mitochondrial reprogramming regulates breast cancer ...pink1, pgc-1a, and mitochondrial...

Biology of Human Tumors

Mitochondrial Reprogramming RegulatesBreast Cancer ProgressionAnbarasu Kannan1, Robert B.Wells2, Subramaniam Sivakumar3, Satoshi Komatsu1,Karan P. Singh4, Buka Samten5, Julie V. Philley6, Edward R. Sauter7, Mitsuo Ikebe1,Steven Idell1,8, Sudeep Gupta9, and Santanu Dasgupta1

Abstract

Purpose: The goal of this study was to understand the role ofaltered mitochondrial function in breast cancer progression anddetermine the potential of the molecular alteration signature indeveloping exosome-based biomarkers.

Experimental Design: This study was designed to characterizethe critical components regulatingmitochondrial function inbreasttumorigenesis. Experiments were conducted to assess the potentialof these molecules for exosome-based biomarker development.

Results: We observed a remarkable reduction in spontaneousmetastases through the interplay in mitochondria by SH3GL2,vesicular endocytosis–associated protein and MFN2, an impor-tant regulator of mitochondrial fusion. Following its overexpres-sion in breast cancer cells, SH3GL2 translocated to mitochondriaand induced the production of superoxide and release of cyto-chrome C from mitochondria to the cytoplasm. These molecular

changeswere accompaniedbydecreased lung and livermetastasesandprimary tumor growth. SH3GL2 depletion reversed the abovephenotypic and associatedmolecular changes in nontumorigenicand tumorigenic breast epithelial cells. Loss of SH3GL2 andMFN2 expression was evident in primary human breast cancertissues and their positive lymph nodes, which was associatedwithdisease progression. SH3GL2 and MFN2 expression was detectedin sera exosomes of normal healthy women, but barely detectablein the majority of the women with breast cancer exhibitingSH3GL2 and MFN2 loss in their primary tumors.

Conclusions: This study identified a new mitochondriareprogramming pathway influencing breast cancer progressionthrough SH3GL2 and MFN2. These proteins were frequentlylost in breast cancer, which was traceable in the circulatingexosomes. Clin Cancer Res; 22(13); 3348–60. �2016 AACR.

IntroductionBreast cancer represents 14.0%of all new cancer cases and is the

second most common cause of cancer-associated morbidityamong the U.S. women (1, 2). In 2015, there will be an estimated231,840 cases and 40,290 deaths (2). Being highly heterogeneousand metastatic, breast cancer poses significant challenges toclinical management (1, 3). Early breast cancer detection has a

better chance of cure or prolonged disease-free survival comparedwith the metastatic disease (4). Although at least one progressionmodel of normal tissue to invasive cancer has been proposedusing cell morphology (5), the molecular drivers behind theinitiation and stage-wise progression of breast cancer are not wellcharacterized.

Continuous proliferation and apoptosis resistance are hall-marks of cancer cells (6). Abnormal mitochondrial function andreprogramming contribute to these hallmarks at least in part andhence are implicated in biomarker development (6, 7). Mito-chondrial fusion is a process of fusion of damaged mitochondriato healthy ones (6, 8). Studies suggest that production in tumorsof normal mitochondria could be tumor suppressive by promot-ing oxidative metabolism and enhanced reactive oxygen species(ROS) production (8). On the other hand, mitochondrial bio-genesis is a process involving replication of the mitochondrialgenome and coordinated expression of both nuclear and mito-chondria-encodedmolecules and assembly of the oxidative phos-phorylation complexes (6, 8, 9). Many factors, including MFN2,PINK1, PGC-1a, and mitochondrial transcription factor A (MT-TFA) play critical role in regulating mitochondrial fusion, bio-genesis, and maintaining mitochondrial integrity (6, 8, 9). Therole of mitochondrial fusion and biogenesis in breast cancerdevelopment and progression remains largely unknown.

Exosomes are 50 to 200 nm, small secreted endocytic vesiclespresent in all cell types andbodyfluids (10–12). Cancer exosomes(CE) carry survival information in the form of nucleic acids andproteins, shuttle constantly between the cancer cells throughthe circulation, and influence growth and progression (10–12).Characterizing the CEs and deciphering the cancer-promoting

1Department of Cellular and Molecular Biology, The University ofTexas Health Science Center at Tyler, Tyler, Texas. 2Department ofPathology, The University of Texas Health Science Center at Tyler,Tyler, Texas. 3Department of Biochemistry, Sri Sankara Arts andScience College, Kanchipuram, Tamil Nadu, India. 4University ofAlabama at Birmingham Comprehensive Cancer Center's Biosta-tistics and Bioinformatics Shared Facility, University of Alabama atBirmingham, Birmingham, Alabama. 5Department of Microbiologyand Immunology, The University of Texas Health Science Center,Tyler, Texas. 6Department of Medicine, The University of TexasHealth Science Center, Tyler, Texas. 7Department of Surgery, TheUniversity of Texas Health Science Center, Tyler, Texas. 8The TexasLung Injury Institute, The University of Texas Health Science Center,Tyler, Texas. 9Department of Medical Oncology, Tata Memorial Cen-ter, Mumbai, Maharashtra, India.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Santanu Dasgupta, The University of Texas HealthScience Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708. Phone: 903-877-7007; Fax: 903-877-7558; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2456

�2016 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 22(13) July 1, 20163348

on April 26, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 17, 2016; DOI: 10.1158/1078-0432.CCR-15-2456

http://clincancerres.aacrjournals.org/

information that they carry have tremendous potential for bio-marker and therapeutic development.

We identified SH3GL2, a vesicular endocytosis–associated pro-tein (13) as a potential breast cancer suppressor. When over-expressed in multiple human breast cancer cells, SH3GL2 trans-located to mitochondria and became phosphorylated. This wasaccompanied by enhanced mitochondrial fusion and expressionof mitochondrial fusion and biogenesis-associated proteinsMFN2, PINK1, and PGC-1a. A concomitant increase in superox-ide (O2

�) production and release of cytochrome C (CYTC) frommitochondria to the cytoplasmwas also evident. An elevated levelof growth-regulatory molecules PTEN, E-cadherin (CDH1), andATG5 was also evident following SH3GL2 overexpression. Thesemolecular changes were associated with reduced growth andprogression of the breast cancer cells in vitro and in vivo. Theorthotopically implanted SH3GL2-overexpressing xenograftsexhibited appreciable epithelial features, increased cell–cell adhe-rens, and reduced metastatic ability to the lung and liver. Thesecellular and molecular changes were reversed following SH3GL2silencing in nontumorigenic and tumorigenic breast epithelialcells. We also observed colocalization and coimmunoprecipita-tion of SH3GL2 with MFN2, PINK1, and PGC-1a in mitochon-dria. Frequent SH3GL2 andMFN2 loss in primary tumor and theirassociation with breast cancer progression was also evident.Compared with normal, the loss of the SH3GL2 and MFN2expression was detected in the sera exosomes of breast cancerpatients.

Materials and MethodsHuman tissue samples and ethical statement

Formalin-fixed paraffin-embedded (FFPE)–deidentified pri-mary breast cancer, matched lymph nodes, normal tissues alongwithmammoplasty tissueswere collected from theDepartment ofPathology, The University of Texas Health Science Center at Tyler(UTHSCT; Tyler, TX). Serum samples from healthy women orwomen with breast cancer were also collected from the Cooper-ative Human Tissue Network. All tissues and serum samples werecollected under an IRB-approved protocol. Relevant demographicdata were collected for necessary clinical correlation analysis. Thedemographic data of all the patients along with the expression

patterns of various altered molecules are represented in Supple-mentary Table S1A.

Cell cultureAuthenticatedMDA-MB-231, MCF-7, SUM-149, andMCF-10A

cells were purchased from ATCC and other suitable vendors andcultured as recommended. The HMLE cell line was kindly pro-videdbyDr.GuojunWu,Wayne StateUniversity (Detroit,MI). Allcell lines were periodically checked for mycoplasma contamina-tion using a Mycoplasma Detection Kit (Sigma # MP-0025; refs.14, 15). All tissue culture media and reagents were purchasedeither from ATCC or Invitrogen.

Antibodies and reagentsSH3GL2 antibody was obtained from Novus Biologicals

(# NBP1-8552). The E-cadherin (#3195P), PTEN (#9559S), P-threonine (#9381S), ATG5 (#2630S), LC3B (#2775S), b-actin(#3700), and ZO-1 (#8193) antibodies were purchased fromCellSignaling Technology. The MFN2 (#ab56889), MT-TFA(#ab119684), PGC-1a (#ab54481), IMMT (#ab110329), cyto-chrome C (#ab90529), and anti-mitochondria (MT-CO2,#ab3298) antibodies were obtained from Abcam Inc. The PINK1antibody (#LS-B3384) was purchased from LS Bioscience Inc.F-Actin antibody (#A12380) was obtained from Invitrogen Inc.Anti-mouse (#115-035-003) and rabbit (#111-035-003) second-ary antibodies were obtained from Jackson ImmunoResearch.Anti-mouse (#A11004) or anti-rabbit (#A11011) Alexa Fluor 568and anti-mouse (#11029) or anti-rabbit (#11008) Alexa Fluor488 secondary antibodies were purchased from Invitrogen. Mito-Tracker Red (#M224250) was obtained from Invitrogen. Far-redsecondary antibody (#A31573)was obtained fromThermoFisherScientific.

Lentiviral transduction of SH3GL2MCF-7, SUM-149, and MDA-MB-231 cells were transduced

with GFP-tagged lentivirus construct encoding SH3GL2(#LVP303171, Applied Biological Materials). A GFP-tagged emp-ty lentivirus construct with the same backbone (#LVP590) wasused as a control. Stable clones were selected in the presence ofpuromycin (10 mg/mL). A single stable clone was expanded andutilized for all subsequent analyses (14, 15).

In the knockdown studies, MCF-10A and MCF-7 cells weretransduced with a GFP-tagged lentivirus SH3GL2-SiRNA pool(#iV022230, Applied Biological Materials). The same lentivirusconstruct harboring scrambled siRNA was used as a control(#LVP015). Stable clones were selected in the presence of puro-mycin (1 mg/mL). A single stable clone was expanded and utilizedfor all subsequent analyses (14, 15). In both gain and loss-of-function studies, na€�ve control cells were used to examine theinfluence of the empty vector on SH3GL2 expression.

IHC and immunofluorescenceIHC and immunofluorescence (IF) analyses were performed as

described previously (14, 15). The IF Images were capturedthrough a Leica TCS SP8 laser-scanning confocal imaging station(Leica Microsystems). Quantitative imaging analysis to assess thecolocalization of the SH3GL2, CYTC, PGC-Ia, and MTCO2 wasperformed using "Intensity Correlation Analysis" function inLeica confocal software. The overlap coefficients generated byPearson correlation coefficient have values between þ1 and �1

Translational Relevance

Developing strategies for molecular early detection, mon-itoring, or surveillance of breast cancer may reduce disease-associatedmorbidity. In this study, we identified SH3GL2 andMFN2 as potential breast cancer suppressors. Frequent loss ofSH3GL2 and MFN2 was observed in primary and metastaticbreast cancer tissues, and their loss was found to be associatedwith disease progression. Functionally, their loss appeared tobe associated with mitochondrial reprogramming favoringtumorigenic progression. The simultaneous loss of SH3GL2and MFN2 was measured through the circulating exosomeprofiling in body fluids of women with invasive breast cancer.Measuring SH3GL2 and MFN2 expression in premalignantlesions or high-risk individuals and their timely profiling incirculating exosomes may improve current strategies for earlydetection, monitoring, and surveillance of breast cancer.

Mitochondria in Breast Cancer

www.aacrjournals.org Clin Cancer Res; 22(13) July 1, 2016 3349




MCF-7EV SH3GL2

EV SH3GL2

EV SH3GL2

SUM-149

MDA-MB-231

MDA-MB-231-SH3GL2 MDA-MB-231

50

40

30

20

10

0

50

40

30

20

10

0

6050403020100

EV SH3GL2

EV SH3GL2

EV SH3GL2

(*P = 0.0003)

(*P = 0.0001)

(*P = 0.0001)

MCF-7 SUM149 MDA-MB-231EV EV EVSH3GL2

SH3GL2

MFN2

PINK1

MT-TFA

IMMT

β-Actin

β-Actin

SH3GL2 SH3GL2

EV EV EVSH3GL2 SH3GL2 SH3GL2

86 kDa

40 kDa

14 kDa CYTC

β-Actin

CYTC

IMMT

14 kDa

84 kDa

45 kDa

45 kDa

86 kDa

50 kDa

29 kDa

84 kDa

45 kDa

50 kDa

29 kDa

45 kDa

MFN2

PINK1

MT-TFA

β-Actin

Mitochondrial lysate

MCF-7 SUM149 MDA-MB-231

Mitochondria

Cytosol

EV SH3GL2

MD

A-M

B-2

31

Total lysate

Mer

ge Mer

ge

GF

P-S

H3G

L2

SH

3GL2

Mito

trac

ker

CY

TC

A

B

F G

C

D

E

Kannan et al.

Clin Cancer Res; 22(13) July 1, 2016 Clinical Cancer Research3350




(þ1 and �1 values indicate that 100% and 0% of both compo-nents of the two images overlap, respectively).

Cell proliferation, invasion, and soft agar colony formationassays

Proliferation of the transduced cells (triplicate)was determinedby a BrdU incorporation assay (#B23151, Life Technologies;refs. 14, 15). Soft agar colony formation and invasion assays(#354481, Corning) were performed as described earlier (14, 15).In all cases, data were presented as mean � SE of duplicateexperiments.

Isolation of mitochondria and determination of O2�

productionMitochondria were isolated from cultured cells and tissues

using kits and protocols from Pierce (#89847 and #89801).O2

� production was measured using MitoSOX Red reagent(#M36008, Invitrogen).

Western blotting and coimmunoprecipitation analysisPreparation of whole-cell or mitochondrial lysates, Western

blotting analysis, and immunoprecipitation were performed fol-lowing protocols described earlier (14, 15).

Docking analysis of SH3GL2, MFN2, PINK1, and PGC-1aThe complete structure of SH3GL2,MFN2, PINK1, andPGC-1a

was predicted using I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/), and appropriate models were selected toperform the docking analysis on the ZDOCK server (http://zdock.umassmed.edu/).

Mitochondrial DNA content analysisGenomic DNA was isolated from the transduced cell lines

followed by qPCR analysis using primers from mitochondria-encodedND4 (MT-ND4) andnuclear-encodedGAPDH. The prim-er sequences are as follows:MT-ND4 (gene ID: 4538): forward (F)-ACTCACAACACCCTAGGCTC; reverse (R)-GCTTCGACATGGGC-TTTAGG.GAPDH(gene ID: 2597): F-TCCTCCACCTTTGACGCTG;R-ACCACCCTGTTGCTGTAGCC. A ratio of mitochondrialDNA/nDNA (MT-ND-4/GAPDH) was used to determine the foldchange among various groups.

In vivo xenograft and metastasis analysesFor tumor growth, 1 � 105 SH3GL2 and empty vector–trans-

duced cells in 1:1mixture of PBS andMatrigel were injected in themammary fat pad of 4- to 6-week-old, female NSGmice (CharlesRiver Laboratories; ref. 16). All experiments were performed inaccordance with the IACUC guidelines. Each group consisted of 8mice. Mice were examined every day, and mice showing any signof morbidity were immediately sacrificed according to the Uni-versity guidelines. All experiments were terminated at week 5 due

to the tumor burden. After 5 weeks, mice were sacrificed, andtumor weights were taken. Lungs and livers were removed for theanalysis of metastasis. Focal tumor nodules were counted in thelung and liver of all the mice from various groups. Tumors wereprocessed for histologic, Western blotting, and immunohisto-chemical analyses. All histopathologic and IHC evaluations of thein vivo tumors were done per pathologic guidance (14, 15). Datawere presented as mean � SE of duplicate experiments.

Exosome preparation from human sera and established cultureExosomes were isolated fromhuman sera or culture supernatant

using commercially available kits and protocols (#EXOQ5A-1 and# EXOTC10A-1, SystemBioscience), followed by protein isolation.Exosomes were treated with proteinase K before protein isola-tion (12).Western blotting analysis was performed using 40 mgof total exosome protein to detect SH3GL2 and MFN2 expres-sion. Syntenin was used as an exosome marker and loadingcontrol (14, 15).

Statistical analysisc2, Fisher exact, or Student t tests were used when appropriate.

AllP valueswere two sided, and all confidence intervalswere at the95% level. Computation for all the analyses was performed usingthe Statistical Analysis System.

ResultsSH3GL2 translocates to mitochondria, stimulatesmitochondrial apoptosis, and inhibits breast cancerprogression

A single study, so far, identified allelic loss of SH3GL2 in breastcancer tissues (17). However, the precise role of SH3GL2 in breastcancer has not yet been evaluated. We stably overexpressedSH3GL2 in three breast cancer cell lines MCF-7, SUM-149, andMDA-MB-231 (Supplementary Fig. S1A). The introduction ofSH3GL2 reduced proliferation (P ¼ 0.001–0.004), anchorage-independent growth (P ¼ 0.0002–0.0016), and invasion (P ¼0.0003–0.0006) of these breast cancer cells compared with thecontrol (Supplementary Fig. S1B–S1D). The SH3GL2-overexpres-sing cells also produced high level of O2

� (P ¼ 0.0001–0.0003; Fig. 1A). This was accompanied by an enhanced cyto-plasm/mitochondria ratio of CYTC expression (Fig. 1B). Releaseof CYTC frommitochondria to the cytoplasm is the central step inapoptosis (18). These results suggest for a possible interplaybetween SH3GL2 and mitochondria for triggering downstreamapoptotic signaling. In the SH3GL2 overexpressing cells, weobserved an increased distribution of mitochondrial fusion bod-ies (Fig. 1C), accompanied by enhanced expression of MFN2,PINK1, and MT-TFA (Fig. 1D). As MFN2, PINK1, and MT-TFApredominantly function in mitochondria (6, 8), it is likely thatSH3GL2 cooperates with them in themitochondria. We observed

Figure 1.SH3GL2 translocation to mitochondria induces ROS production and modulates mitochondria function. A, SH3GL2-overexpressing breast cancer cellsproduced significantly higher amount of superoxides (P ¼ 0.0001–0.0003), exhibited an enhanced expression of CYTC in the cytoplasm compared withmitochondria (B), and demonstrated increased numbers of mitochondrial fusion bodies compared with the empty vector–treated cells (C). D and E, enhancedexpression of MFN2, PINK1, and MT-TFA in the whole-cell or mitochondrial lysate of the SH3GL2-transduced groups compared with the empty vector–treated groups. EV, empty vector transduced; SH3GL2, SH3GL2-transduced cells. b-Actin and IMMT were used as cytosolic and mitochondrial loading controls.F, confocal imaging demonstrating colocalization of GFP-tagged SH3GL2 and MitoTracker Red–labeled mitochondria in MDA-MB-231 cells. Scale bar, 50 mm;magnification, 200� (A) and 400� (C and F). G, confocal imaging showing colocalization of endogenous SH3GL2 and mitochondria in the na€�veMDA-MB-231 cells (arrowheads). Scale bar, 20 mm; magnification, 400�.






SH3GL2 expression and associated increase inMFN2, PINK1, andMT-TFA expression inmitochondria (Fig. 1E).We did not observeany influence of the empty vector on SH3GL2orMFN2expressionin these cells (Supplementary Fig. S1E). The SH3GL2 constructhas a GFP tag, and IF analysis of the GFP-SH3GL2–expressingMDA-MB-231 cells labeled withMitoTracker Red confirmedGFP-SH3GL2 and mitochondrial colocalization (Fig. 1F). We alsoobserved colocalization of the endogenous SH3GL2 and mito-chondria in the na€�ve MDA-MB-231 cells (Fig. 1G). The overlapcoefficient value between SH3GL2 and mitochondrial markerCYTC (Fig. 1G) was 0.8936 (89% colocalization).

In the SH3GL2-overexpressing cells, we also observed anenhanced expression of PINK1 regulator PTEN (SupplementaryFig. S2A; ref. 19). The breast cancer lines that we used poorlyexpress CDH1, a regulator of cellular growth and metastasis(20, 21). We observed appreciable reversal of CDH1 expressionfollowing SH3GL2 introduction in these cells (SupplementaryFig. S2A). We also examined the expression of ATG5 and LC3B,proteins associated with mitophagy and apoptosis in these cells(22–25). An enhanced expression of ATG5, but not LC3B, exceptfor LC3B-I in MDA-MB-231 cells, was observed (SupplementaryFig. S2B).

SH3GL2 was physically associated with various proteins inmitochondria

The above mentioned results suggest that SH3GL2 mightphysically be associated with MFN2 and PINK1 in mitochondria.The IF analyses confirmed colocalization of SH3GL2 and MFN2,as well as SH3GL2 and PINK1, in these cells (SupplementaryFig. S2C–S2D). On the basis of the docking analysis, the trans-membrane domain and near coiled–coiled region of MFN2appears to interact with the SH3 domain of the SH3GL2 protein(Fig. 2A and Supplementary Fig. S2E; Supplementary TableS1B). On the other hand, the amino acid residues 34–41 and255–263 of PINK1 seem to participate in the association withSH3GL2 (Fig. 2B and Supplementary Fig. S2F; SupplementaryTable S1C). We then performed coimmunoprecipitation anal-ysis using lysates prepared from intact mitochondria, assumingthat they were physically associated in mitochondria. We couldpull down MFN2 or PINK1 with SH3GL2 and vice versa inmitochondria (Fig. 2C and D).

Other than MT-TFA, PGC-1a also plays a key role in mito-chondrial biogenesis (26). We observed enhanced expression ofPGC-1a in the SH3GL2-overexpressing cells (Fig. 2E). Augmentedexpression of PGC-1a was confirmed in mitochondria (Fig. 2F).This observation suggests that SH3GL2 may interplay with PGC-1a inmitochondria as well. The confocal imaging analysis using amitochondria-specific marker (MTCO2) further demonstratedtheir colocalization in the mitochondria of the SH3GL2-over-expressing MDA-MB-231 cells (Fig. 2G). The values of overlapcoefficient between SH3GL2 and MTCO2 were 0.8782; PGC-Iaand MTCO2: 0.6558; and SH3GL2 and PGC-1a: 0.6603, whichconfirmed their colocalization. On the other hand, dockinganalysis predicted two interacting regions, namely amino acid190–204 and 627–637 in PGC-1a for SH3GL2 and PGC-1ainteraction (Fig. 2H; Supplementary Table S1D). Through coim-munoprecipitation and Western blotting, we could pull downSH3GL2 with PGC-1a and vice versa in the mitochondria of theSH3GL2-overexpressing MDA-MB-231 cells (Fig. 2I).

The above results raised our interest to determine whetherSH3GL2 is phosphorylated in mitochondria. Bioinformatic anal-

ysis predicted several serine, threonine, and tyrosine phosphor-ylation sites in the BAR and SH3 domains of SH3GL2 (Supple-mentary Fig. S3A). The phosphorylation potential appeared to behigher for serine and threonine residues (SupplementaryFig. S3B). Several serine and threonine residues are conservedamong different species (Supplementary Fig. S3C and S3D).Next,we performed Western blotting analysis with an anti-phospho-threonine antibody using SH3GL2 protein immunoprecipitatedfromwhole cells or mitochondrial lysates of SH3GL2-overexpres-sing cells. Phosphothreonine-positive SH3GL2 was detected inthe total and mitochondrial lysates prepared from the SH3GL2-transduced cells (Fig. 3A). To compare SH3GL2 phosphorylationspecifically in mitochondria of both empty vector and SH3GL2-overexpressing cells, we performedWestern blotting analysis withthe same anti-phosphothreonine antibody. Phosphothreonine-positive SH3GL2 was detectable inmitochondria of the SH3GL2-overexpressing cells but barely detectable, or undetectable, in theempty vector–treated cells (Fig. 3B).

Depletion of SH3GL2 promotes progression ofnontumorigenic breast epithelial cells

To assess whether altered SH3GL2 expression was necessary toprevent growth and progression, we stably silenced SH3GL2 innontumorigenic breast epithelial cells MCF-10A (Fig. 3C). Appre-ciable depletion of SH3GL2 in these cells markedly reduced theexpression of MFN2, PINK1, PTEN, and CDH1 (Fig. 3C) mole-cules, which were induced following SH3GL2 overexpression inthe breast cancer cell (Fig. 1 and Supplementary Fig. S2A). Thesecells exhibited a high mitochondria/cytoplasm ratio of CYTCexpression (Fig. 3D). This was accompanied by amarked increasein cellular proliferation (P ¼ 0.0007), anchorage-independentgrowth (P ¼ 0.0003), and invasion (P ¼ 0.0003; Fig. 3E–G).Moreover, reduction (P ¼ 0.0003) in O2

� production was alsoevident in the SH3GL2-depletedMCF-10A cells (Fig. 3H).We alsosilenced SH3GL2 in tumorigenicMCF-7 cells. Similar toMCF-10Acells, SH3GL2-silenced MCF-7 cells demonstrated a decreasedexpressionofMFN2, PINK1, andCDH1, accompaniedby ahighermitochondria/cytoplasm ratio of CYTC expression (Supplemen-tary Fig. S4A and S4B). An increase in proliferation (P¼ 0.0001),invasion (P ¼ 0.0002), and concomitant decrease in O2

� pro-duction (P ¼ 0.0001) were also noted in these cells (Supplemen-tary Fig. S4C–S4E). The empty vector had negligible influence onSH3GL2 or MFN2 expression in MCF-10A cells (SupplementaryFig. S4F).

Mitochondrial SH3GL2 halted primary tumor growth anddistant metastases in vivo

We next evaluated the impact of SH3GL2 overexpression on invivo growth and progression using an orthotopic breast cancermodel (16). SH3GL2 or empty vector–transduced MDA-MB-231cells were implanted in the mammary fat pad of 4- to 6-week-oldfemale NSG mice (N ¼ 8/group). Mice were sacrificed at week 5due to the tumor burden, and average tumor weights were taken.Mean tumor weight was lower (P ¼ 0.001) in the SH3GL2-overexpressing group compared with the control (Fig. 4A). TheGFP-SH3GL2–overexpressing breast cancer cells appeared moreepithelial and differentiatedwith notable cell–cell adherence (Fig.4B).The control GFP-empty vector–expressing cells appearedmore mesenchymal with disrupted cell–cell contact representingthe poorly differentiated features of the parental MDA-MB-231

Kannan et al.






105 kDa PGC1α PGC1α

IMMTβ-Actin

β-Actin

IB:SH3GL240 kDa

IB:PGC1α105 kDa

IB:PGC1α105 kDa

IB:SH3GL240 kDa

45 kDa

105 kDa

45 kDa

84 kDa

Total lysate


SUM149MCF-7 MCF-7MDA-MB-231EV EV EVSH3GL2 SH3GL2 SH3GL2

SUM149 MDA-MB-231

MDA-MB-231-SH3GL2

MDA-MB-231-SH3GL2

MDA-MB-231-SH3GL2

IP:M

FN

2

IP:M

FN

2IP

:PG

C1α

IP:S

H3G

L2

IP:S

H3G

L2

IP:S

H3G

L2

IB:SH3GL2

Mitochondrial protein

A B

C D

E

G H

I

F

Mitochondrial protein

IB:MFN2

IB:MFN2

IB:SH3GL2

40 kDa

IB:SH3GL240 kDa

86 kDa

86 kDaIB:PINK150 kDa

IB:PINK150 kDa

40 kDa IB:SH3GL240 kDa

Input (5%) Input (5%)IgG IgG

SH3GL2 SH3GL2

MFN2 PINK1

SH3GL2

PGC1

SH3GL2 MTCO2

PGC-1α MERGE

Lysate Lysate


Input (5%) IgG Lysate

MDA-MB-231-SH3GL2

Figure 2.SH3GL2 is physically associatedwithMFN2, PINK1,and PGC-1a. A and B, docking analysispredicted several interacting sites betweenSH3GL2, MFN2, and PINK1 (arrows). C and D,SH3GL2 was coimmunoprecipitated with MFN2and PINK1 and vice versa using total proteinsprepared from intact mitochondria. IgG was usedas a control. E and F, enhanced PGC-1a expressionin the whole-cell or mitochondrial lysate of theSH3GL2-overexpressing breast cancer cellscompared with the control. EV, empty vector;SH3GL2, SH3GL2-transduced cells. b-Actin andIMMT were used as cytosolic and mitochondrialloading controls. G, colocalization of PGC-1a(cyan), SH3GL2 (green), andmitochondria (red) inthe SH3GL2-overexpressing MDA-MB-231 cells(arrowheads). Scale bar, 20 mm; magnification,400�. H, SH3GL2 and PGC-1a docking complex(arrows) as predicted by bioinformatic analysis.I, coimmunoprecipitation of SH3GL2 and PGC-1aand vice versa in mitochondria of the SH3GL2-overexpressing MDA-MB-231 cells. IgG was usedas a control. IP, immunoprecipitation; IB,immunoblotting.






cells (Fig 4B). The expression and distribution pattern of ZO-1,CDH1, and F-actin proteins in these tissues further support thisobservation (Fig. 4C). Due to the restoration of epithelial phe-notype following SH3GL2 overexpression, the fluorescence sig-nals of cortical actin were also enhanced in the SH3GL2-trans-duced cells (Fig. 4C). The extent of lung and livermetastases of theSH3GL2-overexpressing MDA-MB-231 cells also decreased. Thenumber of visible lung tumor nodules was lower (P¼ 0.0001) inthe SH3GL2-overexpressing group compared with the control

(Fig. 4D). Histologic analysis of lung tissues from multiple micerevealednumerous largemacroscopic tumor foci in the lungof thecontrol group (Fig. 4E). However, in the SH3GL2-overexpressinggroup, small and relatively few micrometastases were noted (Fig.4E). Macroscopic tumor nodules and histologic metastases of theMDA-MB-231 cellswere noted in the liver of the controlmice (Fig.4F and G). However, no visible tumor nodule or metastaticinfiltration was observed histologically in the liver of theSH3GL2-overexpressing group (Fig. 4F and G).

IP:SH3GL2

IB:P

-Threonine

IB:P

-Threonine

IB:S

H3G

L2

IB:S

H3G

L2

IP:SH3GL2

200150

100

37

37

2015

10

200150100

75

75

50

50

25

20

1510

C C

40 kDa SH3GL2

0C KD

C KD

C KD

20

40

0

01020304050607080

20

30

10

40

C KD0

40

60

20

Mea

n nu

mbe

r of

col

onie

sM

ean

num

ber o

f inv

aded

cel

lsM

ean

num

ber o

f pos

itive

cel

ls

60(*P = 0.0007)

(*P = 0.0003)

(*P = 0.0003)

(*P = 0.0003)

Mea

n nu

mbe

r of

Brd

U-p

ositi

ve c

ells

MFN2

PINK1

CDH1

PTEN

β-Actin

β-Actin

β-Actin

CYTC

CYTC

IMMT

86 kDa

50 kDa

135 kDa

54 kDa

45 kDa

Total lysate

Cytosol

Mitochondria

45 kDa

45 kDa

84 kDa

14 kDa

14 kDa

KD KD

25

200150

100

37

37

2015

10

200

150100

75

75

50

50

25

201510

25

Total protein

A

C

D

E

F

G

H

B

Mitochondrial proteinMitochondrial protein

MCF-7EV EV EVGL2 GL2 GL2

SUM-149 MDA-MB-231

Figure 3.SH3GL2 is phosphorylated inmitochondria, and its silencing invokesgrowth and progression ofnontumorigenic breast epithelial cell. A,detection of phosphorylated SH3GL2 inwhole-cell or mitochondrial lysate ofSH3GL2-overexpressing cells using aP-threonine antibody. B, enhancedP-threonine–positive SH3GL2 signaldetection in mitochondria of theSH3GL2-overexpressing cells, whichwas low or barely detected in theempty vector–treated groups. EV,empty vector; GL2, SH3GL2transduced. C, silencing of SH3GL2 innontumorigenic breast epithelialMCF-10A cells markedly reduced theexpression of MFN2, PINK1, CDH1, andPTEN. D, mitochondria/cytoplasm ratioof CYTC was higher in the SH3GL2-depleted cells compared with theempty vector–treated cells. E–G,increased proliferation (P ¼ 0.0007),anchorage-independent growth(P ¼ 0.0003), and invasion (P ¼0.0003) of the SH3GL2-depleted MCF-10A cells compared with the emptyvector–treated control. H, low O2

�

production (P ¼ 0.0003) by theSH3GL2-depleted cells compared withthe control scrambled SiRNA-treatedcells. C, control SiRNA–treated cells;KD, SH3GL2-specific SiRNA-treatedcells. b-Actin and IMMT were used ascytoplasmic and mitochondrialloading controls, respectively. Scalebar, 50 mm; magnification, 200�.IP, immunoprecipitation; IB,immunoblotting; BrdU,bromodeoxyuridine.

Kannan et al.





Immunohistochemical analysis of the in vivo FFPE tissuesrevealed enhanced expression of SH3GL2 (P ¼ 0.0002) andCDH1 (P ¼ 0.01) in the SH3GL2-overexpressing MDA-MB-231cells compared with the control (Supplementary Fig. S5A).Increased CDH1 expression was further confirmed by Westernblotting analysis in SH3GL2-overexpressing tumor tissues frommultiple mice (Supplementary Fig. S5B). Enhanced expression ofSH3GL2, MFN2, and PINK1 was also confirmed in mitochondriaof SH3GL2-overexpressing implants from multiple mice com-

pared with the control by Western blotting analysis (Supplemen-tary Fig. S5C).

SH3GL2 andMFN2 expression is frequently lost and associatedwith breast cancer progression

The expression pattern of SH3GL2 in paired (normal/tumormatched) breast cancer tissues has not yet been evaluated. Weexamined SH3GL2 expression pattern in 51 primary humanbreast cancer tissues with and without lymph node metastasis

A

D F

GE

B CS

H3G

L2E

mpt

y ve

ctor

Number of mice

Tum

or w

eigh

t (m

g)

00

0.20.40.60.8

11.21.41.61.8

2 4 6 8 10

SH3GL2

SH3GL2 SH3GL2

Empty vector

Empty vectorEmpty vector

SH3GL2Empty vector

F-A

ctin

F-A

ctin

ZO

-1

CD

H1

SH3GL2

SH3GL2 SH3GL2Empty vector Empty vector Empty vector

SH3GL2Empty vector

Mou

se 1

Mou

se 2

Mou

se 3

Mou

se 1

Mou

se 2

Mou

se 3

Number of mice examinedNumber of lung examined

Num

ber

of n

odul

es

Num

ber

of n

odul

es

00

1

2

3

4

5

6

2 4 6 8 10 12

30

25

20

15

10

5

00 5 10 15 20

Figure 4.SH3GL2 overexpression reduced primary growth and metastases of MDA-MB-231 cells in vivo. A, mean tumor weight was significantly lower (P ¼ 0.001) in theSH3GL2-overexpressingmice group comparedwith the control group. Empty vector, cells transducedwith the empty vector; SH3GL2, cells transducedwith SH3GL2.B, epithelial-like appearance of the GFP-SH3GL2–overexpressing MDA-MB-231 mammary implants, with considerable cell–cell contact (arrowheads) comparedwith the GFP-empty vector–treated group exhibiting pronounced disruption of the epithelial morphology and cell–cell adhesion. C, IF analysis of the SH3GL2-overexpressing xenografts with ZO-1, CDH1, and F-actin demonstrates pronounced epithelial-like appearance and cell–cell adhesion (arrowheads) compared withthe control group. Scale bar, 20 mm; magnification, 400�. D and E, the number of visible lung tumor nodules (encircled and scatter plot) and histologicmacrometastases (arrows) were markedly lower (P ¼ 0.0001) in the SH3GL2-overexpressing group compared with the empty vector–treated group. F andG, visible tumor nodules and histologic macrometastases (encircled and scatter plot) were detected only in the liver of the empty vector–treated group, butnot in the SH3GL2-overexpressing group at 5 weeks. Scale bar, 50 mm; magnification, 200� (B, D–G).






(LNM; Supplementary Table S1A). Overall, loss of SH3GL2 wasdetected in 61% (31/51, P ¼ 0.001–0.003) of primary breastcancer tissues (Fig. 5A). Thirty four of these 51 cases had LNM(Table S1A). SH3GL2 loss was detected in 79% (27/34, P ¼0.001–0.002) of the primary tumors positive for LNM. Corre-sponding positive lymph node tissues were available for 20 of 51

cases (Supplementary Table S1A). Ninety percent (18/20) of theLNM tumors had a loss (P ¼ 0.003–0.004) of SH3GL2.

To our knowledge, the expression pattern of MFN2 and itsassociation with breast cancer progression is unknown. The samecohort of 51 patients described above was analyzed to determineMFN2expression (Supplementary Table S1A).WedetectedMFN2

SH3GL2

MFN2

Pat

ient

1P

atie

nt 2

Pat

ient

2P

atie

nt 1

C E F G

B

A

D

N

N

IDC LNM

IDC LNM

N IDC LNM

N IDC LNM

IA I

III

IIIIA

IIB

IIIA

IIIC

No

No No

Yes

YesYes

NSC NSCNSC NSC NSC

SL SLSL SL SL

Stage (P = 0.003) Grade (P = 0.009)Lymph node metastasis (P = 0.0002) Lymph node metastasis (P = 0.016) Lymph node metastasis (P = 0.007)

TT

Figure 5.SH3GL2 and MFN2 expression wasfrequently lost and associated withbreast cancer progression. A, SH3GL2expression was significantly low(P¼ 0.001–0.003) in primary invasivebreast cancer and corresponding LNMtissues compared with matchednormal. Representative examplesfrom two patients were shown. B, theexpression level of MFN2 was alsosignificantly lower (P¼0.002–0.004)in primary invasive breast cancer andcorresponding LNM tissues comparedwith matched normal. Representativeexamples from the same two patientswere shown. N, normal tissues; IDC,invasive ductal carcinoma; T: areacontaining tumor cells. Magnification,200�. C–E, loss of SH3GL2 expressionwas associatedwith stage (P¼0.003,C), grade (P ¼ 0.009, D), and LNM(P ¼ 0.0002, E). F, MFN2 loss wasassociated with lymph nodemetastasis (P¼ 0.016). G, loss of bothSH3GL2 and MFN2 was associatedwith LNM (P ¼ 0.007). Grade I, welldifferentiated; grade II, moderatelydifferentiated; grade III, poorlydifferentiated; no, negative for LNM;yes, positive for LNM; NSC, nosignificant change; SL, significant loss.

Kannan et al.





loss in 55% (28/51, P ¼ 0.002–0.004) of primary breast cancertissues (Fig. 5B). MFN2 loss was detected in 65% (22/34, P ¼0.0003–0.001) of the primary tumors positive for LNM. Eightypercent (16/20) of the available LNM from these patients exhib-ited loss (P ¼ 0.001–0.002) of MFN2. Notably, loss of coexpres-sion of SH3GL2 and MFN2 was detected in 37% (19/51) cases(Supplementary Table S1A). Of the 34 LNM cases, loss of theircoexpression was evident in 47% (16/34) cases (SupplementaryTable S1A). Thirteen of 20 LNM tumors (65%) had a simulta-neous loss of SH3GL2 and MFN2 (Supplementary Table S1A).Loss of SH3GL2was associated with stage (P¼ 0.003), grade (P¼0.009), and LNM (P ¼ 0.0002; Fig. 5C–E). Loss of MFN2 expres-sion was associated with LNM alone (P ¼ 0.016; Fig. 5F) or incombination with SH3GL2 (P ¼ 0.007; Fig. 5G). No associationwas found between MFN2 loss and stage (P ¼ 0.028; Supple-mentary Fig. S6A) or grade alone (P ¼ 0.99; Supplementary Fig.S6B) or in combination with SH3GL2 for stage (P ¼ 0.09;Supplementary Fig. S6C) or grade (P ¼ 0.22; Supplementary Fig.S6D).Of note, other than the tumor adjacent normal, we detectedhigh SH3GL2 and MFN2 expression in 100% (4/4) of normalhuman breast ductal epithelial tissues obtained from cancer-freenormal women undergoing mammoplasty (Supplementary Fig.S6E).We also confirmed the presence of SH3GL2 inmitochondriaof the above four breast tissues obtained from normal healthywomen (Supplementary Fig. S6F).

An SH3GL2 and MFN2 loss was detectable in the circulatingexosomes

SH3GL2 was overexpressed in three breast cancer cell lines(Supplementary Fig. S1A). To determine whether the cancer cellexosomes carry this information, we performed Western blottinganalysis using the culture supernatant–derived exosomes. Wecould detect a low level of SH3GL2 expression in the controlculture-derived exosomes (Fig. 6A). On the other hand, theenhanced expression level of SH3GL2 was readily detectable inthe exosomes derived from the SH3GL2-overexpressing cultures(Fig. 6A). MFN2 expression was induced in these cell linesfollowing SH3GL2 overexpression (Fig. 1). The induced expres-sion level of MFN2 was also detectable in the culture-derivedexosomes of the SH3GL2-overexpressing cells compared with thecontrol (Fig. 6A).

To determine SH3GL2 and MFN2 expression pattern in thecirculating exosomes, we performed Western blotting analysis.Sera exosomes from 27 women with invasive breast cancer and 6normal, healthy women were scrutinized to determine SH3GL2and MFN2 expression. All but 2 breast cancer cases (CE12 andCE21) exhibited a low or barely detectable expression of SH3GL2in the sera exosomes (Fig. 6B). An appreciable level of SH3GL2expression was detectable in the circulating exosomes derivedfrom normal sera (Fig. 6B; NE1–NE6). Similarly, all but 4 breastcancer cases (CE1, CE2, CE18, and CE20) exhibited a low orbarely detectable expression ofMFN2 in the circulating exosomes(Fig. 6C). An appreciable level ofMFN2 expressionwas detectablein the normal sera–derived exosomes (Fig. 6C; NE1–NE6).

DiscussionRecent reviews of the cancer genome landscape implicated

that cancer mortality can be reduced to more than 70% by earlydetection and prevention (27). Other than mammography,MRI, and ultrasound-based screening, no other suitable method

is clinically available for the early diagnosis and monitoring ofbreast cancer (28–30). However, these methods are limited todetect early genetic changes and predict the biologic behaviorof the tumor. From the treatment standpoint, targeted cancertherapy is often dependent on overexpressed and activated onco-genes such as HER2/neu for breast cancer (31). However, in themajority of the solid tumors, tumor suppressor genes (TSG)act as the drivers of cancer development and progression (27).For example, p53 is the most frequently mutated TSG among thetop 21 altered genes in breast cancer (32). Therapeutic targeting ofTSGs is difficult as they are already inactivated by allelic loss,genetic mutation, or various other mechanisms. Thus, molecularcharacterization of the key TSGs could aid to develop strategies forearly detection,monitoring, and surveillance. Our studies definedpreviously unidentified loss and function of SH3GL2 in breastcancer progression. SH3GL2 is expressed predominantly in thecytoplasm and altered in various malignancies (33, 34). Thetranslocation and phosphorylation of SH3GL2 in mitochondriaappears to trigger the intrinsic apoptotic pathway through induc-tion of O2

� production, mitochondrial fusion, and CYTC release.Although, there was some basal and low level of SH3GL2 expres-sion in the breast cancer cells, its exogenous introduction inducedthe phenotypic and associatedmolecular changes. It could be dueto relatively inactive endogenous SH3GL2 and its inability toaugment the mitochondrial proteins and release CYTC in thecytoplasm, as evident from our studies. CYTC release from mito-chondria to the cytosol is critical in the intrinsic apoptotic path-way (18), whereas production in tumors of normalmitochondriathrough mitochondrial fusion could be tumor suppressive bypromoting oxidative metabolism and enhanced ROS production(6, 8). At the molecular level, cross-talk between SH3GL2, MFN2,and PINK1 in mitochondria appeared to be the key event inopposing progression in vitro and in vivo. Their colocalizations alsoindicate that the interplay occurs in mitochondria. PINK1 over-expression was shown to reduce anchorage-independent growthof breast cancer cells, and its mRNA expression was associatedwith better survival in adrenocortical tumors (35). A number ofstudies demonstrated a tumor-suppressive role of MFN2 in lung,bladder, liver, gastric, and colon carcinomas by reducing prolif-eration and triggering apoptosis (36–41). In lung cancer cells,enhanced MFN2 expression induced apoptosis by triggering ROSproduction in vitro and reduced in vivo growth by augmentingmitochondrial fusion network (36). An in vitro study also dem-onstrated a marked decrease in migration and invasion of breastcancer cells through enhanced MFN1 and MFN2 expression andmitochondrial fusion (42). In this context, the induction of ATG5,PTEN, and CDH1 might also have contributed to the onset ofapoptosis and growth inhibition. ATG5 was shown to induceapoptosis (43), and enhanced expression of ATG5 was associatedwith favorable disease-free survival of breast cancer patients (44).So far, the functional role of ATG5 in breast cancer is unknown.On the other hand, PTEN is a well-defined TSG and the regulatorof PINK1 (19). CDH1 is a key molecule of the cell–cell adhesioncomplex, and its loss promotes metastasis (20, 21). In a recentstudy, simultaneous loss of CDH1 and PTEN was shown toaccelerate cellular invasiveness and angiogenesis in the mouseuterus (45). Thus, concurrent augmentation of these moleculesfollowing SH3GL2 overexpression might have contributed inpreventing primary growth and metastasis by promoting apop-tosis. The well-differentiated morphology of the SH3GL2-over-expressing in vivo implants with marked cell–cell contact as






confirmed by various epithelial and mesenchymal markerscould be due to the rescued expression of CDH1 and othermolecules. On the other hand, SH3GL2 knockdown in nontu-morigenic as well as tumorigenic breast epithelial cells con-firmed the involvement of mitochondrial pathway proteins,along with PTEN–CDH1 niche for opposing tumor growth andprogression.

Despite the increased expression of key mitochondrial biogen-esis–associatedmoleculesPGC-1a andMT-TFA,wedidnotobserve

an increase in mitochondrial DNA content (data not shown).However, an increase in overall mitochondrial mass was observedas demonstrated by an enhanced mitochondrial fusion network.PGC-1a is a key coordinator of mitochondrial function, includingregulationofMT-TFA transcription (26). Loss of expressionofPGC-1a alone, and in combination with MT-TFA, was found to beassociated with highly aggressive clear-cell ovarian carcinoma pro-gression and its resistance to chemotherapy (26). In addition,frequent truncatingmutationsofMT-TFA resulted inmitochondrial

NE

1N

E2

NE

3N

E4

NE

5N

E6

CE

1

CE

4

CE

5

CE

6

CE

7

CE

8

CE

9

CE

10

CE

11

CE

12

CE

4

CE

5

CE

6

CE

7

CE

8

CE

9

CE

10

CE

11

CE

12

CE

13

CE

14

CE

15

CE

16

CE

17

CE

18

CE

19

CE

20

CE

13

CE

14

CE

15

CE

16

CE

17

CE

18

CE

19

CE

20

CE

21

CE

22

CE

23

CE

24

CE

25

CE

26

CE

27

CE

21

CE

22

CE

23

CE

24

CE

25

CE

26

CE

27

CE

2C

E3

NE

1N

E2

NE

3N

E4

NE

5N

E6

CE

1C

E2

CE

3

SH3GL2SH3GL2

SH3GL2

40 kDa

40 kDa

SH3GL240 kDa

SH3GL240 kDa

33 kDa

33 kDa

Syntenin

Syntenin

33 kDaSyntenin

33 kDaSyntenin

33 kDa

86 kDaMFN2

86 kDa5075

100

50

75100

50

50

503725

503725

503725

503725

Cytosol

SH3GL2

SH3GL2

Mitochondria

Mitochondrial fusion

CYTC

Release

Apoptosis

O2–

PINK1

MFN2

SH3GL2PTENp

p

75

75

100

100

MFN2

86 kDa

86 kDa

33 kDa

40 kDa

A

C

D

B

MFN2

MFN2

86 kDaMFN2

Syntenin

33 kDaSyntenin

33 kDaSyntenin

Syntenin

33 kDaSyntenin


MCF-7 SUM149 MDA-MB-231

Figure 6.Detection of SH3GL2 and MFN2signature in the exosomes. A,enhanced expression of SH3GL2 andMFN2 in the culture supernatant–derived exosomes of the SH3GL2-overexpressing cells. EV, empty vectortransduced; SH3GL2, SH3GL2-transduced cells. Synteninwas used asan exosome marker and loadingcontrol. B, compared with the normalhealthy women, all but 2 breast cancercases (CE12 and CE21) demonstrated alow or barely detectable expressionof SH3GL2 in the circulating exosomes.C, all but 4 breast cancer cases (CE1,CE2, CE18, and CE20) exhibited a lowor barely detectable expression ofMFN2 in the circulating exosomes,compared with the normal controls.NE, exosomes isolated from sera ofcancer-free normal healthy women;CE, exosomes isolated from sera ofwomen with invasive ductalcarcinomas. D, model depicting thepossible interplay between thephosphorylated SH3GL2, MFN2, andPINK1 in mitochondria, whichtriggered mitochondrial fusionnetwork, O2

� production, and releaseof CYTC, leading to apoptoticinduction.


Kannan et al.




DNAdepletion andapoptotic resistance in colon cancer (46). Thus,augmentation of both PGC-1a and MT-TFA and a physical asso-ciation between PGC-1a and SH3GL2 strongly suggest for thepossibility of functional cooperation among these molecules lead-ing to normalized mitochondrial function and apoptosis.

SH3GL2 is located on human chromosome 9p22, a frequentlydeleted region in breast cancer (47). In addition to SH3GL2, theexpression pattern of MFN2 in breast cancer progression remainsunknown. Loss of coexpression of both of these potential TSGsand their association with disease progression implicate for acausative role in breast tumorigenesis. The normal expression of aTSG may be affected in cancer-adjacent normal tissues due to the"field cancerization effect". However, high and comparable levelof SH3GL2 and MFN2 expression in the mammoplasty tissuesindicated that these are abundantly expressed proteins in normalbreast ductal epithelial cells. Notably, the detection of SH3GL2 inmitochondria of these normal breast tissues further suggests a roleof SH3GL2 in regulating mitochondrial function or biogenesis.However, due to the lackof premalignant tissue samples,we couldnot evaluate the expression pattern of SH3GL2 and MFN2 at thistime.

Exosomes are emerging as a promising biomarker tool as theycarry specific genetic information and influence tumor growthand progression (10–12). Syntenin is among the top 20 proteinsmost abundantly expressed in the exosomes and epithelial cellcancers (48–50). Thus, syntenin could serve as a reliable exosomemarker. The detection of enhanced SH3GL2 and MFN2 expres-sion in the SH3GL2-overexpressing cell–derived exosomes sug-gests that thesemolecules perform important regulatory functionsthrough the exosomes. Otherwise, we would not have seen theirelevated expression in the culture-derived exosomes followingSH3GL2 overexpression. The tumor suppressive effect that wehave seen following SH3GL2 overexpression in the breast cancercells could partly be due the "paracrine effect" of these exosomesenriched in SH3GL2 and MFN2 proteins. As these molecules arepotential TSGs and normally carried by the exosomes as evidentfromour studies, their early inactivation or loss could favor cancerinitiation and progression and vice versa. Possibly as a reason, theexpression of these molecules was barely detectable in the circu-lating exosomes of the majority of the women with invasivedisease. To our knowledge, no studies so far reported the presenceof SH3GL2 and MFN2 proteins in the circulating exosomes ofnormal, healthy women and their loss of expression in breast

cancer patients. Taken together, our study uncovered a novelmitochondrial reprogramming pathway regulating breast cancerdevelopment and progression. In this signaling cascade, activatedSH3GL2 appears to induce PTEN and interacts with MFN2 andPINK1 in mitochondria (Fig. 6D). This interplay normalizesmitochondrial function and triggers apoptosis by releasing O2

�

and CTYC from the mitochondria (Fig. 6D). Molecular profilingof SH3GL2 and MFN2 alterations in circulating exosomes couldbe a feasible approach for noninvasive early detection, monitor-ing, and surveillance of breast cancer.

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

Authors' ContributionsConception and design: K.P. Singh, E.R. Sauter, S. DasguptaDevelopment of methodology: A. Kannan, S. DasguptaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Kannan, S. DasguptaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Kannan, S. Sivakumar, S. Komatsu, K.P. Singh,B. Samten, M. Ikebe, S. Idell, S. DasguptaWriting, review, and/or revision of the manuscript: A. Kannan, S. Sivakumar,E.R. Sauter, M. Ikebe, S. Idell, S. Gupta, S. DasguptaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Kannan, J.V. Philley, S. DasguptaStudy supervision: S. DasguptaOther (pathologic evaluation): R.B. Wells

AcknowledgmentsThis work is dedicated to the memory of Jnan Ranjan Dasgupta for his brave

fight against cancer. The authors thank Zane Robertson for his generous helpwith the histopathology and Karen Durham, a Susan G. Komen Scholar andadvocate, for critical reading of the manuscript and support of our research.The authors also thank Henry James at the UTHSCT Animal Facility for his helpon the animal work.

Grant supportThe study was supported by the UTHSCT and Chamblee Foundation

(to S. Dasgupta).The costs of publication of this articlewere defrayed inpart by the payment of

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

Received October 13, 2015; revised January 21, 2016; accepted February 6,2016; published OnlineFirst February 17, 2016.

References1. Eccles SA, Aboagye EO, All S, Anderson AS, Armes J, Berditchevski F,

et al. Critical research gaps and translational priorities for the success-ful prevention and treatment of breast cancer. Breast Cancer Res 2013;15:R92.

2. National Cancer Institute. Available from: www.cancer.gov.3. Polyak K. Breast cancer: origin and evolution. J Clin Invest 2007;117:

3155–63.4. Dos Anjos Pultz B, da Luz FA, de Faria PR, Oliveira AP, de Ara�ujo RA, Silva

MJ. Far beyond the usual biomarkers in breast cancer: a review. J Cancer2014;5:559–71.

5. Bombonati A, Sgori DC. The molecular pathology of breast cancer pro-gression. J Pathol 2011;223:310–317.

6. Archer SL. Mitochondrial dynamics- mitochondrial fission and fusion inhuman diseases. New Eng J Med 2013;369:2236–51.

7. He Y, Wu J, Dressman DC, Iacobuzio-Donahue C, Markowitz SD, Velcu-lescu VE, et al. Heteroplasmic mitochondrial DNA mutations in normaland tumour cells. Nature 2010;464:610–14.

8. Boland ML, Chourasia AH, Macleod KF. Mitochondrial dysfunction incancer. Front Oncol 2013;3:1–18.

9. Da silva AF,Mariotti FR,Maximo V, Campello S.Mitochondria dynamism:of shape, transport and cell migration. Cell Mol Life Sci 2014;71:2313–24.

10. Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis,and drug resistance: a comprehensive review. Cancer Metastasis Rev2013;32:623–42.

11. Kahlert C, Kalluri R. Exosomes in tumor microenvironment influencecancer progression and metastasis. J Mol Med 2013;91:431–37.

12. Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, et al.Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis andPromote Tumorigenesis. Cancer Cell 2014;26:707–21.

13. Kjaerulff O, Brodin L, Jung A. The structure and function of endophilinproteins. Cell Biochem Biophys 2011;60:137–54.

14. Dasgupta S, Menezes ME, Das SK, Emdad L, Janijic A, Bhatia S, et al. Novelrole of MDA-9/syntenin in regulating urothelial cell proliferation bymodulating EGFR signaling. Clin Cancer Res 2013;19:4621–33.






15. Oyesanya RA, Bhatia S, Menezes ME, Dumur CI, Singh KP, Bae S, et al.MDA-9/Syntenin regulates differentiation and angiogenesis pro-grams in head and neck squamous cell carcinoma. Oncoscience2014;1:725–37.

16. Iorns E, Drews-Elger K, Ward TM, Dean S, Clarke J, Berry D, et al. A newmouse model for the study of human breast cancer metastasis. PLoS One2012;7:e4799.

17. Sinha S, Chunder N, Mukherjee N, Alam N, Roy A, Roychoudhury S, et al.Frequent deletion and methylation in SH3GL2 and CDKN2A loci areassociated with early- and late-onset breast carcinoma. Ann Surg Oncol2008;15:1070–80.

18. Dasgupta S, Hoque MO, Upadhyay S, Sidransky D. Forced Cytochrome Bgene mutation expression induces mitochondrial proliferation and pre-vents apoptosis in human uroepithelial SV-HUC-1 cells. Int J Cancer2009;125:2829–35.

19. O'Flanagan CH, O'Neill C. PINK1 signaling in cancer biology. BiochimBiophys Acta 2014;1846:590–8.

20. Onder TT,Gupta PB,Mani SA, Yang J, Lander ES,Weinberg RA, et al. Loss ofE-cadherin promotes metastasis via multiple downstream transcriptionalpathways. Cancer Res 2008;68:3645–54.

21. Berx G, Van Roy F. The E-cadherin/catenin complex: an important gate-keeper in breast cancer tumorigenesis and malignant progression. BreastCancer Res 2001;3:289–93.

22. LuH, LiG, Liu L,WangX, JinH.Regulationof and functionofmitophagy indevelopment and cancer. Autophagy 2013;9:1720–36.

23. Gomes LC, Scorrano L. Mitochondrial morphology in mitophagy andmacroautophagy. Biochim Biophys Acta 2013;1833:205–12.

24. Dolman NJ, Chambers KM, Mandavilli B, Batchelor RH, Janes MS. Toolsand techniques to measure mitophagy using fluorescence microscopy.Autophagy 2013;9:1653–62.

25. Zhu J, Wang ZQ, Chu CT. Mitochondrial biogenesis, mitophagy and cellsurvival. Autophagy 2013;9:1663–76.

26. Gabrielson M, Bjorklund M, Carlson J, Shoshan M. Expression ofmitochondrial regulators pgc-1a and tfam as putative markers ofsubtype and chemoresistance epithelial ovarian carcinoma. PLoS One2014;9:e107109.

27. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, KinzlerKW. Cancer genome landscapes. Science 2013;339:1546–58.

28. American Cancer Society. Available from: http://www.cancer.org/cancer/breastcancer/moreinformation/breastcancerearlydetection/breast-cancer-early-detection-toc.

29. Susan G. Komen for the Cure. Available from: http://ww5.komen.org/AboutBreastCancer/ScreeningandEarlyDetection/ScreeningTestsandDiag-nosticTools/ScreeningTestsDiagnosticTools.html

30. Hirata BKB, Oda JMM, Guembarovski RL, Ariza CB, Coral de Oliveira CE,Watanabe MA. Molecular markers for breast cancer: prediction on tumorbehavior. Dis Markers 2014;2014:513158.

31. Wang WJ, Lei YY, Mei JH, Wang CL. Recent progress in HER2 associatedbreast cancer. Asian Pac J Cancer Prev 2015;16:2591–600.

32. Polyak K, Filho OM. Snapshots: breast cancer. Cancer Cell 2012;22:562.33. Dasgupta S, Jang JS, Shao C, Mukhopadhyay ND, Sokhi UK, Das SK, et al.

SH3GL2 is frequently deleted in non-small cell lung cancer and down-

regulates tumor growth by modulating EGFR signaling. J Mol Med 2013;91:381–93.

34. Majumdar S, Gong EM, Di Vizio D, Dreyfuss J, Degraff DJ, Hager MH, et al.Loss of Sh3gl2/endophilin A1 is a common event in urothelial carcinomathat promotes malignant behavior. Neoplasia 2013;15:749–60.

35. Gomes LC, Scorrano L. Mitochondrial morphology in mitophagy andmacroautophagy. Biochim Biophys Acta 2013;1833:205–12.

36. 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.

37. Jin B, Fu G, Pan H, Cheng X, Zhou L, Lv J, et al. Anti-tumor efficacy ofmitofusin-2 in urinary bladder carcinoma. Med Oncol 2011;28 Suppl 1:S373–S380.

38. Wang W, Lu J, Zhu F, Wei J, Jia C, Zhang Y, et al. Pro-apoptotic and anti-proliferative effects of mitofusin-2 via bax signaling in hepatocellularcarcinoma cells. Med Oncol 2012;29:70–6.

39. Wang W, Xie Q, Zhou X, Yao J, Zhu X, Huang P, et al. Mitofusin-2 triggersmitochondria Ca2þ influx from the endoplasmic reticulum to induceapoptosis in hepatocellular carcinoma cells. Cancer Lett 2015;358:47–58.

40. ZhangGE, JinHL, Lin XK,ChenC, Liu XS, ZhangQ, et al. Anti-tumor effectsof mfn2 in gastric cancer. Int J Mol Sci 2013;14:13005–21.

41. Cheng X, Zhou D, Wei J, Lin J. Cell-cycle arrest at G2/M and proliferationinhibition by adenovirus-expressed mitofusin-2 gene in human colorectalcancer cell lines. Neoplasma 2013;60:620–26.

42. 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.

43. Luo S, Rubinszztein DC. Atg5 and bcl-2 provide novel insight into theinterplay between apoptosis and autophagy. Cell Death Differ 2007;14:1247–50.

44. Wang L, Yao L, Zheng YZ, Xu Q, Liu XP, Hu X, et al. Expression ofautophagy-related proteins atg5 and fip200 predicts favorable disease-freesurvival in patients with breast cancer. Biochem Biophys Res Commun2015;458:816–22.

45. LindbergME, StoddenGR, KingML,MacLean JA 2nd,Mann JL, DeMayo FJ,et al. Loss of CDH1 and Pten accelerates cellular invasiveness and angio-genesis in the mouse uterus. Biol Reprod 2013;89:8.

46. Guo J, Zheng L, LiuW,Wang X, Wang Z, Wang Z, et al. Frequent truncatingmutation of TFAM induces mitochondrial DNA depletion and apoptoticresistance in microsatellite-unstable colorectal cancer. Cancer Res 2011;71:2978–87.

47. Brenner AJ, Aldaz CM. Cancer Res. Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortalbreast epithelial cells. Cancer Res 1995;55:2892–95.

48. Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, et al.Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat CellBiol 2012;14:677–85.

49. Tomlinson PR, Zheng Y, Fischer R, Heidasch R, Gardiner C, Evetts S, et al.Identification of distinct circulating exosomes in Parkinson's disease.Ann Clin Transl Neurol 2015;2:353–61.

50. Philley JV, Kannan A, Dasgupta S. MDA-9/Syntenin control. J Cell Physiol2016;231:545–50.


Kannan et al.




2016;22:3348-3360. Published OnlineFirst February 17, 2016.Clin Cancer Res Anbarasu Kannan, Robert B. Wells, Subramaniam Sivakumar, et al. ProgressionMitochondrial Reprogramming Regulates Breast Cancer

Updated version

10.1158/1078-0432.CCR-15-2456doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2016/02/17/1078-0432.CCR-15-2456.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/22/13/3348.full#ref-list-1

This article cites 47 articles, 5 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://clincancerres.aacrjournals.org/content/22/13/3348To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-2456

http://clincancerres.aacrjournals.org/content/suppl/2016/02/17/1078-0432.CCR-15-2456.DC1

http://clincancerres.aacrjournals.org/content/22/13/3348.full#ref-list-1

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

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/22/13/3348


mitochondrial reprogramming regulates breast cancer ...pink1, pgc-1a, and mitochondrial...

Documents