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Molecular and Cellular Pathobiology

MYC Is a Crucial Mediator of TGFb-InducedInvasion in Basal Breast CancerMagdalena A.Cichon1, Megan E. Moruzzi1,Tiziana A. Shqau1, ErinMiller1, ChristineMehner1,Stephen P. Ethier2, John A. Copland1, Evette S. Radisky1, and Derek C. Radisky1

Abstract

Basal subtype breast cancers have a particularly poor prognosis,with high invasiveness and resistance to most targeted therapies.TGFb and MYC drive central features of basal breast cancer: TGFbis an autocrine and paracrine signaling factor that drives cellinvasion andmetastasis, andMYC is a central regulator of cellularproliferation that is upregulated in many cancer types. We showhere that genetic or pharmacologic inhibition ofMYC inMCF10Abasal breast cells results in increased sensitivity to TGFb-stimu-lated invasion and metastasis and also show that this signalingloop is dependent on activation of SRC. Analysis of human breast

cancer datasets and additional experiments with breast cancercell lines further suggest the relevance of this signaling loopin basal, but not luminal, breast cancers. Our results implyprecaution should be taken when utilizing therapeutic inhi-bitors of MYC with basal breast cancer patients as this couldlead to increased metastasis; however, simultaneous phar-macologic inhibition of SRC and MYC for these patientscould facilitate the antiproliferative effects of MYC inhibi-tion while blocking the consequent promotion of metastasis.Cancer Res; 76(12); 3520–30. �2016 AACR.

IntroductionBreast cancers can be classified into clinically relevant groups

with distinct therapeutic responsiveness on the basis of theirintrinsic molecular subtypes (1, 2). Human breast cancer celllines can also be clustered according to these intrinsic molecularsubtypes, which are associatedwith differences in phenotypic andgenetic features, as well as differences in responsiveness to drugtreatments (1, 3). Both basal subtype breast cancers and basal celllines are associated with aggressive features, invasiveness, highergrade, and poor prognosis, and as basal breast cancers overlapwith the triple-negative histologic phenotype (ER�, PR�, HER2�),there exist few effective targeted therapies for this groupof patients(4, 5). Thus, there is a need to better understand the distinctmolecular pathways controlling progression of basal breast can-cers to develop new effective therapeutic approaches.

MYC is a pleiotropic transcription factor that regulates cellularproliferation, growth, differentiation, apoptosis, adhesion, andmigration (6). Elevated or deregulated expression of MYC hasbeen found inmultiple types of cancer, including breast cancer (7,8).MYC induces cell proliferation by targeting genes that promote

progression of the cell cycle, as well as by inhibiting transcriptionof growth-inhibitory genes (9). It also plays roles in inhibition ofapoptosis, induction of angiogenesis, and epithelial–mesenchy-mal transition (7). In breast cancer, MYC gene amplification isassociated with higher tumor grade, poor clinical prognosis, riskof relapse, and ER/PR negativity (7), and inhibitors of MYC arebeing evaluated for antitumor activity in clinical trials (10).However, despite its role as a pleiotropic oncogene, a recent studyof MYC has shown that it can also behave as a tumor suppressorunder certain circumstances through inhibition of cancer cellinvasion and metastasis (11).

Integrins are heterodimeric cellmembrane proteins that consistof a and b transmembrane subunits; acquisition of malignancy isassociated with cancer subtype–dependent increased expressionof specific integrins that facilitate cell invasion and metastasis(12). Integrin avb3 is often upregulated in breast cancer and hasbeen found to be associated with increased invasion and metas-tasis (12). A recent study identified a tumor suppressor activity forMYC that was dependent upon specific regulation of integrins avand b3; specifically, while overexpression of MYC in mammaryepithelial cells induced cell proliferation in culture and led toformation of larger tumors in mice, direct inhibition of integrinsubunits av and b3 by MYC was shown to inhibit cell invasionand metastasis (11).

TGFb is a multifunctional cytokine that can act as a tumorsuppressor, inhibiting proliferation and suppressing stromalmitogens, as well as a tumor promoter, allowing evasion ofimmune surveillance and driving invasion and metastatic colo-nization (13–15). The consequences of TGFb signaling are highlydependent on cellular context (16), and it is a prominent playerduring tumorigenesis (17, 18). Early findings that growth inhi-bition by TGFb occurred through transcriptional inhibition ofMYC (19)were supported by identification of a SMAD-responsiveelement in the MYC promoter (20) and demonstration of directbinding of SMAD3 to theMYCpromoter (21). As downregulation

1Department of Cancer Biology, Mayo Clinic Cancer Center, Jackson-ville, Florida. 2Department of Pathology and Laboratory Medicine,Medical University of South Carolina, Charleston, South Carolina.

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

Current address for M.E. Moruzzi: The University of Manchester, Faculty of LifeSciences, Oxford Road, Manchester M13 9PT, United Kingdom.

Corresponding Author: Derek C. Radisky, Mayo Clinic Cancer Center, 4500 SanPablo Ave, Jacksonville, FL 32224. Phone: 904-953-6913; Fax: 904-953-0277;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-3465

�2016 American Association for Cancer Research.

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of MYC also leads to increased breast cancer cell invasion andmetastasis, we hypothesized that TGFb-induced inhibition ofproliferation and promotion of invasion could be linked throughdownregulation of MYC in breast cancer cells.

Here, we show that TGFb decreases expression of MYC andconsequently leads to upregulation of integrin avb3 and that thissignaling axis can drive invasion andmetastasis. We further showthat this effect is specific for the basal subtype of breast cancer, andfind that MYC knockdown (KD) or inhibition can drive theinvasive phenotype, but that simultaneous inhibition of SRC canblock this effect. Our study reveals a hidden danger of MYCinhibition:while suppressing cancer cell proliferation, these drugscan also stimulate invasion andmetastasis of basal subtype breastcancer cells. Importantly, bydefining themechanistic basis for thisphenomenon, we have also identified a solution: the simulta-neous inhibition of MYC and SRC can block the proliferativecapacity of MYC while abrogating its SRC-dependent proinvasiveactivity. On the basis of our findings, we propose a novel ther-apeutic strategy for these patients through simultaneous targetedinhibition of MYC and SRC. This strategy has the potential toblock the proliferative capacity of MYC while abrogating theactivation of invasion and metastasis mediated through SRCsignaling.

Materials and MethodsCell culture

MCF10A and MCF12A cells were grown in DMEM/F12(Gibco), supplemented with 5% horse serum (Gibco), 20 ng/mLEGF (Peprotech), 0.5 mg/mL hydrocortisone (Sigma), 100 ng/mLcholera toxin (Sigma), 10 mg/mL insulin (Sigma), and 100 mg/mLgentamicin (Gibco) andmaintained as described previously (22).MCF10CA1h cells were grown in DMEM/F12 (Gibco), supple-mentedwith 5%horse serum (Gibco) and 100 mg/mL gentamicin(Gibco). HCC38, BT-474, BT-549, T47D, and MCF7 cells weremaintained in RPMI1640 (Gibco), MDA-MB-231 and MDA-MB-468 cells were grown in DMEM (Gibco), and SKBR3 cells inMcCoy's 5A (Gibco), all supplementedwith 10%heat-inactivatedFBS (Thermo Scientific) and 100 mg/mL gentamicin (Gibco).SUM149 cells were grown in Ham's F12 medium (Gibco), sup-plemented with 5% FBS (Thermo Scientific), 5 mg/mL insulin(Sigma), 1 mg/mL hydrocortisone (Sigma), 10 mmol/L HEPES(Gibco), 25mg/mLgentamicin (Gibco), and2.5mg/mLFungizone(Gibco). SUM149 cells were maintained at 37�C in 10% CO2. Allother cell lines were maintained at 37�C in 5% CO2.

293FT cells, used for lentivirus production, were purchasedfrom Invitrogen and maintained in DMEM (Gibco), supplemen-ted with 10% heat-inactivated FBS (Thermo Scientific), 1�MEMnon-essential amino acids (Gibco) and 500 mg/mL G418 sulfate(CellGro). No G418 sulfate was present in the medium used forlentivirus production. Pheonix-A cells, used for retrovirus pro-duction, were grown in DMEM (Gibco), supplemented with 10%heat-inactivated FBS (Thermo Scientific) and 100 mg/mL genta-micin (Gibco).

For TGFb treatments, recombinant human TGFb1 (R&D Sys-tems) was used, and unless otherwise indicated, cells were treatedwith TGFb at 25 ng/mL for 18 hours. For drug treatments,following compounds were used: dasatinib (Selleckchem); PP2(Calbiochem); c-Myc inhibitor 2 (MI2; Calbiochem), and JQ1(Cayman Chemical). Dasatinib treatments were performed at50 nmol/L.

All relevant human cell lines used in experiments wereobtained from ATCC, which authenticates using short tandemrepeat profiling.

Lentivirus production and shRNA-mediated gene knockdownand gene expression

Specific gene knockdowns were achieved by lentiviral trans-duction with Mission shRNAs (Sigma). Third-generation lenti-viruses were produced usingHEK293FT cells. Briefly, 6� 106 cellswere plated in 10-cmplates and simultaneously transfectedwith 3mg shRNA plasmid and 3 mg of each packaging plasmid: pMD2.G(Addgene, #12259), pRSV-Rev (Addgene, #12253), pMDLg/pRPE(Addgene, #12251; ref. 23); lipofectamine 2000 (Invitrogen)was used for transfection, according to the manufacturer'sinstructions. Medium was changed after 24 hours, and lenti-virus-containing medium was collected 48 hours posttransfec-tion. For lentiviral transduction in a 6-well format, 600 mL viralsupernatant, 400 mL regular medium, and 6 mg/mL polybrene(Millipore) were used. Following shRNAs were used: MYCNM_002467.3-1661s21c1 (TRC2), targeting sequence 50-CCA-GAGGAGGA ACGAGCTAAA-30; ITGAV NM_002210.x-2551s1c1 (TRC1/1.5), targeting sequence 50-CTCTGTTGTA-TATCCTTCATT-30; ITGB3 NM_000212.x-2084s1c1, targetingsequence 50-GTCGTCAGATTCCAGTACTAT-30 (TRC1/1.5); SRCNM_198291.1-648s21c1 (TRC1/1.5), targeting sequence 50-GACAGACCTGTCC TTCAAGAA-3. Nontarget shRNAs wereused as a control, specifically SHC002 for TRC1/1.5 libraryconstructs and SHC202 for TRC2 library constructs, both withthe same nonmammalian hairpin sequence 50-CCGGCAAC-AAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGT-TGTTTTT-30. In experiments involving double knockdown withconstructs from both TRC1/1.5 and TRC2 libraries, both non-targets were used as a control. The same lentivirus productionand lentiviral transduction protocols were used for specific geneexpression. Refer to "Plasmids and cloning" section for con-struct details.

For MCF10A cells, following antibiotic concentrations wereused for selection, where indicated: puromycin 1 mg/mL, neomy-cin (G418) 200 mg/mL. For MDA-MB231 cells, puromycin wasused at 1 mg/mL for lentiviral transduction selection and at 0.25mg/mL for retroviral transduction selection.

Retrovirus production and transductionRetrovirus for expressionofMycwas producedusing Pheonix-A

cells and pMSCV/puro Myc plasmid (11). Retrovirus with anempty vector was used as a control. Briefly, 3 � 106 cells wereplated in 10-cmplates and transfected the following daywith 6-mgof plasmid DNA, using Fugene6 transfection reagent (Promega),following the manufacturer's instructions. Retrovirus-containingmedium was collected 48 hours posttransfection. For retroviraltransduction of MBA-MB231 and MCF10A cells, 1 � 106 cellswere plated in 10-cm plates the day before transduction; 1.8 mLviral supernatant, 4.2 mL regular medium, and 10 mg/mL poly-brene (Millipore)were used.Mediumwas changed after 24hours,and antibiotic selection using 0.25 mg/mL puromycin was started48 hours posttransduction.

RNA extraction, cDNA synthesis, and qRT-PCR analysisRNAwas isolated using TRIzol Reagent (Invitrogen), following

the manufacturer's instructions. Briefly, the cells were lysed with

MYC Mediates Basal Breast Cancer Invasion and Metastasis

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TRIzol, followed by phenol–chloroform phase separation. RNAwas precipitated with isopropanol, washed with 75% ethanol,and dried RNA pellet was resuspended in DEPC water. RNAfrom mouse tissue was isolated using RNeasy Mini Kit (Invi-trogen), according to the instruction manual. cDNA was syn-thesized using the High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems). Gene expression levels were assayedby qRT-PCR using 7900 HT Fast Real-Time PCR System, Taq-Man Fast Universal Master Mix, and TaqMan probes for specificgenes (Applied Biosystems): ITGAV Hs00233808_m1, ITGB3Hs00173978_m1, MYC Hs00153408_m1, SERPINE (PAI-1)Hs01126607_g1, GAPDH Hs99999905_m1. All assays wereperformed in triplicate, and analysis was performed using RQManager software (Applied Biosystems) and the 2�DDC

t methodto obtain relative quantitation values, with GAPDH used asendogenous control.

Plasmids and cloningCloning was performed utilizing Gateway cloning approach.

MYC was cloned from pMSCV/puro MYC (11). Constitutivelyactive TGFb receptor I (CA TGFBR1) was cloned from pCMV5TBRI-HA T204D (Addgene, #19162). For obtaining inserts in anentry vector pENTR/D-TOPO, cloning kit with One Shot Mach1-T1Chemically Competent E. coli (Invitrogen)was used, followingthe manufacturer's instructions. PCR of inserts for the TOPOreaction was done using PfuUltra High-Fidelity DNA Polymerase(Stratagene). Following primers were used: for MYC: forward, 50-CACCATGCCCCTCAACGTTAGCTT-30; reverse, 50-TTACGCA-CAAGAGTTCCG TAG-30; for CA TGFBR1: forward 50-CACCATG-GAG GCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCC-TC-30; reverse, 50-CT AGAGGCTAGCATAATCAGGA-30; for YFP:forward, 50-CACCATGGTGAGCAA GGGCGAGGA-30; reverse, 50-TTACTTGTACAGCTCGTCCATGCC-30. After verifying the insertsin entry vectors by sequencing, the LR recombination reaction(defined as the recombination between attL and attR sites) wasperformed with Gateway LR Clonase II Enzyme Mix (Invitro-gen) and specific destination vectors. MYC was cloned intopLenti CMV Puro DEST (Addgene, #17452; ref. 24) vector, forconstitutive expression, and into pLenti CMVtight Neo DEST(Addgene, #26432) vector, for tetracycline-inducible expres-sion. FUW-M2rtTA (Addgene, #20342) plasmid was used forsubsequent tetracycline-inducible expression experiments. CATGFBR1, as well as YFP, were cloned into pLenti 6.3/V5-DEST(Invitrogen). One Shot Stbl3 chemically competent E.coli(Invitrogen) was used for transformation and plasmid ampli-fication after the LR reaction. All final insert sequences wereverified by sequencing.

Luciferase assayFor luciferase assays, Dual-Luciferase Reporter Assay System

(Promega) was used, according to the manufacturer's instruc-tions. Briefly, 48 hours posttransfection the cells, grown in 6-wellplates, were lysed by scraping in 250 mL passive lysis buffer. Thelysateswere clearedbybrief centrifugation, and10mLof lysatewasused per measurement. Luminescence measurements were per-formed in Veritas Microplate Luminometer (Turner BioSystems),with a 2-second premeasurement delay and a 10-second signalintegration time. The Firefly luciferase activity was normalized toeither Renilla luciferase activity. All samples were assayed intriplicates.

Immunoblot analysisCells were lysed by scraping in lysis buffer (1% Triton X-100,

150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA,1mmol/L EGTA in water) with addedHalt Phosphatase InhibitorCocktail (Thermo Scientific) and Halt Protease Inhibitor Cocktail(Thermo Scientific). Lysates were cleared by centrifugation at13,000 rpm � 10 minutes � 4�C. Protein concentrations weredetermined using Bicinchoninic Acid (BCA) Protein Assay Kit(Pierce), following the manufacturer's instructions, and usingSpectraMax M5 Microplate Reader (Molecular Devices). Equalprotein amounts were loaded onto Mini-PROTEAN TGX 4%–

20% gradient gel, and SDS-PAGE electrophoresis was performedat 150 V. Transfer was done at 100 V for 90 minutes usingImmobilon-P PVDF membrane with pore size 0.45 mm (Milli-pore). Membrane was blocked in 5% non-fat dry milk in TBS þ1% Tween20 for 90 minutes and probed with primary antibodyovernight at 4�C. Following primary antibodies were used: c-Myc(9E10) mouse monoclonal (Santa Cruz Biotechnology, #sc-40)1:250; integrin avmousemonoclonal (BD Biosciences #611012)1:5,000; integrin b3 mouse monoclonal (BD Biosciences#611140) 1:200; Src rabbit monoclonal (Cell Signaling Technol-ogy, #2123) 1:2,000; Phospho-Src (Tyr416) rabbit polyclonal(Cell Signaling Technology, #2101) 1:1000; PAI-1 mouse mono-clonal (American Diagnostica, #380) 1:200; HA-tag (HRP-con-jugated) mouse monoclonal (Cell Signaling Technology #2999)1:1,000. Secondary antibody incubations were performed for 1hour at room temperature using goat anti-mouse IgG (HþL)cross-adsorbed secondary antibody at 1:5,000 dilution (ThermoScientific, #31432) or goat anti-rabbit (Invitrogen #656120)1:7,000. GAPDH was used as loading control probing withGAPDH rabbit monoclonal (Meridian, #H86504M) 1:500,000for 15 minutes, followed by incubation in secondary goat anti-mouse antibody 1:10,000 for 30 minutes, both at room temper-ature. All antibody incubations were performed in blockingbuffer. All washes were performed in TBS þ 1% Tween20. EitherAmersham ECL Plus Western Blotting Detection Reagent (GEHealthcare) or Clarity Western ECL Substrate (Bio-Rad) was usedfor detection. Blue Devil film (Genesee Scientific) was developedin KODAK X-OMAT 2000A (Kodak).

ImmunofluorescenceCells were fixed in 3% paraformaldehyde in PBS for 30

minutes, permeabilized with 0.2% Triton X-100 in PBS for 5minutes, and blocked in 5% non-fat dry milk in PBS for 10minutes, all done at room temperature. Washes in betweenwere done with 10 mmol/L glycine in PBS. Incubation withprimary antibodies was performed overnight at 4�C, followedby secondary antibody incubation for 30 minutes at roomtemperature, both in blocking buffer. Nuclei were stained withHoechst 33342 (Invitrogen, #H3570) 1:10,000 for 10 minutesat room temperature. The following primary antibodies anddilutions were used: anti-vinculin mouse mAb (Sigma,#V9131) 1:100, anti-b-tubulin mouse monoclonal (Sigma,#T4026) 1:200. The following secondary antibodies were used:goat anti-mouse Alexa Fluor 488 (Invitrogen, #A11017) 1:200,goat anti-mouse Alexa Fluor 546 (Invitrogen, #A11030) 1:500.Aqua-Poly/Mount (Polysciences) was used to mount coverslipson glass slides. Images were acquired using Olympus IX71microscope, equipped with Olympus LUCPLanFLN objectives(20� NA 0.45, 40� NA 0.6, 60� NA 0.7) and a QuantiFire XIcamera (Optronics).

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Proliferation assaysClick-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen,

#C10337) was used to directly detect proliferation by immu-nofluorescence of newly synthesized DNA. Briefly, cells wereincubated with 10 mmol/L 5-ethynyl-20-deoxyuridine (EdU) for2 hours to allow EdU incorporation into the DNA, followed byfixation in 3% paraformaldehyde in PBS for 15 minutes andpermeabilization in 0.5% Triton X-100 for 20 minutes at roomtemperature. EdU detection was performed by incubation inClick-iT reaction cocktail containing for 30 minutes at roomtemperature. Nuclei were stained with Hoechst 33342 (Invi-trogen, #H3570) 1:10,000 for 10 minutes at room tempera-ture. Images were acquired using Olympus IX71 microscope,an Olympus LUCPLanFLN 20� NA 0.45 objective and aQuantiFire XI camera (Optronics). Proliferation was deter-mined as percentage of EdU-positive cells among Hoechst-stained cells.

Invasion assayCell culture inserts (8 mm pore size, 24-well format; BD Bio-

sciences) were coated with 50 mg/well growth factor–reducedMatrigel (BD Biosciences) diluted with plain DMEM/F12 medi-um to a total volume of 100 mL. The invasion chambers wereplaced in a cell culture incubator for 3 hours, after which, 750 mLof complete MCF10A growth medium was added to the bottomwells. MCF10A (5 � 104) cells resuspended in 300 mL assaymedium, which contained a reduced 2% serum amount and noEGF, as described in ref. 22, were seeded in the invasion chambersand incubated at 37�C.After 24hours, noninvading cells on topof

the invasion inserts were removed with a cotton swab. Theinvaded cells on the bottom of the inserts were fixed withmethanol at�20�C for 30 minutes and stained with 0.1% crystalviolet in 200 mmol/L MES at room temperature for 1 hour. Theinvasion chambers were photographed using Olympus IX51microscope equipped with a PlanN 2� NA 0.06 objective, andthe total invaded cells per chamber were counted. The pre-sented data represent triplicates � SEM for each experimentalcondition.

Flow cytometry analysis and FACSThe cells were trypsinized, counted, and washed three times

with sterile 2% BSA in PBS. For antibody incubation, 1 � 106

cells (scaled up for FACS) were resuspended in 100 mL "wash"buffer and incubated with indicated antibodies for 30 minutesat 4�C. Following antibodies were used: APC-CD49f (BioLe-gend, #313615) 5 mL/reaction, FITC-EpCAM (BD Biosciences,#347197) 20 mL/reaction, FITC-CD49f (BioLegend, #313606)5 mL/reaction, and APC-EpCAM (BD Biosciences, #347200) 5mL/reaction. The cells were then washed three times andsieved through a cell strainer. Flow cytometry analysis andFACS were performed using FACSAria II Flow Cytometer (BDBiosciences).

Orthotopic xenograft model of breast cancer metastasisAll animal procedureswereperformedunder the guidanceof an

approved Mayo Clinic Institutional Animal Care and Use Com-mittee protocol (protocol no. A330-13). Eight- to 10-week-old

Figure 1.MYC overexpression inhibits TGFb-inducedmetastasis. A, high MYC expression is associatedwith longermetastasis-free survival. Kaplan–Meier analysis for n¼ 346lymph node–positive (pos.) breast cancer patients stratified into two equally sized groups by MYC expression levels. B, CA TGFBR1 (R1) acts as a surrogate forexogenous TGFb in activating TGFb-responsive luciferase reporter construct. C, bioluminescence analysis of decreasing cell numbers of MDA-MB-231 cellstransduced with a luciferase (luc.) construct, with either control (Cont.), R1 or MYC, and R1. D, growth of orthotopic xenografts was monitored by bioluminescentimaging of the animals (n¼ 7/group). Representative images of mice after 6 weeks. E, immunohistochemical staining of representative tumors with H&E or an anti-HA tag antibody to detect R1 expression (left image of pair: scale bar, 2 mm; right image of pair: scale bar, 100 mm). F and G, transcript levels ofPAI-1 (F; as an indicator of TGFb signaling) and MYC expression in primary tumors (G) presented as relative quantification, normalized to GAPDH. H, samplebioluminescence images of lungs. I, MYC overexpression decreases number of lung metastases induced by CA TGFBR1. Results are presented as number ofmetastases per section in each group. Error bars, SEM; � , P < 0.5; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






female NOD/SCID mice were anesthetized with ketamine 100mg/kg and xylazine 10 mg/kg by intraperitoneal injection. Themice also received buprenorphine for pain relief at 0.1mg/kg. Theabdomen was shaved and sterilized using povidone iodine swab-sticks and ethanol. The left number four mammary fat pad wasexposed by performing a small Y-shaped incision in the skin.Suspension of MDA-MB-231 cells was prepared in a mix of 50%DMEM (Gibco) and 50% phenol red–free Matrigel (BD Bio-sciences). MDA-MB-231 cells (1 � 105) in 50 mL were injectedinto the number four fat pad using a 28-gauge insulin syringe (BDBiosciences). The incision was closed with 7-mm surgical clips(CellPoint Scientific). Mice were subcutaneously injected with0.9%NaCl (Baxter) andplacedon awarmingbedduring recovery.Mice were sacrificed at 6 weeks postsurgery. Tissues (primary

tumors and lungs) were formalin fixed and paraffin embeddedfor further analysis.

Biophotonic imaging of miceMice were injected intraperitoneally with 200 mg/kg body

weight of D-luciferin (Gold Biotechnology) in PBS, anesthetizedwith 1.5% to 2% isoflurane (Piramal Healthcare) using XGI-8anesthesia system (Xenogen) and imaged in IVIS 200 ImagingSpectrum System biophotonic imager (Xenogen). Imaging wasperformed within 5 to 10 minutes after injection. A region ofinterest (ROI) was drawn around each tumor and quantified.At the time of experimental endpoints, animals were injectedwith D-luciferin prior to sacrifice, and primary tumor and organswere removed and imaged. Analysis was performed using Living

Figure 2.TGFb modulates integrin avb3 levelsthrough regulation of MYC in basalsubtype breast cancers. A, MCF10Acells treated with TGFb decreasedtranscription of MYC and increasedtranscription of av and b3 integrins.Results are presented as relativequantification, normalized to GAPDH.Error bars, SEM. B, CA TGFBR1 (R1)acts as a surrogate for exogenousTGFb and decreases MYC whileincreasing integrin av and b3. C, MYCoverexpression under control of adoxycycline (DOX)-induciblepromoter inhibits TGFb-inducedupregulation of integrin subunits avand b3 in a dose-dependent manner.D–F, meta-analysis of MYC expressionusing GOBO; number of patientsindicated above plots. MYCexpressionby breast cancer stage (D; P ¼ 1e�5),by ER status (E; P < 1e�5), and intrinsicsubtype classified by PAM50(F; P < 1e�5). G and H, expression ofMYC, integrin aV (ITGAV), andintegrin b3 (ITGB3) in response toMYC knockdown (KD) and TGFbtreatment in basal cell lines MCF10A,MCF10CA1h, MCF12A, and SUM149 (G)and luminal cell lines BT474, MCF7,SKBR3, and T47D (H). Error bars, SEM;� , P < 0.5; �� , P < 0.01; �� , P < 0.001.

Cichon et al.





Image software (Xenogen). Luminescence is shown as photons/sec/ROI.

IHCFormalin-fixed, paraffin-embedded primary tumors and lungs

were sectioned and stained, either with hematoxylin and eosin(H&E) or specific antibodies. Briefly, the tissue sections mountedon glass slides were deparaffinized and rehydrated with distilledwater, followed by a 25-minute incubation in Target RetrievalSolution pH 6 (Dako) heated to 100�C. After a 25-minuteincubation at room temperature, the sections were blocked in 3%H2O2 for 5 minutes, then in serum-free protein block (Dako) foranother 5minutes. Primary antibody incubationswere performedat room temperature for 1 hour. Detection of exogenous TGFBRIused anti-HA tag rat monoclonal (Roche, #11867423001) 1:100.EnVisionþ System HRP DABþ (Dako, #K4007) anti-mouse andRat-on-Mouse Secondary Antibody Kit (Biocare, #RT517) wereused for secondary antibody incubations, performed for 30 min-utes, followed by a 5-minute incubation with the DABþ chro-mogen, and counterstained with hematoxylin, all at room tem-perature. Stained sectionswere scanned and imageswere capturedusing the T2 ScanScope console and Aperio ImageScope software(Aperio Technologies).

Statistical analysisData analysis was performed using Microsoft Excel and Graph-

Pad Prism. Bar graphs represent means � SEM, as indicated.Student t test or Mann–Whitney test was performed, as indicated,to assess statistical significance.

ResultsTGFb-induced downregulation of MYC expression leads toincreased metastasis

In a recent study, we found that MYC inhibits breast cancermetastasis in experimental models (11). Corroborating the clin-ical relevance of this tumor suppressor activity of MYC, weperformed a meta-analysis of published datasets using the Gene

Expression-Based Outcome for Breast Cancer Online (GOBO;ref. 25) and found that MYC expression in node-positive breastcancers is associated with improved distant metastasis-free sur-vival (Fig. 1A). Because TGFb is a key driver of breast cancermetastasis, we hypothesized that MYC downregulation isrequired for TGFb-induced breast cancer metastasis, and thusthat forced overexpression of MYC would inhibit TGFb-inducedmetastasis. We tested this hypothesis using metastatic MBA-MB-231 breast cancer cells in an orthotopic xenograft model. As asurrogate for exogenous TGFb in the in vivo experiments, wetransduced cells with the HA-tagged CA TGFBR1 (R1; ref. 26),which effectively activated a SMAD-responsive luciferase reporterconstruct (Fig. 1B). MDA-MB-231 cells were transduced with aretrovirus conferring constitutive expression of MYC or retroviralcontrol, then transduced with CA TGFBR1 lentivirus or control,and then injected into the mammary gland of immunocompro-mised mice. All cells were additionally transduced with lentiviralFirefly luciferase to enable tumor monitoring (Fig. 1C and D).Primary tumors from mice sacrificed after 6 weeks showed sus-tained tumor expression of HA-tagged CA TGFBR1 (Fig. 1E)and increased expression of TGFb-responsive plasminogen acti-vator inhibitor-1 (PAI-1) transcript levels (Fig. 1F) in tumorstransduced with the CA TGFBR1 construct, as well as sustainedMYC overexpression in the MYC-transduced cells (Fig. 1G).Consistent with our hypothesis, MYC expression significantlydecreased the number of lung metastases induced by CA TGFBR1(Fig. 1H and I), showing that TGFb-induced metastasis can beinhibited by expression of MYC.

TGFb-induced downregulation of MYC is necessary forinduction of integrin avb3

TGFb is known to be able to downregulate MYC (19, 20), andMYC has been recently shown to decrease expression of integrinavb3 in breast cancermodels (11).We found that these effects arelinked: inMCF10Abreast epithelial cells, an establishedmodel forTGFb-dependent regulation of MYC (27), TGFb treatment led todownregulation of MYC transcription and protein expression,

Figure 3.MYC reduction potentiates TGFb-induced upregulation of integrin avb3and an invasive cellular phenotype.A and B, TGFb treatment and MYCknockdown of MCF10A cells stimulateintegrin avb3 through a potentiationeffect that is dependent uponexpression of integrin avb3, assessedby protein expression (A) and cellinvasion (B). C–E, MCF10A cellscontinuously exposed to TGFb orexposed for a short period of time andthen cultured in the absence ofexogenous TGFb showed persistentexpression of MYC, integrin av(ITGAV) and integrin b3 (ITGB3; C),and TGFb-responsive reporterconstruct (D) through consistentactivation of TGFb expression (E).Transcriptional results are presentedas relative quantification, normalizedto GAPDH. Error bars, SEM; � , P < 0.5;�� , P < 0.01; �� , P < 0.001.






while upregulating transcript and protein levels of both integrinav and b3 (Fig. 2A). Similar results were obtained by exogenousexpression of CA TFBR1 (26), although expression of this trans-gene was less effective at regulating MYC, ITGAV, and ITGB3 thantreatment with TGFb (Fig. 2B). Of note, exogenous expression ofMYC inhibited TGFb-induced upregulation of integrin subunitsav and b3 in a dose-dependent manner (Fig. 2C), demonstratingthat TGFb-induced upregulation of integrin subunits av and b3 isdependent upon downregulation of MYC.

TGFb-dependent regulation of integrinavb3 is specific to basalbreast cancer cells

To begin to evaluate the clinical impact of TGFb-dependentdownregulation of MYC on breast cancer metastasis, we per-formed ameta-analysis ofMYCexpression using theGOBOserverand found that MYC expression was significantly increased withcancer grade (Fig. 2D) andwas elevated in ER� cancers versus ERþ

cancers (Fig. 2E). Strikingly, MYC expression was significantlyelevated in basal breast cancers compared with other subtypes inthese datasets (Fig. 2F), as has been noted previously (28, 29). Toevaluate the impact of subtype-dependent differences on TGFb-and MYC-dependent regulation of integrin avb3, we tested theeffect of MYC knockdown and TGFb treatment in a selection ofbreast cancer cell lines.We found that knockdownofMYC inbasalsubtype MCF10A and breast cancer cell lines led to increasedlevels of integrin subunits av and b3 (Fig. 2G), whereas similareffects were not observed in luminal subtype cell lines (Fig. 2H).Responsiveness of cell lines to TGFb was verified by checkingtranscriptional levels of PAI-1 (Supplementary Fig. S1).

Simultaneous TGFb treatment and MYC knockdownpotentiates cell invasion through highly amplified levels ofintegrin avb3

In evaluating TGFb treatment and MYC knockdown in basalsubtype breast cancer cell lines, we made the unexpected

discovery that MYC knockdown can potentiate the TGFb-induced increase in integrin av and b3 levels (Figs. 2Gand 3A). The striking increase in av and b3 levels induced bycombined TGFb treatment and MYC knockdown in MCF10Acells was reflected in equally striking potentiation of cellularinvasiveness in transwell assays (Fig. 3B; similar results withSUM149 cells, Supplementary Fig. S3). We further found thatpotentiation of invasion in MCF10A cells was specificallydependent on expression of integrin avb3, as the effect couldbe blocked by knocking down integrin av (Fig. 3A and B), andwe verified that the observed differences in cell invasion wereindependent of MYC-driven effects on cell proliferation (Fig. 3Band Supplementary Fig. S2A and S2B), which are not impactedby integrin av expression level.

The invasion assays of TGFb pretreated cells were performedin the absence of exogenous TGFb; thus, the observed TGFb-stimulated invasion suggested a persistent mechanism of sig-naling after its removal from the media. We found that theeffects of TGFb on downregulation of MYC, upregulation ofintegrin avb3, and SMAD-dependent signaling in MCF10A cellspersisted after removing exogenous TGFb from the cell culturemedium (Fig. 3C and D) and were associated with endogenousTGFb transcription that persisted once induced by exogenousTGFb (Fig. 3E). These results suggest that the effects of TGFb onMYC-dependent inhibition of proliferation and increased inva-sion, once induced, will require active targeting of susceptiblepathways to reverse.

Treatment of mammary epithelial cells with TGFb has beenshown to induce features of a claudin-low phenotype (30, 31),including highly increased invasiveness, acquisition of pro-genitor-like characteristics, and altered expression of manytranscripts, including downmodulation of the epithelial mark-er EpCAM (32). Flow analysis of MCF10A cells treated for3 days with TGFb revealed the induction of an EpCAM-lowpopulation; when the TGFb-treated cells were sorted by

Figure 4.MYC mediates TGFb-induced generationof EpCAM-high and EpCAM-low cellpopulations. A, MCF10A cells exposed toTGFb for 3 days were sorted into EpCAM-high and EpCAM-low populations,cultured for 6 additional days in theabsence of TGFb, and then reanalyzed forEpCAM expression. B, TGFb treatmentand MYC knockdown for 3 days stimulatedevelopment of the EpCAM-lowpopulation of MCF10A cells. C, MYCoverexpression under control of adoxycycline-inducible promoter inhibitsTGFb-induced development of theEpCAM-low population in a dose-dependent manner after 4-day treatmentof MCF10A cells. Insets in histograms,percent of population below threshold.

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EpCAM expression and cultured in the absence of TGFb for6 additional days, we found that the EpCAM-low populationdifferentiated into EpCAM-high parental-like cells, while theEpCAM-high cells maintained their differentiated status(Fig. 4A). We further found that MYC KD in MCF10A cellsinduced a similar induction of the EpCAM-low populationand that this was potentiated by TGFb (Fig. 4B), and that theTGFb-induced effect was blocked by doxycycline-dependentexogenous expression of MYC (Fig. 4C). These results suggestthat modulation of MYC levels is fundamental to the pheno-typic alterations induced by TGFb.

MYC knockdown potentiates TGFb-induced, integrin avb3–mediated invasion in a SRC-dependent mechanism

It has been previously shown that integrinavb3 can lead to SRCactivation and that integrin b3–dependent SRC activation canlead to activation of TGFb signaling (33–38).On the basis of theseprevious findings, we hypothesized that decreased MYC andconsequent increased expression of integrin b3 could activate an

SRC-dependent positive feedback loop (Fig. 5A). Consistent withthis hypothesis, we found that MYC knockdown led to potenti-ation of TGFb-induced increase of the SMAD-dependent PAI-1gene expression levels in MCF10A cells and that this effect couldbe inhibited by reduction of integrin avb3 (Fig. 5B) or SRC (Fig.5C). We also found that the potentiating effect of MYC knock-down on TGFb-induced integrin av and b3 expression could beinhibited either by SRC knockdown (Fig. 5D) or by pharmaco-logic inhibition of SRC using dasatinib (Fig. 5E and Supplemen-tary Fig. S4A), or by the alternative SRC inhibitor PP2 (Supple-mentary Fig. S4B). Furthermore, cellular invasion induced byMYC knockdown and TGFb treatment could also be inhibitedby dasatinib (Fig. 5F).

Pharmacologic MYC inhibition upregulates integrin avb3 andincreases cell invasion in an SRC-dependent manner

As inhibition of MYC represents a potential therapeuticstrategy for breast cancer, we next considered whether pharma-cologic inhibitors of MYC might also potentiate avb3-driven

Figure 5.MYC knockdown potentiates TGFb-induced upregulation of integrin avb3through a SRC-dependentmechanism. A, schematic of theproposed model of signaling. B and C,TGFb/MYC KD potentiated PAI-1expression is blocked by integrin av(ITGAV) KD (B) or SRC KD (C) inMCF10A cells. Transcriptional resultsare presented as relativequantification, normalized to GAPDH(C and D), SRC knockdown (D), ortreatment with SRC kinase inhibitordasatinib (E). Inhibition of MYCknockdown induced potentiation ofTGFb-induced increase in integrinavb3 in MCF10A cells. F, dasatinibinhibits MYC knockdown–inducedpotentiation of TGFb-inducedinvasion in MCF10A cells. Error bars,SEM; �, P < 0.5; ��, P < 0.01; ��,P < 0.001. ITGB3, integrin b3.






cellular invasion. We evaluated the effects of two different MYCinhibitors on MCF10A cells. Similar to the effects with MYCknockdown, we found that both JQ1 (Fig. 6A) andMI2 (Fig. 6B)led to dose-dependent increases in av and b3 integrin subunits(Supplementary Fig. S5A–S5C), with comparable results foundwith another basal subtype cell line MCF12A (SupplementaryFig. S5D). Also similar to the effects of MYC knockdown,pharmacologic inhibition of MYC with dasatinib potentiatedTGFb-induced upregulation of avb3 in an SRC-dependent fash-ion in MCF10A cells (Fig. 6C). Furthermore, MYC inhibitionwith MI2 stimulated cell invasion, and this effect could also beblocked by dasatinib (Fig. 6D), which also enhanced the anti-proliferative effects of MYC inhibition (Fig. 6E and F). Theseresults suggest that inhibition of MYC in basal breast cancercould have optimal therapeutic benefit when combined withtherapeutic inhibition of SRC.

DiscussionBasal subtypes of breast cancer have poor prognosis, with

high breast cancer mortality within the first few years afterdiagnosis and the highest risk of recurrence after therapy (39,40). The poor outcomes for basal breast cancer patients arerelated to the lack of available targeted therapies, in contrast tothe more common luminal subtypes for which standard ther-apies have been developed, as also to the intrinsically aggressivenature of the basal breast cancer disease (41). As a consequence,considerable effort has been devoted to identifying signalingpathways in basal subtype tumors that can be therapeuticallytargeted with sufficient specificity so as to minimize adverseeffects in normal tissue, toward development of personalized

therapy for women with basal breast cancer. Here, we haveidentified a signaling axis connecting TGFb, MYC, and integrinavb3 that controls both cellular proliferation and invasiveness,the key features of malignancy that underlie mortality, and weshow that this pathway is specific for basal breast cancer cells.We found that TGFb-induced suppression of MYC transcriptand protein levels specifically increased abundance of integrinavb3, thus promoting invasion and metastasis (Figs. 1 and 2).We demonstrated that knockdown or pharmacologic inhibi-tion of MYC effectively activates the feedback loop drivinginvasive and metastatic properties in basal breast cancer cells(Figs. 2–6), that this feedback loop can be interrupted by SRCinhibition (Fig. 5), and that pharmacologic inhibition of SRC issufficient to inhibit the proinvasive effects while maintainingthe antiproliferative characteristics of MYC inhibitors (Fig. 6).Our findings have important implications for using MYC as atherapeutic target and suggest that in certain cases MYCinhibition could potentially result in deleterious clinicalresponses, by promoting increased tumor metastasis. On thebasis of our findings, we propose a novel therapeutic strategyfor patients who have the basal subtype of breast cancerthrough simultaneous targeted inhibition of MYC and SRC.We have demonstrated that this strategy has the potential toblock the proliferative capacity of MYC while abrogating theactivation of invasion and metastasis mediated through SRCsignaling.

We found that the TGFb/MYC–dependent regulation of integ-rin avb3, including the potentiating effects of MYC knockdownand active TGFb signaling, were specific to basal subtype breastcancer cells lines (Fig. 2G and H). In luminal cell lines, not onlydid TGFb fail to downregulateMYC and increase levels of integrin

Figure 6.Pharmacologic MYC inhibitionupregulates integrin avb3 andincreases cell invasion in a SRC-dependent fashion. A and B, MYCinhibition by JQ1 (A) and MI2 (B)increases av and b3 protein levels andpotentiates theTGFb-induced effect inMCF10A cells. C, potentiation of TGFb-induced upregulation of integrin avand b3 by MI2-inhibited MYC isblocked by dasatinib in MCF10A cells.D, dasatinib (DAS) blocks MI2-dependent activation of invasion. Eand F, dasatinib further activates MI2-dependent inhibition of proliferation inMCF10A cells, assessed by EdU(images, E; quantification, F). Scalebars, 50 mm. Error bars, SEM; � , P <0.05; �� , P < 0.01. ITGAV, integrin av;ITGB3, integrin b3; ns, not significant.

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av or b3, but MYC knockdown itself had no effect on integrinavb3 (Fig. 2H). One possible contributor to the observed spec-ificity of this signaling axis is the increased expression of integrinb3 in basal breast cancer cell lines as compared with luminal-derived cell lines; of note, we have previously reported thatintegrin b3 is required for MYC knockdown–induced cellularinvasiveness (11).

One limitation of the study is the focus on the basal subtype ofbreast cancer, while at present, clinical decisions are made on thebasis of histologic analyses of marker expression. Breast cancersthat lack expression of ER, PR, and Her2, designated as triple-negative cancers, are frequently also basal subtype, while themajority of basal subtype cancers are also histologically triplenegative (41). At present, the biology and treatment of basalsubtype cancers that express hormone receptors or Her2 has notbeen clearly defined, although preliminary data suggesting thatbasal subtype tumors that are not triple negative behave similarlyto the larger population of triple-negative basal subtype tumors(42). Another limitation is that it is not clear whether the TGFb–MYC–integrin avb3 prometastatic pathway is limited to breastcancers or whether it may also be found in tumors developing inother organs. Systematic analyses directly comparing multiplecancer types have confirmed that basal subtype breast cancersare substantially different from luminal subtype breast cancers,reinforcing concepts that these represent very different diseaseswith distinct etiologies (43). These studies suggest that basalbreast cancers share many molecular similarities with serousovarian cancers, including a central role for MYC signaling; thepotential that the TGFb–MYC–integrin avb3 signaling axiscould exist in serous ovarian cancer cells offers an intriguingdirection for future study.

Our results have particular relevance for clinical studies thattherapeutically target MYC, particularly for basal breast cancerpatients. The majority of current clinical trials as listed on www.clinicaltrials.gov that target MYC employ agents that inhibitbromodomain and extraterminal (BET) family members. Selec-tive inhibitors of BET bromodomains, of which the agent JQ1used in our investigation is one of the best studied (44),constitute a family of agents that regulate a broad range oftranscriptional events, although inhibition of MYC transcrip-tion is believed to underlie their therapeutic effect (45, 46).Several other current clinical trials are testing the utility of

DCR-MYC, a siRNA targeting MYC (47), and these have shownpromising initial results for patient tolerance (10). Our findingspredict that although these agents may effectively inhibit basalbreast cancer cell proliferation, there is likely to be increasedincidence of relapse with metastasis. Importantly, our resultsalso point to a solution to this problem in that inhibitors ofMYC expression can be combined with agents that target SRC,such as dasatinib. Although dasatinib has shown limited effi-cacy as a single agent for triple0negative (48) and hormonereceptor–positive breast cancer patients (49), our results sug-gest that combination therapy of a MYC inhibitor with dasa-tinib could confer both antiproliferative and antimetastaticeffects. Clinical trials evaluating such a combination therapyin basal breast cancer patients will be required to test thisstrategy.

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

Authors' ContributionsConception and design: M.A. Cichon, M.E. Moruzzi, D.C. RadiskyDevelopment of methodology: M.A. Cichon, M.E. Moruzzi, T.A. Shqau,D.C. RadiskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.A. Cichon, M.E. Moruzzi, E. Miller, C. Mehner,S.P. Ethier, D.C. RadiskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.A.Cichon,M.E.Moruzzi, T.A. Shqau, J.A. Copland,E.S. Radisky, D.C. RadiskyWriting, review, and/or revision of the manuscript: M.A. Cichon, S.P. Ethier,J.A. Copland, E.S. Radisky, D.C. RadiskyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.A. Shqau

Grant SupportThis workwas supported by theNCI (CA187692) and theMayoClinic Breast

Cancer SPORE (CA116201).The costs of publication of this articlewere defrayed inpart by the payment of

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

Received December 19, 2015; revised March 2, 2016; accepted March 18,2016; published OnlineFirst April 13, 2016.

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2016;76:3520-3530. Published OnlineFirst April 13, 2016.Cancer Res Magdalena A. Cichon, Megan E. Moruzzi, Tiziana A. Shqau, et al. Breast Cancer

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