Mitochondrial Superoxide Dismutase Has a Protumorigenic Role in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinomas (OCCC) represent approximately 10%–25 of all epithelial ovarian cancer (EOC), depending on ethnic background ( 1 ). It is now evident that OCCC differs widely from the more common high-grade serous adenocarcinoma. While the primary tumor mass of OCCC is found on the ovary, its origin is not thought to be the ovary or fallopian tube, but rather stem from endometrioid tissue and endometriosis. Due to the ROS stress associated with endometrioisis OCCC has been characterized as a stress-responsive cancer ( 2 , 3 ). Gene expression studies have shown increased expression of a number of stress-related and metabolic genes, in particular those related to hypoxic insult, glycolysis and antioxidant defense mechanisms ( 4 ). The Nrf2 stress response pathway has been implicated in driving some of these changes, including the enhanced expression of mitochondrial manganese-containing superoxide dismutase (Sod2) ( 5 ). Sod2 is a nuclear-expressed mitochondria-targeted antioxidant enzyme, which catalyzes the conversion of two molecules of superoxide anion (O 2 •− ) to hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). Since a small amount of O 2 •− leakage occurs during normal oxidative phosphorylation from the mitochondrial electron transport chain, Sod2 is of importance in preventing redox-mediated damage of mitochondrial proteins and preserving mitochondrial function. Enhanced O 2 •− production can occur in response to stress, such as hypoxia, and has been linked to a number of cancer types. For this reason, Sod2 was initially characterized as a tumor suppressor gene ( 6 ). However, recent data now point to a dichotomous role of Sod2 during tumor progression. Evidence suggests that Sod2 expression is often increased during metastatic progression, potentially as an adaptation to enhanced levels of intra- and extracellular ROS ( 7 ). While Sod2 may initially prevent reactive oxygen species (ROS)-mediated DNA damage to facilitate tumor initiation, increased Sod2 expression appears to conversely contribute to the metastatic phenotype by altering redox-signaling pathways ( 8 ). We and others have observed that increased Sod2 expression correlates with a shift to higher intracellular H 2 O 2 levels, contributing to pro-metastatic behavior ( 7 , 9 , 10 ). In the present study we set out to interrogate the role of Sod2 in OCCC and found a dual function for this mitochondrial antioxidant in OCCC tumorigenicity. Sod2 not only provides a protective role in scavenging mitochondrial O 2 •− , thereby maintaining high mitochondrial function and proliferation, but also alters the steady-state ROS balance to drive H 2 O 2 -mediated migration and metastasis of OCCC cells.

Mining publicly available expression data ( Oncomine.org ) revealed that Sod2 mRNA levels are elevated in OCCC compared to other ovarian cancer histological subtypes ( Supplementary Fig. S1 ). Two representative data sets displayed in Figure 1A and B show that levels of this antioxidant enzyme are statistically higher than in any other ovarian cancer subtype. Similarly, significantly higher Sod2 mRNA expression was observed by microarray analysis in a panel of OCCC cell lines when compared to cell lines of high grade serous adenocaricnoma origin ( Fig. 1C ). This was further verified by assessing Sod2 expression in cultured ovarian cancer cell lines, using semi quantitative real-time RT-PCR and western blotting ( Fig. 1D and E ). To further investigate the role of Sod2 we utilized two OCCC cell lines, ES-2 and TOV-21-G. Sod2 expression was inhibited by shRNA and siRNA transfection, which was demonstrated to lead to a concomitant decrease in Sod2 enzyme activity ( Fig. 2A , Supplementary Fig. S2 & S3A ). Following Sod2 expression knockdown, ES-2 cell proliferation rate ( Fig. 2B , Supplementary Fig. S3B ) and clonogenicity ( Fig. 2C , Supplemental Fig. 3SC ) were significantly attenuated. This appeared to be Sod2 concentration dependent. A 30% reduction in Sod2 levels mediated by stable shRNA transfection reduced clonogenicity by approximately 50% (shSod2_#1), while a 70% Sod2 decrease almost completely abrogated the cells ability to survive in this assay (shSod2_#2; Supplemental Fig. S3C ). Analysis of PARP cleavage and Annexin V staining suggested that the decrease in cell viability observed in cells with 30% Sod2 knock-down is not related to a significant increase in apoptosis ( Supplemental Fig. S4 ). This cell line, referred to as shSod2 in subsequent Figures, was chosen for subsequent studies to achieve pathophysiologically relevant changes in Sod2 expression (rather than complete loss), which also closely reflects Sod2 levels observed in non-OCCC cell lines OVCA433 and OVCA429 ( Fig. 1E ). The CAM ex ovo model was used to further test the role of Sod2 on ES-2 tumorigenicity. A significant decrease in tumor size and weight was observed in tumors grown from ES-2 cells with reduced Sod2 expression (shSod2; Fig. 2D ). In addition, the shRNA-Sod2 tumors exhibited less vascularization compared to the control groups ( Fig. 2D ), suggesting that Sod2 may contribute to both proliferation and the recruitment of blood vessels to the tumor. We previously demonstrated that OCCC cell lines are highly energetic, and depend on both oxidative phosphorylation and glycolysis for their energy needs ( 13 ). Given that Sod2 has a primary role in protecting mitochondria from excess O 2 •− , the effect of Sod2 knock-down on mitochondrial respiration was assessed. Oxygen consumption rate (OCR), representing mitochondrial oxidative phosphorylation, and extracellular acidification rate (ECAR), correlating with glycolytic activity, was measured using extracellular flux analysis in both ES-2 and TOV-21-G OCCC cell lines following Sod2 knock-down ( 13 , 16 ). Stable shRNA-Sod2 decreases in ES-2 and transient siRNA-mediated knock-down of Sod2 in TOV-21-G cells resulted in significant reduction in basal OCR compared to control scramble RNAi transfected cells ( Fig. 3A–D , Supplementary Fig. S5A ). Further, respiratory reserve capacity, a measure of the cells’ ability to enhance respiration in response to physiological cues and stress, was significantly inhibited with reduced Sod2 levels ( Fig. 3B & D ), suggesting that Sod2 plays a major role in maintaining mitochondrial health to support maximal respiration. Although slight, a consistent negative effect on OCR was observed in stable shRNA-Sod2 cells following FCCP treatment ( Fig. 3B ). A wide range of FCCP concentrations were tested on these cells, but none were able to enhance OCR with Sod2 loss. FCCP can be inhibitory at high concentrations and it has been speculated that this may be due to a loss in the ability of mitochondria to accumulate respiratory substrates ( 16 ). While not tested here, it is possible that sustained Sod2 expression decreases, and concomitant increases in mitochondrial O 2 •− levels, may exacerbate this FCCP-depended OCR inhibition, thereby influencing mitochondrial membrane integrity and substrate transport. No significant increases in ECAR were observed between control and sh- or si-RNA transfected cells, suggesting that decreases in Sod2 expression do not influence a compensatory shift towards glycolysis ( Supplementary Fig. S5B–D ). The above observations show that both si- and shRNA mediated decreases in Sod2 reduced basal OCR and respiratory reserve capacity, indicating that Sod2 is important in maintaining mitochondrial respiration in OCCC. To assess whether a decrease in Sod2 expression result in compromised O 2 •− scavenging, which may be one of the causes of compromised mitochondrial function, the presence of mitochondrial O 2 •− was evaluated using the mitochondria-targeted redox-sensitive dye MitoSox. As expected, increased oxidation and consequential enhanced fluorescence of MitoSox were observed in the Sod2 knockdown cells compared to controls ( Fig. 3E & F ). Addition of the Sod2-mimetic porphyrin (MnTnBuOE-2-PyP5+), which acts as a O 2 •− scavenger, reduced MitoSox oxidation and in both control and shRNA-Sod2 groups ( Fig. 3E & F ). We have previously demonstrated that enhanced Sod2 expression is implicated with metastatic progression ( 7 , 8 , 17 ). To investigate the role of Sod2 during OCCC metastasis, the appearance of metastatic lesions of GFP-labeled ES-2 cells were investigated in the CAM tumor model. Single cells and micrometer-sized cellular clusters were highly abundant throughout the membrane in the control group, which could be observed 2–3 cm from the tumor ( Fig. 4A ). In contrast, metastatic spread from shRNA-Sod2 knockdown tumors was limited to the appearance of single cells in the membrane confined to an approximate 1–1.5 cm radius from the tumor ( Fig. 4A ). Further, lung metastases in the chick embryo were observed in 11 of the 12 controls compared to only 5 of 10 embryo’s in the shRNA-Sod2 group ( Fig. 4B ). Further, 10 of 12 control tumors metastasized into the liver, whereas only 4 of10 liver metastases were observed in the shRNA-Sod2 group ( Fig. 4B ). While clusters of five or more cells were found in the lungs and livers of control groups, only single cells were detected in the Sod2 knockdown group ( Fig. 4B ). Due to the significant abrogation of metastatic spread in response to Sod2 expression decreases, the role of Sod2 on cell migration was further investigated. Cell migration, assessed by wound healing assays, was significantly inhibited with reduced Sod2 expression in ES-2 cells ( Fig. 5A ; Supplementary Fig. S6 ). Further, the ability of cellular spheroid clusters to attach and cells to migrate from the spheroid onto collagen-I and un-coated surfaces was also compromised with reduced Sod2 expression ( Fig 5B ). Anchorage independent spheroid formation is a commonly observed phenotype of ovarian cancer cells metastasizing via the transcoelomic route through the IP cavity and these have been shown the ability to attach on the peritoneum to form metastatic lesions. These data suggest that Sod2 plays an important role in tumor spheroid metastasis ( Fig. 5B ). While spheroids of equal size were chosen for this assay, it should be noted that Sod2 knock-down also decreased ES-2 spheroid growth in anchorage independence (data not shown). To gain mechanistic insights into the signaling pathways that may be altered by Sod2-mediated metastasis, phosphorylation profiles of Akt, p130cas and focal adhesion Kinase (FAK) were investigated. These were chosen based on previous observations of their redox regulation and involvement in tumor cell migration ( 8 , 9 , 18 , 19 ). shRNA-Sod2 cells exhibited a 50% decrease in phospho-Akt levels compared to scramble control cells, while there was no appreciable change observed in phosphorylation of FAK or the focal adhesion adapter protein p130cas ( Fig. 5C ). The effects on Akt phosphorylation were also Sod2 concentration dependent, where cells with lower Sod2 expression demonstrated a more striking decrease in Akt phosphorylation ( Supplementary Fig S6 ). These data suggest that Akt signaling may be important in driving Sod2-mediated tumrigenicity and metastasis of OCCC. Sod2 is the primary enzyme involved in converting O 2 •− to H 2 O 2 within the mitochondria. While it serves as a protective mechanism to maintain mitochondrial function ( Fig. 3 ) by removal of damaging O 2 •− , a shift towards increasing levels of H 2 O 2 has also been observed in response to enhanced Sod2 expression ( 8 , 10 , 18 , 20 , 21 ). Due to its relative stability and ease in traversing cellular membranes, H 2 O 2 can mediate redox signaling, including events that drive migration ( 9 ). To test if Sod2 changes the steady-state H 2 O 2 levels in OCCC, we assessed intracellular H 2 O 2 status in control and shRNA-Sod2 ES-2 cells using a biochemical assay based on the irreversible inhibition of catalase by aminotriazole ( 15 , 17 ). Steady-state levels of H 2 O 2 were reduced approximately 2-fold in Sod2 knockdown cells compared to controls ( Fig. 6A ), while baseline catalase activity and protein expression were comparable ( Fig. 6B ). In order to test whether OCCC cell migration is H 2 O 2 depended, wound-healing assays were carried in the presence of catalase, which catalyzes the conversion of H 2 O 2 to H 2 O and O 2 . As previously demonstrated, exogenous application of recombinant catalase resulted in accumulation of catalase within ES-2 cells ( Supplementary Figure S7 ) ( 9 ). Catalase significantly reduced the migration of both control and shRNA-Sod2 cells, suggesting that H 2 O 2 is a promoter of ES-2 cell migration ( Fig. 6C ). Conversely, treatment with low levels of H 2 O 2 (5 µM) significantly reversed the slow migration of shRNA-Sod2 cells ( Fig. 6C ). Further, 5 and 50 µM H 2 O 2 treatment was able to increase Phospho-Akt levels ( Fig. 6D ), while catalase expression abrogated Akt phosphorylation ( Supplementary Fig. S8 ), suggesting that this may be an important redox-dependent signaling pathway in OCCC. It was noted that ES-2 control cells were not able to tolerate long-term exposure to low dose H 2 O 2 during migration assays ( Fig. 6C ). To examine this further, cell viability was assessed in response to H 2 O 2 . A significant reduction in cell survival in response to H 2 O 2 was observed in the control group compared to cells with decreased Sod2 expression ( Fig. 7A ). This suggests that a higher intracellular steady-state H 2 O 2 milieu in OCCC predisposes cells to enhanced killing by additional exposure to low level exogenous H 2 O 2 . The above data imply that high Sod2 expression provides several advantages to OCCC, by protecting mitochondrial function through scavenging of O 2 •− and driving H 2 O 2 dependent migration. While these attributes are advantageous for OCCC survival and metastatic progression, an enhanced intracellular steady-state H 2 O 2 level presents a double-edged sword, as these cells are consequentially more susceptible to H 2 O 2 toxicity ( Fig. 7B ).

Although the five different EOC histological subtypes share the same primary tumor location on the ovaries, it is now evident that these are distinct diseases with vastly different tissue origins, and genetic and epigenetic profiles ( 2 , 11 ). In the present study, we show that Sod2 is highly expressed in OCCC compared to other EOC histological subtypes, and that this mitochondrial antioxidant plays a significant role in OCCC tumorigenicity and metastasis. Intracellular ROS are maintained within a narrow range, tightly regulated by the balance of the rate of ROS production and ROS scavenging/detoxifying by antioxidant enzymes. This balance is often disrupted in the context of cancer, due to high ROS production as a consequence of changes in metabolism or the tumor environment (e.g. hypoxia), and the resulting changes in antioxidant expression. Since the mitochondrial respiratory chain is the major site of O 2 •− generation within cells, Sod2 plays an important role in maintaining cellular ROS balance. Based on the above findings Sod2 appears to play a dual role in enhancing OCCC tumorigenicity; first by protecting cells from mitochondrial O 2 •− damage; and second, by shifting the steady-state ROS balance towards H 2 O 2 . We recently demonstrated that a distinguishing feature of OCCC is their unique metabolic phenotype. Compared to serous adenocarcinoma cells, OCCC cell lines were significantly more energetic, displaying both very high levels of mitochondrial oxidative phosphorylation and glycolytic flux ( 13 ). Our data suggest that Sod2 is intricately involved in maintaining this high rate of oxygen consumption, potentially by preserving mitochondrial function as a consequence of O 2 •− scavenging ( Fig. 3 ). By preventing damage mediated by O 2 •− or secondary products of mitochondrial electron transport chain complexes, Sod2 likely supports the high rate of OCCC proliferation, clonogenicity and tumor growth. The present study suggests that inhibiting mitochondrial antioxidant defenses may provide and alternate strategy to therapeutically target OCCC. In addition to scavenging O 2 •− and maintaining mitochondrial health for optimal cell proliferation, we believe that Sod2 has an additional role in promoting the aggressiveness of OCCC, by shifting steady-state H 2 O 2 levels and driving pro-metastatic behavior ( Fig. 4 – 6 ). It has been previously shown that high expression of Sod2 is associated with metastatic progression ( 8 , 10 , 17 , 20 – 23 ), and that dependence of cancer cell migration is related to cellular H 2 O 2 production ( 9 , 19 , 24 , 25 ). For example, we have shown that steady-state increases in H 2 O 2 can lead to induction of the focal adhesion kinase pathway and migration of metastatic bladder cancer cells and cells with enforced Sod2 expression ( 9 , 19 ). This was mediated by oxidation-dependent inhibition of the phosphatase PTPN12, leading to enhanced phosphorylation of p130cas and Rac1 activation. In addition, work from the Melendez’ group has shown that Sod2 expression significantly contributes to the expression of the matrix-degrading enzyme MMP-1 in a H 2 O 2 -dependent manner ( 21 ), and that the Sod2/H 2 O 2 -dependent inhibition of the dual lipid protein tyrosine phosphatase PTEN enhances Akt/GSK3β/VEGF-dependent angiogenesis ( 18 ), both processes contributing significantly to metastasis. Our present data suggest that Sod2 may similarly contribute to metastatic progression of OCCC by activating Akt signaling ( Fig 5 and 6 ). In addition to its pro-survival function, Akt has been shown to influence metastasis and cell migration by regulating cytoskeletal rearrangement, pro-metastatic cell signaling and gene transcription ( 26 ). These results are of specific importance to OCCC, which, unlike other ovarian cancer histological subtypes, has been characterized by high frequency Akt pathway activation. 70% of early and 68% of late stage OCCC cases have been shown to display phospho-Akt (S473) staining ( 27 ). About 38% of OCCC cases show PTEN loss ( 28 ) and 40% of cases PI3K activating mutations ( 29 ). Our data imply that Sod2-dependent Akt phosphorylation may also contribute to high activation of Akt signaling in OCCC. Since Akt-phosphorylation was highly susceptible to H 2 O 2 treatment, it suggests that this signaling pathway is redox regulated in OCCC, with a plausible mechanism for this being the oxidation of PTEN ( 18 ). Although an increase in Sod should theoretically not result in higher levels of H 2 O 2 production based on the kinetic properties of Sod2 ( 30 ), a number of studies have demonstrated increases in H 2 O 2 levels that correlate with Sod2 expression ( 18 , 31 – 33 ). While the reason for this observation in OCCC has not been investigated, there are plausible explanations for this increase in steady-state H 2 O 2 as a consequence of Sod expression. These primarily relate to changes in the reaction rates within the mitochondrial electron transport chain (ETC). For example, it has been proposed that Sod2 in the mitochondria may alter the flux of O 2 ·− from some quinone/semiquinone/hydroquinone triads, such as coenzyme Q, thereby driving the reaction into the direction of O 2 ·− production, potentially leading to enhanced localized dismutation to H 2 O 2 by Sod2 ( 31 , 34 ). Alternatively, inhibition of cytochrome c oxidase by nitric oxide, arising as a consequence of Sod2 expression may influence the reduction state of the ETC and drive O 2 ·− and H 2 O 2 production ( 35 ). Our observation that Sod2 knock-down also decreases H 2 O 2 levels, suggests that Sod2 is involved in regulating H 2 O 2 balance within cells and this may contribute to H 2 O 2 -mediated redox signaling. While Sod2 appears to contribute to H 2 O 2 -mediated metastatic progression, an enhanced steady-state H 2 O 2 milieu may also present a disadvantage to OCCC cells. Our data suggest that cells with high Sod2 levels and concomitant increases in intracellular H 2 O 2 are more susceptible to exogenous sources of redox stress ( Fig. 7A ). This likely puts cells closer to the cytotoxic threshold of H 2 O 2 , which is reached once cells are further challenged by exogenous ROS. Interestingly, OCCC do not appear to have enhanced expression of Catalase to provide additional scavenging of excess H 2 O 2 ( Fig. 6A ). While sub-lethal levels of H 2 O 2 have been shown to contribute to redox signaling, high levels of H 2 O 2 can elicit tumor cell death by a number of pathways, including apoptosis, protein/DNA damage and mitochondrial dysfunction ( 36 – 38 ). Further, it was recently reported that H 2 O 2 exposure of tumor cells with enforced Sod2 expression can result in Sod2 peroxidase activity, leading to mitochondrial damage and dysfunction ( 39 ). An increased H 2 O 2 steady-state has been observed in a number of cancer cells ( 17 , 40 – 42 ) and lends credence to the idea that this higher H 2 O 2 threshold may be exploited therapeutically. In this regard, the use of high-dose ascorbic acid, which is oxidized within tumor cells to produce H 2 O 2 , has recently been revisited for use in cancer treatment ( 43 – 45 ) and has shown promise in early clinical trials in advanced stage cancers ( 46 , 47 ). Ascorbate and concomitant H 2 O 2 – mediated DNA damage and apoptosis were shown to enhance ovarian cancer cell death and increased chemosensitivity ( 48 ). While that study was not focused on OCCC, this type of treatment may be of particular benefit to this histological subtype given the high expression of Sod2. It is important to highlight that cancer cells with enhanced Sod2 expression may respond differently to ROS-producing agents, depending on both the type and cellular location of the ROS/reactive nitrogen species generated. For instance, Sod2 may enhance scavenging of O 2 •− , and therefore provide chemoresistance benefits to the tumor cells in response to these ROS. Conversely, while an increase in steady-state H 2 O 2 facilitates redox-signaling beneficial to the cancer cells, this higher threshold may facilitate H 2 O 2 -mediated OCCC cell death in response to further insult by exogenous sources of H 2 O 2 . Understanding the complex interaction of antioxidants and ROS in OCCC is therefore of importance and may provide novel therapeutic avenues to pursue for this histological subtype of ovarian cancer.

Oncomine.org was used to screen Sod2 expression in ovarian cancer histological subtypes ( Supplementary Fig. S1 ). Two representative data sets are shown in Fig. 1A & B (GEO Accession no. GSE2109 & GSE6008 ). Microarray data of the following ovarian cancer cell lines was obtained using the GeneChip Human Genome U133A 2.0 Array (affymetrix; GEO Accession no.: GSE25428 ) ( 4 , 11 ). Data represents expression of Sod2 probe 215223_s_at (log2 RMA normalized). OCCC: JHOC-5, JHOC-7, JHOC-8, JHOC-9, KOC-5C, KOC-7C, OVISE, OVTOKO, RMG-1, RMG-2, RMG-5, TAYA, TOV-21-G. Serous adenocarcinoma: CAOV3, Fuov1, HEY, Hey-A8, Hey-Ce, JHOS-2, JHOS-3, JHOS-4, M41, M41-cisR, OV90, OVARY1847, OVCA420, OVCA429, OVCA432, OVCAR3, PEO1, PEO4, SKOV3. Mucinous: JHOM-1, JHOM-2B, MCAS, OMC-3. Endometrioid: OVK-18, TOV-112D. Adenocarcinoma: A2780 (A2780-J), A2780J-cisR, DOV13, OVCAR2, OVCAR5, OVCAR8. Teratocarcinoma: CH1, PA1. Undifferentiated: TYK-nu, TYK-nu cisR. Prior to microarray analysis, cell lines were authenticated by STR analysis at the Fragment Analysis Facility, Johns Hopkins University (PowerPlex 1.2 System; Promega) or at the University of Colorado Cancer Center (AmpFℓSTR Identifier Plus PCR Kit, Applied Biosystems) ( 11 ). At commencement of this study ES-2 and TOV-21-G cells were newly obtained from American Type Culture Collection (ATCC, Manassas, VA). Authenticity was verified by ATCC using STR analysis. ES-2 cells were maintained in McCoy’s 5A media + 10% FBS and TOV-21-G cells in 40% Media199/40% MCBD supplemented with 20% FBS and sodium bicarbonate. Cells were maintained at 37°C with 5% CO 2 . Scramble non-targeting control and Sod2-specific siRNA oligonuecliotides were synthesized by Life Technologies/Dharmacon. 5’- CAACAGGCCUUAUUCCACU-3’ and 5’- AAGUAAACCACGAUCGUUA-3’ sequences were used as siSod2_#1 and siSod2_#2 respectively ( Supplementary Fig.S2 ) and 10pmol transfected into cells using lipofectamine RNAiMax (Invitrogen). Short hairpin RNA (shRNA) with non-targeting scramble sequence or targeting Sod2 (shSod2_#1: 5’-CTGACGGCTGCATCTGTTGGTGTCCAAGG-3’, and shSod2_#2: 5’-ACCTGAACGTCACCGAGGAGAAGTACCAG-3’) in pGFP-V-RS vector (Origene; TG309190) were used to stably transfect ES-2 cells ( Fig. 2 ; Supplementary Fig.S3 ). The clone expressing shSod2_#1 was used in Figures 2 – 7 . Protein expression was analyzed by standard western blotting using antibodies Antibodies were from Cell Signaling Technology (Boston, MA; pAkt-s473, Akt, pFAK-Y397, FAK, p-p130cas-Y165, p130cas) or Abcam (Cambridge, MA; Sod2). Primary Antibodies were diluted in blocking solution (5% nonfat milk in TBS with 0.1% tween 20, 1:1000), and incubated overnight at 4 °C. Blots were visualized using Femto and Pico ECL chemiluminescence substrate (Thermo scientific, Rockford, IL) and imaged using a ChemiDoc MP system (BioRad). Densitometric analysis was performed using ImageJ software (NIH). Each protein band was normalized to the respective GAPDH or β-Actin loading control band. Sod2 activity was analyzed using Sod2 in-gel zymography as previously described ( 12 ). Briefly, cell lysates were loaded on non-denaturing acrylamide gels, followed by electrophoresis. Sod2 activity is visualized by the inhibition of nitroblue tetrazolium reduction. Single cell survival clonogenicity assays were performed as previously described ( 13 ). Briefly, 100 cells were plated in each well of a 6 well plate colonies visualized after 10 days using crystal violet. Viability was assessed by cell counting using trypan blue (1%) staining or crystal violet uptake assays ( 13 ). Each CAM was inoculated with 5× 10 5 ES-2 cells stably expressing either scramble-shRNA-GFP or Sod2-shRNA_#1-GFP that were suspended in 50 µl PBS (with 1 mM MgCl 2 , 0.5 mM CaCl 2 , 100 U/mL penicillin, and 100 µg/mL streptomycin), essentially as previously described ( 14 ). Tumors were allowed to form for 7 days prior to termination of the experiments by sacrificing the chick embryo. Tumors on the CAM were removed and measured. Chorioallantoic membrane and chick embryo organs (liver and lung) were collected for tumor metastasis analysis by surveying for GFP-labeled cells. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and mitochondria stress tests were measured using the Seahorse XF24 3 Extracellular Flux Analyzer (Seahorse Bioscience; Billerica, MA), as described previously ( 13 ). Cells were plated at a density of 40,000 cells/well and media replaced with XF media the following day 1hr prior to the assay. Three measurements of OCR and ECAR were taken at baseline and after each injection of the following mitochondrial stress test compounds: oligomycin (1µM; complex V inhibitor); FCCP (0.75µM; proton gradient uncoupler); antimycin A (1µM; complex III inhibitor). Basal and maximal respiration were normalized by subtracting non-mitochondrial OCR (i.e. after Antimycin A addition). Respiratory reserve capacity was calculated as the difference between maximal and basal OCR. ATP-linked OCR was derived as the difference between basal and Oligomycin A inhibited OCR. Data was normalized to total protein content in each well. Cell migration was assessed in serum free media by wound healing assays using Ibidi inserts (Martisried, Germany) and quantified after 72hrs. Ibdi inserts were removed from a monolayer of GFP-labeled cells to expose the cell-free wound area. Fluorescence images were taken after 72 hrs of migration and overlayed with corresponding images at time 0hr. Pixels representing GFP-labeled cells were quantified within the wound area using Image J and corrected by subtracting any GFP-detected cells in the same area at time 0. Cells were plated at a density of 1000 cells per well in ultra-low attachment 96 well plates (Corning) and incubated for 5 days. Spheroids were transferred to 24 well plates with or without Collagen I coating. Percentage outgrowth was calculated by subtracting the area covered by migrating cells onto the collagen matrix from the area of the spheroid at time 0h. As an indicator of mitochondrial O 2 •− the oxidation and fluorescence of the redox-sensitive MitoSox Red dye (Life Technologies) was monitored by live cell imaging according to manufacture’s instructions. Cells were imaged to detect oxidation and fluorescence of MitoSox using a Leica SP5 II AOBS confocal microscope following incubation with dye in HBSS for 30 min at 37°C. The Manganese Porphyrin O 2 •− scavenger ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-2-PyP5+) was generously provided by Dr. Ines Batinic Haberle (Duke University). Concentration of cellular H 2 O 2 was determined by measuring the rate of inactivation of catalase with amino1,2,4-triazol (ATZ), according to Yusa et al. ( 15 ). ATZ irreversibly inactivates catalase by covalently binding with intermediate compound I, formed following oxidation of catalase by H 2 O 2 . Briefly, cells were treated with 20 mM ATZ for different time intervals (15 and 30 min). Cells were washed with PBS and protein lysates were prepared in 50 mM phosphate buffer (pH=7.4) with protease inhibitors. Decomposition of H 2 O 2 by catalase in protein lysates was analyzed using ultraviolet spectroscopy at 240nm wavelength. H 2 O 2 concentration was determined using the equation [H 2 O 2 ] = k/k 1, where k is the empirically-determined pseudo first order rate constant of catalase inactivation in the cells by ATZ ( Figure 6A ), while k 1 is the rate of compound I formation (1.7 × 10 7 M −1 s −1 ). All data presented are representative of at least three independent experiments and expressed as mean ± SEM, unless otherwise stated. Statistical data analysis (ANOVA with Tukey’s post-test or student t-test) was performed using GraphPad Prism Software v6. p < 0.05 was considered to be significant.