Bo-hyun Choi

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.

The Sensitivity of Cancer Cells to Pheophorbide a-Based Photodynamic Therapy Is Enhanced by NRF2 Silencing

Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.

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.

Beneficial Effects of Sarpogrelate and Rosuvastatin in High Fat Diet/Streptozotocin-Induced Nephropathy in Mice.

Chronic kidney disease (CKD) is a major complication of metabolic disorders such as diabetes mellitus, obesity, and hypertension. Comorbidity of these diseases is the factor exacerbating CKD progression. Statins are commonly used in patients with metabolic disorders to decrease the risk of cardiovascular complications. Sarpogrelate, a selective antagonist of 5-hydroxytryptamine (5-HT) 2A receptor, inhibits platelet aggregation and is used to improve peripheral circulation in diabetic patients. Here, we investigated the effects of sarpogrelate and rosuvastatin on CKD in mice that were subjected to a high fat diet (HFD) for 22 weeks and a single low dose of streptozotocin (STZ, 40 mg/kg). When mice were administrated sarpogrelate (50 mg/kg, p.o.) for 13 weeks, albuminuria and urinary cystatin C excretion were normalized and histopathological changes such as glomerular mesangial expansion, tubular damage, and accumulations in lipid droplets and collagen were significantly improved. Sarpogrelate treatment repressed the HFD/STZ-induced CD31 and vascular endothelial growth factor receptor-2 expressions, indicating the attenuation of glomerular endothelial proliferation. Additionally, sarpogrelate inhibited interstitial fibrosis by suppressing the increases in transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor-1 (PAI-1). All of these functional and histological improvements were also seen in rosuvastatin (20 mg/kg) group and, notably, the combinatorial treatment with sarpogrelate and rosuvastatin showed additive beneficial effects on histopathological changes by HFD/STZ. Moreover, sarpogrelate reduced circulating levels of PAI-1 that were elevated in the HFD/STZ group. As supportive in vitro evidence, sarpogrelate incubation blocked TGF-β1/5-HT-inducible PAI-1 expression in murine glomerular mesangial cells. Taken together, sarpogrelate and rosuvastatin may be advantageous to control the progression of CKD in patients with comorbid metabolic disorders, and particularly, the use of sarpogrelate as adjunctive therapy with statins may provide additional benefits on CKD.

Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres

Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters.

NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA)-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages.

Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS) are known to be an important contributor to monocytes’ differentiation and macrophages’ function. NF-E2-related factor 2 (NRF2), a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA). In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNFα) were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1) was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER) stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-ҡB (NFҡB) p50 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFҡB or ERK1/2 largely blocked PMA-induced IL-1β and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFҡB axis is involved in PMA-facilitated differentiation of NRF2-silenced U937 cells.

High CD44 expression mediates p62-associated NFE2L2/NRF2 activation in breast cancer stem cell-like cells: Implications for cancer stem cell resistance

Cluster of differentiation 44 (CD44) is the most common cancer stem cell (CSC) marker and high CD44 expression has been associated with anticancer drug resistance, tumor recurrence, and metastasis. In this study, we aimed to investigate the molecular mechanism by which CD44 and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a key regulator of antioxidant genes, are linked to CSC resistance using CD44high breast CSC-like cells. NRF2 expression was higher in CD44high cell populations isolated from doxorubicin-resistant MCF7 (ADR), as well as MCF7, MDA-MB231, and A549 cells, than in corresponding CD44low cells. High NRF2 expression in the CD44highCD24low CSC population (ADR44P) established from ADR cells depended on standard isoform of CD44. Silencing of CD44 or overexpression of CD44 resulted in the reduction or elevation of NRF2, respectively, and treatment with hyaluronic acid, a CD44 ligand, augmented NRF2 activation. As functional implications, NRF2 silencing rendered ADR44P cells to retain higher levels of reactive oxygen species and to be sensitive to anticancer drug toxicity. Moreover, NRF2-silenced ADR44P cells displayed tumor growth retardation and reduced colony/sphere formation and invasion capacity. In line with these, CD44 significantly colocalized with NRF2 in breast tumor clinical samples. The molecular mechanism of CD44-mediated NRF2 activation was found to involve high p62 expression. CD44 elevation led to an increase in p62, and inhibition of p62 resulted in NRF2 suppression in ADR44P. Collectively, our results showed that high CD44 led to p62-associated NRF2 activation in CD44high breast CSC-like cells. NRF2 activation contributed to the aggressive phenotype, tumor growth, and anticancer drug resistance of CD44high CSCs. Therefore, the CD44-NRF2 axis might be a promising therapeutic target for the control of stress resistance and survival of CD44high CSC population within breast tumors.

High NRF2 level mediates cancer stem cell-like properties of aldehyde dehydrogenase (ALDH)-high ovarian cancer cells: inhibitory role of all- trans retinoic acid in ALDH/NRF2 signaling

Aldehyde dehydrogenase 1A1 (ALDH1A1) is one of cancer stem cell (CSC) markers, and high ALDH1 expression has been related to drug resistance and facilitated tumor growth. In this study, we investigated the potential involvement of nuclear factor erythroid 2-like 2 (NFE2L2/NRF2) in CSC-like properties of ALDH-high ovarian CSCs. Our experimental system, ALDH1A1-high (ALDH-H) subpopulation, was isolated and stabilized using doxorubicin-resistant ovarian cancer A2780 cells. ALDH-H exerted CSC-like properties such as drug resistance, colony/sphere formation, and enhanced tumor growth along with high levels of CSCs markers compared to ALDH1A1-low (ALDH-L). Levels of NRF2 and subsequent target genes substantially increased in ALDH-H cells, and the increase in ALDH1A1 and p62 was associated with NRF2 upregulation. ALDH1A1-silencing blocked increases in NRF2, drug efflux transporters, and p62, along with CSC markers in ALDH-H cells. The inhibition of p62, which was elevated in ALDH-H, suppressed NRF2 activation. High NRF2 level was confirmed in the ALDH1-high subpopulation from colon cancer HCT116 cells. The functional implication of NRF2 activation in ovarian CSCs was verified by two experimental approaches. First, CSC-like properties such as high CSC markers, chemoresistance, colony/sphere formation, and tumor growth were significantly inhibited by NRF2-silencing in ALDH-H cells. Second, all-trans retinoic acid (ATRA) suppressed ALDH1 expression, inhibiting NRF2 activation, which led to the attenuation of CSC-like properties in ALDH-H cells but not in ALDH-L cells. These results provide insight into the molecular basis of the ALDH1A1-mediated development of CSC-like properties such as stress/treatment resistance, and further suggest the therapeutic potential of ATRA in ALDH-high ovarian CSCs.

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.