SOCS1 Gene Therapy Improves Radiosensitivity and …...(19–22). Moreover, SOCS1 gene therapy was shown to induce potent antitumor effects in a preclinical patient-derived ESCC xenograft

Therapeutics, Targets, and Chemical Biology

SOCS1 Gene Therapy Improves RadiosensitivityandEnhances Irradiation-InducedDNADamage inEsophageal Squamous Cell CarcinomaTakahito Sugase1,2,3, Tsuyoshi Takahashi1,2, Satoshi Serada2,3, Minoru Fujimoto2,3,Kosuke Hiramatsu2,3, Tomoharu Ohkawara2,3, Koji Tanaka1, Yasuhiro Miyazaki1,Tomoki Makino1, Yukinori Kurokawa1, Makoto Yamasaki1, Kiyokazu Nakajima1,Tadamitsu Kishimoto4, Masaki Mori1, Yuichiro Doki1, and Tetsuji Naka2,3

Abstract

STAT3hasbeen implicated recently in radioresistance in cancer.In this study, we investigated the association between STAT3 andradioresistance in esophageal squamous cell carcinoma (ESCC).Strong expression of activated phospho-STAT3 (p-STAT3) wasobserved in 16/22 ESCC patients with preoperative chemora-diotherapy (CRT), compared with 9 of 24 patients with surgeryalone, where the prognosis of those with CRT was poor. Expres-sionof p-STAT3and the antiapoptotic proteinsMcl-1 and survivinwas strongly induced in ESCC cells by irradiation. Ectopic STAT3expression increased radioresistance, whereas expression of theSTAT3 negative regulator SOCS1 via an adenoviral vectorimproved radioresponse. Inhibiting the STAT3–Mcl-1 axis by

SOCS1 enhanced DNA damage after irradition and inducedapoptosis. Combining SOCS1 with radiotherapy enhanced anti-tumor responses in amurine xenograft model compared with theindividual therapies. Tumor repopulation occurred transientlyafter treatment by irradiation but not the combination SOCS1/radiotherapy. Tumors subjected to this combination expressedhigh levels of gH2AX and low levels of Ki-67, which was main-tained after cessation of treatment. Overall, we demonstrated thatinhibiting the STAT3–Mcl-1 signaling axis by ectopic SOCS1improved radiosensitivity by inducing apoptosis and enhancingDNA damage after radiotherapy, offering a mechanistic rationalefor a new ESCC treatment. Cancer Res; 77(24); 1–12. �2017 AACR.

IntroductionEsophageal cancer is the eighth most common cancer and the

sixth most common cause of cancer-related mortality in theworld (1). In Asia, esophageal squamous cell carcinoma(ESCC) is the predominant histologic form of esophagealcancer. Although multimodal treatments, including surgery,radiotherapy, and chemotherapy are currently used, patientswith ESCC still face with poor prognosis (2, 3). Definitivechemoradiotherapy (CRT) is a standard treatment option forlocally advanced or unresectable ESCC (4, 5). However, locor-egional recurrence after CRT is observed in 40% to 60% ofpatients (6, 7). In addition, salvage surgery after CRT is asso-ciated with a high complication rate (50%–77%) and high

mortality (approximately 15%; refs. 7–9). Therefore, outcomesfor these patients are presently not satisfactory. To improveprognosis, the development of new multimodal treatments forthe initial treatment of advanced ESCC is necessary.

A recent report suggested that members of the signal transdu-cers and activators of transcription (STAT) family of proteins, andmost prominently STAT3, are promising target candidates forcombined CRT treatment (10). STAT3 is a DNA-binding factorthat selectively binds the IL6-responsive element of promoters ofacute-phase genes in IL6–stimulated hepatocytes (11). STAT3 actsas an important transcriptional mediator of proinflammatorycytokine signaling pathways and contributes to oncogenesis bypreventing apoptosis and enhancing cell proliferation (12, 13).Recently, a number of reports showed that irradiation (IR) acti-vates STAT3, as indicated by increased phosphorylation (14), andthat STAT3 mediates CRT resistance; thus, inhibition of STAT3holds great promise for future multimodal treatment concepts inoncology (10).

Suppressor of cytokine signaling (SOCS) is a negative regu-lator of inflammatory cytokine signaling, including the JAK/STAT pathway (15). Among the SOCS family, which is char-acterized by a central src homology 2 (SH2) domains and aconserved C-terminal SOCS box, SOCS1 is the most potentnegative regulator of proinflammatory cytokine signaling (16).SOCS1 interacts with phosphotyrosine residues to interferewith activation of STAT or other signaling intermediates(17, 18). We previously reported that SOCS1 overexpressionusing an adenovirus vector (AdSOCS1) results in antitumoreffects by targeting the JAK/STAT and other signaling pathways

1Department of Gastroenterological Surgery, Osaka University Graduate Schoolof Medicine, Suita, Japan. 2Laboratory of Immune Signal, National Institute ofBiomedical Innovation, Health and Nutrition, Ibaraki, Japan. 3Center for Intrac-table Immune Disease, Kochi University, Nankoku, Japan. 4Laboratory ofImmune Regulation, Osaka University Graduate School of Frontier Biosciences,Osaka, Japan.

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

CorrespondingAuthors:Tsuyoshi Takahashi, OsakaUniversityGraduate Schoolof Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. Phone: 816-6879-3251; Fax: 816-6879-3259; E-mail: [email protected]; andTetsuji Naka, [email protected]

doi: 10.1158/0008-5472.CAN-17-1525

�2017 American Association for Cancer Research.

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(19–22). Moreover, SOCS1 gene therapy was shown to inducepotent antitumor effects in a preclinical patient-derived ESCCxenograft mouse model by targeting the JAK/STAT and FAK/ERK signaling pathway in ESCC (23).

Thus, we investigated the association between STAT3 activationand IR-resistance, and further examined the antitumor effects andassociated mechanisms of radiotherapy combined with SOCS1gene therapy for ESCC. In addition,we evaluated the expressionofactivated STAT3 in ESCC patients who underwent preoperativeCRT, and examined changes in expression as it relates toprognosis.

Patients and MethodsPatients

Forty-six patients with thoracic advanced ESCC (surgeryalone, N ¼ 24; preoperative CRT, N ¼ 22) who underwentcurative resection between 2005 and 2012 at Osaka UniversityHospital were eligible for this retrospective study. This studywas conducted in accordance with Declaration of Helsinki andperformed after approval by an Institutional Review Boardat Osaka University Hospital (No. 08226-6). CRT consistedof two courses of 5-fluorouracil (5-FU), cisplatin (CDDP),and adriamycin (ADM) and radiation (50.4 Gy). The dataregarding patient characteristics, histologic examination, andsurvival were reviewed from medical reports. Follow-up wasperformed through routine visits for clinical assessment, andthis information was obtained from outpatient records. Patientstatus was assessed at the time of the last follow-up, andsurvival was evaluated. We obtained informed written consentfrom all patients.

IHCThe expression of phospho-STAT3 (p-STAT3) was examined by

IHC staining using formalin-fixed and paraffin-embedded ESCCtissue sections using an anti-p-STAT3 (Tyr705) antibody (#9145,Cell Signaling Technology).

Formalin-fixed paraffin-embedded sections (5-mm-thick)were collected for IHC experiments. Briefly, after deparaffiniza-tion, antigen retrieval using HistoVT One (Nacalai Tesque)treatment, and blocking using peroxidase-Blocking Solution(DAKO) and Blocking One (Nacalai Tesque), sections wereincubated with an anti–p-STAT3 antibody at 4�C overnight.After washing, tissue sections were treated using the Dako REALEnVision Detection System (K5007, DAKO), counterstainedwith 10% Mayer's hematoxylin, dehydrated, and mounted.Nuclear staining was observed for p-STAT3. Staining was con-sidered strong positive when >25% of tumor cell nuclei–stainedpositive for p-STAT3. Specimens in which � 25% of cellsstained positive for p-STAT3 were considered weakly immu-noreactive. IHC staining was evaluated by two independentindividuals (T. Sugase and T. Takahashi).

Subcutaneously implanted tumors were harvested andembedded in paraffin for immunohistochemical analysis usinganti-SOCS1 (#18381, IBL), anti-gH2AX (#9718, Cell SignalingTechnology), and anti-Ki-67 (Novocastra Laboratories) anti-bodies, as described previously. Terminal dUTP nick-end label-ing (TUNEL) assays (with DAPI nuclear counterstaining) wereperformed using an ApopTag Fluorescein In Situ ApoptosisDetection Kit (Chemicon International), according to the man-ufacturer's instructions.

Cell lines and irradiationThree human ESCC cell lines, namely, TE4 (RCB2097), TE8

(RCB2098), andTE14 (RCB2101),were obtained from theRIKENBRC cell bank (Tsukuba, Japan). The identity of each cell line wasconfirmed by DNA fingerprinting via short tandem repeat pro-filing on Jan 21, 2014. All cell lines were maintained in RPMI-1640 medium supplemented with 10% FBS (HyClone Labora-tories), 100 U/mL penicillin, and 100 mg/mL streptomycin(PS; Nacalai Tesque, Japan) at 37�C, in a humidified atmosphereof 5% CO2. The cells and mice were irradiated using the MBR-1520R-3 system (Hitachi Medico Technology). We generatedESCC cells stably expressing high levels of STAT3 using thepEB-Multi-constitutive-STAT3 (c-STAT3) vector. TE8 and TE14cells were seeded at 5 � 105 cells per well in 6-well plates andtransfected with pEB-Multiconstitutive-STAT3 (c-STAT3). G418sulfate was used at a concentration of 200 mg/mL.

Western blotting analysisESCC cell lines were harvested and lysed as described previ-

ously (23). The following antibodies were used: anti-phospho-STAT3 (#9145, 1:1,000 dilution), anti-Mcl-1 (#5453, 1:1,000dilution), anti-survivin (#2808, 1:1,000 dilution), anti-cleavedcaspase-3 (#9664, 1:500 dilution), anti-gH2AX (#9718, 1:1,000dilution), all from Cell Signaling Technology, as well as anti-STAT3 (sc-482, 1:1,000 dilution) and anti-GAPDH (sc-32233,1:2,000 dilution), both from Santa Cruz Biotechnology, and anti-SOCS1 (#18381, 1:1,000 dilution; IBL).

Clonogenic assaysCells were seeded at optimum densities (1 � 102–104 cells) in

6-well plates and allowed to adhere overnight. Subsequently, cellswere irradiated with a single dose of 2, 4, 6, and 8 Gy of X-rays.Clonogenic assay procedures were performed as described previ-ously (24). Experiments were performed using three replicatewells for each sample, and the data are presented as averages.

Adenoviral vectorsA replication-defective recombinant adenoviral vector expres-

sing the mouse SOCS1 gene (AdSOCS1) was provided byDr. Hiroyuki Mizuguchi (Osaka University) and was constructedusing an improved in vitro ligation method, as described previ-ously (25). An adenoviral vector expressing the LacZ gene(AdLacZ) was constructed using similar methods. After incubat-ing ESCC cells for 24 hours, they were infected with adenoviralvectors at a multiplicity of infection (MOI).

Cell proliferation assaysESCC cell lines were plated in 96-well plates at a density

of 2 � 103 cells per well and incubated for 24 hours. Cellproliferation was evaluated using WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tet-razolium, monosodium salt] assays (Cell Counting Kit-SF;Nacalai Tesque) at the indicated times after treatment. Theabsorption of WST-8 was measured at a wavelength of450 nm with a reference wavelength of 630 nm using amicroplate reader (Model 680; Bio-Rad Laboratories). Thegrowth rate was expressed as the percentage of absorbance oftreated cells versus that of control cells. Experiments wereperformed using six replicate wells for each sample, and thedata are presented as averages.

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Caspase-3/7 activity assaysESCC cell lines were seeded in 96-well white plates at a density

of 2� 103 cells per well. Irradiation (IR) was performed 24 hoursafter infection with AdLacZ or AdSOCS1. Caspase-3 and -7activities in cell culture were detected 24 hours after IR usingCaspase Glo 3/7 Assays (Promega), according to the manufac-turer's instructions. Luminometer readings were recorded 1 hourafter reagent addition using a Spectra Max Gemini EMMicroplateReader (Molecular Devices).

Small-interfering RNA transfectionThe following siRNA ON-TARGET Plus SMART pools were

purchased from Thermo Scientific Dharmacon: nontargetingsiRNA (D-001810-10-20) and human MCL1 siRNA (L-004501-00). ESCC cells were seeded in antibiotic-free medium. The nextday, cells were transfected with nontargeting or MCL1 siRNAusing Lipofectamine RNAiMAX transfection reagent (Invitrogen),according to the manufacturer's instructions.

ESCC cell xenograft mouse modelsAll animal experiments were conducted according to the insti-

tutional ethical guidelines for animal experimentation of theNational Institutes of Biomedical Innovation, Health and Nutri-tion (Osaka, Japan). Female ICR nu/nu mice (6–8-weeks of age)were obtained from Charles River Laboratories, Japan. For cellinoculation, 2.5 � 106 TE14 cells in 100 mL of 1:1 (v/v) PBS/Matrigel (Becton Dickinson) were injected subcutaneously intotheflanks of thesemice. Animalsweremonitored several times perweek for tumor growth. To evaluate changes in tumor volumeafter each therapeutic regimen, we continued to measure tumorvolume twice per week until day 32 from the start of therapy.Tumor volumes were determined by measuring the tumor lengthand width; volume ¼ (width2 � length)/2

Statistical analysisOverall survival (OS) was evaluated using the Kaplan–Meier

method, and assessed by the log-rank test. For in vitro experiments,data are shown asmeans� standard deviations (SD) based on theindicated number of experiments. For xenograft mouse models,data are shown as means � standard errors of the means (SEM).To test for statistically significant differences between two groups,

unpaired Student t tests were performed. Two-sided P values lessthan 0.05 were considered significant. These analyses were per-formed using JMP version 12.0 (SAS Institute).

ResultsStrong expression of p-STAT3 is confirmed in ESCC patientsafter CRT

We examined the expression of p-STAT3 in advanced ESCCpatients who underwent surgery alone or preoperative CRT byimmunohistochemistry. p-STAT3 positivity was noted in thenuclei. Of 46 ESCC patient samples analyzed in this study, 25(54%) specimens strongly expressed this marker in ESCC tissue,but not in normal tissue (Fig. 1A). Significantly stronger expres-sion of p-STAT3was observed in samples fromESCCpatientswhounderwent preoperative CRT (N ¼ 16, 64%) compared withexpression in those from patients undergoing surgery alone(N ¼ 9, 36%; P ¼ 0.015). There were no significant differencesbetween age, gender, pT, or pStage (UICC 7th edition) and STAT3expression in ESCC patients who underwent preoperative CRT(Table 1). The total 5-year overall survival (5-year OS) rate ofESCC patients undergoing preoperative CRT was 32.7%. ForESCC patients who underwent preoperative CRT, poorer OS wasassociated with those exhibiting strong p-STAT3 expression com-pared with those with weak expression (5-year OS rate, 13.4% vs.66.7%; log rank P ¼ 0.0362) and this difference was significant(Fig. 1B).

STAT3 activation is induced by IR and involved in IR-resistanceWe confirmed that p-STAT3 is strongly induced by IR using

three ESCC cell lines (TE4, TE8, and TE14). In addition to theactivation of STAT3, anti-apoptotic proteins such as Mcl-1 andsurvivin were also induced by IR (Fig. 2A). To evaluate theassociation between STAT3 activation and IR-resistance, we gen-erated ESCC cells stably expressing high levels of STAT3 using thepEB-multiconstitutive-STAT3 (c-STAT3) vector, as previouslydescribed (Fig. 2B; ref. 26). ESCC cells (TE8 and TE14) withc-STAT3 showed no significant differences in cell proliferationcompared with that in parental and mock-transfected cells(Fig. 2C). However, ESCC cells with c-STAT3 showed significantlyincreased colony forming ability after IR compared with that inparental and mock-transfected cells (Fig. 2D).

Figure 1.

Immunohistochemical staining for p-STAT3 in ESCC patient samples. A, Typical weak and strong p-STAT3 IHC staining. Scale bars, white, 100 mm; black, 25 mm.B, Survival curves based on p-STAT3 expression levels in ESCC patients treated with preoperative CRT (N ¼ 22).

SOCS1 Improves Radiosensitivity and Enhances DNA Damage

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Combining radiotherapy with AdSOCS1 enhances thetherapeutic effect of IR

We next examined the therapeutic effect of combining radio-therapy with AdSOCS1, as compared with each monotherapy, inESCC cell lines. Combining radiotherapy with AdSOCS1 resultedin a remarkable decrease in colony forming ability comparedwiththat in untreated ESCC cells and those with AdLacZ, in TE4, TE8,and TE14 cells (Fig. 3A). In terms of proliferation, although IRmonotherapy resulted in a growth inhibitory effect of approxi-mately 70% to 80%, combining radiotherapy with AdSOCS1significantly enhanced this antitumor effect at a low AdSOCS1dose (10 MOI in TE4 and TE14 and 20 MOI in TE8 cells; Fig.3B). Figure 3C shows the antitumormechanisms after combiningradiotherapy with AdSOCS1 in TE14 cells, as assessed byWesternblotting. The expression of p-STAT3 was markedly inhibited inresponse to SOCS1 overexpression after infection with AdSOCS1.Although the expression of Mcl-1 and survivin were enhanced inESCC cells treated with IR compared with that in untreated cells,combining radiotherapy with AdSOCS1 suppressed this effect,and cells expressed markedly high levels of cleaved-caspase-3(Fig. 3C). To evaluate the induction of apoptosis after combiningradiotherapy with AdSOCS1, wemeasured caspase-3/7 activity inTE4, TE8, and TE14 cells using luminescence-based assays.Although there were no significant differences between non-IRand IR-treated groups for both uninfected (Non-Ad) and com-bining radiotherapy with AdLacZ groups, caspase-3/7 activity wassignificantly higher upon combining radiotherapywith AdSOCS1compared with that with IR or AdSOCS1monotherapy (Fig. 3D).In TE14 cells, caspase-3/7 activity increased according to IR-dosewhen radiotherapy was combined with AdSOCS1 (Fig. 3E).

AdSOCS1 inhibits Mcl-1, enhances IR-induced DNA damage,and induces apoptosis

To evaluate IR-induced DNA damage during combining radio-therapy with AdSOCS1, we evaluated gH2AX as an indicator ofdouble-strand breaks (DSB) after IR. Although there was unde-tectable expression of gH2AX without IR in TE14 cells, theexpression of this factor reached its maximum after 0.5 hours ofIR (5 Gy and 10 Gy) treatment and gradually decreased (Fig. 4A).Combining radiotherapy with AdLacZ also resulted in the sametendency. Combining radiotherapy with AdSOCS1 resulted indecreased Mcl-1 expression and enhanced gH2AX expression at1.5 and 4 hours post-IR, compared with that with other therapies

(Fig. 4B). On the basis of the results in Figure 4B, we examined ifMcl-1 in ESCC is associated withDNAdamage.We inhibitedMcl-1 expression in TE14 cells using siRNA.Mcl-1 knockdown in TE14cells resulted in markedly enhanced gH2AX expression 4 hourspost-IR compared to that in parental and control-siRNA cells.Mcl-1 knockdown had no effects on p-STAT3 levels (Fig. 4C). Thisresult suggested that Mcl-1 inhibition might be involved in DNArepair.

Next, we evaluated the induction of apoptosis through Mcl-1inhibition by AdSOCS1 because combining radiotherapy withAdSOCS1 resulted in Mcl-1 inhibition and the induction ofcleaved-caspase-3 induction by SOCS1 overexpression (Fig.3C). Mcl-1 knockdown in TE14 cells resulted in markedlyenhanced cleaved-caspase-3 expression 24 hours post-IR com-pared with that in parental and control-siRNA cells. IncreasedgH2AX signals from apoptosis at later time points post-IR withAdSOCS1were also observed inMcl-1 knockdown cells (Fig. 4D).In addition, although therewere no significant changes in caspase-3/7 activity after IR in parental and control-siRNA cells comparedwith that in non-IR cells, the induction of caspase-3/7 activity inTE14 cells was stronger in Mcl-1 knockdown cells compared tothat in parental and control-siRNA cells (Fig. 4E). The proliferativeactivity of TE14 cells with Mcl-1 siRNA and IR significantlydecreased compared with that with Mcl-1 siRNA and no treat-ment, but was unchanged in parental cells (Fig. 4F). Finally, ESCCcells with stable high c-STAT3 expression showed increasedMcl-1expression and decreased gH2AX signals after IR compared withthat in parental and mock-transfected cells (Fig. 4G).

Combining radiotherapy with AdSOCS1 results in therapeuticeffects in ESCC xenograft mice

We next evaluated the therapeutic effects of combining radio-therapywith AdSOCS1 in ESCC in vivo. For this, we established anESCC xenograft mouse model with subcutaneously-implantedTE14 cells (2.5 � 106 cells) in ICR nu/nu mice (6–8-week-old,females). When tumor volume reached approximately 100mm3,1 � 108 plaque-forming units of AdSOCS1 or AdLacZ wereinjected intratumorally twice per week, six times. Before injectionof the adenovirus vector, mice were irradiated with 2 Gy (6 frac-tions; total ¼ 12 Gy) using an X-ray irradiator (Fig. 5A). Theinjection of AdSOCS1 significantly suppressed tumor growthcompared with that in mice injected with AdLacZ. In addition,radiotherapy significantly suppressed tumor growth comparedwith that in untreatedmice, resulting in a therapeutic effect similarto that with AdSOCS1 injection. On day 32, the tumors ofirradiated mice exhibited marked repopulation compared withthat in mice injected with AdSOCS1. In contrast, mice with thiscombination radiotherapy showed little increase and no repop-ulation in tumor volume after treatment (Fig. 5B). IHC confirmedthe overexpression of SOCS1 in mice treated with AdSOCS1, butnot in mice administered control therapy. The tumors of irradi-ated mice also exhibited increased expression of p-STAT3 com-paredwith that in tumors of untreatedmice. Tumors ofAdSOCS1-injected mice had little expression of p-STAT3 (Fig. 5C).

Combining radiotherapy with AdSOCS1 enhances DNAdamage and induces apoptosis in ESCC xenograft mouse

We then examined the therapeutic effects of combining radio-therapy with AdSOCS1 in vivo by examining pathological changesin tumor tissues. The expression of gH2AX significantly increasedin tumor tissue from irradiated mice compared with that in

Table 1. Correlation between p-STAT3 and various clinicopathologicalparameters in advanced ESCC patients with preoperative CRT

Weak(N ¼ 6)

Strong(N ¼ 16) P

Age, years, median (range) 70 (44–84) 70 (48–72) 0.821Gender, n (%) 0.910Male 5 (83) 13 (81)Female 1 (17) 3 (19)

pT, n (%) 0.2622 3 (50) 4 (25)3 3 (50) 12 (75)

pN, n (%) 0.7930 3 (50) 7 (44)1–3 3 (50) 9 (56)

pStagea, n (%) 1.000I/II 3 (50) 8 (50)III/IV 3 (50) 8 (50)

aUICC 7th edition.

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tumors fromuntreatedmice, andwas highest in tissues frommicetreatedwith combining radiotherapy with AdSOCS1. In addition,the expression of gH2AX in the tumors of irradiated micedecreased on day 32, whereas that in tumors from animals treatedwith combining radiotherapy with AdSOCS1 was sustained (Fig.6A and B). We next examined the expression of Ki-67 to evaluatethe proliferative index of xenograftmouse tumors. The expressionof Ki-67 was significantly suppressed in tumors from irradiatedmice and animals treated with AdSOCS1 compared with that inuntreated mice and AdLacZ-injected mice, and the lowest expres-sion was observed in mice treated with combining radiotherapywith AdSOCS1. In addition, the expression of Ki-67 in irradiatedmouse tumors increased on day 32, whereas that in tumors fromanimals treated with combining radiotherapy with AdSOCS1remained low (Fig. 6C and D). TUNEL staining showed that

combining radiotherapy with AdSOCS1 induced the highestlevels of apoptosis of all treatments in vivo (Fig. 6E).

DiscussionCRT has been established as an effective treatmentmodality for

many human cancers such as breast, esophageal, and lung.However, treatment resistance represents a substantial and com-plex problem, as more and more patients are treated with CRTalone or in combination with other modalities. Increasing evi-dence has demonstrated that STAT3, which is an indicator of poorprognosis in various cancers (27, 28), plays a critical role inmediating resistance to CRT because of its diverse functions incontrolling various (patho-) physiological cellular processes.STAT3 suppresses the transcription of proapoptotic genes (29)

Figure 2.

Relationship between p-STAT3 expression and irradiation resistance. A, Expression levels of p-STAT3, Mcl-1, and survivin in TE4, TE8, and TE14 ESCC cell lineswere analyzed by Western blotting. Cell lysates were prepared every 24 hours after irradiation (2 Gy). STAT3 expression (B), cell proliferative activity (C),and colony forming activity (D) were evaluated in TE8 and TE14 cells transfected with pEB-multiconstitutive-STAT3 (c-STAT3).






Figure 3.

Antitumor effect of combining radiotherapy with AdSOCS1 in ESCC cell lines. A, Colony forming activity; IR: 0, 2, 4, 6, 8 Gy; AdLacZ and AdSOCS1: 5 MOI.B, Cell proliferative activity 48 hours after IR and 24 hours after AdSOCS1 or AdLacZ infection; IR: 10 Gy; AdLacZ and AdSOCS1: 10 MOI for TE4 and TE14 cells;20 MOI for TE8 cells. C, Expression levels of SOCS1, p-STAT3, Mcl-1, survivin, and cleaved-caspase-3 24 hours after IR (0, 2, 5, and 10 Gy) of TE14 cells and24 hours after AdSOCS1 or AdLacZ infection (10 MOI). D, Caspase-3/7 activity 24 hours after IR (5 Gy) of TE14 cells and 24 hours after AdSOCS1 or AdLacZinfection (TE4, TE14: 10 MOI; TE14: 20 MOI). E, Caspase-3/7 activity compared with that in Non-Ad–treated TE14 cells. Statistical analyses were performedusing Student t tests (�� , P < 0.01).

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and induces the expression of proliferative genes (30), in additionto stemness-promoting or stemness-preserving genes (31). Fur-thermore, stress response pathway components regulate STAT3activity and are regulated by it, and several DNA repair genes are

also targets of STAT3 (32). Spitzner and colleagues (10) reviewedthat STAT3 is a promising therapeutic target that can augment thetreatment of CRT-resistant tumors. In our study, the analysis ofresected specimens from ESCC patients showed that the

Figure 4.

Evaluation of DNA damage after combining radiotherapy with AdSOCS1. A, The expression of gH2AX after IR (5 Gy and 10 Gy) in time-dependent manner(0, 0.5, 1.5, 4, 12, and 24 hours). B, Time-dependent expression of SOCS1, p-STAT3, Mcl-1, and gH2AX in TE14 cells and 36 hours after AdSOCS1 or AdLacZ infectionand after IR (5 Gy). C, Expression of Mcl-1, gH2AX, and p-STAT3 4 hours after IR (5 Gy) of Mcl-1-knockdown TE14 cells. D, Expression of Mcl-1, cleaved-caspase-3,gH2AX, and p-STAT3 24 hours after IR (5 Gy) of Mcl-1-knockdown TE14 cells. E, Caspase-3/7 activity 24 hours after IR (5 Gy) of Mcl-1-knockdown TE14 cells.F, Cell proliferative activity 72 hours after IR (5 Gy) of Mcl-1-knockdown TE14 cells. Mcl-1 expression was suppressed by siRNA. G, Expression of Mcl-1 andgH2AX 24 hours after IR (5 Gy) of parental, mock-transfected, and c-STAT3-expressing cells. Statistical analyses were performed using Student t tests (� , P < 0.05).






expression of p-STAT3 is significantly higher after CRT comparedwith expression in those without CRT. Moreover, among ESCCpatients with preoperative CRT, prognosis was significantlypoorer in those with strong expression of p-STAT3 comparedwith those with weak expression. We also demonstrated thatSTAT3 activation might be induced by IR in vitro and in vivo andthat ESCC cells with constitutive STAT3 expression are associatedwith poor therapeutic outcome after IR. In contrast, there were nosignificant changes in p-STAT3andMcl-1 induction after exposureto chemotherapy (CDDP and 5-FU) in vitro (SupplementaryFig. S1A–S1C).

From this, we considered that STAT3 activation is involved intherapeutic resistance to radiotherapy in ESCC, and thus thedevelopment of new modalities that combine radiotherapy withdrugs that target STAT3 activation could improve the therapeuticefficacy of CRT against advanced ESCC.

Mcl-1 is an antiapoptotic member of the Bcl-2 family that wasoriginally isolated from the ML-1 human myeloid leukemia cellline during phorbol ester-induced differentiation to the mono-cyte/macrophage lineage (33). Similar to other Bcl-2 familymembers, Mcl-1 localizes to mitochondria, interacts with andantagonizes proapoptotic Bcl-2 family members (34, 35), and

inhibits apoptosis induced by a number of cytotoxic stimuli (36).A recent report showed that Mcl-1 inhibition, either alone or incombination with other anti-cancer drugs, is effective againstseveral solid cancer-derived cell lines (37). In ESCC cell lines,the expression of antiapoptotic proteins such as Mcl-1 and survi-vin was strongly induced after IR, similar to the induction ofSTAT3 activation. We also demonstrated that Mcl-1 was stronglyinduced after IR in ESCC cells stably high expression levels ofSTAT3, which also conferred IR-resistance. Therefore, enhancedactivation of the STAT3–Mcl-1 axis might be involved in IR-resistance and repopulation after IR. Apoptosis was not readilyinduced by IR monotherapy, and this might have been caused byenhanced activity of the STAT3–Mcl-1 axis after IR, which wouldhave been mitigated by SOCS1 gene therapy.

We previously reported that the overexpression of SOCS1,which is a negative regulator of inflammatory cytokine signaling,including JAK/STAT signaling has potent antitumor effects invarious cancers including ESCC (19–23). Tagami-Nagata andcolleagues (22) demonstrated that the repression of antiapoptoticproteins such as Mcl-1 by AdSOCS1 was one mechanism ofgrowth suppression. In this study, we showed that the expressionof antiapoptotic proteins such asMcl-1 and survivinwasmarkedly

Figure 5.

Antitumor effect of combiningradiotherapy with AdSOCS1 in anESCC xenograft mouse model.A, Summary of model using the TE14ESCC cell line; female ICR nu/numice (6–8 weeks of age) wereinjected with 2.5 � 106 TE14 cells.When tumor volumes reachedapproximately 100 mm3, AdSOCS1 orAdLacZ was injected intratumorallyand IR was performed six times, twiceper week. B, Tumor volumes weredetermined twice per week.C, Immunohistochemical analysis ofSOCS1 and p-STAT3 in xenograftmice; scale bar, 25 mm.

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suppressed by SOCS1 expression in ESCC cell lines similar to thatobserved in a previous report (22). In addition, we also demon-strated that Mcl-1 knockdown strongly induces apoptosis andsignificantly suppresses cell proliferation after IR. From theseresults, we revealed that SOCS1 gene therapy for ESCC stronglyinduces apoptosis by suppressing the STAT3–Mcl-1 axis. There-fore, we hypothesize that this approach could be clinically usefulin combination with radiotherapy. Indeed, we demonstrated thatcombining radiotherapy with AdSOCS1 improved IR-resistanceby suppressing the STAT3–Mcl-1 axis, which was induced after IR,and had additional antitumor effects pertaining to the inductionof apoptosis compared to that with radiation monotherapy. In amouse model, combining radiotherapy with AdSOCS1 showedmarked antitumor effects compared with that with each mono-therapy. After each monotherapy, tumor volumes were repopu-lated, especially in the IR group. However, the therapeutic effectwas maintained with combining radiotherapy with AdSOCS1,

and repopulation was not observed. Chemotherapy is currentlyused for combination therapy to improve the efficacy of radio-therapy. However, regrowth and local recurrence after CRT is acommon clinical problem for ESCC (6, 7). Therefore, the resultsof this our study are considered noteworthy.

In accordance with other mechanisms through which SOCS1gene therapy improved the therapeutic effect of IR, we focused onthe association between STAT3 activation and DNA damage afterIR, in addition to the aforementioned effect on apoptosis. Severalreports have shown that STAT3 modulates the DNA damageresponse pathway (32, 38, 39). Accordingly, inhibition of acti-vated STAT3 decreases DNA repair activity after IR (40, 41). In thisstudy, we demonstrated that the expression of gH2AX,which is anindicator of double-strand DNA breaks after IR, is enhancedin vitro and in vivo after combining radiotherapy with SOCS1gene therapy, as compared with that with each monotherapy. Inthe mouse model, although tumors exposed to AdSOCS1

Figure 6.

A, Immunohistochemical analysis ofgH2AX in tumors from xenograft miceafter each therapy on day 22 and day32. B, gH2AX staining was recorded asthe ratio of positively stained cells to thetotal number of tumor cells based on5 fields (magnification, �400).C, Immunohistochemical analysis ofKi-67 in xenograft mouse samples aftereach therapy on day 22 and day 32.D, Ki-67 staining was recorded as theratio of positively stained cells to thetotal number of tumor cells based onfive fields (magnification, �400).Statistical analyses were performedusing Student t tests (� , P < 0.05 and�� ,P<0.01). Values shown represent themeans � SDs. E, Analysis of apoptosisby TUNEL staining (blue fluorescence,DAPI staining for nuclei; cyanfluorescence, TUNEL-positive staining)in xenograft mice after each therapy.






monotherapy exhibited little DNA damage, those treated withcombination radiotherapy showed significantly stronger DNAdamage compared with that in tumors exposed to IR monother-apy. Interestingly, DNA damage was restored with time in tumorsexposed to IR monotherapy. However, this was maintained inthose treated with combination radiotherapy. In addition, theextent of DNA damage was associated with the expression of Ki-67, and anegative correlationwas observedwith IRmonotherapy.Previously, we reported that AdSOCS1 monotherapy inhibits theexpression of Ki-67 (19–23). Therefore, the combining radiother-apywithAdSOCS1modalitymight reduce tumormalignancy.Wenext examined the association between the STAT3–Mcl-1 axis andDNA damage in vitro. Mattoo and colleagues (42) revealed thatMCL-1 depletion increases 53BP1 and RIF1 colocalization atDSBs, which in turn inhibits BRCA1 recruitment and sensitizescells to IR-induced DSBs or stalled replication forks that requirehomologous recombination for DNA repair. In our study,enhanced expression of gH2AX was observed in ESCC cell lineswith low Mcl-1 expression as a result of SOCS1 gene therapy orMcl-1–siRNA. Thus, we revealed for the first time that suppressionof the STAT3–Mcl-1 axis by SOCS1 gene therapy stronglyenhances DNA damage. Calabrese and colleagues (43) showedthe regulation of p53 signaling by SOCS1 in response to DNAdamage. However, the p53 gene was mutated inmost esophagealcancer specimens and the ESCC cell lines used in this study alsoharboredmutant p53 (44). Therefore, thep53-related response byIR and/or SOCS1overexpressionmight not have beenobserved inour study (Supplementary Fig. S2A–S2E).

There are some clinical problems associated with current radio-therapy with respect to the treatment of ESCC. Although variousradiotherapy methods have been devised to minimize the impacton nontumor tissues, the side effects of radiotherapy on normalorgans and other adjacent tissue remain a clinical problem.The location of the esophagus, specifically, in a very narrowmediastinum, results in clinical complications after radiationexposure in adjacent organs such as the trachea, bronchi, lung,and aorta (45, 46). AdSOCS1,whichwehave used as a therapeuticagent, can be easily administered, in a tumor-specific manner,through local endoscopic injection, to treat ESCC. In addition,this gene therapy is expected to improve the SOCS1 expressionarea (i.e., cancer tissue), relative to the SOCS1non-expressing area(i.e., normal tissue), based on our results, which showed thatSOCS1 gene therapy has the potential to improve radiosensitivity.Therefore, combining radiotherapy with SOCS1 gene therapy hasthe potential to increase tumor specificity, which could decreasethe required radiation dose and improve the local therapeuticeffects in ESCC. Second, tumor relapse and repopulation afterradiotherapy is also a problem (47–49). Options for local therapyin response to tumor relapse and repopulation after radiotherapyonly include surgical resection. Considering the associationbetween STAT3 activation and resistance to radiotherapy, whichwas clarified in this study, SOCS1 gene therapy might representone effective option for salvage therapy in the future. Spitzner andcolleagues reviewed that STAT3 is involved in CRT resistance inbrain cancer, breast cancer, colon cancer, prostate cancer, bladdercancer, head and neck cancer, lung cancer and skin cancer, whichare currently treated with CRT (11). Considering our results, webelieve that SOCS1 gene therapy might lead to improved radia-tion resistance and mitigate some of the clinical problems asso-ciated with radiotherapy in various cancers.

However, there are some problems associated with applyingSOCS1 gene therapy in a clinical setting. First, immune cellsuppression might occur through the inhibition of the JAK/STAT signaling by SOCS1 overexpression. In addition, becausethe recombinant adenovirus vector is replication-defective andhas been modified to prevent intracellular growth (except in293 cells), the therapeutic efficacy is limited to the injectionarea of the tumor in this system. Therefore, a better drugdelivery system to enhance tumor cell selectively might beneeded to improve the therapeutic efficacy of this agent. How-ever, local disease control in advanced ESCC is also importantto improve prognosis, as is treatment of lymph node anddistant metastases (48–50). Therefore, combining radiationwith SOCS1 gene therapy is expected to result in therapeuticefficacy at lower doses and reduce the side effects associatedwith each monotherapy. Regarding SOCS gene therapy, we arescheduled to begin human clinical trials, and the treatmentefficacy and safety results are awaited.

In conclusion, we demonstrated that adenovirus-based SOCS1gene therapy enhances the therapeutic efficacy of radiotherapy forESCC in vitro and in vivo. We revealed for the first time that theSTAT3–Mcl-1 axis is associated with IR-resistance in ESCC andthat inhibition of this axis through SOCS1 expression improvedradiosensitivity by inducing apoptosis and enhancing DNA dam-age after IR. These results suggest that combining radiotherapywith SOCS1 gene therapy targeting STAT3 activation will becomea promising therapeutic option for ESCC in the future.

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

Authors' ContributionsConception and design: T. Sugase, T. Takahashi, M. Yamasaki, T. Kishimoto,Y. Doki, T. NakaDevelopment of methodology: T. Sugase, T. Takahashi, S. Serada, T. NakaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Sugase, M. YamasakiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Sugase, T. Takahashi, S. Serada, M. Fujimoto,K. Hiramatsu, T. Ohkawara, K. Tanaka, Y. Miyazaki, T. Makino, Y. Kurokawa,M. Yamasaki, K. Nakajima, M. Mori, T. NakaWriting, review, and/or revision of the manuscript: T. Sugase, T. Takahashi,S. Serada, K. Tanaka, M. Yamasaki, T. Kishimoto, M. Mori, Y. Doki, T. NakaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Takahashi, S. Serada, M. Fujimoto,K. Hiramatsu, T. Ohkawara, T. Makino, Y. KurokawaStudy supervision: T. Takahashi, M. Yamasaki, T. Kishimoto, Y. Doki, T. Naka

AcknowledgmentsWe would like to thank Editage (www.editage.jp) for English language

editing.

Grant SupportThis research was partially supported by Research for Practical Research for

Innovative Cancer Control from the Japan Agency for Medical Research andDevelopment, AMED (15ck0106106h0002 to T. Naka).

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 June5, 2017; revised September 5, 2017; acceptedOctober 11, 2017;published OnlineFirst October 17, 2017.

Sugase et al.




www.editage.jp


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Sugase et al.




Published OnlineFirst October 17, 2017.Cancer Res Takahito Sugase, Tsuyoshi Takahashi, Satoshi Serada, et al. Squamous Cell CarcinomaEnhances Irradiation-Induced DNA Damage in Esophageal SOCS1 Gene Therapy Improves Radiosensitivity and

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SOCS1 Gene Therapy Improves Radiosensitivity and …...(19–22). Moreover, SOCS1 gene therapy was shown to induce potent antitumor effects in a preclinical patient-derived ESCC xenograft