Acta Oto-Laryngologica. 2014; 134: 238–243
ORIGINAL ARTICLE
The oncoprotein, gankyrin, is up-regulated in middle ear cholesteatoma
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KI HYUN KIM1, HYE JIN LIM1, YEON JU KIM1, SEUNG WON KIM1, YOUNG SUN KIM1, CHUNJIE TIAN1, KEEHYUN PARK1, TAE JUN PARK2 & YUN-HOON CHOUNG1 1
Department of Otolaryngology and 2Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Republic of Korea
Abstract Conclusion: Gankyrin seems to be a better biomarker for cholesteatoma compared with Ki-67. Objective: Gankyrin is an oncoprotein, and occurs in cancers but not in benign diseases. The goal of this study was to compare expression of gankyrin, p53, and a proliferation marker (Ki-67) in cholesteatoma and retroauricular skin (RAS), and to evaluate their significance as clinical parameters. Methods: The levels of expression of gankyrin, Ki-67, and p53 in 10 cholesteatoma and 10 paired samples of normal RAS were evaluated by immunohistochemical staining and Western blot. The results were compared with clinical profiles to investigate a correlation. Results: The expression of gankyrin, Ki-67, and p53 proteins was observed in both basal and suprabasal layers of cholesteatoma. The intensity of gankyrin expression was ‘positive’ in two cases (20%) and ‘strongly positive’ in eight cases (80%); p53 expression in the suprabasal layer was ‘positive’ in 70% of cases; and the Ki-67 staining was ‘focal’ in 80% of cases. In RAS, these proteins were expressed dominantly in the basal layer. Western blot analysis showed that the gankyrin band was more intense in cholesteatoma than in RAS for three of four cases (p < 0.05). However, there was no significant difference in the expression of gankyrin, Ki-67, and p53 according to clinical variables.
Keywords: p53, Ki-67, hyperproliferation
Introduction Middle ear cholesteatoma is a benign, hyperproliferative, and hyperdifferentiated disease characterized by the accumulation of excessive keratin debris released by excessive numbers of keratinocytes in the epidermis. In addition, cholesteatoma has characteristics of migration and local invasion similar to a neoplasm, but it seems to be modulated by a delicate balance of proliferation and apoptosis. Together with the development of molecular biology techniques, many efforts have so far been made to assess the mechanism and to examine biologic behaviors of cholesteatoma, particularly using antibodies such as antibodies against beta-defensins and Ki-67 [1,2]. Even though cholesteatoma has been explained by the hyperproliferative nature of epithelia due to decreased regulatory
function in the cell cycle [3], its exact pathogenesis remains elusive. In a normal epidermis, the proliferation and differentiation of cells are dependent on the control of the cell cycle. Oncogenes and tumor suppressor genes are tightly regulated for the homeostasis of the cell cycle. Gankyrin is a p28 oncoprotein, which has recently been identified in human malignant tumors such as hepatocellular carcinoma, colorectal cancer, and oral squamous cell carcinoma [4,5]. It binds with murine double minute 2 (MDM2) and facilitates p53 degradation to sustain cell cycle progression [5]. p53, one of the tumor suppressor proteins, is known to initiate apoptosis and promote cell differentiation by checkpoint control. However, different results have been presented for p53 expression in cholesteatoma epithelium. Huisman et al. [6]
Correspondence: Yun-Hoon Choung MD DDS PhD, Department of Otolaryngology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. Tel: +82 31 219 5263. Fax: +82 31 219 5264. E-mail: [email protected] Tae Jun Park MD PhD, Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. E-mail: [email protected]
(Received 5 July 2013; accepted 1 September 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.844362
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Gankyrin expression in cholesteatoma reported that the expression of p53 protein in cholesteatoma was more prominent than in retroauricular skin (RAS), with statistical significance (p = 0.007), whereas Motamed et al. [7] observed minimal difference in immunoreactivity between cholesteatoma and deep meatal skin. Ki-67 has been studied as a marker for cell proliferation in cholesteatoma [1], and its expression pattern is dependent on the cell cycle. Ki-67 protein is usually detected in active phases of the cell cycle, while not detected during G0 phase, thus the staining of Ki-67 protein varies depending on the stage of the cell division cycle [8]. Since the nature of cholesteatoma has never been evaluated before by means of anti-gankyrin antibody, we thought that the study of gankyrin expression might help elucidate its role in the pathogenesis of cholesteatoma. To understand the correlation between gankyrin expression in cholesteatoma and RAS, we also evaluated the expression of p53 and Ki-67. Material and methods Ten cholesteatoma samples and 10 paired RAS samples were collected from the middle ears of patients with cholesteatoma who were treated at the Department of Otolaryngology, Ajou University Hospital between March 2011 and May 2012. RAS samples served as normal controls. All patients signed the informed consent before the middle ear operations, and the study was performed with the approval of the Institutional Review Board at Ajou University Hospital (IRB number: AJIRB-MED-SMP-11-198). Immunohistochemical staining Specimens were fixed in 4% paraformaldehyde for immunohistochemical staining and embedded in paraffin. They were serially sliced at a 4.5 mm thickness and then 1:100 anti-gankyrin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53 (Santa Cruz Biotechnology), and anti-Ki-67 (Bioss Inc., Woburn, MA, USA) antibodies, respectively, were applied to the sections. After overnight incubation at 4 C, the slides were rinsed three times with phosphate-buffered saline (PBS) and incubated with secondary antibodies for 25 min and streptavidin-horseradish peroxidise (HRP) from the Cap-Plus Detection Kit (Invitrogen, Carlsbad, CA, USA) for 25 min. After washing with PBS, the samples were exposed to 3,3’-diaminobenzidine tetrahydrochloride (DAB) for up to 5 min and counterstained with hematoxylin. According to its brown colorization, the intensity of staining on blinded slides was classified as ‘negative’ (no immunoreactivity), ‘focal’ (75% positive cells) by two independent observers [9]. Western blot Samples of both freshly frozen cholesteatoma and RAS were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% sodium dodecylsulfate) and left on ice for 30 min. The homogenate was centrifuged at 13 000 rpm at 4 C for 30 min. The supernatants were then collected and their protein content was measured by protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates were resolved on 12% SDS-PAGE and the gels were then transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) before incubation with the following antibodies at room temperature for 1 h, or at 4 C overnight. The antibodies anti-gankyrin and anti-p53 were obtained from Santa Cruz, and anti-Ki-67 was from Bioss Inc. Densitometric analysis of bands was performed by ImageJ software. Clinical evaluation The bone destruction grade and disease extent were measured by CT scan and intraoperative findings. For measurement of the severity of bone destruction, we allocated 1 point when only one ossicle was involved. If two or more ossicles were involved, it was counted as 2 points. Also, 2 points were allocated for tegmen erosion, 2 points for facial nerve canal erosion, and 2 points for bony lateral semicircular canal erosion. Therefore, if erosion was shown in all the ossicles and tegmen, 4 points were allocated. Otorrhea was confirmed by otoscopy in the outpatient clinic and swabs were taken to the Department of Laboratory Medicine in Ajou University Hospital to identify the microorganism. Statistical analysis The SPSS 12.0 software package (SPSS, Chicago, IL, USA) was used for statistical analysis. All numerical values were expressed as means ± standard deviation (SD) and a p Value < 0.05 was considered to be statistically significant. The Mann–Whitney test was used to compare the means of gankyrin expression in Western blot between RAS and cholesteatoma samples. Results Among the 10 cholesteatoma samples in this study, 8 cases were diagnosed as having acquired cholesteatoma and 2 cases were congenital. The male to female
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Table I. Immunohistochemical expression of gankyrin, Ki-67, and p53 in middle ear cholesteatoma and retroauricular skin (RAS). Staining
Gankyrin Cholesteatoma
Ki-67 RAS
p53
Cholesteatoma
RAS
Cholesteatoma
RAS
Negative
0
0
0
3
0
0
0
4
0
0
0
7
Focal
0
0
0
4
8
8
0
3
0
1
0
3
Positive
2
2
0
1
1
1
2
2
6
7
1
0
Strongly positive
8
8
10
2
1
1
8
1
4
2
9
0
ratio was 4:6, and ages ranged from 4.3 to 73 years old (mean age 37.9 ± 23.5 years). Bone destruction grade ranged from 1 to 4 (mean grade 2.9 ± 1.2), and three patients had infection, showing microorganisms on the otorrhea cultures. Immunohistochemical staining of gankyrin, Ki-67, and p53 Immunohistochemical staining of gankyrin in cholesteatoma samples showed brownish colorization in the stratum basale, spinosum, and granulosum. Table I shows that gankyrin expression was detected in both
the basal and suprabasal layers of cholesteatoma epithelia. The intensity was more than positive, and both nuclear and cytoplasmic staining was observed (Figure 1B). Furthermore, immunoreactivity was more intense in basal and spinous layers than in the granular layer of the cholesteatoma samples, whereas staining of the corneal layer was negative (Figure 1B). In the RAS samples, the gankyrin expression in the basal layer was strongly positive in 100% (10/10) of cases (Figure 1F). p53 and Ki-67 were mainly expressed in the basal layer of RAS specimens and the intensity was positive and strongly positive, respectively. p53 and Ki-67
Gankyrin
Ki-67
p53
B
C
D
F
G
H
Chole
A
E-1
E-2
RAS
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Intensity of staining Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal layer layer layer layer layer layer layer layer layer layer layer layer
Figure 1. Clinical profiles with immunohistochemical staining of gankyrin, Ki-67, and p53 in serial sections of cholesteatoma and paired retroauricular skin (RAS) samples. (A) Otorrhea and attic destruction were revealed in the right ear as the otoscopic appearance in the outpatient clinic. (B, C, and D) Gankyrin, Ki-67, and p53 expression of cholesteatoma was mainly detected in the stratum basale, spinosum, and granulosum. The intensity of gankyrin and p53 expression was stronger than that of Ki-67. (E-1 and E-2) On CT scan, cholesteatoma extended to the ossicles and mastoid cavity in the right ear. (F, G, and H) In RAS, the antibodies were stained in the basal layer. Chole, cholesteatoma. Insets show high magnification fields. Arrows indicate the representative staining cells. Scale bars = 50 mm.
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Gankyrin expression in cholesteatoma
A
B
Gankyrin (28 kDa) β-actin
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Skin chole Patient 1
Skin chole Patient 2
Skin chole Patient 3
Skin chole Patient 4
Relative gankyrin level (%)
*
*
*
*
140 120 100 80 60 40 20 0
Skin chole Skin chole Skin chole Skin chole Patient 1 Patient 2 Patient 3 Patient 4
Figure 2. Western blot analysis of gankyrin in cholesteatoma and paired retroauricular skin (RAS) samples. (A) The protein levels of gankyrin were up-regulated in most of the cholesteatoma samples. (B) Comparison of gankyrin expression in cholesteatoma and the corresponding RAS. Data analysis was performed by the Mann–Whitney test. Chole, cholesteatoma; skin, retroauricular skin. *p < 0.05.
expression in the middle ear cholesteatoma was found in the stratum basale, spinosum, and granulosum, which was similar to the pattern of gankyrin expression. Antibodies to both p53 and Ki-67 were detected in the suprabasal layer, but the intensity of the Ki-67 staining was focal in 80% (8/10) of cholesteatoma cases. p53 expression in the suprabasal layer of cholesteatoma was positive in 70% (7/10) of cases, and the expression patterns of p53 and Ki-67 in cholesteatoma samples tended to be more diffuse than in RAS samples (Figure 1C, D, G, H). Western blot analysis of gankyrin Western blot analysis was carried out on 10 paired samples of cholesteatoma and RAS. Among them, appropriate results were identified in four paired samples, which were identified as otorrhea and showed 4 points on the bone destruction grade. Intense bands of gankyrin with approximately 28 kDa protein were detected in cholesteatoma for three paired samples (p < 0.05; Figure 2A), whereas gankyrin protein was faintly detected in RAS. However, a reverse result was found in one paired sample, indicating that the gankyrin protein rather increased in RAS compared with cholesteatoma (p < 0.05; Figure 2B). There was no significantly specific relationship between the level of gankyrin protein and clinical parameters. Clinical correlation We attempted to correlate the findings for expression of gankyrin, p53, and Ki-67 in cholesteatoma and RAS samples with clinical profiles, such as bone destruction grade, otorrhea, and recurrence [10]. In all, 80% (8/10) of cholesteatoma cases showed otorrhea. In both basal and suprabasal layers of the eight otorrhea cases, the expression of gankyrin was
detected as strongly positive in six cases and positive in the other two cases. However, the gankyrin protein was also expressed as strongly positive in two nonotorrhea cases, each showing 1 and 2 points on the bone destruction grade, respectively. Of the eight otorrhea cases, Ki-67 expression was focal in 75% of cases (6/8). The expression of p53 protein was positive in 62.5% (5/8) and strongly positive in 25% (2/8) of otorrhea cases; however, the two non-otorrhea cases also showed positive staining. Two cases were diagnosed as having recurrent cholesteatoma and the expression of gankyrin was strongly positive in the basal and suprabasal layers. The expression patterns of the relevant antibodies showed no significant differences according to clinical variables. Discussion Pathophysiologically, middle ear cholesteatoma may be caused by altered growth capacity of the epithelial basal layer, which was originally considered to have a proliferative property. Unlike malignant cancers, cholesteatoma not only proliferates, but apoptosis may also be increased [11]. Recently, the proliferative capacity of cholesteatoma has been evaluated by using Ki-67 and proliferating cell nuclear antigen (PCNA), and its apoptotic feature was estimated by studying tumor suppressor genes such as connexin 26, connexin 43, and p53 [1,12]. However, despite considerable efforts, the pathogenesis of cholesteatoma remains largely unknown. To the best of our knowledge, the present study is the first attempt to elucidate a possible role of gankyrin expression as a mechanism of cholesteatoma. Gankyrin, which has recently been introduced as a protein with ankyrin repeats in malignant cancers, especially in hepatocellular carcinoma, interacts with cyclin-dependent kinase 4 to degrade the phosphorylated retinoblastoma protein. It also activates
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E2F-dependent transcription factors of DNA synthesis and functions in the proteosomal degradation and ubiquitination of p53 protein by binding the ubiquitin ligase [5]. Therefore, during the development of a malignancy from a benign status, gankyrin inhibits two important tumor suppressor proteins to facilitate cell cycle progression and anti-apoptosis. A study conducted on gankyrin expression in otolaryngology, which is related to human oral cancer, showed that immunoreactivity of gankyrin in oral squamous cell carcinoma was stronger than in normal oral epithelium [13]. In the present study, we compared cholesteatoma and RAS in terms of gankyrin protein expression and found that gankyrin protein was overexpressed in cholesteatoma epithelia compared with RAS samples. This suggests that up-regulation of gankyrin may play a proliferative role as regards keratinocytes in cholesteatoma epithelia, thus participating in the pathogenesis of cholesteatoma. To sustain appropriate cell cycle progression, normal epithelium maintains a balance between a proliferation and apoptosis [12], and a proliferation of keratinocytes in cholesteatoma is considered to be in a delicate equilibrium status with an apoptosis [14]. Unlike in malignant tumors, p53 expression in cholesteatoma was found to be much more intense than that in RAS samples. Since gankyrin mediates the degradation of p53 in hepatocellular carcinomas [15], an increased level of gankyrin could possibly decrease p53 in cholesteatoma, as shown in some samples in the present study. However, the level of p53 expression in cholesteatoma may vary in different clinical situations. For example, since cholesteatoma is considered to be a benign, chronic inflammatory disease, cell proliferation could elevate the level of the p53 protein [16] and increased levels of reactive oxygen species could also activate p53 [17]. Furthermore, MDM2 protein expression is down-regulated in the basal layer compared with the suprabasal layer in the skin [18]. MDM2 is E3 ubiquitin ligase and involved in p53 protein degradation. Gankyrin binds with MDM2 protein and is involved cooperatively in p53 protein degradation [5]. Cholesteatoma has hyperproliferative characteristics of the basal layer and we speculated that gankyrin could not degrade p53 protein because of insufficient MDM2 protein in cholesteatoma. This is one of the reasons that gankyrin and p53 proteins, which are thought to have opposite functions, showed similar cellular localization in cholesteatomas. The present study indicated that the apoptotic feature of p53 was still functioning in cholesteatoma, and gankyrin was highly expressed, implying that cholesteatoma still maintains its benign characteristic.
In the present study, the expression of gankyrin was also detected in RAS, especially in the basal layer. Basal keratinocytes in normal skin, which is separated from the dermis by the basement membrane, may progress throughout the proliferation and differentiation by epidermal stem cells and move up to constitute the suprabasal epithelial cell layer. Gankyrin is known as a subunit of the 26S proteasome complex. In normal cells, 26S proteasome acts as a protease to degrade many intracellular ubiquitinated proteins to establish cell viability. This implies that gankyrin in RAS plays a fundamental role in the normal cell cycle and maintains its homeostasis [13]. Ki-67 is recognized in actively proliferating cells during the G1, S, and G2 phases of the cell cycle, but is absent in the G0 phase. The expression of anti-Ki67 antibody in cholesteatoma has previously been reported as a proliferation marker, compared with normal deep meatal skin and RAS [10]. In the present study, however, the intensity of Ki-67 staining was weaker than that of gankyrin in most cholesteatoma samples, although some specimens showed a positive expression. Furthermore, clinical profiles demonstrated that otorrhea was reported in 8 (80%) of the 10 cases and at least 2 ossicles were eroded in 9 (90%) of the 10 cases. Among these nine cases, the tegmen, facial nerve canal, and bony lateral semicircular canal were also found to be eroded in five cases. Earlier it was proposed that Ki-67 is immunoreactively positive, especially on actively growing keratinocytes; therefore, the results of the present study suggest that the keratinocytes of the samples with otorrhea and a higher grade of bone destruction may occur in cellular senescence or inactive status after hyperproliferation of cholesteatoma epithelium is completed at some point of the cell cycle. Conclusion Gankyrin seems to be overexpressed more than Ki-67 and p53 in middle ear cholesteatoma, compared with RAS. The results of the present study suggest that up-regulated gankyrin may be one of the markers for hyperproliferation of cholesteatoma and may be related to the pathogenesis of cholesteatoma. The role of gankyrin expression in cholesteatoma needs to be further investigated to gain an insight into its treatment.
Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry
Gankyrin expression in cholesteatoma of Education Science and Technology (20100010678). Declaration of interest: The authors declare no conflict of interest.
[9]
[10]
[11]
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References [1] Mallet Y, Nouwen J, Lecomte-Houcke M, Desaulty A. Aggressiveness and quantification of epithelial proliferation of middle ear cholesteatoma by MIB1. Laryngoscope 2003; 113:328–31. [2] Park K, Moon SK, Choung YH, Choi HS. Expression of beta-defensins in human middle ear cholesteatoma. Acta Otolaryngol 2003;123:236–40. [3] Byun JY, Yune TY, Lee JY, Yeo SG, Park MS. Expression of CYLD and NF-kB in human cholesteatoma epithelium. Mediators Inflamm 2010;2010:1–6. [4] Wakamori M, Umehara T, Yokoyama S. A series of bacterial co-expression vectors with rare-cutter recognition sequences. Protein Exp Purif 2010;74:88–98. [5] Higashitsuji H, Higashitsuji H, Itoh K, Sakurai T, Nagao T, Sumitomo Y, et al. The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell 2005;8:75–87. [6] Huisman MA, De Heer E, Grote JJ. Cholesteatoma epithelium is characterized by increased expression of Ki-67, p53 and p21, with minimal apoptosis. Acta Otolaryngol 2003;123:377–82. [7] Motamed M, Powe D, Kendall C, Birchall JP, Banerjee AR. p53 Expression and keratinocyte hyperproliferation in middle ear cholesteatoma. Clin Otolaryngol Allied Sci 2002;27:505–8. [8] Raynov AM, Moon SK, Choung YH, Hong SP, Park K. Nucleoplasm staining patterns and cell cycle-associated
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expression of Ki-67 in middle ear cholesteatoma. Am J Otolaryngol 2005;26:296–301. Sudhoff H, Dazert S, Gonzales AM, Borkowski G, Park SY, Baird A, et al. Angiogenesis and angiogenic growth factors in middle ear cholesteatoma. Am J Otol 2000;21:793–8. Vitale RF, Pereira CSB, Alves AL, Fregnani JHTG, Ribeiro FQ. TNF-R2 expression in acquired middle ear cholesteatoma. Braz J Otorhinolaryngol 2011;77:531–6. Jung MH, Lee JH, Cho JG, Jung HH, Hwang SJ, Chae SW. Expressions of caspase-14 in human middle ear cholesteatoma. Laryngoscope 2009;118:1047–50. Choung YH, Park K, Kang SO, Markov Raynov A, Ho Kim C, Choung PH. Expression of the gap junction proteins connexin 26 and connexin 43 in human middle ear cholesteatoma. Acta Otolaryngol 2006;126:138–43. Li J, Knobloch TJ, Kresty LA, Zhang Z, Lang JC, Schuller DE, et al. Gankyrin, a biomarker for epithelial carcinogenesis, is overexpressed in human oral cancer. Anticancer Res 2011;31:2683–92. Kojima H, Tanaka Y, Tanaka T, Miyazaki H, Shiwa M, Kamide Y, et al. Cell proliferation and apoptosis in human middle ear cholesteatoma. Arch Otolaryngol Head Neck Surg 1998;124:261–4. Iakova P, Timchenko L, Timchenko NA. Intracellular signaling and hepatocellular carcinoma. Semin Cancer Biol 2011;21:28–34. Allred DC, Clark GM, Elledge R, Fuqua SA, Brown RW, Chamness GC, et al. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in nodenegative breast cancer. J Natl Cancer Inst 1993;85:200–6. Macip S, Igarashi M, Berggren P, Yu J, Lee SW, Aaronson SA. Influence of induced reactive oxygen species in p53-mediated cell fate decisions. Mol Cell Biol 2003;23:8576–85. Ganguli G, Abecassis J, Wasylyk B. MDM2 induces hyperplasia and premalignant lesions when expressed in the basal layer of the epidermis. EMBO J 2000;19:5135–47.
ORIGINAL ARTICLE
The oncoprotein, gankyrin, is up-regulated in middle ear cholesteatoma
Acta Otolaryngol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
KI HYUN KIM1, HYE JIN LIM1, YEON JU KIM1, SEUNG WON KIM1, YOUNG SUN KIM1, CHUNJIE TIAN1, KEEHYUN PARK1, TAE JUN PARK2 & YUN-HOON CHOUNG1 1
Department of Otolaryngology and 2Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Republic of Korea
Abstract Conclusion: Gankyrin seems to be a better biomarker for cholesteatoma compared with Ki-67. Objective: Gankyrin is an oncoprotein, and occurs in cancers but not in benign diseases. The goal of this study was to compare expression of gankyrin, p53, and a proliferation marker (Ki-67) in cholesteatoma and retroauricular skin (RAS), and to evaluate their significance as clinical parameters. Methods: The levels of expression of gankyrin, Ki-67, and p53 in 10 cholesteatoma and 10 paired samples of normal RAS were evaluated by immunohistochemical staining and Western blot. The results were compared with clinical profiles to investigate a correlation. Results: The expression of gankyrin, Ki-67, and p53 proteins was observed in both basal and suprabasal layers of cholesteatoma. The intensity of gankyrin expression was ‘positive’ in two cases (20%) and ‘strongly positive’ in eight cases (80%); p53 expression in the suprabasal layer was ‘positive’ in 70% of cases; and the Ki-67 staining was ‘focal’ in 80% of cases. In RAS, these proteins were expressed dominantly in the basal layer. Western blot analysis showed that the gankyrin band was more intense in cholesteatoma than in RAS for three of four cases (p < 0.05). However, there was no significant difference in the expression of gankyrin, Ki-67, and p53 according to clinical variables.
Keywords: p53, Ki-67, hyperproliferation
Introduction Middle ear cholesteatoma is a benign, hyperproliferative, and hyperdifferentiated disease characterized by the accumulation of excessive keratin debris released by excessive numbers of keratinocytes in the epidermis. In addition, cholesteatoma has characteristics of migration and local invasion similar to a neoplasm, but it seems to be modulated by a delicate balance of proliferation and apoptosis. Together with the development of molecular biology techniques, many efforts have so far been made to assess the mechanism and to examine biologic behaviors of cholesteatoma, particularly using antibodies such as antibodies against beta-defensins and Ki-67 [1,2]. Even though cholesteatoma has been explained by the hyperproliferative nature of epithelia due to decreased regulatory
function in the cell cycle [3], its exact pathogenesis remains elusive. In a normal epidermis, the proliferation and differentiation of cells are dependent on the control of the cell cycle. Oncogenes and tumor suppressor genes are tightly regulated for the homeostasis of the cell cycle. Gankyrin is a p28 oncoprotein, which has recently been identified in human malignant tumors such as hepatocellular carcinoma, colorectal cancer, and oral squamous cell carcinoma [4,5]. It binds with murine double minute 2 (MDM2) and facilitates p53 degradation to sustain cell cycle progression [5]. p53, one of the tumor suppressor proteins, is known to initiate apoptosis and promote cell differentiation by checkpoint control. However, different results have been presented for p53 expression in cholesteatoma epithelium. Huisman et al. [6]
Correspondence: Yun-Hoon Choung MD DDS PhD, Department of Otolaryngology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. Tel: +82 31 219 5263. Fax: +82 31 219 5264. E-mail: [email protected] Tae Jun Park MD PhD, Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. E-mail: [email protected]
(Received 5 July 2013; accepted 1 September 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.844362
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Gankyrin expression in cholesteatoma reported that the expression of p53 protein in cholesteatoma was more prominent than in retroauricular skin (RAS), with statistical significance (p = 0.007), whereas Motamed et al. [7] observed minimal difference in immunoreactivity between cholesteatoma and deep meatal skin. Ki-67 has been studied as a marker for cell proliferation in cholesteatoma [1], and its expression pattern is dependent on the cell cycle. Ki-67 protein is usually detected in active phases of the cell cycle, while not detected during G0 phase, thus the staining of Ki-67 protein varies depending on the stage of the cell division cycle [8]. Since the nature of cholesteatoma has never been evaluated before by means of anti-gankyrin antibody, we thought that the study of gankyrin expression might help elucidate its role in the pathogenesis of cholesteatoma. To understand the correlation between gankyrin expression in cholesteatoma and RAS, we also evaluated the expression of p53 and Ki-67. Material and methods Ten cholesteatoma samples and 10 paired RAS samples were collected from the middle ears of patients with cholesteatoma who were treated at the Department of Otolaryngology, Ajou University Hospital between March 2011 and May 2012. RAS samples served as normal controls. All patients signed the informed consent before the middle ear operations, and the study was performed with the approval of the Institutional Review Board at Ajou University Hospital (IRB number: AJIRB-MED-SMP-11-198). Immunohistochemical staining Specimens were fixed in 4% paraformaldehyde for immunohistochemical staining and embedded in paraffin. They were serially sliced at a 4.5 mm thickness and then 1:100 anti-gankyrin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53 (Santa Cruz Biotechnology), and anti-Ki-67 (Bioss Inc., Woburn, MA, USA) antibodies, respectively, were applied to the sections. After overnight incubation at 4 C, the slides were rinsed three times with phosphate-buffered saline (PBS) and incubated with secondary antibodies for 25 min and streptavidin-horseradish peroxidise (HRP) from the Cap-Plus Detection Kit (Invitrogen, Carlsbad, CA, USA) for 25 min. After washing with PBS, the samples were exposed to 3,3’-diaminobenzidine tetrahydrochloride (DAB) for up to 5 min and counterstained with hematoxylin. According to its brown colorization, the intensity of staining on blinded slides was classified as ‘negative’ (no immunoreactivity), ‘focal’ (75% positive cells) by two independent observers [9]. Western blot Samples of both freshly frozen cholesteatoma and RAS were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% sodium dodecylsulfate) and left on ice for 30 min. The homogenate was centrifuged at 13 000 rpm at 4 C for 30 min. The supernatants were then collected and their protein content was measured by protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates were resolved on 12% SDS-PAGE and the gels were then transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) before incubation with the following antibodies at room temperature for 1 h, or at 4 C overnight. The antibodies anti-gankyrin and anti-p53 were obtained from Santa Cruz, and anti-Ki-67 was from Bioss Inc. Densitometric analysis of bands was performed by ImageJ software. Clinical evaluation The bone destruction grade and disease extent were measured by CT scan and intraoperative findings. For measurement of the severity of bone destruction, we allocated 1 point when only one ossicle was involved. If two or more ossicles were involved, it was counted as 2 points. Also, 2 points were allocated for tegmen erosion, 2 points for facial nerve canal erosion, and 2 points for bony lateral semicircular canal erosion. Therefore, if erosion was shown in all the ossicles and tegmen, 4 points were allocated. Otorrhea was confirmed by otoscopy in the outpatient clinic and swabs were taken to the Department of Laboratory Medicine in Ajou University Hospital to identify the microorganism. Statistical analysis The SPSS 12.0 software package (SPSS, Chicago, IL, USA) was used for statistical analysis. All numerical values were expressed as means ± standard deviation (SD) and a p Value < 0.05 was considered to be statistically significant. The Mann–Whitney test was used to compare the means of gankyrin expression in Western blot between RAS and cholesteatoma samples. Results Among the 10 cholesteatoma samples in this study, 8 cases were diagnosed as having acquired cholesteatoma and 2 cases were congenital. The male to female
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Table I. Immunohistochemical expression of gankyrin, Ki-67, and p53 in middle ear cholesteatoma and retroauricular skin (RAS). Staining
Gankyrin Cholesteatoma
Ki-67 RAS
p53
Cholesteatoma
RAS
Cholesteatoma
RAS
Negative
0
0
0
3
0
0
0
4
0
0
0
7
Focal
0
0
0
4
8
8
0
3
0
1
0
3
Positive
2
2
0
1
1
1
2
2
6
7
1
0
Strongly positive
8
8
10
2
1
1
8
1
4
2
9
0
ratio was 4:6, and ages ranged from 4.3 to 73 years old (mean age 37.9 ± 23.5 years). Bone destruction grade ranged from 1 to 4 (mean grade 2.9 ± 1.2), and three patients had infection, showing microorganisms on the otorrhea cultures. Immunohistochemical staining of gankyrin, Ki-67, and p53 Immunohistochemical staining of gankyrin in cholesteatoma samples showed brownish colorization in the stratum basale, spinosum, and granulosum. Table I shows that gankyrin expression was detected in both
the basal and suprabasal layers of cholesteatoma epithelia. The intensity was more than positive, and both nuclear and cytoplasmic staining was observed (Figure 1B). Furthermore, immunoreactivity was more intense in basal and spinous layers than in the granular layer of the cholesteatoma samples, whereas staining of the corneal layer was negative (Figure 1B). In the RAS samples, the gankyrin expression in the basal layer was strongly positive in 100% (10/10) of cases (Figure 1F). p53 and Ki-67 were mainly expressed in the basal layer of RAS specimens and the intensity was positive and strongly positive, respectively. p53 and Ki-67
Gankyrin
Ki-67
p53
B
C
D
F
G
H
Chole
A
E-1
E-2
RAS
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Intensity of staining Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal Basal Suprabasal layer layer layer layer layer layer layer layer layer layer layer layer
Figure 1. Clinical profiles with immunohistochemical staining of gankyrin, Ki-67, and p53 in serial sections of cholesteatoma and paired retroauricular skin (RAS) samples. (A) Otorrhea and attic destruction were revealed in the right ear as the otoscopic appearance in the outpatient clinic. (B, C, and D) Gankyrin, Ki-67, and p53 expression of cholesteatoma was mainly detected in the stratum basale, spinosum, and granulosum. The intensity of gankyrin and p53 expression was stronger than that of Ki-67. (E-1 and E-2) On CT scan, cholesteatoma extended to the ossicles and mastoid cavity in the right ear. (F, G, and H) In RAS, the antibodies were stained in the basal layer. Chole, cholesteatoma. Insets show high magnification fields. Arrows indicate the representative staining cells. Scale bars = 50 mm.
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Gankyrin expression in cholesteatoma
A
B
Gankyrin (28 kDa) β-actin
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Skin chole Patient 1
Skin chole Patient 2
Skin chole Patient 3
Skin chole Patient 4
Relative gankyrin level (%)
*
*
*
*
140 120 100 80 60 40 20 0
Skin chole Skin chole Skin chole Skin chole Patient 1 Patient 2 Patient 3 Patient 4
Figure 2. Western blot analysis of gankyrin in cholesteatoma and paired retroauricular skin (RAS) samples. (A) The protein levels of gankyrin were up-regulated in most of the cholesteatoma samples. (B) Comparison of gankyrin expression in cholesteatoma and the corresponding RAS. Data analysis was performed by the Mann–Whitney test. Chole, cholesteatoma; skin, retroauricular skin. *p < 0.05.
expression in the middle ear cholesteatoma was found in the stratum basale, spinosum, and granulosum, which was similar to the pattern of gankyrin expression. Antibodies to both p53 and Ki-67 were detected in the suprabasal layer, but the intensity of the Ki-67 staining was focal in 80% (8/10) of cholesteatoma cases. p53 expression in the suprabasal layer of cholesteatoma was positive in 70% (7/10) of cases, and the expression patterns of p53 and Ki-67 in cholesteatoma samples tended to be more diffuse than in RAS samples (Figure 1C, D, G, H). Western blot analysis of gankyrin Western blot analysis was carried out on 10 paired samples of cholesteatoma and RAS. Among them, appropriate results were identified in four paired samples, which were identified as otorrhea and showed 4 points on the bone destruction grade. Intense bands of gankyrin with approximately 28 kDa protein were detected in cholesteatoma for three paired samples (p < 0.05; Figure 2A), whereas gankyrin protein was faintly detected in RAS. However, a reverse result was found in one paired sample, indicating that the gankyrin protein rather increased in RAS compared with cholesteatoma (p < 0.05; Figure 2B). There was no significantly specific relationship between the level of gankyrin protein and clinical parameters. Clinical correlation We attempted to correlate the findings for expression of gankyrin, p53, and Ki-67 in cholesteatoma and RAS samples with clinical profiles, such as bone destruction grade, otorrhea, and recurrence [10]. In all, 80% (8/10) of cholesteatoma cases showed otorrhea. In both basal and suprabasal layers of the eight otorrhea cases, the expression of gankyrin was
detected as strongly positive in six cases and positive in the other two cases. However, the gankyrin protein was also expressed as strongly positive in two nonotorrhea cases, each showing 1 and 2 points on the bone destruction grade, respectively. Of the eight otorrhea cases, Ki-67 expression was focal in 75% of cases (6/8). The expression of p53 protein was positive in 62.5% (5/8) and strongly positive in 25% (2/8) of otorrhea cases; however, the two non-otorrhea cases also showed positive staining. Two cases were diagnosed as having recurrent cholesteatoma and the expression of gankyrin was strongly positive in the basal and suprabasal layers. The expression patterns of the relevant antibodies showed no significant differences according to clinical variables. Discussion Pathophysiologically, middle ear cholesteatoma may be caused by altered growth capacity of the epithelial basal layer, which was originally considered to have a proliferative property. Unlike malignant cancers, cholesteatoma not only proliferates, but apoptosis may also be increased [11]. Recently, the proliferative capacity of cholesteatoma has been evaluated by using Ki-67 and proliferating cell nuclear antigen (PCNA), and its apoptotic feature was estimated by studying tumor suppressor genes such as connexin 26, connexin 43, and p53 [1,12]. However, despite considerable efforts, the pathogenesis of cholesteatoma remains largely unknown. To the best of our knowledge, the present study is the first attempt to elucidate a possible role of gankyrin expression as a mechanism of cholesteatoma. Gankyrin, which has recently been introduced as a protein with ankyrin repeats in malignant cancers, especially in hepatocellular carcinoma, interacts with cyclin-dependent kinase 4 to degrade the phosphorylated retinoblastoma protein. It also activates
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E2F-dependent transcription factors of DNA synthesis and functions in the proteosomal degradation and ubiquitination of p53 protein by binding the ubiquitin ligase [5]. Therefore, during the development of a malignancy from a benign status, gankyrin inhibits two important tumor suppressor proteins to facilitate cell cycle progression and anti-apoptosis. A study conducted on gankyrin expression in otolaryngology, which is related to human oral cancer, showed that immunoreactivity of gankyrin in oral squamous cell carcinoma was stronger than in normal oral epithelium [13]. In the present study, we compared cholesteatoma and RAS in terms of gankyrin protein expression and found that gankyrin protein was overexpressed in cholesteatoma epithelia compared with RAS samples. This suggests that up-regulation of gankyrin may play a proliferative role as regards keratinocytes in cholesteatoma epithelia, thus participating in the pathogenesis of cholesteatoma. To sustain appropriate cell cycle progression, normal epithelium maintains a balance between a proliferation and apoptosis [12], and a proliferation of keratinocytes in cholesteatoma is considered to be in a delicate equilibrium status with an apoptosis [14]. Unlike in malignant tumors, p53 expression in cholesteatoma was found to be much more intense than that in RAS samples. Since gankyrin mediates the degradation of p53 in hepatocellular carcinomas [15], an increased level of gankyrin could possibly decrease p53 in cholesteatoma, as shown in some samples in the present study. However, the level of p53 expression in cholesteatoma may vary in different clinical situations. For example, since cholesteatoma is considered to be a benign, chronic inflammatory disease, cell proliferation could elevate the level of the p53 protein [16] and increased levels of reactive oxygen species could also activate p53 [17]. Furthermore, MDM2 protein expression is down-regulated in the basal layer compared with the suprabasal layer in the skin [18]. MDM2 is E3 ubiquitin ligase and involved in p53 protein degradation. Gankyrin binds with MDM2 protein and is involved cooperatively in p53 protein degradation [5]. Cholesteatoma has hyperproliferative characteristics of the basal layer and we speculated that gankyrin could not degrade p53 protein because of insufficient MDM2 protein in cholesteatoma. This is one of the reasons that gankyrin and p53 proteins, which are thought to have opposite functions, showed similar cellular localization in cholesteatomas. The present study indicated that the apoptotic feature of p53 was still functioning in cholesteatoma, and gankyrin was highly expressed, implying that cholesteatoma still maintains its benign characteristic.
In the present study, the expression of gankyrin was also detected in RAS, especially in the basal layer. Basal keratinocytes in normal skin, which is separated from the dermis by the basement membrane, may progress throughout the proliferation and differentiation by epidermal stem cells and move up to constitute the suprabasal epithelial cell layer. Gankyrin is known as a subunit of the 26S proteasome complex. In normal cells, 26S proteasome acts as a protease to degrade many intracellular ubiquitinated proteins to establish cell viability. This implies that gankyrin in RAS plays a fundamental role in the normal cell cycle and maintains its homeostasis [13]. Ki-67 is recognized in actively proliferating cells during the G1, S, and G2 phases of the cell cycle, but is absent in the G0 phase. The expression of anti-Ki67 antibody in cholesteatoma has previously been reported as a proliferation marker, compared with normal deep meatal skin and RAS [10]. In the present study, however, the intensity of Ki-67 staining was weaker than that of gankyrin in most cholesteatoma samples, although some specimens showed a positive expression. Furthermore, clinical profiles demonstrated that otorrhea was reported in 8 (80%) of the 10 cases and at least 2 ossicles were eroded in 9 (90%) of the 10 cases. Among these nine cases, the tegmen, facial nerve canal, and bony lateral semicircular canal were also found to be eroded in five cases. Earlier it was proposed that Ki-67 is immunoreactively positive, especially on actively growing keratinocytes; therefore, the results of the present study suggest that the keratinocytes of the samples with otorrhea and a higher grade of bone destruction may occur in cellular senescence or inactive status after hyperproliferation of cholesteatoma epithelium is completed at some point of the cell cycle. Conclusion Gankyrin seems to be overexpressed more than Ki-67 and p53 in middle ear cholesteatoma, compared with RAS. The results of the present study suggest that up-regulated gankyrin may be one of the markers for hyperproliferation of cholesteatoma and may be related to the pathogenesis of cholesteatoma. The role of gankyrin expression in cholesteatoma needs to be further investigated to gain an insight into its treatment.
Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry
Gankyrin expression in cholesteatoma of Education Science and Technology (20100010678). Declaration of interest: The authors declare no conflict of interest.
[9]
[10]
[11]
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