Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

Hypoxia, oxidative stress, and the interplay of HIFs and NRF2 signaling in cancer.

Oxygen is crucial for life and acts as the final electron acceptor in mitochondrial energy production. Cells adapt to varying oxygen levels through intricate response systems. Hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, orchestrate the cellular hypoxic response, activating genes to increase the oxygen supply and reduce expenditure. Under conditions of excess oxygen and resulting oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF2) activates hundreds of genes for oxidant removal and adaptive cell survival. Hypoxia and oxidative stress are core hallmarks of solid tumors and activated HIFs and NRF2 play pivotal roles in tumor growth and progression. The complex interplay between hypoxia and oxidative stress within the tumor microenvironment adds another layer of intricacy to the HIF and NRF2 signaling systems. This review aimed to elucidate the dynamic changes and functions of the HIF and NRF2 signaling pathways in response to conditions of hypoxia and oxidative stress, emphasizing their implications within the tumor milieu. Additionally, this review explored the elaborate interplay between HIFs and NRF2, providing insights into the significance of these interactions for the development of novel cancer treatment strategies.

Emerging Role of NRF2 Signaling in Cancer Stem Cell Phenotype.

Cancer stem cells (CSCs) are a small population of tumor cells characterized by self-renewal and differentiation capacity. CSCs are currently postulated as the driving force that induces intra-tumor heterogeneity leading to tumor initiation, metastasis, and eventually tumor relapse. Notably, CSCs are inherently resistant to environmental stress, chemotherapy, and radiotherapy due to high levels of antioxidant systems and drug efflux transporters. In this context, a therapeutic strategy targeting the CSC-specific pathway holds a promising cure for cancer. NRF2 (nuclear factor erythroid 2-like 2; NFE2L2) is a master transcription factor that regulates an array of genes involved in the detoxification of reactive oxygen species/electrophiles. Accumulating evidence suggests that persistent NRF2 activation, observed in multiple types of cancer, supports tumor growth, aggressive malignancy, and therapy resistance. Herein, we describe the core properties of CSCs, focusing on treatment resistance, and review the evidence that demonstrates the roles of NRF2 signaling in conferring unique properties of CSCs and the associated signaling pathways.

Association of NRF2 with HIF-2α-induced cancer stem cell phenotypes in chronic hypoxic condition.

The acquisition of the cancer stem cell (CSC) properties is often mediated by the surrounding microenvironment, and tumor hypoxia is considered an important factor for CSC phenotype development. High levels of NRF2 (Nuclear Factor Erythroid 2-Like 2; NFE2L2), a transcription factor that maintains cellular redox balance, have been associated with facilitated tumor growth and therapy resistance. In this study, we investigated the role of NRF2 in hypoxia-induced CSC phenotypes in colorectal cancer cells. Chronic hypoxia for 72 h resulted in CSC phenotypes, including elevation of krupple-like factor 4 (KLF4) and octamer-binding transcription factor 4 (OCT4), and an increase in cancer migration and spheroid growth with concomitant hypoxia-inducible factor 2α (HIF-2α) accumulation. All these chronic hypoxia-induced CSC properties were attenuated following HIF-2α-specific silencing. In this chronic hypoxia model, NRF2 inhibition by shRNA-based silencing or brusatol treatment blocked HIF-2α accumulation, which consequently resulted in decreased CSC marker expression and inhibition of CSC properties such as spheroid growth. In contrast, NRF2 overactivation by genetic or chemical approach enhanced the chronic hypoxia-induced HIF-2α accumulation and cancer migration. As a molecular mechanism of the NRF2-inhibition-mediated HIF-2α dysregulation, we demonstrated that miR-181a-2-3p, whose expression is elevated in NRF2-silenced cells, targeted the HIF-2α 3'UTR and subsequently suppressed the chronic hypoxia-induced HIF-2α and CSC phenotypes. The miR-181a-2-3p inhibitor treatment in NRF2-silenced cells could restore the levels of HIF-2α and CSC markers, and increased cancer migration and sphere formation under chronic hypoxia. In line with this, the miR-181a-2-3p inhibitor transfection could increase tumorigenicity of NRF2-silenced colorectal cancer cells. Collectively, our study suggests the involvement of NRF2/miR181a-2-3p signaling in the development of HIF-2α-mediated CSC phenotypes in sustained hypoxic environments.

High Levels of Hyaluronic Acid Synthase-2 Mediate NRF2-Driven Chemoresistance in Breast Cancer Cells.

Hyaluronic acid (HA), a ligand of CD44, accumulates in some types of tumors and is responsible for tumor progression. The nuclear factor erythroid 2-like 2 (NRF2) regulates cytoprotective genes and drug transporters, which promotes therapy resistance in tumors. Previously, we showed that high levels of CD44 are associated with NRF2 activation in cancer stem like-cells. Herein, we demonstrate that HA production was increased in doxorubicin-resistant breast cancer MCF7 cells (MCF7-DR) via the upregulation of HA synthase-2 (HAS2). HA incubation increased NRF2, aldo-keto reductase 1C1 (AKR1C1), and multidrug resistance gene 1 (MDR1) levels. Silencing of HAS2 or CD44 suppressed NRF2 signaling in MCF7-DR, which was accompanied by increased doxorubicin sensitivity. The treatment with a HAS2 inhibitor, 4-methylumbelliferone (4-MU), decreased NRF2, AKR1C1, and MDR1 levels in MCF7-DR. Subsequently, 4-MU treatment inhibited sphere formation and doxorubicin resistance in MCF7-DR. The Cancer Genome Atlas (TCGA) data analysis across 32 types of tumors indicates the amplification of HAS2 gene is a common genetic alteration and is negatively correlated with the overall survival rate. In addition, high HAS2 mRNA levels are associated with increased NRF2 signaling and poor clinical outcome in breast cancer patients. Collectively, these indicate that HAS2 elevation contributes to chemoresistance and sphere formation capacity of drug-resistant MCF7 cells by activating CD44/NRF2 signaling, suggesting a potential benefit of HAS2 inhibition.

The multifaceted role of NRF2 in cancer progression and cancer stem cells maintenance.

The transcription factor nuclear factor erythroid 2-like 2 (NEF2L2; NRF2) plays crucial roles in the defense system against electrophilic or oxidative stress by upregulating an array of genes encoding antioxidant proteins, electrophile/reactive oxygen species (ROS) detoxifying enzymes, and drug efflux transporters. In contrast to the protective roles in normal cells, the multifaceted role of NRF2 in tumor growth and progression, resistance to therapy and intratumoral stress, and metabolic adaptation is rapidly expanding, and the complex association of NRF2 with cancer signaling networks is being unveiled. In particular, the implication of NRF2 signaling in cancer stem cells (CSCs), a small population of tumor cells responsible for therapy resistance and tumor relapse, is emerging. Here, we described the dark side of NRF2 signaling in cancers discovered so far. A particular focus was put on the role of NRF2 in CSCs maintenance and therapy resistance, showing that low ROS levels and refractory drug response of CSCs are mediated by the activation of NRF2 signaling. A better understanding of the roles of the NRF2 pathway in CSCs will allow us to develop a novel therapeutic approach to control tumor relapse after therapy.

Negative correlation of urinary miR-199a-3p level with ameliorating effects of sarpogrelate and cilostazol in hypertensive diabetic nephropathy.

The prevalence of chronic kidney disease is increasing globally; however, effective therapeutic options are limited. In this study, we aimed to identify urinary miRNAs reflecting the effect of therapeutic intervention in rats with comorbid hypertension and diabetes. Additionally, the potential beneficial effects of anti-platelet sarpogrelate and cilostazol were investigated. Nephropathy progression in streptozotocin (STZ)-treated spontaneously hypertensive rats (SHRs), including albuminuria, collagen deposition, and histopathological changes, was alleviated by sarpogrelate and antihypertensive agent telmisartan. Global analysis of urinary miRNAs identified that miR-199a-3p was commonly reduced by sarpogrelate and telmisartan treatment. In vitro analysis suggested CD151 as a target gene of miR-199a-3p: miR-199a-3p overexpression repressed CD151 levels and miR-199a-3p interacted with the 3'-untranslated region of the CD151 gene. In addition, we demonstrated that the miR-199a-3p/CD151 axis is associated with the transforming growth factor-ß1 (TGF-ß1)-induced fibrogenic pathway. TGF-ß1 treatment led to miR-199a-3p elevation and CD151 suppression, and miR-199a-3p overexpression or CD151-silencing enhanced TGF-ß1-inducible collagen IV and α-smooth muscle actin (α-SMA) levels. In vivo analysis showed that the decrease in CD151 and the increase in collagen IV and α-SMA in the kidney from STZ-treated SHR were restored by sarpogrelate and telmisartan administration. In an additional animal experiment using cilostazol and telmisartan, there was a correlation between urinary miR-199a-3p reduction and the ameliorating effects of cilostazol or combination with telmisartan. Collectively, these results indicate that urinary miR-199a-3p might be utilized as a marker for nephropathy treatment. We also provide evidence of the benefits of antiplatelet sarpogrelate and cilostazol in nephropathy progression.

Role of CD133/NRF2 Axis in the Development of Colon Cancer Stem Cell-Like Properties.

Cancer stem cells (CSCs) exhibit intrinsic therapy/stress resistance, which often cause cancer recurrence after therapy. In this study, we investigated the potential relationship between the cluster of differentiation (CD)-133, a CSC marker of colon cancer, and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a master transcription factor for the regulation of multiple antioxidant genes. In the first model of CSC, a sphere culture of the colorectal cell line HCT116, showed increased levels of CD133 and NRF2. Silencing of CD133 reduced the levels of CSC markers, such as Kruppel-like factor 4 (KLF4) and ATP-binding cassette subfamily G member 2 (ABCG2), and further suppressed the expression levels of NRF2 and its target genes. As a potential molecular link, CD133-mediated activation of phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT) signaling appears to increase the NRF2 protein levels via phosphorylation and the consequent inhibition of glycogen synthase kinase (GSK)-3ß. Additionally, NRF2-silenced HCT116 cells showed attenuated sphere formation capacity and reduced CSC markers expression, indicating the critical role of the NRF2 pathway in the development of CSC-like properties. As a second model of CSC, the CD133high cell population was isolated from HCT116 cells. CSC-like properties, including sphere formation, motility, migration, colony formation, and anticancer resistance, were enhanced in the CD133high population compared to CD133low HCT116 cells. Levels of NRF2, which were elevated in CD133high HCT116, were suppressed by CD133-silencing. In line with these, the analysis of The Cancer Genome Atlas (TCGA) database showed that high levels of CD133 expression are correlated with increased NRF2 signaling, and alterations in CD133 gene or expression are associated with unfavorable clinical outcome in colorectal carcinoma patients. These results indicate that the CD133/NRF2 axis contributes to the development of CSC-like properties in colon cancer cells, and that PI3K/AKT signaling activation is involved in CD133-mediated NRF2 activation.

NRF2 level is negatively correlated with TGF-ß1-induced lung cancer motility and migration via NOX4-ROS signaling.

Transforming growth factor-ß1 (TGF-ß1) is a multifaceted factor in cancer biology that regulates cell proliferation and migration. Overactivation of nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) in cancers has been associated with facilitated tumor growth and therapy resistance; however, role in cancer migration has not been clearly explained yet. In this study, we investigated the role of NRF2 on TGF-ß1-induced cell motility/migration. In NRF2-silenced lung cancer A549 cells, both basal and TGF-ß1-inducible cell motility/migration increased compared to those in A549. SMAD transcription activity and phosphorylated SMAD2/3 levels were higher in TGF-ß1-treated NRF2-low A549 cells than those in A549. Notably, the levels of reactive oxygen species (ROS) that were elevated by TGF-ß1 treatment were higher in the NRF2-low A549 than those in control cells, and treatment with ROS scavenger blocked TGF-ß1-induced cell motility. As an underlying molecular link, NADPH oxidase 4 (NOX4) was associated with higher ROS elevation and cell motility of NRF2-low A549. NOX4 and TGF-ß1-inducible NOX4 levels were higher in NRF2-low A549 cells than those in A549. Moreover, the pharmacological inhibition of NOX4 blocked the TGF-ß1-induced motility of NRF2-low A549 cells. Collectively, these results indicate that TGF-ß1-induced cell motility/migration is facilitated in NRF2-inhibited lung cancer cells and that high levels of NOX4/ROS are associated with enhanced motility/migration.

Inhibition of cytochrome P450 2B6 by Astragalus extract mixture HT042.

Astragalus extract mixture (AEM) HT042 is a functional food approved by the MFDS (Korean FDA) for increasing height. It comprises a mixture of three standardized extracts from Astragalus membranaceus root, Eleutherococcus senticosus stem, and Phlomis umbrosa root. In this study, drug-functional food interaction was analyzed using six major human cytochrome P450 enzymes. The inhibitory effect of AEM HT042 on P450 activities was studied using a P450-NADPH P450 reductase reconstitution system. Among the six P450 enzymes (1A2, 2A6, 2B6, 2D6, 2C9, and 3A4), only P450 2B6 activity was markedly decreased by AEM HT042 addition. The bupropion hydroxylation activity of P450 2B6 was analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A calculated IC50 value of 10.62 µg/ml was obtained. To identify the inhibitory compounds in the mixture, four active compounds in AEM HT042 were analyzed. Shanzhiside methylester exhibited inhibitory effects on P450 2B6, whereas formononetin, eleutheroside E, and sesamoside did not affect P450 2B6 activity at all. Our results suggest that shanzhiside methylester in AEM HT042 is responsible for the inhibitory effect on P450 2B6 metabolism. Characterization of the inhibitory effect on P450 can help determine the safe administration of functional foods along with many clinical drugs that are metabolized by P450.

Bardoxolone ameliorates TGF-ß1-associated renal fibrosis through Nrf2/Smad7 elevation.

Transforming growth factor-ß (TGF-ß) is a potent pathogenic factor of renal injury through the upregulation of extracellular matrix (ECM) expression and facilitation of renal fibrosis. Nuclear factor erythroid 2-like 2 (Nfe2l2; Nrf2), a master regulator of antioxidant and detoxifying systems, is mainly controlled by the binding with cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) and subsequent proteasomal degradation. The protective effect of Nrf2 on renal injury has been attributed to its antioxidant role, where it aids in coping with oxidative stress-associated progression of renal disease. In this study, we investigated the effect of Nrf2 activation on ECM production and TGF-ß/Smad signaling using Keap1-silenced MES-13 cells (a genetic glomerular mesangial cell model with Nrf2 overexpression). The TGF-ß1-inducible expression of fibronectin and α-smooth muscle actin (α-Sma) was suppressed and Smad2/3 phosphorylation was blocked in Nrf2-high mesangial cells as compared with that in control cells. Notably, in these Nrf2-high mesangial cells, levels of TGF-ß1 receptor 1 (TßR1) were substantially diminished, and the protein levels of Smad7, an inhibitor TGF-ß1/Smad signaling, were increased. Nrf2-mediated Smad7 elevation and its anti-fibrotic role in Keap1-silenced cells were confirmed by studies with Nrf2-or Smad7-silencing. As a molecular link for Smad7 elevation in Nrf2-high cells, the reduction of Smad-ubiquitination-regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was notable. Silencing of Smurf1 increased Smad7 in the control mesangial cells; however, forced expression of Smurf1 repressed Smad7 levels in Keap1-silenced cells. Additionally, we demonstrate that bardoxolone (BARD; CDDO-methyl), a pharmacological activator of Nrf2, increased Smad7 levels and attenuated TGF-ß/Smad/ECM expression in MES-13. Moreover, in an aristolochic acid (AA)-mediated nephropathy mouse model, the renal expression of Nrf2 and Smad7 was elevated by BARD treatment, and AA-induced tubular necrosis and interstitial fibrosis were substantially ameliorated by BARD. Collectively, these results indicate that the Nrf2-Smad7 axis plays a key role in the protection of TGF-ß-induced renal fibrosis, and further suggest a novel molecular mechanism of beneficial effect of BARD on renal disease.

Impairment of HIF-1α-mediated metabolic adaption by NRF2-silencing in breast cancer cells.

Hypoxia, a common element in the tumor environment, leads to Hypoxia-Inducible Factor-1α (HIF-1α) stabilization to modulate cellular metabolism as an adaptive response. In a previous study, we showed that inhibition of the nuclear factor erythroid 2-like-2 (NFE2L2; NRF2), a master regulator of many genes coping with electrophilic and oxidative stress, elevated the level of miR-181c and induced mitochondrial dysfunction in colon cancer cells. In this study, we demonstrate that NRF2-silencing hindered HIF-1α accumulation in hypoxic breast cancer cells and subsequently suppressed hypoxia-inducible expression of glycolysis-associated glucose transporter-1, hexokinase-2, pyruvate dehydrogenase kinase-1, and lactate dehydrogenase A. HIF-1α dysregulation in NRF2-silenced cancer cells was associated with miR-181c elevation. Overexpression of miR-181c in breast cancer cells blocked HIF-1α accumulation and diminished hypoxia-inducible levels of glycolysis enzymes, whereas the inhibition of miR-181c in NRF2-silenced cells restored HIF-1α accumulation. In a subsequent metabolomic analysis, hypoxic incubation increased the levels of metabolites involved in glycolysis and activated the pentose phosphate pathway (PPP) in control cells. However, these elevations were less pronounced in NRF2-silenced cells. In particular, hypoxic incubation increased the levels of amino acids, which implies a shift to catabolic metabolism, and the increased levels were higher in control cells than in NRF2-silenced cells. Concurrently, hypoxia activated BCL2 interacting protein 3 (BNIP3)-mediated autophagy in the control cells and miR-181c was found to be involved in this autophagy activation. Taken together, these results show that hypoxia-induced metabolic changes to glycolysis, the PPP, and autophagy are inhibited by NRF2-silencing through miR-181c-mediated HIF-1α dysregulation. Therefore, targeting NRF2/miR-181c could be an effective strategy to counteract HIF-1α-orchestrated metabolic adaptation of hypoxic cancer cells.

Regulatory crosstalk between the oxidative stress-related transcription factor Nfe2l2/Nrf2 and mitochondria.

Mitochondria play essential roles in cellular bioenergetics, biosynthesis, and apoptosis. During the process of respiration and oxidative phosphorylation, mitochondria utilize oxygen to generate ATP, and at the same time, there is an inevitable generation of reactive oxygen species (ROS). As excess ROS create oxidative stress and damage cells, the proper function of the antioxidant defense system is critical for eukaryotic cell survival under aerobic conditions. Nuclear factor, erythroid 2-like 2 (Nfe2l2/Nrf2) is a master transcription factor for regulating basal as well as inducible expression of multiple antioxidant proteins. Nrf2 has been involved in maintaining mitochondrial redox homeostasis by providing reduced forms of glutathione (GSH); the reducing cofactor NADPH; and mitochondrial antioxidant enzymes such as GSH peroxidase 1, superoxide dismutase 2, and peroxiredoxin 3/5. In addition, recent research advances suggest that Nrf2 contributes to mitochondrial regulation through more divergent intermolecular linkages. Nrf2 has been positively associated with mitochondrial biogenesis through the direct upregulation of mitochondrial transcription factors and is involved in the mitochondrial quality control system through mitophagy activation. Moreover, several mitochondrial proteins participate in regulating Nrf2 to form a reciprocal regulatory loop between mitochondria and Nrf2. Additionally, Nrf2 modulation in cancer cells leads to changes in the mitochondrial respiration system and cancer bioenergetics that overall affect cancer metabolism. In this review, we describe recent experimental observations on the relationship between Nrf2 and mitochondria, and further discuss the effects of Nrf2 on cancer mitochondria and metabolism.

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.

Overexpression of CD44 Standard Isoform Upregulates HIF-1α Signaling in Hypoxic Breast Cancer Cells.

Cluster of differentiation 44 (CD44), a cell surface receptor for hyaluronic acid (HA), is involved in aggressive cancer phenotypes. Herein, we investigated the role of the CD44 standard isoform (CD44s) in hypoxia-inducible factor-1α (HIF-1α) regulation using MCF7 overexpressing CD44s (pCD44s-MCF7). When pCD44s-MCF7 was incubated under hypoxia, levels of HIF-1α, vascular endothelial growth factor, and the HIF-1α response element-derived luciferase activity were significantly increased compared to those in the control MCF7. Incubation of pCD44s-MCF7 cells with HA further increased HIF-1α accumulation, and the silencing of CD44s attenuated HIF-1α elevation, which verifies the role of CD44s in HIF-1α regulation. In addition, the levels of phosphorylated extracellular signal-regulated kinase (ERK) was higher in hypoxic pCD44s-MCF7 cells, and HIF-1α accumulation was diminished by the pharmacological inhibitors of ERK. CD44s-mediated HIF-1α augmentation resulted in two functional outcomes. First, pCD44s-MCF7 cells showed facilitated cell motility under hypoxia via the upregulation of proteins associated with epithelialmesenchymal transition, such as SNAIL1 and ZEB1. Second, pCD44s-MCF7 cells exhibited higher levels of glycolytic proteins, such as glucose transporter-1, and produced higher levels of lactate under hypoxa. As a consequence of the enhanced glycolytic adaptation to hypoxia, pCD44s-MCF7 cells exhibited a higher rate of cell survival under hypoxia than that of the control MCF7, and glucose deprivation abolished these differential responses of the two cell lines. Taken together, these results suggest that CD44s activates hypoxia-inducible HIF-1α signaling via ERK pathway, and the CD44s-ERK-HIF-1α pathway is involved in facilitated cancer cell viability and motility under hypoxic conditions.

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.

NFE2L2/NRF2 Activity Is Linked to Mitochondria and AMP-Activated Protein Kinase Signaling in Cancers Through miR-181c/Mitochondria-Encoded Cytochrome c Oxidase Regulation.

AIMS: The nuclear factor (erythroid-derived 2)-like 2 (NFE2L2; NFE2L2/NRF2) pathway contributes to the environmental resistance of cancers by enhancing the antioxidant capacity. Here, we explored the potential connection between NFE2L2/NRF2 and mitochondrial function in cancers. RESULTS: Global miRNA expression analysis of HT29 and HCT116 human colon cancer cells identified that NFE2L2/NRF2 silencing upregulated miR-181c through nuclear factor-κB signaling, and this increase was associated with the reduction in mitochondria-encoded cytochrome c oxidase subunit-1 (MT-CO1), a catalytic core subunit of the complex IV of the electron transport chain (ETC). As a result of ETC dysfunction, NFE2L2/NRF2-silenced cancer cells exhibited the decreases in the mitochondrial membrane potential, oxygen consumption rate, and cellular adenosine triphosphate (ATP) contents. Notably, these changes induced adenosine monophosphate (AMP)-activated protein kinase-α (AMPKα) activation and subsequent metabolic adaptation signaling, including the inhibition of fatty acid and sterol biosynthesis enzymes. As supportive evidence of AMPKα-driven adaption, NFE2L2/NRF2-silenced cells were more vulnerable to AMPKα inhibition-induced growth suppression. Similarly, mouse tumor xenografts derived from NFE2L2/NRF2-silenced HT29 exhibited MT-CO1 reduction and AMPKα activation, thereby increasing responsiveness to the AMPK inhibitor treatment. The association of NFE2L2/NRF2 with MT-CO1 and AMPKα was confirmed in breast cancer cells. INNOVATION: We demonstrated the significance of NFE2L2/NRF2 in cancer mitochondria by elucidating the involvement of miR-181c/MT-CO1 as underlying molecular events. We also provide evidence of the crosstalk between NFE2L2/NRF2 and AMPKα as an adaptive link in cancers. CONCLUSION: Therefore, it may be an effective strategy to inhibit both NFE2L2/NRF2 and AMPKα signaling to overcome adaptive behaviors of cancer. Antioxid. Redox Signal. 27, 945-961.

Tempo-spatial Activation of Sequential Quadruple Stimuli for High Gene Expression of Polymeric Gene Nanocomplexes.

The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications.

Amelioration of high fat diet-induced nephropathy by cilostazol and rosuvastatin.

Multiple comorbidities of metabolic disorders are associated with facilitated chronic kidney disease progression. Anti-platelet cilostazol is used for the treatment of peripheral artery disease. In this study, we investigated the potential beneficial effects of cilostazol and rosuvastatin on metabolic disorder-induced renal dysfunctions. C57BL/6 mice that received high fat diet (HFD) for 22 weeks and a low dose of streptozotocin (STZ, 40 mg/kg) developed albuminuria and had increased urinary cystatin C excretion, and cilostazol treatment (13 weeks) improved these markers. Histopathological changes, including glomerular mesangial expansion, tubular vacuolization, apoptosis, and lipid accumulation were ameliorated by cilostazol treatment. Tubulointerstitial fibrosis that was indicated by the increases in collagen and transforming growth factor-ß1 subsided by cilostazol. Renoprotective effects were also observed in rosuvastatin-treated mice, and combinatorial treatment with cilostazol and rosuvastatin demonstrated enhanced ameliorative effects in histopathological evaluations. Notably, repressed renal heme oxygenase-1 (Ho-1) level in HFD/STZ mice was restored in cilostazol group. Further, we demonstrated that cilostazol enhanced Nrf2/Ho-1 signaling in cultured proximal tubular epithelial cells. Collectively, these results suggest the potential advantageous use of cilostazol as an adjunctive therapy with statins for the amelioration of metabolic disorder-associated renal injury.

NFE2L2/NRF2 silencing-inducible miR-206 targets c-MET/EGFR and suppresses BCRP/ABCG2 in cancer cells.

The nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) plays a critical role in the expression of multiple antioxidant and detoxifying enzymes. Herein, we provide evidence of the molecular links between NRF2 and oncogenic signaling hepatocyte growth factor receptor (HGFR/c-MET) and epidermal growth factor receptor (EGFR). Interfering RNA-induced stable inhibition of NRF2 in ovarian carcinoma SKOV3 and renal carcinoma A498 reduced the levels of c-MET and EGFR. MicroRNA-206 (miR-206) that was increased in both NRF2-silenced cells was predicted as a dual regulator of c-MET and EGFR. As experimental evidence, miR-206 decreased c-MET and EGFR levels through a direct binding to the 3'-untranslated region of the c-MET and EGFR genes. The treatment of NRF2-knockdown cells with the miR-206 inhibitor could restore c-MET and EGFR levels. The miR-206-mediated c-MET/EGFR repression resulted in two outcomes. First, presumably through the inhibition of c-MET/EGFR-dependent cell proliferation, overexpression of miR-206 inhibited tumor growth in SKOV3-inoculated nude mice. Second, reduced c-MET/EGFR in NRF2-silenced cells affected breast cancer resistance protein (BCRP/ABCG2) levels. The pharmacological and genetic inhibition of c-MET or EGFR, as well as the miR-206 mimic treatment, repressed BCRP levels and increased cellular accumulation of doxorubicin. In line with these, treatment of NRF2-silenced SKOV3 with the miR-206 inhibitor elevated BCRP levels and consequently made these cells more resistant to doxorubicin treatment. Collectively, our results demonstrated that the NRF2 silencing-inducible miR-206 targeted both c-MET and EGFR, and subsequently suppressed the BCRP level in cancer cells.