Journal of Pharmacy And Bioallied Sciences
Journal of Pharmacy And Bioallied Sciences Login  | Users Online: 1472  Print this pageEmail this pageSmall font sizeDefault font sizeIncrease font size 
    Home | About us | Editorial board | Search | Ahead of print | Current Issue | Past Issues | Instructions | Online submission




 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 5  |  Page : 205-210  

Immunohistochemical expression of ezrin in oral potentially malignant disorders-A descriptive study


1 Department of Oral Pathology and Microbiology, Madha Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Oral Pathology and Microbiology, Saveetha Dental College, Saveetha University, Chennai, Tamil Nadu, India

Date of Web Publication27-Nov-2017

Correspondence Address:
Raghini Mohanraj
30/14, Broddies Road, Karayanchavadi, Poonamallee, Chennai - 600 056, Tamil Nadu
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.JPBS_139_17

Rights and Permissions
   Abstract 


Introduction: Ezrin, also known as cytovillin, is a member of the ERM family of protein. Ezrin cross-links actin filament with the plasma membrane. They are involved in the formation of microvilli, cell–cell adhesion, maintenance of cell shape, cell motility, and membrane trafficking. Recent analysis reveals their involvement in signaling pathways. Ezrin is highly expressed in several types of human cancers, and correlation between its immunoreactivity and histopathological data as well as the patient outcome has previously been studied. Objective: The objective of the study was to analyze the immunohistochemical expression pattern of ezrin in oral potentially malignant disorders (OPMDs), namely, oral submucous fibrosis (OSMF) with different grades and clinically leucoplakia (hyperkeratosis with various degree of dysplasia) and its use as a predictive marker for malignant transformation. Subjects and Methods: Sample size n = 43, histopathologically confirmed cases of OPMDs (13 cases of OSMF with different grades and 30 cases of clinically leukoplakia) were retrieved from the Department of Oral and Maxillofacial Pathology. Immunohistochemistry was done using anti-ezrin antibody, and the expression was graded in terms of proportion and intensity. Results: There was a significant expression of ezrin in OPMDs, and its cytoplasmic shift can be used as a predictive marker for malignant transformation. Conclusion: The findings of the current study revealed that the expression of ezrin in OPMDs may be related to the progression of the disease.

Keywords: Cytovillin, ezrin, immunohistochemistry, leukoplakia, oral potentially malignant disorders, oral submucous fibrosis


How to cite this article:
Mohanraj R, Ramani P, Premkumar P, Natesan A, Sherlin HJ, Sukumaran G. Immunohistochemical expression of ezrin in oral potentially malignant disorders-A descriptive study. J Pharm Bioall Sci 2017;9, Suppl S1:205-10

How to cite this URL:
Mohanraj R, Ramani P, Premkumar P, Natesan A, Sherlin HJ, Sukumaran G. Immunohistochemical expression of ezrin in oral potentially malignant disorders-A descriptive study. J Pharm Bioall Sci [serial online] 2017 [cited 2019 Jul 22];9, Suppl S1:205-10. Available from: http://www.jpbsonline.org/text.asp?2017/9/5/205/219273




   Introduction Top


Head and neck cancer is the sixth most common human cancer, representing 3% of all types of cancer. They are located in the oral cavity in 48% of cases and 90% of these are oral squamous cell carcinoma.[1] Important risk factors related to the carcinoma itself that are associated with a poor prognosis include large size of the tumor at the time of diagnosis, the presence of metastases in regional lymph nodes, and a deep invasive front of the tumor.[2]

Leukoplakia and oral submucous fibrosis (OSMF) are the most common potentially malignant disorders of the oral mucosa. Studies revealed that many oral carcinomas develop from these common oral potentially malignant disorders (OPMDs) of the oral cavity. To prevent the malignant transformation of these potentially malignant disorders, multiple screening and detection techniques are used. The early detection of cancer is of critical importance because survival rate is markedly improved when the oral lesion is identified at an early stage.

Ezrin, a membrane-cytoskeleton linker protein, participates in many physiological functions that includes cell survival, cell motility, extracellular matrix interactions, cell–cell communication, apoptosis, and signal transduction.[3],[4] Recent studies have revealed that ezrin may have an important role in tumorogenesis, development, invasion, and metastasis, probably through regulation of adhesion molecules, participation in signal transduction, and signalling to other cell membrane channels in the tumor.[5] Overexpression of ezrin is correlated with the metastatic potential and poor prognosis of various tumors. The aim of the current study is to evaluate the expression of ezrin in OPMDs including oral submucous fibrosis with different grades and clinically leukoplakia (hyperkeratosis with various degree of dysplasia). The expression of ezrin which is a marker of invasion might help to identify patients vulnerable to malignant transformation.


   Subjects and Methods Top


The study material consists of 43 formalin-fixed paraffin-embedded samples of OPMDs. The tissue samples were retrieved from the archives of the Department of Oral and Maxillofacial Pathology, between the years 2010 and 2014. The specimens were selected after re-confirmation of the diagnosis on histopathological examination. Histopathologically confirmed cases of hyperkeratosis with different grades of dysplasia (clinically leukoplakia) and oral submucous fibrosis with different stages were included in the study. All the selected cases were subjected to immunohistochemical analysis for ezrin along with positive procedure controls. The antibodies and reagents used for immunohistochemical analysis were obtained from Abcam and Leica Microsystems, New Castle, United Kingdom. The primary antibody was Monoclonal Mouse Anti-ezrin 3C12 antibody in liquid form, and the secondary antibody was Novolink Mini Polymer Detection System (RE7290-K) containing Peroxidase Block, Protein Block, Post Primary Block, Novolink Polymer, DAB chromogen, Novolink DAB substrate buffer, and hematoxylin. The sections were de-waxed using xylene and hydrated through two changes of graded alcohol (100% and 90%) 5 min each. Antigen retrieval was done using pressure cooker method. Sections were immersed in 0.01 ml sodium citrate buffer at the pH of 6.0 and boiled for 3 min in a 5 L stainless steel pressure cooker. The pressure cooker was allowed to cool down to room temperature with the slides remaining in the buffer itself for 15–20 min. Slides were allowed to cool down in the citrate buffer till the pressure on the lid came down completely and then were washed in distilled water. Tris buffer saline (TBS) at the pH of 7.6 was used as immunohistochemistry wash buffer. Sections were incubated with peroxide block for 5 min. Wash sections in TBS for 1 × 5 min. Incubate with protein block for 5 min. Wash in TBS for 2 × 5 min. Incubate with primary antibody for 30 min. Wash in TBS for 2 × 5 min. Incubate with postprimary block for 30 min. Wash in TBS for 2 × 5 min. Incubate with NovoLink Polymer for 30 min. Wash in TBS for 2 × 5 min. Develop peroxidase activity with DAB working solution for 5 min (Prepare DAB working solution by adding 50ul of DAB chromogen to 1 ml of DAB buffer). Rinse slides in running water. Counterstain with hematoxylin. Rinse slides in water for 5 min. Dehydrate, clear, and mount sections with DPX. Immunohistochemical staining of ezrin was done, and the localization, intensity, and the proportionality of staining were determined both in the epithelium and in the connective tissue. The staining characteristics were observed semi-quantitatively by two independent observers and were assessed from three fields. A scale of − to +++ was used. The grading was based on the intensity of the stain and the area of positive staining. Descriptive statistics such as mean, median, and its 95% confidence interval (CI) were calculated.


   Results Top


A total of 43 cases of OPMDs including OSMF with different grades and clinically leukoplakia (hyperkeratosis with various degrees of dysplasia) were evaluated for ezrin expression. [Table 1] summarizes the mean, median, and standard deviation (SD) value of ezrin expression in OPMDs. The mean value is 2.23 with 95% CI: 1.67–2.79 and SD of 1.875. The median value is 2.00 with 1st quartile 0.00 and 3rd Quartile 4.00. [Table 2] summarizes the overall intensity index and proportionality index of ezrin expression in OPMDs and its descriptive statistics. The mean value of intensity index is 1.28 with 95% CI: 0.94–1.62 and SD of 1.120. The median value is 1.00 with 1st Quartile 0.00 and 3rd Quartile 2.00. The mean value of proportionality index is 0.95 with 95% CI: 0.71–1.19 and SD of 0.815. The median value is 1.00 with 1st quartile 0.00 and 3rd quartile 2. [Table 3] summarizes the expression of ezrin in percentage with 95% CI. Ezrin expression was found to be negative in 34.9% of cases with 95% CI: 21.8–50.7. Mild expression of ezrin was found to be 16.3% with 95% CI: 7.7–31.1. Moderate expression of ezrin was found to be 34.9% with 95% CI: 21.8–50.7. Severe expression of ezrin was found to be 14.0% with 95% CI: 6.2–28.5. [Table 4] summarizes the staining location of ezrin in percentage and its 95% CI. Ezrin expression was found to be membranous in 67.9% of cases with 95% CI: 48.0–82.8. Membranous and cytoplasmic expression in 28.5% of cases with 95% CI: 14.5–48.4 and cytoplasmic in 3.6% of cases with 95% CI: 0.5–22.9.
Table 1: Overall expression of ezrin in oral potentially malignant disorders and descriptive statistics such as mean, median, and its 95% confidence interval

Click here to view
Table 2: Intensity index and proportionality index of ezrin expression in oral potentially malignant disorders

Click here to view
Table 3: Overall percentage of ezrin expression in oral potentially malignant disorders

Click here to view
Table 4: Staining location of ezrin expression in oral potentially malignant disorders

Click here to view



   Discussion Top


Many oral squamous cell carcinomas are preceded by clinically evident OPMDs. It is very important to prevent a malignant change in people diagnosed with OPMDs, but the hazard ratios of various OPMDs are not well known. The OPMDs include hyperkeratosis with epithelial dysplasia and OSMF. The malignant transformation rates of OPMDs show a great variation and it is difficult to determine the frequency of OPMD transforming to cancer based on the clinical and histological factors.[6] New approaches, such as interventions with molecular-targeted agents in high-risk individuals, are undoubtedly the need of the hour to mitigate the devastating worldwide consequences of oral malignancy.[7]

Ezrin belongs to the ERM family of proteins which plays a positive role in maintaining cell shape and cell polarity as well as in membrane trafficking pathways, cell migration, cell signaling, growth regulation, and differentiation. Due to its unique function, ezrin is also actively involved in the biology of tumor development.[8] Aberrant expression of ezrin is well correlated with poor survival in tumors including head and neck squamous cell carcinoma, osteosarcomas, cervical carcinoma, breast carcinoma, and prostatic carcinomas.[9] Exhaustive literature search suggests that the present study is the first attempt to determine the expression of ezrin in OPMDs, namely, OSMF with different grades and clinically leukoplakia (hyperkeratosis with various degree of dysplasia).

Among the cases of OPMDs, the pattern of ezrin expression was found to be predominantly membranous in 67.9% of cases and cytoplasmic in 3.6% of cases [Figure 1] and both membranous and cytoplasmic in 28.5% of cases [Figure 2]. The results are in accordance with the study conducted by Ling et al., who found the expression of ezrin seems to show increased cytoplasmic staining in dysplastic and esophageal squamous cell carcinoma in contrast to normal esophageal mucosa and hyperplasias.[10]
Figure 1: Photomicrograph showing dysplastic cells with Strong cytoplasmic positivity of ezrin expression (×40)

Click here to view
Figure 2: Photomicrograph showing dysplastic cells with strong membranous and cytoplasmic positivity of ezrin expression (×40)

Click here to view


The possible reason could be that certain circumstances can stimulate the translocation of ezrin, and there is evidence suggesting that the subcellular redistribution of ezrin significantly correlates with tumorigenesis.[11],[12] Darwish et al. state that loss of membranous localization and cytoplasmic accumulation are fundamental in keratinocyte transformation.[13] Gamei et al. explained the reason for the cytoplasmic shift could be attributed to decrease on activating signals or increase of inactivating signals.[14]

During the transformation from benign cell to malignant cell, the location of ezrin protein also changed, and it was distributed in the cytoplasm of malignant tumor. This transformation may weaken intercellular adhesion of normal cells and enhance the adhesion between tumor cells and normal cells, which promote tumor metastasis.[15] According to the author Zhai et al., the activation of Rac 1 pathway by ezrin causes hepatocyte growth factor to induce tyrosine phosphorylation of E-catenin and X-catenin, inhibits the function of E-cadherin, and leads to redistribution of E cadherin in cytoplasm and on cell membrane.[16]

The pattern of ezrin expression was found to be membranous in the spinous layer of the epithelium in 17.8% of cases and in the superficial layer in 7.14% of cases. The finding well correlated to the study by Madan et al., who observed that ezrin normally localizes to the membrane in nonproliferating squamous cells.[17]

The present study reveals that 20.05% cases of leukoplakia with membranous and cytoplasmic ezrin expression were confined to the basal layer of the epithelium. The shift in localization of ezrin from membranous to cytoplasm in the basal layer of the epithelium could be attributed to the potentially malignant transformation of the basal cells. Saito et al. indicate that ezrin could enhance the growth of cancer cells by supporting cell division and cell cycle progression from G0/G1 to the S and G2/M phases.[9]

The cell cycle analysis revealed that ezrin depletion increased the G0/G1 fraction but decreased the G2-M fraction by interfering with cell mitosis and cell cycle progression.[9] Ezrin can also support the survival of tumor cells by activating the antiapoptotic pathways. Phosphorylation of ezrin at its tyrosine residues activates the PI3-kinase/Akt pathway, thereby prevents apoptosis of tumor cells.[15] Schlecht et al. observed that high cytoplasmic ezrin expression indicates the altered expression of antiapoptotic genes (BIRC2, CCDC50, and TGM2) and proapoptotic genes (EDARADD), resulting in survival rather than cell death.[18] Thus, the above findings signify the role of ezrin in tumorigenesis by its cell proliferative and antiapoptotic activity.

The present study also reveals that 3.6% of advanced OSMF shows only cytoplasmic pattern of ezrin expression. The above finding was supported by Zeng et al., who observed that, in low-grade dysplasia, ezrin expression seems to be membranous whereas, in advanced dysplastic conditions, ezrin expression was found to be predominantly cytoplasmic which could be equated with the invasiveness of the tumor in later stages. He also stated that the shift in localization of ezrin from membrane to cytoplasm is seen in the development of early squamous cell carcinomas.[19] This would imply that ezrin not only acts as a linker protein but also involves in cytoskeletal reorganization and cell differentiation.[19]

Ling et al. state that ezrin mediates aberrant linkage of the cytoskeleton to various proteins, including CD44 and LAMP-1, inducing marked changes in the general framework of cellular function. This also leads to aberrant engagement with the extracellular microenvironment that is directly involved in metastatic behavior of tumor cells.[10]

The cytoplasmic expression of ezrin helps in understanding the various biological behaviors of the neoplastic cells such as tumorigenesis, cell motility, and invasiveness. Xie et al. observed in his studies state that ezrin regulates the growth and invasiveness of cancer cells through regulating the MAPK and TGF-β pathways. In TGF-β pathway, ezrin might bind to Smad2/3 and then affect the activity of the pathway.[8] Ezrin phosphorylates the ERK protein in MAPK pathway and thereby regulates the growth and invasiveness of the tumor cells. This was confirmed by studies on cell migration assay that reveals knockdown of ezrin led to decreased migration and hence decreased invasiveness of the cancer cells.[20]

From the existing literature, it has been known that the cytoplasmic positivity of ezrin has been linked to its invasive potential in various squamous cell carcinomas.[8] In the present study, some of the mild dysplasia cases showed cytoplasmic shift of ezrin expression that might probably represent an increased potential for malignant transformation of these cases. This finding further supports the fact that the grading of dysplasia does not always correlate to its malignant transformation potential.

The study also reveals negative ezrin expression in 34.9% of cases. Gao et al. explain that the reason for decreased or negative expression of ezrin in certain dysplastic conditions was not clear which could be explained by a study on ezrin mRNA level by reverse-transcriptase-polymerase chain reaction (RT-PCR) and to determine the role of mRNA level and its interaction with upstream regulators and promoters modulating the ezrin expression.[21] Zeng et al. explain that ezrin downregulation in tumor cells can be explained partly by a corresponding alteration on the mRNA level, as shown by RT-PCR.[19]

This study is a maiden attempt to assess the pattern of ezrin expression in OPMDs, namely, OSMF with different grades and clinically leukoplakia (hyperkeratosis with various degree of dysplasia). In our study, we observed a shift of ezrin expression from membrane to cytoplasm in 28.5% of cases which render us in profiling the malignant transformation potential of OPMDs. Thus, ezrin is a promising marker that could be used to assess the high-risk patients in earlier stages and necessitates the periodic follow-up and proper treatment planning for better prognosis of the patients.


   Conclusion Top


In the cell membrane, ezrin plays a role in cell adhesion whereas its cytoplasmic localization might confer a cell with migratory properties. This might act as a prognostic marker in predicting malignant transformation of OPMDs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Tanaka T, Tanaka M, Tanaka T. Oral carcinogenesis and oral cancer chemoprevention: A review. Patholog Res Int 2011;2011:431246.  Back to cited text no. 1
    
2.
Feller L, Lemmer J. Oral squamous cell carcinoma: Epidemiology, clinical presentation and treatment. J Cancer Ther 2012;3:263-8.  Back to cited text no. 2
    
3.
Turunen O, Wahlström T, Vaheri A. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994;126:1445-53.  Back to cited text no. 3
    
4.
Xie J-J, Zhang F-R, Tao L-H, et al. Expression of Ezrin in Human Embryonic, Fetal, and Normal Adult Tissues. Journal of Histochemistry and Cytochemistry. 2011;59:1001-8.  Back to cited text no. 4
    
5.
Han K, Qi W, Gan Z, Shen Z, Yao Y, Min D, et al. Prognostic value of ezrin in solid tumors: A meta-analysis of the literature. PLoS One 2013;8:e68527.  Back to cited text no. 5
    
6.
Ho PS, Chen PL, Warnakulasuriya S, Shieh TY, Chen YK, Huang IY. Malignant transformation of oral potentially malignant disorders in males: A retrospective cohort study. BMC Cancer 2009;9:260.  Back to cited text no. 6
    
7.
Tanaka T, Ishigamori R. Understanding carcinogenesis for fight ing oral cancer. J Oncol 2011;2011:603740.  Back to cited text no. 7
    
8.
Xie JJ, Xu LY, Wu ZY, Zhao Q, Xu XE, Wu JY, et al. Prognostic implication of ezrin expression in esophageal squamous cell carcinoma. J Surg Oncol 2011;104:538-43.  Back to cited text no. 8
    
9.
Saito S, Yamamoto H, Mukaisho K, Sato S, Higo T, Hattori T, et al. Mechanisms underlying cancer progression caused by ezrin overexpression in tongue squamous cell carcinoma. PLoS One 2013;8:e54881.  Back to cited text no. 9
    
10.
Ling ZQ, Mukaisho K, Yamamoto H, Chen KH, Asano S, Araki Y, et al. Initiation of malignancy by duodenal contents reflux and the role of ezrin in developing esophageal squamous cell carcinoma. Cancer Sci 2010;101:624-30.  Back to cited text no. 10
    
11.
Tokunou M, Niki T, Saitoh Y, Imamura H, Sakamoto M, Hirohashi S, et al. Altered expression of the ERM proteins in lung adenocarcinoma. Lab Invest 2000;80:1643-50.  Back to cited text no. 11
    
12.
Moilanen J, Lassus H, Leminen A, Vaheri A, Bützow R, Carpén O, et al. Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients. Gynecol Oncol 2003;90:273-81.  Back to cited text no. 12
    
13.
Darwish AM, et al. The prognostic values of the immunohistochemical expression of ezrin and large safety margins in basal cell carcinoma of the face. Egypt J PlastReconstr Surg 2011;35:245-51.  Back to cited text no. 13
    
14.
Gamei MM, Abd el Naby NM, El-Ashmawy AA, Shareef MM. Prognostic value of ezrin expression in common epithelial tumors: An immunohistochemical study. J Microsc Ultrastruct 2014;2:67-76.  Back to cited text no. 14
    
15.
Wang DS, Pan CC, Lai HC, Huang JM. Expression of HMGA1 and ezrin in laryngeal squamous cell carcinoma. Acta Otolaryngol 2013;133:626-32.  Back to cited text no. 15
    
16.
Zhai JW, Yang XG, Yang FS, Hu JG, Hua WX. Expression and clinical significance of ezrin and E-cadherin in esophageal squamous cell carcinoma. Chin J Cancer 2010;29:317-20.  Back to cited text no. 16
    
17.
Madan R, Brandwein-Gensler M, Schlecht NF, Elias K, Gorbovitsky E, Belbin TJ, et al. Differential tissue and subcellular expressionof ERM proteins in normal and malignant tissues: Cytoplasmic ezrin expression has prognostic significance for head and neck squamous cell carcinoma. Head Neck 2006;28:1018-27.  Back to cited text no. 17
    
18.
Schlecht NF, Brandwein-Gensler M, Smith RV, Kawachi N, Broughel D, Lin J, et al. Cytoplasmic ezrin and moesin correlate with poor survival in head and neck squamous cell carcinoma. Head Neck Pathol 2012;6:232-43.  Back to cited text no. 18
    
19.
Zeng H, Xu L, Xiao D, Zhang H, Wu X, Zheng R, et al. Altered expression of ezrin in esophageal squamous cell carcinoma. J Histochem Cytochem 2006;54:889-96.  Back to cited text no. 19
    
20.
Xie JJ, Xu LY, Xie YM, Zhang HH, Cai WJ, Zhou F, et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer 2009;124:2549-58.  Back to cited text no. 20
    
21.
Gao W, Zhang C, Feng Y, Chen G, Wen S, Huangfu H, et al. Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma. PLoS One 2012;7:e50710.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
   Subjects and Methods
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed772    
    Printed5    
    Emailed0    
    PDF Downloaded45    
    Comments [Add]    

Recommend this journal