mtbp is overexpressed in triple-negative breast cancer and ... · triple-negative breast cancer...

Cell Death and Survival

MTBP Is Overexpressed in Triple-Negative Breast Cancerand Contributes to Its Growth and Survival

Brian C. Grieb1, Xi Chen2, and Christine M. Eischen1

AbstractTriple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to

therapeutics that have been successful in increasing survival in patients with estrogen receptor–positive (ERþ) andHER2þ breast cancer. As such, identifying factors that contribute to poor patient outcomes andmediate the growthand survival of TNBC cells remain important areas of investigation. MTBP (MDM2-binding protein), a genelinked to cellular proliferation and a transcriptional target of the MYC oncogene, is overexpressed in humanmalignancies, yet its contribution to cancer remains unresolved. Evaluation of mRNA expression and copy numbervariation data from The Cancer Genome Atlas (TCGA) revealed thatMTBP is commonly overexpressed in breastcancer and 19% show amplification of MTBP. Increased transcript or gene amplification of MTBP significantlycorrelated with reduced breast cancer patient survival. Further analysis revealed that while MTBP mRNA isoverexpressed in both ERþ and HER2þ breast cancers, its expression is highest in TNBC. MTBP mRNA andprotein levels were also significantly elevated in a panel of humanTNBC cell lines. Knockdown ofMTBP in TNBCcells induced apoptosis and significantly reduced TNBC cell growth and soft agar colony formation, which wasrescued by expression of shRNA-resistantMtbp.Notably, inducible knockdown ofMTBP expression significantlyimpaired TNBC tumor growth, in vivo, including in established tumors. Thus, these data emphasize thatMTBP isimportant for the growth and survival of TNBC and warrants further investigation as a potential novel therapeutictarget.

Implications:MTBP significantly contributes to breast cancer survival and is a potential novel therapeutic target inTNBC. Mol Cancer Res; 12(9); 1216–24. �2014 AACR.

IntroductionThe use of the estrogen receptor (ER), progesterone

receptor (PR), and human epidermal growth factor receptor2 (HER2) as biomarkers is standard practice in the clinicalmanagement of breast cancer. Their expression directs theuse of targeted therapeutics, such as tamoxifen and trastu-zumab, that have dramatically improved patient survival.Unfortunately, such improvements in clinical outcomeshave not been realized in the management of triple-negativebreast cancer (TNBC), a subset of breast cancers lackingHER2 amplification and expression of ER and PR (1).TNBC comprises 10% to 20% of breast cancer cases andis more commonly identified in younger women and thosewith African-American or Hispanic heritage (2). It is clin-

ically aggressive, correlating with an increased risk of distantrecurrence within 3 years following treatment and a signif-icant decrease in overall patient survival, compared withreceptor-positive cases (3, 4). While there has been somesuccess in exploiting novel molecular targets, such as PARPinhibitors in BRCA1-mutant tumors with errors in DNAbreak repair (5, 6), these cases are isolated and applicable toonly select TNBCs. Other targets such as mTOR, Src, andHER1 tested in phase II clinical trials have shown onlyminimal success (7–9). Thus, there is a need to identify andtest the therapeutic efficacy of novel molecular targets inTNBC.The Mdm2 (Two)-binding protein (MTBP) was first

identified as a potential tumor suppressor that binds Mdm2,a negative regulator of p53 (10). However, subsequentgenetic studies indicated it functions independent of Mdm2and, instead, contributes to tumor development induced bythe Myc oncogene (11–13). Recently, MTBP has beenimplicated in regulating proliferation and cell-cycle progres-sion (12, 14, 15).MTBP is a transcriptional target of MYC,and its protein expression increased in response to propro-liferative signals and decreased upon growth factor with-drawal (12). In mouse models, Mtbp heterozygosity led toreduced levels of Mtbp protein and this inhibited Myc-inducedB-cell proliferation, resulting in a significant delay in

1Department of Pathology, Microbiology and Immunology, VanderbiltUniversity Medical Center, Nashville, Tennessee. 2Department of Biosta-tistics, Vanderbilt University Medical Center, Nashville, Tennessee.

Corresponding Author: Christine M. Eischen, Vanderbilt University Med-ical Center, Department of Pathology, Microbiology and Immunology,C3321 MCN, 1161 21st Avenue South, Nashville, TN 37232. Phone:615-322-3234; Fax: 615-343-1633; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-14-0069

�2014 American Association for Cancer Research.

MolecularCancer

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lymphoma development (12). Furthermore, siRNA-medi-ated knockdown of MTBP was reported to delay cell-cycleprogression through the S and G2–Mphases of the cell cycle(14, 15). Therefore, MTBP appears to contribute to thedevelopment and possibly the maintenance of tumorsthrough regulation of proliferation, but further investigationis needed.Here, we report thatMTBP is overexpressed and amplified

in breast cancer, correlating with decreased patient survival.Notably, MTBP mRNA expression was highest in TNBC.shRNA-mediated knockdown of MTBP in human TNBCcell lines inhibited their expansion and induced apoptosis, invitro, as well as significantly reduced tumor growth, in vivo.Our data reveal that MTBP significantly contributes tobreast cancer and is a potential novel therapeutic target inthe treatment of TNBC.

Materials and MethodsPatient dataPatient survival and gene expression data for 844 breast

cancers was accessed from The Cancer Genome Atlas(TCGA) public data portal (https://tcga-data.nci.nih.gov/tcga/) January–April 2013. For Kaplan–Meier survivalcurves, normalized RNA-Seq data (version 2, level 3) wasused as gene expression values and the median was used toclassify samples into high and low expression groups. Log-rank tests were used to compare survival between groups.Box and whisker plots (box represents first and third quar-tiles, thick band is median value, and bars extend to �1.58the interquartile range divided by the square root of thenumber of samples) were applied to describe MTBP geneexpression values. Groups were compared using a Wilcoxonrank-sum test. Gene copy number alteration (CNA) andsurvival data for 913 breast cancers was obtained from thecBioPortal for Cancer Genomics (http://www.cbioportal.org/public-portal/) May 2013.

Cell culture, vectors, transfection, and infectionThe human cell lines MDA-MB-231, HCC1806, and

HCC1937were cultured as described by the American TypeCulture Collection and were provided by Dr. JenniferPietenpol. Cells were transfected with Effectene (Qiagen)or were infected with retroviruses, as previously described(16). MTBP shRNA 19 mer sequences (shRNA1 GGA-GAGTGTTCTAGCTATT or shRNA2 GAAACACAG-TATTACCGAG) and nontargeting control (GACTTAC-GAGATCAGAAAG) were used in pSuper constitutiveexpression constructs (Oligoengine) and were adapted tothe doxycycline-inducible system (pInducer) generouslyprovided by Dr. Thomas Westbrook (17) using the RNAicentral shRNA retriever (http://cancan.cshl.edu/RNAi_-central/RNAi.cgi?type ¼ shRNA).

Proliferation, cell cycle, apoptosis, and transformationassaysFor measurement of proliferation, 1,000 to 5,000 cells

were plated in triplicate and MTT assays were performed asper manufacture's protocol (Sigma). Cell cycle (Dean–Jett–

Fox analysis) and apoptosis (sub-G1 DNA content) wereevaluated with FlowJo software (TreeStar Inc.) followingDNA staining with propidium iodide and flow cytometry.Apoptosis was also evaluated by flow cytometric analysis ofAnnexin V–APC binding (Life Technologies) and caspase-3cleavage by Western blotting (see below). Cell viability wasassessed by Trypan Blue Dye exclusion. Soft agar assays wereperformed as previously described (18). For doxycycline-inducible shRNA experiments, 0.5 to 1 mg/mL of doxycy-cline was added to the cultures.

MiceFemale athymic nude mice (5–6 week old; Harlan) were

injected subcutaneously in the flank with 3 � 106

HCC1806 cells. Mice were housed with drinking watersupplemented with 5% sucrose with or without 2 g/L ofdoxycycline (Research Products International Corp.) thatwas changed every 48 hours beginning on the day ofinjection or 10 days later. Tumor volume was calculatedfrom measurements with electronic calipers. At time ofsacrifice, mice were photographed, and tumors wereextracted, photographed, and weighed. A piece of eachtumor was frozen for Western blot analysis. All experimentswere approved by the Vanderbilt Institutional Animal Careand Use Committee and followed all federal and state rulesand regulations.

Western blotting and quantitative real-time PCRanalysisForWestern blotting, cells or tumors that were infected or

transiently transfected (see above) were harvested after 48hours or at indicated times and were lysed as previouslyreported (16, 18). Equal amounts of proteinwere resolved bySDS-PAGE and Western blotted using antibodies specificforMTBP (B5, Santa Cruz Biotechnology), cleaved caspase-3 (D175, Cell Signaling Technology), MYC (C33, SantaCruz Biotechnology), and b-ACTIN (AC15, Sigma). Toevaluate mRNA expression, total RNA was isolated, cDNAwas generated, and qRT-PCR for MTBP and b-ACTINlevels was performed as previously described (12). mRNAdata are relative to b-ACTIN levels.

Statistical evaluationWilcoxon rank-sum test (Figs. 1A and 2A), log-rank tests

(Fig. 1B and C), the Student t-test (Figs. 2–6), and Coxregression analysis were used to compare data. Error barsrepresent SD (Figs. 2–5) or SEM (Fig. 6).

ResultsMTBP is overexpressed in human breast cancer andcorrelates with decreased patient survival and triple-negative statusWe previously detected MTBP/Mtbp overexpression in

human lymphoma cell lines and primary murine lymphomas(12). MTBP was also reported amplified in colorectal carci-noma andmultiplemyeloma (19, 20), as well as several humancancer cell lines (21), suggesting that its overexpression

MTBP Regulates Breast Cancer Survival

www.aacrjournals.org Mol Cancer Res; 12(9) September 2014 1217




contributes to human cancers. To specifically evaluateMTBPexpression in human breast cancer, mRNA expression andpatient survival data for 844 breast cancers from TCGA wereassessed. MTBP was significantly elevated in breast cancersamples comparedwithnormal breast tissue (P¼ 2.2� 10�16;Fig. 1A). When cancers were separated by their MTBPexpression, those patients whose breast cancers had elevatedMTBP expression exhibited reduced overall survival com-pared with patients whose breast cancers had lower levels ofMTBP (P ¼ 0.0337; Fig. 1B). A Cox regression analysis

also showed that increased MTBP levels are significantlylinked with worse patient survival (P ¼ 0.033). Moreover,MTBP was amplified in 19% of breast cancers, and thisamplification decreased overall patient survival comparedwith tumors without amplified MTBP (P ¼ 0.01955;Fig. 1C; refs. 22, 23). These data indicate that MTBPoverexpression is common and thus, likely selected forduring breast cancer formation and/or progression. Theresults also show that elevated levels of MTBP correlatewith reduced breast cancer patient survival.

Figure 1. MTBPoverexpression in breast cancer decreases survival. A andB,RNA-SeqmRNAexpression data for normal and breast cancer tissue fromTCGAdatabase. A, box and whisker plot of relative MTBP mRNA expression in normal and cancerous breast tissue with n indicating the number of samples.B, Kaplan–Meier survival curves for patients with breast cancer divided by themedian value into low and highMTBPmRNAexpression (n¼ 421 per group). C,Kaplan–Meier survival curves ofMTBP gene copy number in breast cancer samples with (n¼ 171) and without (n¼ 742) amplifiedMTBP from the cBio Portalfor Cancer Genomics (22, 23). P values calculated using a Wilcoxon rank-sum test for A and a log-rank test for B and C.

Figure 2. MTBP is overexpressed in TNBC. A, box and whisker plot of relative MTBP mRNA expression from TCGA RNA-Seq mRNA expression data,representing normal breast tissue and breast cancers divided into ERþ, HER2þ, or triple-negative (TN: ER�, PR�, HER2�). The n indicates the number ofsamples. B, expression ofMTBPmRNAmeasured by qRT-PCR in TNBC cell lines and normal HMECs. (�,P� 0.0007). C,Western blot analyses of whole-celllysates of TNBC cell lines and HMECs for the proteins indicated.

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To assess MTBP levels in different subtypes of breastcancers, we separated the TCGA breast cancer patientsamples into clinically relevant subgroups: ER-positive(ERþ), HER2-positive (HER2þ), and triple-negative(TN: ER�, PR�, HER2�) tumors. While MTBP mRNAwas elevated significantly in all 3 subgroups comparedwith normal breast tissue, TNBC expressed significantlymore MTBP than the ERþ or HER2þ subgroups (Fig.2A). This finding was supported by the observation thatMTBP mRNA was also significantly elevated in a panel ofhuman TNBC cell lines compared with normal humanmammary epithelial cells (HMEC; Fig. 2B). To determinewhether the increased mRNA levels translated intoincreased protein, the levels of MTBP protein wereassessed. MTBP protein levels were elevated in all of theTNBC cells (Fig. 2C). In comparison, the oncogenictranscription factor MYC, which has previously beenshown to positively regulate MTBP expression and to beelevated in aggressive breast cancers (12, 24), was alsoelevated in these same cells (Fig. 2C). Therefore, MTBPmRNA levels are the highest in patient samples of theclinically aggressive TNBC subtype, and TNBC cell lineshave high levels of MTBP mRNA and protein.

Reducing MTBP levels inhibit TNBC cell proliferationConsidering Mtbp expression increases in response to

proproliferative factors (12), and MTBP is highly over-expressed in TNBC, we questioned whether reducing levelsof MTBP in human TNBC cells would alter their ability toproliferate. To begin to test this concept, we knocked down

MTBP expression with 2 different MTBP shRNAs in theMDA-MB-231 and HCC1806 TNBC cell lines. Whileboth cell lines overexpress MTBP (Fig. 2B), they representdistinct subtypes of TNBC (mesenchymal-like and basal-like, respectively), and HCC1806 cells have an MTBPamplification (21, 25). In both cell lines, reduced MTBPexpression resulted in a decrease in proliferation that corre-lated with the amount of MTBP protein present, whereMTBP shRNA1 was more effective at reducing MTBPprotein levels than MTBP shRNA2 (Fig. 3A). Similarly,anchorage-independent growth in soft agar was significantlyreduced for both the MDA-MB-231 and the HCC1806cells when MTBP was knocked down (Fig. 3B). To ensurethe observed effects were due to reduced MTBP expression,shRNA resistantmurineMtbpwas co-expressed withMTBPshRNA1. The murine Mtbp rescued the ability of MDA-MB-231 cells to form colonies in soft agar in the presence ofMTBP shRNA1, whereas cells cotransfected with vectorcontrol and MTBP shRNA1 still showed decreased colonyformation (Fig. 3C). These results indicate that knockdownof MTBP inhibits TNBC expansion and anchorage-inde-pendent growth.To further evaluate the antiproliferative effects of

MTBP knockdown in TNBC cells, MTBP shRNA1 wasadapted to a lentiviral doxycycline-inducible system (17).Using the 3 TNBC cell lines MDA-MB-231, HCC1806,and the basal-like HCC1937 cells that also overexpressMTBP (Fig. 2C), we observed that doxycycline-inducedMTBP shRNA1 resulted in reduced MTBP proteinexpression within 24 hours (Fig. 4A) and significantly

Figure 3. MTBP knockdown inhibitsproliferation and colony growth.MDA-MB-231 or HCC1806 cellswere transfected withconstitutively expressing MTBPshRNA1, shRNA2, or nontargeting(NT) control shRNA vectors. A,whole-cell lysates were Westernblotted, andcellswere subjected toMTT assays at 24-hour intervals(for MDA-MB-231 at 48–72 hoursP < 0.001 for NT vs. shRNA1 orshRNA2 and P < 0.01 for shRNA1vs. shRNA 2; for HCC1806P < 0.01for NT vs. shRNA1 or shRNA2 at24–96 hours and P ¼ 0.0013 forshRNA1 vs. shRNA2 at 96 hours).B, cells were subjected to soft agarcolony formation assay (MDA-MB-231: �, P ¼ 0.0004;��, P ¼ 0.0021 and HCC1806:�, P ¼ 0.0036; ��, P ¼ 0.0136for NT vs. shRNA1 or shRNA2,respectively). C, MDA-MB-231cells transiently transfected withMTBP shRNA1 or NT shRNA andshRNA1-resistant murine Mtbp orvector control were subjected tosoft agar colony formation assay(�, P < 0.0001 for NT vs. shRNA1and shRNA1 vs. shRNA1 þ Mtbp).






decreased (37%–40% reduced) proliferation within 72hours (Fig. 4B) in all three lines. The steady-state levels ofMYC remained unchanged (Fig. 4A). Expression of theshRNA-resistant murine Mtbp allowed MTBP shRNA1-expressing MDA-MB-231 cells to continue growing atrates analogous to that of cells treated with vehicle control(Fig. 4C). Therefore, disruption of MTBP expression withconstitutive or inducible shRNA caused a reduction in theability of TNBC cells to form colonies in soft agar and toproliferate.

MTBP knockdown induces apoptosis in TNBC cellsTo investigate the biologic reason thatMTBP knockdown

inhibited the expansion of TNBC cells, HCC1806 cellsexpressing the doxycycline-inducibleMTBP shRNA1 or thenontargeting shRNA control were cultured with or withoutdoxycycline for 72 hours. At this time, there were visiblyfewer adherent and more floating doxycycline-treatedMTBP shRNA1-expressing cells compared with vehiclecontrol-treated cells and nontargeting shRNA-expressingcells cultured with or without doxycycline (Fig. 5A). Eval-uation of the cell cycle revealed no significant difference inG1, S, or G2–M distribution of the cells where MTBPshRNA1 had been induced with doxycycline compared withvehicle control (Fig. 5B). In contrast, doxycycline-treated

MTBP shRNA1-expressing HCC1806 cells had an increasein the percentage of cells with sub-G1 DNA content(Fig. 5B). There was also a significant decrease in viability(Fig. 5C) and an increase in Annexin V–positive (Fig. 5D)MTBP shRNA1-containing cells. Moreover, in HCC1806cells withMTBP shRNA1, cleaved caspase-3 was visible afterthe addition of doxycycline compared with vehicle control orwith those cells with the nontargeting shRNA control (Fig.5E). Thus, shRNA-mediated knockdown of MTBP inTNBC cells induced apoptosis, without detectable altera-tions in phases of the cell cycle.

MTBP loss inhibits TNBC growth, in vivoTo evaluate whether MTBP knockdown would alter

TNBC growth in vivo, HCC1806 cells expressing doxycy-cline-inducible MTBP shRNA1 were subcutaneouslyinjected into the flanks of athymic nude mice. Their drink-ing water was supplementedwith or without doxycycline. Byday 7, tumors in mice receiving doxycycline to induceMTBP shRNA1 showed a statistically significant decreasein volume compared with tumors in control mice notreceiving doxycycline (24 vs. 43 mm3; P < 0.0001), andthis difference continued to increase through the duration ofthe experiment (Fig. 6A). At the time of sacrifice (day 21),tumors that expressed MTBP shRNA1 due to doxycycline

Figure 4. Inducible MTBP shRNAinhibits cell expansion. MDA-MB-231, HCC1806, and HCC1937cells expressing doxycycline (dox)-inducible MTBP shRNA1 ornontargeting (NT) shRNA control.A, Western blot analyses of whole-cell lysates not exposed todoxycycline or at intervals after theaddition of doxycycline. B, cellswere cultured with doxycycline orvehicle (veh) control andproliferation was monitored byMTT assay at 24-hour intervals(�, P < 0.01 shRNA1 þ veh vs.shRNA1 þ dox). C, MTT assay ofMDA-MB-231 cells expressingdoxycycline-inducible MTBPshRNA1 and constitutivelyexpressing shRNA1-resistantmurine Mtbp or empty vectorcontrol after 72 hours in thepresence of doxycycline orvehicle control (no dox;�, P ¼ 0.0027).

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exposure were smaller in volume and weighed significantlyless than the tumors from control mice (Fig. 6A–C). Therewas a 70% reduction in tumor volume in themice expressingMTBP shRNA1 compared with controls. These tumorsshowed reduced levels of MTBP protein, verifying MTBPshRNA1 expression persisted over the course of the exper-iment (Fig. 6C). MYC protein levels were similar in alltumors (Fig. 6C).We also tested whether established TNBC tumors would

be affected by knockdown ofMTBP. Specifically,HCC1806cells expressing the doxycycline-inducible MTBP shRNA1were injected into the flanks of nudemice at the same time ascells for the experiment described above and were allowed togrow. After 10 days when the tumors averaged 100 to 150mm3, these mice were given doxycycline to induce MTBPshRNA1 expression. Within 72 hours these tumors weresignificantly smaller than tumors that were not exposed todoxycycline (188 vs. 350 mm3; P¼ 0.0057), yet tumors didnot completely disappear (Fig. 6A). Instead, after the initialdecrease in tumor size, a significant reduction in the rate of

tumor growth was observed that was analogous to the rate oftumor growth for the mice that received doxycycline on dayone. At sacrifice (day 21), the tumors from the mice thatreceived doxycycline to induceMTBP shRNA1 after tumorshad established were smaller and weighed significantly lessthan the tumors from mice that never received doxycycline(Fig. 6B and C). The tumors were similar in size and weightto the tumors frommice that been exposed to doxycycline forthe entire experiment. These data collectively indicate thattargetingMTBP, reducing its expression, significantly limitsthe growth of TNBC cells in vivo, including establishedTNBC tumors.

DiscussionStudies have linked MTBP to cancer (11, 12, 19, 20, 26),

but little was known aboutMTBP in established cancer cells,particularly how it influenced proliferation, cellular survival,and patient outcomes. Here, we show MTBP is overex-pressed in human breast cancer, and this correlated withsignificantly decreased patient survival. Notably, among the

Figure 5. MTBP knockdowninduces apoptosis. HCC1806 cellsexpressing doxycycline (dox)-inducible MTBP shRNA1 ornontargeting (NT) shRNA controlincubated with doxycycline orvehicle control (no dox) for 72hours. A, representative lightmicroscopy images. B, thepercentage of cells with sub-G1

DNA content, following staining ofDNA with propidium iodide, wasmeasured by flow cytometry.Representative histograms withthe percentage of sub-G1 DNAindicated (left); themeanof thedataobtained shown on the right(�, P ¼ 0.0008). C, cell viability wasdetermined by Trypan Blue dyeexclusion (�, P � 0.0003 MTBPshRNA doxycycline vs. MTBPshRNA no doxycycline or NTdoxycycline). D, the percentage ofAnnexin V–positive cells relative tosamples at time0wasmeasuredbyflow cytometry (�, P ¼ 0.034;��, P ¼ 0.0069). E, Western blotanalyses for the indicated proteinswere performed on whole-celllysates.






different breast cancer subtypes, we determined thatMTBPexpression was highest in the TNBC subtype, which lackstargeted therapies and is known for being clinically aggressive(1, 3, 4). Experiments also revealed that reducing MTBPexpression in human TNBC cell lines with shRNA signif-icantly inhibited cell expansion by inducing apoptosis. Thegrowth-inhibitory effects of MTBP knockdown in TNBCcells were also observed in vivo, in xenografts, and, impor-tantly, in established TNBC tumors. Therefore, this studyidentifies MTBP as an important indicator of poor breastcancer patient prognosis and triple-negative status as well asbeing critical for the growth and survival of TNBC cells. The

results of this study support further investigation intoMTBPas a novel therapeutic target in TNBC.This study reveals that MTBP overexpression contributes

in a significant way to human breast cancer and increasesunderstanding of MTBP in cancer. Specifically, we previ-ously, reported that MTBP is overexpressed in human andmurine B-cell lymphomas (12). Others have shown that theregion of the genome encoding MTBP is amplified incolorectal cancer and multiple myeloma (19, 20). Similarly,evaluation of copy number variation data from TCGAindicates that MTBP is amplified in many types of humancancer (13, 22, 23). For breast cancer, we determinedMTBPamplification occurred in 19% of the tumors, and thissignificantly correlated with decreased patient survival.Because most breast cancer deaths are associated with metas-tasis, the current analysis suggests thatMTBP overexpressionis a potential novel indicator of aggressive breast cancers withincreasedmetastatic potential that are more likely to result inpatient death. However, this concept conflicts with exper-imental data that indicate decreased MTBP expressionincreases cell migration, invasion, and metastasis (11, 26).It is possible that both are correct if, as has been shown forMYC (27), MTBP is temporarily downregulated whencancer cells move and is then upregulated after cancer cellsseedmetastatic sites and begin to proliferate again.However,there is also one report showing that decreased MTBPexpression in a narrow subset of head and neck cancercorrelated with reduced survival (28). Thus, althoughMTBP could have a tissue-specific or cancer cell muta-tion–specific function, much of the data suggest that inmultiple hematopoietic and nonhematopoietic human can-cers, including breast cancer, MTBP overexpression isselected for and contributes to cancer development andprogression. The current study links MTBP overexpressionin breast cancer to advanced disease and poor patientprognosis.Data from multiple groups, including our own, suggest

that MTBP has a critical function in proliferation, and thatthis significantly contributes to tumor development (12,13, 15). Specifically, we previously determined that anMtbphaploinsufficiency suppressed proliferation mediated by theMyc oncogene, significantly inhibiting the ability of Myc toinduce B-cell lymphoma development (12). Moreover,MTBPmRNA and protein expression increased in responseto oncogene expression (MYC and E2F1) or growth factorexposure, and MTBP was shown to be a transcriptionaltarget of MYC (12). Others have indicated that MTBPcontributes to cell-cycle progression by linking MTBP toDNA replication origins andmitotic progression (14, 15). InAddition, we recently determined that elevated levels ofMTBP resulted in enhanced cellular proliferation and trans-formation, in vitro and in vivo (13). Here we showMTBP isoverexpressed in breast cancers and its expression is thehighest in the TNBC subtype. TNBCs are reported to havea higher proliferative index than receptor-positive high-gradeinvasive carcinomas (29). In addition, elevated MYC tran-scriptional activity, which is correlated with decreased breastcancer patient survival, is linked to increased proliferation in

Figure 6. Knockdown of MTBP inhibits breast cancer growth in vivo. A,HCC1806cellswere injected subcutaneously into the flanks of nudemice(n ¼ 10 per group) on day 0. Mice received drinking water with (þdox)or without doxycycline (no dox) at day 0 or water with doxycyclinestarting at day 10 (� dox; indicated by arrow). Tumor volume wasmeasured at intervals (P < 0.001 for shRNA1þ no dox vs. shRNA1þ doxfor days 7–21 and for shRNA1 þ no dox vs. shRNA1 � dox for days13–21). B, on day 21, mice were sacrificed and photographed. Arepresentative photo of all 3 treatment groups is shown with the tumorsoutlined in black. C, tumors were extracted, photographed, and weighed(�, P ¼ 0.0058 and ��, P ¼ 0.0024 compared with no doxycycline). Arepresentative photograph is shown. Protein lysates from representativetumors were Western blotted for the indicated proteins.

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breast cancer, and the TNBC subtype has the highest MYCtranscriptional activity (24). We recently determinedpatients with breast cancers who express high levels of bothMYC and MTBP have a worse prognosis than those withjust high MYC expression (13), suggesting cooperationbetween MYC and MTBP overexpression in breast cancer.Therefore, MTBP appears to be a proproliferative factorwhere its overexpression supports the increased proliferativecapacity of cancer cells, which is associated with poor patientsurvival in many human cancers (29–35).Deletion ofMtbp is embryonic lethal inmice, indicating it

has an indispensable function in development (11). Here weshow that MTBP also has an essential function in breastcancer cell survival. Knockdown ofMTBP in human TNBCcell lines using constitutive or inducible MTBP shRNAseverely limited TNBC growth, in vitro and in vivo, due toinduction of apoptosis. This is similar to the oncogeneaddiction–mediated cell death observed when an oncogene,such as MYC, is knocked down in cancer cells (36–38),suggesting that TNBC cancer cells can become reliant onMTBP for their continued growth and survival. Support ofthis concept was reported when knockdown ofMTBP withsiRNA inHeLa cells delayedDNA replication ormitosis andalso led to cell death (14, 15). Although we did not detectcell-cycle changes with MTBP knockdown in TNBC cells,as was reported for HeLa cells (14, 15), we did observesignificant apoptosis resulting in reduced TNBC cell sur-vival. The apoptosis that occurred uponMTBP knockdownresulted in a reduction in the ability of the TNBC cells togrow in soft agar and in vivo in mice. Notably, usinginducible MTBP shRNA revealed that, in vivo, establishedbreast cancers rely on MTBP for their continued growth.Therefore, our data identify MTBP as a protein TNBC cells

need to survive and grow. Thus, MTBP is a potential noveltherapeutic target in TNBC warranting further investiga-tion. Moreover, additional studies are needed to examinewhether MTBP has a similar essential function in otherhuman malignancies, including receptor-positive subtypesof breast cancers.

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

Authors' ContributionsConception and design: B.C. Grieb, C.M. EischenDevelopment of methodology: B.C. Grieb, C.M. EischenAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): B.C. Grieb, C.M. EischenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): B.C. Grieb, X. Chen, C.M. EischenWriting, review, and or revision of the manuscript: B.C. Grieb, C.M. EischenAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): C.M. EischenStudy supervision: C.M. Eischen

AcknowledgmentsThe authors thank Brandon Metge for technical assistance, Dr. Jennifer Pietenpol

for the breast cancer cell lines, Dr. Thomas Westbrook for pInducer, and members ofthe Eischen laboratory for helpful discussion and review of the manuscript.

Grant SupportThese studies were supported by F30AG039164 (B.C. Grieb), the Vanderbilt

MSTP T32 GM007347 (B.C. Grieb), R01CA148950 (C.M. Eischen), the Vander-bilt Breast Cancer SPORE CA098131, and the CTSA UL1TR000445 from theNational Center for Advancing Translational Sciences, and the NCI Cancer CenterSupport Grant P30CA068485 using the Flow Cytometry and the TranslationalPathology Shared Resources.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received February 5, 2014; revised April 15, 2014; accepted May 16, 2014;published OnlineFirst May 24, 2014.

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Grieb et al.




2014;12:1216-1224. Published OnlineFirst May 27, 2014.Mol Cancer Res Brian C. Grieb, Xi Chen and Christine M. Eischen Contributes to Its Growth and SurvivalMTBP Is Overexpressed in Triple-Negative Breast Cancer and

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