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MiR-200a inhibits epithelial-mesenchymal transition of pancreatic cancer stem cell

Background Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT. Methods Cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs. Results Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs. Conclusion In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.

Background
Pancreatic cancer is one of the most aggressive cancers, which is usually diagnosed in an advanced state for which there are few or no effective therapies [1]. One of the major hallmarks of pancreatic cancer is its extensive local tumor invasion and early systemic dissemination [2]. Over the past two decades, numerous efforts have been made in improving treatment and survival of pancreatic cancer patients but the outcome has been disappointing [3]. Emerging evidence suggest that the resistance could in fact be due to the enriched existence of tumor initiating cells, also classified as cancer stemlike cells in a tumor mass. The CSCs have the capacity of self-renewaland the potential to regenerate into all types of differentiated cells giving rise to heterogeneous tumor cell populations in a tumor mass, which contributes to tumor aggressiveness [4,5]. The existence of CSCs or cancer stem-like cells in a tumor mass is believed to be responsible for tumor recurrence because of their intrinsic and extrinsic drug-resistance characteristics [3].
Oct4 and Nanog are transcription factors essential for maintaining stem cell phenotypes. Oct4 is a POU domain-containing transcription factor. It is involved in the regulation of cell growth and differentiation in a variety of tissues. Nanog is a homeobox-containing transcription factor. Its overexpression is associated with the pluripotency and self-renewing nature of embryonic stem cells. And Oct4 and Nanog expression is associated with early stages of pancreatic carcinogenesis [6].
It has recently become clear that EMT is associated with drug resistance and cancer cell metastasis [7]. During this process, the expression of E-cadherin is downregulated, which is a transmembrane protein essential for the stable adherens junctions, and the expression of the mesenchymal molecules vimentin, fibronectin, and/or N-cadherin are up-regulated [8,9]. In pancreatic cancer cells, EMT is also reported to be a crucial step for tumor cell migration and invasion [10]. Recent studies have demonstrated that EMT plays a great role not only in tumor metastasis, but also in tumor recurrence that is believed to be tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. However, the mechanisms by which EMT cells generate the stem-like cells remain to be elucidated [11][12][13]. Importantly, emerging evidence implicated the critical role of microRNAs because they are key regulatory molecules in biological and pathologic processes including EMT [7]. MicroRNAs are small and non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either degradation of mRNA or inhibition of their translation to functional proteins [7,14]. Members of the miR-200 family are downregulated in human cancer cells and tumors due to aberrant epigenetic gene silencing and play a critical role in the suppression of EMT, tumor cell adhesion, migration, invasion and metastasis, by targeting and repressing the expression of key mRNAs that are involved in EMT (ZEB1 and ZEB2), and participates in a signalling network with the E-cadherin transcriptional repressors ZEB1/deltaEF1 and ZEB2/SIP1, and TGF-β2 that is postulated to facilitate maintenance of stable epithelial or mesenchymal states but also allow reversible switching between these states in response to EMT effectors (such as TGF-β) [15,16]. In ovarian and breast cancer, low expression of miRNA-200 plays important roles in cancer metastasis [14,17,18]. MiR-200 changed the tumor environment, inhibiting the process of EMT and metastasis [19,20]. These findings hypothesizes that the expression of miR-200 in pancreatic cancer cell is correlated with stemness, EMT and metastasis.
Since miR-200 is associated with EMT, which is believed to be associated with cancer stem cells or cancer stem-like cells, we investigated the effects of miR-200 family on pancreatic CSC functions in this study. We identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD24, CD44 and ESA in pancreatic adenocarcinoma cell line PANC-1. And CD24 + CD44 + ESA + cells in PANC-1 were sorted by BD FACS Aria II for further study. Then, we analysed the miR-200 family and transcription factors Oct4 and Nanog expression in CSCs of pancreatic cancer cell line PANC-1, and determined their relationships with EMT markers and repressors of E-cadherin transcription. In order to study the role of miR-200a for EMT in CSCs, miR-200a mimic was transformed into CSCs. In addition, invasion and metastasis were determined in CSCs and transformed CSCs.

Flow cytometry
Dissociated cells were counted and transferred to a 5 ml tube, washed twice with PBS, counted and resuspended in PBS at 1 × 10 6 cell/100 μl. Then, the antibodies APC anti-human CD44 (Becton Dickinson, USA), PE antihuman CD24 (Becton Dickinson, USA) and FITC antihuman ESA (Becton Dickinson, USA) (each at a dilution of 1: 40) were added and incubated for 20 min on ice in dark. The respective isotype control antibodies were used at the same concentrations according to the manufacturer's instructions. After washing twice with PBS, samples were resuspended in 500 μl PBS and analyzed on a flow cytometer (FACSAriaII, USA). Side-scatter and forward-scatter profiles were used to eliminate cell doublets. Cells were routinely sorted twice, and the cells were reanalyzed for purity, which typically was > 97%. Data were analyzed with BD FACS Diva software.

miR expression analysis
Total RNA was reverse transcribed using the miScript Reverse Transcription Kit (Qiagen, Valencia, CA). RT-qPCR was performed using the miScript PCR Kit (Qiagen). Experiments were normalized to U6. Results were reported as RQ with respect to a calibrator sample using the 2-ΔΔCt method. MiR-200a, miR-200b, miR-200c and U6 kits were purchased from Biomics biotech (NanTong).

Western blot analysis
Cells were lysed in RIPA lysis buffer and the protein concentration was determined. Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 5% skim milk in TBST buffer containing 0.1% Tween 20 and then incubated with indicated primary antibodies (Rabbit anti-N-cadherin, 1: 1000, Millipore; Mouse anti-Vimentin, 1: 500, Millipore; Rabbit anti-Ecadherin, 1: 1000, Abgent; Rabbit Anti-ZEB1, 1: 1000, Cell Signaling) for 2 h at room temperature. HRPconjugated secondary antibodies (HRP Goat anti-Rabbit IgG Antibody, 1: 5000, Abgent; HRP Goat anti-Mouse IgG Antibody, 1: 5000, Abgent) were incubated at room temperature (RT) for 1 h and detected using the enhanced chemiluminesence detection system.Total protein was extracted from untreated and the cells treated by transfecting miR-200a and subjected to western blot analysis as described to evaluate the expression of E-cadherin, N-cadherin, ZEB1 and vimentin. The data was adjusted against loading control using β-actin.

Transwell migration assay
For transwell migration assays, 1 × 10 5 pancreatic CSCs were plated in the top chamber onto the noncoated membrane (24-well insert; pore size, 8 mm; Corning Costar) and allowed to migrate toward serum-containing medium in the lower chamber. Cells were fixed after 48 hours of incubation with methanol and stained with 0.1% crystal violet (2 mg/mL, Sigma-Aldrich). The number of cells invading through the membrane was counted under a light microscope (three random fields per well).

Transwell invasion assay
For invasion assay, 1 × 10 5 cells were plated in the top chamber onto the Matrigel coated Membrane (24-well insert; pore size, 8 μm; Corning Costar). Each well was coated freshly with Matrigel (60 mg; BD Bioscience) before the invasion assay. Cells were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemo-attractant in the lower chamber. The cells were incubated for 48 hours and cells that did not invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were fixed with methanol and stained with crystal violet. Thenumber of cells invading through the membrane was counted under a light microscope (three random fields per well).

Statistical analyses
All values were expressed as means ± S.E.M. The statistical significance of differences among groups was determined by a one-way analysis of variance (ANOVA) followed by the Tukey's post hoc multiple comparison tests. A P < 0.05 was considered significant. Each experiment consisted of at least three replicates per condition. All statistical analyses were conducted with a STATA 7.0 software package (Stata Corp, College Station, TX) experiments.

Isolation and characterization of human pancreatic CSCs
Flow cytometric analysis was used to determine the presence of CD44, CD24 and ESA on the cell surface of the pancreatic adenocarcinoma cell lines PANC-1 ( Figure 1A and B). In PANC-1, 0.7%-2.2% of cells were CD44 + CD24 + , and 0.1%-0.9% of cells were CD44 + CD24 + ESA + . The morphology of PANC-1 cell line and CD44 + CD24 + ESA + CSCs were shown in ( Figure 1C and D). CD44 + CD24 + ESA + CSCs formed spheroid in CSC medium × 200 magnification ( Figure 1D). Nanog and Oct4 are required for the maintenance of pluripotency in embryonic stem cells and induce cancer stem cell-like properties [21]. To determine stem cell characteristics of CD44 + CD24 + ESA + cells, real time-PCR was used to determine the expressions of transcription factors Oct4 and Nanog in CSCs. The results showed that Oct4 and Nanog expressions were significantly increased in CSCs ( Figure 1E).

MIR-200a and EMT markers expression in CSC of PANC-1
MiR-200 family is known as tumor suppressor and they are usually down-regulated in EMT in some cancer stem cells, such as in prostate cancer and breast cancer [22,23]. MiR-200/ZEB2 pathway in mesenchymal-toepithelial transition and induced pluripotent stem cell generation [24]. We determined the expression levels of miR-200a, miR-200b and miR-200c in PANC-1 cell line and CSCs by real time-PCR. Real time-PCR result showed that miR-200a were significantly downregulated in CSCs (Figure 2A), while miR-200b and miR-200c expressions were similar with PANC-1 cell line (data was not shown). EMT is accompanied by E-cadherin downregulation and N-cadherin up-regulation. We found the expressions of N-cadherin, vimentin and ZEB1 were upregulated, while the expression of E-cadherin exhibited down-regulation at mRNA levels in CSCs of pancreatic cancer cell line PANC-1. These results suggested that CSCs showed EMT characteristics which was defined by the expression of specific miRNA and its target genes ( Figure 2B).

MIR-200a regulated EMT of CSCs in pancreatic cancer
Members of miR-200 family are known to be tumor suppressors, which are usually downregulated in some tumors. Down-regulation of miR-200 by siRNA technique has been shown to be associated with EMT phenotype while overexpression of miR-200a can result in the reversal of EMT phenotype [22]. To investigate the role of miR-200a in the reversal of EMT phenotype of CSCS, we transfected miR-200a mimics or non-specific control miRNA mimic (NC mimic) into CSCs ( Figure 3A). We found that the overexpression of miR-200a in the CSCs resulted in the up-regulation of epithelial marker E-cadherin and down-regulation of mesenchymal markers ZEB1, N-cadherin and Vimentin at mRNA level ( Figure 3B). These results suggest that the loss of miR-200a is critical for the acquisition of EMT characteristics and that the overexpression of miR-200a could reverse the EMT phenotype of CSCs. After transfection, the morphology of miR-200a transfected CSCs was partially changed from elongated fibroblastoid to epithelial cobblestone-like appearance, and the  Figure 3C-E). By westernblot analysis, we found that the overexpression of miR-200a in the CSCs resulted in the up-regulation of epithelial marker E-cadherin and down-regulation of mesenchymal markers ZEB1 and N-cadherin, but not Vimentin at protein level ( Figure 4A and 4B).

MiR-200a overexpression decreased the ability of invasion and migration of CSCs
EMT induction in cancer cells results in the acquisition of invasive and metastatic properties [25]. To evaluate the invasive and migration potential of miR-200 overexpression CSCs, an assay was performed using Transwell inserts. The results showed that the number of CSCs  transfected with miR-200a mimic which invaded and migrated to the lower side of the membrane was significantly decreased than CSCs or NC mimic by about 0.33 fold in invasion and 0.22 fold in migration, respectively ( Figure 5). These results clearly suggested that overexpression of miR-200a inhibits migration and invasion of CSC cells through the reversal of EMT to MET phenotype.

Discussion
Pancreatic cancer is the fourth to fifth leading cause of cancer-related death in western societies with an average overall 5-year survival of less than 5% and a median survival period of less than 6 months [26]. CSCs are a small subpopulation of cells capable of self-renewal and differentiation and have been identified in a variety of tumors. CSCs may be responsible for tumor initiation, progression, metastasis and resistance to therapy. The expression patterns of the stem cell surface markers CD44, CD24 and ESA in pancreatic adenocarcinoma cell lines are diverse and not all CSCs sorted from pancreatic adenocarcinoma cell lines develop cell spheres [27], PANC-1 is a common pancreatic adenocarcinoma cell line used for study of CSCs [28][29][30][31][32]. In this study, we sorted CD44 + CD24 + ESA + CSCs in pancreatic adenocarcinoma cell line PANC-1. Stem cell features are controlled by a small group of transcription factors, like Nanog and Oct4 [33]. Coexpression of Oct4 and Nanog is associated with pancreatic carcinogenesis and is negatively correlated with the survival prognosis of oral squamous cell carcinoma patients [34]. Oct4 and Nanog induce cancer stem cell-like properties and have correlations with EMT [35]. Our study also demonstrates that the expressions of Oct4 and Nanog were increased in CSCs of PANC-1. EMT induction in cancer cells results in the acquisition of invasive and metastatic properties. Recent reports indicate that the emergence of CSCs occurs in part as a result of EMT, for example, through cues from tumor stromal components. CSCs and EMT-type cells, which shares molecular characteristics with CSCs, have been believed to play critical roles in drug resistance and early cancer metastasis as demonstrated in several human malignancies including pancreatic cancer. Interestingly, recent studies have also shown that miR-200 family could regulate the processes of EMT by targeting E-box binding protein ZEB1 and ZEB2 [36]. Thus, the discovery of molecular knowledge of drug resistance and metastasis in relation to miR-200 family, CSCs and EMT in pancreatic cancer are becoming an important area of research, and such knowledge is likely to be helpful in the discovery of newer drugs as well as designing novel therapeutic strategies for the treatment and/or prevention of pancreatic cancer with better outcome. E-cadherin, occludin and cytokeratin are downregulated during EMT, while N-cadherin, vimentin, fibronectin, SNAI1/SAIL, SNAI2/ SLUG, ZEB2/SIP1, and TWIST1 are upregulated class switch from E-cadherin to Ncadherin results in the loss of epithelial phenotype and the acquisition of mesenchymal phenotype. Transcriptional repression of E-cadherin gene or functional repression of E-cadherin protein is the critical step for EMT. Upregulation of EMT regulators is associated with more malignant phenotypes in a variety of human cancer, such as gastric cancer, pancreatic cancer, breast cancer, and ovarian cancer [37]. In the present study, miR-200a inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, N-cadherin and ZEB1, and also retards CSCs migration and invasion. The inhibition of EMT markers by these agents suggests that they could inhibit early metastasis of pancreatic CSCs.
The EMT is an embryonic key developmental program that is often activated during cancer invasion and metastasis. It is a process by which cells undergo a morphological switch from the epithelial polarized phenotype to the mesenchymal fibroblastoid phenotype. Many signalling pathways have contributed to the induction of EMT, including TGFβ-1, Wnt, Hedgehog, Notch, and nuclear factor-kappa B (NF-kB) [38][39][40][41][42]. Kyoung-Ok Hong et al. have shown that activation of PI3K/Akt axis is one of the key mechanisms in the process of EMT and it seems that its inhibition by treatment with phosphatidylinositol ether lipid analogues (PIA) may regulate the reverse process MET leading to the re-expression of both E-cadherin and β-catenin, and reducing expression of vimentin, mesenchymal marker, in oral squamous lines carcinoma stabilized [43]. Further stuty was needed to probe the signalling pathways participatived in MET of CSCs.

Conclusion
In summary, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD24, CD44 and ESA in pancreatic adenocarcinoma cell line PANC-1. MiR-200a played important roles in the MET process of CSCs by changing EMT markers expressions and cell migration and invasion. Thus, further studies are needed to elucidate how miR-200a eliminates the stem-like cells by reversing EMT phenotypic cells and to assess whether miR-200a could be useful for the prevention of pancreatic cancer tumorigenesis and metastasis.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Global DNA Methylation of Ischemic Stroke Subtypes

Ischemic stroke (IS), a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA) of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA), small-artery disease (SAD), and cardio-aortic embolism (CE). A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204) were included, distributed across 3 IS subtypes: LAA (78/281, 59/204), SAD (97/281, 53/204), and CE (106/281, 89/204). In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.

Introduction
Ischemic stroke (IS) is a complex disease with outcomes of high mortality and long-term disability. Despite current attention to stroke risk factors and preventive treatment, the number of stroke cases has been rising in recent decades, likely because the aging population has increased as well [1]. Stroke pathogenesis involves a number of different disease processes as well as interactions between environmental, vascular, systemic, genetic, and central nervous system factors. About 80% of strokes are ischemic, as opposed to 20% hemorrhagic. Our current research focuses on ischemic stroke and its most common subtypes: large-artery atherosclerosis (LAA), small-artery disease (SAD), and cardioaortic embolism (CE) [2,3].
Substantial evidence from studies in twins, families and animal models points to a genetic risk component [4][5][6] associated with stroke and recent genome-wide association studies have identified new variants associated with IS and specific genetic variants to IS subtypes [7][8][9]. These genetic factors can contribute to conventional risk factors such as hypertension, diabetes, or homocysteine concentrations (which have a known genetic component), and may interact with environmental factors, such as smoking habit, or contribute to triggering an intermediate phenotype, such as atherosclerosis. As a result, they may contribute to stroke latency, infarct size, functional recovery, and outcome. However, stroke risk is not completely explained by these genetic factors [10]. Epidemiological data have provided evidence for a genetic component of the disease, but knowledge about its contribution to IS occurrence and characteristics is limited. There is a need to find new biomarkers for stroke risk, and epigenetic involvement is still largely unknown.
Epigenetics is a promising field of growing interest, both because it may help in the study of complex diseases and because it may generate useful biomarkers. Epigenetic mechanisms, such as DNA methylation, regulate high-order DNA structure and gene expression. DNA methylation is the most widely studied epigenetic modification, and is a marker of genomic DNA because it adds a methyl group to the 5-carbon position of cytosine, in a 59-CpG-39 context. This dinucleotide is quite rare in mammalian genomes (,1%) and clusters in regions known as CpG islands. The methylation of the CpG-island is associated with gene silencing [11].
Global DNA methylation (GDM) has been widely used in epidemiological studies because it is cost-effective, has a highthroughput, and provides quantitative results. Luminometric methylation assay (LUMA) measures levels of 5-m C residing in the -CCGG-motif [12,13]. This motif, which represents 8% of all CpG sites and occurs throughout the genome [14], is used as a proxy marker to estimate GDM.
As global DNA methylation changes over time, an association with aging processes and with modulation in the risk of several pathologies has been suggested [15][16][17]. Aberrant GDM has been associated with atherosclerosis, cancer, hypertension, and coronary heart disease [18][19][20][21]. It also has been reported that stroke patients show global DNA hypomethylation compared with healthy individuals [19]. However, stroke was analyzed disregarding its etiology, and the relationship between GDM and IS stroke subtypes remains unknown.
In the present study, we analyzed global DNA methylation using LUMA in 3 different ischemic stroke subtypes: large-artery atherosclerosis, small-artery disease, and cardio-aortic embolism. Taking into account the differences in pathogenesis, our hypothesis was that some differences in methylation status would exist.

Ethics Statement
All aspects of the study were approved by the local institutional review board/institutional ethics committee for each cohort, the Clinical Research Ethics Committee of Parc de Salut Mar and the Ethics Committee of the Vall d'Hebron Hospital, Barcelona. All participants or their approved proxy provided their written informed consent for participation.

Study Participants
The study included 2 independent prospective cohorts of Caucasian IS patients from 2 hospitals in Barcelona, Spain (n = 281 and n = 204), analyzed retrospectively, and 99 healthy participants. Common inclusion criteria were as follows: (1) firstever IS, (2) brain imaging with CT or MRI, (3) availability of the clinical data supporting the assigned stroke subtype according to TOAST classification [2], and (4) absence of intracranial hemorrhage, neoplasms, demyelinating and autoimmune diseases, and vasculitides. All patients were assessed and classified by a neurologist. Consenting patients recruited from 2005 to 2012 with a diagnosis of IS fulfilling World Health Organization criteria were considered for inclusion. The subjects were recruited in IMIM-Hospital del Mar (HM cohort) from those enrolled in BasicMar (Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III; FIS No. PI051737), an ongoing prospective registry of Ischemic Stroke [22], and in the Neurovascular Research Group of Hospital Vall d'Hebron (VH cohort).

Stroke Subtype Classification
Using TOAST criteria, patients were classified into 5 categories: (1) large-artery atherosclerosis (LAA), (2) small-artery disease (SAD), (3) cardio-aortic embolism (CE), (4) stroke of other determined etiology, and (5) stroke of undetermined etiology. Diagnoses were based on clinical features and on data collected by methods such as brain imaging (CT/MRI) and cardiac imaging [2]. Only those individuals classified in LAA, SAD and CE subtype were included in this study.

Demographic and Vascular Risk Factor Variables
In both cohorts, risk factors were collected in a structured questionnaire, as follows: arterial hypertension (evidence of at least 2 elevated blood pressure measurements, systolic . 140 mm Hg or diastolic .90 mm Hg, recorded on different days before stroke onset; a physician's diagnosis; or use of medication); diabetes (a physician's diagnosis or use of medication); hyperlipidemia (a physician's diagnosis, use of medication, serum cholesterol concentration .220 mg/dL, LDL cholesterol .130 mg/dL, or serum triglyceride concentration .150 mg/dl); coronary artery disease (documented history of angina pectoris or myocardial infarction); and atrial fibrillation (documented history or diagnosis during hospitalization). We also recorded age, sex, lymphocyte count, and current smoking habits.

Peripheral Blood Collection and DNA Extraction
DNA samples were extracted from whole peripheral blood collected in 10 mL EDTA tubes. The Chemagic Magnetic Separation Module I system (Chemagen) was used for DNA isolation at Hospital del Mar and PuregenTM (Gentra Systems) at Hospital Vall d'Hebron. At each site, all DNA extractions were performed at the same time and stored together at 220uC. DNA concentrations were quantified using Picogreen assay and nanodrop technology. The quality of DNA samples was visualized in agarose gels.

Luminometric Methylation Assay (LUMA)
The assay was carried out as described previously [12]. Genomic DNA (300 ng) was cleaved with HpaII + EcoRI or MspI + EcoRI (New England Biolabs) in 2 parallel reactions containing 2 ml of Tango buffer (Fermentas) and 5U of each restriction enzyme in a final volume of 20 ml. The reactions were set up in a 96-well format and incubated at 37uC for 4 hours. Then 20 ml annealing buffer (20 mM Tris-acetate, 2 mM Mg-acetate pH 7.6) was added to the cleavage reactions. The original LUMA assay was modified by changing the nucleotide dispensing order to eliminate any background and unspecific digestions of DNA samples as previously described [15]. The samples were placed in a PyroMark Q96 ID System (Qiagen) with the following dispensation order: GTGTCACAGTGT. Percentage of DNA methylation was expressed as [12(HpaII + EcoRI SG/ST)/(MspI + EcoRI SG/ST)]*100.

Statistical Analysis
We tested the association between LUMA global DNA methylation and IS subtypes. Calculation of sample size for the population analysis was based on results from previous analysis (methylation results and dispersion of the variable), in order to achieve a statistical power of 80%, calculated using GRANMO v7.12. LUMA GDM measurements were expressed as a continuous variable and did not show normal distribution by Kolmogorov-Smirnov test. We first used global DNA methylation as a continuous variable expressed in percentage and tested for univariate associations using Kruskal-Wallis or Mann-Whitney U test statistic for categorical variables and Spearman correlations for continuous data. Global DNA methylation was also categorized into quartiles (Q1-Q4) and x 2 and Kruskal-Wallis test were applied. Variables that differed significantly between IS subtypes in univariate analyses (age, sex, hyperlipidemia, and smoking habit) were included in a multivariate model. Coronary artery disease and atrial fibrillation were not included in the multivariate analyses because they were only present in the CE subtype in HV cohort. We tested for differences in LUMA GDM between IS subtypes by regression models, taking LAA subtype and Q1 of global DNA methylation as reference. All statistical analyses were performed using SPSS version 18.0. Statistical significance was set at a p-value of 0.05.

Results
A total of 281 ischemic stroke patients from HM cohort and 204 from HV cohort were studied. For each IS subtype, patients were distributed as follows: HM cohort, LAA n = 78, SAD n = 97, and CE n = 106; HV cohort, LAA n = 59, SAD n = 56 and CE n = 89. The clinical and demographic characteristics of the study population are shown in Table 1.
In order to replicate the previously published work of Baccarelli et al. [19], we analyzed the GDM differences between 281 ischemic stroke patients and 99 healthy individuals, both from the HM cohort. The demographic characteristics of the controls are shown in Table S1. In univariate analysis, methylation showed significant association to age and hypertension in controls, and significant differences between IS patients and controls, p = 0.008 ( Figure S1 and Table S2). In multivariate analyses, adjusted by age and hypertension, hypomethylation was associated to IS patients (p = 0.048; OR = 2.5 (95% CI = 2.3-2.7)).
In univariate analyses ( Table 1), there were significant differences in age, female sex, coronary artery disease, and atrial fibrillation between IS subtypes in both cohorts. Hyperlipidemia (p = 0.016) and smoking habit (p,0.001) were significant only in the HM cohort. There were no significant differences in GDM between IS subtypes in either cohort, as a continuous variable, or in quartile categories of LUMA methylation.
In multivariate analyses ( Table 2), only female sex was independently associated with CE subtype, in both cohorts. Advanced age was associated with CE subtype in HV cohort and showed a statistical trend among HM patients. However, global DNA methylation was not associated with ischemic stroke subtypes in either cohort, neither as a continuous variable nor in Table 2. Multivariate analysis for ischemic stroke subtypes.  quartile categories of LUMA methylation ( Figure 1). Despite borderline differences in the HV cohort, these were not replicated in the other cohort. Our results were adjusted by the percentage of lymphocytes in all multivariate regression models, with no changes in the final results (data not shown). Additionally, we performed univariate analysis of methylation as a dependent variable ( Table 3). Methylation was not associated with IS subtype in either cohort. In the HM cohort, LUMA hypomethylation was associated with diabetes (p = 0.03) and had a nearly significant association with advanced age (p = 0.08), hyperlipidemia (p = 0.06) and CAD (p = 0.08). In the HV cohort, hypomethylation was associated only with male sex (p = 0.03).

Discussion
Ischemic stroke is a heterogeneous disease with different etiological subtypes as the result of differences in physiopathology and genetic variants [7]. Global DNA hypomethylation of stroke patients has been reported compared with healthy individuals [19] and we replicated, as well. This study is the first to analyze global DNA methylation differences between IS subtypes. Contrary to what might be expected, there were no consistent GDM differences between large-artery atherosclerosis, small-artery disease, and cardio-aortic embolism strokes.
Changes in DNA methylation contribute to inter-individual phenotypic variation and are associated with cancer development and other complex diseases [21,23]. However, there is GDM variability in blood DNA, which has been associated with age, sex, alcohol consumption, white blood cell counts, and method used for DNA methylation measurement [24][25][26][27]. These variables must be considered both in the statistical analysis and in interpreting the results. Differences in the proportion of white blood cell types and cell type methylation profiles might be a confounder in DNA methylation analysis [25,27]. For this reason, we adjusted by percentage of lymphocytes but our results were unchanged.
Loss of genomic DNA methylation has been found in a variety of common aging-related diseases, including stroke. Risk of stroke is associated with cardiovascular risk factors and underlying diseases such as atherosclerosis, atrial fibrillation, and hypertension. Atherosclerosis is characterized by global hypomethylation [28,29], and reports of low genomic meth-ylation have been described in patients with cardiovascular disease [19,29,30]. These results enhance the interest in GDM as a potential biomarker of cardiovascular disease risk. However, it has not still been clarified whether GDM is causally linked to cardiovascular disease and atherogenesis.
There are only two previous publications about stroke global methylation, both referring to the same cohort of individuals [19,31]. Those studies showed that hypomethylation of the repetitive LINE-1 elements analyzed in blood DNA are associated with ischemic heart disease and stroke. However, a limited number of stroke patients were included in these analyses (55 white males), and stroke subtypes were not specified. In our cohort, we replicate their results.
In our study, ischemic stroke cases were classified, according to TOAST criteria, as LAA, SAD, and CE and no significant differences in global DNA methylation were observed using LUMA. Methylation differences in 4 variables (age, male sex, diabetes and hyperlipidemia) showed a trend or a borderline significance in at least one of the cohorts. These results are consistent with previous evidences in global methylation, with different methodologies revealing a decrease in DNA methylation during aging [15,17,19,32].
Our study has a larger sample size than previous studies in the field, and we did not identify GDM differences between IS subtypes. In a post-hoc analysis, we estimated the sample size needed to achieve statistical significance with a small GDM differences. In the most optimistic scenario, assuming the highest difference in global DNA methylation between groups in one of the cohorts as the difference to find (73.6% to 74.0%) with alfa error of 0.05 and a statistical power of 80%, we would require n = 2383 individuals for each subgroup (n = 7149 for each cohort, n = 14298 for the global study). This may have an uncertain clinical and biological significance in a nonspecific epigenetic determination.
Absence of differences between IS subtypes could be explained by detecting, with LUMA, mainly the effect of aging, vascular risk factors, and the baseline diseases of IS patients in the global methylation profile. Although Baccarelli et al. [19] showed that global hypomethylation is already determined in stroke patients and statistically significant compared with healthy individuals, it could result in a similar global hypomethylation between the IS subtypes analyzed in our study. For these reasons, applying new technologies such as genome bisulphate sequencing of specific genome regions and methylation microarrays might help to determine gene-specific methylation patterns underlying GDM and show a distinctive methylation profile between the stroke subtypes. The limitations of the study are related, first, to its retrospective design. Second, we were unable to adjust for alcohol consumption because of an excess of missing data across one of the cohorts. However, in the other cohort we were able to include alcohol in the analyses but this adjustment did not make any difference.
This study described for the first time that, using LUMA, there is no significant methylation difference between three ischemic stroke subtypes, large-artery atherosclerosis, small-artery disease, and cardio-aortic embolism. Further work is required to establish the role of epigenetic mechanisms in this complex disease. Future studies may benefit from powerful and specific new technologies such as methylation microarrays that determine genome-wide DNA methylation profiles and whole genome bisulphate sequencing.


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A new variant produced by Rhizoctonia solani AG1-IC isolate CH-1 with a new type of nuclei

Background Isolate CH-1 of Rhizoctonia solani Kühn was commonly used in our studies of the pathogenicity and genetics of this pathogen. During the preparation of homokaryons through protoplast regeneration and tuft formation, a defective homokaryon was detected and a new variant was obtained. Results When tuft formation was used to identify the karyotic nature of single protoplast regenerants (SPRs) of Rhizoctonia solani AG1-IC isolate CH-1, one homokaryon type designated as A type and the parental heterokaryon designated as AB type were obtained. The homokaryon B type was not found. Various approaches were used to obtain SPRs, including from fast or slow growing protoplast regenerants, and from regenerants of protoplasts released from mycelia grown in different nutrient broths or at different temperatures. Without exception, all these SPRs were either homokaryon A or heterokaryon AB. Moreover, the SPRs obtained from different generations of SPRs, and from different generations of SPRs treated with lytic enzymes 3 to 4 times also were invariably either homokaryon A or heterokaryon AB. When single hyphal isolates were obtained from the tuft resulting from the pairing between homokaryon A and heterokaryon AB, only the heterokaryon and a variant were obtained. The variant did not form tuft when paired with parental heterokaryon AB or homokaryon A. Its protoplast regenerants gave rise to heterokaryon AB, homokaryon A and the variant, indicating that it is a new kind of heterokaryon. Conclusion Inability to obtain homokaryon B despite numerous attempts suggests that the B type nuclei are probably defective and are dependent on A type nuclei for their multiplication. This is the first report of a heterokaryotic R. solani strain carrying a defective type of nuclei. A new variant which is a new kind of heterokaryon was obtained from the tuft resulting from the paring between the homokaryon A and the parental heterokaryon AB. Electronic supplementary material The online version of this article (doi:10.1186/s40529-014-0069-z) contains supplementary material, which is available to authorized users.

Background
Protoplast released from mycelia of fungi have been used as experimental tools in physiological and biochemical studies and protoplast fusion as a means of establishing genetic crosses for genetic study and strain improvements (Peberdy 1989). Recently, protoplasts of fungi and oomycetes also have been used in transfer of nuclei (Sivan et al. 1990;Vagvolgyi and Ferenczy 1992;Gu and Ko 1998, 2000a, 2001Wang et al. 2005) and mitochondria (Gunge and Sakaguchi 1979;Yoshida 1979;Sulo et al. 1989;Ko 2000b, 2005;Ko 2007). Organelle transfer of fungi and oomycetes is still in the early stage of development, and its possible application in the biological and genetical studies remains to be exploited.
Rhizoctonia solani Kühn is a widespread soil-borne plant pathogen causing economically important diseases in a wide range of crops (Anderson 1982;Adams 1988). It is desirable to apply organelle transfer to the studies of pathogenicity and genetics of this important fungal plant pathogen. A project was, therefore, initiated for the study of organelle transfer of the isolate CH-1 of Rhizoctonia solani AG1-IC commonly used in our research (Liu et al. 2010(Liu et al. , 2011Tsai et al. 2012). For organelle transfer, it is preferable to use homokaryotic isolates. In R. solani, homokaryotic isolates are normally derived from single basidiospores (Whitney and Parmeter 1963) or protoplasts (Phillips 1993). Since R. solani AG1-IC is heterothallic (Qu et al. 2008) and our numerous attempts to induce basidiospore formation of isolate CH-1 also were not successful, protoplasts were used for preparation of homokaryons. In the process of homokaryon preparation, it was found that colonies derived from protoplasts of isolate CH-1 consisted of heterokaryon and one type of homokaryon only. The other type of homokaryon was assumed to be defective and not viable. Moreover, a new colony type variant was obtained from the tuft resulting from the pairings between the homokaryon and the heterokaryotic parent. Details of the study are reported herein.

Source of R. solani
Isolate CH-1 of Rhizoctonia solani AG1-IC was provided by Dr. J. W. Huang of the Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan. The Culture was deposited at the Bioresouce Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan (BCRC 34905), and maintained on potato dextrose agar (PDA).

Protoplast formation
The method of Liu et al. (2010) was modified for protoplast formation and regeneration. A 5-mm culture disk of the fungus used was placed near the edge of a PDA plate (9 cm diam). After incubation at 24°C for 3 days, four culture disks were obtained from the advancing margin of the colony and placed on a cellophane disk (9 cm diam) laid on V-8 beef extract agar containing 1% V-8 juice, 0.1% beef extract and 2% agar. After incubation at 24°C for 12 h, the culture disks were cut off with a sterile scalpel and the cellophane disk was placed in 300 ml sterile distilled water in a 500-ml beaker. The mycelial mat was transferred to 25 ml V-8 broth consisting of 5% V-8 juice centrifuged at 1000 g for 5 min and adjusted to pH 6 with 1 N NaOH in a 250-ml-flask. After incubation at 24°C under light for 24 h, the mycelial mat with numerous young hyphae was transferred to a sterile 8-cm screen (Buckbee Mears, St. Paul, MN) with 150-μm pore size, rinsed with 200 ml sterile distilled water and placed in 5 ml stabilizer solution I (0.6 M manitol, 0.04 M CaCl 2 , and 0.01 M Tis-HCl, pH 7.5) mixed with 50 mg Drislase (D-9515, Sigma, St. Louis, MO, USA) and 25 mg lysing enzymes (L-1412, Sigma) in a 15-ml centrifuge tube. Stabilizer solution with enzymes was sterilized by filtration through 0.22 μm membrane filter (Millex, Millipore, Co Cork, Ireland). The centrifuge tube containing the mixture was incubated at 37°C for 15 min and 34°C for 105 min with gentle shaking to release protoplasts. Protoplasts were separated from non-digested mycelial fragments by filtration through a Whatman No. 113 filter paper (30-μm pore size) and the filtrate was collected in another centrifuge tube. To remove the lytic enzymes, 1 ml 0.8 M mannitol solution was pipetted onto the bottom of the centrifuge tube containing the protoplast suspension before centrifugation at 1000 g for 10 min. The supernatant containing the lytic enzymes was discarded and protoplasts retained in the bottom portion were used for regeneration.

Preparation of single-protoplast regenerants (SPRs)
Protoplasts in the centrifuge tube were mixed with 1 ml of stabilizing solution II consisting of 0.1 M sucrose, 0.1 M CaCl 2 and 0.1 M Ca(NO 3 ) 2 , supplemented with 1% clarified V-8 juice. The mixture was equally dispensed into two 1.5-ml centrifuge tubes. After incubation at 24°C for 24 h in darkness, the concentration of regenerated protoplasts was adjusted to 2 to 3 protoplasts per 10-μl drop with a Pipetman microliter pipette (West Coast Scientific, CA, USA) (Ann et al. 2010;Ko et al. 1973). Twenty drops of the protoplast suspension were evenly distributed on a PDA plate. After incubation at 24°C for 24 h under light, colonies originated from SPRs were each transferred to a PDA slant under a dissecting microscope and stored at 24°C for subsequent study.

Determination of karyotic nature
Karyotic nature of SPRs or single hyphal isolates was identified by tuft formation. At the initial test, 10 SPRs were each paired with the parental isolate on PDCYA consisting of PDA amended with 0.1% charcoal and 0.5% yeast extract modified from the medium described by Butler and Bolkan (1973), and incubated at 24°C under light for 4 days. Six SPRs did not form tuft indicating that they were heterokaryons just like the parental isolate, while 4 SPRs formed tuft indicating that they were homokaryons. When one of the homokaryons was paired separately with the other three homokaryons, none of the parings showed tuft formation, indicating that all of them belonged to the same type of homokaryon. Since R. solani AG1-IC has been reported to produce two types of homokaryons (Ou et al. 2008), this type of homokaryon was designated as A, while the other not yet found homokaryon type was designated as B. The heterokaryon was designated as AB. Subsequently in search for the B type homokaryon, the karyotic nature of SPRs was determined by paring each SPR with an A type homokaryon and the parental heterokaryon AB. The B type homokaryon should form tuft when paired with A type homokaryon and heterokaryon AB.

Isolation of single hyphal isolates from tuft
Homokaryon A and heterokaryotic parent were paired on PDCYA as described for tuft formation. After incubation at 24°C for 3 days, mycelial mat was obtained from the tuft with a sterile scalpel and placed on the center of a plate containing 2% water agar. After incubation at 24°C for 24 h, single hyphal tip isolates were isolated as described by Whitney and Parmeter (1963), and were each transferred to a PDA plate for colony formation.

Confirmation of karyotic nature of SPRs
During the initial test, the 10 SPRs obtained consisted of 4 putative homokaryon A and 6 putative heterokaryon AB based on the tuft formation in the pairing with parental isolate. Randomly selected 3 putative homokaryon A and 3 putative heterokaryon AB were subjected to protoplast formation and regeneration, and the karyotic nature of each SPR was determined by tuft formation in pairing with the parental isolate. All 3 SPRs of putative homokaryon A produced only homokaryon A type SPRs (Table 1), confirming that they were true A type homokaryon. All the 3 SPRs of putative heterokaryon AB produced both homokaryon A type and heterokaryon AB type SPRs, confirming that they were true heterokaryon AB.

Searching for B type homokaryon
During the initial search, five independent experiments were carried out and ranging from 16 to 48 SPRs per experiment were obtained. All the experiments produced A type homokaryon and heterokaryon AB with the exception of Exp. 4 which produced only heterokaryon AB (Table 2). Since all five experiments failed to produce B type homokaryon, one more experiment was carried out to generate a total of 80 SPRs. Again only A type homokaryon and heterokaryon AB were obtained.
To test whether fast or slow growth was the reason for failure to obtain B type homokaryon, SPRs were isolated after 10 or 72 h regeneration in comparison with the original 24 h regeneration. All the tests gave only A type homokaryon and heterokaryon AB (Table 3). To test if certain nutrient may favor the multiplication of B type nuclei, the fungus was grown in 10% V-8 juice broth or potato dextrose broth supplemented with 0.1% yeast extract in comparison with the original 5% V-8 juice broth before enzyme treatment to release protoplasts. All SPRs obtained were still either A type homokaryon or heterokaryon AB (Table 4). To test if high or low temperature may encourage the multiplication of B type nuclei, the fungus was grown at 12 or 32°C in comparison with the original 24°C before enzyme treatment for release of protoplasts. All SPRs obtained in each treatment were similar, containing only A type homokaryon and heterokaryon AB (Table 5).
To test if regenerants of protoplasts released from multiple generations of SPRs may contain B type homokaryon, from 3rd to 6th generation, 5 SPRs each were used to produce SPRs and their karyotic nature was determined. All the SPRs contained A type homokaryon and heterokaryon AB with the exception of no. 5 of the 6th generation of SPR which gave rise to only heterokaryon AB (Table 6). To test if B type nuclei may require larger hole for exit from mycelia, protoplasts were    obtained from mycelia of parental isolate and different generations of SPRs after repeated enzyme treatments. Even after the mycelia were treated with lytic enzymes 4 times, the protoplasts released were either A type homokaryon or heterokaryon AB (Table 7).

New variant formation
Ranging from 10 to 30 single hyphal tip isolates were obtained from tufts resulting from three independent pairings between homokaryon A and heterokaryotic parent. Colony morphology of these isolates was mostly of the heterkaryotic parent type. However, colony morphology of about 7 to 40% of single hyphal tip isolates was different from homokaryon A and heterokaryotic parent, and appeared to be a new variant (Table 8). The new variants did not form tuft when paired with either homokaryon A or heterokaryotic parent. When protoplasts were obtained from the variants homokaryon A, heterokaryotic parent or the variant type of colony morphology was found among the protoplast regenerants of each variant (Table 9). Homokaryon B was not detected as before.
On PDA, the heterokaryotic parent grew faster than the homokaryon A and the variant. The variant was the slowest in growth, about two times slower than the parent. On V-8 agar, the heterokaryotic parent also grew faster than the other two. However, the homokaryon A became the slowest in growth, only about one third of the parent (Table 10, Figure 1).

Discussion
Confirmation of homokaryons of R. solani obtained through studies of basidiospores has been reported (Whitney and Parmeter 1963;Carza-Chapa and Anderson 1966;Anderson et al. 1972;Adams and Butler 1982) because not all basidiospores of this fungus are mononucleate (Flentje et al. 1963;Anderson et al. 1972). Since not all protoplasts of R. solani are mononucleate (Hashiba and Yamada 1982;Phillips 1993;Liu et al. 2010), confirmation of karyotic nature of SPRs was also performed in this study. The result showed that the tuft formation method for determining the karyotic nature of SPRs is reliable. Inability to obtain B type homokaryons from regenerants of protoplasts released from mycelia with various kinds of treatments suggests that the B type nuclei are probably defective and are dependent on A type nuclei for their multiplication. This may explain why SPRs contained only A type homokaryon and heterokaryon AB. Protoplasts containing only B type nuclei are probably not viable. The observation that only 48-79% protoplasts of this fungus were able to regenerate (Liu et al. 2010) is compatible with this explanation. To our best knowledge, this is the first report of a heterokaryotic R. solani strain carrying a defective type of nuclei. The molecular difference between the defective and the normal nuclei and the effect of the defective nuclei on the pathogenicity of this fungus remain to be investigated.
When the heterokaryotic parent was paired with the homokaryon A, a variant was isolated from the tuft. The variant isolates did not form tuft when paired with the heterokaryotic parent or homokaryon A. The mechanism leading to such phenomenon remains to be investigated. When protoplasts were obtained from the variant isolates, the regenerants gave rise to heterokaryotic parent, homokaryon A or the variant, indicating that they belong to a new kind of heterokaryon.

Conclusion
Inability of isolate CH-1 of R. solani AG1-IC to produce homokaryon B despite numerous attempts may suggests that B type nuclei are probably defective and are dependent on A type nuclei for their multiplication. This is the first report of a heterokaryotic R. solani strain carrying a defective type of nuclei. From this study a new variant which is a new kind of heterokaryon was also obtained from the tuft resulting from the pairing between the homokaryon A and the parental heterokaryon AB.


extfieldsofstudy: Medicine Chemistry Biology

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MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signaling in cancer

Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

INTRODUCTION
Advances in the understanding of the molecular basis of cancer have placed mitochondria [1] at the center of numerous abnormalities observed in tumor cell metabolism [2][3][4], differentiation [5], proliferation [6] and survival [7,8]. Either due to its direct impact on the cellular metabolism or its role as a hub for signal transduction, deregulation of intrinsic mitochondrial processes combined with failure to halt cell cycle progression results in the genesis and progression of tumors [9][10][11][12][13]. Among the many abnormal features of cancerous cells, a form of metabolism reliant on aerobic glycolysis is remarkable [4] as it enables cell survival in the near absence of oxygen and provides the necessary building blocks to support vigorous proliferation. Recently, a growing number of studies aimed at defining mechanisms of mitochondrial deregulation in cancer have indicated that a deeper understanding of tumor cell metabolism will likely impact therapeutics by enabling the development of targeted treatments with fewer complications and increased efficacy in preventing recurrence post therapy [7,[14][15][16][17][18].
In parallel with glycolytic metabolism [19][20][21][22], high MnSOD expression [23][24][25] is a distinctive feature of tumors particularly notable at advanced stages [26,27]. In healthy mitochondria, MnSOD directly regulates the metabolism of superoxide radical anions generated as a by-product of the electron transport chain. In isolation, MnSOD converts the diffusion-restricted, mild-oxidant superoxide radical into the diffusible, strong oxidant hydrogen peroxide (H 2 O 2 ) and thereby critically changes mitochondria-driven signaling in the cell. Thus, MnSOD does not always act as a first line mitochondrial antioxidant defense. Recently, a study by our group demonstrated that in the absence of matched upregulation of mechanisms of H 2 O 2 removal, MnSOD overexpression is actually detrimental to the integrity of mitochondria and the maintenance of its energetic functions [28]. This indicates that either directly or indirectly [29,30] MnSOD regulates mitochondrial energetic and signaling functions. Using mitochondria-depleted cancer cells it was established that the abrogation of mitochondria-dependent regulatory functions results in the appearance of highly invasive, aggressive, glycolytic cellular phenotypes [31]. Taken together these observations indicate that progressive MnSOD upregulation, which results in mitochondrial dysfunction, could participate in the appearance of malignant cellular phenotypes characterized by glycolytic metabolism.
In this report, results are presented showing that mitochondrial MnSOD upregulation leads to the activation of AMPK, a cellular metabolic master switch [32,33] that directly enhances glycolysis. We also establish that in cancer cells, mtH 2 O 2 released from mitochondria consequentially to MnSOD upregulation is the signal that engages AMPK to produce and sustain the Warburg effect, thereby enabling cancer cell survival.

MnSOD upregulated in cancer cells promotes glycolysis
In luminal breast cancer samples stratified by stage, MnSOD expression was present at significantly elevated levels in progressing tumor stages ( Figure 1A-D). The levels of MnSOD increased with histologic tumor grade being highest at histologic grade III and lowest in healthy and hyperplastic benign tissue (Figure1D). Elevated MnSOD levels were also observed in advanced prostate ( Supplementary Fig. 1A) and colon ( Supplementary Fig.  1B) cancer tissue as compared to healthy tissue samples. In breast cancer, MnSOD levels were noted to be highest in triple negative and Her2 subtypes (Supplementary Fig. 2A), elevated in luminal cancers and lowest in healthy control tissue, indicating an association between high MnSOD expression and tumor aggressiveness. This association was further strengthened by the epidemiologic analysis of published data [34] on 5-year breast cancer survival which negatively correlatedwith levels of MnSOD expression. Supplementary Fig.  2B, shows the Kaplan-Meier distribution of 5-year breast cancer survival indicating a clear inverse relationship between survival and MnSOD levels. In all cancer types analyzed (breast, prostate and colon), MnSOD expression was also correlated with expression of lactate dehydrogenase (LDH, Figure1B; Supplementary Fig. 1), a surrogate of glycolytic metabolism [35,36] that is associated with poor prognosis. Quantification of total cell MnSOD and LDH fluorescence in breast ( Figure 1B), prostate and colon tissue (Supplementary Fig. 1C and 1D, respectively) indicated these changes are significant and support a role for MnSOD upregulation in the switch to glycolysis, a common feature of aggressive cancer subtypes.
To assess whether MnSOD upregulation occurs during the progression towards malignancy, MCF10A (non tumorigenic mammary epithelial cells) expressing oncogenic v-Src kinase under the control of an inducible estrogen receptor promoter were used. Activation of v-Src expression by exposure of MCF10-Er-Src cells to tamoxifen resulted in MnSOD upregulation early during the process of malignant transformation (12h, Supplementary Fig.  3) and progressively increased. Fully transformed cells (48-72 h) [37,38] expressed the highest levels of MnSOD. The increase in MnSOD levels during transformation paralleled a strong metabolic shift towards glycolysis with notable reduction in the steady state levels of ATP ( Supplementary Fig. 3).
To begin to assess the impact of MnSOD upregulation on breast cancer cells metabolism, 5 isogenic cell lines stably overexpressing MnSOD were obtained. The control cell line expressing the empty vector (neo) and four additional clonal lines Mn1, Mn44, Mn11 and Mn28 expressing MnSOD at various levels ( Figure 2A) within the range observed in human breast cancer ( Figure 1D) were used to assess the impact of MnSOD expression on O 2dependent metabolism and glycolysis. In accordance with recent observations [39], MnSOD activity increased with enhanced expression levels but not in a directly proportional fashion ( Figure 2B). However, incremental increase in MnSOD dampened O 2 uptake ( Figure 2C) in parallel to dissipating the mitochondrial electrochemical potential as assessed by JC-1 ( Figure 2D) and CMXROS ( Figure 2E). Importantly, only when MnSOD levels were higher than 5-6 fold over basal (such as in the Mn44 clone) the electrochemical potential dropped in parallel with the reduction in the steady state levels of ATP ( Figure 2F) and strong activation of compensatory glycolysis as assessed by lactate production ( Figure 2G). Importantly, the elevated MnSOD expression levels were associated to progressive enhancement of H 2 O 2 production ( Figure 2H) indicating a redirection in oxygen utilization from ATP production to H 2 O 2 synthesis. Collectively, the findings summarized in Figure 2 support the hypothesis that enhanced MnSOD expression sustains the metabolic switch in cancer cells. Further experiments using the H 2 O 2 biosensor, roGFP targeted to mitochondria, indicated that H 2 O 2 is locally generated in the organelle and that the increase in H 2 O 2 , as expected, paralleled the loss of mitochondrial electrochemical potential ( Figure  2H).
Using extracellular flow analysis (Seahorse Biosciences), we further examined the effect of MnSOD expression on mitochondrial respiration and the glycolytic rate. In agreement with the data shown in Figure 2, cells expressing MnSOD at basal levels (neo) had significantly higher antimycin-inhibitable oxygen consumption rates (OCR) in comparison to Mn44 (moderate MnSOD expression) and Mn11 (high MnSOD expression Figure 3A). Reserve respiratory capacity (the difference between maximal and basal respiratory rates) in all three cell lines was found to be significantly compromised indicating that in all cases mitochondria were functioning at the limit of their respiratory capacity even at baseline MnSOD activity in neo cells. Further confirmation of the increasing dependence of cells expressing high MnSOD levels on glycolysis was obtained in experiments where the extracellular acidification rate (ECAR) was measured ( Figure 3B). ECAR is a direct measure of lactic acid production by cells and thus of glycolytic activity. As shown in Figure 3C, the OCR/ECAR values for neo cells indicate that these cells are predominantly utilizing mitochondrial metabolism for energy. Higher levels of MnSOD expression in Mn44 and Mn11 promoted an increasing in glycolysis ( Figure 3D). The relevance of this finding was highlighted by the observation that in the metastatic and heavily glycolytic breast cancer cell line (MDA-MB-231), MnSOD expression levels were higher than those found in neo (MCF7 background) ( Figure 4A). In comparison to MCF7/neo, glycolytic and metastatic cells (MDA-MB231/U2OS) had MnSOD expression that matched those obtained in the Mn11 clone. Silencing MnSOD in MDA-MB231 cells ( Figure 4B) reduced the glycolytic rate of these cells with a parallel decrease in ATP steady state levels ( Figure 4E and 4F) indicating that MnSOD-driven glycolysis significantly contributes to the maintenance of ATP production in phenotypically aggressive breast cancer cells.

MnSOD upregulation activates AMP-activated kinase
AMPK is a key energetic sensor and regulator of cellular metabolism that is activated by either a reduction in the intracellular ratio ATP/AMP or by the elevation of intracellular H 2 O 2 levels [40]. Because both a reduction in ATP and elevation of H 2 O 2 were found to occur in parallel with MnSOD overexpression, we examined whether AMPK was activated in cells expressing incremental levels of MnSOD. Figure 5A shows that increasing levels of MnSOD expression increased AMPK activity as assessed both by the phosphorylation of active site Thr172 and the phosphorylation of the downstream target Acetyl CoA Carboxylase (ACC). Challenging the cells with exogenous H 2 O 2 , the product of MnSOD activity, significantly increased the phosphorylation and activity of AMPK in neo and Mn1 cells ( Figure 5A). Conversely, H 2 O 2 only minimally increased AMPK phosphorylation and activity in Mn44 cells and did not further augment the phosphorylation or activity of AMPK in cells expressing high MnSOD levels (Mn11 and Mn28, Figure 5A). These results indicated that the increase in MnSOD expression beyond levels found in Mn44 cells maximally activated AMPK by endogenously generating sufficiently high steady fluxes of H 2 O 2 ( Figure 5A). We also confirmed that mtH 2 O 2 was the molecular agent generated by MnSOD that led to AMPK activation. Neo cells were exposed to (0-20 μM) H 2 O 2 , and AMPK phosphorylation, the phosphorylation of its downstream target ACC and the intracellular steady state levels of ATP were assessed in side by side experiments (Supplementary Fig. 4). Low H 2 O 2 levels insufficient to cause changes in ATP fully activated AMPK, indicating that H 2 O 2 can produce AMPK activation independently of variations in ATP levels. To further demonstrate that when MnSOD activity is elevated mtH 2 O 2 mediates the activation of AMPK we expressed mitochondria-targeted catalase (mtcatalase) that efficiently dismutates H 2 O 2 into O 2 and H 2 O in mitochondria. Restricting mtH 2 O 2 outflow resulted in a marked decrease in AMPK activation, especially in cells expressing higher levels of MnSOD ( Figure 5B). In accordance with our prediction that high MnSOD expression levels lead to a predominantly glycolytic metabolism (in order to maintain ATP production), mt-catalase did not change steady state concentrations of ATP in neo cells but significantly reduced ATP levels by an additional ~ 75% in Mn11 cells ( Figure  5C). Addition of mt-catalase also resulted in decreased glycolysis as assessed using lactate accumulation and the overall decrease in basal OCR/ECAR measured by extracellular flow analysis ( Figure 5D and 5E). Importantly, the increase in AMPK activity observed in the cells expressing high levels of MnSOD was also observed in breast, colon and prostate cancers ( Figure 5F and 5G). Importantly, AMPK activation paralleled MnSOD upregulation during the course of MCF10-Er-Src transformation ( Supplementary Fig. 3C), was enhanced in MDA-MB-231 in comparison to MCF7/neo ( Figure 4) and was suppressed following silencing of MnSOD in MDA-MB-231 ( Figure 4D). Together, these findings provide evidence for the involvement of MnSOD in promoting the glycolytic switch of aggressive breast cancer cells in an AMPK-dependent manner.
In view of recent reports implicating AMPK in the maintenance of NADPH levels in cancer cells, we also examined whether the MnSOD-directed activation of AMPK maintained steady state NADPH levels in MnSOD overexpressing cells ( Supplementary Fig. 5). The levels of NADPH did not significantly change in cells overexpressing MnSOD when grown in complete media. However, in the absence of glucose, NADPH levels were diminished in MnSOD overexpressing cells consistent with the high demand for NADPH in these cells that may have been sustained by the chronically elevated levels of H 2 O 2 production. These results also indicate that MnSOD, in a glucose-dependent manner, contributes to activate NADPH synthesis possibly via AMPK ( Supplementary Fig. 5), thereby sustaining cancer cell biosynthetic capacity, viability and proliferation. AMPK phosphorylation and activity (as assessed by ACC phosphorylation) were found to parallel MnSOD expression in a tumor-stage dependent manner ( Supplementary Fig. 6). AMPK activity was also found to be significantly increased in early malignant breast tumors as compared to control tissue and to increase progressively with tumor stage (Supplementary Fig. 6). Also, in aggressive breast cancer subtypes, the elevation of MnSOD expression was strongly correlated with an increase in AMPK activation ( Supplementary Fig. 6). This observation taken together with findings shown in Figure 5F and G (that AMPK activation also parallels MnSOD upregulation in prostate and colon cancer) indicates the potential relevance of this finding as a mechanism to sustain aggressive cancer cell metabolism in multiple types of cancer.

MnSOD/AMPK direct the metabolic shift towards glycolysis
The activating effect of MnSOD upregulation on AMPK and the subsequent shift from cellular oxidative respiration to glycolysis was confirmed by silencing of MnSOD expression in MCF7/neo, Mn44 and Mn11 using siRNA. Suppression of MnSOD expression in Mn44 and Mn11 was controlled to match levels of MnSOD expression equivalent to those found in resting MCF7/neo cells ( Figure 6A). AMPK phosphorylation analyzed by Western blot was observed to be reduced in Mn44 and in Mn11 cells transfected with MnSOD siRNA. Silencing of MnSOD caused significant suppression of glycolysis in Mn44 and Mn11 ( Figure 6B) while not significantly affecting MCF7/neo. Directly interfering with AMPK using a pharmacologic inhibitor (Compound C) significantly inhibited glycolysis in MCF7/neo, Mn44 and Mn11 cells as expected based on the prominent role of AMPK in activating glycolysis, but only affected ATP levels in Mn44 (mildly, ~ − 30%) and Mn11 (heavily ~ − 85%), Figure 6D. Thus, while neo cells were capable of using different routes for ATP synthesis, cells expressing higher levels of MnSOD appeared to have increasing dependence on glycolysis. This finding was further examined in cells devoid of AMPK's catalytic subunit AMPKα1. To investigate the effect of MnSOD/AMPK in the induction of the Warburg effect, MnSOD was transfected into MCF-7 cells constitutively expressing AMPKα1 siRNA vs. scrRNA. Results shown in Figure 6E and Figure 6F show that MnSOD overexpression in AMPK-competent cells recapitulated the strong repression of O 2 uptake observed in Mn11 cells in parallel with an increase in the extracellular acidification rate (an index of increased glycolytic metabolism). Conversely, overexpression of MnSOD in cells devoid of AMPKα1 produced only marginal effects on cellular metabolism that remained predominantly aerobic.
The dependence of MnSOD overexpressing cells on glucose was also exemplified by the finding that Mn44 and Mn11 could only maintain steady levels of NADPH in glucose rich medium (supplementary Fig. 5) consistent with the expectation that higher intracellular H 2 O 2 leads to accelerated NADPH turnover. These observations are in agreement with recent published work demonstrating that one of the many important functions of AMPK activation in cancer is to maintain NADPH recycling and availability [41] ( Supplementary  Fig. 5).

MnSOD overexpression upregulates glycolytic enzymes
Additional insight into the importance of the MnSOD/AMPK pathway for the glycolytic shift of cancer cells was obtained by defining whether MnSOD levels affected the expression and activity of key glycolytic enzymes. Hexokinases are enzymes that catalyze glucose phosphorylation for entry in the glycolytic and pentose phosphate pathways and phosphofructokinases (PFK) catalyze the rate limiting step in the glycolytic pathway. Hexokinase and phosphofructokinase activity were found to be enhanced in cells expressing high MnSOD levels in comparison to neo controls. PFK activity was increased consistent with a greater reliance of MnSOD overexpressing cells on glycolysis ( Supplementary Fig. 7) and the direct activating effect of AMPK on PFK2. In fact, it was confirmed that MnSOD overexpression in AMPK-competent cells resulted in remarkable phosphorylation of PFK2 on Ser466, an activating AMPK-dependent phosphorylation site [42] (Supplementary Fig.  7B). MnSOD overexpression in AMPK-deficient cells did not result in increased PFK2 phosphorylation supporting our hypothesis that MnSOD sustains the glycolytic activity of cells where it is overexpressed by engaging AMPK-dependent signaling ( Supplementary  Fig. 7). Notably, a marked reduction in the expression levels of mitochondrial electron transfer chain complexes II, III, IV and V ( Supplementary Fig. 8A) was observed in cells overexpressing MnSOD consistent with the observations of reduced mitochondrial potential and O 2 uptake. In parallel, we analyzed the expression of key antioxidant enzymes in order to gain additional understanding of the mechanisms underlying the persistent elevation in H 2 O 2 due to MnSOD upregulation. Catalase, glutathione reductase, thioredoxin and thioredoxin reductase were examined and only thioredoxin was significantly reduced in Mn44, Mn11 and Mn28 cells ( Supplementary Fig. 8B). These results indicate that alterations in the expression and functioning of electron transfer chain complexes in MnSOD overexpressing cells enhance mtH 2 O 2 production unopposed by the major H 2 O 2 scavenging systems.

CaMKII, a redox sensitive kinase that activates AMPK
Data presented above indicated that the constitutive elevation of cellular H 2 O 2 maintained AMPK activation thereby promoting the irreversible conversion of the cellular metabolism to glycolytic. The mechanism of MnSOD-dependent AMPK constitutive activation was next investigated with a focus on CaMKII, a kinase responsive to H 2 O 2 -driven oxidative activation upstream of AMPK [43,44]. Oxidation of CaMKII at residues M281 and M282 had previously been shown to result in chronic activation, and this constitutive activation of CaMKII was apparent even in cells overexpressing low levels of MnSOD (2 -3 fold over basal, Mn1), Figure 7A. Consistent with the hypothesis that AMPK is critical for maintaining increased glycolysis and steady state ATP levels in MnSOD overexpressing cells; the CaMKII inhibitor KN93 dampened glycolysis and reduced the steady state ATP levels in Mn44 and Mn11 cells ( Figure 7B). Importantly, inhibition of CaMKII resulted in robust dephosphorylation of AMPK at the active site Thr172 ( Figure 7C). Decreased AMPK phosphorylation was accompanied, as expected, by a reduction in AMPK activity as indicated by lower levels of phosphorylated ACC ( Figure 7C), a downstream target of AMPK. This observation indicates that CaMKII oxidation by mtH 2 O 2 leads to its constitutive activation and consequentially the activation of AMPK. Relevantly, inhibition of CaMKII markedly reduced the viability of cells expressing high MnSOD levels ( Figure  8). The potential clinical importance of this finding was furthered by comparing the effect of CaMKII inhibitor KN93 with two commonly used breast cancer chemotherapeutic compounds, paclitaxel (a mitotic inhibitor) and 5-fluorouracil (a thymidylate synthase inhibitor). Exposure to KN93 resulted in 60-70% cell death of Mn44 and Mn11 cells while only minimally reducing the viability of MCF7/neo cells by 20% approximately, which was in clear contrast to the effect of paclitaxel and 5-flurouracil which were far more efficient in killing MCF7/neo (Figure 8). This observation indicated that cells expressing high MnSOD levelsare far more sensitive to CaMKII inhibition than to paclitaxel or 5-fluorouracil.

Glycolysis maintains cells overexpressing MnSOD viable
The dependence of cells expressing high MnSOD levels on glycolysis was further examined by conducting experiments where glycolysis was inhibited using 2-deoxy-D-glucose (2-DG). MCF7/neo, Mn44 and Mn11 cells were treated with 2-DG(a potent glucose analog that cannot be metabolized) and then apoptosis was measured by flow cytometry (Figure 9). 2-DG produced marked cell death among Mn11 cells compared to MCF7/neo controls. The inhibition of glycolysis dramatically lowered steady state ATP levels in Mn44 and Mn11 cells, while only moderately inhibiting ATP production in neo cells ( Figure 9B). These results indicate that glycolysis significantly contributes toATP production in MCF7/neo cells, but accounts for most, if not all, of the ATP production in cells expressing high MnSOD levels. Accordingly, we found that supplementation of media with substrates that support glycolytic metabolism (pyruvate and uridine) stimulated Mn11 and Mn28 proliferation without affecting MCF7/neo or Mn1 growth rates (Supplementary Fig. 9). The proliferation rate of Mn11 and Mn28 in the presence of pyruvate and uridine was comparable to the baseline proliferation rate of MCF7/neo, dismissing the concept that MnSOD per se is a tumor suppressor. Rather, MnSOD imposes a form of metabolism that requires glycolysis-supportive substrates to maintain rapid cellular proliferation.

Activation of the MnSOD/AMPK axis suppresses apoptosis
MnSOD is known to promote cell survival and inhibit apoptosis by mechanisms that remain poorly defined [45]. Since AMPK has been implicated in upregulating Bcl-2/survivin expression [46], we examined the possibility that in addition to the metabolic effects describd above, MnSOD/AMPK fostered cancer cell survival by upregulating anti-apoptotic factors such as survivin and Bcl-2. Cells expressing high MnSOD levels also had concurrent increases in survivin and Bcl-2 ( Figure 10A and 10B). To test the involvement of AMPK in the induction of survivin and Bcl-2 expression by MnSOD, AMPK expression was reduced using siRNA which resulted in decreased Bcl-2 and survivin levels in both Mn44 and Mn11 cells. This observation implicates AMPK as a critical mediator of MnSOD-induced antiapoptosis ( Figure 10C). Expression of MnSOD and survivin/Bcl-2 were also similarly increased in human cancer tissues compared to control tissue ( Figure 9D). Furthermore, KN93 was observed to be a potent inducer of apoptosis of MnSOD overexpressing cells, likely because of additive effects in suppressing AMPK-driven metabolic and anti-apoptotic routes ( Figure 8). These findings support a critical role for MnSOD/oxCaMKII/AMPK in the maintenance of glycolytic cancer cell survival while adding mechanistic insight to the known anti-cancer effect of CaMKII inhibition [45,47]. MnSOD upregulation also repressed the cleavage of effector caspases 3 and 7 while activating caspase 9 and downregulating p53 expression ( Supplementary Fig. 10). These findings further substantiate the critical role of MnSOD in adaptative responses that favor cancer cell survival. Our studies also indicate that MnSOD upregulation produced phenotypes that metabolically and functionally resemble more malignant cells such as those that occur in late stage and aggressive subtypes of breast cancers. This conclusion was further supported by experiments using MCF10-Er-Src cells where MnSOD upregulation was shown to be essential for the survival of transformed cells ( Supplementary Fig. 11). Silencing MnSOD prior to induction of MCF10A-Er-Src transformation induced by tamoxifen led to > 70% cell death 48 h after tamoxifen stimulation. Although MnSOD ectopic expression did not significantly increase metastasis of xenografts produced in NOD/SCID mice, enhanced MnSOD expression in breast cancer cells conferred characteristics of aggressive cancer cells such as glycolytic metabolism (Figure 2 and 3), drug resistance (Figure 8), and enhanced capacity to grow in soft agar ( Supplementary Fig. 12).

DISCUSSION
Investigations dedicated to unraveling the role of MnSOD in tumorigenesis and cancer progression have yielded conflicting results in regard to the nature and potential effects of MnSOD in influencing cancer risk, prognosis, survival, susceptibility to therapy, and likelihood of relapse. Recently the consideration of MnSOD expression in specific contexts along with the functional status of other cellular antioxidant systems led to the more encompassing idea that MnSOD contributes to the progression of tumors towards aggressive phenotypes in which MnSOD enhances anti-apoptotic defenses and increases invasion [47,48]. The role of MnSOD as a primary participant and director of the malignant transformation process has been less appreciated; however, MnSOD and its product H 2 O 2 have recently been shown to play critical roles in controlling oxygen consumption rates and glucose uptake in mouse embryonic fibroblasts as these cells progress through the cell cycle from quiescence to proliferative states [49] and in a variety of other systems where MnSOD and H 2 O 2 affect survival and cellular function [50][51][52][53][54][55][56]. Our results provide direct evidence in support of the concept that MnSOD strongly suppresses mitochondrial oxidative metabolism while bolstering glycolysis by mechanisms that involve the rerouting of O 2 towards the production of a steady flow of mtH 2 O 2 . MnSOD expression is regulated by inflammatory [57,58], metabolic, and stress activated transcription factors [59][60][61] and is generally expressed in response to an acute insult [62,63]. While the possibility that MnSOD provides protection against stress when acutely upregulated has been extensively scrutinized [64][65][66], our results indicate that when persistently overexpressed at levels found in human cancers, MnSOD contributes to the progressive deterioration of mitochondrial bioenergetics and the activation of glycolysis via mtH 2 O 2 production and AMPK activation. Interestingly, a similar role for SOD1, a cytosolic superoxide dismutase has been described [67]. Although through a different mechanism (association with two casein kinase 1-gamma homologs) it was demonstrated that H 2 O 2 generation was required to suppress mitochondrial respiration and stimulate glycolysis indicating, as proposed here, that the manipulation of intracellular levels of H 2 O 2 may be of therapeutic utility in cancer. In fact, according to the results shown in Figures 1-5 and Supplementary Figs. 1 and 2, the persistent upregulation of MnSOD with parallel increase in steady state levels of H 2 O 2 seem to be a distinctive feature of transformed cells that appears to satisfy critical energetic and metabolic necessities. Moreover, we found that repressing signals originating from MnSOD overexpression (such as mtH 2 O 2 , oxCaMKII, AMPK) strongly inhibit glycolysis without reactivating oxidative based metabolism in mitochondria. Consequently, the interruption of MnSOD/mtH 2 O 2 / oxCaMKII/AMPK signaling results in the metabolic collapse of cells derived from advanced stage cancers (MDA-MB-231 and transformed MCF10A-Er-Src) resulting in marked cell death. Hydrogen peroxide originating in mitochondria (mtH 2 O 2 ) was identified as the agent that integrates MnSOD-dependent mitochondrial signaling with the activation of AMPKwhich was confirmed in experiments where we attenuated mtH 2 O 2 levels by ectopically expressing mitochondria-targeted catalase which rapidly dismutates H 2 O 2 preventing its long range effects. The effect of mito-catalase in repressing glycolysis in MnSOD-overexpressing cells indicates that there is an aggressive cancer-specific mechanism of glycolysis centered on the mitochondria-driven activation of the MnSOD/ AMPK axis. This conclusion is based on the observation that silencing MnSOD or inhibiting AMPK reduces glycolysis to baseline levels while only marginally affecting ATP levels in cells expressing low levels of MnSOD (MCF7/neo cells). Inhibition of glycolysis with 2-DG reduced steady state ATP levels in all cells lines examined but only killed those expressing elevated MnSOD levels such as Mn44 and Mn11 cells. Taken together with the observation that mitochondria-driven ATP production was severely impaired in Mn44 and Mn11 lines, it is proposed that MnSOD promotes survival independently of oxygen (a clinically relevant problem) at the expense of metabolic flexibility. In this regard, the identification of a specific mitochondria-to-AMPK signaling axis that is activated by MnSOD upregulation and dependent on mtH 2 O 2 in cancer cells can potentially be exploited for cancer therapeutic as thisis not likely to interfere with normal glycolytic processes in healthy cells. This is especially important in view of recent findings demonstrating that AMPK activation is critical for the maintenance of cancer cell biosynthetic activity [68].
On investigating the possible mechanisms by which AMPK is maintained constitutively activated in cells overexpressing MnSOD, we identified CaMKII as the redox sensitive kinase activated upstream of AMPK, thereby translating the persistent increase of intracellular H 2 O 2 into constitutive AMPK activation. The critical role of CaMKII in sustaining metabolic alterations required for cancer cell survival was illustrated by the effectiveness of KN93 in inhibiting AMPK phosphorylation and activity, and by its effectiveness in selectively killing MnSOD overexpressing cells as compared to neo control cells. Collectively, these experiments identified a mechanistic basis for the development of KN93 as an anti-cancer agent.
The likely clinical relevance of the data presented herein is further supported by the strong parallels between the results obtained in cell lines where MnSOD expression was manipulated and tissues obtained from patient biopsies where MnSOD, Bcl-2, and survivin were strongly upregulated in parallel with AMPK activation. The finding of elevated Bcl-2 and survivin expression in tumors has been generally associated with poor prognosis; therefore, mechanisms to suppress the expression of these anti-apoptotic proteins in tumors are generally regarded as attractive therapeutic goals. By specifically targeting the MnSOD/mtH 2 O 2 /AMPK axis, treatments may be developed to limit the expression of Bcl-2 and survivin thereby diminishing the reliance on existing therapies that eliminate cancer cells but have prominent detrimental off-target effects.
MnSOD upregulation, AMPK activation, and a gradual shift towards glycolysis as described in this work are to be regarded as part of a multifaceted process that occur in carcinogenesis. We see the MnSOD/oxCaMKII/AMPK-dependent metabolic switch in cellular metabolism as a necessary occurrence for the successful establishment of malignancy that, based on the results presented herein, permits cells to survive oxygen deprivation while strongly promoting anti-apoptotic defenses. As alterations in cellular metabolism from mitochondria-based to glycolytic encompasses relevant to almost all malignant solid tumors, we think that these data identify the MnSOD/H 2 O 2 /AMPK axis as a primary regulator of the Warburg effect and as such, a valuable new piece to the complex puzzle that is tumor progression.

Human patient sample analysis
Deidentified human tissue samples were obtained from the University of Illinois at Chicago tissue bank according to IRB exemption note 20110082-58687-1/OPRS/UIC. Multiple images were acquired from at least 6 individual normal and 6 cancerous breast, prostate and colon tissues, as identified by Dr. Andre Kajdacsy-Balla, a clinical pathologist. Representative images were used for Figures 1, 4F, 9 and Supplementary Fig. 1.

Fluorescent immunohistochemistry
Slides were blocked with regular fetal calf serum and incubated with primary antibody (MnSOD, LDH, diluted 1:1000 in 1X TBST, Abcam, Cambridge, MA), overnight at 4°C. After rinsing in TBST, sections were incubated with Alexafluor conjugated secondary antibodies (Invitrogen/Life Sciences, Grand Island, NY). Slides were examined on a Nikon ECLIPSE E400 microscope and documented using SPOT Advanced version 4.0.1 software.

TMA Analysis
MnSOD expression in multiple molecular subtypes of breast cancer was assessed using immunofluorescent imaging of tissue micro-array TMA-1005 (Protein Biotechnologies, Ramona, CA). Protein was blocked with normal serum and incubated with anti-MnSOD, anti-pAMPK-Thr172 (diluted 1:100 in 1x TBST containing 0.1% bovine serum albumin, Abcam, Cambridge, MA) overnight at 4°C. Slides were then incubated with Alexafluor conjugated secondary antibodies (Invitrogen/Life Sciences, Grand Island, NY) and examined on an Apotome (Zeiss, Jena, Germany). Relative fluorescent intensity was measured using ImageJ, and RFU values were associated with molecular subtypes described previously [69].

Quantitation of Relative Fluorescent Units
Corrected total fluorescence was calculated as described previously [70]. Three background samples were taken per selection to assure proper calibration.

Measurement of MnSOD activity
MnSOD was immunoprecipitated from cell lysates using a mouse anti-MnSOD antibody conjugated to protein A-bound resin. 20μg or protein from lysates was loaded into PBS solutions containing 20μg anti-MnSOD antibody. MnSOD activity was calculated compared to MCF7/neo based on the ability of MnSOD-bound to resin to inhibit the reduction of 50 μM cytochrome c Fe(III) reduced by hypoxanthine (50μM)/xanthine oxidase (10 mU/ml) system. Incubations of MnSOD-immobilized to resin, cytochrome c and xanthine oxidase/ hypoxanthine were vigorously agitated. Total cytochrome c reduction was measured by absorbance at 550 nm after 30 minutes incubation at room temperature.

6-phosphofructokinase (PFK), pyruvate kinase (PK) and hexokinase (HK) activity assays
Colorimetric assay kits were purchased from Abcam (Cambridge MA). Enzyme activities were measured following manufacturer's instructions. In brief, equal amounts (1-2 × 10 6 cells) of each cell line were lysed with cold assay buffer on ice, and supernatant mixed with freshly prepared reaction mix in 96-well plates. The plates were then incubated at 37°C for 60 minutes, and absorbance was measured every 10 minutes. Two time points (20 min and 60 min) were chosen to calculate the enzyme activity.

Transformation of MCF10A-Er-Src and silencing of MnSOD
For silencing of MnSOD in MCF10A(Er-Src) cells, Lipofectamine LTX (Invitrogen) was used as per manufacturer's instructions. Silencing was performed using control scrRNA and MnSOD siRNA (Santa Cruz Biotechnologies). Briefly, cells were incubated with either scrRNA or siRNA at 37°C for 8 hours, and then treated with 1 μM tamoxifen (Sigma Aldrich). The cells were harvested for analysis 48 hours after tamoxifen was added. For transformation MCF10A-Er-Src cells were plated at 50% confluency and maintained in 1 μM tamoxifen for the duration of the experiment as indicated. MCF10AEr-Src cells were a kind gift from Dr. Kevin Struhl (Harvard University).

Statistical analysis
Statistical analysis was performed with GraphPad InStat by using one-way ANOVA with Student-Newman-Keuls comparison. A value of p<0.05 was considered significant whereas values of p<0.01 and p<0.001 were considered highly significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Quantification of corrected total fluorescence of MnSOD was performed using ImageJ and statistical analysis was performed using one-way ANOVA (GraphPad InStat). **p<0.01; ***p<0.001. For quantification of protein expression levels in patient samples (shown in B and D) a minimum of 15 independent cases were analyzed.    Transfection of mito-catalase into neo and MnSOD overexpressing cells reduced AMPK activation in Mn11 and Mn28 indicating that H 2 O 2 originating from mitochondria activates AMPK. Mito-catalase gene was delivered to MCF7 cells using a lentiviral construct 72h prior to harvesting (C) Treatment with mito-catalase reduced steady state levels of ATP in Mn11 indicating that cells expressing high MnSOD levels depend on mtH 2 O 2 and AMPK activation to support ATP production. Steady state ATP levels were assessed by a chemiluminescence employing luciferin/luciferase mixtures (D) Diminished lactate production by mito-catalase in Mn44 and Mn11 further indicated that MnSOD overexpression via mtH 2 O 2 enhances glycolysis which in turn sustains ATP production. (E) Mito-catalase did not affect the OCR/ECAR ratio in neo cells but significantly altered OCR/  Panel to the right shows quantification by densitometry of oxidized CaMKII detected in three independent experiments. (B) Impact of KN93 (a bona fide CaMKII inhibitor) on lactate production and steady ATP levels in neo, Mn44, Mn11. Water soluble KN93 (5 μM) was diluted into complete media. Cells were harvested at 36 h for assessments of lactate production and steady state levels of ATP as described in methods. (C) The impact of CaMKII inhibition with KN93 on AMPK phosphorylation and activity (pACC was used as a surrogate) was examined. Cells exposed to KN93 (5 μM) were harvested at the indicated time points after KN93 addition to full media, lysed and analyzed for AMPK(Thr172p) and pACC (Ser79) content by Western blot. *p<0.05; **p<0.05; ***p<0.01. Statistical analysis was performed using 6 independent replicates. Cells (neo, Mn44 and Mn11) were exposed to the specified compounds as indicated for 48 h in full media. After 48 h cells were harvested and viability was determined by flow cytometry using YOPRO TM and propidium iodide as described in methods. Experiments show averages of 6 independent experiments. Statistical analysis was performed using oneway ANOVA (GraphPad InStat) *p<0.05; **p<0.01; ***p<0.001.