NNMT contributes to high metastasis of triple negative breast cancer by enhancing PP2A/MEK/ERK/c-Jun/ABCA1 pathway mediated membrane fluidity.

Elucidating the mechanism for high metastasis capacity of triple negative breast cancers (TNBC) is crucial to improve treatment outcomes of TNBC. We have recently reported that nicotinamide N-methyltransferase (NNMT) is overexpressed in breast cancer, especially in TNBC, and predicts poor survival of patients undergoing chemotherapy. Here, we aimed to determine the function and mechanism of NNMT on metastasis of TNBC. Additionally, analysis of public datasets indicated that NNMT is involved in cholesterol metabolism. In vitro, NNMT overexpression promoted migration and invasion of TNBCs by reducing cholesterol levels in the cytoplasm and cell membrane. Mechanistically, NNMT activated MEK/ERK/c-Jun/ABCA1 pathway by repressing protein phosphatase 2A (PP2A) activity leading to cholesterol efflux and membrane fluidity enhancement, thereby promoting the epithelial-mesenchymal transition (EMT) of TNBCs. In vivo, the metastasis capacity of TNBCs was weakened by targeting NNMT. Collectively, our findings suggest a new molecular mechanism involving NNMT in metastasis and poor survival of TNBC mediated by PP2A and affecting cholesterol metabolism.

p38 MAPK Is a Major Regulator of Amyloid Beta-Induced IL-6 Expression in Human Microglia.

The accumulation of amyloid beta (Aß) plaques in the brain is a hallmark of Alzheimer's disease (AD) pathology. Microglial activation-mediated neuroinflammation has been implicated in the pathogenesis of AD and the expression levels of interleukin-6 (IL-6) were increased in the brains of AD patients. However, the mechanisms by which IL-6 expression is regulated in human microglia are incompletely understood. Here, we show that Aß1-40 oligomers (Aß40) dose-dependently stimulate IL-6 expression in HMC3 human microglial cells. Treatment with Aß40 promotes the transcription of IL-6 and tumor necrosis factor α (TNFα) mRNAs in both HMC3 and THP-1 cells. Mechanistic studies reveal that Aß40-induced increase of IL-6 secretion is associated with the activation of p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK by BIRB 796 or SB202190 abrogates Aß40-induced increase of IL-6 production. Through analyzing brain specimens, we found that the immunoreactivity for IL-6 and phosphorylated (the activated form) p38 MAPK was markedly higher in microglia of AD patients than in age-matched control subjects. Moreover, our studies identified the co-localization of IL-6 with phosphorylated p38 MAPK in microglia in the cortices of AD patients. Taken together, these results indicate that p38 MAPK is a major regulator of Aß-induced IL-6 production in human microglia, which suggests that targeting p38 MAPK may represent a new approach to ameliorate Aß accumulation-induced neuroinflammation in AD.

BAY 11-7082 inhibits the secretion of interleukin-6 by senescent human microglia.

The accumulation of senescent cells in aged tissues has been implicated in a variety of age-related diseases, including cancer and neurodegenerative disorders. Recent studies have demonstrated a link between age-associated increase of senescent glial cells in the brain and the pathogenesis of Alzheimer's disease (AD). However, there is a lack of in vitro cellular models of senescent human microglia, which significantly limits our approaches to study AD pathogenesis. Here, we show for the first time that ionizing radiation (IR) dose-dependently induces premature senescence in HMC3 human microglial cells. Senescence-associated ß-galactosidase activity, a well-characterized marker of cellular senescence, was substantially increased in irradiated HMC3 cells compared with control cells. Furthermore, we found that phosphorylated p53 levels and p21 expression levels were markedly higher in IR-induced senescent microglia than in control cells. Senescent human microglia exhibited the senescence-associated secretory phenotype (SASP), as evidenced by the increased secretion of pro-inflammatory cytokine interleukin-6 (IL-6). Treatment with an NF-κB inhibitor, BAY 11-7082, inhibits the secretion of IL-6 by senescent HMC3 cells. Collectively, our studies have established an in vitro cellular model of human microglial senescence and suggest that the NF-κB pathway may play a critical role in regulating the SASP of senescent HMC3 cells.

Nrf2 inhibition sensitizes breast cancer stem cells to ionizing radiation via suppressing DNA repair.

Radiation is widely used for cancer treatment but the radioresistance properties of cancer stem cells (CSCs) pose a significant challenge to the success of cancer therapy. Nuclear factor erythroid-2-related factor 2 (Nrf2) has emerged as a prominent regulator of cellular antioxidant responses and its over-activation is associated with drug resistant in cancer cells. However, the role of Nrf2 signaling in regulating the response of CSCs to irradiation has yet to be defined. Here, we show that exposure of triple-negative breast cancer (TNBC) cells to ionizing radiation (IR) upregulates Nrf2 expression and promotes its nuclear translocation in a reactive oxygen species (ROS)-dependent manner. Ectopic overexpression of Nrf2 attenuates, whereas knockdown of Nrf2 potentiates IR-induced killing of TNBC CSCs. Mechanistically, we found that Nrf2 knockdown increases IR-induced ROS production and impedes DNA repair at least in part via inhibition of DNA-PK. Furthermore, activation of Nrf2 by sulforaphane diminishes, whereas inhibition of Nrf2 by ML385 enhances IR-induced killing of TNBC CSCs. Collectively, these results demonstrate that IR-induced ROS production can activate Nrf2 signaling, which in turn counteracts the killing effect of irradiation. Therefore, pharmacological inhibition of IR-induced Nrf2 activation by ML385 could be a new therapeutic approach to sensitize therapy-resistant CSCs to radiotherapy.

Nrf2-modulation by seleno-hormetic agents and its potential for radiation protection.

The trace element selenium (Se) is an essential component of selenoproteins and plays a critical role in redox signaling via regulating the activity of selenoenzymes such as thioredoxin reductase-1 and glutathione peroxidases. Se compounds and its metabolites possess a wide range of biological functions including anticancer and cytoprotection effects, modulation of hormetic genes and antioxidant enzyme activities. Radiation-induced injury of normal tissues is a significant side effect for cancer patients who receive radiotherapy in the clinic and the development of new and effective radioprotectors is an important goal of research. Others and we have shown that seleno-compounds have the potential to protect ionizing radiation-induced toxicities in various tissues and cells both in in vitro and in vivo studies. In this review, we discuss the potential utilization of Se compounds with redox-dependent hormetic activity as novel radio-protective agents to alleviate radiation toxicity. The cellular and molecular mechanisms underlying the radioprotection effects of these seleno-hormetic agents are also discussed. These include Nrf2 transcription factor modulation and the consequent upregulation of the adaptive stress response to IR in bone marrow stem cells and hematopoietic precursors.

Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1.

ME-344 is a second-generation isoflavone with unusual cytotoxic properties that is in clinical testing in cancer. To identify targets that contribute to its anticancer activity and therapeutic index, we used lung cancer cell lines that are naturally sensitive or resistant to ME-344. Drug-induced apoptosis was linked with enhanced levels of reactive oxygen species and this initiated a nuclear erythroid factor 2-like 2 signaling response, downstream of which, heme oxygenase 1 (HO-1) was also found to be time-dependently inhibited by ME-344. ME-344 specifically bound to, and altered, HO-1 structure and increased HO-1 translocation from the rough endoplasmic reticulum to mitochondria, but only in drug-sensitive cells. These effects did not occur in either drug-resistant or primary lung fibroblasts with lower HO-1 basal levels. HO-1 was confirmed as a drug target by using surface plasmon resonance technology and through interaction with a clickable ME-344 compound (M2F) and subsequent proteomic analyses, showing direct binding of ME-344 with HO-1. Proteomic analysis showed that clusters of mitochondrial proteins, including voltage-dependent anion-selective channels, were also impacted by ME-344. Human lung cancer biopsies expressed higher levels of Nrf2 and HO-1 compared with normal tissues. Overall, our data show that ME-344 inhibits HO-1 and impacts its mitochondrial translocation. Other mitochondrial proteins are also affected, resulting in interference in tumor cell redox homeostasis and mitochondrial function. These factors contribute to a beneficial therapeutic index and support continued clinical development of ME-344. SIGNIFICANCE: A novel cytotoxic isoflavone is shown to inhibit heme oxygenase, a desirable yet elusive target that disrupts redox homeostasis causing cell death.

Oxidative stress induces senescence in breast cancer stem cells.

Cancer stem cells (CSCs) have been shown to be resistant to current anticancer therapies and the induction of oxidative stress is an important mechanism of action for many anticancer agents. However, it is still largely unknown how CSCs respond to hydrogen peroxide (H2O2)-induced oxidative stress. Here, we show that the levels of reactive oxygen species (ROS) are markedly lower in breast CSCs (BCSCs) than that in non-cancer stem cells (NCSCs). A transient exposure of breast cancer cells to sublethal doses of H2O2 resulted in a dose-dependent increase of the epithelium-specific antigen (ESA)+/CD44+/CD24- subpopulations, a known phenotype for BCSCs. Although BCSCs survived sublethal doses of H2O2 treatment, they lost the ability to form tumor spheres and failed to generate colonies as demonstrated by mammosphere-formation and clonogenic assays, respectively. Mechanistic studies revealed that H2O2 treatment led to a marked increase of senescence-associated ß-galactosidase activity but only minimal apoptotic cell death in BCSCs. Furthermore, H2O2 triggers p53 activation and promotes p21 expression, indicating a role for the p53/p21 signaling pathway in oxidative stress-induced senescence in BCSCs. Taken together, these results demonstrate that the maintenance of a lower level of ROS is critical for CSCs to avoid oxidative stress and H2O2-induced BCSC loss of function is likely attributable to oxidative stress-triggered senescence induction, suggesting that ROS-generating drugs may have the therapeutic potential to eradicate drug-resistant CSCs via induction of premature senescence.

Nicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization.

BACKGROUND: Nicotinamide N-methyltransferase (NNMT) is overexpressed in various human tumors and involved in the development and progression of several carcinomas. In breast cancer, NNMT was found to be overexpressed in several cell lines. However, the clinical relevance of NNMT in breast cancer is not yet clear. METHODS: NNMT expression in breast carcinoma was examined by immunohistochemistry, and then, its relationship with patient clinicopathological characteristics was analyzed. The effects of NNMT on chemoresistance in breast cancer cells were assessed by cell viability, colony formation, and apoptosis assay. The NNMT, SIRT1, p53, and acetyl-p53 proteins, which are involved in NNMT-related chemoresistance, were examined by Western blotting. The SIRT1 mRNA was examined by real-time PCR, and its activity was measured by using the SIRT1 deacetylase fluorometric reagent kit. RESULTS: NNMT expression was significantly higher (53.9%) in breast carcinoma than in paracancerous tissues (10.0%) and breast hyperplasia (13.3%). A high level of NNMT expression correlated with poor survival and chemotherapy response in breast cancer patients who received chemotherapy. Ectopic overexpression of NNMT significantly inhibited the apoptotic cell death and suppression of colony formation induced by adriamycin and paclitaxel. Mechanistic studies revealed that NNMT overexpression increased SIRT1 expression and promoted its activity. Either inhibition of SIRT1 by EX527 or knockdown of SIRT1 by siRNA could reverse NNMT-mediated resistance to adriamycin and paclitaxel, which suggests that SIRT1 plays a critical role in NNMT-related chemoresistance in breast cancer. CONCLUSIONS: The results of this study demonstrate a novel correlation between the NNMT expression level and patient survival, suggesting that NNMT has the potential to become a new prognostic biomarker to predict the treatment outcomes of the clinical chemotherapy in breast cancer. Moreover, targeting NNMT or downstream SIRT1 may represent a new therapeutic approach to improve the efficacy of breast cancer chemotherapy.

A seleno-hormetine protects bone marrow hematopoietic cells against ionizing radiation-induced toxicities.

2,2'-diselenyldibenzoic acid (DSBA) is a chemical probe produced to explore the pharmacological properties of diphenyldiselenide-derived agents with seleno-hormetic activity undergoing preclinical development. The present study was designed to verify in vivo the drug's properties and to determine mechanistically how these may mediate the protection of tissues against stress conditions, exemplified by ionizing radiation induced damage in mouse bone marrow. In murine bone marrow hematopoietic cells, the drug initiated the activation of the Nrf2 transcription factor resulting in enhanced expression of downstream stress response genes. This type of response was confirmed in human liver cells and included enhanced expression of glutathione S-transferases (GST), important in the metabolism and pharmacological function of seleno-compounds. In C57 BL/6 mice, DSBA prevented the suppression of bone marrow hematopoietic cells caused by ionizing radiation exposure. Such in vivo prevention effects were associated with Nrf2 pathway activation in both bone marrow cells and liver tissue. These findings demonstrated for the first time the pharmacological properties of DSBA in vivo, suggesting a practical application for this type of Se-hormetic molecules as a radioprotective and/or prevention agents in cancer treatments.

Role of senescence induction in cancer treatment.

Cellular senescence is a form of permanent cell cycle arrest that can be triggered by a variety of cell-intrinsic and extrinsic stimuli, including telomere shortening, DNA damage, oxidative stress, and exposure to chemotherapeutic agents and ionizing radiation. Although the induction of apoptotic cell death is a desirable outcome in cancer therapy, mutations and/or deficiencies in the apoptotic signaling pathways have been frequently identified in many human cancer types, suggesting the importance of alternative apoptosis-independent therapeutic approaches for cancer treatment. A growing body of evidence has documented that senescence induction in tumor cells is a frequent response to many anticancer modalities including cyclin-dependent kinases 4/6 small molecule inhibitor-based targeted therapeutics and T helper-1 cytokine-mediated immunotherapy. This review discusses the recent advances and clinical relevance of therapy-induced senescence in cancer treatment.

MiR-34a modulates ionizing radiation-induced senescence in lung cancer cells.

MicroRNAs (miRNAs) are a new class of gene expression regulators that have been implicated in tumorigenesis and modulation of the responses to cancer treatment including that of human non-small cell lung cancer (NSCLC). However, the role of miR-34a in ionizing radiation (IR)-induced senescence in NSCLC cells remains poorly understood. Here we report that IR-induced premature senescence correlates with upregulation of miR-34a expression in NSCLC cells. Ectopic overexpression of miR-34a by transfection with synthetic miR-34a mimics markedly enhances IR-induced senescence, whereas inhibition of miR-34a by transfection with a synthetic miR-34a inhibitor attenuates IR-induced senescence. Clonogenic assays reveal that treatment with miR-34a mimics augments IR-induced cell killing in human NSCLC cells. Mechanistically, we found that the senescence-promoting effect of miR-34a is associated with a dramatic down-regulation of c-Myc (Myc) expression, suggesting that miR-34a may promote IR-induced senescence via targeting Myc. In agreement with this suggestion, knockdown of Myc expression by RNAi recapitulates the senescence-promoting effect of miR-34a and enhances IR-induced cell killing in NSCLC cells. Collectively, these results demonstrate a previously unrecognized role for miR-34a in modulating IR-induced senescence in human NSCLC cells and suggest that pharmacological intervention of miR-34a expression may represent a new therapeutic strategy for improving the efficacy of lung cancer radiotherapy.

MYC Inhibition Depletes Cancer Stem-like Cells in Triple-Negative Breast Cancer.

There is mounting evidence that cancer stem-like cells (CSC) are selectively enriched in residual tumors after anticancer therapies, which may account for tumor recurrence and metastasis by regenerating new tumors. Thus, there is a critical need to develop new therapeutic agents that can effectively eliminate drug-resistant CSCs and improve the efficacy of cancer therapy. Here, we report that Triptolide (C1572), a small-molecule natural product, selectively depletes CSCs in a dose-dependent fashion in human triple-negative breast cancer (TNBC) cell lines. Nanomolar concentrations of C1572 markedly reduced c-MYC (MYC) protein levels via a proteasome-dependent mechanism. Silencing MYC expression phenocopied the CSC depletion effects of C1572 and induced senescence in TNBC cells. Limited dilution assays revealed that ex vivo treatment of TNBC cells with C1572 reduced CSC levels by 28-fold. In mouse xenograft models of human TNBC, administration of C1572 suppressed tumor growth and depleted CSCs in a manner correlated with diminished MYC expression in residual tumor tissues. Together, these new findings provide a preclinical proof of concept defining C1572 as a promising therapeutic agent to eradicate CSCs for drug-resistant TNBC treatment. Cancer Res; 77(23); 6641-50. ©2017 AACR.

Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition.

Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD), a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM) suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs) function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794) depletes mouse BM Lin(-)Sca-1(+)c-Kit(+) cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU) assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs) in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs) but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs), respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

Catalase inhibits ionizing radiation-induced apoptosis in hematopoietic stem and progenitor cells.

Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme that coverts hydrogen peroxide into hydrogen and water. In this study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells and hematopoietic progenitor cells. Subsequent colony-forming cell and cobble-stone area-forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments, whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism by which catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.

Resveratrol enhances ionizing radiation-induced premature senescence in lung cancer cells.

Radiotherapy is used in >50% of patients during the course of cancer treatment both as a curative modality and for palliation. However, radioresistance is a major obstacle to the success of radiation therapy and contributes significantly to tumor recurrence and treatment failure, highlighting the need for the development of novel radiosensitizers that can be used to overcome tumor radioresistance and, thus, improve the efficacy of radiotherapy. Previous studies indicated that resveratrol (RV) may sensitize tumor cells to chemotherapy and ionizing radiation (IR). However, the mechanisms by which RV increases the radiation sensitivity of cancer cells have not been well characterized. Here, we show that RV treatment enhances IR-induced cell killing in non-small cell lung cancer (NSCLC) cells through an apoptosis-independent mechanism. Further studies revealed that the percentage of senescence-associated ß-galactosidase (SA-ß-gal)-positive senescent cells was markedly higher in cells treated with IR in combination with RV compared with cells treated either with IR or RV alone, suggesting that RV treatment enhances IR-induced premature senescence in lung cancer cells. Comet assays demonstrate that RV and IR combined treatment causes more DNA double-strand breaks (DSBs) than IR or RV treatment alone. DCF-DA staining and flow cytometric analyses demonstrate that RV and IR combined treatment leads to a significant increase in ROS production in irradiated NSCLC cells. Furthermore, our investigation show that inhibition of ROS production by N-acetyl-cysteine attenuates RV-induced radiosensitization in lung cancer cells. Collectively, these results demonstrate that RV-induced radiosensitization is associated with significant increase of ROS production, DNA-DSBs and senescence induction in irradiated NSCLC cells, suggesting that RV treatment may sensitize lung cancer cells to radiotherapy via enhancing IR-induced premature senescence.

Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence.

Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated ß-glactosidase (SA-ß-gal) staining, decreased BrdU incorporation and elevated expression of p16(INK4a) (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53-p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53-p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.

Resveratrol induces premature senescence in lung cancer cells via ROS-mediated DNA damage.

Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV's anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated ß-galactosidase (SA-ß-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage.