The Hsp27-Mediated Ikb Α-Nfkb Signaling Axis Promotes Radiation-Induced Lung Fibrosis

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Author Manuscript Published OnlineFirst on May 24, 2019; DOI: 10.1158/1078-0432.CCR-18-3900 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The Hsp27-Mediated IkBα-NFkB Signaling Axis Promotes Radiation-Induced Lung Fibrosis

Jee-Youn Kim1,#, Seulgi Jeon2,#, Young Jo Yoo2, Hee Jin2, Hee Yeon Won 2, Kyeonghee Yoon2, Eun Sook Hwang2, Yoon-Jin Lee3, Younghwa Na4,*, Jaeho Cho1,*, and Yun-Sil Lee2,*

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

#Equal contribution

Corresponding authors: Yun-Sil Lee, Graduate School of Pharmaceutical Sciences, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul, Republic of Korea; Telephone: (82) 2-3277-3022; Fax: (82) 2-3277- 3051; E-mail: [email protected] Jaeho Cho, Department of Radiation Oncology, Yonsei University Health System, 50, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea; Telephone: (82) 2-2228-8113; E-mail: [email protected] Younghwa Na, College of Pharmacy, CHA University, 120, Haeryong-ro, Pocheon-si, Gyeonggi-do, Republic of Korea; Telephone: (82) 31- 881-7164; E-mail: [email protected]

Running Title: Hsp27-IkBα-NFkB signaling in lung fibrosis Keywords: Heat shock protein 27, Radiation, Lung fibrosis, IkBα-NFkB signaling, Inhibitor

Conflict of interest: The authors declare no potential conflicts of interest.

Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 24, 2019; DOI: 10.1158/1078-0432.CCR-18-3900 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Purpose: Lung fibrosis is a major side effect experienced by patients after lung cancer radiotherapy. However, effective protection strategies and underlying treatment targets remain unclear. In an effort to improve clinical outcomes, pharmacologic treatment of fibrosis is becoming increasingly popular; however, no ideal therapeutic strategy is yet available. Experimental Design/Results: The expression of Hsp27 (Hsp27 in humans and Hsp25 in mice) was increased during radiation (IR)-induced lung fibrosis in a mouse model following IR. Exacerbation of lung fibrosis by IR was also found in Hsp25 transgenic (TG) mice. Knockdown of Hsp27 in lung epithelial cells inhibited IR-mediated epithelial-mesenchymal transition (EMT). J2, a synthetic small molecule inhibitor of Hsp27, significantly alleviated lung fibrosis by IR in control and TG mice, and the therapeutic effects were better than those achieved with pirfenidone and amifostine. The activation of NFkB pathways via direct interaction between Hsp27 and IkBα resulted in increased expressions of Twist, IL-1β, and IL-6 and facilitated IR-mediated EMT, which was identified as an underlying mechanism of Hsp27-mediated fibrosis after IR. Hsp27 was overexpressed in IR-induced lung fibrosis in an orthotopic lung cancer model and was inhibited by J2 treatment. IR-induced lung fibrotic tissues from patients also showed higher expression of Hsp27 than unirradiated lungs. Conclusions: Collectively, IkBα-NFkB signaling activation by Hsp27 is involved in the EMT process that is tightly connected to the development of IR-induced lung fibrosis. Our findings also suggest that inhibition of Hsp27 has the potential to become a valuable therapeutic strategy for IR-induced lung fibrosis.

Translational Relevance Radiation therapy is an important conventional therapy for thoracic malignancies. However, radiation therapy-related pulmonary symptoms occur in up to 30% of patients and effective protection strategies and underlying treatment targets remain unclear. In this study, we used a mouse model simulating clinical stereotactic body radiotherapy (SBRT) and validated the induction of lung fibrosis. We also attempted to identify molecular targets occurring in the process of lung fibrosis development. The expression of heat shock protein (Hsp27) was increased during the induction of radiation (IR)-induced pulmonary fibrosis and Hsp27 overexpression accelerated IR-induced lung fibrosis. Inhibition of Hsp27 using a recently identified small molecule inhibitor that induced crosslinking of Hsp27 attenuated this increase. This study demonstrated the potential of Hsp27 inhibition to improve IR-induced lung fibrosis. Our results support the potential clinical utility of Hsp27 as a novel target for treating IR-induced lung fibrosis.

Introduction

Radiation therapy is a mainstay of lung cancer treatment. However, delivery of high radiation doses to the tumor is often hampered by the risk of radiation (IR)-induced lung injury. Lung damage due to thoracic IR is mediated via acute responses including inflammation and pneumonitis as well as chronic effects such as pulmonary fibrosis (1,2). Fibrosis is the end stage of persistent tissue damage and chronic inflammatory reactions. It is characterized by excessive accumulation of extracellular matrix (ECM) and disruption of normal tissue architecture (3,4). During epithelial-mesenchymal transition (EMT), cells undergo a morphological switch from the epithelial polarized phenotype to the mesenchymal fibroblastoid phenotype. EMT is characterized by the loss of epithelial differentiation markers, and the induction of mesenchymal markers. EMT plays a key role in embryonic development, chronic inflammation, and fibrosis (5,6). Furthermore, EMT was observed during tumor cell invasion and metastasis in various solid tumors, (7). The exact molecular mechanisms leading to the development of IR-induced pulmonary 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 in response to cellular stresses (8). Hsp27 is a critical mediator in cancer progression, preventing apoptosis in transformed cells (9-11). In addition, Hsp27 enhances migration and invasion (12) and mediates EMT in cancer cells (13). It is also an inducer of EMT during fibrosis including idiopathic pulmonary fibrosis (IPF) (14). Overexpression of Hsp27 was reported in patients diagnosed with IPF (15). The upregulation of Hsp27 plays a pivotal role in myofibroblast differentiation and may represent a promising therapeutic target in fibrotic diseases. Hsp27 gene silencing by OGX-427, a second-generation antisense oligonucleotide, inhibited development of bleomycin (BLM)-induced lung fibrosis and EMT via degradation of Snail (14). Accordingly, Hsp27 inhibition is an attractive therapeutic strategy. Three pharmacological treatments for IPF, namely pirfenidone (PFD), nintedanib (BIBF1120), and N- acetylcysteine (NAC) are commercially available. PFD has been shown to prevent the accumulation of hydroxyproline, procollagen I and III, inflammatory cells, and TGF-β1 in bronchoalveolar lavage (BAL), and/or lung tissue (16-22). PFD has also been shown to diminish the fibrocyte pool and the migration of these cells in mouse models of lung fibrosis (23). Nintedanib was discovered as a byproduct in large screening assays targeting cyclin-dependent kinase (CDK4) (24). Nintedanib was systematically developed as a potent angiogenesis inhibitor. However, conclusive evidence is unavailable to support its clinical role in the treatment of pulmonary disease, especially in IR-induced lung fibrosis. Previously, we developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) and validated the induction of lung fibrosis by high-dose IR (25) and this model did not show any difference in the incidence of pulmonary fibrosis according to the radiosensitive and radioresistant mouse strains (26). In this study, we identified the molecular targets in lung fibrosis development. Hsp27 expression was increased during IR-induced lung fibrosis, and functional inhibition of Hsp27 using a small molecule ameliorated lung fibrosis. While investigating mechanisms of Hsp27 in the development of lung fibrosis, we found that IkBα-NFkB signaling activation by direct interaction of IkBα with Hsp27, is involved in the EMT process that is tightly 3

connected to the development of IR-induced lung fibrosis.

Materials and Methods

Animal experiments All procedures were approved by the Animal Care and Use Committees of Yonsei University Medical School (2015-0267) and were performed in accordance with the relevant guidelines. A single dose of 75 or 90Gy was delivered using an X-RAD 320 platform (Precision X-ray, North Branford, CT) as described previously (27). Mice were administered i.p. with J2 (7.5 mg/kg or 15 mg/kg), PFD (100 mg/kg), and amifostine (AMI;100 mg/kg) for 4 weeks on alternate days after IR, and lung tissues (n≥3 per group) were collected at 4 or 6 weeks after IR.

Generation of Hsp25 TG mice Hsp25 mice were generated, interbred, and maintained in pathogen-free conditions at Macrogen, Inc (Seoul, Korea) (see supplementary information).

Establishment of the orthotopic lung tumor model The mouse lung carcinoma LLC1 cells, at 1x106 in 200 ul physiological saline, were injected in the tail vein of 7 weeks old male C57BL/6N mice. Two weeks after the i.v. injection, a single dose of 90 Gy was delivered to the left whole lung using an image-guided small-animal irradiator. The mice were randomly divided into three groups (4-6 mice per group) as follows: (1) LLC1 group - i.v. injection only; (2) LLC1+90 Gy group - mice were exposed to a single dose of 90 Gy delivered to the left whole lung 2 weeks after i.v. injection; (3) LLC1+90 Gy+J2 group – the mice were administered J2 i.p. (15 mg/kg) for 2 weeks on alternate days after irradiation. On week 4, the mice were sacrificed by CO2 asphyxiation, and lung tissues were collected for analysis.

Human tissues analysis The study of patient specimens of radiation-induced lung fibrosis (RILF) was approved by Severance Hospital, Yonsei University. Each patient's tissue contained an irradiated fibrotic and a non-irradiated normal area. The degree of protein expression was compared between the fibrotic and normal areas in each patient's tissue.

Microarray experiment

Total RNA from the mouse lung tissues was extracted using the Easy-SpinTM total RNA extraction kit according to the manufacturer’s instructions (iNtRON Biotechnology, Seoul, Republic of Korea) (See supplementary information). Cell culture and transfection

NCI-H460 (human non-small cell lung cancer cell line), L132 (human normal lung epithelial cell line) and LLC1 (mouse Lewis lung carcinoma) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI or DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) in a 37°C incubator with 5% CO2. Lentiviruses were used to create stable cell lines expressing shRNA for Hsp27 (puromycin resistance gene). The control shRNA lentiviral particle (sc-108080), Hsp27 shRNA lentiviral particle (sc-29350), and polybrene (sc-134220) were obtained from Santa Cruz Biotechnology. To generate the sh-Control cells and sh-Hsp27 cells, cell lines were selected using puromycin (1 µg/mL) for at least one week. Human pulmonary fibroblasts (HPFs) were obtained from PromoCell and used within nine passages. The mesenchymal-like A549 cells (A549TD) which were generated by chronic exposure with TGF- as described previously (28), were obtained from Professor HJ Cha (Seoul National University). Cell lines were tested by BioMycoX Mycoplasma PCR Detection Kit (JCBIO Co., Ltd) to ensure that they were mycoplasma-free.

RNA isolation, qRT-PCR and RT-PCR

Total RNA was isolated from the sample using TRIzol® reagent (Qiagen, Valencia, CA, USA) (See supplementary information). Primer sequences for RT-PCR and qRT-PCR are listed in Supplementary Table S1.

Antibodies and reagents

Immunoblotting were performed as previously described (29) using antibodies: Twist (GeneTex (Irvine, CA, USA) and Abcam (Cambridge, UK)); N-cadherin, p65, Hsp27, LaminB, and β-actin (Santa Cruz Biotechnology, CA, USA); phospho-Hsp27 (Ser82), phospho-IkBα (Ser32/36), phospho-STAT3 (Tyr705), STAT3 (Cell Signaling Technology); IkBα, IL-6, IL-1β and pro-SPC (Abcam, Cambridge, UK); fibronectin, vimentin, and E-cadherin (Becton-Dickinson Laboratories, Mountain View, CA); ZO-1 (Thermo Fisher Scientific, Waltham, MA, USA); α-SMA (Sigma, Saint Louis, MO); Alexa488-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA).

Irradiation Cells in 60 mm and 100 mm petri dishes were exposed to radiation (5 or 10 Gy as a single dose) generated by a 137 Cs gamma-ray source (Elan 3000, Atomic Energy of Canada, Mississauga, Canada) at a dose rate of 3.81 Gy/min. Radiation workers received radiation safety management training annually, provided by the Korea Foundation of Nuclear Safety (KoFONS).

Immunoprecipitation For immunoprecipitation, cells were lysed in lysis buffer (500mM NaCl, 50mM Tris-HCl pH 7.5, 0.5% Triton X-100, 1mM EDTA, and 1mM DTT), clariﬁed by centrifugation, incubated with IkBα antibody, and immunoprecipitated with protein A (Sigma-Aldrich). The precipitates were washed three times and analyzed by western blotting.

Preparation of lung tissues for histology and immunohistochemistry For the histological study, 4 μm tissue sections were stained with hematoxylin and eosin (H & E) and Masson’s trichrome (MT). Immunohistochemical staining was carried out using anti-Twist (1:100 dilution; GTX127310, GeneTex), anti-IL-6 and anti-IL-1β (1:100 dilution; ab6672 and ab9722, Abcam, respectively), and anti-Hsp27 (1:200 dilution; sc-13132, Santa Cruz) at 4°C overnight. Slides were then incubated with avidin- biotin peroxidase complex (ABC kit, Vector Laboratories, CA, USA) and developed using 3, 3′- diaminobenzidine tetrachloride (DAB; Zymed Laboratories, CA, USA).

Immunofluorescence assay

Cells were fixed in 2% paraformaldehyde for 1 h, followed by blocking and incubation with primary antibodies at 4℃ overnight. Anti-α-SMA (A5228, Sigma, 1:200), anti-p65 (sc-8008, Santa Cruz, 1:100), anti-Twist (sc-15393, Santa Cruz, 1:100), and anti-Hsp27 (sc-13132, Santa Cruz, 1:200) were used to detect expression. The morphological change was investigated by Alexa488-conjugated phalloidin staining (A12379, Invitrogen, 1:200). For immunoﬂuorescence staining, tissue sections stained with pro-SPC, α-SMA, and IkBα (1:100 dilution; ab90716, ab7817 and ab32518, Abcam, respectively) were incubated with appropriate ﬂuorescent secondary antibodies and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Images were viewed under a confocal microscope (LSM700, Zeiss, Jena, Germany).

Micro-computed tomographic analysis Micro-computed tomographic analysis was performed as previously described (27).

Functional assessment of the lungs Lung function in irradiated mice was evaluated with the Flexivent system (Flexivent®; SCIREQ©, Montreal, QC, Canada), which measures flow-volume relationships in the respiratory system, including forced oscillation, to distinguish airway and lung tissue variables (30) (See supplementary information and Supplementary Table S2).

Statistical analysis Comparisons of all results were performed by one- or two-way ANOVA and Newman-Keuls test where indicated. The difference was considered statistically significant at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.005. All statistical analyses were performed using GraphPad Prism.

Results

Increased Hsp27 expression during IR-induced lung fibrosis To confirm the fibrosis, lung sections were stained with Masson’s Trichrome to visualize the deposition of blue-colored collagen. At 4 weeks, extensive collagen was observed, correlating with late-stage 6

fibrosis (Figure 1A). Antibody protein arrays showed that irradiation induced the secretion of Hsp27 alone into the blood without increasing the levels of other Hsps such as Hsp90 and Hsp70 (Supplementary Figure S1A). Therefore, we examined the Hsp27 expression in lung tissues by immunohistochemical analysis and found that Hsp27 protein expression was increased during lung fibrosis (Figure 1B). No increase in hspb1 mRNA expression was detected during fibrosis (Supplementary Figure S1B). To elucidate the role of Hsp27 in lung fibrosis directly in vivo, Hsp25 transgenic (TG) mice were used (Supplementary Figure S1C and S1D). IR- induced lung fibrosis was exacerbated after focal exposure to high-dose IR (75 Gy) in Hsp25 TG mice, 6 weeks after IR. Following IR exposure, the Hsp25 TG mice showed an abundance of neutrophils and mononuclear cells in the alveoli, greater destruction of alveolar septa, intra-alveolar hyaline membrane formation, and a marked increase in collagen deposition compared with control C57BL/6N (BL6) mice. CT images may be used to predict fibrosis, and micro-CT is comparable to clinical CT in humans (31). Six weeks after IR, the typical micro-CT manifestations of SBRT-induced lung injury, such as ground-glass opacities and consolidation (32), were observed in the irradiated left lung. These effects were strongly induced in Hsp25 TG mice. Normal lung volume after IR in Hsp25 TG mice was lower compared to that in control BL6 mice (Figure 1C). Functional lung parameters evaluated in this study are listed in Supplementary Table S2. There were significant differences in inspiration capacity (IC), quasi-static compliance (Cst) and tissue damping (G) of the lungs following exposure to IR between BL6 and Hsp25 TG mice. These results reflect the respiratory distress induced by irradiation. The respiratory distress in Hsp25 TG mice appears to have been significantly potentiated (Figure 1D).

Knockdown of Hsp27 inhibited IR-mediated EMT in lung cell lines To elucidate the cellular role of Hsp27 during the development of IR-induced lung fibrosis, we initially examined the morphological changes in L132 cells following IR. Control L132 cells were round or polygonal and exhibited very close cell-cell proximity reminiscent of cellular tight-junctions. IR transformed the cells into a spindle shape. These changes became clearer and more abundant with increased dose. However, cells with Hsp27 shRNA (shHsp27) showed inhibition of these IR-induced morphological features (Supplementary Figure S2A). Western blot results confirmed that IR decreased the expression of the epithelial markers such as ZO-1. However, the expression of mesenchymal markers including Twist, fibronectin and α- SMA increased in L132 cells and shHsp27 attenuated these phenomena. Quantitative RT-PCR for twist1 and fn1 also showed similar patterns of protein expression (Figure 2A). Previously, we identified the functional inhibition of Hsp27 following altered dimerization at the cysteine residue (Cys 137) of Hsp27 using small molecules to ameliorate Hsp27-mediated chemo- or radioresistance in lung cancer cells (29,33). In this study, we investigated the inhibition of IR-mediated EMT using J2, which is a chromone derivative and a small molecule Hsp27 inhibitor. J2 strongly altered the crosslinking of Hsp27 compared to SW15 (xanthone structure derivative). J2 cross-linked Hsp27 even at low concentrations of 0.1 or 0.5 μM and high concentrations of 10 μM, which overcame the chemoresistance, via strongly-altered crosslinking of Hsp27 (Supplementary Figure S2B). J2 treatment of recombinant Hsp27 protein strongly inhibited the formation of large oligomers of Hsp27 in non-reducing gel systems (Supplementary Figure S2C). Pretreatment with J2 mitigated the altered expression of EMT-related proteins such as Twist,

fibronectin, -SMA, vimentin, and ZO-1 induced by IR in L132 cells assayed at 24 and 48 h after IR (Figure 2B and Supplementary Figure S2D). Increased adhesion is a characteristic of cells with a mesenchymal phenotype. Immunofluorescent hair- like fibers stained with phalloidin (green) protruding from cell surfaces into the collagen matrix assembled at the leading edge of irradiated cells, whereas treatment with shHsp27 or J2 reduced these protrusions (Figure 2C). J2 treatment also reduced EMT-related proteins such as fibronectin, N-cadherin, α-SMA, and Twist resulting from IR in human pulmonary fibroblast cells (HPFs) on Western blotting and immunofluorescence images of SMA (Figure 2D). Continuous exposure to TGF-β by A549 lung carcinoma cells (A549 TGF-β– differentiated cell lines [A549TD]) yielded a lower expression of E-cadherin and a higher expression of Hsp27 and vimentin than occurred in the parent A549 cells (Supplementary Figure S3A), without affecting other Hsps such as Hsp70 and Hsp90 (Supplementary Figure S3B), indicating Hsp27-mediated EMT. Moreover, J2 treatment dramatically restored the morphological changes of A549TD cells and decreased the expression of E- cadherin. The expression of vimentin in A549TD cells was also restored by 0.1 and 0.5 μM J2 treatment (Figure 2E and Supplementary Figure S3C), suggesting that Hsp27 inhibition modulated EMT. J2 concentrations (0.05, 0.1, and 0.5 μM) neither induced cellular cytotoxicity in a colony forming assay nor demonstrated cytotoxicity on flow cytometry using L132 lung epithelial cells, NCI-H460 and A549 lung cancer epithelial cells. Cellular protective effects by J2 after IR were observed and the protective effect was predominantly seen in normal cells of L132 rather than in NCI-H460 and A549 cancer cells (Supplementary Figure S3D and S3E).

Hsp27 cross-linker J2 inhibited IR-induced lung fibrosis in mice To elucidate whether a small molecule Hsp27 cross-linker J2 inhibits IR-mediated lung fibrosis in mice, we compared the changes in left lung surface morphology in the control and IR group. In contrast to the brown colored lungs in control mice, the lungs of irradiated mice exhibited a definite white, ring-like appearance. Intraperitoneal injection of J2 resulted in less injury than was apparent in the IR-only mice. Alveolar infiltration of inflammatory cells and the formation of intra-alveolar hyaline membranes in the IR group were significantly greater than in the control group. J2-treated mice exhibited reduced tissue damage. Masson’s trichrome staining revealed a marked increase in collagen deposition in the IR group than in the control group, which was significantly reversed by J2 treatment. Six weeks after IR, ground-glass opacities and consolidation were observed in the irradiated left lung; in contrast, these effects were decreased in J2-treated mice. Normal lung volume in the IR group was lower than in the control mice. However, normal lung volume appeared to significantly recover in J2-treated mice (Figure 3B and Supplementary Figure S4A). Also, there were significant differences in IC, Cst, G, and tissue elastance (H) of the lungs between IR group and control mice. The IC and Cst of the IR group were significantly lower compared to those of the control group. The values of G and H in the IR group were higher than in the control group. Mice treated with 15 mg/kg J2 exhibited significant differences in IC, Cst and G parameters, indicating the protective effect of J2 on IR-induced lung injury (Supplementary Figure S4B). The effects of J2 were more prominent than those of PFD or AMI, even though administration dosage of J2 was less than PFD and AMI. To develop evidence that epithelial cells express a mesenchymal phenotype during IR-induced lung fibrosis, we performed immunofluorostaining for both alveolar epithelial cell-specific protein SPC and 8

myofibroblast-specific marker -SMA. The luminal layer of alveolar cells was immunoreactive for SPC and the SPC-positive cell number was not substantially different among the groups. Moreover, α-SMA expression was increased by IR and SPC/-SMA co-staining cells were also increased by IR, indicating that an EMT process occurred during IR-induced lung fibrosis. However, J2 treatment with IR decreased the SPC/-SMA co-staining cells (Figure 3C). We also investigated the effects of J2 on IR-induced lung fibrosis in Hsp25 TG mice. Similar to the findings of normal BL6 mice, irradiated areas of the left lung clearly exhibited a local injury in BL6 mice. In the Hsp25 TG mice, we observed aggravated lung injury grossly and histologically, which was attenuated by J2 treatment. Masson’s trichrome, Sirius red and immunohistochemical hydroxyproline staining revealed a marked increase in collagen deposition in Hsp25 TG mice after IR compared with IR-treated control BL6 mice and J2 also inhibited collagen deposition in Hsp25 TG mice. Six weeks after IR, the normal lung volume was lower in irradiated Hsp25 TG mice than in irradiated BL6 mice. However, it appears to have been significantly restored in J2-treated Hsp25 TG mice (Figure 3D and Supplementary Figure S5A, B). Increased co-staining of SPC/- SMA after IR was restored by combined treatment with J2, indicating that the IR-induced EMT process was blocked by J2 treatment (Figure 3E). There were significant differences in IC, Cst, G, and H of the lungs between irradiated Hsp25 TG and irradiated control BL6 mice. However, IR-induced respiratory distress in J2- treated mice appeared to be significantly reversed even in Hsp25 TG mice (Supplementary Figure S5C).

NFkB activation by Hsp27 is involved in the expression of IR-induced fibrosis-related genes To identify the underlying mechanisms of inhibition of IR-mediated lung fibrosis by J2, we performed a cDNA microarray of lung tissues after focal exposure to 75 Gy with or without 4 weeks of i.p. of J2. Temporal changes in gene expression were hierarchically clustered (Supplementary Figure S6A). Analysis of fibrosis- related genes in the microarray data revealed upregulation of only twist1, il-6, and il-1β genes by focal irradiation, which was reversed by J2 treatment. We also determined the mRNA levels of twist1, il-6, and il-1β using qRT-PCR (Figure 4A) and protein levels using immunohistochemistry (Figure 4B); the levels were similar to those obtained with cDNA microarray. J2 treatment restored the significant increase induced by focal IR. We also found that J2 did not alter the expression of Hsp25 protein in lung tissues (Supplementary Figure S6B). We next examined whether Hsp27 knockdown or J2 treatment modulated IR-induced twist1, il-6, and il-1β genes in a cell system. The qRT-PCR analysis of L132 lung epithelial cells revealed that the increased expression of twist1, il-6, and il-1β genes by IR was suppressed by shRNA of Hsp27 or J2 pretreatment, based on the results detected at 12 h after IR (Figure 4C). Immunofluorescence data in L132 cells revealed that the basal Twist level was inhibited by shHsp27 and IR-induced Twist activation was also ameliorated by knockdown of Hsp27 (Figure 4D). Since NFkB is a regulator of Twist, IL-1β, and IL-6 (34-36), we investigated the association between p65, one of the components of NFkB, and IR-mediated EMT markers using siRNA and BAY11-7082, an NFkB inhibitor. BAY11-7082 or p65 knockdown dramatically inhibited the Twist protein level (Supplementary Figure S7A and S7B), suggesting Twist as a downstream effector of NFkB. To elucidate whether the binding between Hsp27 and IkBα was affected by IR, immunoprecipitation (IP) was performed. The results indicated an increase in the binding activity between Hsp27 and IkBα by IR, and a decreased binding activity between IkBα and p65 9

(Figure 5A). Moreover, siRNA of twist1 did not inhibit il-6 and il-1β genes in L132 cells, suggesting that NFkB activation by Hsp27 may be a master regulator of twist1, il-6, and il-1β genes (Supplementary Figure S7C). We also investigated whether Hsp27 level regulates NFkB-mediated Twist expression by IR and found that Hsp27 knockdown or Hsp27 cross-linker J2 inhibited IR-mediated Twist expression, accompanied by inhibition of IkBα phosphorylation, at 3 h of IR before Twist activation (24 h of IR). In the case of STAT3 phosphorylation, another transcription factor of Twist, IR slightly induced STAT3 phosphorylation. However, Hsp27 inhibition did not affect the phosphorylation (Figure 5B and 5C). IR-mediated nuclear translocation of p65 was inhibited by shHsp27 or J2 treatment (Figure 5D and Supplementary Figure S7D). The siRNA of p65 inhibited IR- mediated mRNA expression of twist1, il-6, and il-1β as shown in qRT-PCR, suggesting that NFkB activation by IR acted upstream of twist1, il-6, and il-1β (Supplementary Figure S7E). Activation of NFkB was reflected by IkBα degradation and our immunofluorescence results indicated that IkBα expression was lower in irradiated lungs of Hsp25 TG mice compared to the BL6-IR and J2 treatment to Hsp25 TG increased the intensity of fluorescence of IkBα To investigate IkBα-NFkB signaling activation by Hsp27 is connected to the development of IR-induced EMT process in irradiated lungs, the level of IkBα and α-SMA were assessed via co- immunofluorescence staining. Expression of lower IkBα and higher α-SMA was shown in irradiated lungs of Hsp25 TG mice compared to the BL6-IR. J2 treatment to Hsp25 TG reversed the intensity of fluorescence of IkBα and α-SMA. These results indicate that IkBα-NFkB activation by Hsp27 induces IR-induced lung fibrosis through the EMT process (Figure 5E).

Overexpression of Hsp27 in irradiated orthotopic lung cancer models and irradiated human lung tissues To elucidate the expression of Hsp27 in tumor model and human lung tissues, first, orthotropic lung tumors using LLC1 cells were established and 90 Gy IR was irradiated to the left whole lung of mice for 2 weeks. Most of the tumors regressed in mice treated with IR and no detectable residual tumor was observed in mice treated with both IR and J2. Increased collagen deposition in irradiated normal lung lesions of orthotopic mice model was decreased by J2 (Figure 6A). Hsp25 expression was also increased in irradiated normal lungs, while in the tumor lesions, no increase of Hsp25 by IR was observed. Without IR, Hsp25 protein was more abundantly expressed in tumor lesions than non-tumor lesions (Supplementary Figure S8A). Moreover, J2 showed a more positive effect on tumor regression than was seen in the IR-alone group. J2 concentrations (0.05, 0.1, and 0.5 μM) induced cellular cytotoxicity in demonstrated cytotoxicity on flow cytometry using LLC1 mouse Lewis lung adenocarcinoma cells. Microscopic analysis revealed that NCI-H460 lung adenocarcinoma cells are in the form of a polygonal cobblestone and very close together. Following exposure to IR, the cells transformed into a spindle-like shape which was more definite as dose increase. However, cells with Hsp27 shRNA (shHsp27) showed inhibition of IR-induced morphological change (Supplementary Figure S8C). Western blot results confirmed that IR decreased the expression of the epithelial markers such as ZO-1. Also, the expression of mesenchymal markers including Fibronectin, Twist and α-SMA increased in lung cancer cells. However, shHsp27 attenuated these phenomena. Quantitative RT-PCR for twist1 and fn1 also showed similar patterns of protein expression (Supplementary Figure S8D), suggesting that Hsp27 inhibition regulated EMT in cancer cells as well as in normal epithelial cells. Next, we examined whether expression of Hsp27 is increased in RILF tissues of patients. Fibrotic 10

tissues of lung cancer patients who had surgery following radiotherapy for lung adenocarcinoma were selected based on H&E staining. Immunohistochemical staining for Hsp27 and Twist were performed on 14 patient tissues samples of RILF; the fibrotic areas of RILF patient tissues exhibited upregulated Hsp27 expression compared to the normal areas. The increased expression of Twist in the irradiated fibrotic areas of patient tissues was well correlated with Hsp27 expression. Three representative results are shown in Figure 6B and the clinicopathological characteristics of the patients are summarized in Supplementary Table S3.

Discussion

In the present study, we demonstrate the novel mechanisms of Hsp27 in IR-induced lung fibrosis development and propose Hsp27 as a possible therapeutic target for IR-induced lung fibrosis. In the analysis of irradiated lungs, we identified Hsp27 upregulation during IR-induced lung fibrosis. Previous proteomics studies also revealed an upregulation of Hsp27 in lung fibroblast cell lines upon treatment with TGF-β1 and in IPF lung tissues (15,37). Moreover, effective attenuation of BLM-induced lung fibrosis in mice via airway delivery of siRNAs of Hsp27 with downregulation of myofibroblast-associated proteins such as fibronectin, type 1 collagen, and OPN has been reported (38). Another report suggested that Hsp27 antisense oligonucleotide effectively suppressed adenovirus-expressing TGF-β1-induced subpleural fibrosis in rats, which suggested that Hsp27 prevented Snail degradation by the proteasomal system (14). However, no other molecular mechanisms of Hsp27 in lung fibrosis, especially IR-induced lung fibrosis, were reported. IR did not transcriptionally induce Hsp27 or its upstream regulator, HSF1 (data not shown). However, the protein expression of Hsp27 was dramatically increased, suggesting that IR may regulate Hsp27 protein stability rather than its transcriptional activation. Hsp27 protein accumulation in IR-induced fibrotic tissues without affecting Hsp27 transcription suggests that proteasomal inhibition was induced by IR, which may affect the increase in Hsp27 protein levels. The proteasomal inhibitor MG-132 also promotes Hsp27 phosphorylation (39). Hsp27 phosphorylation is catalyzed by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAPK-2), which regulates its expression levels (40,41). IR activates MAPKAPK-2 and phosphorylates Hsp27 (42). Our data also suggest Hsp27 phosphorylation by IR, which may affect Hsp27 protein stability. We used generated Hsp25 TG mice to elucidate whether the increased Hsp27 expression initiates the development of lung fibrosis. Hsp25 TG mice showed increased collagen deposition and defective lung function by IR compared to control mice. When EMT-related genes were evaluated in human lung cell lines, shHsp27 inhibited IR-induced EMT and the small molecule functional inhibitor of Hsp27 (J2), inhibited IR-induced EMT in normal epithelial cells and lung fibrosis in both normal C57BL/6N and Hsp25 TG mice with better lung functions. Moreover, Hsp27 was overexpressed in irradiated fibrotic lung tissues in an orthotopic lung tumor model and non-tumor lesions of human lung tissues after radiotherapy. J2 also inhibited IR-induced lung fibrosis in an orthotopic lung tumor model without affecting therapeutic effects of IR on the tumor, and even more dramatic tumor regression effects than in the IR-alone group. Moreover, Hsp27 inhibition also diminished IR- induced EMT in cancer cells. Therefore, Hsp27 inhibition is an effective strategy for the inhibition of IR- induced lung fibrosis during radiotherapy. To elucidate the role of Hsp27 in the regulation of lung fibrosis, we initially analyzed EMT genes 11

based on the microarray data of lung tissues. The results showed that increased mRNA levels of twist1, il-1β, and il-6 were diminished by treatment with an Hsp27 inhibitor and these genes were downstream molecules in NFkB pathways. Hsp27 is known to directly interact with IkBα and facilitates its proteasome degradation, which is the main mechanism of NFkB activation by Hsp27 (43). IR is also known as an NFkB activator (44). Therefore, Hsp27 may represent a more potent activator of NFkB signaling pathways including twist1, il-1β, and il-6 genes, which are some of the major regulators of Hsp27-mediated EMT progression. Indeed, Hsp27 knockdown or pharmacological inhibition of Hsp27 inhibited NFkB pathway and the expression of twist1, il-1β and il-6 genes in vitro and in vivo. TGF-β1 is a well-known factor in lung fibrosis and several studies suggest that TGF-β1 is produced during IR-mediated lung fibrosis (45). Our previous studies also showed that TGF-β1 expression was overexpressed in our SBRT mimicking fibrotic lungs and serum (46,47). Moreover, J2 treatment inhibited TGF- β1 protein expression in earlier time point, 14 days after IR (47) and our preliminary data also suggested the high expression of TGF-β1 in 75 Gy irradiated lungs was blocked by co-treatment of J2. Therefore, involvement of TGF-β1 in IR-induced lung fibrosis was not ruled out and Hsp27 inhibitor also affect TGF-mediated fibrosis mechanisms. It was recently shown that EMT occurring in peritoneal, kidney, and lung fibrosis, as well as in breast cancer stem cells, was associated with increased Hsp27 expression (48-50), suggesting a rationale for the development of Hsp27 inhibitors in fibrosis treatment. Even though Hsp27 is an attractive therapeutic target in fibrosis, unlike Hsp90 or Hsp70, it lacks an active site or ATP-binding pocket. Hence, only two Hsp27 inhibitors are in the clinical trials. However, the limitations associated with intracellular delivery of OGX427 relate to the small size of the inhibitor and lack of mechanism underlying Hsp27 in the case of RP101 (51-53). Aside from RP101, no small molecules have been developed as Hsp27 inhibitors and clinical trial data of RP101 so far are not good. Even though currently approved therapies for lung fibrosis such as PFD and nintedanib are clinically available, an alternate strategy to address the unmet therapeutic need lung fibrosis is needed. Our results for the first time, demonstrate the increased expression of Hsp27 in IR-induced lung fibrosis and increased Hsp27 aggravated IkBα-NFkB signaling pathways to increase EMT. Therefore, pharmacological Hsp27 inhibitors may be effectively used as inhibitors of IR-induced lung fibrosis, especially after radiotherapy (Figure 6C).

Authors’ Contributions

Conception and design: YS Lee, JH Cho, YJ Lee, ES Hwang Chemical synthesis: Y Na Conducting the experiments: JY Kim, S Jeon, YJ Yoo, H Jin, K Yoon, HY Won Analysis and interpretation of data: YS Lee, JH Cho, YJ Lee, JY Kim Writing and review: YS Lee, YJ Lee, JY Kim, S Jeon Study supervision: YS Lee

Acknowledgments 12

This work was supported by grants from the National Research Foundation of Korea, (NRF- 2017R1A2B2002327, NRF-2017M2A2A702019560, and NRF-2018R1A5A2025286), funded by the Korean government (Ministry of Science and ICT).

Figure Legends

Figure 1: Increased Hsp27 expression during IR-induced lung fibrosis (A) Experimental scheme of focal exposure to high-dose radiation (90 Gy). Lung sections were stained with Masson’s Trichrome for collagen deposition at indicated times. (B) Immunohistochemistry 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’s trichrome-stained lung sections at 6 weeks after focal 75 Gy irradiation (IR) in C57BL/6N and Hsp25 TG mice. Lungs were photographed after complete fixation (middle), horizontal (3rd from the right), trans-axial (2nd from the right), and 3D micro-CT (the most right) images acquired at 6 weeks after IR. Graphs show scores quantifying inflammation, collagen deposition, and normal lung volume. (D) Quantification of inspiratory capacity (IC), Quasi-static compliance (Cst), tissue damping (G) and tissue elastance (H). Data are expressed as mean ± standard error (n≥3, **P < 0.01 vs. BL6-Control; #P < 0.05 vs. BL6-IR).

Figure 2: 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 h after radiation (IR) were analyzed by Western blot and qRT-PCR (24 h of IR), with gapdh mRNA 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 h and 48 h after 5 Gy IR in L132 cells with or without J2 (0.5 μM) pretreatment. (C) L132 cells were pretreated for 2 hours with 0 or 0.5 μM J2 and irradiated with 5 Gy. At 48 hours after IR, the levels of phalloidin (green), DAPI (blue) were assessed via immunoﬂuorescence staining. Magnification at 400 X (D) Western blots and immunofluorescence using HPF cells at 12h after 5 Gy IR with or without J2 (0.5 μM) pretreatment. Magnification at 200 X (E) Western blotting data after treatment with J2 at the indicated concentrations in A549- and TGFdifferentiated A549 cell lines (A549TD).

Figure 3: 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 at 6 weeks after 75 Gy IR. (B) Lung sections of mice at 6 weeks after IR were stained with Masson's trichrome. Quantification of collagen deposition with blue staining (left), inflammatory foci from H&E staining (middle), and volume of normal lung from micro-CT analysis (right) are presented as mean ± standard error. (C) The pro- SPC (red) was used for identifying type II AECs, co-stained with α-SMA (green) in type II AECs. α-SMA

expression levels were upregulated in type II AECs of the irradiated lung tissue. In contrast, J2 (15mg/kg) inhibited the increases in α-SMA expression in these epithelial cells. Magnification at 630x. Scale bar, 20 μm (D) C57BL6N (BL6) and Hsp25 transgenic (TG) mice were sacrificed at 6 weeks after 75 Gy irradiation (IR). The mice were intraperitoneally administered J2 (15 mg/kg) on alternate days after IR for 4 weeks. On week 6, the mice were sacrificed, and Masson's trichrome-stained lung sections were examined. Magnification, 40x (upper), 400x (lower). Quantification of collagen deposition with blue staining (left), inflammatory foci from H&E staining (middle) and volume of normal lung from micro-CT analysis (right) are expressed as mean ± standard error. (E) The pro-SPC (red) was co-stained with α-SMA (green) using lung tissues of BL6 and Hsp25 TG mice. Upregulation of α-SMA expression in epithelial cells by IR was more strongly increased at TG-IR group and significantly decreased at TG-IR+J2 (15mg/kg) group. Magnification at 630 X. Scale bar, 20 μm (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).

Figure 4: Inhibition of Hsp27 blocks the expression of twist1, il-1β, and il-6 The mice were intraperitoneally injected with J2 (15 mg/kg) on alternate days after 75 Gy IR for 4 weeks. Mice were sacrificed at 6 weeks after IR and lungs were subjected to microarray analysis. (A) Microarray data of twist1, il-1and il-6 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 vs. Control; #P < 0.05 vs. IR (B) Immunohistochemical staining of Twist, IL-1β, and IL-6 using lungs from three individual mice. Quantification of stained tissues was performed (n≥3, mean±SD). **P < 0.01 and ***P < 0.001 vs. Control; #P < 0.05 and ##P < 0.01 vs. IR (C) mRNA levels of twist1, il-6, and il-1β using qRT-PCR in L132 cell lines with a stable Hsp27 knockdown (left) or J2 (right) at 12 h after 5 Gy IR; gapdh mRNA was used for # ## normalization. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Control; P < 0.05 and P < 0.01 vs. IR only (D) Immunofluorescence staining for Twist and Hsp27 in L132 cell lines with a stable Hsp27 knockdown at 24 h after 5 Gy IR. Magnification at 400 X

Figure 5: Hsp27 is involved in IkBα -NFkB signaling (A) Co-immunoprecipitation (IP) analysis of IkBα in cells exposed to 5 Gy IR for 4 hr were evaluated for the presence of IkBα, p65, and Hsp27 (left), as indicated. The expression of the same proteins in the corresponding lysates (Input) is reported (right). (B-C) Western blotting analysis using cell lysates at 3 and 24 h after 5 Gy irradiation (IR) in shControl or shHsp27 cells, and with or without J2 (0.5 μM)-pretreated cells. (D) After 8 h of 10 Gy, shCONT and shHsp27 cells with or without J2 (0.5μM) were stained with anti-p65 antibody to investigate subcellular localization. Quantification of nuclear p65 level was performed. Data are expressed as mean ± standard error. *P<0.05 and **P<0.01 vs. shCONT-non treatment; ##P<0.01 vs. shCONT-IR. (E) Immunofluorescence staining for IⱪBα (red) and α-SMA (green) using lung tissues of BL6 and Hsp25 TG mice was performed. Magnification, 100x or 400x. Quantification of IⱪBα and α-SMA expression was presented as † mean ± standard error (***P < 0.001 vs. BL6-Control; #P < 0.05 vs. BL6-IR; P < 0.05 vs. TG-IR).

Figure 6. Hsp27 expression in orthotopic mouse lung tumor models and human RIPF tissues (A) LLC1 group - i.v. injection only group; LLC1+90Gy group - mice were exposed to a single dose of 90 Gy delivered to the left whole lung after 2 weeks of i.v. injection; LLC1+90Gy+J2 group - the mice were intraperitoneally administered J2 (15 mg/kg) for 2 weeks on alternate days after irradiation. On week 4, the mice were sacrificed and the tissues were stained for H&E, Masson’s trichrome, Hsp25 and Twist. Quantification of stained tissues was performed (n≥3, mean±SD). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. LLC1 Control; #P < 0.05 and ##P < 0.01 vs. LLC1+90 Gy (B) Sections from human RILF tissues were stained for H&E, Masson’s 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 in irradiated fibrotic areas from five independent views in each sample. ***P < 0.001 vs. Non-irradiated lesions. Magnification, 100x (C) Schematic illustration of the molecular mechanism of IR-induced lung fibrosis via the Hsp27-NFkB signaling axis.

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The Hsp27-Mediated IkB α-NFkB Signaling Axis Promotes Radiation-Induced Lung Fibrosis

Jee-Youn Kim, Seulgi Jeon, Young Jo Yoo, et al.

Clin Cancer Res Published OnlineFirst May 24, 2019.

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