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

MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism

AbstractRecent studies connect MDM2 with increased cell motility, invasion and/or metastasis proposing an MDM2-mediated ubiquitylation-dependent mechanism. Interestingly, in renal cell carcinoma (RCC) p53/MDM2 co-expression is associated with reduced survival which is independently linked with metastasis. We therefore investigated whether expression of p53 and/or MDM2 promotes aggressive cell phenotypes. Our data demonstrate that MDM2 promotes increased motility and invasiveness in RCC cells (N.B. similar results are obtained in non-RCC cells). This study shows for the first time both that endogenous MDM2 significantly contributes to cell motility and that this does not depend upon the MDM2 RING-finger, i.e. is independent of ubiquitylation (and NEDDylation). Our data suggest that protein–protein interactions provide a likely mechanistic basis for MDM2-promoted motility which may constitute future therapeutic targets

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

Activation of the NRF2 Signaling Pathway by Copper-Mediated Redox Cycling of Para- and Ortho-Hydroquinones

SummaryTranscription factor NF-E2 p45-related factor 2 (Nrf2) mediates adaptation to oxidants and electrophiles through up-regulating genes that contain antioxidant response elements (AREs) in their promoters. Using the stably transfected human AREc32 reporter cell line, we found that copper and other transition metals enhanced induction of ARE-driven luciferase by 2-tert-butyl-1,4-hydroquinone (tBHQ) as a result of increased oxidation to 2-tert-butyl-1,4-benzoquinone (tBQ). Following exposure to tBHQ for 30 min, ARE-luciferase activity measured after 24 hr was dependent on the presence of Cu2+. In contrast, tBQ-induced activity was Cu2+-independent. The metal-catalyzed oxidation of tBHQ to tBQ occured rapidly and stoichiometrically. Compounds that share para- or ortho-hydroquinone structures, such as catechol estrogens, dopamine, and l-DOPA, also induced ARE-driven luciferase in a Cu2+-dependent manner. Thus, the oxidation of para- and ortho-hydroquinones to quinones represents the rate-limiting step in the activation of Nrf2

MDM2 interacts with NME2 (non-metastatic cells 2, protein) and suppresses the ability of NME2 to negatively regulate cell motility

MDM2 expression, combined with increased p53 expression, is associated with reduced survival in several cancers, but is particularly of interest in renal cell carcinoma (RCC) where evidence suggests the presence of tissue-specific p53/MDM2 pathway defects. We set out to identify MDM2-interacting proteins in renal cells that could act as mediators/targets of MDM2 oncogenic effects in renal cancers. We identified the non-metastatic cells 2, protein; NME2 (NDPK-B, NM23-B/-H2), a nucleoside diphosphate kinase, as an MDM2-interacting protein using both a proteomic-based strategy [affinity chromatography and tandem mass spectrometry [MS/MS] from HEK293 cells] and a yeast two-hybrid screen of a renal carcinoma cell-derived complementary DNA library. The MDM2–NME2 interaction is highly specific, as NME1 (87.5% amino acid identity) does not interact with MDM2 in yeast. Specific NME proteins display well-documented cell motility and metastasis-suppressing activity. We show that NME2 contributes to motility suppression under conditions where MDM2 is expressed at normal physiological/low levels. However, up-regulation of MDM2 in RCC cells abolishes the ability of NME2 to suppress motility. Significantly, when MDM2 expression is down-regulated in these cells using small interfering RNA, the motility-suppressing activity of NME2 is rescued, confirming that MDM2 expression causes the loss of NME2 cell motility regulatory function. Thus MDM2 up-regulation in renal cancer cells can act in a dominant manner to abrogate the function of a potent suppressor of motility and metastasis. Our studies identify a novel protein–protein interaction between MDM2 and NME2, which suggests a mechanism that could explain the link between MDM2 expression and poor patient survival in RCC