The Hsp27-Mediated IkBα-NFκB Signaling Axis …...2019/07/12 · Translational Cancer Mechanisms and Therapy The Hsp27-Mediated IkBa-NFkB Signaling Axis Promotes Radiation-Induced

Translational Cancer Mechanisms and Therapy

The Hsp27-Mediated IkBa-NFkB Signaling AxisPromotes Radiation-Induced Lung FibrosisJee-Youn Kim1, Seulgi Jeon2, Young Jo Yoo2, Hee Jin2, Hee Yeon Won2,Kyeonghee Yoon2, Eun Sook Hwang2, Yoon-Jin Lee3, Younghwa Na4,Jaeho Cho1, and Yun-Sil Lee2

Abstract

Purpose: Lung fibrosis is a major side effect experienced bypatients after lung cancer radiotherapy. However, effectiveprotection strategies and underlying treatment targets remainunclear. In an effort to improve clinical outcomes, pharma-cologic treatment of fibrosis is becoming increasingly popular;however, no ideal therapeutic strategy is yet available.

Experimental Design:We used a mouse model to irradiatehigh focal (90 or 75Gy) to 3-mmvolume of the left lung. Lungtissues of mice were subjected to microarray, mRNA expres-sion, and immunohistochemical analysis. Correlations ofradiation (IR)-induced epithelial-mesenchymal transition(EMT) were validated in lung cell lines using appropriatetreatments to activate or inhibit selected pathways.

Results: The expression of Hsp27 was increased during IR-induced lung fibrosis in a mouse model. Inhibition of func-tional Hsp27 using shRNA and a synthetic small molecule

inhibitor (J2) in lung cells alleviated IR-mediated EMT. Theactivation of NFkB pathways via direct interaction betweenHsp27 and IkBa resulted in increased expressions of Twist,IL-1b, and IL-6 and facilitated IR-mediated EMT, which wasidentified as an underlying mechanism of Hsp27-mediatedfibrosis after IR. J2 also inhibited IR-induced lungfibrosis in anorthotopic lung cancer model, and IR-induced lung fibrotictissues from patients showed higher expression of Hsp27 thanunirradiated lungs.

Conclusions: Collectively, IkBa-NFkB signaling activationbyHsp27, which resulted in the facilitation of Twist, IL1b, andIL6 expression, is involved in the EMT process that is tightlyconnected to thedevelopment of IR-induced lungfibrosis.Ourfindings also suggest that inhibitionofHsp27has thepotentialto become a valuable therapeutic strategy for IR-induced lungfibrosis.

IntroductionRadiotherapy is a mainstay of lung cancer treatment.

However, delivery of high radiation doses to the tumor isoften hampered by the risk of radiation (IR)-induced lunginjury. Lung damage due to thoracic IR is mediated via acuteresponses including inflammation and pneumonitis as well aschronic effects such as pulmonary fibrosis (1, 2). Fibrosis is theend stage of persistent tissue damage and chronic inflamma-tory reactions. It is characterized by excessive accumulation of

extracellular matrix (ECM) and disruption of normal tissuearchitecture (3, 4).

During epithelial–mesenchymal transition (EMT), cellsundergo a morphologic switch from the epithelial polarizedphenotype to the mesenchymal fibroblastoid phenotype.EMT is characterized by the loss of epithelial differentiationmarkers, and the induction of mesenchymal markers. EMT playsa key role in embryonic development, chronic inflammation, andfibrosis (5, 6). Furthermore, EMT was observed during tumor cellinvasion and metastasis in various solid tumors, (7). The exactmolecularmechanisms leading to the development of IR-inducedpulmonary fibrosis have yet to be fully identified.

Hsp27 (Hsp27 in humans and Hsp25 in mice) is an ATP-independent molecular chaperone that is highly induced inresponse to cellular stresses (8). Hsp27 is a critical mediatorin cancer progression, preventing apoptosis in transformedcells (9–11). In addition, Hsp27 enhances migration and inva-sion (12) and mediates EMT in cancer cells (13). It is also aninducer of EMT during fibrosis including idiopathic pulmonaryfibrosis (IPF; ref. 14). Overexpression of Hsp27 was reported inpatients diagnosed with IPF (15). The upregulation of Hsp27plays a pivotal role in myofibroblast differentiation and mayrepresent a promising therapeutic target in fibrotic diseases.Hsp27 gene silencing byOGX-427, a second-generation antisenseoligonucleotide, inhibited development of bleomycin-inducedlung fibrosis and EMT via degradation of Snail (14). Accordingly,Hsp27 inhibition is an attractive therapeutic strategy.

Three pharmacologic treatments for IPF, namely pirfenidone,nintedanib (BIBF1120), and NAC are commercially available.

1Department of Radiation Oncology, Yonsei University Health System, Seoul,Korea. 2Graduate School of Pharmaceutical Sciences, EwhaWomans University,Seoul, Korea. 3Korea Institute of Radiological andMedical Science, Seoul, Korea.4College of Pharmacy, CHA University, Pocheon-si, Gyeonggi-do, Korea.

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

J.-Y. Kim and S. Jeon contributed equally to this article.

Corresponding Authors: Yun-Sil Lee, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul KS103, Republic of Korea (South). Phone: 822-3277-3022; Fax: 822-3277-3051; E-mail: [email protected]; Jaeho Cho,Yonsei University Health System, 50, Yonsei-ro, Seodaemun-gu, Seoul 03722,Republic of Korea (South). Phone: 822-2228-8113; E-mail: [email protected]; andYounghwa Na, College of Pharmacy, CHA University, 120, Haeryong-ro,Pocheon-si, Gyeonggi-do, Republic of Korea (South). Phone: 823-1881-7164;E-mail: [email protected]

Clin Cancer Res 2019;XX:XX–XX

doi: 10.1158/1078-0432.CCR-18-3900

�2019 American Association for Cancer Research.

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Pirfenidone has been shown to prevent the accumulation ofhydroxyproline, procollagen I and III, inflammatory cells, andTGFb1 in bronchoalveolar lavage (BAL), and/or lungtissue (16–22). Pirfenidone has also been shown to diminishthe fibrocyte pool and the migration of these cells in mousemodels of lung fibrosis (23). Nintedanib was discovered as abyproduct in large screening assays targeting cyclin-dependentkinase (CDK4; ref. 24). Nintedanib was systematically developedas a potent angiogenesis inhibitor. However, conclusive evidenceis unavailable to support its clinical role in the treatment ofpulmonary disease, especially in IR-induced lung fibrosis.

Previously, we developed a mouse model simulating clinicalstereotactic body radiotherapy (SBRT) and validated the induc-tion of lung fibrosis by high-dose IR (25) and this model didnot show any difference in the incidence of pulmonary fibrosisaccording to the radiosensitive and radioresistant mousestrains (26). In this study, we identified the molecular targetsin lung fibrosis development. Hsp27 expression was increasedduring IR-induced lung fibrosis, and functional inhibition ofHsp27 using a small-molecule ameliorated lung fibrosis. Whileinvestigating mechanisms of Hsp27 in the development of lungfibrosis, we found that IkBa–NFkB signaling activation bydirect interaction of IkBa with Hsp27, is involved in the EMTprocess that is tightly connected to the development of IR-induced lung fibrosis.

Materials and MethodsAnimal experiments

All procedures were approved by the Animal Care andUse Committees of Yonsei University Medical School (Seoul,Korea; 2015–0267) and were performed in accordance with therelevant guidelines. A single dose of 75 or 90 Gy was deliveredusing an X-RAD 320 platform (Precision X-ray) as describedpreviously (27). Mice were administered intraperitoneally withJ2 (7.5 mg/kg or 15 mg/kg), pirfenidone (100 mg/kg), andamifostine (100 mg/kg) for 4 weeks on alternate days after IR,and lung tissues (n� 3 per group) were collected at 4 or 6 weeksafter IR.

Generation of Hsp25 transgenic miceHsp25 mice were generated, interbred, and maintained in

pathogen-free conditions at Macrogen, Inc (see SupplementaryInformation).

Establishment of the orthotopic lung tumor modelThe mouse lung carcinoma LLC1 cells, at 1 � 106 in 200-mL

physiologic saline, were injected in the tail vein of 7-week-oldmale C57BL/6Nmice. Two weeks after the intravenous injection,a single dose of 90Gywas delivered to the leftwhole lung using animage-guided small-animal irradiator. The mice were randomlydivided into three groups (four to six mice per group) as follows:(i) LLC1 group—intravenous injection only; (ii) LLC1þ 90-Gygroup—mice were exposed to a single dose of 90 Gy deliveredto the left whole lung 2 weeks after intravenous injection;(iii) LLC1 þ 90 Gy þ J2 group—the mice were administeredJ2 intraperitoneally (15mg/kg) for 2 weeks on alternate days afterirradiation. On week 4, the mice were sacrificed by CO2 asphyx-iation, and lung tissues were collected for analysis.

Human tissues analysisThe study of patient specimens of radiation-induced lung

fibrosis (RILF) was approved by Severance Hospital, YonseiUniversity (Seoul, Korea). Each patient's tissue contained anirradiated fibrotic and a nonirradiated normal area. The degreeof protein expression was compared between the fibrotic andnormal areas in each patient's tissue.

Microarray experimentTotal RNA from the mouse lung tissues was extracted using the

Easy-SpinTM Total RNA Extraction Kit according to the manu-facturer's instructions (iNtRON Biotechnology; see Supplemen-tary Information).

Cell culture and transfectionNCI-H460 (human non–small cell lung cancer cell line), L132

(human normal lung epithelial cell line), and LLC1 (mouse Lewislung carcinoma) were obtained from the ATCC and cultured inRPMI or DMEM (Gibco) supplemented with 10% FBS (Gibco) ina 37�C incubator with 5% CO2. Lentiviruses were used to createstable cell lines expressing shRNA forHsp27 (puromycin-resistantgene). The control shRNA lentiviral particle (sc-108080), Hsp27shRNA lentiviral particle (sc-29350), and polybrene (sc-134220)were obtained from Santa Cruz Biotechnology. To generate thesh-Control cells and sh-Hsp27 cells, cell lines were selected usingpuromycin (1 mg/mL) for at least 1 week. Human pulmonaryfibroblasts (HPF)were obtained fromPromoCell and usedwithinnine passages. The mesenchymal-like A549 cells (A549TD) thatwere generated by chronic exposure with TGFb as describedpreviously (28), were obtained from Professor H.J. Cha (SeoulNational University, Seoul, Korea). Cell lines were tested byBioMycoX Mycoplasma PCR Detection Kit (JCBIO Co., Ltd) toensure that they were Mycoplasma-free.

RNA isolation, qRT-PCR, and RT-PCRTotal RNA was isolated from the sample using TRIzol reagent

(Qiagen; see Supplementary Information). Primer sequences forRT-PCR and qRT-PCR are listed in Supplementary Table S1.

Antibodies and reagentsImmunoblotting were performed as described previously (29)

using antibodies: Twist (GeneTex and Abcam); N-cadherin, p65,

Translational Relevance

Radiotherapy is an important conventional therapy forthoracic malignancies. However, radiotherapy-related pulmo-nary symptoms occur in up to 30% of patients and effectiveprotection strategies and underlying treatment targets remainunclear. In this study, we used a mouse model simulatingclinical stereotactic body radiotherapy (SBRT) and validatedthe induction of lung fibrosis. We also attempted to identifymolecular targets occurring in the process of lung fibrosisdevelopment. The expression of Hsp27 was increased duringthe induction of radiation (IR)-induced pulmonary fibrosisand Hsp27 overexpression accelerated IR-induced lung fibro-sis. Inhibition of Hsp27 using a recently identified small-molecule inhibitor that induced crosslinking of Hsp27 atten-uated this increase. This study demonstrated the potential ofHsp27 inhibition to improve IR-induced lung fibrosis. Ourresults support the potential clinical utility ofHsp27 as a noveltarget for treating IR-induced lung fibrosis.

Kim et al.

Clin Cancer Res; 2019 Clinical Cancer ResearchOF2




Hsp27, LaminB, and b-actin (Santa Cruz Biotechnology);phospho-Hsp27 (Ser82), phospho-IkBa (Ser32/36), phospho-STAT3 (Tyr705), STAT3 (Cell Signaling Technology); IkBa,IL6, IL1b, and pro-SPC (Abcam); fibronectin, vimentin, andE-cadherin (Becton-Dickinson Laboratories); ZO-1 (ThermoFisher Scientific); a-SMA (Sigma); and Alexa488-conjugatedphalloidin (Invitrogen).

IrradiationCells in 60-mm and 100-mm petri dishes were exposed to

radiation (5 or 10 Gy as a single dose) generated by a 137 Csgamma-ray source (Elan 3000, Atomic Energy of Canada,Mississauga, Canada) at a dose rate of 3.81 Gy/minute. Radiationworkers received radiation safety management training annually,provided by the Korea Foundation of Nuclear Safety (KoFONS).

ImmunoprecipitationFor immunoprecipitation, cells were lysed in lysis buffer

(500 mmol/L NaCl, 50 mmol/L Tris-HCl pH 7.5, 0.5% TritonX-100, 1 mmol/L EDTA, and 1 mmol/L DTT), clarified by centri-fugation, incubated with IkBa antibody, and immunoprecipi-tated with protein A (Sigma-Aldrich). The precipitates werewashed three times and analyzed by Western blotting.

Preparation of lung tissues for histology and IHCFor the histologic study, 4-mm tissue sections were stained with

hematoxylin and eosin (H&E) and Masson trichrome (MT). IHCstaining was carried out using anti-Twist (1:100 dilution;GTX127310, GeneTex), anti-IL6, and anti-IL1b (1:100 dilution;ab6672 and ab9722, Abcam, respectively), and anti-Hsp27(1:200 dilution; sc-13132, Santa Cruz Biotechnology) at 4�Covernight. Slides were then incubated with Avidin–Biotin per-oxidase complex (ABC kit, Vector Laboratories) and developedusing 3, 30-diaminobenzidine tetrachloride (DAB; ZymedLaboratories).

Immunofluorescence assayCells were fixed in 2% paraformaldehyde for 1 hour, followed

by blocking and incubation with primary antibodies at 4�Covernight. Anti-a-SMA (A5228, Sigma, 1:200), anti-p65 (sc-8008,Santa Cruz Biotechnology, 1:100), anti-Twist (sc-15393, SantaCruz Biotechnology, 1:100), and anti-Hsp27 (sc-13132, SantaCruz Biotechnology, 1:200) were used to detect expression. Themorphologic change was investigated by Alexa488-conjugatedphalloidin staining (A12379, Invitrogen, 1:200). For immuno-fluorescence staining, tissue sections stained with pro-SPC,a-SMA, and IkBa (1:100 dilution; ab90716, ab7817 andab32518, Abcam, respectively) were incubated with appropri-ate fluorescent secondary antibodies and counterstained with4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Imageswere viewed under a confocal microscope (LSM700, Zeiss).

Micro-CT analysisMicro-CT analysis was performed as described previously (27).

Functional assessment of the lungsLung function in irradiated mice was evaluated with the Flexi-

vent system (Flexivent; SCIREQ), which measures flow–volumerelationships in the respiratory system, including forced oscilla-tion, to distinguish airway and lung tissue variables (ref. 30; seeSupplementary Information and Supplementary Table S2).

Statistical analysisComparisons of all results were performed by one- or two-way

ANOVA and Newman–Keuls test where indicated. The differencewas considered statistically significant at P � 0.05; P � 0.01;and P � 0.005. All statistical analyses were performed usingGraphPad Prism.

ResultsIncreased Hsp27 expression during IR-induced lung fibrosis

To confirm the fibrosis, lung sections were stainedwithMassonTrichrome to visualize the deposition of blue-colored collagen. At4 weeks, extensive collagen was observed, correlating with late-stage fibrosis (Fig. 1A). Antibody protein arrays showed thatirradiation induced the secretion of Hsp27 alone into the bloodwithout increasing the levels of other Hsps such as Hsp90 andHsp70 (Supplementary Fig. S1A). Therefore, we examined theHsp27 expression in lung tissues by IHC analysis and found thatHsp27 protein expression was increased during lung fibrosis(Fig. 1B). No increase in hspb1 mRNA expression was detectedduring fibrosis (Supplementary Fig. S1B). To elucidate the role ofHsp27 in lung fibrosis directly in vivo, Hsp25 TG mice were used(Supplementary Fig. S1C and S1D). IR-induced lung fibrosis wasexacerbated after focal exposure to high-dose IR (75Gy) inHsp25TG mice, 6 weeks after IR. After IR exposure, the Hsp25 TG miceshowed an abundance of neutrophils and mononuclear cells inthe alveoli, greater destruction of alveolar septa, intra-alveolarhyaline membrane formation, and a marked increase in collagendeposition compared with control C57BL/6N (BL6) mice. CTimages may be used to predict fibrosis, and micro-CT is compa-rable with clinical CT in humans (31). Six weeks after IR, thetypical micro-CT manifestations of SBRT-induced lung injury,such as ground-glass opacities and consolidation (32), wereobserved in the irradiated left lung. These effects were stronglyinduced inHsp25TGmice.Normal lung volumeafter IR inHsp25TG mice was lower compared with that in control BL6 mice(Fig. 1C). Functional lung parameters evaluated in this study arelisted in Supplementary Table S2. There were significant differ-ences in inspiration capacity (IC), quasi-static compliance (Cst),and tissue damping (G) of the lungs following exposure to IRbetween BL6 and Hsp25 TG mice. These results reflect the respi-ratory distress induced by irradiation. The respiratory distress inHsp25 TG mice appears to have been significantly potentiated(Fig. 1D).

Knockdown of Hsp27 inhibited IR-mediated EMT in lungcell lines

To elucidate the cellular role of Hsp27 during the developmentof IR-induced lung fibrosis, we initially examined the morpho-logic changes in L132 cells following IR. Control L132 cells wereround or polygonal and exhibited very close cell–cell proximityreminiscent of cellular tight junctions. IR transformed the cellsinto a spindle shape. These changes became clearer and moreabundant with increased dose. However, cells with Hsp27 shRNA(shHsp27) showed inhibition of these IR-induced morphologicfeatures (Supplementary Fig. S2A).Westernblot results confirmedthat IR decreased the expression of the epithelial markers such asZO-1. However, the expression of mesenchymal markers includ-ing Twist, fibronectin, and a-SMA increased in L132 cells andshHsp27 attenuated these phenomena. Quantitative RT-PCR fortwist1 and fn1 also showed similar patterns of protein expression(Fig. 2A).

Hsp27–IkBa–NFkB Signaling in Lung Fibrosis

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Previously, we identified the functional inhibition ofHsp27 following altered dimerization at the cysteine residue(Cys 137) of Hsp27 using small molecules to ameliorateHsp27-mediated chemo- or radioresistance in lung cancercells (29, 33). In this study, we investigated the inhibition ofIR-mediated EMT using J2, which is a chromone derivativeand a small-molecule Hsp27 inhibitor. J2 strongly altered thecross-linking of Hsp27 compared with SW15 (xanthone struc-ture derivative). J2 cross-linked Hsp27 even at low concentra-tions of 0.1 or 0.5 mmol/L and high concentrations of 10 mmol/L, which overcame the chemoresistance, via strongly alteredcrosslinking of Hsp27 (Supplementary Fig. S2B). J2 treatmentof recombinant Hsp27 protein strongly inhibited the forma-tion of large oligomers of Hsp27 in nonreducing gel systems(Supplementary Fig. S2C). Pretreatment with J2 mitigated thealtered expression of EMT-related proteins such as Twist,fibronectin, a-SMA, vimentin, and ZO-1 induced by IR inL132 cells assayed at 24 and 48 hours after IR (Fig. 2B;Supplementary Fig. S2D).

Increased adhesion is a characteristic of cells with a mesenchy-mal phenotype. Immunofluorescent hair-like fibers stained withphalloidin (green) protruding from cell surfaces into the collagenmatrix assembled at the leading edge of irradiated cells, whereastreatmentwith shHsp27 or J2 reduced these protrusions (Fig. 2C).J2 treatment also reduced EMT-related proteins such as fibronec-tin, N-cadherin, a-SMA, and Twist resulting from IR in HPFcells on Western blotting and immunofluorescence images ofa-SMA (Fig. 2D). Continuous exposure to TGFb by A549 lungcarcinoma cells [A549 TGFb-differentiated cell lines (A549TD)]yielded a lower expression of E-cadherin and a higher expres-sion of Hsp27 and vimentin than occurred in the parent A549cells (Supplementary Fig. S3A), without affecting other HSPssuch as Hsp70 and Hsp90 (Supplementary Fig. S3B), indicatingHsp27-mediated EMT. Moreover, J2 treatment dramaticallyrestored the morphologic changes of A549TD cells and decreasedthe expression of E-cadherin. The expression of vimentin inA549TD cells was also restored by 0.1 and 0.5 mmol/L J2 treat-ment (Fig. 2E; Supplementary Fig. S3C), suggesting that Hsp27

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Increased Hsp27 expression during IR-induced lung fibrosis. A, Experimental scheme of focal exposure to high-dose radiation (90 Gy). Lung sectionswere stained with Masson Trichrome for collagen deposition at indicated times. B, IHC of Hsp27 in mouse lung tissues after IR at each time point(left). The graph shows quantification of Hsp27-positive cells (right). C, H&E and Masson trichrome-stained lung sections at 6 weeks after focal 75-Gyirradiation (IR) in C57BL/6N and Hsp25 TG mice. Lungs were photographed after complete fixation (middle), horizontal (third from the right),trans-axial (second from the right), and 3D micro-CT (the most right) images acquired at 6 weeks after IR. Graphs show scores quantifyinginflammation, collagen deposition, and normal lung volume. D, Quantification of inspiratory capacity (IC), Quasi-static compliance (Cst), tissuedamping (G), and tissue elastance (H). Data are expressed as mean � SE (n � 3; �� , P < 0.01 vs. BL6-Control; #, P < 0.05 vs. BL6-IR).

Kim et al.





inhibition modulated EMT. J2 concentrations (0.05, 0.1, and0.5 mmol/L) neither induced cellular cytotoxicity in a colony-forming assay nor demonstrated cytotoxicity on flow cytometryusing L132 lung epithelial cells, NCI-H460, and A549 lungcancer epithelial cells. Cell-protective effects by J2 after IR wereobserved and the protective effect was predominantly seen innormal cells of L132 rather than in NCI-H460 and A549 cancercells (Supplementary Fig. S3D and S3E).

Hsp27 cross-linker J2 inhibited IR-induced lung fibrosis inmice

To elucidate whether a small-molecule Hsp27 cross-linker J2inhibits IR-mediated lung fibrosis in mice, we compared thechanges in left lung surface morphology in the control and IRgroup. In contrast to the brown colored lungs in control mice, thelungs of irradiated mice exhibited a definite white, ring-likeappearance. Intraperitoneal injection of J2 resulted in less injurythan was apparent in the IR-only mice. Alveolar infiltration ofinflammatory cells and the formation of intra-alveolar hyaline

membranes in the IR group were significantly greater than in thecontrol group. J2-treated mice exhibited reduced tissue damage.Masson trichrome staining revealed amarked increase in collagendeposition in the IR group than in the control group, which wassignificantly reversed by J2 treatment. Six weeks after IR, ground-glass opacities and consolidation were observed in the irradiatedleft lung; in contrast, these effects were decreased in J2-treatedmice. Normal lung volume in the IR group was lower than in thecontrol mice. However, normal lung volume appeared to signif-icantly recover in J2-treated mice (Fig. 3B; Supplementary Fig.S4A). Also, there were significant differences in IC, Cst, G, andtissue elastance (H) of the lungs between IR group and controlmice. The IC and Cst of the IR group were significantly lowercompared with those of the control group. The values of G and Hin the IR groupwere higher than in the control group.Mice treatedwith 15mg/kg J2 exhibited significant differences in IC, Cst, andGparameters, indicating the protective effect of J2 on IR-inducedlung injury (Supplementary Fig. S4B). The effects of J2 were moreprominent than those of pirfenidone or amifostine, even though

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Knockdown of Hsp27 inhibits IR-mediated EMT in lung cell lines. A, Cell lysates of L132 cell lines with shControl (shCONT) or a stable Hsp27 knockdown(shHsp27) after 24 and 48 hours after radiation (IR) were analyzed byWestern blot and qRT-PCR (24 hours of IR), with gapdhmRNA used for normalization.Bars represent means SD. (n� 3; �� , P < 0.01 vs. shCONT-Control; ##, P < 0.01 vs. shCONT-IR, one-way ANOVA). B,Western blots using cell lysates at 24 and48 hours after 5-Gy IR in L132 cells with or without J2 (0.5 mmol/L) pretreatment. C, L132 cells were pretreated for 2 hours with 0 or 0.5 mmol/L J2 and irradiatedwith 5 Gy. At 48 hours after IR, the levels of phalloidin (green), DAPI (blue) were assessed via immunofluorescence staining. Magnification at 400�. D,Westernblots and immunofluorescence using HPF cells at 12 hours after 5-Gy IR with or without J2 (0.5 mmol/L) pretreatment. Magnification at 200�. E,Western blottingdata after treatment with J2 at the indicated concentrations in A549- and TGFb1-differentiated A549 cell lines (A549TD).






administration dosage of J2 was less than pirfenidone andamifostine.

Todevelop evidence that epithelial cells express amesenchymalphenotype during IR-induced lung fibrosis, we performed immu-nofluorostaining for both alveolar epithelial cell–specific proteinSPC andmyofibroblast-specific marker a-SMA. The luminal layerof alveolar cellswas immunoreactive for SPCand the SPC-positivecell number was not substantially different among the groups.Moreover,a-SMA expressionwas increased by IR and SPC/a-SMAcostaining cells were also increased by IR, indicating that an EMTprocess occurred during IR-induced lung fibrosis. However, J2treatment with IR decreased the SPC/a-SMA costaining cells(Fig. 3C).

We also investigated the effects of J2on IR-induced lungfibrosisin Hsp25 TG mice. Similar to the findings of normal BL6 mice,

irradiated areas of the left lung clearly exhibited a local injury inBL6 mice. In the Hsp25 TG mice, we observed aggravated lunginjury grossly and histologically, which was attenuated by J2treatment. Masson trichrome, Sirius red, and IHC hydroxyprolinestaining revealed a marked increase in collagen deposition inHsp25 TG mice after IR compared with IR-treated control BL6mice and J2 also inhibited collagen deposition inHsp25 TGmice.Six weeks after IR, the normal lung volumewas lower in irradiatedHsp25 TG mice than in irradiated BL6 mice. However, it appearsto have been significantly restored in J2-treated Hsp25 TG mice(Fig. 3D; Supplementary Fig. S5A and S5B). Increased costainingof SPC/a-SMA after IR was restored by combined treatment withJ2, indicating that the IR-induced EMT process was blocked by J2treatment (Fig. 3E). Therewere significant differences in IC, Cst,G,and H of the lungs between irradiated Hsp25 TG and irradiated

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Hsp27 cross-linker J2 inhibits IR-induced lung fibrosis in mice. A, Experimental scheme of focal exposure to high-dose radiation (75 Gy). Mice were sacrificed at6 weeks after 75 Gy IR. B, Lung sections of mice at 6 weeks after IR were stained with Masson trichrome. Quantification of collagen deposition with blue staining(left), inflammatory foci from H&E staining (middle), and volume of normal lung frommicro-CT analysis (right) are presented as mean� SE. C, The pro-SPC (red)was used for identifying type II AECs, costained with a-SMA (green) in type II AECs. a-SMA expression levels were upregulated in type II AECs of the irradiatedlung tissue. In contrast, J2 (15 mg/kg) inhibited the increases in a-SMA expression in these epithelial cells. Magnification at 630�. Scale bar, 20 mm (D) C57BL6N(BL6) and Hsp25 transgenic (TG) mice were sacrificed at 6 weeks after 75-Gy irradiation (IR). Themice were intraperitoneally administered J2 (15 mg/kg) onalternate days after IR for 4 weeks. Onweek 6, the mice were sacrificed, and Masson trichrome-stained lung sections were examined. Magnification, 40� (top),400� (bottom). Quantification of collagen deposition with blue staining (left), inflammatory foci from H&E staining (middle), and volume of normal lung frommicro-CT analysis (right) are expressed as mean� SE. E, The pro-SPC (red) was costained with a-SMA (green) using lung tissues of BL6 and Hsp25 TGmice.Upregulation of a-SMA expression in epithelial cells by IR was more strongly increased at TG-IR group and significantly decreased at TG-IRþJ2 (15 mg/kg)group. Magnification at 630�. Scale bar, 20 mm (n� 3, mean� SD; � , P < 0.05; �� , P < 0.01; and �� , P < 0.001 vs. BL6-CONT; #, P < 0.05 vs. BL6-IR; †, P < 0.05 and†††, P < 0.001 vs. TG-IR).

Kim et al.





control BL6 mice. However, IR-induced respiratory distressin J2-treated mice appeared to be significantly reversed even inHsp25 TG mice (Supplementary Fig. S5C).

NFkB activation by Hsp27 is involved in the expression ofIR-induced fibrosis-related genes

To identify the underlying mechanisms of inhibition ofIR-mediated lung fibrosis by J2, we performed a cDNAmicroarrayof lung tissues after focal exposure to 75 Gy with or without4weeks of intraperitoneal administrationof J2. Temporal changesin gene expression were hierarchically clustered (SupplementaryFig. S6A). Analysis of fibrosis-related genes in the microarray datarevealed upregulation of only twist1, il6, and il1b genes by focalirradiation, which was reversed by J2 treatment. We also deter-mined the mRNA levels of twist1, il-6, and il-1b using qRT-PCR(Fig. 4A) and protein levels using IHC (Fig. 4B); the levels weresimilar to those obtained with cDNA microarray. J2 treatmentrestored the significant increase induced by focal IR. We alsofound that J2 did not alter the expression ofHsp25 protein in lung

tissues (Supplementary Fig. S6B). We next examined whetherHsp27 knockdown or J2 treatment modulated IR-induced twist1,il6, and il1b genes in a cell system. The qRT-PCR analysis of L132lung epithelial cells revealed that the increased expression oftwist1, il6, and il1b genes by IR was suppressed by shRNA ofHsp27 or J2 pretreatment, based on the results detected at12 hours after IR (Fig. 4C). Immunofluorescence data in L132cells revealed that the basal Twist level was inhibited by shHsp27and IR-induced Twist activation was also ameliorated by knock-down of Hsp27 (Fig. 4D).

Because NFkB is a regulator of Twist, IL1b, and IL6 (34–36),we investigated the association between p65, one of the compo-nents of NFkB, and IR-mediated EMT markers using siRNA andBAY11–7082, an NFkB inhibitor. BAY11-7082 or p65 knock-down dramatically inhibited the Twist protein level (Supplemen-tary Fig. S7A and S7B), suggesting Twist as a downstream effectorof NFkB. To elucidate whether the binding between Hsp27 andIkBa was affected by IR, immunoprecipitation (IP) was per-formed. The results indicated an increase in the binding activity

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Inhibition of Hsp27 blocks the expression of twist1, il1b, and il6. The mice were intraperitoneally injected with J2 (15 mg/kg) on alternate days after 75-Gy IR for4 weeks. Mice were sacrificed at 6 weeks after IR and lungs were subjected to microarray analysis. A,Microarray data of twist1, il1b, and il6 are shown (top).Confirmation of genes by quantitative RT-PCR using lungs from individual mice (bottom). Each mRNA expression was normalized to gapdh (n� 3, mean� SD).�� , P < 0.001 versus Control; #, P < 0.05 versus IR. B, IHC staining of Twist, IL1b, and IL6 using lungs from three individual mice. Quantification of stained tissueswas performed (n� 3, mean� SD). �� , P < 0.01; �� , P < 0.001 versus Control; #, P < 0.05; ##, P < 0.01 versus IR. C,mRNA levels of twist1, il6, and il1b usingqRT-PCR in L132 cell lines with a stable Hsp27 knockdown (left) or J2 (right) at 12 hours after 5-Gy IR; gapdhmRNAwas used for normalization. � , P < 0.05;�� , P < 0.01; �� , P < 0.001 versus Control; #, P < 0.05; ##, P < 0.01 versus IR only. D, Immunofluorescence staining for Twist and Hsp27 in L132 cell lines with a stableHsp27 knockdown at 24 hours after 5-Gy IR. Magnification at 400�.






between Hsp27 and IkBa by IR, and a decreased binding activitybetween IkBa and p65 (Fig. 5A). Moreover, siRNA of twist1 didnot inhibit il6 and il1b genes in L132 cells, suggesting that NFkBactivation by Hsp27 may be a master regulator of twist1, il6, andil1b genes (Supplementary Fig. S7C).We also investigated wheth-er Hsp27 level regulates NFkB-mediated Twist expression by IRand found that Hsp27 knockdown or Hsp27 cross-linker J2inhibited IR-mediated Twist expression, accompanied by inhibi-tion of IkBa phosphorylation, at 3 hours of IR before Twistactivation (24 hours of IR). In the case of STAT3 phosphorylation,another transcription factor of Twist, IR slightly induced STAT3phosphorylation. However, Hsp27 inhibition did not affect thephosphorylation (Fig. 5B and C). IR-mediated nuclear transloca-tion of p65 was inhibited by shHsp27 or J2 treatment (Fig. 5D;Supplementary Fig. S7D). The siRNA of p65 inhibited IR-mediated mRNA expression of twist1, il-6, and il-1b as shown inqRT-PCR, suggesting thatNFkB activation by IR acted upstreamoftwist1, il6, and il1b (Supplementary Fig. S7E). Activation of NFkBwas reflected by IkBa degradation and our immunofluorescenceresults indicated that IkBa expression was lower in irradiated

lungs of Hsp25 TG mice compared with the BL6-IR and J2treatment to Hsp25 TG increased the intensity of fluorescence ofIkBa. To investigate IkBa–NFkB signaling activation by Hsp27 isconnected to the development of IR-induced EMT process inirradiated lungs, the level of IkBa and a-SMA were assessed viacoimmunofluorescence staining. Expression of lower IkBa andhigher a-SMA was shown in irradiated lungs of Hsp25 TG micecompared with the BL6-IR. J2 treatment to Hsp25 TG reversed theintensity of fluorescence of IkBa and a-SMA. These results indi-cate that IkBa-NFkB activation byHsp27 induces IR-induced lungfibrosis through the EMT process (Fig. 5E).

Overexpression of Hsp27 in irradiated orthotopic lung cancermodels and irradiated human lung tissues

To elucidate the expression of Hsp27 in tumor model andhuman lung tissues, first, orthotropic lung tumors using LLC1cellswere established and90-Gy IRwas irradiated to the leftwholelung of mice for 2 weeks. Most of the tumors regressed in micetreated with IR and no detectable residual tumor was observed inmice treated with both IR and J2. Increased collagen deposition in

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Hsp27 is involved in IkBa–NFkB signaling. A, Coimmunoprecipitation (IP) analysis of IkBa in cells exposed to 5-Gy IR for 4 hours were evaluated for the presenceof IkBa, p65, and Hsp27 (left), as indicated. The expression of the same proteins in the corresponding lysates (Input) is reported (right). B and C,Westernblotting analysis using cell lysates at 3 and 24 hours after 5-Gy irradiation (IR) in shControl or shHsp27 cells, and with or without J2 (0.5 mmol/L)-pretreated cells.D, After 8 hours of 10 Gy, shCONT and shHsp27 cells with or without J2 (0.5 mmol/L) were stained with anti-p65 antibody to investigate subcellular localization.Quantification of nuclear p65 level was performed. Data are expressed as mean� SE. � , P < 0.05; �� , P < 0.01 versus shCONT-non treatment; ##, P < 0.01 versusshCONT-IR. E, Immunofluorescence staining for IkBa (red) and a-SMA (green) using lung tissues of BL6 and Hsp25 TGmice was performed. Magnification, 100�or 400�. Quantification of IkBa and a-SMA expression was presented as mean� SE (�� , P < 0.001 vs. BL6-Control; #, P < 0.05 vs. BL6-IR; †, P < 0.05 vs. TG-IR).

Kim et al.





irradiated normal lung lesions of orthotopic mice model wasdecreased by J2 (Fig. 6A). Hsp25 expression was also increased inirradiated normal lungs, while in the tumor lesions, no increase ofHsp25 by IR was observed. Without IR, Hsp25 protein was moreabundantly expressed in tumor lesions than nontumor lesions(Supplementary Fig. S8A). Moreover, J2 showed a more positiveeffect on tumor regression than was seen in the IR-alone group.J2 concentrations (0.05, 0.1, and 0.5 mmol/L) induced cellularcytotoxicity in demonstrated cytotoxicity on flow cytometry usingLLC1 mouse Lewis lung adenocarcinoma cells. Microscopic anal-ysis revealed that NCI-H460 lung adenocarcinoma cells are in theform of a polygonal cobblestone and very close together. Afterexposure to IR, the cells transformed into a spindle-like shape that

was more definite as dose increase. However, cells with Hsp27shRNA (shHsp27) showed inhibition of IR-inducedmorphologicchange (Supplementary Fig. S8C).Western blot results confirmedthat IR decreased the expression of the epithelial markers such asZO-1. Also, the expression of mesenchymal markers includingFibronectin, Twist, and a-SMA increased in lung cancer cells.However, shHsp27 attenuated these phenomena. QuantitativeRT-PCR for twist1 and fn1 also showed similar patterns of proteinexpression (Supplementary Fig. S8D), suggesting that Hsp27inhibition regulated EMT in cancer cells as well as in normalepithelial cells.

Next, we examinedwhether expression ofHsp27 is increased inRILF tissues of patients. Fibrotic tissues of patients with lung

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Hsp27 expression in orthotopic mouse lung tumor models and human RIPF tissues. A, LLC1 group—intravenous injection only group; LLC1þ 90-Gy group—micewere exposed to a single dose of 90 Gy delivered to the left whole lung after 2 weeks of intravenous injection; LLC1þ 90 Gyþ J2 group—the mice wereintraperitoneally administered J2 (15 mg/kg) for 2 weeks on alternate days after irradiation. Onweek 4, the mice were sacrificed and the tissues were stained forH&E, Masson trichrome, Hsp25, and Twist. Quantification of stained tissues was performed (n� 3, mean� SD). � , P < 0.05; �� , P < 0.01; and �� , P < 0.001 versusLLC1 Control; #, P < 0.05 and ##, P < 0.01 versus LLC1þ 90 Gy. B, Sections from human RILF tissues were stained for H&E, Masson trichrome, Hsp27, and Twist.Three representative images out of 14 specimens are shown. The data from 14 specimens are quantitated and the graph shows the intensity of Hsp27 or Twist inirradiated fibrotic areas from five independent views in each sample. �� , P < 0.001 versus nonirradiated lesions. Magnification, 100�. C, Schematic illustration ofthe molecular mechanism of IR-induced lung fibrosis via the Hsp27–NFkB signaling axis.






cancer who had surgery following radiotherapy for lung adeno-carcinoma were selected on the basis of H&E staining. IHCstaining for Hsp27 and Twist were performed on 14 patienttissue samples of RILF; the fibrotic areas of RILF patient tissuesexhibited upregulated Hsp27 expression compared with the nor-mal areas. The increased expression of Twist in the irradiatedfibrotic areas of patient tissues was well correlated with Hsp27expression. Three representative results are shown in Fig. 6B andthe clinicopathologic characteristics of the patients are summa-rized in Supplementary Table S3.

DiscussionIn this study, we demonstrate the novel mechanisms of Hsp27

in IR-induced lung fibrosis development and propose Hsp27 as apossible therapeutic target for IR-induced lung fibrosis. In theanalysis of irradiated lungs, we identified Hsp27 upregulationduring IR-induced lung fibrosis. Previous proteomics studies alsorevealed an upregulation of Hsp27 in lung fibroblast cell linesupon treatment with TGFb1 and in IPF lung tissues (15, 37).Moreover, effective attenuation of BLM-induced lung fibrosis inmice via airway delivery of siRNAs ofHsp27with downregulationof myofibroblast-associated proteins such as fibronectin, type 1collagen, and OPN has been reported (38). Another report sug-gested that Hsp27 antisense oligonucleotide effectively sup-pressed adenovirus-expressing TGFb1-induced subpleural fibro-sis in rats, which suggested that Hsp27 prevented Snail degrada-tion by the proteasomal system (14). However, no other molec-ular mechanisms of Hsp27 in lung fibrosis, especially IR-inducedlung fibrosis, were reported.

IR did not transcriptionally induce Hsp27 or its upstreamregulator, HSF1 (data not shown). However, the protein expres-sion of Hsp27 was dramatically increased, suggesting that IR mayregulate Hsp27 protein stability rather than its transcriptionalactivation. Hsp27 protein accumulation in IR-induced fibrotictissues without affecting Hsp27 transcription suggests that pro-teasomal inhibition was induced by IR, which may affect theincrease in Hsp27 protein levels. The proteasomal inhibitorMG-132 also promotes Hsp27 phosphorylation (39). Hsp27phosphorylation is catalyzed by MAPK-activated protein kinase2 (MAPKAPK-2), which regulates its expression levels (40, 41).IR activates MAPKAPK-2 and phosphorylates Hsp27 (42). Ourdata also suggest Hsp27 phosphorylation by IR, which mayaffect Hsp27 protein stability.

We used generated Hsp25 TG mice to elucidate whether theincreased Hsp27 expression initiates the development of lungfibrosis. Hsp25 TG mice showed increased collagen depositionand defective lung function by IR compared with control mice.When EMT-related genes were evaluated in human lung cell lines,shHsp27 inhibited IR-induced EMT and the small-molecule func-tional inhibitor of Hsp27 (J2), inhibited IR-induced EMTin normal epithelial cells, and lung fibrosis in both normalC57BL/6N and Hsp25 TG mice with better lung functions.Moreover, Hsp27 was overexpressed in irradiated fibrotic lungtissues in an orthotopic lung tumor model and nontumor lesionsof human lung tissues after radiotherapy. J2 also inhibitedIR-induced lung fibrosis in an orthotopic lung tumor modelwithout affecting therapeutic effects of IR on the tumor, andeven more dramatic tumor regression effects than in the IR-alonegroup. Moreover, Hsp27 inhibition also diminished IR-inducedEMT in cancer cells. Therefore, Hsp27 inhibition is an effective

strategy for the inhibition of IR-induced lung fibrosis duringradiotherapy.

To elucidate the role of Hsp27 in the regulation of lung fibrosis,we initially analyzed EMT genes based on the microarray data oflung tissues. The results showed that increased mRNA levels oftwist1, il1b, and il6 were diminished by treatment with an Hsp27inhibitor and these genes were downstream molecules in NFkBpathways. Hsp27 is known to directly interact with IkBa andfacilitates its proteasome degradation, which is the main mech-anism of NFkB activation by Hsp27 (43). IR is also known as anNFkB activator (44). Therefore, Hsp27 may represent a morepotent activator ofNFkB signalingpathways including twist1, il1b,and il6 genes, which are some of the major regulators of Hsp27-mediated EMT progression. Indeed, Hsp27 knockdown or phar-macologic inhibition of Hsp27 inhibited NFkB pathway and theexpression of twist1, il1b, and il6 genes in vitro and in vivo.

TGFb1 is a well-known factor in lung fibrosis and severalstudies suggest that TGFb1 is produced during IR-mediated lungfibrosis (45). Our previous studies also showed that TGFb1expression was overexpressed in our SBRT mimicking fibroticlungs and serum (46, 47). Moreover, J2 treatment inhibitedTGFb1 protein expression in earlier time point, 14 days afterIR (47), and our preliminary data also suggested the high expres-sion of TGFb1 in 75-Gy–irradiated lungs was blocked by cotreat-ment of J2. Therefore, involvement of TGFb1 in IR-inducedlung fibrosis was not ruled out and Hsp27 inhibitor also affectTGFb-mediated fibrosis mechanisms.

It was recently shown that EMT occurring in peritoneal, kidney,and lung fibrosis, as well as in breast cancer stem cells, wasassociated with increased Hsp27 expression (48–50), suggestinga rationale for the development of Hsp27 inhibitors in fibrosistreatment. Even though Hsp27 is an attractive therapeutic targetin fibrosis, unlike Hsp90 or Hsp70, it lacks an active site orATP-binding pocket. Hence, only two Hsp27 inhibitors are inthe clinical trials. However, the limitations associated with intra-cellular delivery of OGX427 relate to the small size of the inhib-itor and lack of mechanism underlying Hsp27 in the case ofRP101 (51–53). Aside from RP101, no small molecules havebeen developed as Hsp27 inhibitors and clinical trial data ofRP101 so far are not good.

Even though currently approved therapies for lung fibrosissuch as pirfenidone and nintedanib are clinically available, analternate strategy to address the unmet therapeutic need lungfibrosis is needed. Our results for the first time demonstrate theincreased expression of Hsp27 in IR-induced lung fibrosis andincreased Hsp27 aggravated IkBa–NFkB signaling pathways toincrease EMT. Therefore, pharmacologic Hsp27 inhibitors maybe effectively used as inhibitors of IR-induced lung fibrosis,especially after radiotherapy (Fig. 6C).

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

Authors' ContributionsConception and design: Y.-J. Lee, J. Cho, Y.-S. LeeDevelopment of methodology: J. ChoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Jeon, Y.J. Yoo, H. Jin, H.Y. Won, K. Yoon, Y. Na,J. ChoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-Y. Kim, E.S. Hwang, Y. Na, J. Cho, Y.-S. Lee

Kim et al.





Writing, review, and/or revision of the manuscript: J.-Y. Kim, S. Jeon,E.S. Hwang, J. Cho, Y.-S. LeeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.-Y. Kim, S. Jeon, J. ChoStudy supervision: J. Cho, Y.-S. Lee

AcknowledgmentsThis work was supported by grants from the National Research Founda-

tion of Korea, (NRF-2017R1A2B2002327, NRF-2017M2A2A702019560,

and NRF-2018R1A5A2025286), funded by the Korean government(Ministry of Science and ICT).

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.

ReceivedNovember 30, 2018; revised April 12, 2019; acceptedMay 21, 2019;published first May 24, 2019.

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Kim et al.




Published OnlineFirst May 24, 2019.Clin Cancer Res Jee-Youn Kim, Seulgi Jeon, Young Jo Yoo, et al. Radiation-Induced Lung Fibrosis

B Signaling Axis Promotesκ-NFαThe Hsp27-Mediated IkB

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