livin contributes to tumor hypoxia induced ......induce livin upregulation, subsequent antiapoptosis...

Cancer Therapy: Preclinical

Livin Contributes to Tumor Hypoxia–InducedResistance toCytotoxic Therapies inGlioblastomaMultiformeChia-Hung Hsieh1,2,3, Yu-Jung Lin1, Chung-Pu Wu4, Hsu-Tung Lee5,Woei-Cherng Shyu6,7,and Chi-Chung Wang8

Abstract

Purpose: Tumor hypoxia is one of the crucial microenviron-ments to promote therapy resistance (TR) in glioblastoma multi-forme (GBM). Livin, a member of the family of inhibitor ofapoptosis proteins, contributes antiapoptosis. However, the roleof tumor hypoxia in Livin regulation and its impact on TR areunclear.

Experimental Design: Livin expression and apoptosis fortumor hypoxic cells derived from human glioblastoma xeno-grafts or in vitro hypoxic stress-treated glioblastoma cellswere determined by Western blotting, immunofluorescenceimaging, and annexin V staining assay. The mechanism ofhypoxia-induced Livin induction was investigated by chroma-tin immunoprecipitation assay and reporter assay. Genetic andpharmacologic manipulation of Livin was utilized to investi-gate the role of Livin on tumor hypoxia–induced TR in vitro orin vivo.

Results: The upregulation of Livin expression and downregula-tion of caspase activity were observed under cycling and chronichypoxia in glioblastoma cells and xenografts, concomitant withincreased TR to ionizing radiation and temozolomide. However,knockdown of Livin inhibited these effects. Moreover, hypoxiaactivated Livin transcription through the binding of hypoxia-inducible factor-1a to the Livin promoter. The targeted inhibitionof Livin by the cell-permeable peptide (TAT-Lp15) in intracerebralglioblastoma-bearing mice demonstrated a synergistic suppres-sion of tumor growth and increased the survival rate in standard-of-care treatment with radiation plus temozolomide.

Conclusions: These findings indicate a novel pathway thatlinks upregulation of Livin to tumor hypoxia–induced TR inGBMand suggest that targeting Livin using cell-permeable peptidemaybe an effective therapeutic strategy for tumormicroenvironment–induced TR. Clin Cancer Res; 21(2); 460–70. �2014 AACR.

IntroductionHypoxia is well evidenced within most of the solid tumors (1).

The acute, intermittent, or cycling hypoxia is associated withinadequate blood flow, whereas chronic hypoxia is the conse-quence of increased oxygen diffusion distance resulting fromtumor expansion (2). These hypoxic areas can either promotecell death or provoke an adaptive response leading to the selection

for death resistance (1, 3, 4).Once tumor cells become adaptive tohypoxia, they are more resistant to apoptosis and less responsiveto cancer therapy. Tumor hypoxia is thought to be a crucial role inpathologic characteristics of glioblastoma multiforme (GBM),including invasiveness, necrosis, and microvascular hyperplasia.It also contributes the resistance to chemotherapy, immunother-apy, and radiotherapy due to the possibility of dysregulation ofapoptotic pathway or other mechanisms (5). However, the exactmechanisms triggered by hypoxia that lead to antiapoptosis arenot well known.

Tumor resistance to apoptosis is one of the hallmarks of cancer(6). Evasion of apoptosis may contribute to carcinogenesis, tumorprogression and also to therapy resistance (TR). It has beenreported that several gene families are involved in the negativeregulation of apoptosis, including the inhibitor of apoptosisproteins (IAP; refs. 7, 8). IAPs are a family of antiapoptotic proteinswith 70 amino acid baculoviral repeats (BIR) including NAIP, c-IAP1 (MIHB, HIAP-2), c-IAP2 (HIAP-1, MIHC, API2), XIAP (hILP,MIHA, ILP-1), survivin, BRUCE (apollon), ILP-2, and Livin(BIRC7, ML-IAP, KIAP; refs. 9, 10). These proteins can bind andsuppress upstream or downstream caspases and inhibit apoptosis.Among these proteins, Livin is more recently identified and selec-tively binds on the endogenous IAP antagonist SMAC and caspase-3, caspase-7, and caspase-9 (11–13). In addition, a variety ofmalignancies overexpressed Livin, which is correlated with tumorprogression, TR, and poor patient's outcome in some tumors (14).However, the impact of tumormicroenvironment on Livin expres-sion and its regulatory mechanisms remain unclear.

1Graduate Institute of Basic Medical Science,ChinaMedical University,Taichung, Taiwan. 2Department of Medical Research, China MedicalUniversity Hospital, Taichung, Taiwan. 3Department of BiomedicalInformatics, Asia University, Taichung, Taiwan. 4Department of Phys-iology and Pharmacology, Chang Gung University, Tao-Yuan, Taiwan.5Department of Neurosurgery, Taichung Veterans General Hospital,Taichung, Taiwan. 6Department of Neurology, Center for Neuropsy-chiatry, China Medical University and Hospital, Taichung, Taiwan.7Graduate Institute of Immunology, China Medical University, Tai-chung, Taiwan. 8Graduate Institute of Basic Medicine, Fu Jen CatholicUniversity, New Taipei, Taiwan.

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

Corresponding Authors: Chia-Hung Hsieh, China Medical University and Hos-pital, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan. Phone: 886-4-22052121;Fax: 886-4-22333641; E-mail: [email protected]; Woei-CherngShyu, E-mail: [email protected]; and Chi-Chung Wang, Fu Jen CatholicUniversity, No. 510, Zhongzheng Road, New Taipei City 24205, Taiwan. Phone:886-2-29052039; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-0618

�2014 American Association for Cancer Research.

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In this study, we hypothesized that Livin may represent one ofhypoxia-responsive genes and play a role in tumor hypoxia–induced antiapoptotic effect at tumor microenvironment. Ourresults show that both cycling hypoxia and chronic hypoxia caninduce Livin upregulation, subsequent antiapoptosis as well asresistance to cytotoxic therapies in glioblastoma cells and xeno-grafts through a hypoxia-inducible factor 1 (HIF1)-a–dependentpathway. Genetic inactivation and pharmacologic inhibition ofLivin in vitro or in vivo suppress tumor hypoxia–induced TR andgenerate a synergistic suppression of antitumor growth and tumorcell death. These data highlight that tumor hypoxia–induced Livinexpressionmay represent a pathway for resistance of some tumorsto radio- and chemotherapeutics. Besides, the cell-permeablepeptide targeted to Livin derived from this study provides atranslational path for clinical improvement of standard-of-caretherapies in patients with GBM.

Materials and MethodsMolecular and cellular assays

Several molecular and cellular assays were used in this study.These included the following: quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) andWestern blot analyses toassess the expressionof theHIF1a, HIF2a, and Livinproteins;HIF1overexpression and knockdown, promoter analysis, luciferaseassay, and chromatin immunoprecipitation (ChIP) to investigatethe regulation of Livin promoter; genetic inactivation or pharma-cologic inhibition of Livin, caspase-3 activity and apoptosis assays,cells irradiation, clonogenic survival and cytotoxicity assays todetermine the role of Livin in hypoxia-mediated resistance toionizing radiation and temozolomide (TMZ). Detailed procedurescan found in the Supplementary Materials and Methods.

Identification of hypoxia subtypes in glioblastoma xenograftsThe hypoxia subtypes in glioblastoma xenografts were distin-

guished according to the physiologic and molecular characteris-tics of cycling hypoxia. Cycling hypoxia tended to occur in regionswith blood perfusion and HIF1 activation, whereas chronic hyp-oxia located in areas without blood perfusion but with HIF1activation. Perfusion marker, Hoechst 33342, staining, and HIF1activation labeling together with immunofluorescence imagingand FACS were used to isolate hypoxic tumor subpopulationsfromhuman glioblastoma xenografts. Cells with positiveHoechst33342 staining and GFP expression were potential cycling hyp-oxic cells. However, cells thatwere positive for GFP expression but

negative for Hoechst 33342 were mostly chronic hypoxic cells.The detailed methods are described in Supplementary Materialsand Methods.

Animal modelEight-week-oldmale nudemice (Balb/c nu/nu)were purchased

from theAnimal Facility of theNational ScienceCounsel andwereused to establish the orthotopic glioblastoma xenograft modelaccording to the published methods (15). Briefly, 2 � 105

GBM8401/SFFV-LucGFP, GBM8401 and U251 cells were har-vested by trypsinization and injected into the left basal gangliaof anesthetized mice. The tumors developed at 12 days aftertumor implantation for evaluating the efficiency of therapy stud-ies and at 18 days after tumor implantation for in vivo terminaldeoxynucleotidyl transferase–mediated dUTP nick end labeling(TUNEL) and clonogenic survival assays. All animal experimentswere conducted according to Institutional Guidelines of ChinaMedicalUniversity (Taichung, Taiwan) after acquiring permissionfrom the local Ethical Committee for Animal Experimentation.

In vivo treatmentIntracerebral glioblastoma-bearing mice were randomly

assigned to four different therapeutic groups: control (vehicletreatment), two fractions of stereotactic radiation (4Gy each) plustemozolomide (5 mg/kg) by oral gavage 1 hour before radiation,intravenous injection of TAT-K22 or TAT-Lp15 (20mg/kg) beforethe combination of temozolomide, and radiation treatment. Allof the treatments were administered daily on 2 consecutive days.All of treatments were carried out at day 12 after tumor cellinjection for bioluminescent imaging (BLI) and animal survivalassays and at day 18 after tumor cell injection for TUNEL stainingand in vivo/in vitro clonogenic survival assays. Tumor progressionwas monitored by bioluminescence imaging and mice weremonitored daily for survival. Animals were killed at the onset ofneurologic signs or any type of distress.

Bioluminescent imagingMice were imaged with the IVIS Imaging System 200 Series

(Caliper) to record bioluminescent signal emitted from theengrafted tumors. Mice were anesthetized with isoflurane andreceived intraperitoneal injection of D-Luciferin (Caliper) at adose of 270mg/gbodyweight. Imaging acquisitionwas performedat 15 minutes after intraperitoneal injection of luciferin. For BLIanalysis, regions of interest encompassing the intracranial area ofsignal were defined using Living Image software, and the totalnumber of photons per second per steradian per square centime-ter was recorded. To facilitate comparison of growth rates, eachmouse's luminescence readings were normalized against its ownluminescence reading at day 12, thereby allowing each mouse toserve as its own control.

Animal irradiation and in vivo/in vitro clonogenic survival assayMice were locally irradiated (4.25 Gy/minute) at a dose of 4 Gy

under anesthesia using a high-energy X-ray linear accelerator(Varian). Tumors were then excised, minced, and dissociated for30 minutes at 37�C in Hanks' balanced salt solution containing166 U/mL collagenase XI, 0.25 mg/mL protease, and 225 U/mLDNase. Cells were recovered by straining through an 80-mmmeshand centrifuging at 500 g and were resuspended in culturemedium. Cells were counted by hemocytometer using Trypanblue and assessed by in vitro clonogenic survival assay.

Translational Relevance

This study shows that tumor hypoxia can induce Livinupregulation, subsequent antiapoptosis as well as resistanceto cytotoxic therapies through a hypoxia-inducible factor 1(HIF1)-a-dependent pathway. The induction of Livin inresponse to tumor hypoxia is therefore associated with badprognosis. This may be particularly relevant in the selection ofpatients with cancer prone to receive targeting Livin inhibitorsas adjunct treatments. The cell-permeable peptide targeted toLivin derived from this study also provides a tractable path forclinical translation in improvement of Livin-mediated therapyresistance.

Livin in Tumor Hypoxia–Induced Therapy Resistance

www.aacrjournals.org Clin Cancer Res; 21(2) January 15, 2015 461




Figure 1.Tumor hypoxia induces Livin expression. A, time course of LivinmRNAexpression after exposure of GBM8401 cells to<1%O2. LivinmRNA (B) andprotein (C) levels inU251, U87, and GBM8401 cells at 24 hours after normoxic (N.) and hypoxic (H.) treatment (<1% O2). D, representative images of microscopic GBM8401/hif-1-r.Top left, fluorescence overlay image of perfusion marker, Hoechst 33342 (blue), and HIF1 activation labeling, GFP reporter (green), indicating areas with normoxia(Hoechst 33342þ and GFP�), chronic hypoxia (Hoechst 33342� and GFPþ), and cycling hypoxia (Hoechst 33342þ and GFPþ). (Continued on the following page.)

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Statistical analysisOne-way ANOVA with post hoc Scheffe analyses and Kaplan–

Meier survival analysis with Tarone–Ware statistics were carriedout using the SPSS package (version 18.0). The differencesbetween control and experimental groups were determined bythe two-sided, unpaired Student t test. P < 0.05 was consideredsignificant.

ResultsTumor hypoxia induces Livin expression

We first analyzed the time course of Livin mRNA in GBM8401cells with or without hypoxic stress (<1% O2). By 3 hours, LivinmRNA had increased significantly compared with the normoxiccontrol (Fig. 1A). Moreover, Livin mRNA and protein levels wereall increased in the glioblastoma cells, U251, U87, and GBM8401cells, at 24 hours after hypoxic treatment (<1%O2; Fig. 1B and C).Besides, glioblastoma cells experienced an increase in the Livinexpression under both noninterrupted and cycling hypoxic stress(Supplementary Figs. S1 and S2). To better verify endogenoustumor hypoxia–mediated Livin expression in the solid tumor,Hoechst 33342 staining and HIF1 activation labeling togetherwith immunofluorescence imaging and FACS were utilized toisolate hypoxic tumor subpopulations from human glioblastomaxenografts as described in our previous studies (16–18). TheGBM8401/hif-1-r tumors showed highly heterogeneous Hoechst33342 staining and HIF1 activation on immunofluorescenceimages (Fig. 1D and Supplementary Fig. S3). The Livin staining

indicated that Livin expression occurs in the cycling hypoxic(Hoechst 3342þ and GFPþ) and chronic hypoxic (Hoechst3342� and GFPþ) areas. Next, the total RNA of the hypoxic cellsubpopulationsderived fromdisaggregatedorthotopicGBM8401/HIF1-r and U87/HIF1-r xenografts (18) were further analyzed forLivin and vascular endothelial growth factor (VEGF), an HIF1target gene, expression by qRT-PCR. The expression of Livin andVEGF increased significantly in cycling and chronic hypoxic cellscomparedwith that in normoxic cells (Fig. 1E). Besides, the resultsfrom immunofluorescence staining of HIF1a and Livin humanglioblastoma specimens also demonstrate that Livin expressioncolocalized with high expression of HIF1a (Fig. 1F and Supple-mentary Fig. S4), suggesting that endogenous hypoxia leads to theinduction of Livin in glioblastoma.

On the other hand, incubation of normoxic U251, U87, andGBM8401 cells for 24 hours with desferrioxamine (100 mmol/L)or cobalt chloride (50mmol/L),whichmimic hypoxia by inducingtranscription fromHIF1-dependent genes, increased Livin mRNAto a similar level as that found during hypoxia (SupplementaryFig. S5). Moreover, the transcriptional activation of Livin in thehypoxia-treated cells increased significantly after treatment, withmaximal activation occurring 48 hours after hypoxic stress (<1%O2) inGBM8401 cellswhich stably transfectedwith 2510-bp Livinpromoter-driven luciferase reporter gene (Livin-Luc; Supplemen-tary Fig. S6). Taken together, these results that indicate tumorhypoxia, either chronic or cycling hypoxia, can induce Livinexpression and hypoxia-induced Livin expression is associatedwith HIF1 activation.

(Continued.)Top right, fluorescence overlay imageofHoechst 33342 (blue), GFP reporter (green), and Livin (red).White box 1 and 2 indicate cycling hypoxic area andchronic hypoxic area, respectively. Bottom left and right, magnification of white box 1 and 2. White color indicates the colocalization of Hoechst 33342, GFP,and Livin. Yellow color represents the colocalization of GFP and Livin. (Magnification: top, �100; bottom, �200). E, Livin and VEGF mRNA levels in normoxic cells(Hoechst 3342þ and GFP�), chronic hypoxic cells (Hoechst 3342� and GFPþ), and cycling hypoxic cells (Hoechst 3342þ and GFPþ) isolated from disaggregatedGBM8401/hif-1-r and U87/hif-1-r xenografts. Error bars, SD within triplicate experiments. � , P < 0.01, compared with normoxia. #, P <0.01, compared withchronic hypoxia. F, immunofluorescence imaging of Livin andHIF1a expression in primaryGBMs. Top panels show the staining ofDAPI (blue), HIF1a (green), and Livin(red). Bottom panels demonstrate the overlay image of DAPI (blue), HIF1a (green), and Livin (red) and indicate the magnification of white boxes. Yellowcolor indicates the colocalization of HIF1a and Livin. (Magnification: top, �100; bottom, �100, �200, and �400).

Figure 2.Hypoxia-induced Livin expression isdependent on HIF1a. A, verification ofHIF1aorHIF2a knockdownbyHIF1aorHIF2a shRNAs. Livin mRNA (B) andprotein (C) levels in GBM8401 and U87with or without HIF1a or HIF2aknockdown at 24 hours after hypoxictreatment (<1% O2). D, Luciferasereporter assay shows that HIF1ashRNA inhibited the hypoxia-inducedtranscriptional activation of Livin inU251, U87, and GBM8401 glioblastomacells. Error bars, SD within triplicateexperiments. � , P < 0.001, comparedwith normoxia. #, P <0.001, comparedwith scramble (Scr.) shRNA.






Hypoxia-induced Livin expression is dependent on HIF1aIn an attempt to gain insight into the mechanisms of hypoxia-

induced Livin induction, we examined Livin mRNA and proteinlevels in GBM8401 and U87 cells lacking HIF1a or HIF2a expres-sion. After lentiviral transduction with shRNAs against HIF1a andHIF2a, GBM8401 and U87 cells lost hypoxia-induced HIF1a orHIF2a expression compared with that in cells transduced withcontrol scrambled shRNA (Fig. 2A). In nonspecific shRNA controlGBM8401 or U87 cells, hypoxic stress stimulated a significantincrease in Livin mRNA and protein levels (Fig. 2B and C). How-ever, knockdown of HIF1a, but not HIF2a, significantly abrogatedhypoxia-induced Livin expression. Besides, the hypoxia-inducedtranscriptional activation of Livin was inhibited by knockdown ofHIF1a in U251, U87, andGBM8401 cells and treatment with YC-1(a HIF-1a inhibitor; 30 mmol/L) in HEK-293 and GBM8401 cells(Fig. 2DandSupplementary Fig. S7).On the contrary, coexpressionofHIF1a-oxygen–dependentdegradationdomaindeletionmutantand Livin-Luc significantly enhanced the reporter activity but notcontrol plasmids. These results indicate that HIF1a is a crucialtranscription factor for hypoxia-mediated Livin induction.

HIF1a binds directly to the Livin promoter to regulate itsexpression

Next, we sought to determine whetherHIF1a binds to the Livinpromoter for hypoxia-induced its expression. The bioinformaticsanalysis identified two HIF1a-binding sites in the Livin promotersequence from �2000 to þ100 (Fig. 3A), suggesting that HIF1amight regulate Livin expression by directly binding to its pro-moter. To pinpoint the exact binding motifs, we introduce pointmutations into the hypoxia response element (HRE)-1 andHRE-2

of Livin-Luc. The ablation of HRE-1, but not HRE-2, on the Livinpromoter abrogated hypoxia-mediated Livin induction (Fig. 3B).ChIP assays also confirmed the binding of HIF1a to the Livinpromoter (Fig. 3C and D). Collectively, these results suggest thatHIF1a regulates Livin transcription by directly binding to the Livinpromoter in a hypoxia-dependent fashion.

Hypoxia-mediated Livin expression results in antiapoptosisGiven the recognized role of Livin as an apoptosis suppressor,

we determined whether glioblastoma cells with hypoxia-mediat-ed Livin upregulation becamemore resistant to apoptotic insults.Staurosporine STA; 1 mmol/L was chosen as the apoptotic trigger.Tetracycline-regulatable lentiviral vectors encoding shRNAs wereused to stably and specifically knockdown Livin induction inGBM8401 and U87 cells under hypoxia (Fig. 4A and B). Thecaspase activities and apoptotic cells were all decreased signifi-cantly in cycling or noninterrupted hypoxia-treated cells com-pared with normoxic cells (Fig. 4C and D), suggesting thathypoxic cells are resistant to STA-induced apoptosis. After thepretreatment of doxycycline (Dox; 0.04 mg/mL), however, eithercycling or noninterrupted hypoxia-induced antiapoptotic effectswere inhibited by Livin knockdown. These results indicate thatLivin plays an essential role in tumor hypoxia–induced antiapop-tosis in glioblastoma cells.

Hypoxia-mediated Livin expression promotes radioresistanceand chemoresistance

Radiotherapy plus concomitant and adjuvant temozolomide isstandard-of-care treatment for patients with GBM (19). We testedwhether hypoxia-induced Livin expression is associated with the

Figure 3.HIF-1a binds directly to the Livinpromoter to regulate its expression. A,graphic representation of the putativeLivin promoter. Two putative hypoxiaresponse elements (HREs) wereidentified. B, the reporter activities ofLivin promoter in HEK-293 andGBM8401 cells cultured in hypoxia for24 hours in the absence or presence ofYC-1 and in cells co-transfected withcontrol or HIF-1a-ODD deletionmutant plasmids for 48 hours. C,Luciferase reporter plasmids carryingthewild type ormutant Livinpromoterregions were co-transfected with theRenilla luciferase reporter plasmid intoGBM8401 cells; the cells were treatedwith or without hypoxia (<1% O2) for24 hours. D and E, chromatinimmunoprecipitation followedby real-time PCR (ChIP-qPCR) assay of HIF-1abinding in Livin promoter in responseto hypoxia (<1% O2) for 24 hours.Results are expressed as percentageof input. Error bars denote thestandard deviation among triplicateexperiments. � , P < 0.001 comparedwith control or normoxia. #, P < 0.001compared with vehicle or wild type.

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development of radiation and temozolomide resistance via clo-nogenic survival assay and MTT assay. Both noninterrupted andcycling hypoxia pretreatment significantly increased cell resis-tance to ionizing radiation and temozolomide compared withnormoxic controls inGBM8401 andU251 cells (Fig. 5A andB andSupplementary Fig. S8). But, loss of Livin significantly inhibitshypoxia-induced radioresistance and chemoresistance andincreases the radiosensitivity and cytotoxicity of glioblastomacells to temozolomide, suggesting that Livin is a critical effectorinvolved in hypoxia-induced radioresistance and chemoresis-tance in glioblastoma cells.

Next, we generated therapeutic peptide to inhibit Livin activityand test whether this peptide can block hypoxia-induced radio-resistance and chemoresistance in glioblastoma cells. To bindLivin, we constructed a cell-permeable peptide comprising the 23amino acid-terminal residues of Livin-binding peptide, Lp15,derived from a randomized peptide expression library (20). Thetruncated Lp15 and control peptide, K22, were rendered cell-permeant by fusing each to the cell-membrane transductiondomain of the human immunodeficiency virus-type I (HIV-I) Tatprotein to obtain a 35 to 37 amino acid peptide (TAT-Lp15) andcontrol peptide (TAT-K22), respectively (Fig. 5C). The fluorophore

Figure 4.Hypoxia-mediated Livin expressionresults in antiapoptosis. Verification ofLivin knockdown by Tet-regulatablelentiviral knockdown system in mRNA(A) and protein (B) levels. Thelentiviral infected cells were treatedfor 48 hourswith Dox (0.04 mg/mL) toinduce Livin knockdown. Error bars,SD among triplicate experiments.� , P < 0.001, compared with no Doxtreatment. Caspase-3 activities (C)and percentage of apoptotic cells (D)in GBM8401 and U87 cells cultured innormoxia, noninterrupted (Ni.)hypoxia (<1% O2), and cycling (Cy.)hypoxia (<1% O2) with or without Livinknockdown in response tostaurosporine (STA) treatment. Cellswere pretreated for 48 hours with Dox(0.04 mg/mL) to induce Livinknockdown and exposed to hypoxicstresses before STA treatment.Error bars, SD among triplicateexperiments. � , P < 0.01, comparedwith control. #, P < 0.05, comparedwith normoxia with STA treatment.






dansyl chloride was conjugated to TAT-Lp15 and TAT-K22. Con-focal microscopy showed that cells treated with either TAT-Lp15-dansyl or TAT-K22-dansyl exhibited fluorescence in their cyto-plasm (Fig. 5D), indicating intracellular peptide uptake. TAT-Lp15-dansyl accumulation was detectable in cells within 15 min-utes of incubation, peaked during 60 minutes, and remaineddetectable for 8 hours after washing the peptide from medium(Fig. 5E). To assess whether the TAT-Lp15 peptide can sensitizeglioblastoma cells toward apoptosis, a series of concentrations ofTAT-Lp15 peptide were treated to Livin-positive (GBM8401) cellswith or without Livin knockdown and Livin-negative (H1299)cells followed by STA treatment. TAT-Lp15 peptide enhanced STA-induced caspase activities and apoptosis in a dose-dependentmanner in GBM8401 cells but not in H1299 cells or Livin knock-downGBM8401 cells (Supplementary Figs. S9, S10, S11, andS12).

Besides, TAT-Lp15 peptide, but not of a control peptide, signifi-cantly increased the radiosensitivity and cytotoxicity ofU87,U251,and GBM8401 cells to temozolomide and decreased hypoxia-induced radioresistance and chemoresistance in glioblastoma cells(Fig. 5FandG). The combination treatmentofGBM8401cellswithTAT-Lp15 and 4-Gy irradiation could also yield a synergistic effecton tumor cell killing (Supplementary Fig. S13).

Livin blockage enhances the efficiency of radiation plustemozolomide treatment in glioblastoma xenografts

Finally, we investigatedwhether the targeted inhibition of Livinby therapeutic peptide represents a viable approach for inhibitionof tumor microenvironment–mediated TR in GBM. We firstdetermined whether TAT-Lp15 could be delivered into the brainin the intact animal. Fluorescence imaging demonstrated that

Figure 5.Hypoxia-mediated Livin expressionpromotes radioresistance andchemoresistance. A, radiation cellsurvival curves for GBM8401 cells.Cells were pretreated for 48 hourswith Dox to induce Livin knockdownand exposed to hypoxic stress (<1% O2

), either noninterrupted (Ni.) or cycling(Cy.) hypoxia, before irradiation. B,cytotoxicity assay of Livin knockdownrevealed increased cytotoxicity ofcells to TMZ and suppressed hypoxia-mediated TMZ resistance in GBM8401andU251 glioblastoma cells. � , P <0.01,compared with normoxia. #, P < 0.01,compared with untreated Dox groups.C, sequences of TAT-Lp15 amino acidsand TAT-K22 control peptide. D,visualization of intracellularaccumulation of TAT-Lp15-dansyl orTAT-K22-dansyl (10 mmol/L) at 60minutes after incubation to GBM8401cells. E, time course of TAT-Lp15-dansyl (10 mmol/L) fluorescenceafter incubation to GBM8401 cells.Fluorescence intensities weredetermined by flow cytometry.Surviving fraction (F) and cell viability(G) for U251, U87, and GBM8401 cellswith or without TAT-K22 or TAT-Lp15(30 mmol/L) and receiving hypoxicstress (H; <1% O2) followed by 4 Gyirradiation (R) or 100 mmol/L of TMZ(T). Error bars, SD among triplicateexperiments. � , P < 0.001, comparedwith control. #, P < 0.001, comparedwith radiation or TMZ alone., P < 0.001, compared with hypoxia

combined radiation or TMZ groups.

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brains from nude mice injected intravenously with a 500 mmol/Ldose of either TAT-Lp15-dansyl or TAT-K22-dansyl, exhibitedstrong fluorescence in the cortex (Fig. 6A), indicating TAT-Lp15-dansyl or TAT-K22-dansyl enters the brain upon peripheraladministration. Tomimic the clinical setting, we sought to test theefficacy of TAT-Lp15 when added to standard-of-care treatmentwith radiation plus temozolomide. BLI was utilized to assessintracranial tumor growth in the orthotopic GBM8401/SFFV-LucGFP xenograft model (16, 17, 21). Consistent with the in vitrostudies, mice that had TAT-Lp15 treatment before initiation of thecombination of temozolomide and radiation treatment hadsignificantly better tumor growth delay and survival rate thanthose pretreated with TAT-K22 or vehicle (Fig. 6B–D). The com-bination treatment of GBM8401 xenografts with TAT-Lp15 andstandard-of-care treatment with radiation plus temozolomidecould yield a synergistic effect in therapeutic benefits. Moreover,TUNEL assays were done on tumors collected frommice submit-ted to the above experimental conditions on day 3 after treat-ments. TAT-Lp15 treatment during the combination of temozo-lomide and radiation treatment increased the extent of temozo-lomide and radiation-induced apoptosis of tumor cells (Fig. 6Eand F). By pimonidazole/annexin V doubling staining flow-cyto-metric assay, TAT-Lp15 treatment also enhanced the temozolo-mide and radiation-induced apoptosis in tumor hypoxic cells(Fig. 6G). Besides, pretreatment with TAT-Lp15, but not TAT-K22,produced a significant reduction in clonogenicity after temozo-lomide and radiation treatment in both GBM8401 and U251xenografts (Fig. 6H and I). Taken together, these results suggestthat targeted inhibition of Livin by therapeutic peptide enhancesthe efficiency of standard-of-care therapies in vivo in orthotopicglioblastoma xenografts.

DiscussionHypoxic tumormicroenvironment has long been considered as

major challenge in cancer therapy and is one of the primary causesfor cancer recurrence in GBM (22). Understanding the underlyingmechanism governing the nature of therapeutic resistance istherefore critical and may uncover therapeutic interventions. Itis a well-known fact that chronic hypoxia contributes to tumormicroenvironment–mediated TR. Although tumor cycling hyp-oxia is nowawell-recognized phenomenon in animal andhumansolid tumors, however, relatively few studies to date have specif-ically investigated its role andmechanism on TR. In this study, weidentified a novel hypoxia responsive gene, Livin, promotes anti-apoptosis and therapeutic resistance in glioblastoma. Despite thepotential role of other hypoxia-dependent antiapoptotic regula-tion in therapeutic resistance, we discovered a novel aspect by

which HIF1a transactivation of Livin expression is required fortumor chronic and cycling hypoxia-induced antiapoptosis andtherapeutic resistance. On the basis of our findings, we propose amodel in which genetic mutation or hypoxic tumor microenvi-ronment increases HIF1a accumulation through promoting pro-tein synthesis or inhibiting protein degradation. The cytoplasmicHIF1a then translocates to the nucleus, recognizes a cognatesequence on the Livin promoter, induces Livin expression, andpromotes inhibition of caspase activities and antiapoptosis.

IAP family of proteins is known to act as caspase inhibitors,blocking caspase activation and further inhibiting apoptosis (9).Previous studies have shown that a number of different IAPs werehighly expressed in malignant gliomas including XIAP (23),apollon (24), and survivin (25), with an adverse correlation withpatient outcomes. Although less studies are carried out in braintumors, recent studies reported that Livin and survivin are upre-gulated in glioma stem cells (GSC) derived from human glio-blastoma and astrocytoma tissues or glioblastoma cell lines butare the most increased in just differentiated GSC (26, 27). Thesefindings imply that Livin may contribute to antiapoptosis andtherapeutic resistance in glioblastoma. Here, we first demonstratethat the tumormicroenvironmentmediated expression pattern ofLivin. To directly distinguish and isolate hypoxic tumor subpo-pulations from solid tumors, we used a reliable protocol ofcycling- and chronic-hypoxic cell identification derived from ourprevious studies that allows subsequent immunofluorescenceimaging and flow-cytometric analysis of the biosignature of thesecells (16). Our results indicate that both of probable cycling andchronic hypoxic areas are important in tumor microenviron-ment–mediated Livin induction. However, Livin expression tendsto occur in probable cycling hypoxic areas with high HIF1 acti-vation and blood perfusion within the tumor microenvironmentin glioblastoma xenografts. Besides, in vitro studies also demon-strate that cycling hypoxia induced more Livin expression com-pared with noninterrupted hypoxia. These differential Livinexpressions suggest that HIF1a is a major regulator of tumormicroenvironment–mediated Livin induction because Livin isone of the HIF1a target genes. Moreover, the results of Livinexpression colocalized with high expression of HIF1a in humanglioblastoma specimens also support this notion.

Despite frequent reports of Livin overexpression in humancancers and its intrinsic nature in inhibition of caspase activities(28), the impact of Livin contributed to tumor microenviron-ment–mediated antiapoptosis and therapeutic resistance remainsunknown. In contrast with Livin, other IAPs, IAP-2 (29), andsurviving (30), have been identified as hypoxia-responsive genes.Although the detail mechanism is still unclear, IAP-2 can beinduced by severe hypoxia in several mammalian cells via HIF1-

Figure 6.Livin blockage enhances the efficiency of radiation plus TMZ treatment in glioblastomaxenografts. A, detectionof TAT-Lp15-dansyl andTAT-K22-dansyl in the cortexof nude mouse brain 1 hour after intravenous injection. Bars, 100 mm. B, bioluminescent images from control and treated animals including the TAT-K22 orTAT-Lp15 treatment, combination of TMZ (T) and radiation (R) treatment, pretreatment of TAT-K22 plus the combination of TMZ and radiation treatment, andpretreatment of TAT-Lp15 plus the combination of TMZ and radiation treatment on day 20 after tumor implantation. C, the mean normalized BLI values associatedwith longitudinal monitoring of intracranial tumor growth for each treatment group. Error bars, SD among 9mice per group. D, the corresponding survival curves ofGBM8401 xenograft-bearing mice for each treatment group. E, representative sections showing apoptotic cells measured by the TUNEL assay of GBM8401xenografts at 2 days after treatments. Each mean is based on 15 measurements (5 measurements for each of three tumors). F, the percentages of apoptotic cells oncross-sections. � , P < 0.01, compared with control. #, P < 0.001, compared with control TAT-K22 peptide. G, the percentages of apoptotic cells in tumor hypoxic cellswithin GBM8401 xenografts determined by pimonidazole/annexin V doubling staining flow-cytometric assay. �, P < 0.001, compared with control. #, P < 0.01,compared with control TAT-K22 peptide. Surviving fraction (H) and colony images (I) in excised GBM8401 and U251 xenografts after treatments. Error bars, SDamong triplicate experiments. � , P < 0.001, compared with combination of TMZ and radiation treatment.


Hsieh et al.




independent mechanisms. Besides, hypoxia can induce survivinexpression through thebindingofHIF1a to the survivin promoter.Therefore, IAP-2 and survivin are thought to be a role for hypoxia-mediated death resistance. Herein, we provide the first evidence toshow that hypoxia stimulatesHIF1a, but notHIF2a, to bind to thehuman Livin promoter and regulate its transcription. Moreover,our results provide clear evidence that hypoxic tumor microenvi-ronment, either chronic or cycling hypoxia, enhances the expres-sion and function of Livin and further promotes antiapoptosis andresistance to ionizing radiation and temozolomide in glioblasto-ma. Genetic inactivation or pharmacologic inhibition of Livinsensitizes cells to cytotoxic therapies-induced apoptosis in hypoxiccondition or endogenous tumormicroenvironment. These findingare important in the selection of optimal genetic or pharmacologicapproaches to inhibit tumor microenvironment–mediated thera-peutic resistance via the blockade of this mechanism.

Numerous studies suggest that Livin is an attractive target forcancer treatment. The reason is based on several independentpieces of evidence. First, the antiapoptotic ability of Livin cancontribute to the apoptosis-resistant phenotype of cancer cells(13). Second, Livin has been considered critical for tumor pro-gression included its function in cell proliferation, migration,invasion, and epithelial-to-mesenchymal transition (31). Third,Livin is preferentially expressed in tumors but not in normaltissues (14). In this study, we provide a novel rationale for Livin asa potential therapeutic target in cancer, which is hypoxic tumormicroenvironment can induce upregulation of Livin and furthercontributes to inhibition of tumor apoptosis and therapeuticresistance. The strategies to specific Livin intervention for novelcancer therapies have been proposed and developed in the pastdecade such as siRNA (32) and peptide strategies (20). Unfortu-nately, the use of siRNA is still therapeutically impractical. Theideal of targeted inhibitionof Livinby specific targetingpeptides isstill in cell model and not readily translatable to preclinical orclinical trial.

In the present study, we generate a cell-permeable peptide, TAT-Lp15, for targeting Livin and inhibiting its function by fusing Tatdomain fromHIV-I with the truncated peptide derived from linearpeptides that specifically bind to Livin as shown in previous study(20). Our findings demonstrate for the first time the feasibility ofusing cell-penetrating peptide for targeting Livin to sensitize cancercells for proapoptotic stimuli both in vitro and in vivo. We haveshown that the addition of TAT-Lp15 sensitizes glioblastoma celllines to the proapoptotic effects of ionizing radiation and temo-zolomide in vitro. Moreover, the results derived from in vivo studiesdemonstrate that the combination of TAT-Lp15 treatment andcytotoxic therapies has a significant synergistic effect in antiglio-blastoma growth and tumor cell killing above and beyond thatseen with the standard-of-care therapy of radiation plus temozo-lomide. In the absence of a proapoptotic stimulus; however, TAT-Lp15 treatment does not increase the spontaneous apoptosis rateof normal brain tissues (data not shown), suggesting that thistherapeutic peptide may have promising therapeutic potential tosensitize tumors for the cytotoxic therapies without affectingnormal tissues. Besides, our results derived from three differentglioblastoma cell lines with different genetic backgrounds suggestthat the treatment of a Livin-targeting peptide, even in glioblas-tomas contain multiple abnormalities of the apoptotic pathwayupstream of the Livin's or other IAPs' dysregulation from geneticmutation or tumor microenvironment, also appeared to be suffi-cient to initiate apoptosis or enhance therapeutic efficiency of

cytotoxic therapy. When combined with cytotoxic therapy, evenglioblastomas contain multiple abnormalities of the apoptoticpathway upstream of the Livin’s or other IAPs’ dysregulation fromgenetic mutation or tumor microenvironment.

For the molecularly targeted therapy in intracranial tumors,the blood–brain barrier (BBB) is an impediment to drugbioavailability. Here, we find that TAT-Lp15 is able to crosseven the intact BBB in normal brain. Therefore, this peptideoffers a feasible path for translation to the clinic. Previouspreclinical studies validating Livin as therapeutic targets ingliomas have utilized Livin siRNA (33). However, it is notfeasible to use the siRNA employed because of off-target effects,stimulation of the innate immune response, and inefficientdelivery to target organ (34). Besides, a series of Smac mimeticpeptides or compounds derived from the N-terminal tetra-peptide of a mitochondrial protein called Smac/DIABLO (sec-ond mitochondria-derived activator of caspase/direct IAP bind-ing protein with low pI) were designed as IAP antagonists (35).Although these inhibitors can binds to type-II BIR domains ofIAPs and interfere with the interaction between IAPs and activecaspases, the recent study demonstrates that the mechanism ofthese compounds sensitized cells to apoptosis are primarilyindirect due to autoubiquitination and proteasomal degrada-tion of cIAPs rather than depression of caspase activity (36).It is still unclear whether these compounds are able to inhibitLivin function. Although the detail mechanism of Livin-bindingpeptides in inhibition of Livin function is still unknown, noobvious sequence homologies to Smac or caspases and normalLivin expression in intracellular expression of the Livin-targetingpeptides suggest these peptides have distinct mechanisms to resen-sitize cells to apoptosis (20). Therefore, the cell-permeable peptidetargeted to Livin derived from this study provides a unique ther-apeutic drug in treatment of cancer with Livin expression.

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

Authors' ContributionsConception and design: C.-H. Hsieh, C.-P. Wu, W.-C. Shyu, C.-C. WangDevelopment of methodology: C.-H. Hsieh, Y.-J. Lin, W.-C. ShyuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.-H. HsiehAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.-H. Hsieh, Y.-J. Lin, C.-P. Wu, W.-C. ShyugWriting, review, and/or revision of the manuscript: C.-H. Hsieh, C.-P. Wu,W.-C. Shyu, C.-C. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.-H. Hsieh, Y.-J. Lin, H.-T. LeeStudy supervision: C.-H. Hsieh, C.-P. Wu, W.-C. Shyu

AcknowledgmentsThe authors thank theNational RNAiCore Facility, Academia Sinica, Taiwan,

for technical support.

Grant SupportGrant support was provided by the National Science Council of the Republic

of China (grant 102-2314-B-039-029-MY3) and grant CMU103-BC-6 fromChina Medical University.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 12, 2014; revised October 1, 2014; accepted October 28,2014; published OnlineFirst November 4, 2014.






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2015;21:460-470. Published OnlineFirst November 4, 2014.Clin Cancer Res Chia-Hung Hsieh, Yu-Jung Lin, Chung-Pu Wu, et al. Cytotoxic Therapies in Glioblastoma Multiforme

Induced Resistance to−Livin Contributes to Tumor Hypoxia

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