metabolic stress-induced phosphorylation of kap1 ser473 blocks … · molecular and cellular...

Molecular and Cellular Pathobiology

Metabolic Stress-Induced Phosphorylation ofKAP1 Ser473 Blocks Mitochondrial Fusion inBreast Cancer CellsChun-Ting Cheng1,2, Ching-Ying Kuo1, Ching Ouyang3, Chien-Feng Li4,5,Yiyin Chung1, David C. Chan6, Hsing-Jien Kung7,8, and David K. Ann1,2

Abstract

Mitochondrial dynamics during nutrient starvation of cancercells likely exert profound effects on their capability formetastaticprogression. Here, we report that KAP1 (TRIM28), a transcrip-tional coadaptor protein implicated in metastatic progressionin breast cancer, is a pivotal regulator of mitochondrial fusionin glucose-starved cancer cells. Diversemetabolic stresses inducedSer473 phosphorylation of KAP1 (pS473-KAP1) in a ROS-and p38-dependent manner. Results from live-cell imagingand molecular studies revealed that during the first 6 to 8 hoursof glucose starvation, mitochondria initially underwent exten-sive fusion, but then subsequently fragmented in a pS473-KAP1-dependent manner. Mechanistic investigations usingphosphorylation-defective mutants revealed that KAP1 Ser473

phosphorylation limited mitochondrial hyperfusion in glu-cose-starved breast cancer cells, as driven by downregulationof the mitofusin protein MFN2, leading to reduced oxidativephosphorylation and ROS production. In clinical specimensof breast cancer, reduced expression of MFN2 correspondedto poor prognosis in patients. In a mouse xenograft model ofhuman breast cancer, there was an association in the coreregion of tumors between MFN2 downregulation and thepresence of highly fragmented mitochondria. Collectively, ourresults suggest that KAP1 Ser473 phosphorylation acts throughMFN2 reduction to restrict mitochondrial hyperfusion, therebycontributing to cancer cell survival under conditions of sus-tained metabolic stress. Cancer Res; 76(17); 5006–18. �2016 AACR.

IntroductionCancer cells require more nutrients to support their higher

proliferation compared with normal cells. For example, increasedglucose uptake, also known as the Warburg effect, is one of thewell-characterized tumor hallmarks (1). During the course oftumor growth, cancer cells encounter various microenvironmen-tal stresses from transient or fluctuating (acute) to diffusion-limited (chronic) hypoxia and nutrient starvation. In responseto these stress conditions, cells activate a number of signalingpathways to survive diverse types of nutrient deprivation (2). Oneof the most prominent adaptive responses is to regulate mito-

chondrial dynamics (3, 4). Mitochondria undergo dynamicfusion and fission processes that affect cellular biosynthetic andbioenergetic efficiency and emerging evidence suggests that dys-regulated mitochondrial dynamics are associated with humandiseases, including cancers (5). The balance between fragmentedmitochondria and elongated mitochondria is governed by afamily of GTP-dependent dynamin-related "mitochondria-shap-ing" proteins (6). While the dynamic-related protein 1 (DRP1)drives mitochondrial fission, the mitofusin 1/2 (MFN1/2) andoptic atrophy 1 (OPA1) proteins mediate fusion of the mito-chondrial outer and inner membranes, respectively.

Based on the distance from the nearest blood vessel, tumorscontain well-vascularized and poorly vascularized regions,which are thought to utilize mitochondria differently. Aerobicoxidative phosphorylation (OXPHOS) is essential for ATP pro-duction to support cell growth and is also the major intracellularsource of reactive oxygen species (ROS). It has been reportedthat glucose deprivation induces metabolic oxidative stress viaan increase in the steady-state levels of mitochondrial super-oxide and hydrogen peroxide (7–10). In addition, it is also welldocumented that the prolonged depletion of various nutrients,such as glucose, glutamine, or arginine, induces ROS-triggeredcancer cell death (11–14). Considering that mitochondrialfusion maximizes ATP generation under nutrient starvation(3, 4) and that excessive ROS production is detrimental to thecells (15), regional regulation of mitochondrial function insidethe tumors becomes essential not only for bioenergetics but alsofor modulating intracellular redox state. Therefore, the ability oftumor cells to dynamically regulate the balance between mito-chondrial fusion and fission is critical for their adaptation to

1Diabetes and Metabolism Research Institute, City of Hope, Duarte,California. 2Irell and Manella Graduate School of Biological Sciences,City of Hope, Duarte, California. 3Department of Molecular Medicine,BeckmanResearch Institute,City of Hope, Duarte,California. 4Depart-ment of Pathology, Chi-Mei Medical Center, Tainan, Taiwan. 5Depart-ment of Biotechnology, Southern Taiwan University of Science andTechnology, Tainan, Taiwan. 6Department of Biology and BiologicalEngineering, California Institute of Technology, Pasadena, California.7Department of Biochemistry andMolecularMedicine, UCDavis Com-prehensive Cancer Center, Sacramento, California. 8National HealthResearch Institutes, Miaoli, Taiwan.

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

Corresponding Author:David K. Ann, City of Hope Beckman Research Institute,1500 E. Duarte Road, Duarte, CA 91010-3000. Phone: 626-218-4967; Fax: 626-471-7204; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2921

�2016 American Association for Cancer Research.

CancerResearch

Cancer Res; 76(17) September 1, 20165006

on March 10, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 30, 2016; DOI: 10.1158/0008-5472.CAN-15-2921

http://cancerres.aacrjournals.org/

metabolic stress caused by the temporal regional heterogeneity.However, the mechanisms underlying the transition from mito-chondrial fusion to fission during the course of sustainednutrient depletion are not completely understood.

KRAB domain-associated protein 1 (KAP1/TRIM28) is apleiotropic regulator of a diverse range of cellular process(16). Notably, overexpressed KAP1 has emerged as an onco-gene in different types of tumors, including breast cancer (17).Previous work by us and others have shown that KAP1 issubjected to phosphorylation at multiple serine (Ser) residues,including Ser440, Ser473, Ser501, and Ser824 (16). Amongthese, although ATM-mediated Ser824 phosphorylation is asso-ciated with DNA damage response (18–22), the kinase respon-sible for and the role of KAP1 Ser473-phosphorylation (pS473-KAP1) in the context of metabolic stress is poorly understood.Here, we demonstrated that pS473-KAP1 is transiently activat-ed following prolonged starvation by the ROS–p38MAPKpathway, and pS473-KAP1 reduces mitochondrial hyperfusion,promoting colony formation in response to chronic glucosedeprivation. Mechanistically, pS473-KAP1-dependent adaptiveresponse is characterized by a reduction of MFN2. The accu-mulation of fragmented mitochondria in the core region,compared with the periphery, of xenografted tumors supportsthe regional heterogeneity of mitochondrial dynamics. Lastly,reduced MFN2 expression in breast cancer is associated withpoor survival, and metastasis corroborates the concept thatsuppression of MFN2-mediated mitochondrial fusion mayunderline the poor prognosis of breast cancer patients. Takentogether, our studies suggest a paradigm by which the ROS–p38MAPK–pS473-KAP1–MFN2 axis prevents mitochondrialhyperfusion during the course of metabolic stress and enablesbreast cancer cells to survive changes in the tumor nutritionalmicroenvironment to promote tumor growth.

Materials and MethodsCell culture and reagents

MDA-MB-231, MDA-MB-468, MCF7, mouse embryonic fibro-blast (MEF) cells from ATCC, mfn2- and ampk-null MEFs fromcollaborators were obtained during the 6 years of this projectfrom 2010 to 2015. Multiple frozen aliquots were establishedupon the acquisition and all experimental cells were passagedfor fewer than20passages after reviving from liquidN2. Cellswerecultured in DMEM supplemented with 10% fetal bovine serum.MDA-MB-231/shKPA1 and MDA-MB-231/shMFN2 cells werecultured with puromycin (2 mg/mL). MDA-MB-231/shKAP1/KAP1wt, MDA-MB-231/shKAP1/KAP1S473A, MDA-MB-231/shKAP1/KAP1S473D, MDA-MB-231/shKAP1/KAP1S473A/Su9-GFP, and MDA-MB-231/shKAP1/KAP1S473D/COX4-DsRedcells were maintained in the presence of puromycin (2 mg/mL)and G418 (100 mg/mL). MDA-MB-231/MFN2 cells were main-tained in the presence of G418 (100 mg/mL). All nutrientdepletion media were prepared with 10% dialyzed fetal bovineserum (Gibco, 26400). The glucose depletion medium wasDMEM (Gibco, 11966-025) supplemented with sodium pyru-vate (1 mmol/L). The glutamine depletion medium was DMEM(Cellgro, 15-013-CV). The arginine depletion medium wasDMEM (Gibco, A14431-01) supplemented with L-glutamine(4 mmol/L) and L-lysine (0.8 mmol/L). Etomoxir (sc-215009), siRNAs against p38a (sc-29433), p38b (sc-39116),and Mfn2 (sc-43928) were from Santa Cruz biotechnology. N-

acetyl-L-cysteine (A7250), Mdivi-1 (M0199), D–glucose(G7021), oligomycin A (75351), Rotenone (R8875), and FCCP(C2920)were fromSigma-Aldrich. SB203580 (152121-47-6)wasfrom Biovision.

Plasmid and cell engineeringshRNA (TRCN0000017998) against the KAP1 30UTR was

obtained from The RNAi Consortium. MDA-MB-231/shKAP1cells were generated using lentivirus system as described previ-ously (12). FLAG-S473A-KAP1 and FLAG-S473D-KAP1 wereengineered using FLAG-KAP1 as a template (22), respectively, bythe Transformer Site Directed Mutagenesis Kit (Clonetech). TheMFN2 donor vector (HsCD00039867) was obtained from theDNASU Plasmid Repository. All KAP1 constructs were subclonedinto the pDONR201donor vector and recombined into lentiviral-gateway-compatible destination vectors with tetracycline-respon-sive promoter using GATEWAY technology (Invitrogen). Lenti-viruses were generated as described previously (12). All constructswere validated by sequencing analyses.

Western blot analyses and antibodiesWestern blot analysis was conducted as described previously

(23). Antibodies used were phospho-S473-KAP1 (644602, BioLe-gend), phospho-S824-KAP1 (A300-767A, Bethyl), KAP1 (A300-274A, Bethyl), b-actin (MAB1501R), H3K27me3 (07-449) fromMillipore, Tom20 (sc-17764), andGAPDH (sc-25778) from SantaCruz; phospho-T172-AMPKa (2535), AMPKa (2532), phospho-T180/Y182-p38 (9216), and p38 (9212) from Cell SignalingTechnology; MFN1 (GTX116254), MFN2 (GTX102055), OPA1(GTX48589), and FIS1/TTC11 (GTX111010) from GeneTex; H3(39763)andH3K9me3 (61013) fromActiveMotif andMitoprofileOXPHOS Cocktail (MS601) from MitoSciences.

Cell viability, clonogenic cell survival, and apoptosis assayThe acid phosphatase (ACP) assay was used to measure cell

viability as described previously (12). Briefly, 5,000 cells/well inthe 96-well plate were seeded and incubated with different con-centrations of glucose or treated with Mdivi-1 (10 mmol/L) for3 days. Alternatively, clonogenic cell survival was performed48 hours after nutrient deprivation. Cells were trypsinized, com-bined with suspended cells, and total 100 cells were reseededin 6-well plate for 2 weeks as described previously (7). Apoptosiswas analyzed by Annexin V/propidium iodide (PI) staining (BDPharmingen) as described previously (12).

Soft-agar colony formationCells (5,000) were mixed in 0.35% agarose in complete

medium (top layer) and were plated on 0.7% agarose withcomplete medium (bottom layer) in 6-well plates. Extra medi-um with different concentration of glucose was added toeach well, and the medium was changed every 5 days duringthe 2 weeks of cell growth. Colonies were imaged as describedpreviously (12).

Intracellular ROS measurementROS measurement was performed using DCFDA (D6883,

Sigma) staining as described previously (12). Briefly, cells werestained with 1 mmol/L DCFDA for 30 minutes and the resultingDCF was analyzed by flow cytometry with a maximum emissionof �530 nm (Gallios, Beckman Coulter or BD Accuri C6). Oxi-dation-insensitive fluorescent DCF probe was used as a control(35848, Sigma).

pS473-KAP1 Regulates Mitochondrial Dynamics

www.aacrjournals.org Cancer Res; 76(17) September 1, 2016 5007




RNA-seq and quantitative RT-PCR analysesTotal RNA was isolated from cells using the TRIzol reagent

(15596-026, Ambion). RNA-seq was conducted by the Integra-tive Genomics Core at City of Hope. RNA-seq data are availablein the Gene Expression Omnibus (GEO) database (GSE63344).Quantitative RT-PCR for gene expression was performed usingBio-Rad system as described previously (12). MitochondrialD-loop primers, forward: 50-GTGGCTTTGGAGTTGCAGTT-30;reverse: 50-GAAGCAGATTTGGGTACCAC-30. Lamin B primers,forward: 50-ATGAAGCGGATGTCTAAGAAAG-30; reverse: 50-CG-CCTGGGTCCTGTTTACAC-30.

Oxygen consumption rate (OCR)The mitochondrial OCR was measured by the Seahorse Bio-

science XF24 Extracellular Flux Analyzer (Seahorse Bioscience) asdescribed previously (12). Briefly, 6 � 104 cells were seeded inXF24 cell culture microplates overnight. The next day, cells werewashed and incubated with Mito Assay Medium (Seahorse Bio-science) supplemented with glucose (25 mmol/L) and sodiumpyruvate (1 mmol/L). The pH value of the Mito Assay Mediumwas adjusted to 7.4. TheOCRwas assayed by sequential injectionsof oligomycin (1 mmol/L), FCCP (0.5 mmol/L) and Rotenone(2.5 mmol/L) according to the manufacturer's instructions. Afterthe measurements, the cells were trypsinized for cell numbernormalization.

ATP assayATP production was determined using the ENLITEN ATP Assay

System (Promega, FF2000) according to the manufacturer'sinstructions. Briefly, cells were harvested in cold PBS and thenadded to 5% trichloroacetic acid (TCA). Subsequently, tris-acetatebuffer (pH 7.75)was used to neutralize the TCA solution to afinalconcentration of 0.1%. The extracts were further diluted 1:100,and an equal volume of rL/L Reagent (Promega, FF2000) wasadded tomeasure the luminescence using a TD-20e luminometer(Turner). The ATP amount was normalized to cell number, andthe relative ATP level was calculated.

Immunofluorescence staining and imagingTom20 antibody (sc-17764, Santa Cruz) was used for

immunofluorescence staining to visualize mitochondrial mor-phology as described previously (12). Slides were viewed usinga 60� objective on an inverted IX81 microscope (Olympus)or LSM 700 Confocal Microscope (Zeiss). Time lapse imagingwas obtained by Axio Observer Z1 Inverted microscope (Zeiss)with a 20-minute interval for 20 hours. Videos were collectedand processed using ZEN 2012 software. For Electron micros-copy studies, serial block-face scanning electron microscopy(SBF-SEM; Zeiss Sigma VP) was used. Cell pellets were pre-pared by the Electron Microscopy Core at City of Hope asdescribed previously (24). Images were taken with 5,400�magnification. Images were collected and processed usingImage-Pro 6.3 software.

Xenograft and mouse tumor studiesAnimal experiments were approved by the Institutional Animal

Care andUse Committee at City ofHope. A total of 5� 106MDA-MB-231/COX4-DsRed cells were used for subcutaneous injectioninto the flank of 6-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mice. KRAS-driven salivary gland tumors were generatedas described previously (25). The primary tumor cells were

isolated and engineered with COX4-DsRed to visualize mito-chondria. Cells (3 � 106) were injected subcutaneously into theflank of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Mice wereeuthanized on day 28 to harvest tumors. Formalin-fixed, paraffin-embedded (FFPE) sections were subjected to immunohistochem-istry (IHC) staining with MFN2 antibody (WH0009927M3,Sigma) by the pathology core at City of Hope. Tumors weredissected into core and peripheral regions and subjected toWestern blotting analysis as described previously (26). Tumorsfor visualizing COX4-DsRed fluorescence were fixed in para-formaldehyde (4%) at 4�C overnight, and then subjected to15% and 30% sucrose infiltration and optimum cutting tem-perature (OCT) compound embedding. Sections were sliced at8 mm on the cryostat (CM3050S, Leica).

Patient materials and IHCThe institutional review board of the Chi-Mei Medical Center

approved the procurement of formalin-fixed tissue and acquisi-tion of clinical information for this study (IRB10210004). IHCstaining was performed with MFN2 antibody (WH0009927M3,Sigma) as previously described (27) to assess MFN2 expressionon 202 consecutively treated primary breast carcinomas thatunderwent modified radical mastectomy without adjuvant che-motherapy between 1997 and 2002. Pathological staging wasdetermined based on the 7th edition of American Joint Com-mittee on Cancer (AJCC) system. Immuno-expression of MFN2was determined by pathologists (C.-F. Li and Tzu-Ju Chen) usingH-score, defined as H-score ¼ SPi (i þ 1), where i is the intensityof the stained tumor cells (0 to 3þ), and Pi is the percentageof stained tumor cells for each intensity. Tumors with H-scoresno less than the median of all scored cases were classified ashigh MFN2 expression (Table 1), as described previously (28).

BioinformaticsThe Cancer Genome Atlas (TCGA) dataset, the Netherlands

Cancer Institute (NKI) dataset and Prognoscan database (http://www.prognoscan.org/) were used for data mining of mitochon-drial complex genes and KAP1 as well as the clinical relevance(29–32). Histograms were generated by software R or GraphPadPrism 6. Heatmap was generated by GENE-E software.

Statistical analysisData with error bar were presented as mean � SD. Statistical

significance was determined using the two-tailed Student t test.The c2 test was used to evaluate the correlations between MFN2expression and various clinicopathological features. Survivalanalyses were performed for disease-specific and distal metasta-sis-free survival. Survival curves were plotted using the Kaplan–Meier method.

ResultsKAP1 mRNA level is elevated in breast tumors and diversenutrient stresses induce a delayed ROS-p38MAPK-dependentKAP1 phosphorylation at Ser473 in breast cancer cells

Comparative expression analyses, based on breast cancerdatasets from the Netherlands Cancer Institute (NKI; refs.31, 32), revealed that each distinct breast tumor subgroupshowed a unique signature of glycolysis and OXPOS geneexpression (Fig. 1A). Notably, almost all breast tumors,

Cheng et al.

Cancer Res; 76(17) September 1, 2016 Cancer Research5008




Figure 1.

Diverse metabolic stresses induce KAP1 Ser473 phosphorylation in breast cancer cells. A, overexpression of metabolic genes and KAP1 in breast cancers.Genes were selected from 103 NKI samples based on the Kyoto Encyclopedia of Genes and Genomes database to generate heatmap. B, glucose depletioninduces dose- and time-dependent KAP1 Ser473 phosphorylation in TNBC cells. MDA-MB-231 cells were incubated with the indicated concentrations ofglucose for 24 hours (top). MDA-MB-231 and MDA-MB-468 cells were cultured in glucose-depleted medium for different time periods as indicated (2nd and3rd panels). Noncontiguous lanes are separated by a dashed line. C and D, induction of pS473-KAP1 by diverse metabolic stresses in MDA-MB-231 and MCF7cells. C, MDA-MB-231 (right) and MCF7 (left) cells were starved of glucose or glutamine or treated with 2-DG as indicated. D, arginine depletion oretomoxir induces time-dependent KAP1 Ser473 phosphorylation in MDA-MB-231 cells. MDA-MB-231 cells were subjected to arginine depletion (top).MDA-MB-231 and MCF7 cells were treated with increasing concentrations of etomoxir (bottom). E, blocking p38 signaling blunts glucose and glutaminedepletion-induced pS473-KAP1. SB203580 reduced glucose or glutamine depletion-induced pS473-KAP1 (top). p38a knockdown dampened pS473-KAP1induction in response to glucose depletion (bottom). p-p38, T180/Y182-phosphorylated p38MAPK.






irrespective their ER/PR/HER2 status, compared with normal-like samples, exhibited upregulation of messages encodingproteins involved in both glycolysis and OXPHOS as well asKAP1 (Fig. 1A). We further noticed that the expression ofselected mitochondrial OXPHOS genes was positively associ-ated with the KAP1 level (Supplementary Fig. S1). Consistentwith the study by Addison and colleagues, showing that higherKAP1 is associated with poor prognosis in breast cancerpatients (17), an elevated KAP1 expression in breast tumorswas correlated with high grades and poor prognosis (Supple-mentary Fig. S2). We next found that glucose depletioninduced a delayed (at approximately 6 hours) and transient(in some cases) KAP1 phosphorylation at Ser473 (pS473-KAP1), but not Ser824 (pS824-KAP1) in triple-negative breastcancer MDA-MB-231 and MDA-MB-468 cells (Fig. 1B) as wellas ER-positive breast cancer MCF7 cells (Fig. 1C, left). More-over, glutamine starvation (Fig. 1C, right), arginine depriva-tion (Fig. 1D, top) and blockage of fatty acid b-oxidation byetomoxir (a CPT-1 inhibitor; Fig. 1D, bottom) all increasedpS473-KAP1. Together, we proposed that pS473-KAP1 repre-sents a common response to diverse nutrient starvation inbreast cancer cells.

To identify the pathway for pS473-KAP1 induction, we firstfound that the AMPK inhibitor compound C did not alterthe glutamine starvation-induced pS473-KAP1 (SupplementaryFig. S3A, top). In parallel, pS473-KAP1 persisted in glutamine-deprived AMPKa1/AMPKa2 double knockout MEFs (Supple-mentary Fig. S3A, bottom). Similarly, we used the ATM inhibitorKu55933 andknockdownofCHK2 andPKCd by small interfering(si) RNA to exclude the involvement of the ATM-CHK2 (33) andPKCd (34) pathways in inducing pS473-KAP1 under starvation(Supplementary Fig. S3B and S3C).

Based on previous observations that metabolic stress inducesROS production in cells (7, 12, 13), we postulated that ROSinduction activates KAP1 Ser473 phosphorylation during nutri-ent deprivations. Indeed, depletion of either glucose or glutamineled to a surge of oxidation of DCFDA toDCF (Supplementary Fig.S3D, left), but not the oxidative-insensitive DCF analogue (Sup-plementary Fig. S3D, right), and N-acetylcysteine (NAC), a ROSscavenger, impaired starvation-induced pS473-KAP1 (Supple-mentary Fig. S3E). Next, we found that both stress kinasep38MAPK inhibitor SB203580 and siRNA against p38a/b atten-uated starvation-induced pS473-KAP1 (Fig. 1E; SupplementaryFig. S3F and S3G). As expected, SB203580 reducedH2O2-inducedpS473-KAP1, but not pS824-KAP1 (Supplementary Fig. S3H).Lastly, previous studies have shown that NAC prevents glucosedepletion-induced cell death in MCF7 cells (7). Furthermore,NAC rescuedMDA-MB-231 cells from glucose depletion-inducedcell death (Supplementary Fig. S3I and S3J). Overall, our resultswere consistent with those of Spitz and colleagues, who reportedthat H2O2 produced inside cells is mediating the effects (7).

KAP1 Ser473 phosphorylation attenuates mitochondrialOXPHOS

We next explored whether pS473-KAP1 regulates ROS gener-ation in the context of glucose deprivation. To achieve this goal,wild-type (wt)-KAP1, phosphorylation-defective S473A-KAP1,and phosphorylation-mimetic S473D-KAP1 were reexpressed inKAP1-knockdown MDA-MB-231 (MDA-MB-231/shKAP1) cells,respectively (Fig. 2A, bottom). Considering that mitochondrialOXPHOS is the major intracellular ROS source (35), OCR,

reflecting the OXPHOS flux, in different KAP1-expressing cellswas analyzed. As shown in Fig. 2A, reconstitution of wt- andS473A-KAP1 increased both basal and FCCP-induced maximalrespiration to a higher level than S473D-KAP1 did (top) withoutsignificant alteration on overall ATP production among thesecells (Supplementary Fig. S4A, left). One possibility that reduc-ed mitochondrial number would account for the reduced OCRin S473D-KAP1-expressing cells was ruled out (SupplementaryFig. S4A, right).

Given that immediate response to nutrient starvation is mito-chondrial fusion to protect mitochondria from mitophagy andmaximizing OXPHOS for energy generation (3, 4), we exploredwhether pS473-KAP1 counteracts mitochondrial fusion uponnutrient deprivation. First, glucose-depletion (6 hours) led tomitochondrial elongation, similar to the effect of treatment withMdivi-1, a mitochondrial fission inhibitor (Fig. 2B). Time-lapseimaging using MDA-MB-231 cells expressing mitochondria-tar-geted COX4-DsRed further validated this observation (Movies 1and 2). As shown in Supplementary Fig. S4B, mitochondrialfission started between 8 and 9 hours after starvation, followingmitochondrial fusion. Live-cell imaging performed on glucose-starved, cocultured S473A-KAP1/Su9-GFP- and S473D-KAP1/COX4-DsRed-repleted cells revealed that less elongated mito-chondria were observed in the S473D-KAP1-expressing cells thanin the S473A-KAP1-expressing counterparts during the course ofstarvation (Movies 3 and 4). Quantitative analyses based onimages at 0 and 8 hours after glucose starvation (SupplementaryFig. S5A) validated that the expression of S473D-KAP1 aloneshiftedmitochondrial equilibrium toward fragmentedmitochon-dria and that the replacement of S473A-KAP1 favored elongatedmitochondria in glucose-starved MDA-MB-231/shKAP1 cells(Fig. 2C). Scanning electron microscopy (SEM) analyses furtherconfirmed that S473A-KAP1 cells harboredmore elongatedmito-chondria with an increase in total mitochondrial surface area(Supplementary Fig. S5B and S5C).

To assess whether unbalanced mitochondrial fusion sensitizescells toward glucose depletion-induced cell death, we assessed cellviability of glucose-starved MDA-MB-231 cells with (or without)Mdivi-1. Figure 2D showed that both glucose deprivation andMdivi-1 pretreatment increased mitochondrial maximal respira-tion. Moreover, Mdivi-1 pretreatment further increased ROS signal(Fig. 2E) and sensitized cells to glucose starvation-induced celldeath (Supplementary Fig. S5D). Likewise, expression of S473A-KAP1, but not wt- and S473D-KAP1, significantly decreased theviability of glucose-starved cells (Fig. 2F). Together, these resultssupported a positive correlation between mitochondrial hyperfu-sion and elevated OCR. Notably, elevated maximal respirationcould account for increased ROS generation.

pS473-KAP1 downregulates MFN2 to limit mitochondrialfusion

To understand how pS473-KAP1 regulates mitochondrialdynamics, we compared the expression of several mitochondrialproteins during the course of glucose starvation. First, neither thesteady-state levels of respective inner mitochondrial membranecomplex proteins including NDUFB8 (complex I), SDHB (com-plex II), UQCRCII (complex III), COXII (complex IV) and ATP5A(complex V), nor the outer mitochondrial membrane proteinTOM20, changed during 24-hour course of glucose deprivation(Fig. 3A). In contrast, a time-dependent decrease in MFN2, amitochondrial fusion protein, was noted (Fig. 3A). Likewise,

Cheng et al.





Figure 2.

KAP1 Ser473 phosphorylation reduces the level of glucose deprivation-promoted mitochondrial fusion. A, knockdown of KAP1 or repletion with S473D-KAP1reduces basal and maximal mitochondrial respiration. OCR was measured (top) in different KAP1-expressing cells (bottom). Mean � SD from n ¼ 3. B,glucose depletion promotes mitochondrial fusion. MDA-MB-231 cells were pretreated with vehicle or Mdivi-1 (20 mmol/L) for 18 hours prior to glucosestarvation. Arrowhead, tubulated mitochondria; scale bar, 10 mm; n > 3. C, expression of S473A-KAP1 favors mitochondrial fusion in glucose-starvedMDA-MB-231/shKAP1 cells. Representative images of the typical fragmented, intermediate, and tubulated mitochondrial network are shown (left).Quantitation (%) of cells with each mitochondrial morphology (right; n ¼ 80; also see Supplementary Fig. S5A). D, Mdivi-1 treatment mirrors glucosestarvation to induce basal and maximal mitochondrial respiration. MDA-MB-231 cells were pretreated with vehicle or Mdivi-1 (20 mmol/L) for 18 hoursprior to glucose starvation (6 hours); n ¼ 3. E, Mdivi-1 further induces the ROS level after glucose depletion. MDA-MB-231 cells were treated as in D; n ¼ 3.F, repletion of S473A-KAP1 sensitizes MDA-MB-231/shKAP1 cells to glucose depletion. Cells were incubated with the decreasing concentrations ofglucose for 72 hours. Mean � SD from n ¼ 3; � , P < 0.05; �� , P < 0.001.






Figure 3.

KAP1 Ser473 phosphorylation reduces OCR via MFN2 downregulation and regulates mitochondrial gene expression. A, glucose depletion downregulatesMFN2 protein abundance. B, arginine depletion downregulates MFN2 in MDA-MB-231 cells. C, KAP1 S473A substitution retards MFN2 downregulation inglucose-starved MDA-MB-231/shKAP1 cells. Relative MFN2 abundance is calculated by designating the level in control MDA-MB-231 cells (A and B) orMDA-MB-231/shKAP1 cells (C) as 1 after normalization with GAPDH and is shown in italic. D, knockdown of MFN2 reduces basal OCR in different KAP1-expressing cells. MFN2 was knocked down by siRNA in different KAP1-expressing cells. SA, S473A-KAP1; SD, S473D-KAP1. E, the ROS level is higher in S473A-KAP1 reexpressing MDA-MB-231/shKAP1 cells. Cells were cultured in complete (Ctrl) or glucose depletion medium (Glc-) for 6 hours. Relative oxidizedDCF levels were calculated by designating the value in wt-KAP1 reexpressing MDA-MB-231/shKAP1 as 1. F, ROS level is reduced in mfn2�/� MEFs.Relative oxidized DCF levels were calculated by designating the value in wt MEFs as 1. G, knockdown and overexpression of MFN2 have opposite effect on cellviability under different nutrient starvation. Clonogenic survival fraction was calculated by designating cells in complete medium as 1. D–G, mean � SDfrom n ¼ 3; � , P < 0.05; �� , P < 0.01; �� , P < 0.01. H, S473D-KAP1 elevates expression of the mitochondrial complex genes. Relative message abundancewas calculated by designating the respective message abundance in wt-KAP1-expressing MDA-MB-231/shKAP1 cells as 1.

Cheng et al.





arginine depletion decreased MFN2 abundance (Fig. 3B). How-ever, MFN2 abundance did not decrease in the glucose-deprived(8 hours) S473A-KAP1-expressing cells (Fig. 3C, lane 7 versus lane3), suggesting that pS473-KAP1 downregulates MFN2. To ascer-tain the role of MFN2 in regulating OXPHOS, MFN2 was knockeddown in wt-, S473A-, and S473D-KAP1-expressing cells. Clearly,knockdown of MFN2 markedly decreased the basal OCR in allcells and the reduced OCR in wt- and S473A-KAP1-expressingcells was comparable with the basal level of S473D-KAP1-expres-sing cells (Fig. 3D). We also observed higher basal ROS in theS473A-KAP1-expressing cells in the presence or absence of glucose(Fig. 3E). Lastly, the correlation between MFN2 and ROS levelswas established using wild-type and mfn2 knockout MEFs (Fig.3F). We next addressed whether different MFN2 level affects

nutrient sensitivity in MDA-MB-231 cells. As shown in Fig. 3G,MFN2 over-expression and knockdown have opposite effect onnutrient sensitivity. Together with the observations on the mito-chondrial hyperfusion upon arginine starvation in MDA-MB-231cells (Supplementary Fig. S5E and S5F), we proposed that mito-chondrial hyperfusion aggravated the response to nutrient dep-rivation. Analyses of the RNA-seq data further uncovered addi-tional roles for S473D-KAP1 in elevating the expression of mito-chondrial complex genes (Fig. 3H).

Association of MFN2 expression and mitochondrialnetwork in vivo

To validate the alteration in MFN2 in response to starva-tion in vivo, xenografted MDA-MB-231/COX4-DsRed tumors

Figure 4.

MFN2 downregulation is associated with mitochondrial fission in the core region of tumor. A, a gradient decrease of MFN2 staining inwards to thecore region of MDA-MB-231/COX4-DsRed xenografts by IHC. Dotted lines depict borders of different regions as shown in the diagram (inset).Scale bar, 100 mm; n ¼ 2. B, MFN2 is downregulated in the core region of xenografted tumors. P, peripheral region; C, core; n ¼ 2. C, mitochondriaare more fragmented in the core region of tumors. Mitochondrial morphologies were visualized with COX4-DsRed in xenografted MDA-MB-231 cells(top) and K-RASG12V-driven mouse salivary duct carcinoma (bottom). Insets, enlarged views (top); scale bar, 100 mm (top) and 10 mm (bottom).D, repletion of S473A-KAP1 reduces colony formation in MDA-MB-231/shKAP1 cells. n ¼ 3; � , P < 0.05; ��, P < 0.005.






were analyzed. As expected, the tumor core region, generallyconsidered as the area undergoing metabolic stress, showedreduced MFN2 (Fig. 4A and B) and with more fragmentedmitochondria (Fig. 4C, top), supporting the notion thatreduced MFN2 in the core region contributes to the accumu-lation of fragmented mitochondria via suppressed mitochon-drial fusion. Next, we used a KRAS-driven mouse salivary glandtumor model developed in our laboratory recently (25) toindependently validate a higher degree of mitochondrial frag-mentation in the core region of tumors (Fig. 4C, bottom).Elevated H3K9me3 and H3K27me3 marks in the core region ofxenografts (Fig. 4B), indirectly reflecting reduced lysinedemethylases (KDM) activity due to a decrease in their cofac-tor, a-ketoglutarate level, attested to the regional metabolicstress inside tumors. Lastly, we sought to explore the biologi-cal consequence of metabolic stress-induced pS473-KAP1.Figure 4D revealed that S473A-KAP1-expressing cells weremore sensitive to glucose reduction using an anchorage-inde-pendent cell growth assay.

To assess the pathological relevance of the MFN2 level inbreast cancer, we found that the loss of MFN2 expression wasassociated with adverse clinicopathological features and worsepatient outcome (Table 1). Notably, MFN2 low expression wassignificantly associated with increments of primary tumor sta-tus (P ¼ 0.001), nodal metastasis (P ¼ 0.033), and stage (P ¼0.002; Fig. 5A). Multivariate analysis showed that loss of MFN2expression, along with higher stage, remained to be prognos-tically significant for disease-specific (P ¼ 0.0024) and metas-tasis-free (P < 0.0001) survival (Fig. 5B). Consistently, theTCGA breast cancer dataset showed that MFN2 message waslower in breast tumors compared to normal tissues (Fig. 5C).Data mining on the PrognoScan database (http://www.prog-noscan.org/) (29) showed that reduced MFN2 message wascorrelated with poor survival (Fig. 5D–G). Altogether, ourresults highlighted the importance of the p38/pS473-KAP1/MFN2 pathway in promoting tumor cells' survival under met-abolic stress.

DiscussionCancer cells are usually in a state of chronic oxidative stress and

use higher glucose and hydroperoxide metabolism for compen-sation. Therefore, targeting the glycolysis and antioxidant path-

ways could be therapeutic strategies for cancer (9, 36, 37). On theother hand, mitochondrial O2 metabolism is critical for thefurther induction of oxidative stress in cancer upon nutrientstarvation, such as glucose depletion (8, 37). Our current findingsextend the repertoire of critical cellular processes initiated bynutrient starvation, including the induction of pS473-KAP1through the ROS-/p38MAPK-dependent pathway and the subse-quent downregulation of MFN2, to reduce mitochondrial hyper-fusion and OXPHOS in order to ameliorate metabolic oxidativestress. Although our pharmacological and genetic approachessuggested the involvement of p38 in this process, the specificrole of different p38 isoforms in the context of distinct metabolicstress remained obscure, and, as such, whether other kinases alsomediate oxidative stress-induced pS473-KAP1 signaling needsfurther study in the future. Presumably, metabolic stress-triggeredpS473-KAP1 is a general phenomenon present in both normaland tumor cells. However, because proliferating tumor cellsusually exhibit higher oxidative stress and metabolic demands,this signaling pathway could be more prominent in cancer anddepend on cells' nutrient dependency. For example, the fact thatMCF7 cells are more glutamine-independent (38) could partiallyexplainwhy glutamine deprivation-induced pS473-KAP1was lessrobust in MCF7 cells (Fig. 1C). It is likely that primary mammaryepithelial cells are relatively resistant to glucose depletion (36). Asshown in Fig. 6, we propose that pS473-KAP1-mediated MFN2downregulation is crucial for overcoming nutrient stress duringtumor growth.

Upon prolonged metabolic stress, we noted a marked down-regulation of MFN2 that limits the initial hyperfusion response.Although MFN1, another mitofusin, is present, it apparently isunable to compensate for the loss of MFN2. This behavior isconsistent with previous observations that disruption of eithermitofusin gene leads to severe mitochondrial fragmentation(39). Clinically, we found lower MFN2 expression correlatedwith more malignant breast tumors, which is consistent withearlier reports proposing MFN2 as a tumor suppressor (40–42).The mechanisms underlying the control of mitochondrialdynamics in cancer cells received more attention recently. Forexample, Drp1-mediated mitochondrial fission is linked totumor transformation (43, 44). Moreover, more mitochondrialfission is observed in lung cancer compared with normal lungcells, and inhibition of mitochondrial fission induces cancercell death (45). Fragmented mitochondria have been shown topromote breast cancer metastasis (46). These observations andour results together put mitochondrial dynamics controlsquarely in the driver's seat of tumor progression and supportthe notion that MFN2 downregulation represents an additionallayer mechanism of preventing mitochondrial hyperfusionprocess during tumorigenesis.

It has been reported that OCR was elevated under lowglucose (0.75 mmol/L; ref. 47) and that shifting carbon sourcefrom glucose to galactose or acetoacetate induced OCR andsubsequent mitochondrial elongation (48), attesting to ourobservations that short-term (<6-hour) starvation of glucoseincreased OCR. Although upregulation of mitochondrialOXPHOS is beneficial as a short-term response to acute nutri-ent starvation, the increased ROS production from elongatedmitochondria would in turn cause cell death during prolongedstarvation. Collectively, our data provide a new mechanisticinsight into how activation of the KAP1-/MFN2-dependentarm copes with metabolic oxidative stress. Given that most

Table 1. Correlation between MFN2 expression and variousclinicopathological factors

MFN2 expressionParameters Category Cases, n High exp. Low exp. P

Age (years) <60 years 141 73 68 0.444�60 years 61 28 33

Primary tumor (T) T1 82 53 29 0.001a

T2 96 35 61T3–4 24 13 11

Nodal status (N) N0 117 66 51 0.033a

N1–N2 85 35 50Stage I 63 43 20 0.002a

II 115 48 67III 24 10 14

Histological grade Grade I 18 13 5 0.022a

Grade II 141 73 68Grade III 43 15 28

aStatistically significant.

Cheng et al.





Figure 5.

Association of MFN2 with clinical prognosis. A–G, lower MFN2 is associated with adverse clinical features. A, reduced MFN2 expression is correlatedwith malignant breast tumors. Inversed MFN2 expression in low-stage (a) and high-stage (b) breast carcinoma and paired primary breast carcinoma (c)and its nodal deposition (d). B, reduced MFN2 expression is correlated with poor disease-specific and metastasis-free survival. A and B, 202 primarybreast cancer samples from the Chi-Mei Medical Center were used (also see Table 1). C, MFN2 transcript level is downregulated in primary andmetastatic breast tumors compared with normal tissues. The TCGA dataset was used. �� , P < 0.01; �� , P < 0.005. D–G, PrognoScan database-basedKaplan–Meier analysis in breast cancer datasets. The data were retrieved from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), withthe accession numbers GSE9893 (D), GSE11121 (E), GSE1379 (F), and GSE2990 (G), respectively.






cancer cells displayed elevated ROS (49), drugs targeting thisregulatory circuit could have a positive therapeutic impact bysensitizing cancer cells to oxidative stress-based therapeuticstrategies.

To our knowledge, this is the first study demonstrating thatKAP1 can be posttranslationally modified upon metabolicstress. Ideally, we would like to observe a gradient of pS473-KAP1 signals, opposite to the MFN2 levels in solid tumors.However, due to the lack of the availability of IHC-qualitypS473-KAP1 antibody, our repeated efforts failed to detect anypS473-KAP1 signal using FFPE specimens of either primarypatient samples or xenografted tumors. Moreover, whetherother posttranslational modifications of KAP1 also occur undernutrient deprivation to regulate metabolic stress responsedeserves more attention. In the current study, we demonstratedthat pS473-KAP1 is associated with MFN2 downregulationupon metabolic stress. Although the detailed molecular mech-anism is unclear, it is possible that KAP1 directly or indirectlyregulates MFN2 abundance. Based on our unpublished obser-vations, several microRNAs are upregulated in S473D-KAP1-reexpressing cells. Among these, potential starvation-inducedand MFN2-targeting microRNA is identified and underinvestigation.

In conclusion, we show that ROS-/p38MAPK-inducedpS473-KAP1, following the initial induction of mitochondrialfusion, prevents mitochondrial hyperfusion by downregulatingMFN2, thus promoting the survival of metabolically stressedtumor cells. Our findings identify not only a marker potentiallyuseful for breast cancer stratification but also a mechanistic link

for tumor cells to adapt to metabolic stresses during tumorprogression and cancer therapy.

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

Authors' ContributionsConception and design: C.-T. Cheng, C.-Y. Kuo, C.-F. Li, D.K. AnnDevelopment of methodology: C.-T. Cheng, C.-Y. Kuo, Y. Chung, D.K. AnnAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.-T. Cheng, C.-Y. Kuo, C. Ouyang, C.-F. Li, Y. ChungAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.-T. Cheng, C.-Y. Kuo, C. Ouyang, C.-F. Li,D.C. Chan, H.-J. Kung, D.K. AnnWriting, review, and/or revision of the manuscript: C.-T. Cheng, C.-Y. Kuo,C. Ouyang, D.C. Chan, H.-J. Kung, D.K. AnnAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.-T. Cheng, C. Ouyang, D.K. AnnStudy supervision: C.-T. Cheng, D.K. Ann

AcknowledgmentsWe are sincerely grateful to Drs. Emily Wang and Art Riggs for their helpful

suggestions and critical reading ofmanuscript, Ms. Lucy Brown of the AnalyticalCytometry Core for flow cytometry analyses, Dr. Brian Armstrong and Ms. TinaPatel of the LightMicroscopyCore formicroscopy analyses,Mr. AustinChangoufor processing time lapse imaging analyses, Drs. XiweiWu and JinhuiWang andMr. Charles Warden of Functional Genomics Core for RNA-seq data analyses,Ms. Tina Montgomery and Ms. Sofia Loera of Pathology Core for immunohis-tochemical analyses, Dr. Tzu-Ju Chen for pathological evaluation, Drs. MarciaMiller, Zhuo Li, and Ricardo Zerda of Electron Microscopy Core and Mr. KevinChi for EM image analysis and members of Dr. Ann's laboratory for helpfuldiscussions and Dr. Nancy Linford for editing.

Figure 6.

A KAP1/MFN2-dependent arm prevents mitochondrial hyperfusion during the course of metabolic stress. To maximize the utilization of available nutrients,mitochondria undergo fusion, which would generate excessive ROS in tumor cells distant from blood vessel. The elevated oxidative stresssubsequently results in p38MAPK-mediated pS473-KAP1 induction. pS473-KAP1 then suppresses mitochondrial hyperfusion via MFN2 downregulation.

Cheng et al.





Grant SupportThis study was financially supported by National Institute of Health

Research Grants R01DE10742, R01DE14183, and The Mary Kay Founda-tion Research Grant number 005-13 (D.K. Ann), Ministry of Healthand Welfare Research Grant MOHW103-TD-M-111-102001 (C.-F. Li),R01CA165263, R01CA150197, MOHW104-TDU-M-212-13304, MOST102-2320-B-400-018-MY3, MOST104-2321-B-400-009, NHRI05A1-MGPP15-014(H.-J. Kung), and P30CA33572.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 20, 2015; revised May 26, 2016; accepted June 15, 2016;published OnlineFirst June 30, 2016.

References1. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the war-

burg effect: the metabolic requirements of cell proliferation. Science2009;324:1029–33.

2. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. NatRev Cancer 2011;11:85–95.

3. Gomes LC, Benedetto GD, Scorrano L. During autophagy mitochondriaelongate, are spared from degradation and sustain cell viability. Nat CellBiol 2011;13:589–98.

4. Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J. Tubular networkformation protects mitochondria from autophagosomal degradation dur-ing nutrient starvation. Proc Natl Acad Sci U S A 2011;108:10190–5.

5. Grandemange S, Herzig S, Martinou J-C. Mitochondrial dynamics andcancer. Semin Cancer Biol 2009;19:50–56.

6. Lackner L. Shaping the dynamic mitochondrial network. BMC Biol 2014;12:35.

7. Lee YJ, Galoforo SS, Berns CM, Chen JC, Davis BH, Sim JE, et al. Glucosedeprivation-induced cytotoxicity and alterations inmitogen-activated pro-tein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells. J Biol Chem 1998;273:5294–9.

8. Spitz DR, Sim JE, Ridnour LA, Galoforo SS, Lee YJ. Glucose deprivation-induced oxidative stress in human tumor cells: a fundamental defect inmetabolism? Ann N Y Acad Sci 2000;899:349–62.

9. Ahmad IM, Aykin-Burns N, Sim JE,Walsh SA, Higashikubo R, Buettner GR,et al.Mitochondrial andH2O2mediate glucose deprivation-induced stressin human cancer cells. J Biol Chem 2005;280:4254–63.

10. Aykin-Burns N, Ahmad Iman M, Zhu Y, Oberley Larry W, Spitz Douglas R.Increased levels of superoxide and H2O2 mediate the differential suscep-tibility of cancer cells versus normal cells to glucose deprivation. Biochem J2009;418:29–37.

11. Hensley CT, Wasti AT, DeBerardinis RJ. Glutamine and cancer: cellbiology, physiology, and clinical opportunities. J Clin Invest 2013;123:3678–84.

12. Qiu F, Chen Y-R, Liu X, Chu C-Y, Shen L-J, Xu J, et al. Arginine starvationimpairs mitochondrial respiratory function in ASS1-deficient breast cancercells. Sci Signal 2014;7:ra31.

13. Changou CA, Chen Y-R, Xing L, Yen Y, Chuang FYS, Cheng RH, et al.Arginine starvation-associated atypical cellular death involves mitochon-drial dysfunction, nuclear DNA leakage, and chromatin autophagy. ProcNatl Acad Sci U S A 2014;111:14147–52.

14. Graham NA, Tahmasian M, Kohli B, Komisopoulou E, Zhu M, Vivanco I,et al. Glucose deprivation activates ametabolic and signaling amplificationloop leading to cell death. Mol Syst Biol 2012;8:589.

15. Fruehauf JP,Meyskens FL. Reactive oxygen species: a breath of life or death?Clin Cancer Res 2007;13:789–94.

16. Cheng C-T, Kuo C-Y, Ann DK. KAPtain in charge of multiple missions:emerging roles of KAP1. World J Biol Chem 2014;5:308–20.

17. Addison J, Koontz C, Fugett JH, Creighton CJ, Chen D, Farrugia MK, et al.KAP1 promotes proliferation and metastatic progression of breast cancercells. Cancer Res 2014;75:344–55.

18. White DE, Negorev D, Peng H, Ivanov AV, Maul GG, Rauscher FJ. KAP1, anovel substrate for PIKK family members, colocalizes with numerousdamage response factors at DNA lesions. Cancer Res 2006;66:11594–9.

19. Ziv Y, Bielopolski D, Galanty Y, Lukas C, Taya Y, Schultz DC, et al.Chromatin relaxation in response to DNA double-strand breaks is mod-ulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol2006;8:870–6.

20. Nozomi T, Bipasha M, Sandeep B. Distinct roles of ATR and DNAPKcs intriggering DNA damage responses in ATMdeficient cells. EMBO Rep2009;10:629–35.

21. Li X, Lee Y-K, Jeng J-C, Yen Y, Schultz DC, Shih H-M, et al. Role for KAP1serine 824 phosphorylation and sumoylation/desumoylation switch inregulating KAP1-mediated transcriptional repression. J Biol Chem 2007;282:36177–89.

22. Lee Y-K, Thomas SN, Yang AJ, Ann DK. Doxorubicin down-regulatesKr€uppel-associated box domain-associated protein 1 sumoylation thatrelieves its transcription repression on p21WAF1/CIP1 in breast cancerMCF-7 cells. J Biol Chem 2007;282:1595–606.

23. Kuo C-Y, Li X, Kong X-Q, Luo C, Chang C-C, Chung Y, et al. An Arginine-rich motif of ring finger protein 4 (RNF4) oversees the recruitment anddegradation of the phosphorylated and SUMOylated Kr€uppel-associatedbox domain-associated protein 1 (KAP1)/TRIM28 protein during geno-toxic stress. J Biol Chem 2014;289:20757–72.

24. West JB, Fu Z, Deerinck TJ, Mackey MR, Obayashi JT, Ellisman MH.Structure–function studies of blood and air capillaries in chicken lungusing 3D electronmicroscopy. Respir Physiol Neurobiol 2010;170:202–9.

25. Fu Y, Cruz-Monserrate Z, Helen Lin H, Chung Y, Ji B, Lin S-m, et al. Ductalactivation of oncogenic KRAS alone induces sarcomatoid phenotype. SciRep 2015;5:13347.

26. Lin HH, Lin SM, Chung Y, Vonderfecht S, Camden JM, Flodby P, et al.Dynamic involvement of ATG5 in cellular stress responses. Cell Death Dis2014;5:e1478.

27. Li C-F, Chen L-T, Lan J, Chou F-F, Lin C-Y, Chen Y-Y, et al. AMACRamplification and overexpression in primary imatinib-na€�ve gastrointes-tinal stromal tumors: a driver of cell proliferation indicating adverseprognosis. Oncotarget 2014;5:11588–603.

28. Lee S-W, Li C-F, Jin G, Cai Z, Han F, Chan C-H, et al. Skp2-dependentubiquitination and activation of LKB1 is essential for cancer cell survivalunder energy stress. Mol Cell 2015;57:1022–33.

29. Mizuno H, Kitada K, Nakai K, Sarai A. PrognoScan: a new database formeta-analysis of the prognostic value of genes. BMC Med Genomics2009;2:18.

30. Marx V. Drilling into big cancer-genome data. Nat Meth 2013;10:293–7.31. van Vliet M, Reyal F, Horlings H, van de Vijver M, Reinders M, Wessels L.

Pooling breast cancer datasets has a synergetic effect on classificationperformance and improves signature stability. BMC Genomics 2008;9:375.

32. van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AAM, Voskuil DW, et al.A gene-expression signature as a predictor of survival in breast cancer.N Engl J Med 2002;347:1999–2009.

33. HuC, Zhang S, Gao X, Gao X, Xu X, Lv Y, et al. Roles of the KRAB-associatedco-repressor KAP1 S473 phosphorylation in DNA damage response. J BiolChem 2012;287:18937–52.

34. Chang C-W, Chou H-Y, Lin Y-S, Huang K-H, Chang C-J, Hsu T-C, et al.Phosphorylation at Ser473 regulates heterochromatin protein 1 bindingand corepressor function of TIF1beta/KAP1. BMC Mol Biol 2008;9:61.

35. MurphyMP.Howmitochondria produce reactive oxygen species. BiochemJ 2009;417:1–13.

36. Fath MA, Ahmad IM, Smith CJ, Spence J, Spitz DR. Enhancement ofcarboplatin-mediated lung cancer cell killing by simultaneous disruptionof glutathione and thioredoxin metabolism. Clin Cancer Res 2011;17:6206–17.

37. Hadzic T, Aykin-BurnsN, Zhu Y, ColemanMC, Leick K, JacobsonGM, et al.Paclitaxel combined with inhibitors of glucose and hydroperoxide metab-olism enhances breast cancer cell killing via H2O2-mediated oxidativestress. Free Radic Biol Med 2010;48:1024–33.

38. KungH-N,Marks JR, Chi J-T.Glutamine synthetase is a genetic determinantof cell type–specific glutamine independence in breast epithelia. PLoSGenet 2011;7:e1002229.






39. Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC. MitofusinsMfn1 and Mfn2 coordinately regulate mitochondrial fusion and areessential for embryonic development. J Cell Biol 2003;160:189–200.

40. Wu L, Li Z, Zhang Y, Zhang P, Zhu X, Huang J, et al. Adenovirus-expressedhuman hyperplasia suppressor gene induces apoptosis in cancer cells. MolCancer Ther 2008;7:222–32.

41. Chen K-H, Dasgupta A, Ding J, Indig FE, Ghosh P, Longo DL. Role ofmitofusin 2 (Mfn2) in controlling cellular proliferation. FASEB J 2014;28:382–94.

42. Zhang G-E, Jin H-L, Lin X-K, Chen C, Liu X-S, Zhang Q, et al. Anti-tumoreffects of Mfn2 in gastric cancer. Int J Mol Sci 2013;14:13005–21.

43. Serasinghe Madhavika N, Wieder Shira Y, Renault Thibaud T, Elkholi R,Asciolla James J, Yao Jonathon L, et al. Mitochondrial division is requisiteto RAS-induced transformation and targeted by oncogenic MAPKpathway inhibitors. Mol Cell 2015;57:521–36.

44. Kashatus Jennifer A, Nascimento A, Myers Lindsey J, Sher A, ByrneFrances L, Hoehn Kyle L, et al. Erk2 Phosphorylation of Drp1 promotes

mitochondrial fission and MAPK-driven tumor growth. Mol Cell 2015;57:537–51.

45. Rehman J, Zhang HJ, Toth PT, Zhang Y, Marsboom G, Hong Z, et al.Inhibition of mitochondrial fission prevents cell cycle progression in lungcancer. FASEB J 2012;26:2175–86.

46. Zhao J, Zhang J, Yu M, Xie Y, Huang Y, Wolff DW, et al. Mitochondrialdynamics regulates migration and invasion of breast cancer cells. Onco-gene 2013;32:4814–24.

47. Birsoy K, Possemato R, Lorbeer FK, Bayraktar EC, Thiru P, Yucel B, et al.Metabolic determinants of cancer cell sensitivity to glucose limitation andbiguanides. Nature 2014;508:108–12.

48. Mishra P, Carelli V, Manfredi G, Chan David C. Proteolytic cleavage ofOpa1 stimulates mitochondrial inner membrane fusion and couplesfusion to oxidative phosphorylation. Cell Metab 2014;19:630–41.

49. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev DrugDiscov 2009;8:579–91.


Cheng et al.




2016;76:5006-5018. Published OnlineFirst June 30, 2016.Cancer Res Chun-Ting Cheng, Ching-Ying Kuo, Ching Ouyang, et al. Mitochondrial Fusion in Breast Cancer CellsMetabolic Stress-Induced Phosphorylation of KAP1 Ser473 Blocks

Updated version

10.1158/0008-5472.CAN-15-2921doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2016/06/30/0008-5472.CAN-15-2921.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/76/17/5006.full#ref-list-1

This article cites 49 articles, 18 of which you can access for free 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/76/17/5006To 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-15-2921

http://cancerres.aacrjournals.org/content/suppl/2016/06/30/0008-5472.CAN-15-2921.DC1

http://cancerres.aacrjournals.org/content/76/17/5006.full#ref-list-1

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/76/17/5006


metabolic stress-induced phosphorylation of kap1 ser473 blocks … · molecular and cellular...

Documents