Hsp27 Inhibition with OGX-427 Sensitizes Small Cell …...Small Molecule Therapeutics Hsp27 Inhibition with OGX-427 Sensitizes Non–Small Cell Lung Cancer Cells to Erlotinib and Chemotherapy

Small Molecule Therapeutics

Hsp27 Inhibition with OGX-427 SensitizesNon–Small Cell Lung Cancer Cells to Erlotinib andChemotherapyBarbara Lelj-Garolla1, Masafumi Kumano1, Eliana Beraldi1, Lucia Nappi1, Palma Rocchi2,Diana N. Ionescu3, Ladan Fazli1, Amina Zoubeidi1, and Martin E. Gleave1

Abstract

Non–small cell lung cancer (NSCLC) is the most frequentcause of death from cancer worldwide. Despite the availabilityof active chemotherapy regimens and EGFR tyrosine kinaseinhibitors, all advanced patients develop recurrent disease afterfirst-line therapy. Although Hsp27 is a stress-induced chaper-one that promotes acquired resistance in several cancers, itsrelationship to treatment resistance in NSCLC has not beendefined. Understanding adaptive responses of acquired resis-tance will help guide new strategies to control NSCLC. Hsp27levels were evaluated in an HCC827 erlotinib-resistant–derivedcell line (HCC-827Resistant), and sensitivity to erlotinib wasexamined in Hsp27-overexpressing A549 cells. The role ofHsp27 in both erlotinib and cytotoxic treatment resistance wasevaluated in HCC-827 and A549 NSCLC cells using the Hsp27antisense drug OGX-427. The effect of OGX-427 in combina-

tion with erlotinib was also assessed in mice bearing A549xenografts. Hsp27 is induced by erlotinib and protects NSCLCcells from treatment-induced apoptosis, whereas OGX-427sensitizes NSCLC cells to erlotinib. Interestingly, increasedresistance to erlotinib was observed when Hsp27 was increasedeither in HCC827 erlotinib-resistant or overexpressing A549cells. Combining OGX-427 with erlotinib significantlyenhanced antitumor effects in vitro and delayed A549 xenograftgrowth in vivo. OGX-427 also significantly enhanced the activityof cytotoxic drugs used for NSCLC. These data indicate thattreatment-induced Hsp27 contributes to the development ofresistance, and provides preclinical proof-of-principle that inhi-bition of stress adaptive pathways mediated by Hsp27enhances the activity of erlotinib and chemotherapeutics. MolCancer Ther; 14(5); 1–10. �2015 AACR.

IntroductionLung cancer is the most prevalent cancer and the leading cause

of cancer-related death worldwide (1). In North America andEurope, it is the third most common cause of mortality for menand women and is increasingly observed in nonsmokers. Non–small cell lung cancer (NSCLC), with a 5-year survival rate of only15%, accounts for approximately 80% of all lung cancers. Plat-inum-based doublets with gemcitabine or pemetrexed remain themainstay of first-line therapy for advanced NSCLC with modestimprovement in survival and quality of life, and are superior tosingle-agent therapy or three drug combinations (2). EGFR tyro-sine kinase inhibitors (TKI), including afatinib, erlotinib, andgefitinib, are also approved and particularly active in patientswithEGFR activating mutations or overexpression (3). Despite these

active agents, virtually all patients with advanced NSCLC developrecurrent disease after first-line therapy and long-term survivalrates remain low in these patients. Understanding adaptiveresponses that support acquired resistance are needed to developnew strategies to control NSCLC.

The EGFR is a member of the ErbB receptor tyrosine kinasefamily, a transmembrane protein with an intracellular and atyrosine kinase domain that self-phosphorylates upon receptordimerization. Phosphorylation triggers a cascade of intracellularevents, including activation of the PI3K/Akt and the MAPK/RASpathways, leading to cell survival and proliferation (4). Manysolid tumors have constitutively activated EGFR, either by over-expression or by mutation, which leads to excessive signaling.Despite good initial responses to EGFR TKIs, most patientsdevelop resistance and eventually die of metastatic disease.Resistance is often associated with the development of EGFRmutations that interfere with binding to the inhibitors tomaintain EGFR activity (5), which can be partially overcomeby increasing TKI potency or by cotargeting other pathwaysupregulated during treatment. For example, there is evidencethat cotargeting Hsp90, a molecular chaperone known tointeract with EGFR, can increase EGFR molecular degradationeven in the presence of mutations (6).

Other heat shock chaperones, such as Hsp27 (HspB1), play apivotal role in stress responses to treatment. Hsp27 is a stress-induced ATP-independent molecular chaperone that is transcrip-tionally regulated by HSF-1, HIF1a, and other factors influencedby cell type and cell context (7, 8). In addition, Hsp27 oligo-merizes and self-associates in a phosphorylation-dependent

1The Vancouver Prostate Centre and Department of UrologicalSciences, University of British Columbia,Vancouver, British Columbia,Canada. 2Cancer Research Center of Marseille, INSERM, Institut Paoli-Calmettes, and Aix-Marseille Universit�e, Marseille, France. 3PathologyDepartment, BCCancerAgency,Vancouver,BritishColumbia,Canada.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Martin E. Gleave, Department of Urologic Sciences,University of British Columbia, 2775 Laurel Street, Level 6, Vancouver, BritishColumbia, Canada V6H 3Z6. Phone: 604-875-4818; Fax: 604-875-5654; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-0866

�2015 American Association for Cancer Research.

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manner regulated by AKT and MAPK signaling pathways (9).Hsp27 is upregulated in cisplatin-resistant ovarian cancer cells aswell as taxane-resistant prostate cancer cells (10, 11), and shownto be highly expressed and associated with poor prognosis inmany cancers. Hsp27 plays a crucial role in protein homeosta-sis, in stabilizing unfolding proteins, as well as the actin-cyto-skeleton. Hsp27 promotes survival by binding to both cyto-chrome c and caspase-3 to inhibit intrinsic apoptotic pathwayactivation, and facilitates epithelial–mesenchymal transitionand expression of metastatic genes (10, 12–14). Hsp27 is adifficult protein to target because, unlike Hsp90, it lacks anactive site or ATP-binding pocket. Second-generation antisensetechnology enables targeted inhibition of "undruggable" targetslike Hsp27 by downregulating gene expression at the mRNAlevel. The antisense inhibitor of Hsp27, OGX-427 (OncoGeneXPharmaceuticals) potently reduces Hsp27 levels with reportedactivity in preclinical cancer models (15–17) and tolerability assingle agent and in combination with docetaxel chemotherapyin phase I studies. A phase II clinical trial of OGX-427 incastrate-resistant prostate cancer reported single-agent activity,and OGX-427 is currently in multicenter phase II clinical trialsin metastatic CRPC, NSCLC, and bladder cancer (ClinicalTrials.gov, NCT01454089, NCT01829113, and NCT01120470).

In this study, we show that Hsp27 is expressed in nearly 70% to80% of NSCLC patients and that erlotinib or chemotherapyincreases levels of both total and phosphorylated Hsp27 viaactivation of HSF-1. Hsp27 overexpression limited the activityof erlotinib and cytotoxic agents, whereas Hsp27 inhibition withOGX-427 synergistically increased erlotinib-induced apoptosisand inhibition of A549 and HCC827 lung cancer cell growth invitro and in vivo. These results illustrate that cotargeting treatment-induced adaptive responsesmediated byHsp27 can sensitize lungcancer cells to EGFR TKIs and cytotoxic therapies.

Materials and MethodsTumor tissue arrays

Six hundred and nine cases of primary NSCLCs with follow-updata diagnosed between 1978 and 2002 were identified from thearchives of St Paul's Hospital, Vancouver, British Columbia. Theparaffin-embedded tissueblockswere used to construct a duplicatecore Tissue microarray (TMA). After review of the tissue cores, 588cases, consisting of NSCLC met the criteria for inclusion in thisstudy. Carcinoids, atypical carcinoids, large cell neuroendocrinecarcinoma, andmetastatic tumors were all excluded. Hematoxylinand eosin (H&E)-stained sections were reviewed and subclassifiedas follows: 243 adenocarcinoma, 272 squamous cell carcinoma(SCC), 35 large cell carcinoma (LCC), 32 non–small cell carcino-ma, and six other (carcinoma, giant cell carcinoma).

IHCIHC staining was conducted by Ventana autostainer model

Discover XT (Ventana Medical System) with enzyme-labeledbiotin streptavidin system and solvent-resistant DAB Map kit byusing mouse monoclonal HSP27 antibody fromNovocastra nowLeica Biosystems Newcastle Ltd and polyclonal phosphor-HSP27(Ser82) antibody from Cell Signaling Technology. To ensurespecificity of hsp27 and phsp27 antibodies, small cell lungcarcinoma and benign prostate gland basal cells tissues wereincluded as negative and positive controls, respectively (Supple-mentary Fig. S1).

Lung cancer cell lines and reagentsA549 (EGFR wild-type but kRASmutated: G12S) and HCC827

(EGFR mutated: delE746-A750) cells were purchased from theATCC and authenticated with short tandem repeat (STR) profileanalysis in Jan 2013 by Genetic Resources Core Facility at JohnHopkins. Cells weremaintained in RPMI-1640media (InvitrogenLife Technologies) containing 5% FBS (Invitrogen Life Technol-ogies). Erlotinib was purchased from LC Laboratories. A549 celllines overexpressingHsp27 (A549Hsp27)were developed by stablytransfecting cells with an Hsp27 vector by lentivirus transfectionas previously described (8). HCC827Resistant cell lines were devel-opedbymaintainingHCC827Parental in 500nmol/L erlotinib for 3months. Genomic sequencing was done to confirm cell line iden-tity. Antibodies: anti-HSF-1 from Santa Cruz Biotechnology; anti-Hsp27 fromStressGen; anti- phospho-EGFR (Tyr1068), anti-EGFR,anti-cleaved PARP, anti-phospho-Hsp27 (Ser82), anti-cleaved cas-pase-3, anti–caspase-3, anti-phospho-ERK and anti-phospho-Akt(Ser423) were purchased from Cell Signaling Technology; andanti–b-actin from Sigma-Aldrich. Paclitaxel was purchased fromSigma Chemical Co.. Gemcitabine (Lilly Pharmaceuticals), peme-trexed (Lilly Pharmaceuticals), and cis-platinum (Hospira) wereobtained from the B.C. Cancer Agency.

ASO transfectionCells were transfected with antisense as described previously

(18, 19). Hsp27 ASO and scrambled (ScrB) control oligonucle-otide sequences were manufactured by ISIS Pharmaceuticals(Carlsbad) and supplied by OncoGenex Technologies. Thesequence of OGX-427 corresponds to the human Hsp27 trans-lation initiation site (50-GGGACGCGGCGCTCGGTCAT-30). Thesequence of heat shock factor 1 (HSF-1) siRNA corresponds to thehuman HSF-1 site (sc-35611; Santa Cruz Biotechnology). Ascrambled siRNA (50-CAGCGCUGACAACAGUUUCAU-30; Dhar-macon)was used as a control for RNAi experiments. Transfectionswere performed by using Oligofectamine (Invitrogen), as previ-ously described (20).

Western blot analysisTotal proteins were extracted using RIPA buffer (50 mmol/L

Tris, pH 7.2, 1% NP-40, 0.1% deoxycholate, 0.1% SDS, 100mmol/L NaCl, Roche complete protease inhibitor cocktail) andcentrifuged to remove the insoluble material. Western blot anal-ysis was performed as described previously (19).

Quantitative reverse-transcriptase PCRRNA extraction and reverse-transcriptase PCR (RT-PCR) were

carried out as described previously (21). Real-time monitoringof PCR amplification of cDNA was carried out using thefollowing primer pairs and probes: Hsp27 (Hs03044127_g1),HSF1 (Hs00232134_m1), and GAPDH (Hs03929097_g1;Applied Biosystems) on the ABI PRISM 7900 HT SequenceDetection System (Applied Biosystems) with a TaqMan GeneExpression Master Mix (Applied Biosystems). Target geneexpression was normalized to GAPDH levels in the respectivesamples as an internal control. The results are representative ofat least three independent experiments.

In vitro cell growth assayCells were plated in 12-well plates, and transfected with siRNA

for 1 day or ASO for 2 days as indicated followed by erlotinibtreatment at various concentrations for 48 hours. Cell viability

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was then assessed using crystal violet assay as described previously(22). Absorbance was determined with a micro-culture platereader (Becton Dickinson Labware) at 560 nm. Changes in cellviability were calculated as percentage relative to the vehicle-treated cells. Each assay was performed in triplicate. The combi-nation index (CI) was evaluated using CalcuSyn dose–effectanalysis software (Biosoft) as described previously (21). CI wascalculated at ED50, ED75, and ED90.

Cell-cycle analysisCell-cycle populations were analyzed by propidium iodide–

staining using flow cytometry as previously described (18).

Animal manipulation and assessment of in vivo tumorgrowth

For in vivo xenograft studies, 6� 106 A549 cells were inoculateds.c. in the flank of 6- to 8-week-old male athymic nude mice(Harlan Sprague Dawley, Inc.) via a 27-gauge needle underisoflurane anesthesia. When tumors reached 100mm3,mice wererandomly selected for treatment with 50mg/kg erlotinib (formu-lated in 0.5% methylcellulose þ 0.1% Tween-80) orally admin-istered once daily for 5 days per week and 15 mg/kg OGX-427 orScrB injected i.p. once daily for 7 days and three times per weekthereafter. Each experimental group consisted of 8 mice. Tumorvolume measurements were performed once weekly and calcu-lated by the formula length � width � depth � 0.5236. Data

pointswere expressed as average tumor volume� SEM.All animalprocedures were performed according to the guidelines of theCanadian Council on Animal Care and with appropriate institu-tional certification.

Luciferase assayHCC827 and A549 cells were transfected with HSE luciferase

plasmid and pRL-TK as internal control using lipofectin reagent(Invitrogen) followed by erlotinib treatment for 24 hours at theindicated concentrations. The luciferase activity was measuredusing a dual-luciferase reporter assay system (Promega) and amicroplate luminometer (Tecan). The results are representative ofthree independent experiments.

Statistical analysisDifferences between the two groups were compared using

Student t test andMann–WhitneyU test. All statistical calculationswere performed using Statview 5.0 software (Abacus Concepts,Inc.), and P values < 0.05 were considered significant.

ResultsHsp27 and phospho-Hsp27 are highly expressed in humanNSCLC

Hsp27 has been reported to be highly and uniformly expressedin several cancers (23–27). To assess the distribution of Hsp27

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Figure 1.Hsp27 and phosphorylated Hsp27expression in NSCLC. A, paraffin-embedded tissue blocks were used toconstruct a duplicate core tissuemicroarray (TMA). Hsp27 expressionwas evaluated in 440 NSCLC tissues.H&E-stained sections were reviewedand subclassified as follows: 214 SCC,195 adenocarcinoma, and 31 LCC.B, phospho-Hsp27 expression wasevaluated in 427 NSCLC tissues.H&E-stained sections were reviewedand subclassified as follows: 212 SCC,184 adenocarcinoma, and 31 LCC.

Targeting Hsp27 in NSCLC

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and phospho-Hsp27 expression in humanNSCLC, we performedimmunostaining of human NSCLC spotted on TMA. PositiveHsp27 staining was noted in 176 of 214 SCCs (82.2%), 139 of195 adenocarcinomas (71.3%), and 21 of 31 LCCs (67.7%; Fig.1A). Positive phospho-Hsp27 stainingwas observed in 184of 212SCCs (86.8%), 147 of 184 adenocarcinomas (79.9%), and 27 of31 LCCs (87.1%; Fig. 1B). These data suggest that bothHsp27 andphospho-Hsp27 are frequently expressed in NSCLC.

Erlotinib induces Hsp27 expression in A549 and HCC827 cellsvia HSF-1 activation

To evaluate the effect of erlotinib on Hsp27 expression andphosphorylation, we used erlotinib-resistant A549 (IC25 ¼ 4.2

mmol/L) and erlotinib-sensitive HCC827 NSCLC cell lines (IC25

¼ 8 nmol/L). Erlotinib caused a clear decrease of EGFR phos-phorylation associated with a time- (Fig. 2A) and dose (Supple-mentary Fig. S2)- dependent increase of total Hsp27 and phos-pho-Hsp27 with a parallel increase of apoptosis as detected bycleaved PARP. Because HSF-1 is a key regulator of Hsp27 expres-sion, we evaluated the effect of erlotinib on HSF-1 activity.Erlotinib led to increased HSF-1 and Hsp27 both at the protein(Fig. 2B, left) and mRNA levels in both A549 and HCC827 celllines (Fig. 2B, right), together with an increase of HSF-1 activity asmeasured by HSE transactivation assay (Supplementary Fig. S3).Moreover, silencing HSF-1 using siRNA abrogated erlotinib-induced upregulation of Hsp27 (Fig. 2C). Hsp27 can be

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Figure 2.Erlotinib induces Hsp27/phospho-Hsp27 expression via activation of HSF-1. A, A549 (left) andHCC827 (right) cells were treatedwith erlotinib for up to 72 hours, andWestern blot analyses were performed with antibodies against p-Hsp27, total Hsp27, P-EGFR, total EGFR, cleaved PARP. b-actin and vinculin were used as loadingcontrol. B, A549 and HCC827 cells were treated with erlotinib for 48 hours. Left, Western blot analyses were performed against p-EGFR, total EGFR, p-Hsp27,total Hsp27, HSF-1, and b-actin was used as loading control. Right, Hsp27 and HSF-1 mRNA levels were measured by real-time PCR 48 hours after treatment.C, A549 and HCC827 cells were transfected daily with 20 nmol/L HSF-1 siRNA or scramble siRNA for 1 day, followed by erlotinib treatment for 48 hours. Left, HSF-1,pEGFR, total EGFR, Hsp27, and b-actin as loading control were detected byWestern blot analysis. Right, mRNA levelswere analyzed by real-time PCR 48 hours aftertreatments. Bars, SD. �� and � , differ from control P < 0.01 and P < 0.05, respectively.

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phosphorylated by p90Rsk downstream of pAkt and pErk (18),two pathways downstream of pEGFR, and therefore inhibited byerlotinib (data not shown). Although levels of pAkt and pErk donot increase after erlotinib, increase of pHsp27 reflect increasedHSF-1 mediated transcription of total Hsp27. Collectively thesedata indicate that stress-induced activation of HSF-1 by erlotinibleads to increased total Hsp27 levels and a consequent increase inp-Hsp27. This finding, in addition to the positive staining in TMAsamples from a variety of NSCLC, implicates Hsp27 in erlotinibresistance and highlights it is a therapeutic target in combinationtherapy with EGFR TKI.

Hsp27 overexpression reduces erlotinib-induced cell deathWepreviously reported thatHsp27overexpressionpromotes cell

survival and confers resistance to paclitaxel in prostate and bladdercancer cell lines (10, 28). Here, we investigated effects of Hsp27overexpression on A549 cell growth in the presence of erlotinib bycomparing cell viability of a stably transfected A549 cell line(A549Hsp27) to A549Empty controls after increasing doses of erlo-tinib. Cell viability curves in Fig. 3A confirm thatA549Hsp27 cells aremore resistant to erlotinib compared with control A549Empty cells.Moreover, Western blot analysis (Fig. 3B) indicates reduced PARP

cleavage at 10 and 20 mmol/L erlotinib in the A549Hsp27 versusA549Empty cell lines confirming that overexpression of Hsp27protects lung cancer cells from erlotinib-induced apoptosis.

To better evaluate the role of Hsp27 in erlotinib resistance, weestablished an HCC827 resistant cell line (IC25 ¼ 1 mmol/L) bymaintaining HCC827 parental cells in 500 nmol/L erlotinib for 3months (Fig. 3C). Figure 3D shows reduced cleaved PARP andincreased expression of Hsp27 in HCC827resistant cells comparedwith parental. These findings further implicate Hsp27 in erlotinibresistance both when Hsp27 becomes endogenously overex-pressed in response to treatment (HCC827resistant) and when itis highly expressed via stable transfection (A549Hsp27).

OGX-427 sensitizes both HCC827 and A549 to erlotinibBecause the preceding results link Hsp27 to erlotinib resistance,

we hypothesized that cotargeting this chaperone in combinationwith EGFR TKI will increase erlotinib efficacy. Cell viability wasassessed in A549 and HCC 827 cells after erlotinib or OGX-427mono- and combination-therapy. OGX-427 reduces Hsp27 pro-tein expression (Supplementary Fig. S4), and when used in com-bination with erlotinib, leads to increased cell death comparedwith single-agent therapy (Fig. 4A). CI, calculated at a ratio OGX-

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Figure 3.Hsp27 overexpression induces erlotinib resistance. A, A549 cells were stably transfected either with an empty vector (A549Empty) or with a vector containingwild-typeHsp27 (A549Hsp27). Cell viabilitywasdeterminedafter increasing erlotinib doses 72hours after treatment. B, p-EGFR, total EGFR, Hsp27, cleavedPARP, andb-actin as loading control were detected by Western blot analysis in both cell lines after 72 hours erlotinib treatment. C, HCC827 erlotinib-resistant cell lines(HCC827resistant) were established maintaining HCC827parental cells in 500 nmol/L erlotinib for 3 months. Hsp27 levels were measured by Western blotanalyses (insert). Cell viability was determined after increasing erlotinib doses 72 hours after treatment. D, cleaved PARP, Hsp27, p-Hsp27, p-EGFR, total EGFR,and vinculin as loading control were detected by Western blot analyses for resistant cells at 500 nmol/L erlotinib and parental cells at 0 and 72 hours after200 nmol/L erlotinib.






427:erlotinib 1:200 for A549 and 20:1 for HCC827, confirms asynergistic effect for this drug combination (Fig. 4B). Increasedlevels of cleaved PARP and caspase-3 assessed byWestern blotting(Fig. 4C), together with increased subG0 apoptotic fractionassessed by PI staining-based FACS analysis (Fig. 4D) confirmedthat OGX-427 enhances erlotinib-induced apoptosis. These dataconfirm that Hsp27 inhibition increases NSCLC cancer cell sensi-tivity to erlotinib.

Combining OGX-427 with erlotinib enhances tumor growthinhibition in vivo

The combination effect of OGX-427 and erlotinib was nextevaluated in an in vivo model using A549 xenografts. Tumor-bearing mice were randomly assigned to groups treated withScrBþdiluent, ScrBþerlotinib, OGX-427þdiluents, or OGX-427þerlotinib when A549 tumors reached 100 mm3. Meantumor volume were similar in all groups at baseline and alltreatments were performed for 7 weeks. Consistent with in vitrofindings, OGX-427 was able to reduce Hsp27 protein level, and

the combination therapy of OGX-427 plus erlotinib significantlyreduced tumor growth rates (Fig. 5A–C) compared with all othergroups (P ¼ 0.0002, 0.03, and 0.01 against ScrBþdiluent,ScrBþerlotinib, and OGX-427þdiluent, respectively). Moreover,OGX-427plus erlotinib-treated tumors hadhigher apoptotic ratesas shown by increased TUNEL staining compared with the othergroups (Fig. 5D). These studies indicated that this combinationtreatment significantly delays growth and increases apoptoticrates of A549 xenografts.

Combination therapy of OGX-427 plus cytotoxic agentsincreases tumor cell death

Hsp27 is known to be induced by cytotoxic agents in manycancers, and can confer resistance to cytotoxics (29, 30). We nextevaluated effects of OGX-427 in combination with pemetrexed, afirst-line therapy for the treatment of NSCLC. Pemetrexedincreased total and phospho-Hsp27 levels in a time- and dose-dependent manner in A549 cells (Fig. 6A). Combined OGX-427plus pemetrexed reduced A549 cell viability compared to cells

Figure 4.OGX-427 sensitizes both HCC827 and A549 to erlotinib. A, A549 and HCC827 cells were transfected with 50 nmol/L OGX-427 or ScrB for 2 days and treatedwith the indicated concentration of erlotinib for 48 hours after the second transfection. Cell viability was determined by crystal violet assay 48 hours aftererlotinib treatment. B, combination index analysis for erlotinib and OGX-427 treatment in A549 and HCC827 cell lines was performed according to the Chouand Talalay method (49). C, p-EGFR, total EGFR, total Hsp27, cleaved PARP, cleaved caspase-3, caspase-3 and b-actin, as loading control, were detected byWestern blot analysis. Inhibition of erlotinib-induced Hsp27 enhances cell apoptosis in both A549 and HCC827. D, cells were transfected with 50 nmol/L OGX-427 orScrB for 2 days and treated with erlotinib (A549 at 10 mmol/L and HCC827 at 2.5 nmol/L) for 48 hours after the second transfection. Flow-cytometric analysisof A549 and HCC827 shows an increase in sub-G0 and decrease in G0–G1. Bars, SD. �� , differs from control (P < 0.01).

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treated with either drug alone and the CI analysis demonstratedsynergistic effects with combination therapy (Fig. 6B). Similarresults were observed in A549 cells after OGX-427 treatment incombination with cisplatin, paclitaxel, and gemcitabine, all cyto-toxic agents used clinically in patients with NSCLC (Fig. 6C).Western blot analysis in A549 cells indicated marked increase inapoptosis as measured by cleaved PARP with OGX-427 combi-nation regimens compared with single agents (Fig. 6D), support-ing inhibition of Hsp27 as a cotargeting strategy with cytotoxicagents for the treatment of NSCLC.

DiscussionAnticancer treatments induce stress responses that inhibit apo-

ptosis and promote emergence of an acquired treatment-resistantphenotype. Molecular chaperones play key roles in these stressresponses by regulatingmany prosurvival signaling and transcrip-tional networks. Two strategies have recently emerged that arerevolutionizing the treatment landscape in cancer: the use of

molecular-targeted agents and the selective inhibition of adaptiveresponses. In this work, we focus on the role of treatment-inducedHsp27 expression and treatment response in NSCLC, the mostcommon type of lung cancer and the largest cause of cancer-related death worldwide, where chemotherapy or EGFR-TKIs arefirst-line treatment. EGFR belongs to a family of membrane-bound receptors with a tyrosine kinase domain that, when phos-phorylated, activates a number of downstream prosurvival path-ways, including the PI3K/Akt andRAS/MAPKpathways (4). Somepatients, for whom EGFR-TKI is recommended, have a mutatedEGFR that is constitutively activated. For these individuals, the useof EGFR-TKI is recommended over chemotherapy as first line oftreatment (31, 32). Although EGFR TKIs have been shown toprolong life, most patients acquire resistance with a medianprogression-free survival of 10 months (33) and eventually dieof metastatic disease.

Hsp27 is a stress-induced molecular chaperone that regulatesmany prosurvival cellular functions. Many solid tumors, includ-ing bladder, prostate, breast and colorectal cancers, express high

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Figure 5.OGX-427 plus erlotinib delays A549 xenograft growth in vivo. A, A549 cells were inoculated s.c. and, when tumor volume reached 50 to 100mm3, mice weretreated with scrambled (ScrB) controlþdiluent, ScrBþerlotinib, OGX-427þdiluents, or OGX-427þerlotinib as described in Materials and Methods. Each datapoint represents the mean tumor volume in each group containing 8 mice � SEM. � , differs from ScrBþdiluent, ScrBþerlotinib, OGX-427þdiluents, or OGX-427þerlotinib treatment group (P < 0.05). B, tumor volume changes were evaluated 3 weeks after starting the treatment. We set as 0 that at which tumor volumedoubled after starting the treatment. C, Hsp27 downregulation after OGX-427 treatment was evaluated by Western blot analysis. Three animals were (D), Tumorswere collected after 49 days and TUNEL was evaluated by IHC analysis (original magnification: �200). Used as representative samples from each group.






Hsp27 levels that correlate with poor prognosis. Hsp27maintainsprotein homeostasis by stabilizing protein conformation or facil-itating proteasomal degradation (34, 35), inhibits apoptosis byinteracting with proapoptotic molecules such as cytochrome c(36) or pro-caspase 3 (37), and enhances oncogenic signalingpathways such as STAT3, IGFI, androgen receptor (AR), and eIF4E(19, 38, 39). Consistent with these multiple cytoprotective func-tions, overexpression of Hsp27 renders cancer cells resistant tochemo- and hormonal therapy (29, 39).

Because Hsp27 functions as a regulatory "hub" in multipleregulatory pathways, its inhibition will simultaneously suppressmany pathways implicated in cancer progression and resistance tohormone- and chemotherapies. Hsp27 acts through an ATP-independent mechanism and is therefore not amenable to inhi-bition by 17-AAG–derived small molecules. Strategies to inhibitHsp27 at themRNA level are an alternative approach to inhibiting"non-druggable" targets; indeed, siRNA or antisense oligonucleo-tides targeting Hsp27 suppresses proliferation and EMT in pros-tate (10, 39–41) and breast cancer cells (42). Hsp27 silencing alsodecreases clonogenic survival and induces cell senescence inHCT116 human colon cancer (43). Hsp27 silencing chemosen-sitizes SK-BR-3 HR breast cancer cells to Herceptin (44), A549lung cancer cells to 17-AAG (45), MUC-3 bladder cancer cells andPC-3 prostate cancer cells to paclitaxel (28, 40).

In this study, we show that Hsp27 and phospho-Hsp27 arecommonly expressed in a TMA of 440 SCCs, adenocarcinomas,

and LCCs of the lung. We also show that treatment stress witherlotinib or chemotherapy increases Hsp27 expression via thetranscription factor HSF-1 and that high levels of Hsp27 induceresistance to erlotinib. Thesefindings implicate a role forHsp27 incell survival ofNSCLCunder EGFR inhibition, and support testingcotargeting Hsp27 with erlotinib or other chemotherapeutics forthe treatment of NSCLC. Inhibition of treatment-induced Hsp27using OGX-427 synergistically enhanced inhibitory effects oferlotinib on cell proliferation and cell viability in NSCLC celllines in vitro. Similarly, combination therapy with erlotinib plusOGX-427 significantly inhibited A549 tumor growth in vivocompared with monotherapy. Consistent with these results,Hsp27 is upregulated by HSF-1 in c-MET addicted gastric celllines when c-MET is inhibited, and functions to limit the activityof c-MET-targeted therapies (46). In this gastric cell line, theactivation of K-RAS by transfection of G12V K-RAS downstreamof c-MET reverses the effect of c-MET inhibitors on Hsp27 upre-gulation (46). We observed an increase of Hsp27 even in A549lung cancer cells that have a constitutively activated MAPK path-way due to a K-RAS activating mutation (G12S), demonstratingthat in A549 lung cancer cells, Hsp27 upregulation via HSF-1 is aresponse to EGFR inhibition even when the MAPK pathway isconstitutively active.

Because platinum-based doublets with gemcitabine or peme-trexed are the first line of treatment for NSCLC in patients thatdo not carry an EGFR mutation, we also evaluated effects of

Cleaved PARP

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+––––

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Figure 6.Combination of OGX-427 with chemotherapy enhances cytotoxicity in A549 cells. A, time- and dose-dependent induction of Hsp27 expression by pemetrexed inA549 cells. Protein expression levels of p-Hsp27, Hsp27, cleaved PARP, and b-actin as loading control were evaluated by Western blot analysis. (B, left)Cells were treated with OGX-427 for 2 consecutive days followed by incubation with pemetrexed for an additional 72 hours. Cell viability was determined by crystalviolet assay. B, right, combination index at ratio of OGX-427 to pemetrexed of 1:20 was calculated to establish synergy. C, A549 cell viability was evaluated inthe presence of cisplatin, paclitaxel, and gemcitabine in combinationwith ScrB orOGX-427. Bars, SD. �� and � , differ from control (P <0.01 andP <0.05, respectively).D, Hsp27 and cleaved PARP were measured for the combination therapies by Western blot analyses. b-actin expression was used as a loading control.

Lelj-Garolla et al.





Hsp27 inhibition as cotreatment with these cytotoxics. OGX-427 synergistically enhanced growth-inhibitory effects ofpemetrexed, gemcitabine, cisplatin, and paclitaxel in A549 cellsby enhancing treatment-induced apoptosis. These results pro-vide a framework for building new drug combinations based onmechanism-based interventions to overcome drug resistance,and preclinical proof-of-principle supporting clinical trials ofOGX-427 combination regimens for lung and other cancers.OGX-427 has completed single-agent and docetaxel-combina-tion dose-escalation phase I trials in prostate, bladder, breast,and lung cancer (47). OGX-427 is well tolerated, with themajority of the adverse events reported being grade 1 or grade2, although a symptom complex of rigors, pruritus, and ery-thema during or shortly after infusion of drug has requiredsteroid prophylaxis and/or treatment in some patients at higherdoses. Preliminary data from a randomized phase II trial ofOGX-427 plus prednisone in castration-resistant prostate can-cer reported a PSA decline � 30% in 55% of patients and anacceptable safety profile (48). Several randomized phase IItrials of OGX-427 in combination with chemotherapy areactively accruing, including untreated stage IV NSCLC compar-ing carboplatin plus pemetrexed with or without OGX-427(SPRUCE, clinicaltrials.gov NCT01829113).

Disclosure of Potential Conflicts of InterestM.E. Gleave reports receiving commercial research support from, has own-

ership interest (including patents) in, and is a consultant/advisory board

member for OncoGenex. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: B. Lelj-Garolla, P. Rocchi, A. Zoubeidi, M.E. GleaveDevelopment of methodology: B. Lelj-Garolla, P. Rocchi, D.N. Ionescu,A. Zoubeidi, M.E. GleaveAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Rocchi, D.N. Ionescu, M.E. GleaveAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Lelj-Garolla, L. Nappi, M.E. GleaveWriting, review, and/or revision of the manuscript: B. Lelj-Garolla, E. Beraldi,L. Nappi, M.E. GleaveAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Lelj-Garolla, M. Kumano, E. Beraldi, L. NappiStudy supervision: B. Lelj-Garolla,Other (pathology): L. Fazli

Grant SupportThis work was supported by the Terry Fox New Frontiers Program (to M.E.

Gleave) and the Pacific Northwest Prostate Cancer SPORE NCI CA097186(to M.E. Gleave). B. Lelj-Garolla was supported by the Pier Luigi TolainiYoung Investigator Award from the Coalition to Cure Prostate Cancer.

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.

Received October 15, 2014; revised February 11, 2015; accepted February 21,2015; published OnlineFirst March 4, 2015.

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Lelj-Garolla et al.




Published OnlineFirst March 4, 2015.Mol Cancer Ther Barbara Lelj-Garolla, Masafumi Kumano, Eliana Beraldi, et al. Lung Cancer Cells to Erlotinib and Chemotherapy

Small Cell−Hsp27 Inhibition with OGX-427 Sensitizes Non

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