Acta Oto-Laryngologica. 2014; 134: 1121–1127
ORIGINAL ARTICLE
KGFR as a possible therapeutic target in middle ear cholesteatoma
TOMOMI YAMAMOTO-FUKUDA1,2, NAOTARO AKIYAMA2, YASUAKI SHIBATA3, HARUO TAKAHASHI2, TOHRU IKEDA3, MICHIAKI KOHNO4 & TAKEHIKO KOJI1 1
Department of Histology and Cell Biology, 2Department of Otolaryngology-Head and Neck Surgery, Department of Translational Medical Science, 3Department of Oral Pathology and Bone Metabolism, and 4Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract Conclusion: We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. Objectives: Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. Methods: Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. Results: The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.
Keywords: Keratinocyte growth factor, KGF, KGF receptor inhibitor, in vivo model, electroporation
Introduction Middle ear cholesteatoma is a pathological condition associated with otitis media with serious conditions [1]. Although there is currently no more suitable treatment for middle ear cholesteatoma than surgery, it is also true that recurrence is not uncommon after surgery [2]. The recent new therapies for inflammation and cancer include the use of compounds that inhibit various receptors or cellular pathways known to be involved in inflammatory processes, cellular proliferation, and cell survival [3,4]. One of the most important pathological features of middle ear cholesteatoma is active proliferation of epithelial cells, which is considered to be stimulated by growth factors. Although our understanding of the molecular mechanism underlying the pathogenesis of
cholesteatoma is limited, among many possible growth factors, our group has focused on the role of keratinocyte growth factor (KGF) in the pathogenesis, and we have already reported that KGF/fibroblast growth factor (FGF)-7 plays important roles in human middle ear cholesteatoma formation and recurrence [5]. The paracrine actions of KGF are dependent upon KGFR, which is a transmembrane tyrosine kinase receptor with an alternatively spliced variant of FGF receptor-2 (FGFR-2)/bek gene [6]. By binding to KGFR, KGF activates various mitogenactivated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) [7]. Recently, the use of a selective inhibiter of KGFR, SU5402, in an in vitro experiment resulted in inhibition of differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from
Correspondence: Tomomi Yamamoto-Fukuda, MD PhD, Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel: +81 95 819 7026. Fax: +81 95 819 7028. E-mail: [email protected]
(Received 3 February 2014; accepted 18 March 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.907501
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cholesteatoma [8]. SU5402 is an ATP mimetic known as a KGFR-selective inhibitor with 86% homology to the KGFR tyrosine kinase domain and inhibits the tyrosine kinase activity of KGFR by interacting with the catalytic domain of KGFR with binding to the ATP-binding site [9]. The primary purpose of the present study was to analyze the role of KGF/KGFR in the molecular pathology of middle ear cholesteatoma in vivo. The secondary purpose was to investigate the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. The results suggest the promising potential of treatments designed to target KGFR. Material and methods Animals The experiments were conducted in 30 male SpragueDawley (SD) rats (6–7 weeks, 200 g) with normal tympanic membrane (TM). Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with the approval of the Institute of Animal Care and Use Committee (approval nos 0810140707 and 1107141127). Electroporation for KGF cDNA in vivo 3x Flag human KGF (hKGF) vector [10], that cording region was kindly provided by Dr. Jeffrey Rubin from the National Cancer Institute, (Bethesda, MD, USA). p3xFLAG–CMV14 vector was purchased from Sigma Chemical Co., (St Louis, MO, USA). For tissue transfection, pulses generated with a CUY21 Electroporator (Nepa Gene Co., Chiba, Japan) were delivered to the tissuebya pair ofelectrodesattached tothe tip ofcustommade tweezers (Meiwa Shoji, Tokyo, Japan) with six pulsesof50 V for80 ms, eachseparated by 1.0 s [11].The SD rat was anesthetized with pentobarbital, then FlaghKGF DNA plasmid driven by a CMV14 promoter was injected at 50 mg into the epithelial lesion of the right external auditory canal (EAC), injected five times every fourth day. For the left ear, a Flag-plasmid driven by a CMV14 promoter was injected and used as control.
we first administered 200 mM, 2 mM SU5402 in 2% DMSO in PBS, or 200 mM, 2 mM, 20 mM PD0325901 in 0.2% DMSO in PBS by eardrops, 50 ml per day every 24 h from day 0 to day 4 in the right ear of three rats after hKGF cDNA transfection. In three of three (100%) specimens of 200 mM PD0325901-treated ears, two of three (67%) specimens of 2 mM PD0325901-treated ears, and three of three (100%) specimens of 200 mM SU5402-treated ears, diphosphorylated (p)-ERK1/2-positive epithelial cells were detected (Table I). No p-ERK1/2-positive cell was detected in any specimens of 20 mM PD0325901-treated ears (Table I). One of three specimens of 2 mM SU5402-treated ears had p-ERK1/ 2-positive cells, but the number of positive epithelial cells was decreased (Table I). Also, 2 mM SU5402 in 2% DMSO in PBS, or 20 mM PD0325901 in 0.2% DMSO in PBS was administered by eardrops, 50 ml per day every 24 h from day 0 to day 23 in the right ear of five rats after vector transfection. In the control left ears, 50 ml of 2% DMSO in PBS was administered after vector transfection. Otoendoscopic examination In the present study, cholesteatoma formation was evaluated by otoendoscopy. At day 7 after the final injection, and day 23 in the KGFR inhibition experiments, SD rats at each time-point underwent otoendoscopic examination with a rigid rod 0 otoendoscope (AVS Co., Tokyo, Japan). The findings were scored semiquantitatively (Table II). Tissue preparation After tissue transfection, each animal was anesthetized with pentobarbital 150 mg/kg intraperitoneally, Table I. Dose-dependent inhibition of ERK phosphorylation by inhibitors. Group
p-ERK1/2 Positive positive case no. rate (%)
KGF + 200 mM SU5402 (n = 3)
3
100
KGF + 2 mM SU5402 (n = 3)
1
33
KGF + 200 mM PD0325901 (n = 3)
3
100
Administration of KGFR inhibitor or MEK inhibitor in vivo
KGF + 2 mM PD0325901 (n = 3)
2
67
KGF + 20 mM PD0325901 (n = 3)
0
0
The effect of KGFR inhibitor on cholesteatoma formation was examined after eardrop administration of KGFR-selective inhibitor (SU5402; Calbiochem, Darmstadt, Germany) or MEK-inhibitor (PD0325901; prepared by Michiaki Kohno [12]). To determine the effective concentration of inhibitors,
KGF + 2% DMSO (n = 5)
5
100
KGF + SU5402, hKGF expression vector transfected ear with 200 mM or 2 mM SU5402 in 2% DMSO treatment; KGF + PD0325901, hKGF expression vector transfected ear with 200 mM, 2 mM or 20 mM PD0325901 in 0.2% DMSO treatment; KGF + 2% DMSO, hKGF expression vector transfected ears with 2% DMSO treatment.
KGFR inhibitor prevents cholesteatoma formation
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Table II. Criteria for the classification of the pathological state of ears. Otoendoscopic examination
H&E examination
Retraction of TM
Debris
Otorrhea with fungus
Skin erosion
Squamous epithelium of TM
Necrotic and granulation tissue in EAC
Cell infiltration in middle ear
Cholesteatoma
+
+
–
–
+
–
+
Inflammation
–
–
–
+
–
–
+
Malignant external otitis
–
–
+
+
–
+
+
Normal
–
–
–
–
–
–
–
Pathological state
EAC, external auditory canal; H&E, hematoxylin and eosin stain; TM, tympanic membrane.
and the EAC tissues were removed. Half of each sample was fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (RT) overnight and embedded in paraffin, while the other half was frozen immediately on dry ice and later used for western blot analysis. The temporal bones were removed en bloc and fixed with 4% PFA in PBS at 4 C for 18 h. After fixation, the temporal bones were decalcified using 10% ethylenediaminetetraacetic acid at 4 C for 7 days [13] and embedded in paraffin. Sections (5 mm thick) were prepared and then mounted on 3-aminopropyltriethoxysilane-coated glass slides. Hematoxylin and eosin (H&E) staining The sections of temporal bones were stained with H&E. The pathological findings of middle ear cholesteatoma include stratified squamous epithelium with inflamed subepithelium that lines the middle ear cavity [14]. The presence of a thin layer of simple squamous epithelium in the TM was considered to be normal. The presence of infiltrating cells including lymphocytes, plasma cells, and fibroblasts represented inflammation. The presence of necrotic and granulation tissue in the EAC represented malignant external otitis. The details are described in Table II.
was separated and transferred onto PVDF membrane (Immobilon, Millipore Corp., MA, USA). The membranes were blocked with Blocking one (Nacalai tesque, Kyoto, Japan) and then incubated with the 0.5 mg/ml horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-Flag M2 antibody (Sigma). After washing with 0.2% Tween 20 in PBS, signals were visualized by the ECL system (Amersham Biosciences, NJ, USA). For p-ERK reaction, the specimens (150–200 mg each) of EAC tissues were homogenized in EzRIPA Lysis kit (ATTO Co., Tokyo, Japan) on ice. After the supernatants were collected, the protein concentration of each preparation was determined as described above. Immunoprecipitation was done using anti-ERK 1 (K-23) (Santa Cruz Biotechnology, Inc., CA, USA) antibody according to the protocol of the Immunoprecipitation Starter Pack. The membrane was incubated with mouse monoclonal anti-MAPK activated (pERK1&2) antibody (1:5000 dilution; Sigma) overnight, washed with TBS-T, and reacted with ECL anti-mouse IgG for 1 h. After washing with TBS-T, signals were visualized using the Amersham ECL Prime system. As a control, after stripping the antibodies, the same membranes were stained with rabbit polyclonal anti-actin (H-196) antibody (1:1000 dilution; Santa Cruz).
Western blot analysis of Flag expression and p-ERK1/2 Immunohistochemistry for KGF and p-ERK1/2 Western blot analysis was performed to determine the expression of Flag-hKGF after vector transfection in EAC tissues, and to determine the expression of p-ERK1/2 after vector transfection with KGFR inhibitor in EAC tissue. Liver tissue was processed as a negative control. For Flag-hKGF reaction, the specimens (150–200 mg each) were frozen immediately on dry ice and homogenized with a Polytron tissue disrupter (Kinematica, Lucerne, Switzerland) in lysis buffer [15]. The protein concentration of each preparation was determined using a kit from Bio-Rad Laboratories (Tokyo, Japan). Each lysate (50 mg)
Enzyme immunohistochemistry was performed using the paraffin sections to determine the expression of KGF and p-ERK1/2 in other sections. A polyclonal antibody against KGF was prepared by immunization of rabbits against synthetic peptides in cooperation with Nichirei Co. (Tokyo, Japan), as described previously [5]. The sections were incubated with the primary antibody of 0.5 mg/ml rabbit polyclonal anti-KGF antibody [5] or mouse monoclonal anti-pERK1/2 antibody (1:50 dilution) washed with 0.075% Brij 35 in PBS, and reacted with the
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secondary antibody. After washing the slides, HRP sites were visualized with 3,3¢-diaminobenzidine and H2O2. HRP-goat anti-mouse IgG (1:100 dilution; Chemicon International, Inc., CA, USA) or HRPgoat anti-rabbit IgG (1:100 dilution; Medical & Biological Laboratories Co., Nagoya, Japan) was used as the second antibody. For the negative control, normal mouse IgG (Sigma) or normal rabbit IgG (Sigma) was used instead of the primary antibody in every experiment. Statistical analysis The Pearson’s correlation coefficient test was used to compare the rate of pathological state between each groups. All analyses were performed with statistical software packages (JMP 10.0.2; SAS Institute Inc., NC, USA). Results Detection of hKGF vector transfection in EAC To evaluate the expression of transfected cDNA in EAC tissues, we first examined the expression of Flag by western blotting with HRP-conjugated anti-Flag antibody. Analysis of extracts from Flag-hKGF vector-transfected EAC indicated only a single 28 kDa band [16] at day 1 and at day 4 (Figure 1, lanes 1 and 3). In contrast, no specific band was found in extracts from empty vector-transfected EAC on any day (Figure 1, lanes 2 and 4). Actin (43 kDa band) was detected in all extracts at equal levels (data not shown). Immunohistochemical studies of EAC tissues using anti-KGF antibody identified KGF protein in almost all epithelial cells and some stromal cells in sections of KGF-transfected ears at day 1 and at day 4 (Figure 2a and b). In contrast, KGF-positive cells were not found in empty vector-transfected EAC on any day (Figure 2c and d). These results indicated successful in vivo transfection of hKGF vector. PD1
PD4
Expression of p-ERK in EAC of SD rat To determine the effects of KGFR inhibitor and MEK inhibitor against MAPK activation induced by hKGF cDNA transfection, the immunoreactivity of p-ERK1/2 was analyzed. By western blot analysis, only one 44 kDa band was detected in the extracts from the specimen after hKGF vector transfection with 2% DMSO in PBS treatment (Figure 3a, lane 3), the intensity of band was decreased in the extracts from the specimen with 2 mM SU5402 treatment (Figure 3a, lane 1) and no band was detected in the extracts from the specimen with 20 mM PD0325901 treatment (Figure 3a, lane 2) using anti-p-ERK1/2 antibody. Actin (43 kDa band) was detected in all the extracts (data not shown). Virtually no epithelial cells were stained in any of the tissues from hKGF vector-transfected rats treated with 2 mM SU5402 (Figure 3b) or 20 mM PD0325901 (Figure 3c), whereas many intensely stained epithelial cells were detected in tissues from hKGF vectortransfected rats treated with 2% DMSO in PBS (Figure 3d). These results indicated that treatment with 2 mM SU5402 or 20 mM PD0325901 inhibited hKGF vector-induced KGFR phosphorylation. Effect of KGFR signaling inhibition on cholesteatoma formation in vivo To clarify the efficacy of KGFR inhibitor in suppressing cholesteatoma formation, we analyzed the ears by otoendoscopy and histology. At day 7 after the final transfection of hKGF cDNA and treatment with a
b
c
d
KGF Empty KGF Empty Liver 28 kDa
Figure 1. Western blot analysis of Flag on transfected tissues. KGF, lysate of external auditory canal (EAC) of hKGF expression vector transfected ears; Empty, lysate of EAC of empty vector transfected ears; Liver, lysate of liver of Sprague-Dawley rats. Each lysate was obtained from the tissue after vector transfection at day 1 (PD1) and at day 4 (PD4). The total volume applied was 50 mg per lane. Each lane was reacted with 0.5 mg/ml HRPconjugated anti-FlagM2 antibody. Flag proteins were detected in hKGF expression vector transfected ears at day 1 and day 4. hKGF expression vector was successfully transfected.
Figure 2. Immunohistochemistry using anti-KGF antibody. (a, b) hKGF cDNA transfected ear (a, day 1; b, day 4); (c, d) empty vector transfected ear (c, day 1; d, day 4). KGF-positive cells are stained in brown. Intense staining for KGF was detected in stromal cells and some epithelial cells (a, b) and weak staining was detected within the hair follicles (d). Arrows indicate positive cells. Bar = 20 mm.
KGFR inhibitor prevents cholesteatoma formation SU5402
a
PD
DMSO
was simultaneously associated with severe necrotic reaction resulting from a defect in the capillaries of the EAC (Figure 4n) of the MEK inhibitor-treated ear. In contrast, cholesteatoma was detected in five of five SU5402-untreated ears after hKGF vector transfection (Table III, Figure 4l, o).
44 kDa
b
c
*
*
*
*
1125
d
Discussion The major finding of the present study was that treatment with SU5402, a KGFR-selective inhibitor, suppressed the formation of middle ear cholesteatoma without any complications of the middle ear. Previously, animal models of middle ear cholesteatoma have been described and their morphological aspects were almost similar to those in the human [17,18]. In the present study, KGF expression vector was selected based on the understanding of the pathogenesis of cholesteatoma [5]. KGF cDNA transfected repetitively into the cells of EAC induced a thick epithelium with keratin pearl in tympanic membrane (Figure 4o), and 2 months later, cholesteatoma formation expanded into the bulla, and bone resorption was noted at the advancing front of the cholesteatoma (data not shown), similar to the findings of a previous study [18]. The goal of experiments using animal models is to enable analysis of the pathogenesis of human disease and establish new therapies. There is currently no more suitable treatment for middle ear cholesteatoma than surgery. In the present study, we used a selective inhibitor of KGFR, SU5402, which interacts with the catalytic domain of KGFR. It is known that KGF reacts only with KGFR, a tyrosine kinase FGFR-2 IIIb and a spliced variant of FGFR2 [6]. KGF signal transduction requires activation of the Ras/MAPK pathway including the ERK pathway, or can alternatively proceed via the PI3K/Akt pathway [19]. In the present study, we described the effect of KGFR inhibitor, and also investigated the effect of MEK inhibitor (PD0325901) [12] during cholesteatoma
Figure 3. Immunoreactivity of diphosphorylated (p)-ERK1/2. (a) Western blot analysis of ear tissues. SU5402, lysate of hKGF cDNA transfected ear with 2 mM SU5402; PD, lysate of hKGF cDNA transfected ear with 20 mM PD0325901 treatment; DMSO, lysate of hKGF cDNA transfected ear with DMSO treatment. Each lane was reacted with anti- p-ERK1/2 antibody. Note that the intensity of band was decreased in SU5402-treated ear and no band was detected in PD0325901-treated ear. (b–d) Immunohistochemistry using anti-p-ERK1/2 antibody. (b) KGF cDNA transfection with 2 mM SU5402 treatment. (c) KGF cDNA transfection with 20 mM PD0325901 treatment. (d) KGF cDNA transfection with DMSO treatment. Note the lack of staining with anti-p-ERK1/2 antibody in epithelial cells of hKGF cDNA transfected ear treated with SU5402 (b) or PD0325901 (c). In the section of hKGF cDNA transfected ear without inhibitor, p-ERK1/2 positive cells were stained brown (arrows). *: epithelium.
2 mM SU5402, cholesteatoma was not detected in any of the ears (0/5, p = 0.0067, Pearson’s correlation coefficient test) (Table III, Figure 4a–e), although chronic inflammation was found in four of five ears (Table III, Figure 4b–e). Histological examination indicated normal structure of the TM and middle ear in tissue sections from hKGF vector-transfected rats treated with SU5402 (Figure 4m). Only chronic inflammatory changes were noted in three of five ears after empty vector transfection and treatment with SU5402 (Table III, Figure 4f). MEK inhibitor also reduced middle ear cholesteatoma formation in our rat model (0/5, p = 0.0067, Pearson’s correlation coefficient test) (Table III, Figure 4g–k). But this Table III. Summary of the pathological state of ears in inhibition study. Group KGF + SU5402 (n = 5) Empty + SU5402 (n = 5) KGF + PD0325901 (n = 5)
Cholesteatoma
Inflammation
Malignant external otitis
Normal
0*
4
0
1
0
3
0
2
0*
0
5
0
Empty + PD0325901 (n = 5)
0
0
5
0
KGF + 2% DMSO (n = 5)
5
0
0
0
KGF + SU5402, hKGF expression vector transfected ear with 2 mM SU5402 in 2% DMSO treatment; Empty + SU5402, empty vector transfected ears with 2 mM SU5402 in 2% DMSO treatment; KGF + PD0325901, hKGF expression vector transfected ear with 20 mM PD0325901 in 0.2% DMSO treatment; Empty + PD0325901, empty vector transfected ears with 20 mM PD0325901 in 0.2% DMSO treatment; KGF + 2% DMSO, hKGF expression vector transfected ears with 2% DMSO treatment. *p = 0.0003.
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b
c
TM
g
d
e
f
TM TM h
i
TM
j
TM
TM k
l Chole
TM
TM
m
TM
TM
TM
n
*
EAC
o
EAC Chole
EAC
TM
ME ME
Figure 4. Results of experiments conducted in rats for the KGFR inhibitory model. (a–l) Otoendscopic findings of tympanic membrane (TM). (a–e) Ears after hKGF cDNA transfection followed by treatment with SU5402. (f) Ear after empty vector transfection followed by treatment with SU5402. (g–k) Ears after hKGF cDNA transfection followed by treatment with PD0325901. (l) Ear after hKGF cDNA transfection followed by treatment with DMSO. Middle ear cholesteatoma formation was not detected in ears after hKGF cDNA transfection followed by treatment with SU5402 (a–e), although chronic inflammation with crust (arrowheads) was found in four ears (b–e). Only chronic inflammatory changes were noted after empty vector transfection and treatment with SU5402 (f). Note that the cholesteatoma formation was not detected but fungus (arrowheads) was shown in PD0325901-treated ear (g–k). Without inhibitor treatment, cholesteatoma (Chole) with debris was detected (l). Arrows: crusts, arrowheads: fungus. (m–o) H&E staining of paraffin section of bulla of the hKGF-transfected ear with or without KGFR inhibitors. (m) The hKGF-transfected ear with SU5402 treatment indicated the normal external auditory canal (EAC), TM, and middle ear (ME), which contained a thin layer of simple squamous epithelium with thin subepithelium. (n) The hKGF-transfected ear with PD0325901 treatment indicated almost normal TM with thick subepithelial layer (star), and necrotic tissue was shown in the EAC lesion (asterisk). (o) Cholesteatoma (Chole) formation was induced after five times hKGF cDNA transfection. Note the keratin pearl formation within the stratified squamous epithelium and thicker subepithelial layer (see box).
formation. As expected, MEK inhibitor also reduced middle ear cholesteatoma formation in our rat model, but this was simultaneously associated with severe necrotic reaction resulting from a defect in the capillaries of the EAC (Figure 4n). Indeed, it is known that paradoxical MAPK activation through the use of a BRAF inhibitor seems to be involved in the development of certain types of cancer [20]. In experiments using SU5402, we demonstrated that p-ERK was not detected and cholesteatoma formation was reduced without any side effects. We believe that our in vivo study extended the in vitro findings and enhanced our understanding of the pathogenesis of cholesteatoma, especially as regards the interaction between the epithelium and stroma and its role in cholesteatoma formation. In conclusion, overexpression of KGF plays an important role in the pathogenesis of middle ear cholesteatoma. Furthermore, the use of a selective KGFR inhibitor reduced middle ear cholesteatoma in our rats. These results suggest that the key molecules in the KGFR signaling pathway are a potentially suitable therapeutic target in the design of new treatment for human middle ear cholesteatoma. Of course we know
that we should assess the ototoxicity of SU5402 when it is applied to the EAC or middle ear by ear drops; after this step SU5402 treatment may actually become a therapeutic approach for humans.
Acknowledgments The authors thank Dr. Jeffrey S. Rubin from the National Cancer Institute/CCR/LCMB, for providing the human KGF cDNA construct. The study was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (no. 23791906 to T.Y-F.) and by a grant from the Naito Foundation (to T.Y-F.). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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KGFR inhibitor prevents cholesteatoma formation [2] Edelstein DR, Parisier SC. Surgical techniques and recidivism in cholesteatoma. Otolaryngol Clin North Am 1989;22: 1029–40. [3] Hoshino R, Tanimura S, Watanabe K, Kataoka T, Kohno M. Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27Kip1. J Biol Chem 2001;276: 2686–92. [4] Tanimura S, Asato K, Fujishiro S, Kohno M. Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: downregulation of matrix metalloproteinase-3/9/-14 and CD44. Biochem Biophys Res Commun 2003;304: 801–6. [5] Yamamoto-Fukuda T, Aoki D, Hishikawa Y, Kobayashi T, Takahashi H, Koji T. Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma. Lab Invest 2003;83:123–36. [6] Miki T, Bottaro DP, Fleming TP, Smith CL, Burgess WH, Chan AM, et al. Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci USA 1992;89: 246–50. [7] Portnnoy J, Curran-Everett D, Mason RJ. Keratinocyte growth factor stimulates alveolar type II cell proliferation through the extracellular signal-regulated kinase and phosphatidylinositol 3-OH kinase pathways. Am J Respir Cell Mol Biol 2004;30:901–7. [8] Raffa S, Leone L, Scrofani C, Monini S, Torrisi MR, Barbara M. Cholesteatoma-associated fibroblasts modulate epithelial growth and differentiation through KGF/FGF7 secretion. Histochem Cell Biol 2012;138:251–69. [9] Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, Yeh BK, et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 1997;276:955–60. [10] Matsumoto K, Nagayasu T, Hishikawa Y, Tagawa T, Yamayoshi T, Abo T, et al. Keratinocyte growth factor accelerates compensatory growth in the remaining lung after
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ORIGINAL ARTICLE
KGFR as a possible therapeutic target in middle ear cholesteatoma
TOMOMI YAMAMOTO-FUKUDA1,2, NAOTARO AKIYAMA2, YASUAKI SHIBATA3, HARUO TAKAHASHI2, TOHRU IKEDA3, MICHIAKI KOHNO4 & TAKEHIKO KOJI1 1
Department of Histology and Cell Biology, 2Department of Otolaryngology-Head and Neck Surgery, Department of Translational Medical Science, 3Department of Oral Pathology and Bone Metabolism, and 4Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract Conclusion: We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. Objectives: Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. Methods: Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. Results: The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.
Keywords: Keratinocyte growth factor, KGF, KGF receptor inhibitor, in vivo model, electroporation
Introduction Middle ear cholesteatoma is a pathological condition associated with otitis media with serious conditions [1]. Although there is currently no more suitable treatment for middle ear cholesteatoma than surgery, it is also true that recurrence is not uncommon after surgery [2]. The recent new therapies for inflammation and cancer include the use of compounds that inhibit various receptors or cellular pathways known to be involved in inflammatory processes, cellular proliferation, and cell survival [3,4]. One of the most important pathological features of middle ear cholesteatoma is active proliferation of epithelial cells, which is considered to be stimulated by growth factors. Although our understanding of the molecular mechanism underlying the pathogenesis of
cholesteatoma is limited, among many possible growth factors, our group has focused on the role of keratinocyte growth factor (KGF) in the pathogenesis, and we have already reported that KGF/fibroblast growth factor (FGF)-7 plays important roles in human middle ear cholesteatoma formation and recurrence [5]. The paracrine actions of KGF are dependent upon KGFR, which is a transmembrane tyrosine kinase receptor with an alternatively spliced variant of FGF receptor-2 (FGFR-2)/bek gene [6]. By binding to KGFR, KGF activates various mitogenactivated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) [7]. Recently, the use of a selective inhibiter of KGFR, SU5402, in an in vitro experiment resulted in inhibition of differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from
Correspondence: Tomomi Yamamoto-Fukuda, MD PhD, Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel: +81 95 819 7026. Fax: +81 95 819 7028. E-mail: [email protected]
(Received 3 February 2014; accepted 18 March 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.907501
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cholesteatoma [8]. SU5402 is an ATP mimetic known as a KGFR-selective inhibitor with 86% homology to the KGFR tyrosine kinase domain and inhibits the tyrosine kinase activity of KGFR by interacting with the catalytic domain of KGFR with binding to the ATP-binding site [9]. The primary purpose of the present study was to analyze the role of KGF/KGFR in the molecular pathology of middle ear cholesteatoma in vivo. The secondary purpose was to investigate the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. The results suggest the promising potential of treatments designed to target KGFR. Material and methods Animals The experiments were conducted in 30 male SpragueDawley (SD) rats (6–7 weeks, 200 g) with normal tympanic membrane (TM). Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with the approval of the Institute of Animal Care and Use Committee (approval nos 0810140707 and 1107141127). Electroporation for KGF cDNA in vivo 3x Flag human KGF (hKGF) vector [10], that cording region was kindly provided by Dr. Jeffrey Rubin from the National Cancer Institute, (Bethesda, MD, USA). p3xFLAG–CMV14 vector was purchased from Sigma Chemical Co., (St Louis, MO, USA). For tissue transfection, pulses generated with a CUY21 Electroporator (Nepa Gene Co., Chiba, Japan) were delivered to the tissuebya pair ofelectrodesattached tothe tip ofcustommade tweezers (Meiwa Shoji, Tokyo, Japan) with six pulsesof50 V for80 ms, eachseparated by 1.0 s [11].The SD rat was anesthetized with pentobarbital, then FlaghKGF DNA plasmid driven by a CMV14 promoter was injected at 50 mg into the epithelial lesion of the right external auditory canal (EAC), injected five times every fourth day. For the left ear, a Flag-plasmid driven by a CMV14 promoter was injected and used as control.
we first administered 200 mM, 2 mM SU5402 in 2% DMSO in PBS, or 200 mM, 2 mM, 20 mM PD0325901 in 0.2% DMSO in PBS by eardrops, 50 ml per day every 24 h from day 0 to day 4 in the right ear of three rats after hKGF cDNA transfection. In three of three (100%) specimens of 200 mM PD0325901-treated ears, two of three (67%) specimens of 2 mM PD0325901-treated ears, and three of three (100%) specimens of 200 mM SU5402-treated ears, diphosphorylated (p)-ERK1/2-positive epithelial cells were detected (Table I). No p-ERK1/2-positive cell was detected in any specimens of 20 mM PD0325901-treated ears (Table I). One of three specimens of 2 mM SU5402-treated ears had p-ERK1/ 2-positive cells, but the number of positive epithelial cells was decreased (Table I). Also, 2 mM SU5402 in 2% DMSO in PBS, or 20 mM PD0325901 in 0.2% DMSO in PBS was administered by eardrops, 50 ml per day every 24 h from day 0 to day 23 in the right ear of five rats after vector transfection. In the control left ears, 50 ml of 2% DMSO in PBS was administered after vector transfection. Otoendoscopic examination In the present study, cholesteatoma formation was evaluated by otoendoscopy. At day 7 after the final injection, and day 23 in the KGFR inhibition experiments, SD rats at each time-point underwent otoendoscopic examination with a rigid rod 0 otoendoscope (AVS Co., Tokyo, Japan). The findings were scored semiquantitatively (Table II). Tissue preparation After tissue transfection, each animal was anesthetized with pentobarbital 150 mg/kg intraperitoneally, Table I. Dose-dependent inhibition of ERK phosphorylation by inhibitors. Group
p-ERK1/2 Positive positive case no. rate (%)
KGF + 200 mM SU5402 (n = 3)
3
100
KGF + 2 mM SU5402 (n = 3)
1
33
KGF + 200 mM PD0325901 (n = 3)
3
100
Administration of KGFR inhibitor or MEK inhibitor in vivo
KGF + 2 mM PD0325901 (n = 3)
2
67
KGF + 20 mM PD0325901 (n = 3)
0
0
The effect of KGFR inhibitor on cholesteatoma formation was examined after eardrop administration of KGFR-selective inhibitor (SU5402; Calbiochem, Darmstadt, Germany) or MEK-inhibitor (PD0325901; prepared by Michiaki Kohno [12]). To determine the effective concentration of inhibitors,
KGF + 2% DMSO (n = 5)
5
100
KGF + SU5402, hKGF expression vector transfected ear with 200 mM or 2 mM SU5402 in 2% DMSO treatment; KGF + PD0325901, hKGF expression vector transfected ear with 200 mM, 2 mM or 20 mM PD0325901 in 0.2% DMSO treatment; KGF + 2% DMSO, hKGF expression vector transfected ears with 2% DMSO treatment.
KGFR inhibitor prevents cholesteatoma formation
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Table II. Criteria for the classification of the pathological state of ears. Otoendoscopic examination
H&E examination
Retraction of TM
Debris
Otorrhea with fungus
Skin erosion
Squamous epithelium of TM
Necrotic and granulation tissue in EAC
Cell infiltration in middle ear
Cholesteatoma
+
+
–
–
+
–
+
Inflammation
–
–
–
+
–
–
+
Malignant external otitis
–
–
+
+
–
+
+
Normal
–
–
–
–
–
–
–
Pathological state
EAC, external auditory canal; H&E, hematoxylin and eosin stain; TM, tympanic membrane.
and the EAC tissues were removed. Half of each sample was fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (RT) overnight and embedded in paraffin, while the other half was frozen immediately on dry ice and later used for western blot analysis. The temporal bones were removed en bloc and fixed with 4% PFA in PBS at 4 C for 18 h. After fixation, the temporal bones were decalcified using 10% ethylenediaminetetraacetic acid at 4 C for 7 days [13] and embedded in paraffin. Sections (5 mm thick) were prepared and then mounted on 3-aminopropyltriethoxysilane-coated glass slides. Hematoxylin and eosin (H&E) staining The sections of temporal bones were stained with H&E. The pathological findings of middle ear cholesteatoma include stratified squamous epithelium with inflamed subepithelium that lines the middle ear cavity [14]. The presence of a thin layer of simple squamous epithelium in the TM was considered to be normal. The presence of infiltrating cells including lymphocytes, plasma cells, and fibroblasts represented inflammation. The presence of necrotic and granulation tissue in the EAC represented malignant external otitis. The details are described in Table II.
was separated and transferred onto PVDF membrane (Immobilon, Millipore Corp., MA, USA). The membranes were blocked with Blocking one (Nacalai tesque, Kyoto, Japan) and then incubated with the 0.5 mg/ml horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-Flag M2 antibody (Sigma). After washing with 0.2% Tween 20 in PBS, signals were visualized by the ECL system (Amersham Biosciences, NJ, USA). For p-ERK reaction, the specimens (150–200 mg each) of EAC tissues were homogenized in EzRIPA Lysis kit (ATTO Co., Tokyo, Japan) on ice. After the supernatants were collected, the protein concentration of each preparation was determined as described above. Immunoprecipitation was done using anti-ERK 1 (K-23) (Santa Cruz Biotechnology, Inc., CA, USA) antibody according to the protocol of the Immunoprecipitation Starter Pack. The membrane was incubated with mouse monoclonal anti-MAPK activated (pERK1&2) antibody (1:5000 dilution; Sigma) overnight, washed with TBS-T, and reacted with ECL anti-mouse IgG for 1 h. After washing with TBS-T, signals were visualized using the Amersham ECL Prime system. As a control, after stripping the antibodies, the same membranes were stained with rabbit polyclonal anti-actin (H-196) antibody (1:1000 dilution; Santa Cruz).
Western blot analysis of Flag expression and p-ERK1/2 Immunohistochemistry for KGF and p-ERK1/2 Western blot analysis was performed to determine the expression of Flag-hKGF after vector transfection in EAC tissues, and to determine the expression of p-ERK1/2 after vector transfection with KGFR inhibitor in EAC tissue. Liver tissue was processed as a negative control. For Flag-hKGF reaction, the specimens (150–200 mg each) were frozen immediately on dry ice and homogenized with a Polytron tissue disrupter (Kinematica, Lucerne, Switzerland) in lysis buffer [15]. The protein concentration of each preparation was determined using a kit from Bio-Rad Laboratories (Tokyo, Japan). Each lysate (50 mg)
Enzyme immunohistochemistry was performed using the paraffin sections to determine the expression of KGF and p-ERK1/2 in other sections. A polyclonal antibody against KGF was prepared by immunization of rabbits against synthetic peptides in cooperation with Nichirei Co. (Tokyo, Japan), as described previously [5]. The sections were incubated with the primary antibody of 0.5 mg/ml rabbit polyclonal anti-KGF antibody [5] or mouse monoclonal anti-pERK1/2 antibody (1:50 dilution) washed with 0.075% Brij 35 in PBS, and reacted with the
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secondary antibody. After washing the slides, HRP sites were visualized with 3,3¢-diaminobenzidine and H2O2. HRP-goat anti-mouse IgG (1:100 dilution; Chemicon International, Inc., CA, USA) or HRPgoat anti-rabbit IgG (1:100 dilution; Medical & Biological Laboratories Co., Nagoya, Japan) was used as the second antibody. For the negative control, normal mouse IgG (Sigma) or normal rabbit IgG (Sigma) was used instead of the primary antibody in every experiment. Statistical analysis The Pearson’s correlation coefficient test was used to compare the rate of pathological state between each groups. All analyses were performed with statistical software packages (JMP 10.0.2; SAS Institute Inc., NC, USA). Results Detection of hKGF vector transfection in EAC To evaluate the expression of transfected cDNA in EAC tissues, we first examined the expression of Flag by western blotting with HRP-conjugated anti-Flag antibody. Analysis of extracts from Flag-hKGF vector-transfected EAC indicated only a single 28 kDa band [16] at day 1 and at day 4 (Figure 1, lanes 1 and 3). In contrast, no specific band was found in extracts from empty vector-transfected EAC on any day (Figure 1, lanes 2 and 4). Actin (43 kDa band) was detected in all extracts at equal levels (data not shown). Immunohistochemical studies of EAC tissues using anti-KGF antibody identified KGF protein in almost all epithelial cells and some stromal cells in sections of KGF-transfected ears at day 1 and at day 4 (Figure 2a and b). In contrast, KGF-positive cells were not found in empty vector-transfected EAC on any day (Figure 2c and d). These results indicated successful in vivo transfection of hKGF vector. PD1
PD4
Expression of p-ERK in EAC of SD rat To determine the effects of KGFR inhibitor and MEK inhibitor against MAPK activation induced by hKGF cDNA transfection, the immunoreactivity of p-ERK1/2 was analyzed. By western blot analysis, only one 44 kDa band was detected in the extracts from the specimen after hKGF vector transfection with 2% DMSO in PBS treatment (Figure 3a, lane 3), the intensity of band was decreased in the extracts from the specimen with 2 mM SU5402 treatment (Figure 3a, lane 1) and no band was detected in the extracts from the specimen with 20 mM PD0325901 treatment (Figure 3a, lane 2) using anti-p-ERK1/2 antibody. Actin (43 kDa band) was detected in all the extracts (data not shown). Virtually no epithelial cells were stained in any of the tissues from hKGF vector-transfected rats treated with 2 mM SU5402 (Figure 3b) or 20 mM PD0325901 (Figure 3c), whereas many intensely stained epithelial cells were detected in tissues from hKGF vectortransfected rats treated with 2% DMSO in PBS (Figure 3d). These results indicated that treatment with 2 mM SU5402 or 20 mM PD0325901 inhibited hKGF vector-induced KGFR phosphorylation. Effect of KGFR signaling inhibition on cholesteatoma formation in vivo To clarify the efficacy of KGFR inhibitor in suppressing cholesteatoma formation, we analyzed the ears by otoendoscopy and histology. At day 7 after the final transfection of hKGF cDNA and treatment with a
b
c
d
KGF Empty KGF Empty Liver 28 kDa
Figure 1. Western blot analysis of Flag on transfected tissues. KGF, lysate of external auditory canal (EAC) of hKGF expression vector transfected ears; Empty, lysate of EAC of empty vector transfected ears; Liver, lysate of liver of Sprague-Dawley rats. Each lysate was obtained from the tissue after vector transfection at day 1 (PD1) and at day 4 (PD4). The total volume applied was 50 mg per lane. Each lane was reacted with 0.5 mg/ml HRPconjugated anti-FlagM2 antibody. Flag proteins were detected in hKGF expression vector transfected ears at day 1 and day 4. hKGF expression vector was successfully transfected.
Figure 2. Immunohistochemistry using anti-KGF antibody. (a, b) hKGF cDNA transfected ear (a, day 1; b, day 4); (c, d) empty vector transfected ear (c, day 1; d, day 4). KGF-positive cells are stained in brown. Intense staining for KGF was detected in stromal cells and some epithelial cells (a, b) and weak staining was detected within the hair follicles (d). Arrows indicate positive cells. Bar = 20 mm.
KGFR inhibitor prevents cholesteatoma formation SU5402
a
PD
DMSO
was simultaneously associated with severe necrotic reaction resulting from a defect in the capillaries of the EAC (Figure 4n) of the MEK inhibitor-treated ear. In contrast, cholesteatoma was detected in five of five SU5402-untreated ears after hKGF vector transfection (Table III, Figure 4l, o).
44 kDa
b
c
*
*
*
*
1125
d
Discussion The major finding of the present study was that treatment with SU5402, a KGFR-selective inhibitor, suppressed the formation of middle ear cholesteatoma without any complications of the middle ear. Previously, animal models of middle ear cholesteatoma have been described and their morphological aspects were almost similar to those in the human [17,18]. In the present study, KGF expression vector was selected based on the understanding of the pathogenesis of cholesteatoma [5]. KGF cDNA transfected repetitively into the cells of EAC induced a thick epithelium with keratin pearl in tympanic membrane (Figure 4o), and 2 months later, cholesteatoma formation expanded into the bulla, and bone resorption was noted at the advancing front of the cholesteatoma (data not shown), similar to the findings of a previous study [18]. The goal of experiments using animal models is to enable analysis of the pathogenesis of human disease and establish new therapies. There is currently no more suitable treatment for middle ear cholesteatoma than surgery. In the present study, we used a selective inhibitor of KGFR, SU5402, which interacts with the catalytic domain of KGFR. It is known that KGF reacts only with KGFR, a tyrosine kinase FGFR-2 IIIb and a spliced variant of FGFR2 [6]. KGF signal transduction requires activation of the Ras/MAPK pathway including the ERK pathway, or can alternatively proceed via the PI3K/Akt pathway [19]. In the present study, we described the effect of KGFR inhibitor, and also investigated the effect of MEK inhibitor (PD0325901) [12] during cholesteatoma
Figure 3. Immunoreactivity of diphosphorylated (p)-ERK1/2. (a) Western blot analysis of ear tissues. SU5402, lysate of hKGF cDNA transfected ear with 2 mM SU5402; PD, lysate of hKGF cDNA transfected ear with 20 mM PD0325901 treatment; DMSO, lysate of hKGF cDNA transfected ear with DMSO treatment. Each lane was reacted with anti- p-ERK1/2 antibody. Note that the intensity of band was decreased in SU5402-treated ear and no band was detected in PD0325901-treated ear. (b–d) Immunohistochemistry using anti-p-ERK1/2 antibody. (b) KGF cDNA transfection with 2 mM SU5402 treatment. (c) KGF cDNA transfection with 20 mM PD0325901 treatment. (d) KGF cDNA transfection with DMSO treatment. Note the lack of staining with anti-p-ERK1/2 antibody in epithelial cells of hKGF cDNA transfected ear treated with SU5402 (b) or PD0325901 (c). In the section of hKGF cDNA transfected ear without inhibitor, p-ERK1/2 positive cells were stained brown (arrows). *: epithelium.
2 mM SU5402, cholesteatoma was not detected in any of the ears (0/5, p = 0.0067, Pearson’s correlation coefficient test) (Table III, Figure 4a–e), although chronic inflammation was found in four of five ears (Table III, Figure 4b–e). Histological examination indicated normal structure of the TM and middle ear in tissue sections from hKGF vector-transfected rats treated with SU5402 (Figure 4m). Only chronic inflammatory changes were noted in three of five ears after empty vector transfection and treatment with SU5402 (Table III, Figure 4f). MEK inhibitor also reduced middle ear cholesteatoma formation in our rat model (0/5, p = 0.0067, Pearson’s correlation coefficient test) (Table III, Figure 4g–k). But this Table III. Summary of the pathological state of ears in inhibition study. Group KGF + SU5402 (n = 5) Empty + SU5402 (n = 5) KGF + PD0325901 (n = 5)
Cholesteatoma
Inflammation
Malignant external otitis
Normal
0*
4
0
1
0
3
0
2
0*
0
5
0
Empty + PD0325901 (n = 5)
0
0
5
0
KGF + 2% DMSO (n = 5)
5
0
0
0
KGF + SU5402, hKGF expression vector transfected ear with 2 mM SU5402 in 2% DMSO treatment; Empty + SU5402, empty vector transfected ears with 2 mM SU5402 in 2% DMSO treatment; KGF + PD0325901, hKGF expression vector transfected ear with 20 mM PD0325901 in 0.2% DMSO treatment; Empty + PD0325901, empty vector transfected ears with 20 mM PD0325901 in 0.2% DMSO treatment; KGF + 2% DMSO, hKGF expression vector transfected ears with 2% DMSO treatment. *p = 0.0003.
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T. Yamamoto-Fukuda et al. a
b
c
TM
g
d
e
f
TM TM h
i
TM
j
TM
TM k
l Chole
TM
TM
m
TM
TM
TM
n
*
EAC
o
EAC Chole
EAC
TM
ME ME
Figure 4. Results of experiments conducted in rats for the KGFR inhibitory model. (a–l) Otoendscopic findings of tympanic membrane (TM). (a–e) Ears after hKGF cDNA transfection followed by treatment with SU5402. (f) Ear after empty vector transfection followed by treatment with SU5402. (g–k) Ears after hKGF cDNA transfection followed by treatment with PD0325901. (l) Ear after hKGF cDNA transfection followed by treatment with DMSO. Middle ear cholesteatoma formation was not detected in ears after hKGF cDNA transfection followed by treatment with SU5402 (a–e), although chronic inflammation with crust (arrowheads) was found in four ears (b–e). Only chronic inflammatory changes were noted after empty vector transfection and treatment with SU5402 (f). Note that the cholesteatoma formation was not detected but fungus (arrowheads) was shown in PD0325901-treated ear (g–k). Without inhibitor treatment, cholesteatoma (Chole) with debris was detected (l). Arrows: crusts, arrowheads: fungus. (m–o) H&E staining of paraffin section of bulla of the hKGF-transfected ear with or without KGFR inhibitors. (m) The hKGF-transfected ear with SU5402 treatment indicated the normal external auditory canal (EAC), TM, and middle ear (ME), which contained a thin layer of simple squamous epithelium with thin subepithelium. (n) The hKGF-transfected ear with PD0325901 treatment indicated almost normal TM with thick subepithelial layer (star), and necrotic tissue was shown in the EAC lesion (asterisk). (o) Cholesteatoma (Chole) formation was induced after five times hKGF cDNA transfection. Note the keratin pearl formation within the stratified squamous epithelium and thicker subepithelial layer (see box).
formation. As expected, MEK inhibitor also reduced middle ear cholesteatoma formation in our rat model, but this was simultaneously associated with severe necrotic reaction resulting from a defect in the capillaries of the EAC (Figure 4n). Indeed, it is known that paradoxical MAPK activation through the use of a BRAF inhibitor seems to be involved in the development of certain types of cancer [20]. In experiments using SU5402, we demonstrated that p-ERK was not detected and cholesteatoma formation was reduced without any side effects. We believe that our in vivo study extended the in vitro findings and enhanced our understanding of the pathogenesis of cholesteatoma, especially as regards the interaction between the epithelium and stroma and its role in cholesteatoma formation. In conclusion, overexpression of KGF plays an important role in the pathogenesis of middle ear cholesteatoma. Furthermore, the use of a selective KGFR inhibitor reduced middle ear cholesteatoma in our rats. These results suggest that the key molecules in the KGFR signaling pathway are a potentially suitable therapeutic target in the design of new treatment for human middle ear cholesteatoma. Of course we know
that we should assess the ototoxicity of SU5402 when it is applied to the EAC or middle ear by ear drops; after this step SU5402 treatment may actually become a therapeutic approach for humans.
Acknowledgments The authors thank Dr. Jeffrey S. Rubin from the National Cancer Institute/CCR/LCMB, for providing the human KGF cDNA construct. The study was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (no. 23791906 to T.Y-F.) and by a grant from the Naito Foundation (to T.Y-F.). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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