calcineurina y cancer mama

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ORIGINAL ARTICLE

Mitochondrial retrograde signaling induces

epithelialmesenchymal transition and generates

breast cancer stem cellsM Guha1, S Srinivasan1, G Ruthel2, AK Kashina1, RP Carstens3, A Mendoza4, C Khanna4, T Van Winkle5 and NG Avadhani1

Metastatic breast tumors undergo epithelial-to-mesenchymal transition (EMT), which renders them resistant to therapies targeted

to the primary cancers. The mechanistic link between mtDNA (mitochondrial DNA) reduction, often seen in breast cancer patients,

and EMT is unknown. We demonstrate that reducing mtDNA content in human mammary epithelial cells (hMECs) activates

Calcineurin (Cn)-dependent mitochondrial retrograde signaling pathway, which induces EMT-like reprogramming to fibroblastic

morphology, loss of cell polarity, contact inhibition and acquired migratory and invasive phenotype. Notably, mtDNA reduction

generates breast cancer stem cells. In addition to retrograde signaling markers, there is an induction of mesenchymal genes but

loss of epithelial markers in these cells. The changes are reversed by either restoring the mtDNA content or knockdown of CnA a

mRNA, indicating the causal role of retrograde signaling in EMT. Our results point to a new therapeutic strategy for metastatic

breast cancers targeted to the mitochondrial retrograde signaling pathway for abrogating EMT and attenuating cancer stem cells,which evade conventional therapies. We report a novel regulatory mechanism by which low mtDNA content generates EMT and

cancer stem cells in hMECs.

Oncogene advance online publication, 4 November 2013; doi:10.1038/onc.2013.467

Keywords: mitochondrial DNA; mitochondrial retrograde signaling; epithelialmesenchymal transition; breast cancer; ESRP1;

cancer stem cells

INTRODUCTION

Mitochondria are the major sites of energy generation contribut-ing to B80% of cellular ATP in mammalian cells. They also have

an important role in maintaining calcium homeostasis andintegrating both intrinsic and extrinsic apoptosis pathways.1

Each mammalian cell contains 1001000 copies of mitochondrialDNA (mtDNA), which codes for 13 polypeptides that are essentialcomponents of the electron transport chain, in addition to rRNAsand tRNAs. All other mitochondrial proteins are coded by thenuclear genome.2 Therefore, a coordinated expression of bothgenomes is essential for mitochondrial biogenesis and function.Mitochondrial functions can be affected by defects in mtDNA,alterations in nuclear DNA-encoded mitochondrial proteins ordrug-induced mitochondrial membrane damage. Defects inmtDNA, including mutations and low mtDNA copy number,have been implicated in a wide range of disorders such asneurodegenerative disorders, mitochondrial myopathies, agingand cancer.3,4 Reduction in mtDNA copy number results in

disruption of electron transfer chain complexes and loss ofmembrane potential. As a consequence, there is an elevation ofcytosolic Ca2 levels and activationofcalcineurin (Cn)-mediatedretrograde signaling to the nucleus.57

MtDNA defects including deletions and point mutations havebeen reported in many human tumors.810 The progressiveaccumulation of mtDNA mutations and deletions observed insome tumors and the heteroplasmic mtDNA mutations reported

suggest that these perturbations are associated withtumorigenesis.11,12 Transmitochondrial cybrids in HeLa cells withhomoplasmic mtDNA mutations in the mtATP6 gene showed

increased cell growthin vitroand higher tumor incidence in nudemice.13

Reduction in mtDNA copy number has been observed in manycancers including hepatocellular carcinomas, astrocytomas, pros-tate and breast cancers.10,12,1416 Furthermore, chemically inducedmtDNA depletion in colorectal and prostate cancer cells promotesthe emergence of aggressive cancers, suggestinga causative roleof low mtDNA copy number in tumorigenesis.17,18 In support, miceheterozygous for the mitochondrial transcription factor A (TFAM),resulting in reduced mtDNA copy number, exhibit increasedtumor growth in the small intestine when crossed with theadenomatous polyposis coli multiple intestinal neoplasia mousemodel.19

In mammary carcinoma patients, mtDNA mutations and lowmtDNA copy number are associated with increased metastasis

and poor prognosis.12,16

At the onset of metastasis, mammarycarcinomas undergo epithelialmesenchymal transition (EMT), aprocess that involves genetic and phenotypic reprogramming ofepithelial cells to a predominantly mesenchymal phenotype andloss of cell polarity, cellcell and cellextracellular matrix adhe-sions. This transition enables some cells from the primary tumormass to migrate out, intravasate into the blood stream, survive inthe circulation, extravasate from the blood vessels, colonize and

1Department of Animal Biology and Marie Lowe Center for Comparative Oncology, School of Veterinary Medicine, Philadelphia, PA, USA; 2Penn Vet Imaging Core, School of

Veterinary Medicine, Philadelphia, PA, USA; 3Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 4Tumor and Metastasis

Biology Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA and 5Department of Pathobiology, School of Veterinary Medicine, Philadelphia, PA, USA.

Correspondence: Dr NG Avadhani, Department of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia 19104, PA, USA.

E-mail:nara [email protected]

Received 20 June 2013; revised 23 August 2013; accepted 13 September 2013

Oncogene (2013), 113

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form metastases at distant sites.20,21 The cellular reprogrammingin metastatic tumors renders them resistant to therapies targetedto the primary cancer and believed to contribute to the highmortality rates in breast cancer patients.22 Therefore, an increasedunderstanding of pathways that promote such reprogrammingevents is critical for designing therapeutic interventions againsttumor metastasis. Involvement of mtDNA defect in promotingbreast cancer metastasis was suggested in a study in which themetastatic potential of cancer cell line MDA-MB-231 was reversedby replacing its mtDNA with that from normal cells (mtDNAcybrid), while keeping the nuclear background unaltered.23

Even though low mtDNA copy number is reported in 6380%breast cancers,16 its contribution toward EMT and breast cancermetastases has not been previously explored. To investigate thecausal role of low mtDNA copy number in promoting EMT, weused two alternative models: one in which mtDNA content isselectively reduced by treatment with low doses of ethidiumbromide (EtBr), which does not affect nuclear DNA replication,24,25

and second in which mtDNA is depleted by genetic manipulationof TFAM. To delineate the contribution of reduced mtDNA copynumber in tumor initiation and metastatic progression throughEMT, we selected human mammary epithelial cells of non-carcinoma (MCF10A) and carcinoma (MCF7) origin. We showthat the reduction in mtDNA copy number in human mammaryepithelial cells activates a Cn-mediated mitochondrial retrogradesignaling that induces the process of EMT by upregulation ofmesenchymal gene expression, modulation of alternative splicingfactor Esrp1 and generation of breast cancer stem cells.

RESULTS

Mitochondrial respiratory stress induced by reduced mtDNA copynumber in mammary epithelial cells

We used 50 ng/ml of EtBr, which is the minimal concentrationrequired for partial depletion of mtDNA in these cells. Figure 1shows the mtDNA contents of MCF7 and MCF10A cells generatedby EtBr treatment for five passages. These cells will be referred toas mtDNA-reduced cells. Removal of EtBr from the growthmedium allowed for the recovery of mtDNA content to about7080% of the untreated parental cells (Figure 1a). These cells arereferred to as reverted cells. We assessed the relative mtDNAcopy numbers between parental MCF10A (normal mammaryepithelial) and MCF7 (mammary carcinoma epithelial) cells. MCF7cells contain B55% mtDNA copy number compared with that inparental MCF10A cells (Supplementary Figure S1A). It is importantto note that we have not observed any significant difference inthe amplification of the nuclear gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) between the two cell lines or therespective parental, mtDNA-reduced and reverted cell types(Supplementary Figure S1B), indicating that the nuclear genomecopy number remains unchanged.

The mitochondrial functions including oxygen consumptionrate (OCR) and glycolysis (extracellular acidification rate) in

parental, mtDNA-reduced and reverted MCF10A and MCF7 cellswere measured in an extracellular flux analyzer (SeahorseBioscience, Billerica, MA, USA). The complex V inhibitor oligomycinmarkedly inhibited mitochondrial respiration in parental andreverted cells, indicating an ATP-coupled mitochondrial respira-tion. In contrast, ATP-coupled respiration was reduced by morethan 70% in mtDNA-reduced cells compared with parental cells,indicating dysfunctional mitochondria (Figures 1b and c, andSupplementary Figures S1C and D).

Maximum uncoupled respiration is an indicator of thefunctional state of mitochondrial electron transport chain com-plexes. The difference between maximum uncoupled respirationand basal respiration, termed spare respiratory capacity, is used bycells under higher energy demands. Addition of uncouplerdinitrophenol (DNP) resulted in an increase in OCR to the maximal

capacity owing to uncoupling of respiration (Figures 1b and c). Inparental and reverted cells, DNP increased OCR by 120% overbasal rates, indicating a robust spare respiratory capacity. InmtDNA-reduced cells, DNP did not increase the OCR significantly,indicating that the spare respiratory capacity was significantlydiminished (Figures 1b and c). Moreover, in response tomitochondrial stress, cells frequently upregulate glycolysis tocompensate for decreased oxidative phosphorylation. Accord-ingly, mtDNA-reduced cells showed the highest rate of glucose-induced extracellular acidification rate (Supplementary FigureS1D). These results show that mtDNA-reduced MCF10A and MCF7cells exhibit loss of mitochondrial functions that were restored inreverted cells.

Another important indicator of healthy mitochondria is theabundance of filamentous mitochondrial network. Mitochondrialmorphology was examined by confocal fluorescence microscopyof cells stained with antibody to mtDNA-encoded CcO IVi1subunit. Parental MCF10A and MCF7 cells showed an abundantand highly filamentous mitochondrial network, whereas mtDNA-reduced cells had a markedly diminished network and mostlyfragmented mitochondrial particles. As expected, the revertedcells showed a more filamentous structure (Figures 1d and e). Wefurther assessed the mitochondrial transmembrane potential ofthe parental, mtDNA-reduced and reverted cells using a cell-permeant dye tetramethylrhodamine, ethyl ester that accumulatesin functional mitochondria while depolarized mitochondria fail tosequester this dye. As shown in Supplementary Figure S2A,mtDNA-reduced cells have B3045% lower membrane potentialcompared with the parental cells.

Reduction in mtDNA content induces cellular morphologicalchanges

Normal mammary epithelial cells are characterized by their well-organized architecture, polarized morphology, abundant cell

junction proteins, dense packing with negligible intercellularspace and attachment to the basement membrane, whereasepithelial mammary carcinoma cells lose these hallmark epithelialcharacteristics.26 Consistent with this, parental MCF10A cellsexhibit highly ordered cobblestone-shaped epithelial cellmorphology in two-dimensional (2D) culture conditions.However, mtDNA-reduced MCF10A cells lose this morphologyand adopt a spindle-shaped fibroblast-like appearance moretypical of mesenchymal cells (Figure 2a, top panel).

In three-dimensional (3D) cultures, normal mammary epithelialcells form glandular structures that resemble mammary acinifoundin vivo.26,27 Phase contrast images of parental MCF10A cellsgrown in 3D cultures show spherical, well-organized architecturetypical of normal mammary epithelial cells, whereas mtDNA-reduced MCF10A cells formed highly irregular spheroids invadinginto the surrounding matrigel. Reverted MCF10A cells formedspheroids similar to the parental cells (Figure 2a, bottom panel).Phalloidin staining of the 3D spheroids revealed that parental and

reverted cells form well-organized actin filaments forming acinarstructures compared with the mtDNA-reduced cells, which hadhighly irregular actin organization (Figure 2band SupplementaryFigure 3). The irregular 3D clusters of spheroids formed by mtDNA-reduced MCF10A cells are typical of highly invasive cells that havedisrupted the cytoskeletal organization.26,27 This supports the ideathat mitochondrial stress is a potential contributor to mammarycell invasion and metastasis.

MCF7 cells being carcinoma cells in origin do not organize intoa typical cobblestone pattern in monolayer 2D cultures. MtDNA-reduced MCF7 cells exhibited loss of contact inhibition and grewas piled-up colonies, a characteristic of metastatic carcinoma cells(Figure 2c, top panel). Parental MCF7 carcinoma cells grown in 3Dcultures formed organized spheroids that were dense andcompact and had an epithelium-like structure at the periphery

Mitochondrial stress induces EMT in hMECs

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with a necrotic core, which is consistent with their tumorigenicorigin (Figure 2c, bottom panel). MtDNA-reduced MCF7 cells, onthe other hand, formed multiple spheroids that were largelyirregular in architecture with no noticeable necrotic core and hadprotrusions into the matrigel layers consistent with thoseobserved in metastatic tumors.26 Reverted MCF7 cells moreclosely resembled the parental cells in morphology and growthpatterns. These results confirm that changes in morphology andgrowth patterns of these cells are directly related to reduction inmtDNA copy number. A similar alteration to fibroblast-likemorphology was also observed in mtDNA-reduced humanlung adenocarcinoma A549 cells, indicating the generalityof mitochondrial stress-induced morphological alteration(Supplementary Figure S6C).

Mitochondrial stress generates breast cancer stem cells with self-renewal capacity

During EMT, disseminated cancer cells acquire a self-renewalcapacity and stemness, which enable them to colonize distanttissues and form micro-metastases in vivo.20 We thereforeassessed the self-renewal potential of mtDNA-reduced MCF10Aand MCF7 cells in a serial-passaging sphere-forming assay(Figure 3, and Supplementary Figures S4A and B). For thispurpose, we grew the cells in serum-free medium in low-attachment dishes in mammosphere cultures and counted thespheres (termed as Gen1) formed in each cell type. MtDNA-reduced MCF10A and MCF7 cells had eightfold- and fivefold-higher sphere-forming capacity, respectively, compared with theirparental cells (Figures 3a and e). On disseminating these Gen1

Figure 1. Mitochondrial dysfunction in cells with reduced mtDNA content. (a) Relative mtDNA content analyzed by real-time PCRamplification of mtDNA-coded COX I and nuclear-coded COX IV after EtBr treatment in MCF10A (left) and MCF 7 (right). ( b, c) Cellularrespiration indicated as the OCR of parental, mtDNA-reduced and reverted MCF10A ( b) and MCF7 (c) cells measured by Seahorse XF24analyzer using 50 000 cells in each type. Coupled and maximal respiration were determined by sequential addition of oligomycin (2 mg/ml), 3,5DNP (75mM) and rotenone (1mM), respectively, as indicated in the figure. (d,e) Mitochondrial staining of MCF10A (d) and MCF7 (e) cells stainedwith cytochrome oxidase (CcoIVi1) antibody, and nuclei were stained with DAPI. Scale bar, 20mm.


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spheres and further replating them, we observed that mtDNA-reduced MCF10A and MCF7 cells formed markedly higher numberof spheres (termed as Gen 2) that were larger in size (Figures 3a, band eg). Notably, parental and reverted cells mostly grew asmonolayer cultures in Gen2, losing their sphere-forming capacity.Moreover, the spheroids formed by mtDNA-reduced cells survivedforX45 days, whereas those from the parental cells survived p10days. In addition, we observed a significantly higher number of

viable non-adherent floating population of cells in mtDNA-reduced MCF7 cells compared with the parental cells(Supplementary Figure S4A). These disseminated floater cellsfrom the mtDNA-reduced MCF7 mammosphere cultures formedlarger spheroids during serial passaging in 3D cultures, whereasthose from parental cells did not form such spheres. Our resultshere show that mitochondrial stress generates cells with thepotential for continuous self-renewal during serial passagingthrough generations.

We assessed the levels of cell surface markers CD4 4 and CD24that are altered in metastatic breast cancer stem cells.20,28 Proteinlevels of CD44 were markedly higher in mtDNA-reduced MCF10Acells compared with the parental and reverted cells(Supplementary Figure S4C). Flow cytometry analysis demon-strated that mtDNA-reduced MCF10A and MCF7 cells (Figures 3cand h, respectively) contain 10-fold-higher CD44/CD24- popula-tion, which are characteristic of breast cancer stem cells. More-over, 6070% of mtDNA-reduced MCF10A and MCF7 cells(Supplementary Figures S4D and E, respectively) express CD44,with only p10% cells expressing CD24. In contrast, parental cellshave only 1020% expression of CD24 and CD44. RevertedMCF10A and MCF7cells have CD44 and CD24 levels similar to theparental cells. This demonstrates that mitochondrial respiratorystress generates breast cancer stem cells with self-renewalcapacity. Immunostaining patterns of CD24 and CD44 in parental,mtDNA-reduced and reverted MCF10A and MCF7 cells (Figures 3dand i, respectively) further confirm the data from flow cytometryanalysis that mtDNA-reduced cells exhibit higher expression ofCD44 with correspondingly lower levels of CD24.

Acquired migratory potential, invasiveness and metastaticpotential in cells with reduced mtDNA content

Carcinoma cells undergoing EMT acquire an increased migratorycapacity that enables them to detach from the basementmembrane and invade other tissues to promote metastasis.20,21

We assessed the migratory capacity of MCF10A cells (Figures 4ad)as a function of metastatic potential by using an establishedscratch-wound healing assay. Unlike MCF7 cells, parental MCF10Acells grow as a confluent monolayer in 2D culture, which is ideal forthe collective cell migration assay. In this assay, MCF10A cells withreduced mtDNA content were highly motile and migrated into thescratched area within 10 h of plating, whereas the parental andreverted cells showed no migration during this time period. After20 h of plating, the mtDNA-reduced cells completely covered thescratched area, whereas the parental and reverted cells onlypartially migrated toward the cleared region (Figure 4aand time-lapse videos). Moreover, the migration patterns of the threeMCF10A cell types were distinct. The parental and reverted cellsmigrated only along the leading edge in an unidirectional orderedmanner, mostly owing to higher cell division, whereas those withmitochondrial stress were faster and had unorganized trajectorytypical of cells with high metastatic potential. These cells also

showed pseudopodia-like projections characteristic of metastaticcarcinoma cells (Figure 4a and time-lapse videos). The total path,overall displacement and the velocity of mtDNA-reduced MCF10Acells were increased significantly (Figures 4b and c). Windroseplots of cell movement, where all cell tracks are placed at thesame starting point, clearly demonstrated the markedly differentpattern of motion between parental and mtDNA-reducedcells (Figure 4d).

The capacity of cells to migrate across a growth-factor-reducedmatrigel basement membrane in vitro is an indicator of theirin vivo invasive potential.29 In agreement with previousreports,29,30 we observed the inability of parental MCF7 andMCF10A cells to invade the matrigel membrane in a transwellinvasion assay. However, mtDNA-reduced MCF7 and MCF10A cellsreadily invaded the Matrigel membrane within 24 h (Figure 4e).

Figure 2. Morphological changes in mtDNA-reduced MCF10A andMCF7 cells. (a, top panel) Representative bright-field images ofparental, mtDNA-reduced and reverted MCF10A cells grown in 2Dcultures. (a, bottom panel) Phase contrast images ( 20 magnifica-tion) of 3D spheroids formed by parental, mtDNA-reduced andreverted MCF10A cells in 2% matrigel. (b) Two-photon confocalimages ( 40 magnification) of Texas-Red-conjugated Phalloidin(left panel) and DAPI (center panel) staining of spheroids formed byparental, mtDNA-reduced and reverted MCF10A cells grown for 14days under 3D culture conditions using 2% matrigel. (c, top panel)Representative bright-field images ( 20 magnification) of parental,mtDNA-reduced and reverted MCF7 cells grown in 2D cultures.

MtDNA-reduced MCF7 cells showed loss of contact inhibition andformed piled up colonies. (c, bottom panel) Bright-field images( 20 magnification) of spheroids formed in 2% matrigel byparental, mtDNA-reduced and reverted MCF7 cells showingirregular spheres formed by cells with reduced mtDNA content.


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Moreover, the relative inability of reverted cells to invade theMatrigel membrane supports a direct link between mitochondrialrespiratory stress and increased invasiveness.

We assessed the metastatic potential of MCF10A and MCF7 cellsin an ex vivo pulmonary metastatic assay (PuMA) in the native 3Dcollagen network of the mouse lung. This assay mimics the tumorcell microenvironment and stromal interactions that occur in vivo

during metastasis.31 As shown inFigure 4f, at the initial time point(day 0), the mtDNA-reduced MCF10A and MCF7 epithelial cellshad homed-in the lung capillaries with very few parental(MCF10A) and reverted cells and had the histologic appearanceof normal human epithelial cells (larger nucleus:cytoplasmic ratio).Differences in the metastatic phenotype between the parental,mtDNA-reduced and reverted MCF7 and MCF10A cells became

Figure 3. Induction of cancer stem cells with self-renewal capacity by reducing mtDNA copy number. (aande) Number of spheres formed byparental, mtDNA-reduced and reverted MCF10A (a) and MCF7 (e) cells when grown on low attachment surface. The primary spheres (Gen1)were disintegrated by pipetting and replated further, and secondary spheres (Gen 2) formed by the parental, mtDNA-reduced and revertedcells were counted. (b) Bright-field images of mammospheres formed after 14 days in 3D culture (2% matrigel) by parental, mtDNA-reduced

and reverted MCF10A and MCF10A cells. Scale bar, 10 mm. (c, h) CD44 and CD24 expression levels in MCF10A (c) and MCF7 (h) parental,mtDNA-reduced and reverted cells quantified by flow cytometry analysis. (d, i ) Immunofluorescence images ( 40 magnification) showingexpression levels of CD44 and CD24 in parental, mtDNA-reduced and reverted mtDNA-MCF10A (d) and MCF7 (i) cells. Scale bar, 20 mm.(f) Bright-field images of spheres formed by parental, mtDNA-reduced and reverted MCF7 cells grown on low attachment surface for twogenerations. Parental and reverted cells lose their sphere-forming capacity in Gen2 and grow as monolayers. Scale bar, 10 mm. (g) Hematoxylinand eosin staining of the spheres formed by parental and mtDNA-reduced MCF7 cells as described in fabove. Scale bar, 10mm.


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evident by day 7 (Figure 4fand Supplementary Figure S5a) andbecame prominent for MCF10A cells by day 14 in PuMA, with thelung sections injected with mtDNA-reduced cells having numer-ous epithelial cells in the lumen of the capillaries of all sectionsexamined. However, in lung explants from mice injected withparental cells, there was rarely any cell in multiple sectionsexamined. Lung sections from mice injected with reverted cellshad fewer epithelial cells in the capillaries compared with mtDNA-reduced cells (Figure 4f and Supplementary Figure S5A). The

normal mouse lung epithelial cells can be seen lining thebronchioles (indicated by arrows, Supplementary Figure S5B).

Activation of mitochondrial retrograde signaling in humanmammary epithelial cells with reduced mtDNA content

Previously, we reported in C2C12 and A549 cells that mitochon-drial retrograde signaling in response to disruption of mitochon-drial membrane potential (Dcm) involves the activation of

Figure 4. Acquired migratory and invasive potential in epithelial cells with reduced mtDNA copy number. (a) Cell migration patterns ofparental, mtDNA-reduced and reverted MCF10A cells assessed by scratch-wound healing assay. Representative images shown here wereextracted at three time points (0, 10 and 20 h) from the time-lapse recordings (Supplementary Information, Supplementary movie file)captured at 5-min intervals for 20-h duration. (b,c) Individual cells in each category were tracked using the Volocity software (Perkin Elmer) toestimate the maximum velocity of their migration (left panel) and the maximum distance covered during the 20-h migration. Data areexpressed as an average of 10 cells tracked. (d) Winrose plot showing the directionality of migration of four representative MCF10A cells ineach category as indicated. Individual cells tracked are indicated by different colors. (e) Transwell Matrigel invasion patterns of parental,

mtDNA-reduced and reverted MCF10A (top panel) and MCF7 (bottom panel) cells after 24 h of plating. Cells were stained with Hematoxylinand Eosin and imaged under a bright-field microscope ( 10 magnification). (f) Lung sections from PuMA using parental, mtDNA-reducedand reverted MCF10A cells (top two panels) and MCF7 cells (bottom two panels) stained with hematoxylin and eosin. Arrows indicatethe human epithelial cells in the lumen of the capillaries. Images were taken at 40 magnification using a Nikon E600 microscope.Scale bar, 40mm.


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calcium-dependent phosphatase CnA.5,6,32,33 We therefore testedthe roleof this pathway by mtDNA depletion, which also disruptsDcm

6,34 in the activation of the EMT marker genes in breastepithelial cells. The parental MCF10A and MCF7 cells had low Cnactivity, whereas mtDNA-reduced MCF10A and MCF7 cells had a47-fold increase in Cn activity. The Cn activity in reverted cellswas similar to parental cells, confirming that the observed CnAactivation is in response to reduced mtDNA content (Figure 5a). Inaddition, we observed an increase in steady-state levels of CnAainMCF7 cells (Supplementary Figure S6A).

Some of the hallmarks of retrograde signaling include IGF1R, PI-3Kinase and Akt kinase activation, which are critical for thepropagation of the signaling cascade.32,35 We observed thatmtDNA-reduced MCF10A and MCF7 cells have more thanthreefold increased IGF1R (Figures 5b and c, top panels). Wereported that Akt1 is transcriptionally activated and expressed at a

higher steady-state level in response to mitochondrial retrogradesignaling.35 Notably, Akt1 kinase is an important driver of the EMTprocess.36 MtDNA-reduced MCF10A cells had fivefold-higherinduction of Akt, whereas MCF7 cells had only a modestincrease in Akt levels (1.5-fold). This modest increase overcontrol in the latter cell type is not surprising, as MCF7 cells arecarcinoma cells with higher endogenous Akt levels. We alsoobserved higher phospho-Akt (Ser 473) levels in mtDNA-reducedcells, indicating activation of Akt kinase in these cells (Figures 5band c). Notably, the IGF1R and Akt levels in reverted MCF10A andMCF7 cells are markedly lower than mtDNA-reduced cells. Toassess the functional relevance of higher IGF1R expression inmtDNA-reduced cells, we treated parental and mtDNA-reducedcells with a specific IGF1R inhibitor picropodophyllin. Inhibition ofthe IGF1 receptor resulted in a significant increase in cellularapoptosis in mtDNA-reduced MCF10A and MCF7 cells without anynoticeable effect in parental cells (Figures 5d and e), suggestingthat the IGF1R is essential for survival of mtDNA-reduced cells.

These results indicate that the key propagators of mitochondrialretrograde signaling pathway, Cn-IGF1R-Akt, are activated inmtDNA-reduced MCF10A and MCF7 cells.

Mitochondrial retrograde signaling induces nuclear genesinvolved in EMT

We further ascertained the causal role of mitochondrial retrogradesignaling in EMT by analyzing marker gene expression in MCF10Aand MCF7 cells. Reduction in mtDNA content in MCF10A cellscaused a 24-fold increase in transcription factors, Snail, Slug andTwist, whereas mtDNA-reduced MCF7 cells showed 520fold-higher mRNA levels for these factors, which are known totranscriptionally repress the epithelial cell adhesion markere-cadherin (Figures 6a and b). In agreement with these results,we observed that mtDNA-reduced MCF10A and MCF7 cellscontain low levels of mRNA for epithelial form of e-cadherin butfourfold-higher mRNA for the mesenchymal form ofN-Cadherin(Figures 6a and b). In mtDNA-reduced MCF10A and MCF7 cells, weobserved 12- and 5-fold upregulation of matrix metalloproteaseMMP-9, a key factor involved in breakdown and remodeling ofextracellular matrix during loss of cell-basement membraneattachment and extravasation (Figures 6a and b). In addition,the mesenchymal cell-specific extracellular matrix protein fibro-nectin and intermediate filament protein vimentin were upregu-lated in mtDNA-reduced MCF10A and MCF7 cells (Figures 6a andb, and Supplementary Figure S6B). Importantly, the geneexpression patterns in reverted cells were closer to the parentalcells, further supporting a causal role for the low mtDNA copynumber in phenotypic transition to mesenchymal state. Notably,reduction of mtDNA content by 20 or 50% had no appreciableeffect in induction of EMT genes (Supplementary Figure S7),suggesting that a certain level of mtDNA copy number reductionis necessary for inducing this transition process. We also observedan induction of genes involved in EMT of mtDNA-depleted human

lung adenocarcinoma A549 cells (Supplementary Figure S6D).To ascertain the role of mitochondrial retrograde signaling inEMT, we silenced CnAa (90% mRNA knockdown), which is a keyupstream mediator of the retrograde signaling pathway (Figures6c and d). We observed that silencing CnAa in parental MCF10Aand MCF7 cells did not have any significant effect on the levels ofmRNAs forN-Cadherin, VimentinandSnail. This is not surprising, asCnAa is not activated in parental cells, and therefore its mRNAsilencing has no effects on the downstream gene expression. Incontrast, silencing CnAa in mtDNA-reduced cells resulted in amarked decrease in the levels of all of these key mesenchymalgenes, whereas expression of scrambled siRNA had no effect. Wetherefore conclude that CnAa-mediated mitochondrial retrogradesignaling is a driver of the EMT in mtDNA-reduced MCF10A andMCF7 cells.

Figure 5. Induction of hallmarks of mitochondrial retrogradesignaling in mtDNA-reduced MCF10A and MCF7 cells. (a) Calci-neurin activity in parental, mtDNA-reduced and reverted MCF10Aand MCF7 cells. (b,c) Immunoblot showing IGF1 receptor, phospho-Akt (Ser473) and total Akt expression levels in total cell extracts(50mg) of parental, mtDNA-reduced and reverted MCF10A (b) andMCF7 (c) cells. Beta actin was used to assess equal protein loading.(d, e) Percentage of apoptosis in parental, EtBr-treated mtDNA-reduced and Tfam-silenced mtDNA-reduced MCF10A and MCF7cells treated with either DMSO or picropodophyllin.


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MtDNA copy number reduction modulates the expression of thesplicing regulator ESRP and alternative splicing of EMT genes

It is believed that induction of EMT is regulated by changes in theexpression of splicing regulators and alternatively spliced geneproducts.37 Consequently, the modulation of epithelial-specificsplicing regulatory proteins (ESRP), which are expressed only inepithelial cells and downregulated in mesenchymal cells, areimportant indicators of the EMT process.38,39 MtDNA-reducedMCF7 and MCF10A cells showed loss of ESRP1 expression,indicative of their mesenchymal state (Figure 6e). However, wedid not observe any difference in the levels of expression of theESRP2 isoform (data not shown). Finally, ESRP1 expression inreverted cells was similar to that in parental cells, confirming therole of retrograde signaling in the altered expression of this gene.

To ascertain the contribution of the altered expression of ESRP1in the mesenchymal transition of mtDNA-reduced cells, weectopically overexpressed EGFP-ESRP1 in mtDNA-reducedMCF10A and MCF7 cells. ESRP1 overexpression was confirmedby the nuclear GFP (green fluorescent protein) signal in EGFP-ESRP1-expressing cells, in contrast to the cytosolic GFP signal incells expressing the EGFP empty vector (negative control) (Figures6f and g top panels and Supplementary Figures S8A and B). Weobserved a reversal of expression of mesenchymal markersfibronectin, vimentinandN-cadherinwith a corresponding increasein the epithelial marker E-cadherin. These results suggest that themesenchymal phenotype in mtDNA-reduced cells is conferred, atleast in part, by reduced ESRP1 expression, possibly by altering thesplicing patterns of target genes.

Figure 6. Induction of EMT marker genes in mtDNA-reduced cells. (a, b) Real-time PCR analysis showing the mRNA levels of epithelial andmesenchymal marker genes involved in EMT in parental, mtDNA-reduced and reverted MCF10A (a) and MCF 7 (b) cells. (c,d) Real-Time PCRshowing relative mRNA levels of EMT marker genes in parental and mtDNA-reduced MCF10A (c) and MCF7 cells (d) expressing eitherscrambled siRNA or calcineurin Aa siRNA as indicated. Gapdh was used as endogenous control for normalization. (e) Immunoblot showingESRP1 expression levels in 50mg of total extracts from parental, mtDNA-reduced and reverted MCF10A and MCF7 cells. Actin is used as aprotein loading control. (f, g, top panel) Immunofluorescence images ( 40 magnification) showing mtDNA-reduced MCF10A cellsoverexpressing either pMXS-IRES- Puro- EGFP empty vector (GFP-EV) or pMXS-IRES-Puro-Emerald-2xFLAG-ESRP1 (GFP-ESRP1). (f, g, bottompanel) Real-time PCR showing relative mRNA levels (compared with parental cells) of EMT marker genes in mtDNA-reduced MCF10A (f) andMCF7 (g) ectopically expressing either the empty vector or ESRP1. Gapdh was used as endogenous control for normalization.


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ESRP1 modulates the expression of a wide array of genesinvolved in EMT progression by acting both as a negative andpositive splicing regulator.40 We analyzed the splicing patterns ofOSBPL3, ENAH and SLK, which are involved in EMT progressionand are known ESRP1 exon-inclusion targets (SupplementaryFigure S8C). We observed changes in expression of alternativelyspliced isoforms of the three genes in mtDNA-reduced MCF10Acells compared with the parental cells, confirming the role ofESRP1 in EMT.

Induction of retrograde signaling by shRNA-mediated depletionof TFAM

To ascertain the pathophysiological significance of our findingswith EtBr-mediated mtDNA depletion and its effect on EMT, weused a more pathophysiologically relevant approach of modulat-ing mtDNA copy number by shRNA-mediated silencing of TFAM(Figure 7). In both MCF10A and MCF7 cells, Tfam mRNA was

silenced to B90% by expressing specific Tfam shRNA comparedwith parental cells expressing GFP shRNA as a negative control(Figures 7a and f). As TFAM is critical for mtDNA packaging andmaintainence,41 silencing this factor resulted in a 7085% decreasein mtDNA content in both cell types (Figures 7b and g). Similar toEtBr-treated mtDNA-reduced cells, we observed that Tfam-silencedMCF10A cells had aberrant mitochondrial morphology with amarkedly reduced mitochondrial network (Figure 7c). Moreover,Tfam-silenced mtDNA-reduced MCF10A and MCF7 cells hadsignificantly lower maximal respiratory capacity compared withthe parental cells (Figure 7d). The dysfunctional state of mitochon-dria inTfam-silenced cells was further confirmed by growing cells inthe presence of galactose, a non-fermentable carbon source. Weobserved that Tfam-silenced cells have a higher dependence onglucose, as the carbon source and only a small percentage of cellssurvived in the presence of galactose (Supplementary Figure S9).

The key mediator of retrograde signaling, CnAa mRNA, wasincreased by 2- to 2.5-fold in Tfam-silenced MCF10A and MCF7

Figure 7. Induction of mitochondrial stress and EMT in TFAM-silenced cells. (a, f) Real-time PCR showing relative mRNA levels of TFAM andcalcineurin Aain MCF10A (a, left panel) and MCF7 (f) cells expressing shRNA against either GFP (negative control) or TFAM. ( a, right panel)CnA activity in Tfam-silenced MCF10A cells relative to GFPsh MCF10A cells. ( b,g) MtDNA content of MCF10A (b) and MCF7 (g) cells expressingTFAM shRNA compared with the negative control cells expressing GFP shRNA. (c) Mitochondrial staining of MCF10A cells using CcO IVi1antibody, and nuclei are stained with DAPI. (d) Cellular respiration indicated as the OCR of parental (GFP shRNA), Tfam-silenced MCF7 andMCF10A cells measured by Seahorse XF24 analyzer using 50 000 cells in each type. (e) Bright-field images ( 40 magnification) of MCF10Acells expressing shRNA against GFP or TFAM showing fibroblast-like morphology in TFAM shRNA-expressing cells. (h,i) Real-time PCR analysisshowing relative mRNA levels (compared with parental cells) of EMT marker genes in TFAM shRNA-expressing MCF10A (h) and MCF7 (i) cells.Gapdh was used as endogenous control for normalization.


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cells (Figures 7a and f). The CnAaactivity was induced by threefoldinTfam-silenced MCF10A cells compared with cells expressing GFPshRNA (Figure 7b).

Tfam-silenced cells acquired a fibroblast-like spindle-shapedmorphology, whereas those expressing GFP shRNA maintainedtheir characteristic epithelial features (Figure 7e). In addition, inTfam-silenced cells, the mesenchymal markers Vimentin andN-cadherin are markedly induced with a corresponding loss ofE-cadherin, indicative of EMT in these cells (Figures 7h and i).Similar to EtBr-treated mtDNA-reduced cells, Tfam-silenced cellswere also dependent on IGF1 receptor activation, and inhibition ofIGF1 receptor using picropodophyllin resulted in a markedincrease in apoptosis in these cells (Figures 5d and e). Theseresults demonstrate that a reduction in mtDNA copy number byTfamsilencing induces a retrograde signaling and EMT similar toEtBr-treated mammary epithelial cells.

DISCUSSION

Numerous studies support a role for mtDNA defects in a widerange of human cancers. Reduction in mtDNA copy number byincrease in ratios of mutant:wild-type mtDNA owing to time-dependent accumulation of mutant DNA or reduction of mtDNAcopy number owing to defective replication are hallmarks of age-related diseases. The latter has been shown to be a result ofmutations in mtDNA polymerase gamma, the mtDNA helicase

TWINKLE and defective TFAM.4,14,42

We demonstrate here that reducing mtDNA copy numberbelow 7080% either by EtBr treatment or by silencing TFAMinduces cellular reprogramming resembling EMT in humanmammary epithelial cells. The changes include morphological,metabolic and genetic changes that increase the metastaticpotential of tumorigenic MCF7 cells. Interestingly, mtDNA reduc-tion induces tumorigenic markers and promotes EMT in benignMCF10A cells. This is the first mechanistic report establishing thecausal role of mitochondrial retrograde signaling involving Cnactivation in transforming human mammary epithelial cells tohighly invasive phenotype by inducing mesenchymal genesfavoring EMT. EMT is a critical process for breast cancerprogression and metastasis, as it enables epithelial cells to invadeand metastasize. Self-renewal ability, similar to stem cells, is ahallmark of these metastasizing tumor cells.20,28 A key finding hereis that low mtDNA copy number generates breast cancer stemcells, which reportedly contribute to high rates of metastases.

We and others have reported that mitochondrial dysfunction,owing to mtDNA depletion (7080% reduction in mtDNA copynumber) and disruption of membrane potential, initiates a Ca2/Cn-mediated mitochondrial retrograde signaling pathway thatmodulates Ca2 -sensitive transcription factors and altersnuclear gene expression.57,14,17,33,34,43,44 Prior reports suggestthat mitochondrial dysfunction can be a contributor in tumorinduction.5,6,10,19,4549 This study supports a role for mitochondrialretrograde signaling in driving multiple stages of tumorigenesis,

from initiation to progression through EMT (Figure 8). Notably,using two different mitochondria-specific antioxidants, MitoQ andMitoCP, we confirmed that there was no significant mitochondrialreactive oxygen species produced in cells with reduced mtDNAcontent, suggesting that the retrograde signaling in these cells isnot mediated by reactive free radicals (Supplementary Figure S2B).

EMT can be induced by a wide range of cellular signalsinvolving many different pathways. A number of nucleartranscription factors such as Snail, Twist, Slug, FoxC1, TGFb and

Zeb have been reported to act as EMT inducers and metastasispromoters.50 Despite our current knowledge of a number ofnuclear transcription factors and cell adhesion proteins involved inthis transition, the mechanisms driving induction of the EMT arenot completely understood. The present study indicates thatmitochondrial retrograde signaling causes an upregulation of

some of the hallmark EMT-inducing signature factors, such asSnail, SlugandTwist, along with mesenchymal markersN-cadherin,matrix metalloprotease MMP-9, fibronectin and vimentin. This is alsothe first report showing that mitochondrial retrograde signaling isa modulator of epithelial splicing regulatory protein (ESRP)-1expression, which regulates the alternative splicing of a wide arrayof gene sets involved in inducing EMT.39 In addition, we show herethat mitochondrial retrograde signaling in human mammaryepithelial cells induces Akt kinase, which is reported to controlEMT-specific alternative splicing events by phosphorylatingSerine/Arginine-rich splicing regulator SRSF1.51 Together, ourresults suggest that mitochondrial retrograde signaling is anupstream effector of EMT, possibly by regulating the expression ofnuclear genes involved in cellular reprogramming.

We also demonstrate here that reduced mtDNA copy numberand resultant Cn-mediated mitochondrial retrograde signalinghave a causal role in driving EMT in human mammary epithelialcancer cells (Figure 8). Our result showing induction of EMT inmtDNA-reduced MCF10A and MCF7 cells is significant in view ofreports showing that 6380% of breast cancer patients exhibit lowmtDNA copy number and 30% ofpatients carry somatic mutationsin mtDNA D-Loop regions.16,49,52 Furthermore, breast cancer celllines lacking mtDNA (rho zero) showed decreased sensitivity tochemotherapeutic drugs such as doxorubicin, vincristine andpaclitaxel, suggesting that low mtDNA content is a possiblecontributor to the poor prognosis of breast cancer patients.53

Together, our results suggest that mtDNA copy number could be auseful prognostic marker, and mitochondrial stress-specific EMTmarker genes may represent useful therapeutic targets for thetreatment of metastatic breast cancer.

MATERIALS AND METHODS

Cell culture

MCF7 cells were grown in Eagles Minimum Essential Medium containing10% FBS, 0.1% penicillin/streptomycin and 0.01mg/ml insulin. MCF10Acells were grown in DMEM/F12 (1:1) containing 10% FBS, 0.1% 20 ng/mlEGF, 10mg/ml insulin, 100mg/ml hydrocortisone and 10ng/ml choleratoxin. MtDNA content was reduced (relative to the nuclear DNA) type byEtBr treatment (50ng/ml) for five passages and cells were grown in thepresence of 1 mM sodium pyruvate and 50 mg/ml uridine. MtDNA contentmeasured by Real-Time PCR using specific primers for mtDNA codedCytochrome Oxidase I and nuclear DNA coded either Cytochrome OxidaseIV gene or GAPDH gene. Three-dimensional cultures of MCF10A and MCF7cells were grown in 2% growth factor-reduced Matrigel, as describedearlier.26 Details of staining procedures for the 3D MCF10A colonies areprovided in the Supplementary Materials. For self-renewal assays, MCF10Aand MCF7 cells were grown in low-attachment dishes (Corning,Tewksbury,MA, USA) in serum-free growth medium, as described earlier.54 Details onthe gene manipulation experiments are presented in the Supplementarymethods. For assessing the role of IGF1R, cells were treated with IGF1receptor-specific inhibitor picropodophyllin (2.5 mM, 2 h), and cell viabilitywas measured using Guava Viacount (Millipore, Billerica, MA, USA) as perthe manufacturers protocol.

Cellular respiration

Oxygen consumption and extracellular rate of acidification was carried outin a XF24 Seahorse Analyzer (Seahorse Bioscience) using 5 104 cells.Oligomycin (2mg/ml), DNP (75 mM) and Rotenone (1mM) were addedsequentially as indicated in the figures.

Migration (scratch-wound healing) assay

Confluent monolayer of cells were scratched using a pipette tip, and cellsmigrating into this area were observed at 5-min intervals for 20 h under aninverted bright-field microscope. Still images were extracted at 0, 10 and20-h time points. For quantitative analysis, individual cells were trackedand their direction of movement, velocity and distance covered in thedirection of the wound were measured using Volocity software (PerkinElmer, Waltham, MA, USA).


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Matrigel invasion assay

In vitro invasion assays were carried out as described previously.33,35 Cells(5 104) were suspended in 500 ml of growth medium and seeded on topof the Matrigel layer. After 24h, invasive cells were stained withhematoxylin and eosin and observed with bright-field microscopy.

Pulmonary metastasis assay

The ex vivo evaluation of parental, mtDNA-reduced and reverted MCF10Aand MCF7 cells in mouse whole lung cultures was carried out using the(PuMA) as previously described and summarized in the SupplementaryMethods.31

Calcineurin activityCn activity was measured using a Cn activity assay kit (Enzo Life sciences,Farmingdale, NY, USA) according to the manufacturers instructions. Briefly,total phosphatase activity in 5 mg of cell lysates prepared from parental,mtDNA-reduced and reverted MCF10A and MCF7 cells was measured byincubating with Cn-specific RII phosphopeptide substrate in the presenceor absence of EGTA. Free phosphate released was measured usingmalachite green. Phosphatase activity measured in the presence of EGTAwas subtracted from total activity to obtain Cn-specific phosphataseactivity.

Flow cytometry analysis

CD44 and CD24 expression levels in all cell types were quantified by flowcytometry. Non-confluent cells were trypsinized into single-cell suspen-sions, counted and washed with phosphate-buffered saline. Cells were

stained for 15 min at room temperature using CD24-PE and CD44-APCantibodies (BD Biosciences, San Jose, CA, USA) and counted on a BDFACScalibur. Gating during analysis was set with unstained cells.

Statistical analysis

All assays were carried out in triplicate from three independentexperiments. Statistical analysis was carried out using Students t-test.Pvaluep0.001 (indicated as *) was considered highly significant.

ABBREVIATIONS

mtDNA, mitochondrial DNA; EMT, epithelial-to-mesenchymaltransition; EtBr, ethidium bromide; Cn, calcineurin; ESRP1,

epithelial splicing regulatory protein 1; TFAM, mitochondrialtranscription factor A.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

This research was supported by NIH grant CA-22762 and an Endowment from the

Harriet Ellison Woodward Trust to NGA. We thank Benjamin Cieply (Carstens

laboratory) for providing reagents for the ESRP1 experiments; Drs Brett Kaufman and

Jill Kolesar (Kaufman lab) for providing TFAM shRNA constructs; Dr Joseph Baur for

the GFP shRNA plasmid; Dr Mauricio Reginato and members of his laboratory for

sharing protocols and reagents for immunostaining of the MCF10A 3D spheres; and

Figure 8. Schematic outline showing that mtDNA copy number reduction in mammary epithelial cells activates Ca2 /Calcineurin-mediatedretrograde signaling, which leads to transcriptional activation of mesenchymal genes and induction of epithelialmesenchymal transition.


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Drs Christopher Lengner and Qihong Huang for valuable comments on the

manuscript. We thank Dr Leslie King for critical comments and editorial help.

AUTHOR CONTRIBUTIONS

MG and NGA designed research and wrote the paper; MG, SS and AK performed

the research; GR assisted with imaging studies; MG, AM and CK designed and

performed the PuMA experiments; TVM analyzed the histology slides.

REFERENCES

1 Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol 1988; 4:

289333.

2 Ryan MT, Hoogenraad NJ. Mitochondrial-nuclear communications. Annu Rev

Biochem2007; 76: 701722.

3 Greaves LC, Reeve AK, Taylor RW, Turnbull DM. Mitochondrial DNA and disease.

J Pathol2012; 226: 274286.

4 Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE et al. Mito-

chondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.

Science 2005; 309: 481484.

5 Amuthan G, Biswas G, Ananadatheerthavarada HK, Vijayasarathy C, Shephard HM,

Avadhani NG. Mitochondrial stress-induced calcium signaling, phenotypic chan-

ges and invasive behavior in human lung carcinoma A549 cells. Oncogene 2002;

21: 78397849.6 Biswas G, Adebanjo OA, Freedman BD, Anandatheerthavarada HK, Vijayasarathy C,

Zaidi Met al. Retrograde Ca2 signaling in C2C12 skeletal myocytes in response

to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle

crosstalk.EMBO J1999; 18: 522533.

7 Wallace DC. Mitochondria and cancer. Nat Rev Cancer2012; 12 : 685698.

8 Coller HA, Khrapko K, Bodyak ND, Nekhaeva E, Herrero-Jimenez P, Thilly WG. High

frequency of homoplasmic mitochondrial DNA mutations in human tumors can

be explained without selection. Nat Genet2001; 28: 147150.

9 Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A, Imanishi H et al.

ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.

Science 2008; 320: 661664.

10 Lee HC, Yin PH, Lin JC, Wu CC, Chen CY, Wu CW et al. Mitochondrial genome

instability and mtDNA depletion in human cancers.Ann N Y Acad Sci2005; 1042:

109122.

11 Fan W, Lin CS, Potluri P, Procaccio V, Wallace DC. mtDNA lineage analysis of

mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and

intermolecular recombination. Genes Dev2012; 26: 384394.

12 Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J et al. mtDNA

mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci USA 2005;

102: 719724.

13 Shidara Y, Yamagata K, Kanamori T, Nakano K, Kwong JQ, Manfredi G et al.

Positive contribution of pathogenic mutations in the mitochondrial genome to

the promotion of cancer by prevention from apoptosis. Cancer Res 2005; 65:

16551663.

14 Correia RL, Oba-Shinjo SM, Uno M, Huang N, Marie SK. Mitochondrial DNA

depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human

diffusely infiltrating astrocytomas. Mitochondrion 2011; 11: 4853.

15 Horton TM, Petros JA, Heddi A, Shoffner J, Kaufman AE, Graham Jr SDet al. Novel

mitochondrial DNA deletion found in a renal cell carcinoma. Genes Chromosomes

Cancer1996; 15: 95101.

16 Tseng LM, Yin PH, Chi CW, Hsu CY, Wu CW, Lee LM et al. Mitochondrial DNA

mutations and mitochondrial DNA depletion in breast cancer. Genes Chromo-

somes Cancer2006; 45: 629638.17 Guo J , Zheng L , L iu W, W ang X , Wang Z, Wang Z et al. Frequent

truncating mutation of TFAM induces mitochondrial DNA depletion and apop-

totic resistance in microsatellite-unstable colorectal cancer. Cancer Res 2011; 71 :

29782987.

18 Moro L, Arbini AA, Yao JL, di SantAgnese PA, Marra E, Greco M. Mitochondrial

DNA depletion in prostate epithelial cells promotes anoikis resistance

and invasion through activation of PI3K/Akt2. Cell Death Differ 2009; 16:

571583.

19 Woo DK, Green PD, Santos JH, DSouza AD, Walther Z, Martin WD et al.

Mitochondrial genome instability and ROS enhance intestinal tumorigenesis

in APC(Min/ ) mice. Am J Pathol2012; 180: 2431.

20 Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states:

acquisition of malignant and stem cell traits. Nat Rev Cancer2009; 9 : 265273.

21 Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in

development and disease. Cell2009; 139: 871890.

22 Schonberg MA, Marcantonio ER, Li D, Silliman RA, Ngo L, McCarthy EP. Breast

cancer among the oldest old: tumor characteristics, treatment choices, and sur-

vival. J Clin Oncol2010; 28: 20382045.

23 Imanishi H, Hattori K, Wada R, Ishikawa K, Fukuda S, Takenaga K et al. Mito-

chondrial DNA mutations regulate metastasis of human breast cancer cells. PLoS

One 2011; 6 : e23401.

24 Horwitz HB, Holt CE. Specific inhibition by ethidium bromide of mitochondrial

DNA synthesis in physarum polycephalum. J Cell Biol1971; 49: 546553.

25 King MP, Attardi G. Human cells lacking mtDNA: repopulation with exogenous

mitochondria by complementation. Science 1989; 246: 500503.26 Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-

10A mammary epithelial acini grown in three-dimensional basement membrane

cultures.Methods 2003; 30: 256268.

27 Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal

and malignant breast epithelial cells. Nat Methods 2007; 4 : 359365.

28 Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY et al. The epithelial-

mesenchymal transition generates cells with properties of stem cells. Cell 2008;

133: 704715.

29 Bae SN, Arand G, Azzam H, Pavasant P, Torri J, Frandsen TL et al. Molecular

and cellular analysis of basement membrane invasion by human breast

cancer cells in Matrigel-based in vitro assays. Breast Cancer Res Treat 1993; 24:

241255.

30 Gumireddy K, Sun F, Klein-Szanto AJ, Gibbins JM, Gimotty PA, Saunders AJ et al.

In vivo selection for metastasis promoting genes in the mouse. Proc Natl Acad Sci

USA 2007; 104: 66966701.

31 Mendoza A, Hong SH, Osborne T, Khan MA, Campbell K, Briggs Jet al. Modeling

metastasis biology and therapy in real time in the mouse lung. J Clin Invest2010;120: 29792988.

32 Guha M, Srinivasan S, Biswas G, Avadhani NG. Activation of a novel calcineurin-

mediated insulin-like growth factor-1 receptor pathway, altered metabolism, and

tumor cell invasion in cells subjected to mitochondrial respiratory stress. J Biol

Chem 2007; 282: 1453614546.

33 Guha M, Pan H, Fang JK, Avadhani NG. Heterogeneous nuclear ribonucleoprotein

A2 is a common transcriptional coactivator in the nuclear transcription response

to mitochondrial respiratory stress. Mol Biol Cell2009; 20 : 41074119.

34 Butow RA, Avadhani NG. Mitochondrial signaling: the retrograde response. Mol

Cell2004; 14: 115.

35 Guha M, Fang JK, Monks R, Birnbaum MJ, Avadhani NG. Activation of Akt is

essential for the propagation of mitochondrial respiratory stress signaling and

activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

Mol Biol Cell2010; 21: 35783589.

36 Larue L, Bellacosa A. Epithelial-mesenchymal transition in development and

cancer: role of phosphatidylinositol 3

0

kinase/AKT pathways. Oncogene 2005; 24:74437454.

37 Shapiro IM, Cheng AW, Flytzanis NC, Balsamo M, Condeelis JS, Oktay MHet al. An

EMT-driven alternative splicing program occurs in human breast cancer and

modulates cellular phenotype. PLoS Genet2011; 7 : e1002218.

38 Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP. ESRP1 and ESRP2 are

epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 2009; 33:

591601.

39 Warzecha CC, Jiang P, Amirikian K, Dittmar KA, Lu H, Shen S et al. An ESRP-

regulated splicing programme is abrogated during the epithelial-mesenchymal

transition. EMBO J2010; 29: 32863300.

40 Warzecha CC, Shen S, Xing Y, Carstens RP. The epithelial splicing factors ESRP1

and ESRP2 positively and negatively regulate diverse types of alternative splicing

events. RNA Biol2009; 6 : 546562.

41 Campbell CT, Kolesar JE, Kaufman BA. Mitochondrial transcription factor A reg-

ulates mitochondrial transcription initiation, DNA packaging, and genome copy

number. Biochim Biophys Acta 2012; 1819: 921929.

42 Sarzi E, Goffart S, Serre V, Chretien D, Slama A, Munnich Aet al. Twinkle helicase(PEO1) gene mutation causes mitochondrial DNA depletion.Ann Neurol2007;62:

579587.

43 Biswas G, Anandatheerthavarada HK, Avadhani NG. Mechanism of mitochondrial

stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12

myocytes. Cell Death Differ2005; 12 : 266278.

44 Guha M, Tang W, Sondheimer N, Avadhani NG. Role of calcineurin,

hnRNPA2 and Akt in mitochondrial respiratory stress-mediated transcription

activation of nuclear gene targets. Biochim Biophys Acta 2010; 1797:

10551065.

45 Kulawiec M, Safina A, Desouki MM, Still I, Matsui S, Bakin A et al. Tumorigenic

transformation of human breast epithelial cells induced by mitochondrial DNA

depletion. Cancer Biol Ther2008; 7 : 17321743.

46 Magda D, Lecane P, Prescott J, Thiemann P, Ma X, Dranchak PK et al. mtDNA

depletion confers specific gene expression profiles in human cells grown in cul-

ture and in xenograft. BMC Genomics 2008; 9 : 521.


M Guha et al

12



13/13

47 Naito A, Cook CC, Mizumachi T, Wang M, Xie CH, Evans TTet al. Progressive tumor

features accompany epithelial-mesenchymal transition induced in mitochondrial

DNA-depleted cells. Cancer Sci2008; 99: 15841588.

48 Tang W, Chowdhury AR, Guha M, Huang L, Van WT, Rustgi AK et al. Silencing

of IkBbeta mRNA causes disruption of mitochondrial retrograde signaling

and suppression of tumor growth in vivo. Carcinogenesis 2012; 33:

17621768.

49 Yu M, Zhou Y, Shi Y, Ning L, Yang Y, Wei Xet al. Reduced mitochondrial DNA copy

number is correlated with tumor progression and prognosis in Chinese breast

cancer patients. IUBMB Life 2007; 59 : 450457.50 Moreno-Bueno G, Portillo F, Cano A. Transcriptional regulation of cell polarity in

EMT and cancer. Oncogene 2008; 27: 69586969.

51 Blaustein M, Pelisch F, Tanos T, Munoz MJ, Wengier D, Quadrana Let al.Concerted

regulation of nuclear and cytoplasmic activities of SR proteins by AKT.Nat Struct

Mol Biol2005; 12: 10371044.

52 Xia P, An HX, Dang CX, Radpour R, Kohler C, Fokas E et al. Decreased mito-

chondrial DNA content in blood samples of patients with stage I breast cancer.

BMC Cancer2009; 9 : 454.

53 Yu M, Shi Y, Wei X, Yang Y, Zang F, Niu R. Mitochondrial DNA depletion promotes

impaired oxidative status and adaptive resistance to apoptosis in T47D breast

cancer cells. Eur J Cancer Prev2009; 18 : 445457.

54 Fillmore CM, Kuperwasser C. Human breast cancer cell lines contain stem-likecells that self-renew, give rise to phenotypically diverse progeny and survive

chemotherapy. Breast Cancer Res 2008; 10: R25.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)


M Guha et al

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