Survivin Is a Viable Target for the Treatment of Malignant

Cancer Therapy: Preclinical

Survivin Is a Viable Target for the Treatment of MalignantPeripheral Nerve Sheath Tumors

Markus P. Ghadimi1,4, Eric D. Young1,4, Roman Belousov1,4, Yiqun Zhang6, Gonzalo Lopez3,4,7,Kristelle Lusby1,4, Christine Kivlin3,4,7, Elizabeth G. Demicco2, Chad J. Creighton6, Alexander J. Lazar2,4,7,Raphael E. Pollock1,4,7, and Dina Lev3,4,5,7

AbstractPurpose:Toexamine the role of survivin as a therapeutic target inpreclinicalmodels of humanmalignant

peripheral nerve sheath tumors (MPNST)

Experimental Design: Survivin protein expression levels and subcellular localization were examined

immunohistochemically in an MPNST tissue microarray. Human MPNST cells were studied in vitro and in

vivo; real-time PCR, Western blotting, and immunocytochemical analyses were used to evaluate survivin

expression and localization activation. Cell culture assays were used to evaluate the impact of anti-survivin–

specific siRNA inhibition on cell growth and cell-cycle progression and survival. The effect of the small-

molecule survivin inhibitor YM155 on local and metastatic MPNST growth was examined in vivo.

Results: Survivinwas found tobe highly expressed in humanMPNSTs; enhanced cytoplasmic subcellular

localization differentiatedMPNSTs from their plexiform neurofibroma premalignant counterparts. Human

MPNST cell lines exhibited survivin mRNA and protein overexpression; expression in both nuclear and

cytoplasmic compartments was noted. Survivin knockdown abrogated MPNST cell growth, inducing G2

cell-cycle arrest andmarked apoptosis. YM155 inhibited humanMPNST xenograft growth andmetastasis in

severe combined immunodeficient (SCID) mice. Antitumor effects were more pronounced in fast-growing

xenografts.

Conclusions:Our studies show an important role for survivin in human MPNST biology. Patients with

MPNSTs should be considered for ongoing or future clinical trials that evaluate anti-survivin therapeutic

strategies. Most importantly, future investigations should evaluate additional pathways that can be targeted

in combination with survivin for maximal synergistic anti-MPNST effects. Clin Cancer Res; 18(9); 2545–57.

�2012 AACR.

IntroductionCharacterized by aggressive local growth, propensity

for systemic spread, and marked resistance to conven-tional chemo- and radiotherapy, malignant peripheralnerve sheath tumors (MPNST) cause remarkable morbid-ity and mortality in afflicted individuals (1, 2). Develop-ment of more efficacious therapeutic strategies is criticallyneeded and requires more comprehensive knowledge of

molecular constituents driving MPNSTs. The tight asso-ciation between MPNSTs and neurofibromatosis type 1(NF1) is suggested by the reality that more than 50% ofMPNSTs develop against the backdrop of this commongenetic disorder (2), and that 8% to 13% of patients withNF1 will develop MPNSTs (2), suggesting a fundamentalrole for neurofibromin loss of function underlyingMPNST inception (3). Deactivating mutations in the NF1tumor suppressor gene, which encodes for the Ras-GTPase neurofibromin protein, are the hallmark of NF1;plexiform neurofibromas developing in patients with NF1and MPNSTs arising within these typically deep seatedlesions exhibit biallelic NF1 inactivation and consequen-tial enhanced Ras pathway signaling. NF1 mutations alsoare observed in a portion of sporadic, non-NF1–associ-ated MPNSTs (3). While neurofibromin loss and/or Raspathway activation are early molecular events driving thedevelopment of MPNST premalignant stages, additionalgenetic and epigenetic alterations are most likely neces-sary for malignant transformation, disease progression,and metastasis (4). The disappointing results of MPNSTclinical trials, singularly targeting the Ras pathway or

Authors'Affiliations:Departmentsof 1SurgicalOncology, 2Pathology, and3Cancer Biology, 4The Sarcoma Research Center, 5Metastasis ResearchCenter, University of Texas MD Anderson Cancer Center; 6The Division ofBiostatistics, Dan L. Duncan Cancer Center, Baylor College of Medicine;and 7The Graduate School of Biomedical Sciences, Houston, Texas

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

Corresponding Author: Dina Lev, Department of Cancer Biology, Univer-sity of Texas M.D. Anderson Cancer Center, 8515 Fannin, Unit 1104,Houston TX 77054. Phone: 713-792-1637; Fax: 713-563-1185; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-2592

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst March 8, 2012; DOI: 10.1158/1078-0432.CCR-11-2592

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molecules within this signaling cascade (5), highlight theneed to further identify additional MPNST-associatedmolecular aberrations, preferentially those that would beeasily amenable to therapeutic targeting.

Originally identified in 1997 as a member of theinhibitor of apoptosis (IAP) family (6), survivin, encodedby the BIRC5 gene, has since been found to contribute toa multitude of critical biologic functions including cel-lular division, survival, and adaptation to stress (6, 7).While highly expressed during embryogenesis, survivin islargely undetectable in normal adult tissues and isrestricted to the thymus, placenta, stem cell compartment,and basal epithelium of the colon (6, 8). However,survivin reexpression is commonly observed in trans-formed cells, and increased survivin levels have beenfound in multiple cancer types (9). Moreover, increasedsurvivin expression levels have been found to correlatewith adverse patient outcomes and resistance to therapy(10–13). Importantly, survivin has been shown to play acritical role in cancer, functioning as a convergence pointfor multiple signaling pathways controlling tumor main-tenance and growth promotion (8). These attributes haverendered survivin a focus of intense investigation as apotentially worthy target for personalized molecular ther-apeutics (8). Anti-survivin treatment regimens haverecently reached the clinic (http://clinicaltrials.gov/ct2/show/NCT00664677, NCT01186328, NCT01100931, andNCT01250470) with encouraging results noted in earlyhuman cancer clinical trials (14, 15).

While extensively studied in a wide range of humanmalignancies, a role for survivin deregulation in MPNSTbiology has yet to be determined. Several previously pub-lished reports suggest that survivin is aberrantly expressed inMPNSTs (16, 17). Specifically, Storlazzi and colleaguesidentified the BIRC5 genetic region on chromosome 17 ascommonly amplified in humanMPNST samples; high-levelamplification correlated with poor prognosis (17). Over-expression of survivin mRNA in MPNSTs compared withneurofibroma has been observed by 3 independent groups(16, 18, 19). Finally, a recent immunohistochemistry-basedstudy showed survivin protein expression in 52 humanMPNST samples (20). Building on these initial observa-tions, the current study sought to further determine thepotential role of survivin as a MPNST biomarker, to eluci-date the functional consequences of survivin overexpres-sion in these tumors, and, most importantly, to assess theefficacy of survivin blockade as an anti-MPNST therapeuticstrategy.

Materials and MethodsCell lines and reagents

MPNST cell lines used for our studies included the NF1-associated S462 (provided by Dr. Lan Kluwe, UniversityHospital Eppendorf, Hamburg, Germany), ST88-14 (pro-vided by Dr. Jonathan Fletcher, Brigham and Women’sHospital, Boston, MA), the MPNST642 isolated in ourlaboratory (21), and the sporadic MPNST cell lines: STS26T(provided by Dr. Steven Porcelli, Albert Einstein College ofMedicine, Bronx, NY) and MPNST724 (provided by Dr.Jonathan Fletcher); these were propagated and maintainedas previously described (22). Primary human adultSchwann cell cultures established from human caudaequina nerves were provided by Dr. Patrick Wood (MiamiProject, University of Miami, Miami, FL) andmaintained aspreviously described (23). DNA fingerprinting (short tan-dem repeat) was conducted as previously described (21) forall MPNST cell lines, confirming that no cross-contamina-tion has occurred.

The small-molecule survivin inhibitor YM155 was pur-chased from ChemieTek. For in vitro studies, the drug wasdissolved in dimethyl sulfoxide (DMSO) and stored in�20�C. For in vivo experiments, YM155 (dosed at 6 mg/kg/d) was dissolved and diluted in saline immediatelybefore administration. Commercially available antibodieswere used for immunoblotting, immunohistochemical(IHC), or immunocytochemical detection of the full-lengthwild-type (WT) survivin (polyclonal; Abcam), XIAP (poly-clonal; Abcam), cIAP1 (polyclonal; Abcam), cIAP2 (poly-clonal; Abcam), the 85-kDa fragment of cleaved PARP(clone Y34; Abcam), total PARP (clone 46D11; Cell Signal-ing Technology), cleaved caspase-3 (polyclonal; BioCareMedical), a-tubulin (Santa Cruz Biotechnology), Ki67(Thermo/Lab Vision), Lamin A/C (Santa Cruz Biotechnol-ogy), and b-actin (Santa Cruz Biotechnology). Hoechst(Invitrogen) was used as a nuclear stain for immunocyto-chemical analysis.

Translational RelevanceMalignant peripheral nerve sheath tumors (MPNST)

are characterized by aggressive local growth, propensityfor systemic spread, and marked resistance to conven-tional chemo- and radiotherapy. Consequently, afflictedpatients have guarded prognoses and desperately needefficacious treatments. This past decade has witnessedremarkable interests in novel, molecularly targeted ther-apeutic regimens, ultimately mandating comprehensiveknowledge of molecular deregulations driving tumorprogression. Studies reported here show a potential rolefor the protein survivin in MPNST biology. Survivin wasfound to be highly expressed in human MPNSTs;enhanced cytoplasmic subcellular localization differen-tiated MPNSTs from their premalignant plexiform neu-rofibroma counterparts. Survivin knockdown abrogatedMPNST cell growth, inducing G2 cell-cycle arrest andapoptosis. Most importantly, YM155, a small-moleculesurvivin inhibitor, significantly reduced local and met-astatic humanMPNST xenograft growth. Taken together,these data offer new insights into MPNST molecularderegulations and support further development ofanti-survivin–based therapeutic strategies for patientswith MPNSTs.

Ghadimi et al.

Clin Cancer Res; 18(9) May 1, 2012 Clinical Cancer Research2546




Immunohistochemistry and immunocytochemistryA previously reported (24) tissue microarray (TMA) con-

taining specimens retrieved from human MPNSTs andplexiform neurofibroma surgical resections was used toassess survivin expression. After excluding spots with insuf-ficient material, 63 different samples of patients withMPNSTs, 21 plexiform neurofibromas, and 2 normalperipheral nerve samples were available for analysis. Acomprehensive clinical database containing patient, tumor,treatment, and follow-up information linked to the TMAhas previously been constructed and updated to enable thiscurrent analysis. The median follow-up time was 2.5 years(ranging between 3 months and 15.25 years). TMA immu-nostaining, xenograft-derived specimen immunohis-tochemistry, and immunocytochemistry were conductedas previously described (24–26). For TMA analysis, survivinexpression was scored by 2 independent observers (A.J.Lazar and M.P. Ghadimi). Intensity was graded as none(¼ 0), weak/low (¼ 1), moderate (¼ 2), and high (¼ 3);percentage of positively staining cells was also determined.Scoring was conducted separately for the nuclear and cyto-plasmic intracellular compartments.

siRNA transfection proceduressiRNAs (20 nmol/L pools targeting survivin and control

nontargeting constructs; Thermo Scientific) were intro-duced into cells by X-tremeGENE as per manufacturer’sinstructions (Roche). Briefly, 2 � 105 cells were plated ineach well of a 6-well plate and incubated overnight. Amixture of siRNA (20 nmol/L) and X-tremeGENE (6 mL)diluted in 100 mL Dulbecco’s Modified Eagle’s Mediumwasadded for 24 hours, followed by incubation in regularmedium. Cells were harvested at indicated time points forspecific experiments.

In vitro growth assaysMTS assays were conducted with CellTiter96 AQueous

Non-Radioactive Cell Proliferation Assay kit (PromegaCorp) as per manufacturer’s instructions. Absorbance wasmeasured at a wavelength of 490 nm, and the absorbancevalues of treated (or of survivin knocked down) cells arepresented as a percentage of the absorbance of untreatedcells. Colony formation assay was conducted for pretreat-ment analysis; MPNST cells were treated in culture dishesfor 24 hours withDMSO (control) or YM155.One hundredviable cells per well were replated and allowed to grow innormal medium (no drug) for 10 days and then stained for30 minutes at room temperature with a 6% glutaraldehydeand 0.5% crystal violet solution. For continuous treatmentstudies, 100 viable cells per well were plated and treatedwith YM155 or DMSO alone for 10 consecutive days.Pictures were captured digitally and surface area coveredby cells (as surrogate to colony number) was determinedwith ImageJ software. Anchorage-independent growth:MPNST cells were treated with DMSO (control) or YM155for 24 hours in a 6-well plate. A total of 1� 103 viable cellswere plated in a 24-well plate in culturemedium containing0.35% agarose overlying a 0.7% agarose layer. Cells were

incubated for 3 weeks at 37�C. Cells were stained with p-iodonitrotetrazolium violet (1 mg/mL) for 24 hours at37�C.

Cellular assaysWestern blot analyses were conducted by standard meth-

ods (27). Subcellular (nuclear and cytoplasmic) proteinfractionation and isolation was conducted with the Nuclearand Cytoplasmic Extraction Reagents Kit (NE-PER ThermoScientific) as per manufacturer’s instructions. Cell-cycleprogression was measured via propidium iodide (PI) stain-ing/fluorescence-activated cell-sorting (FACS) analysis(27), and apoptosis was measured via Annexin-V/PI stain-ing FACS analysis with the Apoptosis Detection Kit I (BDBiosciences) as permanufacturers’ recommendations. Real-time PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR)were carried out as we have previously described (27);primers (survivin forward: 50-GGACCACCGCATCTCTA-CAT-30 and reverse: 50-GTTCCTCTATGGGGTCGTCA-30)were obtained from Sigma.

In vivo animal modelsAll animal procedures and care were approved by theMD

Anderson Cancer Center (Houston, TX) Institutional Ani-mal Care and Usage Committee. Animals received humanecare as per the Animal Welfare Act and the NIH "Guide forthe Care and Use of Laboratory Animals." For experimentsevaluating the effect of treatment on local tumor growth,trypan blue staining confirmed viableMPNST cells [STS26TandMPNST724; 1� 106 to 2� 106/0.1mLHank’s balancedsalt solution (HBSS)/mouse] was used. Cell suspensionswere injected s.c. into the flank of 6-week-old female hair-less severe combined immunodeficient (SCID) mice (n ¼7–8/treatment group) and growth was measured twiceweekly; after establishment of palpable lesions (averagediameter �4–5 mm), mice were assigned to treatmentgroups as described below. An experimental lungmetastasisMPNST model was used to evaluate the growth of metas-tases. STS26T cells (1 � 106/0.1 mL HBSS/mouse) wereinjected into the tail vein of female SCID mice. Two weeksafter injection, mice were allocated to treatment groups asdescribed below. Therapeutic regimen and dose followedprevious reports (28, 29). YM155 [6 mg/kg/d; several pre-clinical studies (29, 30) have shown antitumor efficacywithdoses of 1 to 10mg/kg/d, with the latter being the maximaltolerated dose] or saline was delivered via a micro-osmoticpump (Alzet model 1003D, DURECT Corporation) thatwas implanted s.c. on day 1 of treatment into each mouse;these pumps enable continuous drug delivery for 3 conse-cutive days. Previous studies have shown that this regimenresults in steady-state levels of YM155 in plasma andenhanced tumor bioavailability (30). YM155 continuousinfusion was found superior to intravenous daily bolus orintermittent schedules (30). The pumps were replaced onday 8 for a total of 6 treatment days. Mice were followed fortumor size, well-being, and body weight and sacrificedwhen control group tumors reached an average of 1.5 cmin their largest dimension. Tumors were resected, weighed,

YM155 Inhibits Local and Metastatic MPNST Growth

www.aacrjournals.org Clin Cancer Res; 18(9) May 1, 2012 2547




and fixed in formalin and paraffin-embedded for immu-nohistochemical studies. For lung metastatic studies, micewere followed for bodyweight andwell-being and sacrificed3 weeks after start of treatment. Lungs were resected, eval-uated macroscopically for tumor load, and weighed andfixed in formalin and paraffin-embedded for hematoxylinand eosin (H&E) staining.

StatisticsSeveral alternative statistical tests were used to determine

the correlation between survivin expression and clinicalfactors such as histology, NF1 status, and disease statusincluding Spearman’s correlation coefficient, c2, and Fish-er’s exact test. Correlation between survivin and othermolecular biomarkers was evaluated by Spearman’s corre-lation coefficient analyses. Kaplan–Meier analyses wereused to determine the potential impact of survivin expres-sion levels on MPNST disease-specific survival. All compu-tations were conducted with SAS for Windows (release 9.2;SAS Institute). Cell culture–based quantitative assays wererepeated at least 3 times andmean� SDwas calculated. Celllines were examined separately. For outcomes that weremeasured at a single time point, two-sample t tests wereused to assess the differences. Differences in xenograftgrowth in vivo were assessed by a 2-tailed Student t test.Significance was set at P � 0.05.

ResultsHuman MPNSTs exhibit increased survivin levels;cytoplasmic expression is significantly enhanced inMPNSTs as compared with plexiform neurofibroma

Survivin overexpression has been previously shown formany cancer types (9). Supported by previous publicationssuggestingBIRC5 gene amplification andenhanced survivinmRNA expression in MPNSTs (16–18), we examined sur-vivin protein expression levels and intracellular localizationin a cohort of human samples. A previously constructed,clinically annotated TMA was used, containing humanMPNST and plexiform neurofibroma samples (24). Survi-vin IHC analysis was conducted (Fig. 1A). Both normalperipheral nerves were negative for survivin expression. AllMPNSTs showed survivin expression; both nuclear andcytoplasmic distributionswere noted (Fig. 1A).Weaknucle-ar expression levelswere found in 31 (49%),moderate in 28(45%), and high in 4 (6%); an average of 73% (�18%) oftumor cells per sample exhibited positive nuclear staining.Cytoplasmic expression levels were low in 3 (5%), moder-ate in 13 (21%), and high in 47 (74%); an average of 83%(�11%) of tumor cells per sample exhibited positive cyto-plasmic staining. Similarly, all plexiform neurofibromaexpressed survivin. Low nuclear levels were found in 9(43%), moderate in 8 (38%), and high in 4 (19%); anaverage of 52% (�23%) of tumor cells per sample exhibited

Figure 1. Survivin is highlyexpressed in human MPNSTspecimens. Cytoplasmic survivinexpression is more pronounced inMPNSTs than in plexiformneurofibroma. A, top, the entireTMA stained for survivin; a range ofexpression levels can be observed.Bottom, representativephotographs of survivin-stainedneurofibroma (left) and MPNSTsspots (middle and right) are shown.Neurofibroma image was capturedin 20�, inset showing normalperipheral nerve negative forsurvivin. MPNSTs imagescaptured at 20� showing bothnuclear (best elucidated in middlepicture; inset picture was taken at40�, arrow marks nuclear staining)and cytoplasmic (best elucidated inright picture; inset picture wastaken at 40�, arrow markscytoplasmic staining) survivinexpression (scale bars areincluded). B, heatmaprepresentation of nuclear andcytoplasmic survivin expressionlevels in each of the evaluable TMAspots. Cytoplasmic survivinexpression was found to bestatistically significantly morepronounced in MPNSTs than inneurofibroma.

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positive nuclear staining. Cytoplasmic expression levelswere low in 8 (38%), moderate in 11 (52%), and high in2 (10%); an average of 80% (�9%) of tumor cells persample exhibited positive cytoplasmic staining. Notably,whereas no difference in nuclear survivin expression wasfound between MPNST and plexiform neurofibroma–derived specimens, a statistically significant enhanced cyto-plasmic survivin expression was identified in MPNSTs (P <0.0001; Spearman’s rank test; Fig. 1B). No correlationbetween survivin expression levels and MPNST diseasestatus (i.e., primary, recurrent, or metastatic) or NF1 versussporadic lesions was identified. Kaplan–Meier analysis wasfurther conducted todeterminewhether survivin expressionlevels correlate with MPNST disease outcome; only scoringresults of evaluable localizedMPNST samples (n¼ 48)wereincluded. No correlation was found between either cyto-plasmic or nuclear survivin expression levels or MPNSTpatient disease-specific survival. In summary, MPNSTs andtheir precursor lesions, plexiform neurofibroma, both com-monly express survivin; cytoplasmic survivin expression ismarkedly more pronounced in malignant lesions. In con-trast to findings in other malignancies, we did not identifysurvivin expression to be a molecular prognosticator ofMPNST disease outcome.Next, we assessed whether there was a correlation (Spear-

man rank correlation analyses) between survivin expressionlevels (cytoplasmic and nuclear) and other biomarkers,with our MPNST TMA, including Ki67 (as a marker ofproliferation), p53 (commonly deregulated in MPNSTs;ref. 31), VEGF (as a marker of angiogenesis), and Ras

pathway signaling effectors including pMEK, pAKT, andthe mTOR downstream target pS6RP. No associationbetween Ki67 or p53 intensity and survivin was identified.However, enhanced cytoplasmic survivin intensity wasfound to directly and statistically significantly correlate withincreased VEGF (r ¼ 0.28, P ¼ 0.013), pMEK (r ¼ 0.48, P <0.0001), pAKT (r¼ 0.29, P¼ 0.012), and pS6RP (r¼ 0.33, P¼ 0.007) expression levels. Interestingly, increased nuclearsurvivin expression was found to inversely correlate withVEGF (r ¼ �0.33, P ¼ 0.004) and pAKT (r ¼ �0.32, P ¼0.008) intensities.

Survivin is highly expressed in humanMPNST cell linesNext, we determined survivin expression levels in a panel

of human MPNST cell lines to confirm that our experimen-tal model recapitulated the findings in human samples andthus could be used to further evaluate the potential functionof this protein in MPNSTs (Fig. 2A). WT survivin mRNAlevels were found to be markedly increased in all MPNSTcell lines as compared with normal human Schwann celland did not correlate with NF1 (S462, ST88, MPNST642)versus sporadic (STS26T and MPNST724) disease back-ground, cell growth (growth rates, STS26T>S462>MPNST724>ST88>MPNST642), or p53 mutational status(WT ¼ ST88 and MPNST642; mutated/null ¼ MPNST724,S462, and STS26T; 29). Similarly, WT survivin proteinexpression was enhanced inMPNST cell lines, and no otherbands were identified to suggest expression of survivinsplice variants. Of note, survivin protein expression andsurvivin mRNA levels did not entirely match, possibly

Figure 2. Survivin is highlyexpressed in human MPNST celllines. A, qRT-PCR (top) and RT-PCR(middle) showing markedlyincreased survivin mRNA expressionin MPNST cell lines as comparedwith normal human Schwann cells(NHSC). Western blot analysis(bottom) further confirming WTsurvivin protein overexpression inMPNST cell lines; B, Western blotanalyses and immunocytochemistry(scale bars are included) showingboth nuclear and cytoplasmicsurvivin expression in MPNST cells.GAPDH, glyceraldehyde-3-phosphate dehydrogenase.






suggesting that additional posttranscriptional factors maycontribute to survivin protein levels, whereas a closer asso-ciation with MPNST cell growth rate was noted. Finally, asobserved in humanMPNST specimens, survivin expressionwas noted in both nuclear and cytoplasmic cellular com-partments of MPNST cells (Fig. 2B).

Survivin promotes MPNST cell growth, cell-cycleprogression, and survival

We next conducted loss of survivin function experimentsto determine its contribution to MPNST cell growth. Anti-survivin siRNA–specific constructs were used to achieveknockdown; a nontargeting construct was used as control.Marked survivin knockdown was noted 48 hours aftertransfection (Fig. 3A). No decrease in the expression of theIAP proteins XIAP (Fig. 3A), cIAP1, or cIAP2 (Supplemen-tary Fig. S1) was noted in response to anti-survivin siRNAtransfection. MTS assays showed significant (P < 0.05)decreases in tumor cell growth at 24 and 48 hours (experi-ments were initiated 24 hours after transfection). PI stain-ing/FACS analyses showed significant (P < 0.05) G2–M cell-cycle arrest 48 hours after transfection (Fig. 3B). Theseassays also showed increased sub-G1 fractions in MPNSTcells, secondary to survivin knockdown. Annexin-V/PIstaining FACS analyses confirmed a statistically significant(P < 0.05) increase in apoptosis 72 hours after siRNAtransfection and Western blot analyses showed increasedcleaved PARP (85-kDa fragment) expression in survivinknocked down cells (Fig. 3C and Supplementary Fig. S2).Taken together, these studies showed a role for survivin inMPNST cell growth, cell-cycle progression, and survival,thereby supporting further investigation of anti-survivintherapeutic strategies in our MPNST experimental models.

YM155, a small-molecule survivin inhibitor, exertsmarked anti-MPNST effects in vitro and in vivo

The anti-MPNST effects of YM155, a small-moleculeinhibitor of survivin currently under clinical investigation,were next considered. YM155 was identified by a cell-basedchemical library screen to specifically inhibit survivin pro-moter activity (30). It is suggested to exert its antitumoreffects through inhibition of survivin mRNA transcription,resulting in decreased protein expression, whereas notimpacting the expression of other IAP proteins such ascIAP1, cIAP2, or XIAP (30). Western blot analyses con-firmed that YM155 induced a dose- (in the nanomolarrange) and treatment time–dependent decrease in survivinexpression inMPNST cell lines (Fig. 4A). No decrease in theexpression of the IAP proteins, XIAP and cIAP1, wasobserved in response to YM155 (Fig. 4A). MTS assayslikewise showed amarked YM155dose-dependent decreasein MPNST cell growth after 48 and 96 hours of treatment(Fig. 4B). YM155 effects were found to correlate withcell line growth rates. A less pronounced effect was shownin MPNST642 cells; among the human MPNST cell linestested, this specific cell line exhibited the slowest growth rateand lowest survivin expression level. On the basis of theseinitial results, YM155 doses used for the remaining cell

culture–based assays ranged between 1and 10 nmol/L;these doses are lower than the YM155 plasma levelsachievable in humans (32, 33), hence clinically relevant.YM155 administration significantly (P < 0.05) abrogatedthe colony-forming capacity and anchorage-independentgrowth of MPNST cells (Fig. 4C and D). Compatible withthe effects observed above in response to survivin knock-down, YM155 treatment resulted in abrogated G2–M cell-cycle progression (P < 0.05) and increased sub-G1 fractionin MPNST cells (Fig. 5A). Finally, a statistically significant(P < 0.05) increase in tumor cell apoptosis was noted inresponse to YM155 (evaluated after 48 hours oftreatment; Fig. 5B) and an increase in the expression ofcleaved PARP (85-kDa fraction) was observed (Fig. 5B andSupplementary Fig. S2).

Todeterminewhether these in vitroobservationsmight berecapitulated in vivo, we carried out a series of therapeuticexperiments with humanMPNST xenograft mouse models.First, we examined the effect of YM155 on STS26T growth(Fig. 6A). YM155 (6 mg/kg/d) therapy was initiated afterestablishment of tumor (�5 mm in largest dimension).Therapy was administered with subcutaneously implantedmicro-osmotic pumps, delivering a 3-day continuous infu-sion; pumpswere implanted ondays 1 and8of treatment asper previously published reports (28, 29). Control micewere treated with saline (carrier) equivalently delivered.YM155 was well-tolerated and no significant weight losswas observed. YM155 treatmentmarkedly abrogated tumorgrowth; the average size of control-treated tumors at studytermination was 1,109 mm3 (�167) versus 251 mm3

(�134) of YM155-treated tumors (P < 0.0001; Fig. 6A).Moreover, treatment with YM155 significantly reducedtumor weight compared with control (P ¼ 0.005). Averagetumorweights at study terminationwere0.82 g (�0.18) and0.36 g (�0.19) in control and YM155 groups, respectively(Fig. 6A). Tumor sections from each experimental armcontaining viable cells were selected for IHC studies. Amarked decrease in survivin expression was observed inYM155-treated tumors (Fig. 6A). Furthermore, a pro-nounced decrease in MPNST cell proliferation (evaluatedvia Ki67 staining) and a demonstrable increase in tumor cellapoptosis (evaluated with cleaved caspase-3 immunohis-tochemistry) were also noted.

We next evaluated the effects of YM155 in a secondMPNST xenograft model derived from s.c. injection ofMPNST724 cells; YM155 doses and regimens were asdescribed above. YM155-treated mice exhibited smallertumors at study termination. The average size and weightof YM155-treated tumors were 702mm3 (�257) and 0.83 g(�0.27) when compared with 1,335mm3 (�362) and 1.53g (�0.58) for the control group (Fig. 6B). While statisticallysignificant (P < 0.05), the effects of YM155 on MPNST724xenografts were less profound than its in vivo anti-STS26Teffects and are probably more consistent with cytostasisthan cytolysis per se. This difference might be related to theinherently slower growth of MPNST724 xenografts thanSTS26T, which was also reflected in the lower baseline Ki67expression in these tumors. Similar to the IHC results

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obtained from the STS26T therapeutic experiments, adecrease in survivin and Ki67 and an increase in cleavedcaspase-3 expression was found in MPNST724 YM155–treated xenografts (Fig. 6B).

Finally, we used the STS26T experimental lungmetastasismodel to evaluate whether YM155 can affect the growth ofMPNST pulmonary metastases. Pumps were implanted 2weeks after tumor cell tail vein injection and then replaced

Figure 3. Survivin knockdown abrogates MPNST cell growth, cell-cycle progression, and survival. A, anti-survivin siRNA (20 nmol/L pool) resulted inpronounced survivin knockdown (KD) inMPNST cell lines 48 hours posttransfection [non targeting (NT-siRNA) constructs were used as control; Western blotanalyses, top]. No decrease in the expression of the IAP protein XIAP was noted. Survivin KD significantly inhibited MPNST cell growth. MTS assays wereconducted for 24 and 48 hours (48 and 72 hours posttransfection, respectively); B, survivin KD resulted in a statistically significant G2 cell-cycle arrest inMPNSTs cells (48 hours posttransfection; graphs represent at least 3 independent experiments). Increased sub-G1 fraction (sG1) was also noted. C, survivinKD induced marked apoptosis in MPNST cells (assays were conducted 72 hours after siRNA transfection) as is shown via Annexin-V/PI staining FACSanalyses (graphs represent at least 3 independent experiments) and increased cleaved PARP expression (cl. PARP; with an antibody directed against the 85-kDa fraction of PARP; Western blot analyses further showmarked survivin KD at this time point). �, statistically significant effects (P < 0.05). FITC, fluoresceinisothiocyanate.






one time 7 days later. Mice were sacrificed 3 weeks aftertreatment initiation. A significant difference in average lungweight was found between control (0.47 � 0.13 g) andtreated mice (0.24 � 0.07 g, P ¼ 0.0004; Fig. 6C). Macro-scopic lung metastasis was observed in all (n ¼ 8) controlmice but not in YM155-treated mice (n ¼ 8; Fig. 6C). H&Estaining identified large lung tumor deposits in all controlmice, whereas only 2 of the YM155-treated mouse lungsexhibited microscopic lesions (Fig. 6C).

DiscussionDriven by the critical need to identify MPNST molecular

deregulations amenable to therapeutic targeting, studies herefocused on themultifunctional protein survivin.More than adecade of intensive research has illuminated the fundamen-tal role survivin plays across a broad range of cancer histol-ogies (9). Multiple molecular mechanisms have been iden-tified that drive aberrant survivin expression in cancer,including amplification of the BIRC5 genetic locus (on

Figure 4. YM155, a small-molecule survivin inhibitor, inhibits MPNST cell growth in vitro. A, Western blot analyses confirm that YM155 induces a dose- (in thenanomolar range) and treatment time–dependent decrease in survivin expression in MPNST cell lines. No decrease in the expression of the IAP proteinsXIAP or cIAP1was observed in response to YM155; B,MTS assays showingmarked YM155 dose–dependent decrease inMPNST cell growth after 48 and 96hours of treatment. A correlation between YM155 sensitivity and cell line growth rate was observed. Of note, a less pronounced effect was shown inMPNST642 cells-–among the human MPNST cell lines tested, this cell line exhibits the slowest growth and lowest survivin expression. C, YM155(pretreatment and continuous treatment) significantly inhibits the colony-forming capacity of MPNST cells (graphs represent at least 3 independentexperiments). D, YM155 significantly inhibits the anchorage-independent growth of MPNST cells (graphs represent at least 3 independent experiments).�, statistically significant effects (P < 0.05).

Ghadimi et al.





chromosome 17q25),BIRC5 demethylation, enhanced tran-scription, and deregulated cellular signaling pathways forwhich survivin acts as a convergence point (34–36). Whilenot previously extensively studied in MPNSTs, publisheddata suggest that BIRC5 amplification is a common molec-ular event in these devastating malignancies (17).The current study further expands these initial observa-

tions showing enhanced survivin protein expression inhuman MPNST specimens. Studies presented here did notidentify survivin expression as a predictor ofMPNST patientdisease-specific survival. This is in contrast to observationsmade in many other solid malignancies including non–small cell lung cancer, gastric cancer, colorectal cancer,breast carcinomas, neuroblastoma, and osteosarcoma,

where survivin expression levels were found to correlatewith poor patient outcome (10–13). However, our negativeresults are potentially confounded by the relatively smallnumber of evaluable localized MPNST samples and shouldperhaps be reexamined with a larger cohort of specimens.

In alignment with its diverse molecular functions, survi-vin has shown a dynamic intracellular expression pattern,localizing to both the cytoplasm and the nucleus of tumorcells (37). Interestingly, nuclear survivin has been shown tobe a predictor of favorable outcome in non–small cell lungcancer and osteosarcoma (18, 38) while portending a poorprognosis in mantle cell lymphoma and squamous cellcarcinomas of the esophagus (38). Enhanced cytoplasmicsurvivin expression has been shown to correlate with

Figure 5. YM155 inhibits MPNST cell-cycle progression and survival in vitro. A, YM155 induces a statistically significant G2 cell-cycle arrest and increases insub-G1 (sG1) fraction inMPNST cells (graphs represent at least 3 independent experiments). B, moreover, YM155 (48 hours of treatment) exerts a statisticallysignificant increase in tumor cell apoptosis (graphs represent at least 3 independent experiments). Increased cleaved PARP (85-kDa fraction) expressioncan also be seen. �, statistically significant effects (P < 0.05). FITC, fluorescein isothiocyanate.






Figure 6. YM155 exerts marked anti-MPNST effects in vivo. A, SCIDmice bearing STS26T xenografts (�4–5mm in average larger dimension) were implantedwith a subcutaneousmicro-osmotic pump delivering YM155 (6mg/kg/d) or vehicle only in a continuous fashion for 3 consecutive days. Pumpswere replacedonce (on day 8; arrows denote days of pump implantation and bolded line days of treatment). YM155 markedly abrogated tumor growth (P < 0.0001; topgraph). Moreover, treatment with YM155 significantly reduced tumor weight compared with control (P ¼ 0.005; bottom graph). IHC analyses showeddecreased survivin and Ki67 and increased cleaved caspase-3 (CC3) expression in YM155-treated xenografts (scale bars are included). B, experimentwas repeated as above for MPNST724 xenografts. YM155 treatment was found to delay tumor growth, a statistically significant decrease in tumor size(P < 0.01; top graph) and tumor weight (P < 0.05; bottom graph) was observed at study termination. IHC analyses results (right) aligned with the STS26Tfindings above (scale bars are included). C, STS26T lung metastases–bearing mice were treated with YM155. A significant (P < 0.001; right) differencein average lung weight between control and YM155-treated mice was found at study termination. Pulmonary metastases were macroscopically observedin all control mice, but not in YM155-treated mice. H&E staining further showed large lung tumor deposits in control mice lungs, but no (n ¼ 6) or only smallmicroscopic lesions (n ¼ 2) in the YM155-treated group (scale bars are included). �, statistically significant effects (P < 0.05).

Ghadimi et al.





shorter disease-free survival in patients with oral squamouscell carcinoma (39). Possibly related to the above observa-tions, it has been previously suggested that the antiapopto-tic effects of survivin are greatest when the protein islocalized in the cytoplasm, whereas nuclear survivin mayplay a more critical role in mitosis (40). Together, it is clearthat the prognostic and functional significance of thesedistinct collections of subcellular survivin are yet to be fullyappreciated and delineated (15). In our study, we notedsurvivin expression in both the nuclear and cytoplasmiccellular compartments of MPNSTs. Interestingly, whereasequivalent nuclear survivin expression levels could beobserved when comparing MPNSTs with their premalig-nant plexiform neurofibroma precursors, cytoplasmicexpression levels were markedly increased in the malignantlesions. It is well established that progression of neurofi-bromas to MPNSTs mandates accumulation of genetic andepigenetic alterations that drive transformation and subse-quent tumor progression (4); our findings possibly justifyevaluating the role of cytoplasmic survivin in this process.Of note, phosphoinositide 3-kinase (PI3K), AKT, mTOR,and MEK/ERK signaling have previously been identified toregulate survivin expression (41). Deregulation of thesepathways is commonly observed in MPNSTs (42); pub-lished data from our laboratory have identified enhancedAKT, mTOR, and MEK activation in human MPNSTs sam-ples compared with plexiform neurofibroma tissues (24).Moreover, in the current study,we found adirect correlationbetween cytoplasmic survivin expression levels and those ofthe activated forms of MEK, AKT, and S6RP. These resultsoffer at least one possiblemolecularmechanismunderlyingenhanced cytoplasmic survivin expression in MPNSTs.In addition to aberrant survivin expression in human

MPNSTs, our data further suggest that survivin plays animportant role in the MPNST protumorigenic phenotypeenhancing tumor cell growth, cell-cycle progression, andsurvival. These survivin-induced effects are not unique toMPNSTs and are similar to those previously shown in othersolid andhematologicmalignancies (6, 30, 35, 41). As such,our study expands previous preclinical observations toinclude the devastating malignancy MPNSTs as a cancertype in which survivin might play a role and further high-lights the potential use of this protein as an attractive andpossibly universal target for molecularly based anticancertherapies. A wide variety of anti-survivin therapeutic strat-egies, including antisense oligonucleotides, ribozymes,siRNA-based approaches, immunotherapy, and smallmolecular weight inhibitors, are currently in various stagesof development (8). YM155 is one such extensively testedinhibitor and has already received clinical attention (15).An imidazolium-based compound originally identified byNakahara and colleagues (30) through a high-throughputcompound screen to specifically inhibit survivin transcrip-tion, the exact mechanisms of YM155 action are yet to beelucidated. This compound induces marked antitumoreffects in multiple preclinical cancer models includingprostate cancer, non–small cell lung cancer, melanoma,and non–Hodgkin lymphoma (30, 43, 44). These encour-

aging results have led to thedevelopment of initial phase I/IIYM155 human clinical studies, which have shown favor-able toxicity and tolerability profiles (32, 33, 45). Further-more, signs of efficacy, albeit modest, have been notedincluding partial responses in non–Hodgkin lymphomaand inheavily pretreated patientswith refractory non–smallcell lung carcinoma. Our studies confirm that humanMPNST cells are highly sensitive to survivin knockdown,resulting in marked cell-cycle arrest and apoptosis. Seekingto establish translational applicability, we examined thepotential efficacy of YM155 in our preclinical model. Thesestudies showed promising YM155-induced anti-MPNSTeffects in vitro, where a dose-dependent decrease in survivinexpression, tumor cell growth, cell-cycle progression, andsurvival were observed at low nanomolar concentrations.Furthermore, YM155 inhibited the local and metastaticgrowth of human MPNST xenografts in SCID mice; anespecially pronounced effect was noted in the STS26T rapidgrowth in vivo model. While these results are very encour-aging (and possibly support the inclusion of patients withMPNSTs in anti-survivin–based clinical trials), it is of notethat the YM155-induced complete regression reported inother tumormodels using similar (or even lower) drug doseand regimen (43, 44) was not observed in our MPNSTxenografts. In that, these latter observations may moreclosely resemble the effects observed in YM155 monother-apy clinical trials, a more efficacious therapeutic strategywill mandate combining anti-survivin strategies with otherMPNST-relevant conventional and/or molecularly targetedagents. Clinical studies combining YM155 with conven-tional chemotherapies (e.g., taxanes and carboplatinum) orbiologic agents (e.g., rituximab) for the treatment of severalcancer types are currently ongoing (ClinicalTrials.gov),buttressed by recent evidence for enhanced antitumor activ-ity of such combinations in preclinical models (46, 47).Future preclinical studies combining YM155 with agentspreviously showing promising effects in MPNSTs such astyrosine kinase inhibitors (e.g.,MET; ref. 48)or inhibitors ofcellular signaling (e.g., AKT, mTOR, and MEK; refs. 24, 49)and others are currently being planned.

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

AcknowledgmentsThe authors thank the expert assistance in figure preparation provided by

Kim Vu.

Grant SupportThemanuscript was supported in part by anNIH/NCI RO1CA138345 (to

D. Lev), an Amschwand Foundation Seed Grant (to D. Lev), a DeutscheForschungsgemeinschaft Fellowship grant (supportingM.P.Gadhimi), andaNIH/NCI 5T32CA009599-21 training grant (supporting K. Lusby). MDACCcell line characterization Core Facility was further supported by an NCICancer Center Support Grant (CA#16672).

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

ReceivedOctober 7, 2011; revised February 7, 2012; accepted February 29,2012; published OnlineFirst March 8, 2012.






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2012;18:2545-2557. Published OnlineFirst March 8, 2012.Clin Cancer Res Markus P. Ghadimi, Eric D. Young, Roman Belousov, et al. Peripheral Nerve Sheath TumorsSurvivin Is a Viable Target for the Treatment of Malignant

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Survivin Is a Viable Target for the Treatment of Malignant