Mitochondrial Superoxide Dismutase Has a Protumorigenic ...cancerres.aacrjournals.org/content/canres/75/22/4973.full.pdf · Tumor and Stem Cell Biology Mitochondrial Superoxide Dismutase

Tumor and Stem Cell Biology

Mitochondrial Superoxide Dismutase Has aProtumorigenic Role in Ovarian Clear CellCarcinomaL.P. Madhubhani P. Hemachandra1, Dong-Hui Shin1,2, Usawadee Dier1, James N. Iuliano3,Sarah A. Engelberth1, Larissa M. Uusitalo1, Susan K. Murphy4, and Nadine Hempel1,2

Abstract

Epithelial ovarian cancer (EOC) is the fourth leading cause ofdeath due to cancer in women and comprises distinct histologicsubtypes, which vary widely in their genetic profiles and tissues oforigin. It is therefore imperative to understand the etiology ofthese distinct diseases. Ovarian clear cell carcinoma (OCCC), avery aggressive subtype, comprises >10% of EOCs. In the presentstudy, we show that mitochondrial superoxide dismutase (Sod2)is highly expressed in OCCC compared with other EOC subtypes.Sod2 is an antioxidant enzyme that converts highly reactivesuperoxide (O2

*�) to hydrogen peroxide (H2O2) and oxygen(O2), and our data demonstrate that Sod2 is protumorigenic andprometastatic in OCCC. Inhibiting Sod2 expression reducesOCCC ES-2 cell tumor growth andmetastasis in a chorioallantoicmembrane (CAM)model. Similarly, cell proliferation,migration,

spheroid attachment and outgrowth on collagen, and Akt phos-phorylation are significantly decreasedwith reduced expression ofSod2. Mechanistically, we show that Sod2 has a dual function insupporting OCCC tumorigenicity and metastatic spread. First,Sod2 maintains highly functional mitochondria, by scavengingO2

*�, to support the high metabolic activity of OCCC. Second,Sod2 alters the steady-state ROS balance to drive H2O2-mediatedmigration. While this higher steady-state H2O2 drives prometa-static behavior, it also presents a doubled-edged sword forOCCC,as it pushed the intracellularH2O2 threshold to enablemore rapidkilling by exogenous sources of H2O2. Understanding the com-plex interaction of antioxidants and ROS may provide noveltherapeutic strategies to pursue for the treatment of this histologicEOC subtype. Cancer Res; 75(22); 4973–84. �2015 AACR.

IntroductionOvarian clear cell carcinomas (OCCC) represent approximately

10% to 25% of all epithelial ovarian cancer (EOC), depending onethnic background (1). It is now evident that OCCCdiffers widelyfrom 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 ratherto stem from endometrioid tissue and endometriosis. Because ofthe reactive oxygen species (ROS) stress associated with endome-trioisis,OCCChas been characterized as a stress-responsive cancer(2, 3). Gene expression studies have shown increased expressionof a number of stress-related and metabolic genes, in particularthose related tohypoxic insult, glycolysis, and antioxidant defense

mechanisms (4). The Nrf2 stress response pathway has beenimplicated in driving some of these changes, including theenhanced expression of mitochondrial manganese-containingsuperoxide dismutase (Sod2; ref. 5).

Sod2 is a nuclear-expressedmitochondria-targeted antioxidantenzyme, which catalyzes the conversion of two molecules ofsuperoxide anion (O2

*�) to hydrogen peroxide (H2O2) andoxygen (O2). Because a small amount of O2

*� leakage occursduring normal oxidative phosphorylation from the mitochon-drial electron transport chain, Sod2 is of importance in preventingredox-mediated damage of mitochondrial proteins and preserv-ingmitochondrial function. EnhancedO2

*� production can occurin response to stress, such as hypoxia, and has been linked to anumber of cancer types. For this reason, Sod2 was initiallycharacterized as a tumor suppressor gene (6). However, recentdata now point to a dichotomous role of Sod2 during tumorprogression. Evidence suggests that Sod2 expression is oftenincreased during metastatic progression, potentially as an adap-tation to enhanced levels of intra- and extracellular ROS (7).While Sod2 may initially prevent ROS-mediated DNA damage tofacilitate tumor initiation, increased Sod2 expression appears toconversely contribute to the metastatic phenotype by alteringredox signaling pathways (8). We and others have observed thatincreased Sod2 expression correlates with a shift to higher intra-cellular H2O2 levels, contributing to prometastatic behavior(7, 9, 10).

In the present study, we set out to interrogate the role of Sod2 inOCCC and found a dual function for this mitochondrial antiox-idant in OCCC tumorigenicity. Sod2 not only provides a protec-tive role in scavenging mitochondrial O2

*�, thereby maintaining

1Nanobioscience Constellation, Colleges of Nanoscale Science andEngineering,SUNYPolytechnic Institute,StateUniversityofNewYork,Albany, New York. 2Department of Pharmacology, Penn State Collegeof Medicine, Hershey, Pennsylvania. 3Department of Chemistry, StonyBrookUniversity, StateUniversityofNewYork, StonyBrook,NewYork.4Department of Obstetrics and Gynecology, Duke University MedicalCenter, Durham, North Carolina.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

L.P.M.P. Hemachandra and D.-H. Shin share first authorship.

Corresponding Author: Nadine Hempel, Department of Pharmacology, PennState College of Medicine, 500 University Drive, MC R130, Hershey, PA 17033.Phone: 717-531-4037; Fax: 717-531-5013; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3799

�2015 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 4973

on June 18, 2018. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 10, 2015; DOI: 10.1158/0008-5472.CAN-14-3799

http://cancerres.aacrjournals.org/

highmitochondrial function and proliferation, but also alters thesteady-state ROS balance to drive H2O2-mediated migration andmetastasis of OCCC cells.

Materials and MethodsOncomine data and ovarian cancer cell line microarray data

Oncomine.org was used to screen Sod2 expression in ovariancancer histologic subtypes (Supplementary Fig. S1). Two repre-sentative datasets are shown in Fig. 1A and B (GEO accession nos.GSE2109 and GSE6008). Microarray data of the following ovar-ian cancer cell lines were obtained using the GeneChip HumanGenome U133A 2.0 Array (Affymetrix; GEO accession no.:GSE25428; refs. 4, 11). Data represent expression of Sod2 probe215223_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 anal-ysis, cell lines were authenticated by STR analysis at the FragmentAnalysis Facility, Johns Hopkins University (Baltimore, MD;PowerPlex 1.2 System; Promega) or at the University of ColoradoCancer Center (Aurora, CO; AmpFlSTR Identifier Plus PCR Kit,Applied Biosystems; ref. 11).

Cell lines and cell culture conditionsAt commencement of this study ES-2 and TOV-21-G cells were

newly obtained from ATCC. Authenticity was verified by ATCCusing STR analysis. ES-2 cells were maintained in McCoy 5Amedia þ 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% CO2.

Sod2 knockdown using RNA interferenceScramble nontargeting control and Sod2-specific siRNA oligo-

nucleotides were synthesized by Life Technologies/Dharmacon.50- CAACAGGCCUUAUUCCACU-30 and 50- AAGUAAACCAC-GAUCGUUA-30 sequences were used as siSod2_#1 andsiSod2_#2, respectively (Supplementary Fig. S2) and 10 pmoltransfected into cells using Lipofectamine RNAiMax (Invitrogen).

Figure 1.Increased Sod2 mRNA expression is observed in OCCC compared with other ovarian carcinoma histologic subtypes. Two representative datasets from ovariancancer microarray studies are displayed as box and whisker plots (data obtained through Oncomine.org). A, clear cell (n ¼ 16), serous (n ¼ 24), mucinous(n ¼ 8), endometrioid (n ¼ 29), serous surface papillary (n ¼ 107); one-way ANOVA (P < 0.0002), Tukey posttest. �� , P < 0.0001; �� , P < 0.001;�� , P < 0.01 (GSE2109). B, clear cell (n ¼ 8), serous (n ¼ 41), mucinous (n ¼ 13), endometrioid (n ¼ 37); one-way ANOVA (P < 0.0001), Tukey posttest.�� , P < 0.001 (GSE6008). C, Sod2 mRNA expression was significantly increased in a panel of OCCC lines, listed in Materials and Methods, compared withserous adenocarcinoma cell lines (Affymetrix array; ANOVA Tukey posttest: �, P < 0.05). D, semiquantitative real-time RT-PCR was performed to assessSod2 mRNA expression in serous ovarian cancer cell lines OVCA433, OVCA429, and OVCAR3 and OCCC cell lines ES-2 and TOV-21-G (data expressed relativeto OVCA433, which displayed lowest Sod2 expression). E, immunoblot analysis and densitometric quantification of Sod2 protein expression in ovariancancer cell lines (for D and E: mean � SEM, n ¼ 3, ANOVA; Tukey posttest. �� , P < 0.0001; �� , P < 0.001; �� , P < 0.01; � , P < 0.05).

Hemachandra et al.

Cancer Res; 75(22) November 15, 2015 Cancer Research4974




shRNA with nontargeting scramble sequence or targeting Sod2(shSod2_#1: 50-CTGACGGCTGCATCTGTTGGTGTCCAAGG-30,and shSod2_#2: 50-ACCTGAACGTCACCGAGGAGAAGTAC-CAG-30) in pGFP-V-RS vector (Origene; TG309190) were usedto stably transfect ES-2 cells (Fig. 2; Supplementary Fig. S3). Theclone expressing shSod2_#1 was used in Figs. 2–7.

ImmunoblottingProtein expression was analyzed by standard Western blotting

using antibodies fromCell Signaling Technology (pAkt-s473, Akt,pFAK-Y397, FAK, p-p130cas-Y165, p130cas) or Abcam (Sod2).Primary antibodies were diluted in blocking solution (5% nonfatmilk in TBS with 0.1% Tween-20, 1:1,000) and incubated over-night at 4�C. Blots were visualized using Femto and Pico ECLchemiluminescence substrate (Thermo scientific) and imagedusing a ChemiDoc MP system (BioRad). Densitometric analysiswas performed using ImageJ software (NIH). Each protein bandwas normalized to the respective GAPDH or b-actin loadingcontrol band.

Sod2 zymographySod2 activity was analyzed using Sod2 in-gel zymography as

previously described (12). Briefly, cell lysates were loaded onnondenaturing acrylamide gels, followed by electrophoresis.Sod2 activity is visualized by the inhibition of nitroblue tetrazo-lium reduction.

Clonogenicity and cell viabilitySingle-cell survival clonogenicity assays were performed as

previously described (13). Briefly, 100 cells were plated in each

well of 6-well plate colonies visualized after 10 days using crystalviolet. Viability was assessed by cell counting using trypan blue(1%) staining or crystal violet uptake assays (13).

Chorioallantoic membrane assayEach chorioallantoic membrane (CAM) was inoculated with 5

� 105 ES-2 cells stably expressing either scramble-shRNA-GFP orSod2-shRNA_#1-GFP that were suspended in 50 mL PBS (with 1mmol/LMgCl2, 0.5mmol/L CaCl2, 100U/mLpenicillin, and 100mg/mL streptomycin), essentially as previously described (14).Tumors were allowed to form for 7 days prior to termination ofthe experiments by sacrificing the chick embryo. Tumors on theCAM were removed and measured. CAM and chick embryoorgans (liver and lung) were collected for tumor metastasisanalysis by surveying for GFP-labeled cells.

Seahorse XF24 extracellular flux analysisOxygen consumption rate (OCR), extracellular acidification

rate (ECAR), and mitochondria stress tests were measured usingthe Seahorse XF243 Extracellular Flux Analyzer (Seahorse Biosci-ence), as described previously (13). Cells were plated at a densityof 40,000 cells per well and media replaced with XF mediathe following day 1 hour prior to the assay. Three measurementsof OCR and ECAR were taken at baseline and after each injectionof the following mitochondrial stress test compounds: oligomy-cin (1 mmol/L; complex V inhibitor); FCCP (0.75 mmol/L; protongradient uncoupler); and antimycin A (1 mmol/L; complex IIIinhibitor). Basal and maximal respiration were normalizedby subtracting nonmitochondrial OCR (i.e., after antimycin Aaddition). Respiratory reserve capacity was calculated as thedifference between maximal and basal OCR. ATP-linked OCR

Figure 2.Inhibition of Sod2 expression abrogates OCCC tumorigenicity A, Sod2 expression and activity were reduced using RNA interference in OCCC cell lines ES-2(shSod2_#1) and TOV-21-G (siRNASod2_#1). Sod2 expression was analyzed using Western blotting (top) and activity determined using in-gel zymography(bottom). B, cell proliferation rate was decreased with reduced Sod2 expression (shSod2_#1), analyzed by cell counting using trypan blue exclusion assay(mean � SEM. ANOVA Tukey posttest; �� , P < 0.0001). C, Sod2 RNA interference decreases single-cell survival of ES-2 cells in clonogenicity assays. D, reducedSod2 levels significantly inhibited the tumor size and weight of ES-2 tumors grown in the ex ovo CAM model. Fertilized chicken eggs were incubated at 37�C for7 days after inoculating the cells (shSod2_#1 or scramble transfected ES-2) onto the CAM, after which tumors were collected and measured (control, n ¼ 12;shSod2, n ¼ 10; mean � SD; Student t test; �� , P < 0.01).

Sod2 Is Protumorigenic in Ovarian Clear Cell Carcinoma

www.aacrjournals.org Cancer Res; 75(22) November 15, 2015 4975




was derived as the difference between basal and oligomycin A–inhibited OCR. Data were normalized to total protein content ineach well.

Wound-healing assayCell migration was assessed in serum-free media by wound-

healing assays using Ibidi inserts (Martinsried) and quantifiedafter 72 hours. Ibidi inserts were removed from a monolayer ofGFP-labeled cells to expose the cell-free wound area. Fluorescentimages were taken after 72 hours of migration and overlayed with

corresponding images at time 0 hour. Pixels representing GFP-labeled cells were quantified within the wound area using ImageJand corrected by subtracting any GFP-detected cells in the samearea at time 0.

Spheroid attachment assayCells were plated at a density of 1,000 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 withoutCollagen I coating. Percentage outgrowth was calculated by

Figure 3.Sod2 preserves OCCC mitochondrialrespiration. A, reduced Sod2 levelssignificantly attenuated themitochondrial oxidativephosphorylation in stably transfectedES-2 cells (shSod2_#1). OCR wasmeasured using extracellular fluxanalysis and pharmacologicmanipulation of mitochondrial activityto interrogate bioenergeticsparameters. OligomycinA (O)was usedto derive ATP-linked OCR, FCCP (F) tostimulate maximal OCR, and antimycinA (A) to inhibit allmitochondrial OCR. B,basal OCR, ATP-linked OCR, maximumOCR, and respiratory reserve capacity(max OCR � basal OCR) weresignificantly decreased with stableshRNA-mediated Sod2 knockdown inES-2 (one representative of threeexperiments is shown, mean � SEM;control, n ¼ 8; shSod2, n ¼ 9. ANOVA,Tukey posttest; ��, P < 0.001; �� , P <0.0001). C, transiently transfectedsiRNA targeting Sod2 in TOV-21-G cellsshowed significantly reducedmitochondrial oxidativephosphorylation (siRNA construct #1).D, basal OCR, maximum OCR, andrespiratory reserve capacity (maxOCR � basal OCR) were significantlydecreased with Sod2 knockdown inTOV-21-G cells. (one representative ofthree experiments is shown; mean �SEM; control, n ¼ 4; siSod2, n ¼ 5;ANOVA, Tukey posttest; � , P < 0.05).E, superoxide-mediated oxidation ofMitoSox redox–sensitive dye wasmeasured in live cells. IncreasedMitoSox fluorescence was observedin ES-2 cells stably transfected withshRNA-Sod2_#1 compared withscramble-transected control cells,suggesting that the amount ofmitochondrial superoxideaccumulation is higher in cells withreduced Sod2 expression. MitoSoxflorescence was abrogated by additionof 10 mmol/L of the porphyrinsuperoxide scavenger ortho tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP5þ),representative of three experiments isshown; mean � SEM, n ¼ 4; ANOVA,ANOVA Tukey posttest; � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

Hemachandra et al.





subtracting the area covered by migrating cells onto the collagenmatrix from the area of the spheroid at time 0 hour.

Live cell imaging of MitoSox oxidationAs an indicator of mitochondrial O2

*�, the oxidation andfluorescence of the redox-sensitive MitoSox Red dye (Life Tech-nologies) were monitored by live cell imaging according tomanufacturer's instructions. Cells were imaged to detect oxida-tion and fluorescence of MitoSox using a Leica SP5 II AOBSconfocal microscope following incubation with dye in HBSS for30 minutes at 37�C. The Manganese Porphyrin O2

*� scavengerortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin(MnTnBuOE-2-PyP5þ) was generously provided by Dr. InesBatinic Haberle (Duke University, Durham, NC).

Measurement of cellular H2O2 using catalase activity assayConcentration of cellular H2O2 was determined by measuring

the rate of inactivation of catalase with amino1,2,4-triazol (ATZ),according to Yusa and colleagues (15). ATZ irreversibly inactivatescatalase by covalently binding with intermediate compound I,formed following oxidation of catalase byH2O2. Briefly, cells weretreatedwith 20mmol/L ATZ for different time intervals (15 and30minutes). Cells were washed with PBS and protein lysates wereprepared in 50 mmol/L phosphate buffer (pH, 7.4) with proteaseinhibitors. Decomposition of H2O2 by catalase in protein lysateswas analyzedusingultraviolet spectroscopyat 240nmwavelength.H2O2 concentrationwas determinedusing the followingequation:

½H2O2� ¼ k/k1;where k is the empirically determinedpseudo first-order rate constant of catalase inactivationin the cells by ATZ (Fig. 6A), whereas k1 is the rate ofcompound I formation [1.7 � 107 (mol/L)�1s�1].

Statistical analysisAll data presented are representative of at least three indepen-

dent experiments and expressed asmean� SEM, unless otherwisestated. Statistical data analysis (ANOVA with Tukey post-test orStudent t test) was performed using GraphPad Prism Software v6.P < 0.05 was considered to be significant.

ResultsSod2 expression is significantly increased in OCCC comparedwith other EOC histologic subtypes

Mining publicly available expression data (Oncomine.org)revealed that Sod2 mRNA levels are elevated in OCCC com-pared with other ovarian cancer histologic subtypes (Supple-mentary Fig. S1). Two representative datasets displayed in Fig.1A and B show that levels of this antioxidant enzyme arestatistically higher than in any other ovarian cancer subtype.Similarly, significantly higher Sod2 mRNA expression wasobserved by microarray analysis in a panel of OCCC cell lineswhen compared with cell lines of high-grade serous adenocar-cinoma origin (Fig. 1C). This observation was further verifiedby assessing Sod2 expression in cultured ovarian cancer celllines, using semiquantitative real-time RT-PCR and Westernblotting (Fig. 1D and E).

Reduced Sod2 levels significantly inhibit OCCC cellproliferation in vitro and tumor formation in the CAM model

To further investigate the role of Sod2, we used two OCCC celllines, ES-2 and TOV-21-G. Sod2 expression was inhibited by

shRNA and siRNA transfection, which was demonstrated to leadto a concomitant decrease in Sod2 enzyme activity (Fig. 2A;Supplementary Figs. S2 and S3A). Following Sod2 expressionknockdown, ES-2 cell proliferation rate (Fig. 2B; SupplementaryFig. S3B) and clonogenicity (Fig. 2C; Supplementary Fig. S3C)were significantly attenuated. This appeared to be Sod2 concen-tration-dependent. A 30% reduction in Sod2 levels mediated bystable shRNA transfection reduced clonogenicity by approximate-ly 50% (shSod2_#1), whereas a 70% Sod2 decrease almostcompletely abrogated the cells' ability to survive in this assay(shSod2_#2; Supplementary Fig. S3C). Analysis of PARP cleavageandAnnexin V staining suggested that the decrease in cell viabilityobserved in cells with 30% Sod2 knockdown is not related to asignificant increase in apoptosis (Supplementary Fig. S4). This cellline, referred to as shSod2 in the following figures, was chosenfor subsequent studies to achieve pathophysiologically relevantchanges in Sod2 expression (rather than complete loss), whichalso closely reflects Sod2 levels observed in non-OCCC cell linesOVCA433 and OVCA429 (Fig. 1E). The CAM ex ovo model wasused to further test the role of Sod2 on ES-2 tumorigenicity. Asignificant decrease in tumor size and weight was observed intumors grown from ES-2 cells with reduced Sod2 expression(shSod2; Fig. 2D). In addition, the shRNA-Sod2 tumors exhibitedless vascularization than the control groups (Fig. 2D), suggestingthat Sod2 may contribute to both proliferation and the recruit-ment of blood vessels to the tumor.

Sod2 maintains OCCC mitochondrial respirationWe previously demonstrated that OCCC cell lines are highly

energetic and depend on both oxidative phosphorylation andglycolysis for their energyneeds (13).Given thatSod2hasaprimaryrole inprotectingmitochondria fromexcessO2

*�, the effect of Sod2knockdown on mitochondrial respiration was assessed. OCR,representingmitochondrial oxidative phosphorylation, and ECAR,correlating with glycolytic activity, were measured using extracel-lular flux analysis in both ES-2 and TOV-21-G OCCC cell linesfollowing Sod2knockdown(13, 16). Stable shRNA-Sod2decreasesin ES-2 and transient siRNA-mediated knockdownof Sod2 in TOV-21-G cells resulted in significant reduction in basal OCR comparedwith control scramble RNAi–transfected cells (Fig. 3A–D; Supple-mentary Fig. S5A). Furthermore, respiratory reserve capacity, ameasure of the ability of cells to enhance respiration in responseto physiologic cues and stress, was significantly inhibited withreduced Sod2 levels (Fig. 3B and D), suggesting that Sod2 playsa major role in maintaining mitochondrial health to supportmaximal respiration. Although slight, a consistent negative effecton OCR was observed in stable shRNA-Sod2 cells following FCCPtreatment (Fig. 3B).Awide rangeofFCCPconcentrationswas testedon these cells, but none were able to enhance OCRwith Sod2 loss.FCCP can be inhibitory at high concentrations, and it has beenspeculated that this may be due to a loss in the ability of mito-chondria to accumulate respiratory substrates (16). While nottested here, it is possible that sustained Sod2 expression decreases,and concomitant increases in mitochondrial O2

*-� levels, mayexacerbate this FCCP-dependentOCR inhibition, thereby influenc-ingmitochondrial membrane integrity and substrate transport. Nosignificant increases in ECAR were observed between control andshRNA- or siRNA-transfected cells, suggesting that decreases inSod2 expression do not influence a compensatory shift towardglycolysis (Supplementary Fig. S5B–S5D). The above observationsshow that both siRNA- and shRNA-mediated decreases in Sod2






reduced basal OCR and respiratory reserve capacity, indicating thatSod2 is important in maintaining mitochondrial respiration inOCCC.

To assess whether a decrease in Sod2 expression results incompromised O2

*� scavenging, which may be one of the causesof compromised mitochondrial function, the presence of mito-chondrial O2

*� was evaluated using the mitochondria-targetedredox-sensitive dyeMitoSox. As expected, increasedoxidation andconsequential enhanced fluorescence of MitoSox were observedin the Sod2-knockdown cells comparedwith controls (Fig. 3E andF). Addition of the Sod2 mimetic porphyrin (MnTnBuOE-2-PyP5þ), which acts as a O2

*� scavenger, reduced MitoSox oxida-tion in both control and shRNA-Sod2 groups (Fig. 3E and F).

Sod2 knockdown attenuates metastasis of cancer cells in theCAM model

We have previously demonstrated that enhanced Sod2 expres-sion is implicated with metastatic progression (7, 8, 17). Toinvestigate the role of Sod2 during OCCCmetastasis, the appear-ance of metastatic lesions of GFP-labeled ES-2 cells was inves-tigated 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 to3 cm from the tumor (Fig. 4A). In contrast,metastatic spread fromshRNA-Sod2 knockdown tumors was limited to the appearanceof single cells in the membrane confined to an approximate 1- to1.5-cm radius from the tumor (Fig. 4A). Furthermore, lungmetastases in the chick embryo were observed in 11 of the 12controls compared with only 5 of 10 embryos in the shRNA-Sod2group (Fig. 4B). Furthermore, 10 of 12 control tumors meta-stasized into the liver, whereas only 4 of 10 liver metastases wereobserved in the shRNA-Sod2 group (Fig. 4B). While clusters offive or more cells were found in the lungs and livers of controlgroups, only single cells were detected in the Sod2-knockdowngroup (Fig. 4B).

Sod2 levels modulate cell migration and tumor spheroidoutgrowth

Because of the significant abrogation of metastatic spread inresponse to Sod2 expression decreases, the role of Sod2 oncell migration was further investigated. Cell migration, assessedby wound-healing assays, was significantly inhibited withreduced Sod2 expression in ES-2 cells (Fig. 5A; SupplementaryFig. S6). Furthermore, the ability of cellular spheroid clusters to

Figure 4.Metastatic spread of ES-2 cells isattenuated with reduced Sod2expression in the ex ovo CAM tumormodel. A, representative images showthe location of the primary tumors andmicrometastasis of GFP-labeled cells inthe membrane (scale bar, 1 mm; CAMtumor study carried out as in Fig. 2).B, reduced Sod2 expression inhibitedtumormetastasis into the chick embryoand CAM (shSod2_#1). Quantificationof percentage metastasis of tumor cellsinto CAM, liver, and lung of chickembryo. Representative images showthe percentage of tumors withmetastatic cancer cells present in thechicken embryo liver and lung (control,n ¼ 12; shSod2, n ¼ 10).

Hemachandra et al.





attach and cells to migrate from the spheroid onto collagen I anduncoated surfaces was also compromised with reduced Sod2expression (Fig. 5B). Anchorage-independent spheroid formationis a commonly observed phenotype of ovarian cancer cellsmetastasizing via the transcoelomic route through the intraper-itoneal cavity, and these have shown the ability to attach on the

peritoneum to form metastatic lesions. These data suggest thatSod2 plays an important role in tumor spheroid metastasis(Fig. 5B). While spheroids of equal size were chosen for thisassay, it should be noted that Sod2 knockdown also decreasedES-2 spheroid growth in anchorage independence (data notshown).

Figure 5.A, cell migration is significantlyinhibited with Sod2 knockdown. A,migration was analyzed by wound-healing assays using Ibidi inserts andthe area covered by cells quantifiedfollowing 72 hours of migration(normalized to day 0). Data from onerepresentative experiment are shown(n¼ 6, mean� SEM; t test; �� , P < 0.01;scale bar, 500 mm). B, representativeimages of attachment and outgrowthof ES-2 spheroids on uncoated andcollagen I–coated surfaces (scale bar,1 mm). Reduced Sod2 levelssignificantly inhibited the outgrowth ofspheroids in both uncoated andcollagen I–coated surfaces. Data fromone representative experiment areshown (n ¼ 4, mean � SEM; ANOVA,Tukey posttest; �� , P < 0.01). C, Aktphosphorylation is dependent on Sod2expression. Protein expression ofphosphorylated and total levels of Akt,p130cas, and FAK were analyzed byWestern blotting. Data from onerepresentative experiment are shown.Levels of phosphorylated proteinswere quantified by densitometry andnormalized to total levels (n ¼ 3,mean � SEM).






To gain mechanistic insights into the signaling pathways thatmay be altered by Sod2-mediated metastasis, phosphorylationprofiles of Akt, p130cas, and focal adhesion kinase (FAK) wereinvestigated. These were chosen on the basis of previous observa-tions of their redox regulation and involvement in tumor cellmigration (8, 9, 18, 19). shRNA-Sod2 cells exhibited a 50%

decrease in phospho-Akt levels compared with scramble controlcells, whereas no appreciable change was observed in phosphor-ylation of FAK or the focal adhesion adapter protein p130cas(Fig. 5C). The effects onAkt phosphorylationwere alsodependenton Sod2 concentration, where cells with lower Sod2 expressiondemonstrated a more striking decrease in Akt phosphorylation

Figure 6.H2O2 contributes to OCCC migration. A, steady-state H2O2 levels decrease with reduced Sod2 expression. Steady-state H2O2 levels were derived in ES-2 cellsstably transfected with either scramble shRNA control (closed circles) or shRNA targeting Sod2 (open circles), using aminotriazole inhibition of catalasekinetics assays, as described in Materials and Methods. First-order decay curves for catalase inhibition by aminotrizaole were used to determine rate constants ofinactivation (k; one representative decay curve from three replicate experiments shown). H2O2 concentrations were determined using the equation [H2O2] ¼ k/k1,where k1 is the rate of catalase compound I formation [1.7 � 107 (mol/L)�1 s�1]. H2O2 data represent an average of three experiments � SD. B, there was nochange in catalase activity and catalase protein expression between control and shRNA-Sod2 cells. C, H2O2 scavenging by catalase abrogates migration ofES-2 cells, whereas H2O2 can stimulate migration in cells with low Sod2 expression. Migration was analyzed by wound-healing assays using Ibidi inserts andthe area covered by cells quantified following 72 hours of migration (normalized to day 0). Scavenging of endogenous H2O2 by catalase significantly reducedthe migration of ES-2 scramble–transfected control cells. Recombinant catalase protein (500 units/mL) was added in serum-free media at time 0 and left oncells for 72 hours. Conversely, migration of shSod2 cells was significantly increased with treatment of 5 mmol/L H2O2, whereas this level of H2O2 was toxicto control cells and effects on migration could therefore not be determined (N/D). Data represent one of two independent cellular migration experiments(n ¼ 5, mean � SEM; ANOVA, Tukey posttest; � , P < 0.05). D, H2O2 can stimulate Akt signaling in OCCC. Dose-dependent increases in phospho-Akt S473 levelswere observed by Western blotting following 2 hours of H2O2 treatment with indicated doses in serum-free media.

Hemachandra et al.





(Supplementary Fig. S6). These data suggest that Akt signalingmay be important in driving Sod2-mediated tumorigenicity andmetastasis of OCCC.

OCCC cell migration is H2O2-dependentSod2 is the primary enzyme involved in converting O2

*� toH2O2 within the mitochondria. While it serves as a protectivemechanism to maintain mitochondrial function (Fig. 3) byremoval of damaging O2

*�, a shift toward increasing levels ofH2O2 has also been observed in response to enhanced Sod2expression (8, 10, 18, 20, 21). Because of its relative stability andease in traversing cellular membranes, H2O2 can mediate redoxsignaling, including events that drive migration (9). To testwhether Sod2 changes the steady-state H2O2 levels in OCCC, weassessed intracellular H2O2 status in control and shRNA-Sod2ES-2 cells using a biochemical assay on the basis of the irreversibleinhibition of catalase by aminotriazole (15, 17). Steady-statelevels of H2O2 were reduced approximately by 50% in Sod2-knockdown cells compared with controls (Fig. 6A), whereasbaseline catalase activity and protein expressionwere comparable(Fig. 6B).

To test whether OCCC cell migration is H2O2-dependent,wound-healing assays were carried out in the presence of catalase,which catalyzes the conversion of H2O2 to H2O and O2. Aspreviously demonstrated, exogenous application of recombinantcatalase resulted in accumulation of catalase within ES-2 cells(Supplementary Fig. S7; ref. 9). Catalase significantly reduced themigration of both control and shRNA-Sod2 cells, suggesting thatH2O2 is a promoter of ES-2 cell migration (Fig. 6C). Conversely,treatment with low levels of H2O2 (5 mmol/L) significantlyreversed the slow migration of shRNA-Sod2 cells (Fig. 6C).Furthermore, 5- and 50-mmol/L H2O2 treatment was able toincrease phospho-Akt levels (Fig. 6D), while catalase expressionabrogated Akt phosphorylation (Supplementary Fig. S8), suggest-ing that this may be an important redox-dependent signalingpathway in OCCC.

It was noted that ES-2 control cells were not able to toleratelong-term exposure to low-dose H2O2 during migration assays(Fig. 6C). To examine this further, cell viability was assessed inresponse to H2O2. A significant reduction in cell survival inresponse to H2O2 was observed in the control group comparedwith cells with decreased Sod2 expression (Fig. 7A). This suggeststhat a higher intracellular steady-state H2O2 milieu in OCCCpredisposes cells to enhanced killing by additional exposure tolow-level exogenous H2O2. The above data imply that high Sod2expression provides several advantages to OCCC by protectingmitochondrial function through scavenging of O2

*� and drivingH2O2-dependent migration. While these attributes are advanta-geous forOCCC survival andmetastatic progression, an enhancedintracellular steady-state H2O2 level presents a double-edgedsword, as these cells are consequentially more susceptible toH2O2 toxicity (Fig. 7B).

DiscussionAlthough the five different EOC histologic subtypes share the

same primary tumor location on the ovaries, it is now evident thatthese are distinct diseases with vastly different tissue origins andgenetic and epigenetic profiles (2, 11). In the present study, weshow that Sod2 is highly expressed inOCCC comparedwith other

EOC histologic subtypes and that this mitochondrial antioxidantplays a significant role in OCCC tumorigenicity and metastasis.

Intracellular ROS are maintained within a narrow range tightlyregulated by the balance of the rate of ROS production and ROSscavenging/detoxifying by antioxidant enzymes. This balance isoften disrupted in the context of cancer, due to high ROS pro-duction as a consequence of changes in metabolism or the tumorenvironment (e.g., hypoxia) and the resulting changes in antiox-idant expression. Because the mitochondrial respiratory chain isthe major site of O2

*� generation within cells, Sod2 plays animportant role in maintaining cellular ROS balance. On the basisof the above findings, Sod2 appears to play a dual role inenhancing OCCC tumorigenicity, first, by protecting cells frommitochondrial O2

*� damage, and second, by shifting the steady-state ROS balance toward H2O2.

We recently demonstrated that a distinguishing feature ofOCCC is their unique metabolic phenotype. Compared withserous adenocarcinoma cells, OCCC cell lines were significantlymore energetic, displaying both very high levels of mitochondrialoxidative phosphorylation and glycolytic flux (13). Our datasuggest that Sod2 is intricately involved in maintaining thishigh rate of oxygen consumption, potentially by preserving mito-chondrial function as a consequence of O2

*� scavenging (Fig. 3).By preventing mitochondrial electron transport chain complexdamage mediated by O2

*� or secondary products, Sod2 likelysupports the high rate of OCCC proliferation, clonogenicity, andtumor growth. The results of the present study suggest thatinhibiting mitochondrial antioxidant defenses may provide analternative strategy to therapeutically target OCCC.

In addition to scavengingO2*� andmaintainingmitochondrial

health for optimal cell proliferation, we believe that Sod2 hasanother role inpromoting the aggressiveness ofOCCC,by shiftingsteady-state H2O2 levels and driving prometastatic behavior(Figs. 4–6). It has been previously shown that high expressionof Sod2 is associated with metastatic progression (8, 10, 17,20–23) and that dependence of cancer cell migration is relatedto cellular H2O2 production (9, 19, 24, 25). For example, we have

Figure 7.Exogenous H2O2 treatment significantly decreases viability of cells withhigher steady-state H2O2 levels. A, ES-2 scramble control cells showsignificantly lower viability than shSod2 ES-2 cells using crystal violetassays, in response to increasing concentrations of H2O2 for 72 hours. Eachdata point represents an average of 6 replicates � SEM. Tukey posttest;� , P < 0.05. B, proposed mechanisms of Sod2-mediated OCCC tumorigenesisand metastasis. OCCC has high Sod2 expression, which provides efficientsuperoxide scavenging and maintenance of high mitochondrial function todrive increased cell proliferation. In addition, Sod2 shifts the intracellularROS balance from O2

*� to H2O2, which drives tumor cell migration andmetastasis. This concomitantly enhances the intracellular H2O2 threshold,enabling more rapid killing by exogenous H2O2.






shown that steady-state increases inH2O2 can lead to induction ofthe FAK pathway andmigration of metastatic bladder cancer cellsand cells with enforced Sod2 expression (9, 19). This effect wasmediated by oxidation-dependent inhibition of the phosphatasePTPN12, leading to enhanced phosphorylation of p130cas andRac1 activation. In addition, work from the Melendez group hasshown that Sod2 expression significantly contributes to theexpression of the matrix-degrading enzyme MMP-1 in an H2O2-dependent manner (21) and that the Sod2/H2O2-dependentinhibition of the dual-lipid protein tyrosine phosphatase PTENenhances Akt/GSK3b/VEGF-dependent angiogenesis (18), bothprocesses contributing significantly to metastasis. Our presentdata suggest that Sod2 may similarly contribute to metastaticprogressionofOCCCbyactivatingAkt signaling (Figs. 5 and6). Inaddition to its prosurvival function, Akt has been shown toinfluence metastasis and cell migration by regulating cytoskeletalrearrangement, prometastatic cell signaling, and gene transcrip-tion (26). These results are of specific importance to OCCC,which, unlike other ovarian cancer histologic subtypes, has beencharacterized by high-frequency Akt pathway activation. Seventypercent of early- and 68% of late-stage OCCC cases have beenshown to display phospho-Akt (S473) staining (27). About 38%of OCCC cases show PTEN loss (28) and 40% of cases PI3Kactivating mutations (29). Our data imply that Sod2-dependentAkt phosphorylationmay also contribute to high activation of Aktsignaling in OCCC. Because Akt phosphorylation was highlysusceptible to H2O2 treatment, it suggests that this signalingpathway is redox-regulated inOCCC,with aplausiblemechanismfor this being the oxidation of PTEN (18).

Although an increase in Sod2 should theoretically not result inhigher levels of H2O2 production based on the enzyme's kineticproperties (30), a number of studies have demonstrated increasesin H2O2 levels that correlate with Sod2 expression (18, 31–33).While the reason for this observation in OCCC has not beeninvestigated, there are plausible explanations for this increase insteady-state H2O2 as a consequence of Sod expression. Theseexplanations primarily relate to changes in the reaction rateswithin the mitochondrial electron transport chain (ETC). Forexample, it has been proposed that Sod2 in the mitochondriamay alter the flux of O2

*� from some quinone/semiquinone/hydroquinone triads, such as coenzyme Q, thereby driving thereaction into the direction ofO2

*� production, potentially leadingto enhanced localized dismutation to H2O2 by Sod2 (31, 34).Alternatively, inhibition of cytochrome c oxidase by nitric oxide,arising as a consequence of Sod2 expression, may influence thereduction state of the ETC and drive O2

*- and H2O2 production(35). Our observation that Sod2 knockdown also decreases H2O2

levels suggests that Sod2 is involved in regulating H2O2 balancewithin cells, and this may contribute to H2O2-mediated redoxsignaling.

While Sod2 appears to contribute toH2O2-mediatedmetastaticprogression, an enhanced steady-state H2O2 milieu may alsopresent a disadvantage to OCCC cells. Our data suggest that cellswith high Sod2 levels and concomitant increases in intracellularH2O2 are more susceptible to exogenous sources of redox stress(Fig. 7A). This likely puts cells closer to the cytotoxic threshold ofH2O2, which is reached once cells are further challenged byexogenous ROS. Interestingly, OCCC cells do not appear to haveenhanced expression of catalase to provide additional scavengingof excess H2O2 (Fig. 6A). While sublethal levels of H2O2 havebeen shown to contribute to redox signaling, high levels of H2O2

can elicit tumor cell death by a number of pathways, includingapoptosis, protein/DNA damage, andmitochondrial dysfunction(36–38). Furthermore, it was recently reported that H2O2 expo-sure of tumor cells with enforced Sod2 expression can result inSod2 peroxidase activity, leading to mitochondrial damage anddysfunction (39). An increased H2O2 steady-state has beenobserved in a number of cancer cells (17, 40–42) and lendscredence to the idea that this higher H2O2 threshold may beexploited therapeutically. In this regard, the use of high-doseascorbic acid, which is oxidized within tumor cells to produceH2O2, 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 H2O2-medi-ated DNA damage and apoptosis were shown to enhance ovariancancer cell death and increase chemosensitivity (48). While thatstudy was not focused onOCCC, this type of treatment may be ofparticular benefit to this histologic subtype given the high expres-sion of Sod2. It is important to highlight that cancer cells withenhanced Sod2 expression may respond differently to ROS-pro-ducing agents, dependingonboth the type and cellular locationofthe ROS/reactive nitrogen species generated. For instance, Sod2may enhance scavenging of O2

*� and therefore provide chemore-sistance benefits to the tumor cells in response to these ROS.Conversely, while an increase in steady-state H2O2 facilitatesredox signaling beneficial to the cancer cells, this higher thresholdmay facilitate H2O2-mediated OCCC cell death in response tofurther insult by exogenous sources of H2O2. Understanding thecomplex interaction of antioxidants and ROS in OCCC is there-fore of importance andmay provide novel therapeutic avenues topursue for this histologic subtype of ovarian cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L.P.M.P. Hemachandra, D.-H. Shin, L.M. Uusitalo,N. HempelDevelopment of methodology: L.P.M.P. Hemachandra, N. HempelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.P.M.P. Hemachandra, U. Dier, J.N. Iuliano,L.M. Uusitalo, S.K. MurphyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.P.M.P. Hemachandra, D.-H. Shin, N. HempelWriting, review, and/or revision of the manuscript: L.P.M.P. Hemachandra,D.-H. Shin, U. Dier, S.A. Engelberth, L.M. Uusitalo, S.K. Murphy, N. HempelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.-H. Shin, S.A. EngelberthStudy supervision: N. Hempel

AcknowledgmentsThe ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-

2-PyP5þ)was generouslyprovidedbyDr. InesBatinic-Haberle (DukeUniversity).The authors thank Dr. J. Andres Melendez (SUNY Poly) for helpful discussionsand Dr. Nate Cady for use of his microscope. They appreciate the help of JeffreyRichards for assistance with spheroid assays.

Grant SupportThis work was supported by NIH/NCI grant R00CA143229.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 23, 2014; revised July 27, 2015; accepted August 19,2015; published OnlineFirst September 10, 2015.

Hemachandra et al.





References1. Karst AM, Drapkin R. Ovarian cancer pathogenesis: a model in evolution.

J Oncol 2010;2010:932371.2. Vaughan S, Coward JI, Bast RC Jr, Berchuck A, Berek JS, Brenton JD, et al.

Rethinking ovarian cancer: recommendations for improving outcomes.Nat Rev Cancer 2011;11:719–25.

3. Mandai M, Matsumura N, Baba T, Yamaguchi K, Hamanishi J, Konishi I.Ovarian clear cell carcinoma as a stress-responsive cancer: influence of themicroenvironment on the carcinogenesis and cancer phenotype. CancerLett 2011;310:129–33.

4. Yamaguchi K,MandaiM,Oura T,MatsumuraN,Hamanishi J, Baba T, et al.Identification of an ovarian clear cell carcinoma gene signature that reflectsinherent disease biology and the carcinogenic processes. Oncogene2010;29:1741–52.

5. Konstantinopoulos PA, Spentzos D, Fountzilas E, Francoeur N, Sanisetty S,Grammatikos AP, et al. Keap1 mutations and Nrf2 pathway activation inepithelial ovarian cancer. Cancer Res 2011;71:5081–9.

6. Bravard A, Sabatier L, Hoffschir F, Ricoul M, Luccioni C, Dutrillaux B.SOD2: a new type of tumor-suppressor gene? Int J Cancer 1992;51:476–80.

7. Hempel N, Carrico PM, Melendez JA. Manganese superoxide dismutase(Sod2) and redox-control of signaling events that drive metastasis. Anti-Cancer Agents Med Chem 2011;11:191–201.

8. Connor KM,HempelN,NelsonKK,Dabiri G,Gamarra A, Belarmino J, et al.Manganese superoxide dismutase enhances the invasive and migratoryactivity of tumor cells. Cancer Res 2007;67:10260–7.

9. Hempel N, Bartling TR, Mian B, Melendez JA. Acquisition of themetastaticphenotype is accompanied byH2O2-dependent activation of the p130Cassignaling complex. Mol Cancer Res 2013;11:303–12.

10. Liu Z, Li S, Cai Y, Wang A, He Q, Zheng C, et al. Manganese superoxidedismutase induces migration and invasion of tongue squamous cellcarcinoma via H2O2-dependent Snail signaling. Free Radic Biol Med2012;53:44–50.

11. Yamaguchi K, Huang Z, Matsumura N, Mandai M, Okamoto T, Baba T,et al. Epigenetic determinants of ovarian clear cell carcinoma biology. Int JCancer 2014;135:585–97.

12. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and anassay applicable to acrylamide gels. Anal Biochem 1971;44:276–87.

13. Dier U, Shin DH, Hemachandra LP, Uusitalo LM, Hempel N. Bioenergeticanalysis of ovarian cancer cell lines: profiling of histological subtypes andidentification of a mitochondria-defective cell line. PLoS One 2014;9:e98479.

14. Deryugina EI, Quigley JP. Chick embryo chorioallantoicmembranemodelsystems to study and visualize human tumor cell metastasis. HistochemCell Biol 2008;130:1119–30.

15. Yusa T, Beckman JS, Crapo JD, Freeman BA. Hyperoxia increases H2O2production by brain in vivo. J Appl Physiol (1985) 1987;63:353–8.

16. Dranka BP, Hill BG, Darley-Usmar VM. Mitochondrial reserve capacity inendothelial cells: The impact of nitric oxide and reactive oxygen species.Free Radic Biol Med 2010;48:905–14.

17. Hempel N, Ye H, Abessi B, Mian B, Melendez JA. Altered redox statusaccompanies progression to metastatic human bladder cancer. Free RadicBiol Med 2009;46:42–50.

18. Connor KM, Subbaram S, Regan KJ, Nelson KK, Mazurkiewicz JE, Bartho-lomew PJ, et al. Mitochondrial H2O2 regulates the angiogenic phenotypevia PTEN oxidation. J Biol Chem 2005;280:16916–24.

19. Hempel N, Melendez JA. Intracellular redox status controls membranelocalization of pro- and anti-migratory signaling molecules. Redox Biol2014;2:245–50.

20. Ranganathan AC, Nelson KK, Rodriguez AM, KimKH, Tower GB, Rutter JL,et al. Manganese superoxide dismutase signals matrix metalloproteinaseexpression via H2O2-dependent ERK1/2 activation. J Biol Chem 2001;276:14264–70.

21. Nelson KK, Ranganathan AC, Mansouri J, Rodriguez AM, Providence KM,Rutter JL, et al. Elevated sod2 activity augments matrix metalloproteinaseexpression: evidence for the involvement of endogenous hydrogen perox-ide in regulating metastasis. Clin Cancer Res 2003;9:424–32.

22. Nonaka Y, Iwagaki H, Kimura T, Fuchimoto S, Orita K. Effect of reactiveoxygen intermediates on the in vitro invasive capacity of tumor cells andliver metastasis in mice. Int J Cancer 1993;54:983–6.

23. Bur H, Haapasaari KM, Turpeenniemi-Hujanen T, Kuittinen O, Auvinen P,Marin K, et al. Oxidative stress markers and mitochondrial antioxidantenzyme expression are increased in aggressive Hodgkin lymphomas.Histopathology 2014;65:319–27.

24. Polytarchou C, Hatziapostolou M, Papadimitriou E. Hydrogen per-oxide stimulates proliferation and migration of human prostatecancer cells through activation of activator protein-1 and up-regula-tion of the heparin affin regulatory peptide gene. J Biol Chem 2005;280:40428–35.

25. Hurd TR, DeGennaro M, Lehmann R. Redox regulation of cell migrationand adhesion. Trends Cell Biol 2012;22:107–15.

26. Xue G, Hemmings BA. PKB/Akt-dependent regulation of cell motility.J Natl Cancer Inst 2013;105:393–404.

27. Sasano T, Mabuchi S, Kuroda H, Kawano M, Matsumoto Y, Takahashi R,et al. Preclinical efficacy for AKT targeting in clear cell carcinoma ofthe ovary. Mol Cancer Res 2015;13:795–806.

28. Hashiguchi Y, Tsuda H, Inoue T, Berkowitz RS, Mok SC. PTEN expres-sion in clear cell adenocarcinoma of the ovary. Gynecol Oncol2006;101:71–5.

29. Kuo KT, Mao TL, Jones S, Veras E, Ayhan A, Wang TL, et al. Frequentactivating mutations of PIK3CA in ovarian clear cell carcinoma. Am JPathol 2009;174:1597–601.

30. Liochev SI, Fridovich I. The effects of superoxide dismutase on H2O2formation. Free Radic Biol Med 2007;42:1465–9.

31. Buettner GR, Ng CF, Wang M, Rodgers VG, Schafer FQ. A new paradigm:manganese superoxide dismutase influences the production of H2O2 incells and thereby their biological state. Free Radic Biol Med 2006;41:1338–50.

32. Dasgupta J, Subbaram S, Connor KM, Rodriguez AM, Tirosh O, BeckmanJS, et al. Manganese superoxide dismutase protects from TNF-alpha-induced apoptosis by increasing the steady-state production of H2O2.Antioxid Redox Signal 2006;8:1295–305.

33. Zhang HJ, Zhao W, Venkataraman S, Robbins ME, Buettner GR, KregelKC, et al. Activation of matrix metalloproteinase-2 by overexpres-sion of manganese superoxide dismutase in human breast cancerMCF-7 cells involves reactive oxygen species. J Biol Chem 2002;277:20919–26.

34. Song Y, Buettner GR. Thermodynamic and kinetic considerations for thereaction of semiquinone radicals to form superoxide and hydrogen per-oxide. Free Radic Biol Med 2010;49:919–62.

35. Buerk DG, Lamkin-Kennard K, Jaron D. Modeling the influence of super-oxide dismutase on superoxide and nitric oxide interactions, includingreversible inhibition of oxygen consumption. Free Radic Biol Med2003;34:1488–503.

36. Hyslop PA, Hinshaw DB, Halsey WA Jr,Schraufstatter IU, Sauerheber RD,Spragg RG, et al. Mechanisms of oxidant-mediated cell injury. The glyco-lytic and mitochondrial pathways of ADP phosphorylation are majorintracellular targets inactivated by hydrogen peroxide. J Biol Chem1988;263:1665–75.

37. Ahmad KA, Iskandar KB, Hirpara JL, Clement MV, Pervaiz S. Hydrogenperoxide-mediated cytosolic acidification is a signal for mitochondrialtranslocation of Bax during drug-induced apoptosis of tumor cells. CancerRes 2004;64:7867–78.

38. Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxidethrough the Fenton reaction in vivo and in vitro. Science 1988;240:640–2.

39. Ansenberger-FricanoK,GaniniD,MaoM,Chatterjee S,Dallas S,MasonRP,et al. The peroxidase activity of mitochondrial superoxide dismutase. FreeRadic Biol Med 2013;54:116–24.

40. Szatrowski TP, Nathan CF. Production of large amounts of hydrogenperoxide by human tumor cells. Cancer Res 1991;51:794–8.

41. Zieba M, Suwalski M, Kwiatkowska S, Piasecka G, Grzelewska-Rzymowska I, Stolarek R, et al. Comparison of hydrogen peroxidegeneration and the content of lipid peroxidation products in lungcancer tissue and pulmonary parenchyma. Respir Med 2000;94:800–5.

42. Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, et al.Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate2005;62:200–7.






43. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, et al.Pharmacologic ascorbic acid concentrations selectively kill cancer cells:action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl AcadSci U S A 2005;102:13604–9.

44. Cameron E, Campbell A. The orthomolecular treatment of cancer. II.Clinical trial of high-dose ascorbic acid supplements in advanced humancancer. Chem Biol Interact 1974;9:285–315.

45. Levine M, Padayatty SJ, Espey MG. Vitamin C: a concentration-functionapproach yields pharmacology and therapeutic discoveries. Adv Nutr(Bethesda, Md) 2011;2:78–88.

46. Hoffer LJ, Levine M, Assouline S, Melnychuk D, Padayatty SJ, Rosadiuk K,et al. Phase I clinical trial of i.v. ascorbic acid in advanced malignancy. AnnOncol 2008;19:1969–74.

47. Monti DA,Mitchell E, Bazzan AJ, Littman S, ZabreckyG, YeoCJ, et al. PhaseI evaluation of intravenous ascorbic acid in combinationwith gemcitabineand erlotinib in patients with metastatic pancreatic cancer. PLoS One2012;7:e29794.

48. Ma Y, Chapman J, Levine M, Polireddy K, Drisko J, Chen Q. High-doseparenteral ascorbate enhanced chemosensitivity of ovarian cancer andreduced toxicity of chemotherapy. Sci Translat Med 2014;6:222ra18.


Hemachandra et al.




2015;75:4973-4984. Published OnlineFirst September 10, 2015.Cancer Res L.P. Madhubhani P. Hemachandra, Dong-Hui Shin, Usawadee Dier, et al. Ovarian Clear Cell CarcinomaMitochondrial Superoxide Dismutase Has a Protumorigenic Role in

Updated version

10.1158/0008-5472.CAN-14-3799doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2015/09/10/0008-5472.CAN-14-3799.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/75/22/4973.full#ref-list-1

This article cites 48 articles, 13 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/75/22/4973.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/75/22/4973To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-14-3799

http://cancerres.aacrjournals.org/content/suppl/2015/09/10/0008-5472.CAN-14-3799.DC1

http://cancerres.aacrjournals.org/content/75/22/4973.full#ref-list-1

http://cancerres.aacrjournals.org/content/75/22/4973.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/75/22/4973


Mitochondrial Superoxide Dismutase Has a Protumorigenic ...cancerres.aacrjournals.org/content/canres/75/22/4973.full.pdf · Tumor and Stem Cell Biology Mitochondrial Superoxide Dismutase

Documents