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DNAJB1 negatively regulates MIG6 to promote epidermal growth factor receptor signaling
Biochimica et Biophysica Acta 1853 (2015) 2722–2730
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr
DNAJB1 negatively regulates MIG6 to promote epidermal growth factor receptor signaling Soo-Yeon Park a,1, Hyo-Kyoung Choi b,1, Jae Sung Seo a, Jung-Yoon Yoo a,c, Jae-Wook Jeong c, Youngsok Choi d, Kyung-Chul Choi e,⁎, Ho-Geun Yoon a,⁎ a
Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Republic of Korea Division of Nutrition and Metabolism Research Group, Korea Food Research Institute, Gyeonggi-do, Republic of Korea Department of Obstetrics, Gynecology & Reproductive Biology, Michigan State University College of Human Medicine, MI, USA d Fertility Center of CHA General Hospital, CHA Research Institute, CHA University, Seoul, Republic of Korea e Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 2 February 2015 Received in revised form 28 July 2015 Accepted 30 July 2015 Available online 1 August 2015 Keywords: DNAJB1 MIG6 EGFR Gefitinib Lung cancer
a b s t r a c t Mitogen-inducible gene 6 (MIG6) is a tumor suppressor implicated in the development of human cancers; however, the regulatory mechanisms of MIG6 remain unknown. Here, using a yeast two-hybrid screen, we identified DnaJ homolog subfamily B member I (DNAJB1) as a novel MIG6-interacting protein. We found that DNAJB1 binds to and decreases MIG6 protein, but not mRNA, levels. DNAJB1 overexpression dosage-dependently decreased MIG6 protein levels. Conversely, DNAJB1 knockdown increased MIG6 protein levels. DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. However, knocking-down of DNAJB1 reduced the ubiquitination of MIG6. DNAJB1 positively regulates the epidermal growth factor receptors (EGFR) signaling pathway via destabilization of MIG6; however, DNAJB1 knockdown diminishes activation of EGFR signaling as well as elevation of MIG6. Importantly, the increased levels of MIG6 by DNAJB1 knockdown greatly enhanced the gefitinib sensitivity in A549 cells. Thus, our study provides a new molecular mechanism to regulate EGFR signaling through modulation of MIG6 by DNAJB1 as a negative regulator. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Mitogen-inducible gene 6 (MIG6/RALT/gene33/Errfi1) is a negative regulator of epidermal growth factor receptors (EGFRs) by inhibition of their tyrosine kinase activities [1]. MIG6 also binds to and inhibits cdc42 activity via its CRIB domain, which leads to inhibition of hepatocyte growth factor-induced cell migration [2]. It was reported that MIG6 is downregulated in human tumors, including breast carcinoma, hepatocellular carcinoma, and thyroid cancer [2–4]. MIG6-deficient mice displayed hyperactivation of EGFR and sustained mitogen-activated protein kinase (MAPK) signaling activation [1,5]. Furthermore, MIG6deficient mice develop spontaneous tumors in various organs and are highly susceptible to chemically-induced skin tumors [6]. Strikingly, inhibition of endogenous EGFR signaling using the EGFR small inhibitor gefitinib (Iressa) or replacement of wild-type EGFR with the kinase-deficient protein encoded by the hypomorphic EGFR allele completely rescued skin Abbreviations: DNAJB1, DnaJ homolog subfamily B member I; MIG6, mitogeninducible gene 6; EGFR, epidermal growth factor receptors ⁎ Corresponding authors at: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea. E-mail addresses: [email protected] (K.-C. Choi), [email protected] (H.-G. Yoon). 1 S.Y.P. and H.K.C. contributed equally to this work.
http://dx.doi.org/10.1016/j.bbamcr.2015.07.024 0167-4889/© 2015 Elsevier B.V. All rights reserved.
defects in MIG6 knockout (KO) mice [6]. Recently, MIG6 was shown to act as a tumor suppressor in endometrial cancers associated with PTEN deficiency by inhibiting estrogen signaling in the uterus [7]. Ablation of MIG6 in the murine uterus leads to endometrial hyperplasia and estrogeninduced endometrial cancer [8]. More recently, it has been shown that MIG6 mediates progesterone action and inhibits ERK1/2 signaling to suppress endometrial cancer development and progression that is associated with PTEN deficiency [9]. These demonstrate that the tumor suppressor MIG6 is a specific negative regulator of EGFR. MIG6 is induced as an immediate early response gene by a variety of stimuli, including growth factors, fetal calf serum, insulin, hypoxia, and stress [10]. It has been also reported that various stressors induce MIG6 transcription [11–14]. Moreover, MIG6 is induced by EGF treatment and functions as a feedback inhibitor of EGFR signaling, regulating receptor activation and stability [1]. Therefore, MIG6 induction is a critical step for cells to sense external stimuli. Although MIG6 clearly acts as a tumor suppressor and its downregulation correlates with human cancer development, the molecular mechanisms regarding how MIG6 levels are regulated remain unknown. The dynamic exchange of multiple chaperone proteins during cellular stress events occurs in important cellular reactions including DNA replication, protein degradation and translocation, cell signaling, and apoptosis [15]. When cells are exposed to environmental stress, such
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as heat shock, alcohol, or oxidative stress, heat shock proteins (HSPs) play a role by regulating the correct folding, degradation, localization, and function of target proteins [16,17]. HSPs are classified based on their relative molecular weight. There are five major HSP proteins: HSP90, HSP70, HSP60, HSP40, and small heat shock proteins [18]. DNAJB1 (also called hDj-1) is a member of the HSP40 protein family [19] and interacts with multiple proteins in the cytoplasm. For example, DNAJB1 interacts with heat HSP70 and induces its ATPase activity, which stimulates the association between HSP70 and Hsc70interacting protein (HIP) [20]. DNAJB1 interacts with Hsc70 to sequester p53 in the cytosol [21]. Moreover, the HSP70-DNAJB1 chaperone was found to prevent nitric oxide-induced apoptosis in both macrophages and COS-7 cells [22,23]. In accordance with these findings, we recently reported that DNAJB1 binds to and destabilizes PDCD5 (programmed cell death 5), ultimately leading to suppression of p53-mediated apoptosis [24]. Collectively, these suggest that DNAJB1 may regulate the stability of various proteins in the cytoplasm to maintain cellular homeostasis. In this study, we found that DNAJB1 binds to and destabilizes the MIG6 protein by enhancing ubiquitin-dependent proteasomal degradation of MIG6, which positively affects the EGFR signaling pathway. Moreover, we provide evidence that DNAJB1 suppresses MIG6 stabilization to enhance lung cancer cell proliferation. We identified DNAJB1 as a negative regulator of MIG6 function and propose that the DNAJB1-MIG6 regulatory network plays an important role in the growth of lung cancer cells. 2. Materials and methods 2.1. Cell culture and reagents Human lung adenocarcinoma cell line A549, human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis. These cells were used within 6 months of purchase. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Hyclone, Logan, UT, USA) at 37 °C under 5% CO2. MG132 and cycloheximide (CHX) were purchased from SigmaAldrich (St. Louis, MO, USA). MG132 and CHX were prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). EGF was purchased from ATGen (Seongnam-si, Gyeonggi-do, Korea) and prepared in 1× PBS. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA) and prepared in DMSO (Sigma-Aldrich). Lipofectamine 2000 was used for overexpression constructs and Lipofectamine RNAi Max was used for siRNA (Life Technologies, Grand Island, NY, USA). 2.2. Plasmids and small-interfering RNAs (siRNAs) Wild-type, full-length MIG6 and DNAJB1 constructs were generated by PCR and cloned into the pSG5-KF2M1-FLAG, -Myc, or -HA (SigmaAldrich), and pGEX4T-1 plasmid vectors (GE healthcare, Piscataway, NJ, USA). All plasmid constructs were verified by DNA sequencing. For siRNA transfection, cells were plated at 60–70% confluency and transfected using Lipofectamine RNAi MAX (Life Technologies) with 20 pM siRNA following the manufacturer's protocol. After 4 h, the media was changed, and cells were incubated for 2 days and treated with indicating reagents. Sequences of siRNAs; Sense 5′-CCUCGUGCCG UUCCAUCAGGUAGUU-3′, Antisense 5′-CUACCUGAUGGAACGGCACG AGGUU-3′ (Negative Control); Sense 5′-CUUUCGUACUGCUGAAUG UUU-3′, Antisense 5′-CAUUCAGCAGUACGAAAGUU-3′ (siDNAJB1 #3); Sense 5′-GGACUAUGGACUCUUUCAAUU 3′, Antisense 5′-UUGAAAGA GUCCAUAGUCCUU-3′ (siDNAJB1 #4); Sense 5′-CCUAACUAGCUCAGAU ACAUU-3′, Antisense 5′-UGUAUCUGACUAGUUAGGUU-3′ (siMIG6 #1); Sense 5′-GUUACUUGGCAACCAUAUUUU-3′, Antisense 5′-AAUA UGGUUGCCAAGUAACUU-3′ (siMIG6 #4); Sense 5′-GAAGCAAUUAGC
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UAUGUGAUU-3′, Antisense 5′-UCACAUAGCUAAUUGCUUCUU-3′ (siMIG6 #5). 2.3. Western blotting, immunoprecipitation, and antibodies Cells were washed with cold phosphate-buffered saline (PBS) and collected. Cell extracts were prepared with lysis buffer [50 mmol/L Tris–Cl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, and protease inhibitors] and incubated on ice for 30 min. The lysates were centrifuged at 20,000 ×g for 10 min at 4 °C. Total cell lysate protein was incubated with the anti-Myc (Cell signaling) or anti-Flag (Sigma) antibodies and with 20 μL of protein A/G agarose overnight at 4 °C. After washing three times with agarose bead washing buffer, the immunoprecipitated protein–antibody complexes were separated on SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked by incubating for 2 h in 5% w/v non-fat DifcoTM skim milk blocking buffer with 1 × PBS with Tween-20 (PBST). The blocked membranes were incubated overnight at 4 °C with the indicated antibodies. After washing with 1 × PBST, the membranes were incubated with the appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Thermo Scientific, Rockford, IL, USA) for 1 h, and visualized using LAS-3000 system (Fujifilm, Stamford, CT, USA) with an enhanced chemiluminescence detection reagent (Thermo Scientific). The antibodies were used anti-Mig6, anti-HSP40, anti-HA (Santacruz), anti-Flag, anti-β-actin (Sigma–Aldrich), anti-Myc, anti-phospho-EGFR (Tyrosine 1068), anti-EGFR, anti-phospho-ERK (Serine 42/44), anti-ERK, and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-PARP1 (BD Transduction Laboratories, Lexington, KY, USA). Western blot bands were quantified using ImageJ software (free download available at http://rsbweb.nih.gov/ij/). Density value was normalized by each β-actin band. 2.4. RNA isolation and quantitative RT-PCR Total RNA was extracted using the Trizol reagent following the standard protocol (TAKARA, Shimogyo-ku, Kyoto, Japan). Then, cDNA was prepared using random hexamer primers (Chromogen). PCR was performed using the following forward and reverse primers: F-5′-GATG GCATGGACTGTGGTCA-3′, R-5′GCAATGCCTCCTGCACCACC-3′ (human GAPDH); F-5′-CTG GAG CAG TCG CAG TGA G-3′, R-5′-GCC ATT CAT CGG AGC AGA TTT G-3′ (human Mig-6); F-5′-TCT TGA TGT CTG GGG AAT CCT T-3′, R-5′-CCA GTC ACC CAC GAC CTT C-3′ (human DNAJB1 RT-1); F-5′-CTC TGG ACG GCA GGA CGA TA-3′, and R-5′-TCT TGA TGT CTG GGG AAT CCT T-3′ (human DNAJB1 RT-2). Concentration of cDNA was normalized using GAPDH. 2.5. In vivo ubiquitination assay HCT116 cells were co-transfected with plasmids encoding HA-Ub, HA-Ub K48, HA-Ub K63, Myc-Mig6 or Flag-DNAJB1. After 48 h, whole cell lysates were treated with MG132 for 6 h and subsequently processed for immunoprecipitation with anti-Myc antibody. Ubiquitination of Myc-MIG6 was visualized by Western blotting with an anti-HA antibody. 2.6. MTT assay Cell viability was determined with the conventional MTT reduction assay. First, 5 × 103–1 × 104 cells were seeded in a 96-well plate. After overnight incubation, cells were transfected with indicated plasmids for 30 h, and the cells were treated with EGF or gefitinib for another 48 h. Cells were then treated with 15 μM MTT solution (Sigma-Aldrich) for 90 min at 37 °C. The formation of formazan was resolved with DMSO solution for 30 min with shaking. The absorbance was recorded at
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570 nm, and a reference was recorded at 630 nm with a microplate reader (Model 550, BIO-RAD Laboratories, Hercules, CA, USA). 2.7. TUNEL assay For the detection of apoptosis in cells, DNA fragmentation was evaluated by a TUNEL assay using the HT Titer TACS Assay Kit (Trivigen,
Gaithersburg, MD, USA) according to the manufacturer's instructions. Briefly, the cells were fixed with 3.7% buffered formaldehyde solution for 7 min, washed with PBS, permeabilized with 100% methanol for 20 min, washed with PBS twice, digested with proteinase K for 15 min, quenched with 3% hydrogen peroxide, washed with distilled water, labeled with deoxynucleotidyl transferase, incubated at 37 °C for 90 min, and then treated with stop buffer. The cells were incubated
Fig. 1. DNAJB1 binds to MIG6 and decreases its protein levels. (A) DNAJB1 interacts with MIG6. Flag-tagged DNAJB1 and Myc-tagged MIG6 were co-transfected into HCT116 cells. Whole cell lysates were imunoprecipitated and immunoblotted with the indicated antibodies (left panel). GST pull downs were performed overnight at 4 °C. Bound proteins were eluted and analyzed using autoradiography (right panel). (B) Endogenous DNAJB1 interacts with MIG6. Immunoprecipitation assays were performed with the anti-MIG6 antibody. (C) The central domain (180–210 a.a.) of DNAJB1 interacts with MIG6. Schematic diagrams of Flag-DNAJB1 constructs for co-immunoprecipitation and mapping analyses. HCT116 cells were cotransfected with the Myc-MIG6 and Flag-DNAJB1 plasmids as indicated. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies.(D) The Cdc42/Rac interaction and binding (CRIB) domain of MIG6 interacts with DNAJB1. Schematic diagrams of Myc-MIG6 constructs for co-immunoprecipitation and mapping analyses. HCT116 cells were co-transfected with the Flag-DNAJB1 and Myc-MIG6 plasmids as indicated. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Representative blots from three independent experiments are shown.
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with TACS-Sapphire substrate, and the colorimetric reaction was stopped with 0.2 N HCl after 30 min. The colorimetric reaction was measured in a microplate reader at absorbance 450 nm.
3. Results
2.8. Statistical analysis
To explore the regulatory mechanisms of MIG6, we first dissected the MIG6 interactome using yeast two-hybrid assays. A human ovary cDNA library was screened using Gal4-fusions of full-length MIG6 (1–462 a.a.). cDNAs were isolated from positive clones and subsequently, DNA sequencing was performed. Based on the sequence analyses, we identified the heat shock protein DNAJB1 as a putative MIG6-interacting protein (Supplementary Table S1). To validate our
Statistical significance was determined using one-way ANOVA followed by post-hoc tests. Values were reported as the mean with 95% confidence intervals (CI. P values b 0.05 were considered significant). Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA).
3.1. DNAJB1 binds to MIG6
Fig. 2. DNAJB1 destabilizes the MIG6 protein. (A) DNAJB1 overexpression reduces the stability of endogenous MIG6 but does not affect its mRNA levels. A549 cells were transfected with either Flag-tagged wild-type DNAJB1 (DNAJB1) or mutant DNAJB1Δ180–210. Cell lysates were immunoblotted with the indicated antibodies. (B) DNAJB1 knockdown increases MIG6 stability. si-DNAJB1s (10 pM) were transfected into HCT116 cells, and cell lysates were immunoblotted with the indicated antibodies (C–D) DNAJB1 reduces the stability of MIG6. HCT116 cells were transfected with the indicated plasmids (D) or siRNAs (E). 48 h after transfection, cells were treated with 10 μM cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. *P b 0.01, **P b 0.05, and ***P b 0.001 compared to transfection control, ANOVA analysis.
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screening data, we performed in vitro and in vivo pull-down assays. Coimmunoprecipitation (co-IP) and GST pull down analyses demonstrated that DNAJB1 or MIG6 specifically bound each other, but not the control (Fig. 1A). Moreover, we verified an endogenous interaction between MIG6 and DNAJB1 (Fig. 1B). To elucidate the structure and function of the MIG6-DNAJB1 interaction, we next mapped the DNAJB1 interaction domains for MIG6. For these experiments, the DNAJB1 protein was divided into seven fragments, and their interaction with MIG6 was evaluated using co-IP assays (Fig. 1C). The central domain (180–210 a.a.) of DNAJB1 was interacted with MIG6. On the other hand, the MIG6 protein was divided into four fragments, and their interaction with DNAJB1 was evaluated using co-IP assays (Fig. 1D). We found that the Cdc42/Rac interaction and binding (CRIB) domain of MIG6 was interacted with DNAJB1.
These data collectively indicates that the central domain (180–210 a.a.) of DNAJB1 is interacted with the CRIB domain of MIG6. 3.2. DNAJB1 destabilized MIG6 levels via protein destabilization We further validated this interaction using dose-dependent overexpression or knockdown of DNAJB1 on MIG6 level. Overexpression of wild-type DNAJB1 (DNAJB1) substantially decreased MIG6 levels. However, overexpression of the MIG6-interaction defective mutant DNAJB1Δ180–210 failed to reduce the MIG6 protein levels. Importantly, DNAJB1 overexpression did not affect the level of Mig6 mRNA (Fig. 2A). Similarly, DNJAB1 knockdown substantially increased the level of endogenous MIG6 protein, but not its mRNA (Fig. 2B).
Fig. 3. DNAJB1 enhances ubiquitin-dependent proteosomal degradation of MIG6. (A) DNAJB1 overexpression abrogates the effect of MG132 on the stabilization of MIG6. HCT116 cells were transfected with Flag-tagged DNAJB1. Two days after transfection, cells were treated with MG132 (10 μM) for 6 h. Total lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. (B) DNAJB1 overexpression increases MIG6 ubiquitination. HCT116 cells were transfected with HA-tagged ubiquitin, Flag-tagged DNAJB1 or DNAJB1Δ180–210, and Myc-tagged MIG6 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. (C) DNAJB1 knockdown diminishes MIG6 ubiquitination. HCT116 cells were transfected with si-DNAJB1. After 24 h, cells were transfected with HA-tagged Ub and Myc-tagged MIG6 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. (D) DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination. HCT116 cells were transfected with the constructs as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvest. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. Representative blots from three independent experiments are shown.
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Time-course experiments with cycloheximide (CHX; an inhibitor of protein biosynthesis) treatment showed that DNAJB1 overexpression further enhanced the effect of CHX on MIG6 destabilization (Fig. 2C). Notably, DNAJB1 knockdown greatly diminished the negative effect of CHX on the stability of MIG6 protein, verifying a critical role for DNAJB1 in MIG6 destabilization (Fig. 2D). 3.3. DNAJB1 enhances K48-linked ubiquitination of MIG6 Since DNAJB1 decreased the MIG6 protein level, not mRNA expression, we next examined whether DNAJB1 reduces the stability of the MIG6 protein. Treatment with MG132 efficiently increased MIG6 levels, whereas DNAJB1 overexpression diminished the effect of MG132 on MIG6 protein stabilization (Fig. 3A). This result indicates that DNAJB1 reduces MIG6 levels via protein destabilization. Fiorini et al. reported that MIG6 protein is ubiquitynylation target for proteasome-dependent degradation [25]. Because DNAJB1 overexpression greatly decreased MIG6 protein stability, we next investigated whether DNAJB1 decreases MIG6 stability by altering the ubiquitin-dependent proteasomal degradation pathway. As shown in Fig. 3B, overexpression of DNAJB1 dramatically increased MIG6 ubiquitination. Importantly, overexpression of DNAJB1Δ180–210 failed to enhance the levels of MIG6 ubiquitination. Consistently, DNAJB1 knockdown substantially reduced MIG6 ubiquitination (Fig. 3C). It has been reported that several ubiquitin linkages (K6, K11, K27, K29, K33, K48, K63) are typically associated with different cellular functions [26]. Because K48-linked polyubiquitin chains are involved in proteasomal degradation, whereas K63-linked polyubiquitination leads to nonproteasomal modification [27], we next examined whether DNAJB1-induced ubiquitination of MIG6 is mediated by K48-linked ubiquitination. HCT116 cells were transfected with Flag-DNAJB1 and either wild-type HA-tagged ubiquitin or ubiquitin mutants harboring
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K48- or K63-specific ubiquitination [28]. As shown in Fig. 3D, DNAJB1induced MIG6 ubiquitination is induced in the presence of the K48specific ubiquitin mutant but not with the K63-specific mutant. These results suggest that DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. 3.4. DNAJB1 is a positive regulator of EGFR signaling pathway via reduction of MIG6 levels MIG6 is a negative regulator of EGFR signaling pathway [1]. Therefore, we next examined whether DNAJB1-mediated negative regulation of MIG6 affects EGF receptor signaling. As consistent with previous reports [2,6], overexpression of MIG6 dose-dependently decreases EGFR levels as well as ERK1/2 phosphorylation (Fig. 4A, left panel). However, MIG6 knockdown increased EGFR protein and ERK1/2 phosphorylation levels (Fig. 4A, right panel). DNAJB1 overexpression reduced MIG6 protein levels and increased the levels of EGFR protein and ERK1/2 phosphorylation. However, mutant DNAJB1Δ180–210 did not affect the levels of both EGFR protein and ERK1/2 phosphorylation (Fig. 4B). DNAJB1 knockdown increased MIG6 levels, which corresponded to reduced EGFR level and ERK1/2 phosphorylation (Fig. 4C). These results indicate that DNAJB1 is a positive regulator of EGFR signaling pathway via the reduction of MIG6 levels. 3.5. DNJAB1 knockdown enhances the sensitivity of A549 cells to gefitinib We next explored the role of DNAJB1 during activation of EGFR signaling by EGF treatment. As expected, MIG6 is induced rapidly upon EGFR activation, which is believed to be part of a negative feedback loop that regulates EGFR activation and stability (Fig. 5A). Importantly, DNAJB1 overexpression significantly increased EGFR stability,
Fig. 4. DNAJB1 positively regulates EGFR signaling pathway via MIG6 destabilization. (A) MIG6 inhibits EGFR signaling. A549 cells were transfected with Myc-tagged MIG6 or siRNAs against MIG6. Cell lysates were immunoblotted with the indicated antibodies. (B) The mutant DNAJB1Δ180–210 failed to suppress EGFR signaling. Either DNAJB1 or DNAJB1Δ180–210 was transfected into A549 cells. Cell lysates were immunoblotted with the indicated antibodies. (C) DNAJB1 knockdown reduces EGFR signaling. A549 cells were transfected siDNAJB1. Cell lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. *P b 0.01, **P b 0.05, and ***P b 0.001 compared to transfection control, ANOVA analysis.
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phosphorylation, and ERK1/2 phosphorylation with reduced MIG6 expression. On the other hand, mutant DNAJB1Δ180–210 did not affect to EGF-induced EGFR activation signaling (Fig. 5B). DNAJB1 knockdown leads to downregulation of EGFR signaling by increasing the levels of MIG6 (Fig. 5C), verifying that DNAJB1 is a negative regulator of MIG6 in the regulation of EGF signaling. Aberrant activation of EGFR signaling leads to tumor growth and progression [29]. Because we observed a
positive role for DNAJB1 in the regulation of EGFR activation, we next examined the function of DNAJB1 on the growth of A549 lung cancer cells, which exhibit genomic amplification of wild-type EGFR. EGF treatment of A549 cells increased cell proliferation. However, DNAJB1 knockdown fully abrogated the positive effect of EGF on the growth of A549 cells (Fig. 5D). A recent study demonstrated that MIG6 overexpression overcomes gefitinib resistance by inhibiting EGFR signaling [30]. Finally,
Fig. 5. DNAJB1 knockdown increases the sensitivity of A549 cells to gefitinib. (A) EGF rapidly activates the EGFR signaling pathway. A549 cells were treated with 100 ng/ml EGF for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (B) Overexpression of DNAJB1 enhances EGFR activation via reduction of MIG6. Cells were transfected with indicated plasmids. After 48 h, cells were treated with EGF, and cell lysates were analyzed by western blotting using the indicated antibodies. (C) DNAJB1 knockdown diminishes EGFR activation. A549 cells were transfected with siDNAJB1. After 48 h, cells were treated with EGF, and cell lysates were analyzed by western blotting with the indicated antibodies. Representative blots from three independent experiments are shown. (D) DNAJB1 knockdown diminishes the EGF effect on the viability of A549 cells. A549 cells were transfected with siRNAs. After 24 h, cells were treated with EGF, and cell viability was determined using MTT assays. Error bars indicate 95% confidence intervals;**,***P b 0.0001 using one-way ANOVA. (E) DNAJB1 knockdown enhanced the gefitinib-induced apoptosis. After 24 h transfection with siRNA, cells were treated with 50 μM gefitinib for 48 h. Apoptosis was measured by Tunel assay kit. ***P b 0.0001 using one-way ANOVA. (F) Knockdown of DNAJB1 increased the gefitinib-induced PARP-1 and Caspase-3 cleavage. A549 cells were transfected with DNAJB1 siRNA, treated with 50 μM gefitinib for 48 h, and analyzed by western blotting using indicated antibodies. Representative blots from independent experiments are shown. (G) DNAJB1 overexpression abrogates the gefitinib sensitivity of A549 cells. A549 cells were transfected with indicated plasmids. After 24 h, cells were treated with gefitinib (50 μM), and cell viability was determined using MTT assays. Error bars indicate SD (n = 3). ***P b 0.0001 using one-way ANOVA. (H) DNAJB1 knockdown enhanced the gefitinib sensitivity of A549 cells. After 48 h transfection with DNAJB1 siRNAs, cells were treated with gefitinib (50 μM) for 24 h and cell viability was determined using MTT. *** P b 0.0001 using one-way ANOVA.
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we examined the possibility that knockdown of DNAJB1 may enhance the sensitivity of A549 cells to gefitinib by increasing MIG6 protein levels. As expected, gefitinib-induced apoptosis were enhanced by DNAJB1 knockdown (Fig. 5E). In addition, gefitinib-induced PARP-1 cleavage and active caspase-3 were increased after knockdown of DNAJB1 (Fig. 5F). DNAJB1 suppression promotes MIG6-mediated apoptosis. Overexpression of DNAJB1 abolished the sensitivity of A549 cells to gefitinib; whereas the DNAJB1Δ180–210 mutant had negligible effects on gefitinib sensitivity (Fig. 5G). Importantly, DNAJB1 knockdown significantly enhanced the sensitivity of A549 cells to gefitinib (Fig. 5H). Collectively, these data demonstrates that reduction of DNAJB1 enhances the sensitivity of lung cancer cells to gefitinib. 4. Discussion HSPs have an important role in the unfolded protein response [31]. HSP dysregulation is implicated in a variety of human diseases, including cancer. Thus, HSPs are highly expressed in human tumors and often associated with increased chemoresistance and poor patient prognosis [31]. In this context, selective inhibition of HSPs using specific inhibitors has been proposed as a strategy to chemosensitize cancer cells [32,33]. DnaJ/HSP40 proteins are well-known co-chaperones that regulate the ATP hydrolysis activity of HSP70 [20]. However, recent research has revealed that they function as independent chaperones, particularly in various types of cancers [19]. Although the exact role of DnaJ/HSP40 proteins and the molecular mechanisms by which they are involved in cancer biology need further investigation, recent studies have provided critical clues. Our group very recently showed that DNAJB1 negatively regulates p53 signaling through ubiquitin-mediated proteosomal degradation of the tumor suppressor gene PDCD5 [24]. It appears possible that the DnaJ/HSP40 protein family is involved in cancer development by directly regulating the stability of proteins such as tumor suppressors. Moreover, data from several studies support the notion that DnaJ/HSP40 proteins may mediate ubiquitin-dependent proteasomal degradation of target proteins. The DNAJ/HSP40 proteins human Hsj1 (DNAJ-like-1) and yeast Ydj1 have two putative ubiquitin-interacting motifs (UIMs) based on bioinformatics analyses and biological experiments [34–36]. Previous data have demonstrated that MIG6 acts as a tumor suppressor. Thus, its downregulation is observed in multiple human tumors [7,37,38]. Tumor size was also inversely correlated with MIG6 expression in some cancers [38]. Based on cDNA microarray analyses, MIG6 is downregulated relative to normal tissues in patients with shorter survival times [3]. These data strongly suggest that MIG6 expression is closely related with poor prognoses in some types of human cancer. However, despite the strong association between MIG6 expression and cancer, it remained unknown how MIG6 protein levels are regulated. In this study, we identified a novel protein, DNAJB1, which affected MIG6 protein stabilization without changing its mRNA levels. These results suggested that MIG6 expression appears to be regulated by DNAJB1 at the post-translational level. In this study, we found that MIG6 was dramatically stabilized in the presence of MG132, and overexpression of DNAJB1 effectively abrogated stabilization of MIG6 stabilization. Moreover, DNAJB1-enhanced proteosomal degradation of MIG6 was mediated by polyubiquitin chains linked through the K48 residue of ubiquitin, which are most commonly associated with proteins targeted for proteosomal degradation [27]. These results provide evidence that support previous studies addressing the role of DnaJ/HSP proteins in proteosomal-dependent degradation pathways, as well as describe a novel protein involved in MIG6 destabilization. The tumor suppressor role of MIG 6 is important in EGFR signaling because it is a known negative feedback regulator of EGFR [1]. EGFR is large family of receptor tyrosine kinases (RTK) that are highly expressed in several types of cancer, including lung, esophageal, and breast cancer [29]. MIG6 inhibits EGFR via a two-tiered mechanism that involves receptor degradation and kinase suppression, thereby causing activation
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of ERK signaling [1,39,40]. Our data demonstrate that DNAJB1 positively affects EGFR signaling by mediating MIG6 degradation. EGFR signaling is closely related to drug resistance [29]. Selective small molecule tyrosine kinase inhibitors (TKIs) that target EGFR, such as gefitinib and erlotinib, are currently used to treat various human cancers [41]. Although these drugs have improved progression-free and overall survival, primary as well as acquired drug resistance eventually occurs in most, if not all, treated patients [42,43]. Thus, drug resistance remains a problem that affects patient survival. However, although MIG6 is involved in signaling, experimental evaluation of the role of MIG6 in drug resistance in human cancers has not been addressed [38]. A recent study showed that MIG6 is overexpressed in prostate cancer cell lines with high gefitinib sensitivity, but not in gefitinib-resistant NSCLC cell lines [44]. Based on previous reports, we hypothesized that stabilizing MIG6 by attenuating DNAJB1 function may improve gefitinib resistance. Indeed, we observed that DNAJB1 knockdown significantly improved the sensitivity of A549 lung cancer cell to gefitinib. These data suggest that modulation of DNAJB1 could be a beneficial effect for gefitinib-resistant lung cancer. Collectively, our data reveals the new molecular mechanism involved in regulation of EGFR signaling pathway by manipulation of MIG6 protein via DNAJB1 as its negative regulator. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2015.07.024. Conflict of interest All authors confirm that they have no potential conflicts of interest regarding this publication. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (NRF-2013R1A1A2059010; K.-C.C.), the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A6A3A04041010) (H.-K.C.), and the NRF grant funded by the Korea Government (MSIP) (No. NRF-2015R1A2A1A05000899 & NRF-2011-0030086) (H.-G.Y.). References [1] P.O. Hackel, M. Gishizky, A. Ullrich, Mig-6 is a negative regulator of the epidermal growth factor receptor signal, Biol. Chem. 382 (2001) 1649–1662. [2] M. Reschke, I. Ferby, E. Stepniak, N. Seitzer, D. Horst, E.F. Wagner, et al., Mitogeninducible gene-6 is a negative regulator of epidermal growth factor receptor signaling in hepatocytes and human hepatocellular carcinoma, Hepatology 51 (2010) 1383–1390. [3] S. Anastasi, G. Sala, C. Huiping, E. Caprini, G. Russo, S. Iacovelli, et al., Loss of RALT/ MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin, Oncogene 24 (2005) 4540–4548. [4] C.I. Lin, J. Du, W.T. Shen, E.E. Whang, D.B. Donner, N. Griff, et al., Mitogen-inducible gene-6 is a multifunctional adaptor protein with tumor suppressor-like activity in papillary thyroid cancer, J. Clin. Endocrinol. Metab. 96 (2011) E554–E565. [5] C. Ballaro, S. Ceccarelli, C. Tiveron, L. Tatangelo, A.M. Salvatore, O. Segatto, et al., Targeted expression of RALT in mouse skin inhibits epidermal growth factor receptor signalling and generates a waved-like phenotype, EMBO Rep. 6 (2005) 755–761. [6] I. Ferby, M. Reschke, O. Kudlacek, P. Knyazev, G. Pante, K. Amann, et al., Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation, Nat. Med. 12 (2006) 568–573. [7] T.H. Kim, D.K. Lee, S.N. Cho, G.D. Orvis, R.R. Behringer, J.P. Lydon, et al., Critical tumor suppressor function mediated by epithelial Mig-6 in endometrial cancer, Cancer Res. 73 (2013) 5090–5099. [8] J.W. Jeong, H.S. Lee, K.Y. Lee, L.D. White, R.R. Broaddus, Y.W. Zhang, et al., Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 8677–8682.
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DNAJB1 negatively regulates MIG6 to promote epidermal growth factor receptor signaling Soo-Yeon Park a,1, Hyo-Kyoung Choi b,1, Jae Sung Seo a, Jung-Yoon Yoo a,c, Jae-Wook Jeong c, Youngsok Choi d, Kyung-Chul Choi e,⁎, Ho-Geun Yoon a,⁎ a
Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Republic of Korea Division of Nutrition and Metabolism Research Group, Korea Food Research Institute, Gyeonggi-do, Republic of Korea Department of Obstetrics, Gynecology & Reproductive Biology, Michigan State University College of Human Medicine, MI, USA d Fertility Center of CHA General Hospital, CHA Research Institute, CHA University, Seoul, Republic of Korea e Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 2 February 2015 Received in revised form 28 July 2015 Accepted 30 July 2015 Available online 1 August 2015 Keywords: DNAJB1 MIG6 EGFR Gefitinib Lung cancer
a b s t r a c t Mitogen-inducible gene 6 (MIG6) is a tumor suppressor implicated in the development of human cancers; however, the regulatory mechanisms of MIG6 remain unknown. Here, using a yeast two-hybrid screen, we identified DnaJ homolog subfamily B member I (DNAJB1) as a novel MIG6-interacting protein. We found that DNAJB1 binds to and decreases MIG6 protein, but not mRNA, levels. DNAJB1 overexpression dosage-dependently decreased MIG6 protein levels. Conversely, DNAJB1 knockdown increased MIG6 protein levels. DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. However, knocking-down of DNAJB1 reduced the ubiquitination of MIG6. DNAJB1 positively regulates the epidermal growth factor receptors (EGFR) signaling pathway via destabilization of MIG6; however, DNAJB1 knockdown diminishes activation of EGFR signaling as well as elevation of MIG6. Importantly, the increased levels of MIG6 by DNAJB1 knockdown greatly enhanced the gefitinib sensitivity in A549 cells. Thus, our study provides a new molecular mechanism to regulate EGFR signaling through modulation of MIG6 by DNAJB1 as a negative regulator. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Mitogen-inducible gene 6 (MIG6/RALT/gene33/Errfi1) is a negative regulator of epidermal growth factor receptors (EGFRs) by inhibition of their tyrosine kinase activities [1]. MIG6 also binds to and inhibits cdc42 activity via its CRIB domain, which leads to inhibition of hepatocyte growth factor-induced cell migration [2]. It was reported that MIG6 is downregulated in human tumors, including breast carcinoma, hepatocellular carcinoma, and thyroid cancer [2–4]. MIG6-deficient mice displayed hyperactivation of EGFR and sustained mitogen-activated protein kinase (MAPK) signaling activation [1,5]. Furthermore, MIG6deficient mice develop spontaneous tumors in various organs and are highly susceptible to chemically-induced skin tumors [6]. Strikingly, inhibition of endogenous EGFR signaling using the EGFR small inhibitor gefitinib (Iressa) or replacement of wild-type EGFR with the kinase-deficient protein encoded by the hypomorphic EGFR allele completely rescued skin Abbreviations: DNAJB1, DnaJ homolog subfamily B member I; MIG6, mitogeninducible gene 6; EGFR, epidermal growth factor receptors ⁎ Corresponding authors at: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea. E-mail addresses: [email protected] (K.-C. Choi), [email protected] (H.-G. Yoon). 1 S.Y.P. and H.K.C. contributed equally to this work.
http://dx.doi.org/10.1016/j.bbamcr.2015.07.024 0167-4889/© 2015 Elsevier B.V. All rights reserved.
defects in MIG6 knockout (KO) mice [6]. Recently, MIG6 was shown to act as a tumor suppressor in endometrial cancers associated with PTEN deficiency by inhibiting estrogen signaling in the uterus [7]. Ablation of MIG6 in the murine uterus leads to endometrial hyperplasia and estrogeninduced endometrial cancer [8]. More recently, it has been shown that MIG6 mediates progesterone action and inhibits ERK1/2 signaling to suppress endometrial cancer development and progression that is associated with PTEN deficiency [9]. These demonstrate that the tumor suppressor MIG6 is a specific negative regulator of EGFR. MIG6 is induced as an immediate early response gene by a variety of stimuli, including growth factors, fetal calf serum, insulin, hypoxia, and stress [10]. It has been also reported that various stressors induce MIG6 transcription [11–14]. Moreover, MIG6 is induced by EGF treatment and functions as a feedback inhibitor of EGFR signaling, regulating receptor activation and stability [1]. Therefore, MIG6 induction is a critical step for cells to sense external stimuli. Although MIG6 clearly acts as a tumor suppressor and its downregulation correlates with human cancer development, the molecular mechanisms regarding how MIG6 levels are regulated remain unknown. The dynamic exchange of multiple chaperone proteins during cellular stress events occurs in important cellular reactions including DNA replication, protein degradation and translocation, cell signaling, and apoptosis [15]. When cells are exposed to environmental stress, such
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as heat shock, alcohol, or oxidative stress, heat shock proteins (HSPs) play a role by regulating the correct folding, degradation, localization, and function of target proteins [16,17]. HSPs are classified based on their relative molecular weight. There are five major HSP proteins: HSP90, HSP70, HSP60, HSP40, and small heat shock proteins [18]. DNAJB1 (also called hDj-1) is a member of the HSP40 protein family [19] and interacts with multiple proteins in the cytoplasm. For example, DNAJB1 interacts with heat HSP70 and induces its ATPase activity, which stimulates the association between HSP70 and Hsc70interacting protein (HIP) [20]. DNAJB1 interacts with Hsc70 to sequester p53 in the cytosol [21]. Moreover, the HSP70-DNAJB1 chaperone was found to prevent nitric oxide-induced apoptosis in both macrophages and COS-7 cells [22,23]. In accordance with these findings, we recently reported that DNAJB1 binds to and destabilizes PDCD5 (programmed cell death 5), ultimately leading to suppression of p53-mediated apoptosis [24]. Collectively, these suggest that DNAJB1 may regulate the stability of various proteins in the cytoplasm to maintain cellular homeostasis. In this study, we found that DNAJB1 binds to and destabilizes the MIG6 protein by enhancing ubiquitin-dependent proteasomal degradation of MIG6, which positively affects the EGFR signaling pathway. Moreover, we provide evidence that DNAJB1 suppresses MIG6 stabilization to enhance lung cancer cell proliferation. We identified DNAJB1 as a negative regulator of MIG6 function and propose that the DNAJB1-MIG6 regulatory network plays an important role in the growth of lung cancer cells. 2. Materials and methods 2.1. Cell culture and reagents Human lung adenocarcinoma cell line A549, human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis. These cells were used within 6 months of purchase. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Hyclone, Logan, UT, USA) at 37 °C under 5% CO2. MG132 and cycloheximide (CHX) were purchased from SigmaAldrich (St. Louis, MO, USA). MG132 and CHX were prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). EGF was purchased from ATGen (Seongnam-si, Gyeonggi-do, Korea) and prepared in 1× PBS. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA) and prepared in DMSO (Sigma-Aldrich). Lipofectamine 2000 was used for overexpression constructs and Lipofectamine RNAi Max was used for siRNA (Life Technologies, Grand Island, NY, USA). 2.2. Plasmids and small-interfering RNAs (siRNAs) Wild-type, full-length MIG6 and DNAJB1 constructs were generated by PCR and cloned into the pSG5-KF2M1-FLAG, -Myc, or -HA (SigmaAldrich), and pGEX4T-1 plasmid vectors (GE healthcare, Piscataway, NJ, USA). All plasmid constructs were verified by DNA sequencing. For siRNA transfection, cells were plated at 60–70% confluency and transfected using Lipofectamine RNAi MAX (Life Technologies) with 20 pM siRNA following the manufacturer's protocol. After 4 h, the media was changed, and cells were incubated for 2 days and treated with indicating reagents. Sequences of siRNAs; Sense 5′-CCUCGUGCCG UUCCAUCAGGUAGUU-3′, Antisense 5′-CUACCUGAUGGAACGGCACG AGGUU-3′ (Negative Control); Sense 5′-CUUUCGUACUGCUGAAUG UUU-3′, Antisense 5′-CAUUCAGCAGUACGAAAGUU-3′ (siDNAJB1 #3); Sense 5′-GGACUAUGGACUCUUUCAAUU 3′, Antisense 5′-UUGAAAGA GUCCAUAGUCCUU-3′ (siDNAJB1 #4); Sense 5′-CCUAACUAGCUCAGAU ACAUU-3′, Antisense 5′-UGUAUCUGACUAGUUAGGUU-3′ (siMIG6 #1); Sense 5′-GUUACUUGGCAACCAUAUUUU-3′, Antisense 5′-AAUA UGGUUGCCAAGUAACUU-3′ (siMIG6 #4); Sense 5′-GAAGCAAUUAGC
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UAUGUGAUU-3′, Antisense 5′-UCACAUAGCUAAUUGCUUCUU-3′ (siMIG6 #5). 2.3. Western blotting, immunoprecipitation, and antibodies Cells were washed with cold phosphate-buffered saline (PBS) and collected. Cell extracts were prepared with lysis buffer [50 mmol/L Tris–Cl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, and protease inhibitors] and incubated on ice for 30 min. The lysates were centrifuged at 20,000 ×g for 10 min at 4 °C. Total cell lysate protein was incubated with the anti-Myc (Cell signaling) or anti-Flag (Sigma) antibodies and with 20 μL of protein A/G agarose overnight at 4 °C. After washing three times with agarose bead washing buffer, the immunoprecipitated protein–antibody complexes were separated on SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked by incubating for 2 h in 5% w/v non-fat DifcoTM skim milk blocking buffer with 1 × PBS with Tween-20 (PBST). The blocked membranes were incubated overnight at 4 °C with the indicated antibodies. After washing with 1 × PBST, the membranes were incubated with the appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Thermo Scientific, Rockford, IL, USA) for 1 h, and visualized using LAS-3000 system (Fujifilm, Stamford, CT, USA) with an enhanced chemiluminescence detection reagent (Thermo Scientific). The antibodies were used anti-Mig6, anti-HSP40, anti-HA (Santacruz), anti-Flag, anti-β-actin (Sigma–Aldrich), anti-Myc, anti-phospho-EGFR (Tyrosine 1068), anti-EGFR, anti-phospho-ERK (Serine 42/44), anti-ERK, and anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-PARP1 (BD Transduction Laboratories, Lexington, KY, USA). Western blot bands were quantified using ImageJ software (free download available at http://rsbweb.nih.gov/ij/). Density value was normalized by each β-actin band. 2.4. RNA isolation and quantitative RT-PCR Total RNA was extracted using the Trizol reagent following the standard protocol (TAKARA, Shimogyo-ku, Kyoto, Japan). Then, cDNA was prepared using random hexamer primers (Chromogen). PCR was performed using the following forward and reverse primers: F-5′-GATG GCATGGACTGTGGTCA-3′, R-5′GCAATGCCTCCTGCACCACC-3′ (human GAPDH); F-5′-CTG GAG CAG TCG CAG TGA G-3′, R-5′-GCC ATT CAT CGG AGC AGA TTT G-3′ (human Mig-6); F-5′-TCT TGA TGT CTG GGG AAT CCT T-3′, R-5′-CCA GTC ACC CAC GAC CTT C-3′ (human DNAJB1 RT-1); F-5′-CTC TGG ACG GCA GGA CGA TA-3′, and R-5′-TCT TGA TGT CTG GGG AAT CCT T-3′ (human DNAJB1 RT-2). Concentration of cDNA was normalized using GAPDH. 2.5. In vivo ubiquitination assay HCT116 cells were co-transfected with plasmids encoding HA-Ub, HA-Ub K48, HA-Ub K63, Myc-Mig6 or Flag-DNAJB1. After 48 h, whole cell lysates were treated with MG132 for 6 h and subsequently processed for immunoprecipitation with anti-Myc antibody. Ubiquitination of Myc-MIG6 was visualized by Western blotting with an anti-HA antibody. 2.6. MTT assay Cell viability was determined with the conventional MTT reduction assay. First, 5 × 103–1 × 104 cells were seeded in a 96-well plate. After overnight incubation, cells were transfected with indicated plasmids for 30 h, and the cells were treated with EGF or gefitinib for another 48 h. Cells were then treated with 15 μM MTT solution (Sigma-Aldrich) for 90 min at 37 °C. The formation of formazan was resolved with DMSO solution for 30 min with shaking. The absorbance was recorded at
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570 nm, and a reference was recorded at 630 nm with a microplate reader (Model 550, BIO-RAD Laboratories, Hercules, CA, USA). 2.7. TUNEL assay For the detection of apoptosis in cells, DNA fragmentation was evaluated by a TUNEL assay using the HT Titer TACS Assay Kit (Trivigen,
Gaithersburg, MD, USA) according to the manufacturer's instructions. Briefly, the cells were fixed with 3.7% buffered formaldehyde solution for 7 min, washed with PBS, permeabilized with 100% methanol for 20 min, washed with PBS twice, digested with proteinase K for 15 min, quenched with 3% hydrogen peroxide, washed with distilled water, labeled with deoxynucleotidyl transferase, incubated at 37 °C for 90 min, and then treated with stop buffer. The cells were incubated
Fig. 1. DNAJB1 binds to MIG6 and decreases its protein levels. (A) DNAJB1 interacts with MIG6. Flag-tagged DNAJB1 and Myc-tagged MIG6 were co-transfected into HCT116 cells. Whole cell lysates were imunoprecipitated and immunoblotted with the indicated antibodies (left panel). GST pull downs were performed overnight at 4 °C. Bound proteins were eluted and analyzed using autoradiography (right panel). (B) Endogenous DNAJB1 interacts with MIG6. Immunoprecipitation assays were performed with the anti-MIG6 antibody. (C) The central domain (180–210 a.a.) of DNAJB1 interacts with MIG6. Schematic diagrams of Flag-DNAJB1 constructs for co-immunoprecipitation and mapping analyses. HCT116 cells were cotransfected with the Myc-MIG6 and Flag-DNAJB1 plasmids as indicated. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies.(D) The Cdc42/Rac interaction and binding (CRIB) domain of MIG6 interacts with DNAJB1. Schematic diagrams of Myc-MIG6 constructs for co-immunoprecipitation and mapping analyses. HCT116 cells were co-transfected with the Flag-DNAJB1 and Myc-MIG6 plasmids as indicated. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Representative blots from three independent experiments are shown.
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with TACS-Sapphire substrate, and the colorimetric reaction was stopped with 0.2 N HCl after 30 min. The colorimetric reaction was measured in a microplate reader at absorbance 450 nm.
3. Results
2.8. Statistical analysis
To explore the regulatory mechanisms of MIG6, we first dissected the MIG6 interactome using yeast two-hybrid assays. A human ovary cDNA library was screened using Gal4-fusions of full-length MIG6 (1–462 a.a.). cDNAs were isolated from positive clones and subsequently, DNA sequencing was performed. Based on the sequence analyses, we identified the heat shock protein DNAJB1 as a putative MIG6-interacting protein (Supplementary Table S1). To validate our
Statistical significance was determined using one-way ANOVA followed by post-hoc tests. Values were reported as the mean with 95% confidence intervals (CI. P values b 0.05 were considered significant). Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA).
3.1. DNAJB1 binds to MIG6
Fig. 2. DNAJB1 destabilizes the MIG6 protein. (A) DNAJB1 overexpression reduces the stability of endogenous MIG6 but does not affect its mRNA levels. A549 cells were transfected with either Flag-tagged wild-type DNAJB1 (DNAJB1) or mutant DNAJB1Δ180–210. Cell lysates were immunoblotted with the indicated antibodies. (B) DNAJB1 knockdown increases MIG6 stability. si-DNAJB1s (10 pM) were transfected into HCT116 cells, and cell lysates were immunoblotted with the indicated antibodies (C–D) DNAJB1 reduces the stability of MIG6. HCT116 cells were transfected with the indicated plasmids (D) or siRNAs (E). 48 h after transfection, cells were treated with 10 μM cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. *P b 0.01, **P b 0.05, and ***P b 0.001 compared to transfection control, ANOVA analysis.
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screening data, we performed in vitro and in vivo pull-down assays. Coimmunoprecipitation (co-IP) and GST pull down analyses demonstrated that DNAJB1 or MIG6 specifically bound each other, but not the control (Fig. 1A). Moreover, we verified an endogenous interaction between MIG6 and DNAJB1 (Fig. 1B). To elucidate the structure and function of the MIG6-DNAJB1 interaction, we next mapped the DNAJB1 interaction domains for MIG6. For these experiments, the DNAJB1 protein was divided into seven fragments, and their interaction with MIG6 was evaluated using co-IP assays (Fig. 1C). The central domain (180–210 a.a.) of DNAJB1 was interacted with MIG6. On the other hand, the MIG6 protein was divided into four fragments, and their interaction with DNAJB1 was evaluated using co-IP assays (Fig. 1D). We found that the Cdc42/Rac interaction and binding (CRIB) domain of MIG6 was interacted with DNAJB1.
These data collectively indicates that the central domain (180–210 a.a.) of DNAJB1 is interacted with the CRIB domain of MIG6. 3.2. DNAJB1 destabilized MIG6 levels via protein destabilization We further validated this interaction using dose-dependent overexpression or knockdown of DNAJB1 on MIG6 level. Overexpression of wild-type DNAJB1 (DNAJB1) substantially decreased MIG6 levels. However, overexpression of the MIG6-interaction defective mutant DNAJB1Δ180–210 failed to reduce the MIG6 protein levels. Importantly, DNAJB1 overexpression did not affect the level of Mig6 mRNA (Fig. 2A). Similarly, DNJAB1 knockdown substantially increased the level of endogenous MIG6 protein, but not its mRNA (Fig. 2B).
Fig. 3. DNAJB1 enhances ubiquitin-dependent proteosomal degradation of MIG6. (A) DNAJB1 overexpression abrogates the effect of MG132 on the stabilization of MIG6. HCT116 cells were transfected with Flag-tagged DNAJB1. Two days after transfection, cells were treated with MG132 (10 μM) for 6 h. Total lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. (B) DNAJB1 overexpression increases MIG6 ubiquitination. HCT116 cells were transfected with HA-tagged ubiquitin, Flag-tagged DNAJB1 or DNAJB1Δ180–210, and Myc-tagged MIG6 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. (C) DNAJB1 knockdown diminishes MIG6 ubiquitination. HCT116 cells were transfected with si-DNAJB1. After 24 h, cells were transfected with HA-tagged Ub and Myc-tagged MIG6 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. (D) DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination. HCT116 cells were transfected with the constructs as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvest. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. Representative blots from three independent experiments are shown.
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Time-course experiments with cycloheximide (CHX; an inhibitor of protein biosynthesis) treatment showed that DNAJB1 overexpression further enhanced the effect of CHX on MIG6 destabilization (Fig. 2C). Notably, DNAJB1 knockdown greatly diminished the negative effect of CHX on the stability of MIG6 protein, verifying a critical role for DNAJB1 in MIG6 destabilization (Fig. 2D). 3.3. DNAJB1 enhances K48-linked ubiquitination of MIG6 Since DNAJB1 decreased the MIG6 protein level, not mRNA expression, we next examined whether DNAJB1 reduces the stability of the MIG6 protein. Treatment with MG132 efficiently increased MIG6 levels, whereas DNAJB1 overexpression diminished the effect of MG132 on MIG6 protein stabilization (Fig. 3A). This result indicates that DNAJB1 reduces MIG6 levels via protein destabilization. Fiorini et al. reported that MIG6 protein is ubiquitynylation target for proteasome-dependent degradation [25]. Because DNAJB1 overexpression greatly decreased MIG6 protein stability, we next investigated whether DNAJB1 decreases MIG6 stability by altering the ubiquitin-dependent proteasomal degradation pathway. As shown in Fig. 3B, overexpression of DNAJB1 dramatically increased MIG6 ubiquitination. Importantly, overexpression of DNAJB1Δ180–210 failed to enhance the levels of MIG6 ubiquitination. Consistently, DNAJB1 knockdown substantially reduced MIG6 ubiquitination (Fig. 3C). It has been reported that several ubiquitin linkages (K6, K11, K27, K29, K33, K48, K63) are typically associated with different cellular functions [26]. Because K48-linked polyubiquitin chains are involved in proteasomal degradation, whereas K63-linked polyubiquitination leads to nonproteasomal modification [27], we next examined whether DNAJB1-induced ubiquitination of MIG6 is mediated by K48-linked ubiquitination. HCT116 cells were transfected with Flag-DNAJB1 and either wild-type HA-tagged ubiquitin or ubiquitin mutants harboring
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K48- or K63-specific ubiquitination [28]. As shown in Fig. 3D, DNAJB1induced MIG6 ubiquitination is induced in the presence of the K48specific ubiquitin mutant but not with the K63-specific mutant. These results suggest that DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. 3.4. DNAJB1 is a positive regulator of EGFR signaling pathway via reduction of MIG6 levels MIG6 is a negative regulator of EGFR signaling pathway [1]. Therefore, we next examined whether DNAJB1-mediated negative regulation of MIG6 affects EGF receptor signaling. As consistent with previous reports [2,6], overexpression of MIG6 dose-dependently decreases EGFR levels as well as ERK1/2 phosphorylation (Fig. 4A, left panel). However, MIG6 knockdown increased EGFR protein and ERK1/2 phosphorylation levels (Fig. 4A, right panel). DNAJB1 overexpression reduced MIG6 protein levels and increased the levels of EGFR protein and ERK1/2 phosphorylation. However, mutant DNAJB1Δ180–210 did not affect the levels of both EGFR protein and ERK1/2 phosphorylation (Fig. 4B). DNAJB1 knockdown increased MIG6 levels, which corresponded to reduced EGFR level and ERK1/2 phosphorylation (Fig. 4C). These results indicate that DNAJB1 is a positive regulator of EGFR signaling pathway via the reduction of MIG6 levels. 3.5. DNJAB1 knockdown enhances the sensitivity of A549 cells to gefitinib We next explored the role of DNAJB1 during activation of EGFR signaling by EGF treatment. As expected, MIG6 is induced rapidly upon EGFR activation, which is believed to be part of a negative feedback loop that regulates EGFR activation and stability (Fig. 5A). Importantly, DNAJB1 overexpression significantly increased EGFR stability,
Fig. 4. DNAJB1 positively regulates EGFR signaling pathway via MIG6 destabilization. (A) MIG6 inhibits EGFR signaling. A549 cells were transfected with Myc-tagged MIG6 or siRNAs against MIG6. Cell lysates were immunoblotted with the indicated antibodies. (B) The mutant DNAJB1Δ180–210 failed to suppress EGFR signaling. Either DNAJB1 or DNAJB1Δ180–210 was transfected into A549 cells. Cell lysates were immunoblotted with the indicated antibodies. (C) DNAJB1 knockdown reduces EGFR signaling. A549 cells were transfected siDNAJB1. Cell lysates were immunoblotted with the indicated antibodies. Mean ± SEM of three independent experiments. *P b 0.01, **P b 0.05, and ***P b 0.001 compared to transfection control, ANOVA analysis.
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phosphorylation, and ERK1/2 phosphorylation with reduced MIG6 expression. On the other hand, mutant DNAJB1Δ180–210 did not affect to EGF-induced EGFR activation signaling (Fig. 5B). DNAJB1 knockdown leads to downregulation of EGFR signaling by increasing the levels of MIG6 (Fig. 5C), verifying that DNAJB1 is a negative regulator of MIG6 in the regulation of EGF signaling. Aberrant activation of EGFR signaling leads to tumor growth and progression [29]. Because we observed a
positive role for DNAJB1 in the regulation of EGFR activation, we next examined the function of DNAJB1 on the growth of A549 lung cancer cells, which exhibit genomic amplification of wild-type EGFR. EGF treatment of A549 cells increased cell proliferation. However, DNAJB1 knockdown fully abrogated the positive effect of EGF on the growth of A549 cells (Fig. 5D). A recent study demonstrated that MIG6 overexpression overcomes gefitinib resistance by inhibiting EGFR signaling [30]. Finally,
Fig. 5. DNAJB1 knockdown increases the sensitivity of A549 cells to gefitinib. (A) EGF rapidly activates the EGFR signaling pathway. A549 cells were treated with 100 ng/ml EGF for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (B) Overexpression of DNAJB1 enhances EGFR activation via reduction of MIG6. Cells were transfected with indicated plasmids. After 48 h, cells were treated with EGF, and cell lysates were analyzed by western blotting using the indicated antibodies. (C) DNAJB1 knockdown diminishes EGFR activation. A549 cells were transfected with siDNAJB1. After 48 h, cells were treated with EGF, and cell lysates were analyzed by western blotting with the indicated antibodies. Representative blots from three independent experiments are shown. (D) DNAJB1 knockdown diminishes the EGF effect on the viability of A549 cells. A549 cells were transfected with siRNAs. After 24 h, cells were treated with EGF, and cell viability was determined using MTT assays. Error bars indicate 95% confidence intervals;**,***P b 0.0001 using one-way ANOVA. (E) DNAJB1 knockdown enhanced the gefitinib-induced apoptosis. After 24 h transfection with siRNA, cells were treated with 50 μM gefitinib for 48 h. Apoptosis was measured by Tunel assay kit. ***P b 0.0001 using one-way ANOVA. (F) Knockdown of DNAJB1 increased the gefitinib-induced PARP-1 and Caspase-3 cleavage. A549 cells were transfected with DNAJB1 siRNA, treated with 50 μM gefitinib for 48 h, and analyzed by western blotting using indicated antibodies. Representative blots from independent experiments are shown. (G) DNAJB1 overexpression abrogates the gefitinib sensitivity of A549 cells. A549 cells were transfected with indicated plasmids. After 24 h, cells were treated with gefitinib (50 μM), and cell viability was determined using MTT assays. Error bars indicate SD (n = 3). ***P b 0.0001 using one-way ANOVA. (H) DNAJB1 knockdown enhanced the gefitinib sensitivity of A549 cells. After 48 h transfection with DNAJB1 siRNAs, cells were treated with gefitinib (50 μM) for 24 h and cell viability was determined using MTT. *** P b 0.0001 using one-way ANOVA.
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we examined the possibility that knockdown of DNAJB1 may enhance the sensitivity of A549 cells to gefitinib by increasing MIG6 protein levels. As expected, gefitinib-induced apoptosis were enhanced by DNAJB1 knockdown (Fig. 5E). In addition, gefitinib-induced PARP-1 cleavage and active caspase-3 were increased after knockdown of DNAJB1 (Fig. 5F). DNAJB1 suppression promotes MIG6-mediated apoptosis. Overexpression of DNAJB1 abolished the sensitivity of A549 cells to gefitinib; whereas the DNAJB1Δ180–210 mutant had negligible effects on gefitinib sensitivity (Fig. 5G). Importantly, DNAJB1 knockdown significantly enhanced the sensitivity of A549 cells to gefitinib (Fig. 5H). Collectively, these data demonstrates that reduction of DNAJB1 enhances the sensitivity of lung cancer cells to gefitinib. 4. Discussion HSPs have an important role in the unfolded protein response [31]. HSP dysregulation is implicated in a variety of human diseases, including cancer. Thus, HSPs are highly expressed in human tumors and often associated with increased chemoresistance and poor patient prognosis [31]. In this context, selective inhibition of HSPs using specific inhibitors has been proposed as a strategy to chemosensitize cancer cells [32,33]. DnaJ/HSP40 proteins are well-known co-chaperones that regulate the ATP hydrolysis activity of HSP70 [20]. However, recent research has revealed that they function as independent chaperones, particularly in various types of cancers [19]. Although the exact role of DnaJ/HSP40 proteins and the molecular mechanisms by which they are involved in cancer biology need further investigation, recent studies have provided critical clues. Our group very recently showed that DNAJB1 negatively regulates p53 signaling through ubiquitin-mediated proteosomal degradation of the tumor suppressor gene PDCD5 [24]. It appears possible that the DnaJ/HSP40 protein family is involved in cancer development by directly regulating the stability of proteins such as tumor suppressors. Moreover, data from several studies support the notion that DnaJ/HSP40 proteins may mediate ubiquitin-dependent proteasomal degradation of target proteins. The DNAJ/HSP40 proteins human Hsj1 (DNAJ-like-1) and yeast Ydj1 have two putative ubiquitin-interacting motifs (UIMs) based on bioinformatics analyses and biological experiments [34–36]. Previous data have demonstrated that MIG6 acts as a tumor suppressor. Thus, its downregulation is observed in multiple human tumors [7,37,38]. Tumor size was also inversely correlated with MIG6 expression in some cancers [38]. Based on cDNA microarray analyses, MIG6 is downregulated relative to normal tissues in patients with shorter survival times [3]. These data strongly suggest that MIG6 expression is closely related with poor prognoses in some types of human cancer. However, despite the strong association between MIG6 expression and cancer, it remained unknown how MIG6 protein levels are regulated. In this study, we identified a novel protein, DNAJB1, which affected MIG6 protein stabilization without changing its mRNA levels. These results suggested that MIG6 expression appears to be regulated by DNAJB1 at the post-translational level. In this study, we found that MIG6 was dramatically stabilized in the presence of MG132, and overexpression of DNAJB1 effectively abrogated stabilization of MIG6 stabilization. Moreover, DNAJB1-enhanced proteosomal degradation of MIG6 was mediated by polyubiquitin chains linked through the K48 residue of ubiquitin, which are most commonly associated with proteins targeted for proteosomal degradation [27]. These results provide evidence that support previous studies addressing the role of DnaJ/HSP proteins in proteosomal-dependent degradation pathways, as well as describe a novel protein involved in MIG6 destabilization. The tumor suppressor role of MIG 6 is important in EGFR signaling because it is a known negative feedback regulator of EGFR [1]. EGFR is large family of receptor tyrosine kinases (RTK) that are highly expressed in several types of cancer, including lung, esophageal, and breast cancer [29]. MIG6 inhibits EGFR via a two-tiered mechanism that involves receptor degradation and kinase suppression, thereby causing activation
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of ERK signaling [1,39,40]. Our data demonstrate that DNAJB1 positively affects EGFR signaling by mediating MIG6 degradation. EGFR signaling is closely related to drug resistance [29]. Selective small molecule tyrosine kinase inhibitors (TKIs) that target EGFR, such as gefitinib and erlotinib, are currently used to treat various human cancers [41]. Although these drugs have improved progression-free and overall survival, primary as well as acquired drug resistance eventually occurs in most, if not all, treated patients [42,43]. Thus, drug resistance remains a problem that affects patient survival. However, although MIG6 is involved in signaling, experimental evaluation of the role of MIG6 in drug resistance in human cancers has not been addressed [38]. A recent study showed that MIG6 is overexpressed in prostate cancer cell lines with high gefitinib sensitivity, but not in gefitinib-resistant NSCLC cell lines [44]. Based on previous reports, we hypothesized that stabilizing MIG6 by attenuating DNAJB1 function may improve gefitinib resistance. Indeed, we observed that DNAJB1 knockdown significantly improved the sensitivity of A549 lung cancer cell to gefitinib. These data suggest that modulation of DNAJB1 could be a beneficial effect for gefitinib-resistant lung cancer. Collectively, our data reveals the new molecular mechanism involved in regulation of EGFR signaling pathway by manipulation of MIG6 protein via DNAJB1 as its negative regulator. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2015.07.024. Conflict of interest All authors confirm that they have no potential conflicts of interest regarding this publication. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (NRF-2013R1A1A2059010; K.-C.C.), the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A6A3A04041010) (H.-K.C.), and the NRF grant funded by the Korea Government (MSIP) (No. NRF-2015R1A2A1A05000899 & NRF-2011-0030086) (H.-G.Y.). References [1] P.O. Hackel, M. Gishizky, A. Ullrich, Mig-6 is a negative regulator of the epidermal growth factor receptor signal, Biol. Chem. 382 (2001) 1649–1662. [2] M. Reschke, I. Ferby, E. Stepniak, N. Seitzer, D. Horst, E.F. Wagner, et al., Mitogeninducible gene-6 is a negative regulator of epidermal growth factor receptor signaling in hepatocytes and human hepatocellular carcinoma, Hepatology 51 (2010) 1383–1390. [3] S. Anastasi, G. Sala, C. Huiping, E. Caprini, G. Russo, S. Iacovelli, et al., Loss of RALT/ MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin, Oncogene 24 (2005) 4540–4548. [4] C.I. Lin, J. Du, W.T. Shen, E.E. Whang, D.B. Donner, N. Griff, et al., Mitogen-inducible gene-6 is a multifunctional adaptor protein with tumor suppressor-like activity in papillary thyroid cancer, J. Clin. Endocrinol. Metab. 96 (2011) E554–E565. [5] C. Ballaro, S. Ceccarelli, C. Tiveron, L. Tatangelo, A.M. Salvatore, O. Segatto, et al., Targeted expression of RALT in mouse skin inhibits epidermal growth factor receptor signalling and generates a waved-like phenotype, EMBO Rep. 6 (2005) 755–761. [6] I. Ferby, M. Reschke, O. Kudlacek, P. Knyazev, G. Pante, K. Amann, et al., Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation, Nat. Med. 12 (2006) 568–573. [7] T.H. Kim, D.K. Lee, S.N. Cho, G.D. Orvis, R.R. Behringer, J.P. Lydon, et al., Critical tumor suppressor function mediated by epithelial Mig-6 in endometrial cancer, Cancer Res. 73 (2013) 5090–5099. [8] J.W. Jeong, H.S. Lee, K.Y. Lee, L.D. White, R.R. Broaddus, Y.W. Zhang, et al., Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 8677–8682.
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