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PAK1 tyrosine phosphorylation is required to induce epithelial-mesenchymal transition and radioresistance in lung cancer cells EunGi Kim 1 , HyeSook Youn 2,3 , TaeWoo Kwon 1 , Beomseok Son 1 , JiHoon Kang 1 , Hee Jung Yang 1 , Ki Moon Seong 4 , Wanyeon Kim 2,3 and BuHyun Youn 1,2,3,* 1 Department of Integrated Biological Science, Pusan National University, Busan, 609-735, South Korea, 2 Department of Biological Sciences, Pusan National University, Busan, 609- 735, South Korea, 3 Nuclear Physics and Radiation Technology Institute, Pusan National University, Busan, 609-735, South Korea, 4 Department of Planning & Research for Radiological Emergency, National Radiation Emergency Medical Center, Korea Institute of Radiological & Medical Sciences, Seoul, 139-706, South Korea Running title: Radioresistance mediated by Tyr phosphorylation of PAK1 Key words: PAK1, Tyrosine phosphorylation, Protein stability, Epithelial-mesenchymal transition, Radioresistance Grant Support: This work was supported by the Radiation Technology R&D Program through the National Research Foundation of Korea funded by the Ministry of Science, ITC & Future Planning (2013M2A2A7042502), a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1320100), and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2013R1A1A2059832 to W Kim). * Correspondence should be addressed to BuHyun Youn, Ph.D. Department of Biological Sciences, Pusan National University Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 609-735, South Korea Tel: 82-51-510-2264; Fax: 82-51-581-2962; E-mail: [email protected] Word count: 4996 Total numbers of figures and tables: 7 Conflict of interest: The authors declare that they have no conflict of interest. on July 12, 2018. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 14, 2014; DOI: 10.1158/0008-5472.CAN-14-0735

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  • PAK1 tyrosine phosphorylation is required to induce epithelial-mesenchymal transition

    and radioresistance in lung cancer cells

    EunGi Kim1, HyeSook Youn2,3, TaeWoo Kwon1, Beomseok Son1, JiHoon Kang1, Hee

    Jung Yang1, Ki Moon Seong4, Wanyeon Kim2,3 and BuHyun Youn1,2,3,*

    1Department of Integrated Biological Science, Pusan National University, Busan, 609-735,

    South Korea, 2Department of Biological Sciences, Pusan National University, Busan, 609-

    735, South Korea, 3Nuclear Physics and Radiation Technology Institute, Pusan National

    University, Busan, 609-735, South Korea, 4Department of Planning & Research for

    Radiological Emergency, National Radiation Emergency Medical Center, Korea Institute of

    Radiological & Medical Sciences, Seoul, 139-706, South Korea

    Running title: Radioresistance mediated by Tyr phosphorylation of PAK1

    Key words: PAK1, Tyrosine phosphorylation, Protein stability, Epithelial-mesenchymal

    transition, Radioresistance

    Grant Support: This work was supported by the Radiation Technology R&D Program

    through the National Research Foundation of Korea funded by the Ministry of Science, ITC

    & Future Planning (2013M2A2A7042502), a grant from the National R&D Program for

    Cancer Control, Ministry for Health and Welfare, Republic of Korea (1320100), and the

    Basic Science Research Program through the National Research Foundation of Korea funded

    by the Ministry of Education, Science and Technology (2013R1A1A2059832 to W Kim).

    *Correspondence should be addressed to

    BuHyun Youn, Ph.D.

    Department of Biological Sciences, Pusan National University

    Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 609-735, South Korea

    Tel: 82-51-510-2264; Fax: 82-51-581-2962; E-mail: [email protected]

    Word count: 4996

    Total numbers of figures and tables: 7

    Conflict of interest: The authors declare that they have no conflict of interest.

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  • Abstract

    PAK1 kinase has an essential role in tumorigenesis and cell survival in many cancers

    but its regulation is not fully understood. In this study, we showed that in response to

    irradiation of lung cancer cells, PAK1 was upregulated, tyrosine phosphorylated and

    translocated to the nucleus. Tyrosine phosphorylation relied upon JAK2 kinase activity and

    was essential for PAK1 protein stability and binding to Snail. This radiation-induced

    JAK2/PAK1/Snail signaling pathway increased epithelial-mesenchymal transition (EMT) by

    regulating epithelial and mesenchymal cell markers. Notably, JAK2 inhibitors mediated

    radiosensitization and EMT blockade in a mouse xenograft model of lung cancer. Taken

    together, our findings offered evidence that JAK2 phosphorylates and stabilizes functions of

    PAK1 that promote EMT and radioresistance in lung cancer cells, with additional

    implications for the use of JAK2 inhibitors as radiosensitizers in lung cancer treatment.

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  • Introduction

    Radiotherapy can be a preferential management strategy for patients with inoperable

    cancer types, including advanced stage of non-small cell lung cancer (NSCLC), in the

    absence of more effective targeted therapies (1). Nevertheless, therapeutic outcomes are not

    fully satisfactory due to the emergence of radioresistance which is considered to be a critical

    obstacle that leads to the failure of radiation therapy and consequently increases mortality in

    NSCLC patients (2). So far, a large number of studies have been conducted to find way to

    control radioresistance and develop potent adjuvants for radiotherapy that enhance treatment

    efficacy (3, 4). A profound understanding of the molecular events associated with therapeutic

    resistance would greatly advance the discovery of drugs enabling to modulate radiation-

    induced signaling responses to improve the prognosis of NSCLC patient (5-7).

    We previously found that the p21-activated Ser/Thr kinase 1 (PAK1) is

    overexpressed in radioresistant NSCLC cells exposed to ionizing radiation (IR) (8). PAK1 is

    known to be broadly expressed in various normal tissues, but PAK1 is also significantly

    overexpressed in some types of cancer, such as ovarian, breast, and bladder cancers (9).

    Several functional studies have reported that PAK1 is highly associated with cell

    transformation and tumorigenesis, supported by the development of premalignant lesions and

    tumor formation in mammary glands induced by PAK1 overexpression and hyperactivation

    (10, 11). This evidence implies that PAK1 may contribute to tumor initiation and acquisition

    of tumor aggressiveness under certain cellular conditions. In addition, other reports have

    shown that activated PAK1 can protect against chemotherapy- and anoikis-induced cell death

    (12, 13). Genomic analysis has shown that gene amplification and overexpression of PAK1

    can be detected in the cytoplasm and nucleus of cells from NSCLC patients, and selective

    inhibition targeting PAK1 can negatively regulate cell proliferation both in vitro and in vivo

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  • (13). Many undefined mechanisms may be involved, and the effects of PAK1 on the

    apoptosis of human NSCLC cells have to be thoroughly investigated.

    It is known that PAK1 activity is dependent on phosphorylation of Ser/Thr residues

    (9). The role of phosphorylation at Tyr residues in the regulation of PAK1 function has not

    been fully elucidated compared to phosphorylation at Ser/Thr sites. Prolactin-activated JAK2

    phosphorylates the three Tyr residues in PAK1 and subsequently increases PAK1 kinase

    activity in Nb2 cells (14). In addition to JAK2, the non-receptor tyrosine kinase ETK/Bmx, a

    member of the Tec family, can interact with PAK1, leading to the phosphorylation and

    activation of PAK1 (15). Furthermore, PAK1 can form multifactor complexes with several

    proteins, including Shc, Grb2, and Nck, and mediates specific responses governed by

    signaling induced by Epithelial growth factor receptor (EGFR), a well-known receptor Tyr

    kinase (16). Altogether, these data serves as the critical evidence demonstrating that the

    modulation of PAK1 activity by Tyr phosphorylation may play a significant role in the

    control of cellular responses to various stimuli.

    JAK2 is a Tyr kinase, a Janus kinase family member. JAKs are well-known to

    activate STAT signaling, leading to regulation of the gene expression that influences

    processes associated with tumor malignancy including cell cycle progression, angiogenesis,

    metastasis, and immune evasion (17, 18). Many investigations have demonstrated that JAKs

    and STATs are highly activated in cases of lung cancer and implied that JAKs as upstream

    regulators can be promising targets for lung cancer therapy (19, 20). For this, small-molecule

    compounds targeting JAKs and their signaling activity have been currently developed as

    chemotherapeutic reagents. However, only a few studies have reported a relationship JAKs

    and radiation-associated signaling. It has been demonstrated that the induction of c-jun

    expression in irradiated cells requires the activation of JAK3, but is impervious to the

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  • activation status of JAK1 and JAK2 (21). A recent study using TG101209, a small-molecule

    inhibitor of JAK2, found that JAK2 inhibition could be a promising strategy for lung cancer

    treatment (22). Therefore, it is important to define the role of the JAK2 pathway in irradiated

    solid tumors, since better therapies are desperately needed to treat these malignancies.

    The purpose of the present study was to elucidate the mechanisms and key molecules

    that confer radioresistance in NSCLC cells. We demonstrated that IR-dependent

    phosphorylation of PAK1 on Tyr residues by JAK2 and its subsequently increased stability

    play an important role in survival signal transduction in NSCLC cells. Our findings provide a

    possible explanation of how NSCLC cells can acquire and regulate resistance to radiation.

    Furthermore, we suggest that JAK2 and PAK1 could be attractive pharmaceutical targets for

    overcoming radioresistance of NSCLC and ultimately contribute to the effective radiation

    treatment of lung cancer.

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  • Materials and Methods

    Chemicals, antibodies and reagents

    Chemicals, antibodies, and reagents used are described in Supplementary Materials

    and Methods.

    Cell lines, cell culture, irradiation and drug treatment

    A549 and NCI-H460 cells were acquired from the American Type Culture Collection

    (ATCC, Manassas, VA), authenticated, and maintained in early passages, no more than 6

    months after receipt from ATCC. Cells were grown in RPMI-1640 medium supplemented

    with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin at 37C in 95% air/5%

    CO2. The cells were exposed to a single dose of -rays using a Gamma Cell-40 Exactor

    (Nordion International, Inc., Kanata, Ontario, Canada) at a dose rate of 0.81 Gy/min. Flasks

    containing the control cells were placed in the irradiation chamber but not exposed to

    radiation. The cells were treated with the indicated drug dissolved in dimethyl sulfoxide

    (DMSO) for 4 h.

    Northern blot analysis

    Northern blot analysis was performed as described previously (23). The detailed

    conditions and primers used for mRNA expression are listed in Supplementary Materials and

    Methods.

    Western blot analysis, immunoprecipitation (IP), and in vivo kinase assay

    Following the experimental treatment, Western blot analysis, IP, and in vivo kinase

    assay studies were performed as previously described (4, 6, 23). Detailed procedures are

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  • described in Supplementary Materials and Methods.

    Transient transfection

    For transient transfection, cells were plated at a density of 5 105 cells in 6-well

    dishes and incubated for 4 h. The cells were transiently transfected with the indicated plasmid

    using Lipofectin (Invitrogen, Carlsbad, CA) or the siRNA oligonucleotides (10 nM) targeting

    EGFR, c-Abl, JAK1, JAK2, and JAK3 using DharmaFECT 1 (Dharmacon, Chicago, IL)

    according to the manufacturers instructions.

    Interactome analysis

    For interactome analysis, cells were transfected with Myc-PAK1 and harvested after

    being irradiated (4 Gy, 2 h). Nuclear extract was then used for double immunoaffinity

    purification of PAK1-containing complexes as previously described (24). Detailed

    procedures are summarized in Supplementary Materials and Methods.

    Luciferase reporter gene assay

    Following co-transfection with E-cadherin (E-cad-Luc) or occludin Luciferase

    reporter gene (Occlu-Luc) and specific combination of plasmids as indicated, the medium

    was changed and the cells were treated with siRNA, drug and/or irradiation. After 1 h, the

    cells were washed twice with cold PBS and lysed in reporter lysis buffer (Promega, Madison,

    WI). After vortexing the lysates and centrifugation at 12,000 g for 1 min at 4C, the cell

    extract and luciferase assay reagent (Promega) were mixed at room temperature, and placed

    in a luminometer (AutoLumat LB 953, EG & G Berthold, Bad Widbad, Germany) to measure

    luciferase activity.

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  • Cell assay (3D culture) and immunofluorescence (IF) staining

    The 3D acini culture was conducted to detect morphologic changes of the epithelial

    acini as previously described (25). Detailed conditions are described in Supplementary

    Materials and Methods.

    Transwell cell migration assay

    A cell migration assay was performed using a 24-well Transwell chamber (Corning,

    Corning, NY). Cells (1 104 in serum-free RPMI-1640 medium) cultured with or without

    specific treatments (transfection, IR and/or drugs) for 60 h (A549) or 72 h (NCI-H460) were

    seeded in the upper chamber of a 5 m pore-size insert. The lower chamber was filled with

    RPMI-1640 medium containing 2% FBS. After 6 h, the upper membrane surface was wiped

    with a cotton swab to remove cells that had not migrated into the lower chamber. Migrated

    cells attached to the lower membrane surface were fixed with 4% paraformaldehyde, stained

    with hematoxylin, and counted. The migration index was calculated and normalized to the

    number of untreated cells that had migrated and the results were based on the relative number

    of cells in a randomly selected field from three representative experiments.

    Wound healing assay

    Cell monolayers were scratched using a 200 L pipette tip after the cells had reached

    70% confluency in RPMI-1640 medium supplemented with 1% FBS. The cells were further

    incubated with fresh medium with or without treatment for 18 h and 30 h (A549) or 24 h and

    48 h (NCI-H460). Photomicrographs were then taken at 100 magnification with an Olympus

    IX71 fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

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  • Real-time quantitative RT-PCR (qRT-PCR)

    The level of EMT-related gene expression was measured using qRT-PCR as

    previously described (23). The detailed conditions and primers used for mRNA expression

    are listed in Supplementary Materials and Methods including Table S1.

    Tumor xenografts in nude mice

    Six-week-old male BALB/c athymic nude mice (Central Lab Animals Inc., Seoul,

    South Korea) were injected with 2 106 A549 or NCI-H460 cells into the flank and tumors

    were allowed to develop. Upon identification of a palpable tumor (minimal volume of 200

    mm3), DMSO or drug (200 g/kg body weight) was administered intraperitoneally every day

    for 25 d. The animals were also irradiated with 10 Gy once a week for 3 wk. Tumor length (L)

    and width (W) were measured with a caliper and tumor volumes were calculated with the

    formula (L W2)/2. At the end of the treatment period, the animals were euthanized and the

    tumors were used for biochemical studies. Animal care protocol is detailed in Supplementary

    Materials and Methods.

    Statistical analysis

    All numeric data are presented as the mean SD or SEM from at least three

    independent experiments. The results were analyzed using a one-way ANOVA for ranked

    data followed by Tukey's honestly significant difference test, and the two-way ANOVA for

    ranked data followed by a Bonferroni post test. Prism 4 software (GraphPad Software,

    SanDiego, CA) was used to conduct all statistical analyses. A p-value < 0.05 was considered

    to be statistically significant.

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  • Results

    IR induces overexpression, Tyr phosphorylation, and nuclear localization of PAK1 in

    NSCLC cells

    Nuclear and cytoplasmic PAK1 is strongly expressed in squamous NSCLC cells (13).

    Based on this information and our previous transcriptome analysis (8), we measured the

    expression of PAK1 in irradiated A549 and NCI-H460 cells in the present study. As shown in

    Fig. 1A and B, IR-exposure of A549 and NCI-H460 cells dramatically induced both mRNA

    and protein expression of PAK1 in a dose-dependent manner. Next, IR-dependent molecular

    modification of PAK1 was investigated since PAK1 was known to be phosphorylated for its

    activity. In both NSCLC cells, IR-induced PAK1 phosphorylation on Tyr sites was observed,

    whereas phosphorylation at Ser/Thr sites was not detected (Fig. 1C and Supplementary Fig.

    S1A). Based on the literature and 3-dimensional structure of PAK1 (PDB ID: 1F3M), to

    further determine which residues are the targets of Tyr kinase in PAK1, we used each mutant

    (Y153F, Y201F, Y295F, or Y3F) as a substrate for an in vivo kinase assay (14, 26). Tyr

    phosphorylation was reduced in Y153F, Y201F, or Y285F mutants of PAK1, and completely

    eliminated from the PAK1 Y3F (Y153F/Y201F/Y285F) mutant (Fig. 1D and Supplementary

    Fig. S1B). These findings indicated that Y153, Y201, and Y285 of PAK1 are required for Tyr

    phosphorylation in irradiated A549 and NCI-H460 cells. It has been reported that PAK1

    contains nuclear localizing signal sequences and signal-dependent nuclear localization is

    required for transcriptional modulation of PAK1 (27, 28). To determine whether IR can

    change the subcellular localization of PAK1, the protein levels of PAK1 in the cytoplasmic or

    nuclear region were measured in irradiated A549 and NCI-H460 cells. As shown in Fig. 1E,

    IR increased the amount of nuclear PAK1 in a time-dependent manner. To further examine

    whether IR-induced Tyr phosphorylation affects the nuclear localization of PAK1, we

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  • conducted an IP assay of the cytoplasmic or nuclear fraction from irradiated A549 and NCI-

    H460 cells after transfection with HA-PAK1 (WT or Y3F). We found that IR-induced nuclear

    localization of the PAK1 Y3F mutant was significantly inhibited compared to that of PAK1

    WT (Fig. 1F). Collectively, these data suggest that IR induces overexpression, Tyr

    phosphorylation, and, subsequently, nuclear localization of PAK1.

    JAK2 phosphorylates PAK1 in irradiated NSCLC cells

    Since we found that IR induced the phosphorylation of PAK1 on Tyr residues in

    NSCLC cells, we further examined upstream kinases of PAK1. SiRNA specific for each Tyr

    kinase (EGFR, c-Abl, JAK1, JAK2, and JAK3) was prepared and an in vivo kinase assay was

    performed on the cells expressing these siRNA molecules (Fig. 2A, inset). Inhibition of JAK2

    expression led to complete abolishment of PAK1 phosphorylation in irradiated A549 and

    NCI-H460 cells; JAK2 was therefore selected as a candidate for upstream kinase of PAK1

    (Fig. 2A). We next assessed the interaction between JAK2 and PAK1 in both irradiated

    NSCLC cells. It was observed that JAK2 was activated by irradiation and interacted with

    PAK1 in cytoplasm (Fig. 2B and Supplementary Fig. S2). Moreover, PAK1 was

    phosphorylated at Tyr sites by JAK2 WT but not by JAK2 kinase-dead (KD) after IR

    exposure (Fig. 2C). To further investigate whether the Y153, Y201, and Y285

    phosphorylation sites of PAK1 are targets of JAK2, an in vivo kinase assay was performed

    with the PAK1 Y3F mutant. As shown in Fig. 2D, JAK2-induced PAK1 phosphorylation was

    eliminated by the Y3F mutation in PAK1, indicating that the suggested three residues of

    PAK1 are required for JAK2-mediated phosphorylation in irradiated A549 and NCI-H460

    cells. Direct phosphorylation of PAK1 by JAK2 was confirmed by treating A549 and NCI-

    H460 cells with two JAK2-specific inhibitors, TG101209 and TG101348. IR-induced PAK1

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  • phosphorylation by JAK2 was significantly diminished following treatment with TG101209

    and TG101348, but not AG1478 (an EGFR inhibitor) or STI-571 (a c-Abl inhibitor) (Fig. 2E).

    Therefore, we propose that IR-activated PAK1 phosphorylation at Tyr residues is directly

    mediated by JAK2 in NSCLC cells.

    Tyr phosphorylation is required for maintaining PAK1 protein stability in irradiated

    NSCLC cells

    To explore the biological effects of PAK1 Tyr phosphorylation, both HA-PAK1 WT

    and HA-PAK1 Y3F were expressed in NSCLC cells by transient transfection. As shown in

    Fig. 3A, HA-PAK1 Y3F was expressed at a lower level under IR compared to the WT

    construct. Treatment with proteasome inhibitors (PI) restored the mutant protein level,

    indicating that Tyr phosphorylation of PAK1 modulates protein stability. Indeed, the

    estimated half-life of PAK1 WT (12 h) was significantly longer than that of the Y3F mutant

    (4 h; Fig. 3B). These results indicate that cellular PAK1 is subject to proteasome-dependent

    degradation and that phosphorylation on Tyr residues prevents PAK1 degradation. In line

    with these findings, proteasome inhibitors caused a more pronounced accumulation of poly-

    ubiquitinated PAK1 Y3F than PAK1 WT (Fig. 3C). Previously, we observed that PAK1 is

    phosphorylated by JAK2 after irradiation (Fig. 2). To test whether the same mechanism also

    mediates the poly-ubiquitination that is associated with PAK1 Y3F, JAK2 activation was

    blocked by a JAK2-specific inhibitor. The results showed that JAK2 inhibition did increase

    the level of poly-ubiquitinated PAK1 WT, indicating that the protein stability of PAK1 is

    controlled by a signaling pathway that is dependent on Tyr phosphorylation of PAK1 induced

    by JAK2 after irradiation in NSCLC cells (Fig. 3D). According to these data, we propose that

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  • IR-activated PAK1 Tyr phosphorylation by JAK2 is directly related to PAK1 protein

    stabilization in NSCLC cells.

    PAK1 interacts with Snail in irradiated NSCLC cells

    Since we found that PAK1 was phosphorylated by IR-activated JAK2 and localized

    to the nucleus, we further set out to identify a PAK1-interacting proteome in irradiated

    NSCLC cells to elucidate the molecular functions of PAK1 in the nucleus. The strategy used

    to identify nuclear proteins associated with nuclear PAK1 is presented in Fig. 4A. Nuclear

    extracts were prepared from A549 cells cultured with IR and soluble nuclear protein

    complexes were separated to reduce sample complexity. Complexes containing PAK1 were

    purified using double immunoaffinity purification, and the identity of PAK1 interactors was

    determined by repeated rounds of Peptide mass fingerprint (PMF) (29). As shown in Fig. 4B,

    several IR-dependent PAK1 interacting proteins were identified with high confidence. Based

    on information from published databases of physical and functional interactions, we focused

    on Snail (indicated band (a) in Fig. 4B), a key regulator of the epithelial-mesenchymal

    transition (EMT) (30). We subsequently confirmed the interaction between PAK1 and Snail

    (Fig. 4C and Fig. 4D). Unlike PAK1 Y3F, PAK1 WT interacted with Snail, suggesting that

    the interaction of PAK1 with Snail in irradiated NSCLC cells is phosphorylation-dependent.

    A previous report has shown that the phosphorylation of Snail on S246 by PAK1 is essential

    for Snail function by promoting transcriptional repression of several genes, including

    epithelial marker proteins (E-cadherin), in breast cancer cell lines (30). As shown in Fig. 4E,

    PAK1-mediated Snail phosphorylation was detected and eliminated by an S246A mutation in

    Snail, indicating that S246 of Snail is required for PAK1-mediated phosphorylation in

    irradiated NSCLC cells. To explore a potential effect of PAK1 signaling on the functions of

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  • Snail, we next examined the influence of PAK1 on the ability of Snail to repress E-cadherin

    promoter activity. Our results demonstrated that IR-activated PAK1 had an inhibitory effect

    on E-cadherin promoter activity (Fig. 4F). We next showed that inhibition of the PAK1

    pathway by PAK1-specific siRNA resulted in increased transcriptional activity of the E-

    cadherin promoter in irradiated NSCLC cells. The involvement of phosphorylated PAK1 in

    the ability of Snail to repress E-cadherin promoter activity was confirmed by treatment with a

    JAK2-specific inhibitor, TG101209 (Fig. 4F). To support the generality of our findings, we

    assessed whether PAK1 activity affects another Snail-regulated gene, Occludin (31). It was

    observed that IR-activated PAK1 also promoted the Snail-induced transcriptional repression

    of Occludin-promoter reporter constructs (Fig. 4F). According to these results, we propose

    that IR-activated PAK1 directly interacts with Snail and regulates transcriptional repression

    activity of Snail by Ser phosphorylation in NSCLC cells.

    Inhibition of Tyr phosphorylation of PAK1 reduces IR-dependent EMT in NSCLC cells

    Several lines of evidence have demonstrated the critical role of PAK1 and Snail in

    the EMT (30, 32). To explore a potential role for PAK1 phosphorylation in Snail-mediated

    EMT and the effect of a JAK2 inhibitor as an EMT suppressor, we monitored morphologic

    changes of NSCLC cells. A549 and NCI-H460 cells were grown on a thick layer of Matrigel

    to form epithelial acini (3D culture model). This system has been used extensively to identify

    morphological changes that can perturb the normal cancer growth architecture (33, 34). In

    this 3D culture model, irradiated NSCLC cells were distinguishable from the control cells and

    formed an increased number of acini that invaded the Matrigel-containing 3D matrix.

    Interestingly, the observed morphological changes of the epithelial acini were abrogated by

    the addition of TG101209, indicating that the inhibition of PAK1 Tyr phosphorylation

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  • reduced IR-dependent EMT in the NSCLC cells (Fig. 5A). Next, we measured the migration

    capacity and EMT marker expression of NSCLC cells treated with TG101209. NSCLC cells

    treated with the JAK2 inhibitor exhibited reduction of motility as determined by both

    Transwell cell migration and wound healing assays (Fig. 5B and C). Rhamnetin and cirsiliol

    were administered as positive controls of EMT inhibitors (35). TG101209 treatment

    alleviated IR-induced EMT by increasing the expression of E-cadherin (an epithelial marker)

    while decreasing the expression of Vimentin and Fibronectin (two mesenchymal markers) at

    both mRNA and protein levels (Fig. 5D and E). Reduced EMT was also observed in the

    NSCLC cells in which PAK1 expression was knocked down. Taken together, these results

    suggest that the inhibition of PAK1 activation in NSCLC cells suppresses the EMT.

    JAK2-inhibitor increases in vivo radiosensitization and decreases in vivo EMT in a

    xenograft mouse model

    In our wound healing assay, we observed that TG101209 treatment prevented not

    only the EMT but also the proliferation of IR-activated NSCLC cells (Fig. 5C). To evaluate

    the combined effects of JAK2 inhibition with IR on tumor growth in vivo, a xenograft mouse

    model was established (Fig. 6A). In vivo data from nude mice bearing tumors formed by

    A549 and NCI-H460 cells indicated that TG101209 had an in vivo radiosensitization effect

    (Fig. 6B). Tumor volumes of the mice treated with IR and TG101209 were significantly

    reduced by approximately 57% (the group with tumors formed by A549 cells) and 50% (the

    group injected with NCI-H460 cells) on day 25 compared to mice receiving radiation alone.

    Moreover, IR-induced expression of PAK1 and EMT-related proteins was significantly

    reduced in the extracted tumor tissue lysates when TG101209 was administered in the mice

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  • (Fig. 6C). Thus, we suggest that the JAK2 inhibitor significantly increased in vivo

    radiosensitization while inhibiting the EMT.

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  • Discussion

    Many researchers have focused on developing potent radiosensitizers to overcome

    radioresistance and to increase the therapeutic efficacy in NSCLC patients. Given the

    inherent insensitivity to cytotoxic agents, identifying molecules that drive the growth,

    survival and metastasis of lung cancer cells is critical for the development of novel

    therapeutic reagents. In the same context with identification of the biomarkers, we found that

    JAK2 and PAK1 are promising pharmacological targets for the radiosensitization of NSCLC

    cells because Tyr phosphorylation of PAK1 by JAK2 was shown to prevent cell death and to

    promote cell invasion. In the present study, PAK1 mRNA and protein expression was up-

    regulated, and the protein localized to the nucleus in irradiated NSCLC cells. In addition to

    nuclear localization, the stability of PAK1 protein increased through IR-dependent Tyr

    phosphorylation of PAK1 by JAK2. We also found that activation of the JAK2/PAK1/Snail

    pathway by IR was important for radioresistance primarily via EMT induction (Fig. 7). Based

    on these findings, we suggest that JAK2 can be a potent target as an upstream factor for Tyr

    phosphorylation of PAK1 associated with radioresistant signaling in NSCLC cells.

    PAK1 has an autoregulatory region (including a p21-binding domain, dimerization

    segment, and inhibitory switch domain) and several motifs for interaction with adaptor

    proteins (Nck, Grb2, and PIX) at the N-terminus, and a catalytic domain at the C-terminus

    (26). PAK1 activity is known to be inhibited by the formation of a resting homodimer

    through interaction of the inhibitory switch domain (26). The inhibitory PAK1 homodimer

    can be disrupted and rearranged by interaction with activated Cdc42/Rac1 (26, 36).

    Monomeric PAK1 can then undergo autophosphorylation at several residues including S57,

    S144, S149, S199/S203, and T423, leading to PAK1 activation (26, 36-38). In the present

    investigation, we could only observe PAK1 phosphorylation on Tyr residues and not Ser/Thr

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  • residues in response to irradiation (Fig. 1). According to our data and those from several

    studies, we assumed that Tyr phosphorylation of PAK1 could provide additional properties

    compared to Ser/Thr phosphorylation. First of all, PAK1 phosphorylation on three Tyr

    residues, especially Y201, might sufficiently disrupt the inhibitory conformation similar to

    phosphorylation at S199/S203. Additionally, maintenance of the PAK1 active conformation

    could be achieved, at least partially, by phosphorylation at Y153 since this residue also

    resides nearby the autophosphorylation sites, S144 and S149 (especially S144 is responsible

    for maximal kinase activity of PAK1) (39). Phosphorylation at Y285 could also affect kinase

    activity since this residue is present in the N-lobe of the C-terminal catalytic domain. A

    previous structural study revealed that PAK1 could form an active conformation through an

    interaction between K299 at the N-lobe, E315 at the C-helix, and ATP, leading to exposure

    to a substrate binding site and phosphorylation of Ser/Thr residues of substrates (40). Based

    on the location of Y285 at the ATP binding site, we expect that phosphorylation of this

    residue might help stabilize the interaction between K299 and an ATP molecule. Although it

    should be confirmed by further structural studies, phosphorylation of PAK1 at three Tyr

    residues might directly or indirectly help PAK1 to form and maintain its catalytically active

    conformation, resulting in the phosphorylation of downstream substrates in response to

    irradiation.

    We observed that Tyr phosphorylation of PAK1 was mediated by JAK2, a Tyr kinase

    (Fig. 2). Most substrates bind to Tyr kinases through their SH2 domains. According to

    previous structural studies, PAK1 does not contain an SH2 domain but has a proline-rich

    motif for binding to SH3 domains (26, 36, 37). This structural information implies that PAK1

    could be phosphorylated on Tyr residues by JAK2 through interaction with SH2/SH3-

    containing adaptor proteins that provide a bridge for facilitating spatial proximity between

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  • JAK2 primarily in near membrane and PAK1 in the cytoplasm. PAK1 has several known

    adaptor proteins including Nck, Grb2, and PIX (41, 42). These proteins enable membrane

    localization of PAK1 to be activated by other kinases including JAK2, Cdc42, and Rac1 (41,

    43). Based on previous studies, we believed that Nck might be involved in JAK2-mediated

    PAK1 Tyr phosphorylation in response to irradiation (28, 41). Nck shuttles PAK1 from the

    cytoplasm to the plasma membrane. After membrane localization, PAK1 would be

    susceptible to binding to JAK2 and, subsequently activated PAK1 would dissociate from Nck.

    PAK1 activated by JAK2 could be therefore released from the plasma membrane into the

    cytoplasm and even translocate into the nuclear. In this study, we observed that activated

    PAK1 affected Snail-dependent cell migration in the nucleus (Fig. 4 and 5).

    In addition to activating PAK1, we showed that Tyr phosphorylation increased PAK1

    stability as well (Fig. 3). To understand how PAK1 stabilization is mediated by Tyr

    phosphorylation, it was important to determine which E3 ligase is responsible for PAK1

    degradation. Although a PAK1-specific E3 ligase has not been identified, Cdc42 homologous

    protein (Chp) is a Rho-family GTPase that mediates ubiquitination-dependent degradation of

    PAK1 (44). A previous report indicated that kinase activity of PAK1 is critical for PAK1

    degradation (44). According to this study, Chp could mediate PAK1 degradation only when

    PAK1 was activated by autophosphorylation on several Ser/Thr residues including S57, S144,

    S149, S199/S203, and T423. Alanine substitutions at these sites led to increased PAK1

    stability. Thus, we believe that Y201 phosphorylation of PAK1 might have structurally

    similar property of phosphorylated moiety as phosphorylation of S199/S203, expecting

    PAK1 destabilization. However, PAK1 was stabilized after being Tyr-phosphorylated as

    shown in Fig. 3. One possible explanation is that Chp might not recognize Tyr-

    phosphorylated PAK1 due to different autophosphorylation patterns. Thus, JAK2-induced

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  • Tyr-phosphorylation, especially at Y201, increased PAK1 activity and stability instead of

    promoting PAK1 degradation. Our data are supported by findings from other investigations

    demonstrating that protein degradation could be inhibited by phosphorylation at Tyr residues

    (45, 46). For example, ubiquitination and degradation were more rapid for a mutant form of

    c-Kit (Y823F) than in c-Kit WT (45). In this case, mutant c-Kit-mediated downstream

    pathways involving Akt and Erk were negatively affected, leading to the suppression of cell

    survival and proliferation. However, the Y823F mutation has no effect on kinase activity of

    c-Kit. Another study showed that proliferating cell nuclear antigen (PCNA) was stabilized by

    phosphorylation on Y211, leading to increased functional activity on DNA replication (46).

    We showed that PAK1 without Tyr phosphorylation is susceptible to proteasome-dependent

    degradation in Fig. 3. Similar to c-Kit and PCNA, phosphorylation on Tyr residues is

    responsible for maintaining PAK1 stability and activity, leading to cell proliferation and

    invasion induced by Snail phosphorylation.

    Several investigations conducted by Diakonova et al. have suggested that Tyr-

    phosphorylated PAK1 could be crucially involved in the regulation of cancer cell migration

    (14, 28, 47). Based on the mechanisms underlying EMT regulation, we focused on a nuclear

    factor as a downstream target of PAK1 that could confer radioresistant properties on NSCLC

    cells. We found that Snail interacted with PAK1 in the nucleus according to our interactome

    analysis in Fig. 4. Although this interaction was observed in the cytoplasm as well, it is

    unclear whether PAK1 and Snail translocated into the nucleus together or individually.

    Nevertheless, the interaction between PAK1 and Snail could be responsible for EMT

    induction through the suppression of E-cadherin expression in irradiated NSCLC cells. This

    finding could be supported by a previous study demonstrating that Snail phosphorylation at

    S246 by PAK1 led to nuclear accumulation of Snail and promotes cell invasiveness (30).

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  • Additionally, constitutively active PAK1 (T423E mutant) enhanced Snail activation

    compared to wild-type PAK1. T423 of PAK1 is located in the catalytic domain necessary for

    kinase activity. As we mentioned above, phosphorylation of PAK1 on three Tyr residues

    might have similar effects on kinase activity due to exposure of the catalytic domain through

    phosphorylation of residues in the inhibitory switch domain. In this study, even when

    phosphorylation at Ser/Thr residues (such as T423) of PAK1 was not detected, PAK1 could

    interact with Snail in response to irradiation, consequently leading to increasing cell motility

    through reduced E-cadherin expression.

    In addition to regulating cell motility, Snail also promotes cell survival and

    proliferation. Snail regulates the expression of several genes mainly involved in cell invasion

    and migration including matrix metalloproteinase (MMP) family members (MMP1, MMP2,

    and MMP7) and cell-cell or cell-matrix interacting proteins (E-cadherin, integrin 6, and

    tissue inhibitor of metalloproteinase 3) (48). Furthermore, this factor is responsible for

    suppressing the expression of other genes including caspase-8 and tumor suppressor CYLD

    (49). It has been reported that CYLD could be repressed in a Snail-dependent manner

    resulting in increased expression of cyclin D1 and N-cadherin, thus enhancing the

    proliferation and migration of melanoma cells (49). These findings could explain, at least in

    part, the hyper-growth of cells (especially NCI-H460 cells at 48 h) treated with irradiation

    alone compared to that of cells treated with irradiation and each drug observed in the present

    study (Fig. 5C). Taken together, our findings and data from the literature indicate that PAK1-

    activated Snail might directly or indirectly confer radioresistance on NSCLC cells by

    inducing EMT and cell proliferation.

    The exact molecular mechanism underlying radioresistance in NSCLC has been still

    largely elusive. In the present study, we demonstrated that Tyr phosphorylation of PAK1

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  • could be a critical factor for conferring radioresistance. We have provided the first evidence

    of a novel regulatory mechanism of radioresistance including the functional involvement of

    JAK2, PAK1, and Snail in irradiated NSCLC cells (Fig. 7). We also propose that JAK2

    inhibitors might be potential radiosensitizers that suppress Tyr phosphorylation of PAK1. In

    this study, our results demonstrated that regulation of Tyr phosphorylation of PAK1 with

    pharmacological agents in combination with radiotherapy could overcome radioresistance

    and eventually enhance the efficiency of radiotherapy for treating NSCLC.

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  • Authors Contributions

    Conception and design: E. Kim, B. Youn

    Development of methodology: E. Kim, H. Youn, W. Kim, B. Youn

    Acquisition of data (provided animals, acquired and managed patients, provided facilities,

    etc.): E. Kim, H. Youn, T. Kwon, B. Son, J. Kang, H.J. Yang, K.M. Seong

    Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

    analysis): E. Kim, H. Youn, H.J. Yang, K.M. Seong, W. Kim, B. Youn

    Writing, review, and/or revision of the manuscript: E. Kim, W. Kim, B. Youn

    Administrative, technical, or material support (i.e., reporting or organizing data,

    constructing databases): W. Kim, B. Youn

    Study supervision: B. Youn

    Acknowledgements

    The authors thank Dr. Maria Diakonova (University of Toledo, Toledo, OH) for

    providing the mutant PAK1 constructs.

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  • Figure legends

    Figure 1. IR induces overexpression, Tyr phosphorylation, and nuclear localization of

    PAK1 in NSCLC cells. A, IR-induced PAK1 mRNA expression was detected by Northern

    blot analysis. After irradiation (0, 2, 4, or 6 Gy), the cells were harvested and total RNA was

    extracted. Total RNA was analyzed using an [-32P]-labeled PAK1 cDNA probe. GAPDH was

    used for normalization. B, IR-induced PAK1 protein expression was detected by Western blot

    analysis. C, IR-induced PAK1 phosphorylation was verified by an in vivo kinase assay. After

    irradiation (4 Gy) for 0, 1, 2, or 3 h, the cells were harvested and cell lysates were subjected

    to an IP assay with an anti-PAK1 antibody followed by Western blot analysis for pSer/Thr

    and pTyr. D, IR-induced PAK1 phosphorylation at three Tyr residues (Y153, Y201, and Y285)

    was confirmed by an in vivo kinase assay using PAK1 Y153F, Y201F, Y285F, and Y3F

    mutants (WCL, whole cell lysates). Cells were harvested after irradiation (4 Gy) for 2 h and

    cell lysates were immunoprecipitated with an anti-HA antibody and analyzed by Western blot

    analysis for pTyr. E, IR-induced translocation of PAK1 from the cytoplasm into the nucleus

    was assayed by Western blot analysis after cytoplasmic or nuclear fractionation (CE,

    cytoplasmic extract; NE, nuclear extract). Tubulin and Lamin A/C were used as markers for

    the CE and NE, respectively. F, Involvement of IR-activated nuclear localization and

    phosphorylation of PAK1 was determined by an IP assay and Western blot analysis. After

    irradiation, the cells transiently transfected with PAK1 (WT or Y3F mutant) were harvested

    to conduct cytoplasmic or nuclear fractionation. Each extract was immunoprecipitated with

    an anti-HA antibody and detected by Western blot analysis using an anti-pTyr antibody.

    Figure 2. JAK2 phosphorylates PAK1 in irradiated NSCLC cells. A, Tyr phosphorylation

    of PAK1 by JAK2 was measured by an in vivo kinase assay using each Tyr kinase-specific

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  • siRNA molecule (EGFR, c-Abl, JAK1, JAK2, and JAK3). The inset shows that siRNA

    oligonucleotides specific for Tyr kinase significantly reduced Tyr kinase expression. B, In

    vivo binding of IR-activated PAK1 with JAK2 was measured with an IP assay. After

    irradiation for 2 h, cell lysates were immunoprecipitated with an anti-FLAG antibody and

    subjected to Western blot analysis using an anti-PAK1 antibody. C, Phosphorylation of PAK1

    by JAK2 was measured with an in vivo kinase assay with a JAK2 KD mutant. After

    irradiation for 2 h, cells transfected with JAK2 WT or KD were harvested and

    immunoprecipitated with an anti-PAK1 antibody followed by Western blot analysis using an

    anti-pTyr antibody. D, JAK2-mediated PAK1 phosphorylation at Y153, Y201, and Y285 was

    confirmed by an in vivo kinase assay with a PAK1 Y3F mutant. Cells were harvested after

    irradiation (4 Gy) for 2 h. Immunoprecipitant of HA (PAK1) was subjected to Western blot

    analysis using an anti-pTyr antibody. E, JAK2-mediated PAK1 phosphorylation was

    reconfirmed using JAK2-specific inhibitors (TG101209 or TG101348), an EGFR inhibitor

    (AG1478), and a c-Abl inhibitor (STI-571). After treatment of each inhibitor and irradiation

    for 2 hr, cells were harvested and were immunoprecipitated with an anti-PAK1 antibody

    followed by Western blot analysis using an anti-pTyr antibody.

    Figure 3. Tyr phosphorylation is required for maintaining PAK1 protein stability in

    irradiated NSCLC cells. A, Effects of Tyr phosphorylation on the protein stability of PAK1

    were measured by Western blot analysis. Ectopic expression of PAK1 WT or the Y3F mutant

    in NSCLC cells with or without IR (4 Gy; 6 h incubation) and PI (a mix of 5 M MG115 and

    5 M MG262, 24 h incubation). B, The half-life of IR-activated PAK1 WT or Y3F mutant

    with cycloheximide (CHX; 50 M) in the presence of PI was estimated by Western blot

    analysis. Cells were treated with PI at different time points after irradiation (0, 4, 8, 12, or 16

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  • h). Cells were harvested and cell lysates were detected by Western blot analysis using an anti-

    HA antibody. The level of HA-PAK1 WT or Y3F mutant protein at each time point measured

    in Fig. 3B was plotted by densitometric analysis. The level of HA-PAK1 WT or Y3F mutant

    expression at time point 0 (lane 3) was set at 100%. C, The accumulation of poly-

    ubiquitinated IR-activated PAK1 Y3F mutant was measured. NSCLC cells expressing PAK1

    WT or Y3F mutant were treated with or without PI as described in Fig. 3A. HA-PAK1 WT or

    Y3F mutant protein was then immunoprecipitated with an anti-HA antibody and Western blot

    analysis was performed with an anti-ubiquitin antibody. D, The direct relationship between

    JAK2-dependent phosphorylation of PAK1 and poly-ubiquitination with irradiation in

    NSCLC was confirmed by the same experiment described in Fig. 3C with a JAK2-specific

    inhibitor, TG101209.

    Figure 4. PAK1 interacts with Snail in the nucleus in irradiated NSCLC cells. A,

    Schematic presentation of the strategy used to identify the PAK1 nuclear interactome in A549

    cells. B, Purified PAK1 complexes in non-irradiated or irradiated A549 cells were separated

    by PAGE and visualized by silver staining. The silver stained gel was analyzed by mass

    spectrometry (peptide mass fingerprint). It was shown that three bands especially increased

    by irradiation. The bands indicated by an arrowhead (a, b, and c) correspond to Snail, ER,

    and FKHR, respectively. C, In vivo binding of IR-activated PAK1 with Snail in the nucleus of

    NSCLC cells was confirmed by an IP assay. After irradiation, the cells transiently transfected

    with PAK1 WT or Y3F mutant were harvested to conduct cytoplasmic or nuclear

    fractionation. Each extract was subjected to an IP assay with an anti-PAK1 antibody followed

    by Western blot analysis for Snail. D, Interaction of endogenous PAK1 with endogenous

    Snail was measured by an IP assay. After irradiation, the cells were harvested to conduct

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  • cytoplasmic or nuclear fractionation. Each extract was subject to an IP assay with an anti-

    PAK1 antibody followed by Western blot analysis using an anti-Snail antibody. E, PAK1-

    mediated Snail phosphorylation at S246 was confirmed by an in vivo kinase assay with a

    Snail S246A mutant. The cells transiently transfected with Snail (WT or S246A mutant) were

    harvested to conduct cytoplasmic or nuclear fractionation. Each extract was subjected to an IP

    assay with an anti-FLAG antibody (Snail) followed by Western blot analysis using an anti-

    pSer/Thr antibody. F, Effect of PAK1 on the ability of Snail to repress E-cadherin (or

    Occludin) promoter activity was assessed with a luciferase assay. Error bars represent SD

    (n = 3); p < 0.05 compared with E-cad (or Occlu)-Luc plasmid-transfected non-irradiated

    cells, p < 0.05 compared with E-cad (or Occlu)-Luc plasmid-transfected irradiated cells

    (two-way ANOVA, Bonferroni post test).

    Figure 5. Inhibition of PAK1 Tyr phosphorylation reduces IR-dependent EMT in

    NSCLC cells. A, Morphological changes of NSCLC cells treated with a JAK2 inhibitor were

    monitored with a 3D culture model. NSCLC cells treated with or without IR and TG101209

    were grown in 3D cultures and fixed after 20 h (A549) or 28 h (NCI-H460). The cells were

    permeabilized and stained for tubulin (green) and with DAPI (blue). B, The inhibitory effects

    of a JAK2 inhibitor on IR-induced migration of NSCLC cells were measured with a

    Transwell migration assay. The results are expressed as fold-increase of migration compared

    to the control group and are based on the relative number of cells in a randomly selected field

    from three representative experiments. Rhamnetin and cirsiliol were used as positive EMT

    inhibitor controls. C, The inhibitory effects of a JAK2 inhibitor on IR-induced migration of

    NSCLC cells were measured with a wound healing assay. After the treatment with each

    inhibitor for 4 h and irradiation, cells were wounded and incubated for 18 h and 30 h (A549)

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  • or 24 h and 48 h (NCI-H460). D, Effects of a JAK2 inhibitor on mRNA expression of E-

    cadherin, Vimentin, and Fibronectin in irradiated NSCLC cells were analyzed by qRT-PCR.

    E, Effects of a JAK2 inhibitor on the protein expression of E-cadherin, Vimentin, and

    Fibronectin in irradiated NSCLC cells were analyzed by Western blot analysis. Error bars

    represent SD (n = 3); *p < 0.05 compared with non-irradiated cells, **p < 0.05 compared

    with irradiated cells (one-way ANOVA, Tukey's honestly significant difference test, or two-

    way ANOVA, Bonferron post test).

    Figure 6. JAK2-inhibitor acts as an in vivo radiosensitizer and decreases in vivo EMT in

    a xenograft mouse model. A, An experimental protocol to determine whether JAK2-

    inhibitor acts as an in vivo radiosensitizer and decreases in vivo EMT in a xenograft mouse

    model. B, The effect of a JAK2 inhibitor on in vivo radiosensitization was measured in a

    xenograft mouse model. C, The in vivo effects of a JAK2 inhibitor on the expression of PAK1

    and EMT-related proteins were evaluated by Western blot analysis. Error bars represent

    SEM (n = 3 with four animals per group); *p < 0.05 compared with tumor volume on day 25

    in mice treated with radiation alone (one-way ANOVA, Tukey's honestly significant

    difference test).

    Figure 7. A schematic diagram illustrates JAK2-pPAK1-pSnail signaling in response to

    irradiation and the radiosensitizing effects of a JAK2 inhibitor in NSCLC cells. PAK1 is

    up-regulated and phosphorylated on Tyr residues by JAK2, consequently leading to

    translocation into the nucleus after radiation exposure. IR-induced Tyr phosphorylation of

    PAK1 is required to maintain PAK1 protein stability in NSCLC cells. After phosphorylation

    on Tyr residues, PAK1 binds with Snail, thus promoting the transcriptional repression

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  • activity of Snail by Ser phosphorylation. Additionally, activation of the JAK2-pPAK1-pSnail

    signaling axis by irradiation promotes the EMT and cell proliferation. Treatment with a JAK2

    inhibitor enhances EMT inhibition and radiosensitization in vitro and in vivo.

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  • Published OnlineFirst August 14, 2014.Cancer Res EunGi Kim, HyeSook Youn, TaeWoo Kwon, et al. cancer cellsepithelial-mesenchymal transition and radioresistance in lung PAK1 tyrosine phosphorylation is required to induce

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