NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

Transcription factor NRF2 is an important regulator of oxidative stress. It is involved in cancer progression, and has abnormal constitutive expression in acute myeloid leukaemia (AML). Posttranscriptional regulation by microRNAs (miRNAs) can affect the malignant phenotype of AML cells. In this study, we identified and characterised NRF2-regulated miRNAs in AML. An miRNA array identified miRNA expression level changes in response to NRF2 knockdown in AML cells. Further analysis of miRNAs concomitantly regulated by knockdown of the NRF2 inhibitor KEAP1 revealed the major candidate NRF2-mediated miRNAs in AML. We identified miR-125B to be upregulated and miR-29B to be downregulated by NRF2 in AML. Subsequent bioinformatic analysis identified putative NRF2 binding sites upstream of the miR-125B1 coding region and downstream of the mir-29B1 coding region. Chromatin immunoprecipitation analyses showed that NRF2 binds to these antioxidant response elements (AREs) located in the 5′ untranslated regions of miR-125B and miR-29B. Finally, primary AML samples transfected with anti-miR-125B antagomiR or miR-29B mimic showed increased cell death responsiveness either alone or co-treated with standard AML chemotherapy. In summary, we find that NRF2 regulation of miR-125B and miR-29B acts to promote leukaemic cell survival, and their manipulation enhances AML responsiveness towards cytotoxic chemotherapeutics

Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53

BACKGROUND: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. METHODS: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. RESULTS: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. CONCLUSION: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients

Mechanism of Chemical Activation of Nrf2

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation

Modalities for image- and molecular-guided cancer surgery

Background: Surgery is the cornerstone of treatment for many solid tumours. A wide variety of imaging modalities are available before surgery for staging, although surgeons still rely primarily on visual and haptic cues in the operating environment. Image and molecular guidance might improve the adequacy of resection through enhanced tumour definition and detection of aberrant deposits. Intraoperative modalities available for image- and molecular-guided cancer surgery are reviewed here. Methods: Intraoperative cancer detection techniques were identified through a systematic literature search, with selection of peer-reviewed publications from January 2012 to January 2017. Modalities were reviewed, described and compared according to 25 predefined characteristics. To summarize the data in a comparable way, a three-point rating scale was applied to quantitative characteristics. Results: The search identified ten image- and molecular-guided surgery techniques, which can be divided into four groups: conventional, optical, nuclear and endogenous reflectance modalities. Conventional techniques are the most well known imaging modalities, but unfortunately have the drawback of a defined resolution and long acquisition time. Optical imaging is a real-time modality; however, the penetration depth is limited. Nuclear modalities have excellent penetration depth, but their intraoperative use is limited by the use of radioactivity. Endogenous reflectance modalities provide high resolution, although with a narrow field of view. Conclusion: Each modality has its strengths and weaknesses; no single technique will be suitable for all surgical procedures. Strict selection of modalities per cancer type and surgical requirements is required as well as combining techniques to find the optimal balance

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors

Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes:demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds

To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated ≥2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to ∼80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1–NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human