NRF2 regulates HER1 signaling pathway to modulate the sensitivity of ovarian cancer cells to lapatinib and erlotinib

NF-E2-related factor 2 (NRF2) regulates the transcription of a battery of metabolic and cytoprotective genes. NRF2 and epidermal growth factor receptors (EGFRs/HERs) are regulators of cellular proliferation and determinants of cancer initiation and progression. NRF2 and HERs confer cancers with resistance to several therapeutic agents. Nevertheless, there is limited understanding of the regulation of HER expression and activation and the link between NRF2 and HER signalling pathways. We show that NRF2 regulates both basal and inducible expression of HER1, as treatment of ovarian cancer cells (PEO1, OVCAR3, and SKOV3) with NRF2 activator tBHQ inducing HER1, while inhibition of NRF2 by siRNA knockdown or with retinoid represses HER1. Furthermore, treatment of cells with tBHQ increased total and phosphorylated NRF2, HER1, and AKT levels and compromised the cytotoxic effect of lapatinib or erlotinib. Treatment with siRNA or retinoid antagonised the effect of tBHQ on NRF2 and HER1 levels and enhanced the sensitivity of ovarian cancer cells to lapatinib or erlotinib. Pharmacological or genetic inhibition of NRF2 and/or treatment with lapatinib or erlotinib elevated cellular ROS and depleted glutathione. This extends the understanding of NRF2 and its regulation of HER family receptors and opens a strategic target for improving cancer therapy

A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target

Role of G{alpha}12 and G{alpha}13 as Novel Switches for the Activity of Nrf2, a Key Antioxidative Transcription Factor

G{alpha}12 and G{alpha}13 function as molecular regulators responding to extracellular stimuli. NF-E2-related factor 2 (Nrf2) is involved in a protective adaptive response to oxidative stress. This study investigated the regulation of Nrf2 by G{alpha}12 and G{alpha}13. A deficiency of G{alpha}12, but not of G{alpha}13, enhanced Nrf2 activity and target gene transactivation in embryo fibroblasts. In mice, G{alpha}12 knockout activated Nrf2 and thereby facilitated heme catabolism to bilirubin and its glucuronosyl conjugations. An oligonucleotide microarray demonstrated the transactivation of Nrf2 target genes by G{alpha}12 gene knockout. G{alpha}12 deficiency reduced Jun N-terminal protein kinase (JNK)-dependent Nrf2 ubiquitination required for proteasomal degradation, and so did G{alpha}13 deficiency. The absence of G{alpha}12, but not of G{alpha}13, increased protein kinase C {delta} (PKC {delta}) activation and the PKC {delta}-mediated serine phosphorylation of Nrf2. G{alpha}13 gene knockout or knockdown abrogated the Nrf2 phosphorylation induced by G{alpha}12 deficiency, suggesting that relief from G{alpha}12 repression leads to the G{alpha}13-mediated activation of Nrf2. Constitutive activation of G{alpha}13 promoted Nrf2 activity and target gene induction via Rho-mediated PKC {delta} activation, corroborating positive regulation by G{alpha}13. In summary, G{alpha}12 and G{alpha}13 transmit a JNK-dependent signal for Nrf2 ubiquitination, whereas G{alpha}13 regulates Rho-PKC {delta}-mediated Nrf2 phosphorylation, which is negatively balanced by G{alpha}12

Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to β-lapachone

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent β-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to β-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of β-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to β-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to β-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to β-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to β-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations

Viral delivery of antioxidant genes as a therapeutic strategy in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no effective treatment to date. Despite its multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of antioxidant genes that are downregulated in the presence of mutant SOD1 may serve as a therapeutic strategy for motor neuronal protection. Lentiviral vectors expressing either PRDX3 or NRF2 genes were tested in the motor neuronal-like NSC34 cell line, and in the ALS tissue culture model, NSC34 cells expressing the human SOD1(G93A) mutation. The NSC34 SOD1(G93A) cells overexpressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40 and 50% respectively compared with controls, whereas cell survival was increased by 30% in both cases. The neuroprotective potential of those two genes was further investigated in vivo in the SOD1(G93A) ALS mouse model, by administering intramuscular injections of adenoassociated virus serotype 6 (AAV6) expressing either of the target genes at a presymptomatic stage. Despite the absence of a significant effect in survival, disease onset or progression, which can be explained by the inefficient viral delivery, the promising in vitro data suggest that a more widespread CNS delivery is needed

Spongionella secondary metabolites protect mitochondrial function in cortical neurons against oxidative stress

Accepted: 8 January 2014 This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acknowledgments The research leading to these results has received funding from the following FEDER cofunded-grants: From Ministerio de Ciencia y Tecnología, Spain: AGL2009-13581-CO2-01, AGL2012-40485-CO2-01. From Xunta de Galicia, Spain: 10PXIB261254 PR. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement Nos. 265896 BAMMBO, 265409 µAQUA, and 262649 BEADS, 315285 CIGUATOOLS and 312184 PHARMASEA. From the Atlantic Area Programme (Interreg IVB Trans-national): 2009-1/117 Pharmatlantic. MER thanks the Government of the Arab Republic of Egypt for a PhD Scholarship. MJ thanks the Scottish University Life Science Alliance which provided funding to set up the compound library.Peer reviewedPublisher PD

Quantitative analysis of NRF2 pathway reveals key elements of the regulatory circuits underlying antioxidant response and proliferation of ovarian cancer cells

Cells are constantly exposed to Reactive Oxygen Species (ROS) produced both endogenously to meet physiological requirements and from exogenous sources. While endogenous ROS are considered as important signalling molecules, high uncontrollable ROS are detrimental. It is unclear how cells can achieve a balance between maintaining physiological redox homeostasis and robustly activate the antioxidant system to remove exogenous ROS. We have utilised a Systems Biology approach to understand how this robust adaptive system fulfils homeostatic requirements of maintaining steady-state ROS and growth rate, while undergoing rapid readjustment under challenged conditions. Using a panel of human ovarian and normal cell lines, we experimentally quantified and established interrelationships between key elements of ROS homeostasis. The basal levels of NRF2 and KEAP1 were cell line specific and maintained in tight correlation with their growth rates and ROS. Furthermore, perturbation of this balance triggered cell specific kinetics of NRF2 nuclear–cytoplasmic relocalisation and sequestration of exogenous ROS. Our experimental data were employed to parameterise a mathematical model of the NRF2 pathway that elucidated key response mechanisms of redox regulation and showed that the dynamics of NRF2-H2O2 regulation defines a relationship between half-life, total and nuclear NRF2 level and endogenous H2O2 that is cell line specific

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury

FAM129B, an antioxidative protein, reduces chemosensitivity by competing with Nrf2 for Keap1 binding.

BackgroundThe transcription factor Nrf2 is a master regulator of antioxidant response. While Nrf2 activation may counter increasing oxidative stress in aging, its activation in cancer can promote cancer progression and metastasis, and confer resistance to chemotherapy and radiotherapy. Thus, Nrf2 has been considered as a key pharmacological target. Unfortunately, there are no specific Nrf2 inhibitors for therapeutic application. Moreover, high Nrf2 activity in many tumors without Keap1 or Nrf2 mutations suggests that alternative mechanisms of Nrf2 regulation exist.MethodsInteraction of FAM129B with Keap1 is demonstrated by immunofluorescence, colocalization, co-immunoprecipitation and mammalian two-hybrid assay. Antioxidative function of FAM129B is analyzed by measuring ROS levels with DCF/flow cytometry, Nrf2 activation using luciferase reporter assay and determination of downstream gene expression by qPCR and wester blotting. Impact of FAM129B on in vivo chemosensitivity is examined in mice bearing breast and colon cancer xenografts. The clinical relevance of FAM129B is assessed by qPCR in breast cancer samples and data mining of publicly available databases.FindingsWe have demonstrated that FAM129B in cancer promotes Nrf2 activity by reducing its ubiquitination through competition with Nrf2 for Keap1 binding via its DLG and ETGE motifs. In addition, FAM129B reduces chemosensitivity by augmenting Nrf2 antioxidative signaling and confers poor prognosis in breast and lung cancer.InterpretationThese findings demonstrate the important role of FAM129B in Nrf2 activation and antioxidative response, and identify FMA129B as a potential therapeutic target. FUND: The Chang Gung Medical Foundation (Taiwan) and the Ministry of Science and Technology (Taiwan)