, our results indicate that the expression of AKRs is highly dependent on NRF2 in human cells; therefore AKRs can be an effective biomarker for predicting NRF2 activity in human cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2066. doi:1538-7445.AM2012-2066