Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 2  |  Page : 105-109

Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition


1 Professor and Head of the Department, Department of Oral Pathology and Oral Microbiology, Vinayaka Missions Sankarachariyar Dental, VMRF (DU), Salem, Tamil Nadu, India
2 Professor, Department of Periodontology, Vinayaka Missions Sankarachariyar Dental, VMRF (DU), Salem, Tamil Nadu, India
3 Professor, Department of Oral Pathology, Rajamuthiah Dental College and Hospital, Chidambaram, Tamil Nadu, India
4 Senior Lecturer, Department of Oral Pathology and Oral Microbiology, Vinayaka Missions Sankarachariyar Dental, VMRF (DU), Salem, Tamil Nadu, India
5 Professor and Head, Department of Oral and Maxillofacial Surgery, Butterworth Penang, Malaysia

Date of Submission18-Nov-2019
Date of Decision10-Dec-2019
Date of Acceptance10-Dec-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr B. Sekar
Professor and Head of the Department, Department of Oral Pathology and Oral Microbiology, Vinayaka Missions Sankarachariyar Dental, VMRF (DU), Sankakiri Main Road, Salem- 636308, Salem, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_140_19

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  Abstract 


Introduction: For the past decades long-term survival of oral squamous cell carcinoma (OSCC) patients remain unchanged which provides a wide platform for research. Caldesmon (CaD), an actin-myosin binding protein, has a contributing role in cytoskeleton modulation and cell motility. The aim of this study is to evaluate the expression of CaD in metastatic and non-metastatic OSCC and to discuss the possible role of CaD in epithelial mesenchymal transition. Materials and Methods: Archival blocks of 25 metastatic and 25 non-metastatic OSCC patients are included where CaD expression is evaluated immunohistochemically. Results: Overall expression and staining intensity of CaD were statistically significant in different grades of metastatic and non-metastatic OSCC. In summary, high expression of CaD is observed in increasing grades of OSCC but the metastatic potential of the tumour doesn’t seem to have any relation with CaD.

Keywords: Caldesmon, epithelial mesenchymal transition, metastatic, non-metastatic, oral squamous cell carcinoma


How to cite this article:
Sekar B, Saranyan R, Nirmal RM, Kumaresan I, Murugesan A, Kamaraj L. Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition. J Orofac Sci 2019;11:105-9

How to cite this URL:
Sekar B, Saranyan R, Nirmal RM, Kumaresan I, Murugesan A, Kamaraj L. Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition. J Orofac Sci [serial online] 2019 [cited 2020 May 31];11:105-9. Available from: http://www.jofs.in/text.asp?2019/11/2/105/276716




  Introduction Top


Head and neck squamous cell carcinoma is the sixth most common cancer worldwide. Cancer is a complex system comprising of tumour cells, surrounding stroma, various types of mesenchymal cells, and extra-cellular matrix (ECM), which are collectively known as tumor microenvironment (TME).[1] The stroma comprises numerous non-cancerous cells such as fibroblasts, endothelial cells, pericytes, immune regulatory cells, and cytokines.[2] These activated fibroblasts reprogram and resemble like the normal myofibroblast which is the basic component in healing granulation tissue and chronic inflammation. The recruited myofibroblasts tend to aggregate peritumorally and encircle the carcinoma cells invading adjacent normal tissues.[3],[4]

Cell adhesion and migration, responsible for various normal and pathological events such as malignancy, are controlled by a complex change in the cytoskeleton. In various studies Ca2+ dependant regulation of smooth muscle and non-smooth muscle cells showed caldesmon (CaD) in the cytoskeleton as a regulatory protein functioning as actomyosin contractile system. [5],[6] CaD, encoded from CALD1 gene, has two different isoforms: a high molecular weight protein (h-CaD) restricted to actomyosin contractile structures in visceral and vascular smooth muscle. In migrating cells, low molecular weight (l-CaD) in non-smooth muscle cells localized in stress fibres and membrane ruffles.[7],[8] Myofibroblast usually is negative for smooth muscle antigen such as myosin chains, CaD, and desmin but studies are done where myofibroblast showed positive expression of smooth muscle antigen in areas surrounding more advanced stage of tumour cell differentiation.[9],[10] Increased expression of mesenchymal markers, and loss of cell-cell adhesion and epithelial cell markers, is a key event in tumour cell progression and invasion which contributes epithelial-mesenchymal transition (EMT).[11] This event in EMT is chiefly because of alteration in extracellular proteins and cell adhesion molecules in tumour cell.[12] Selective studies have discussed the overexpression of CaD in OSCC suggesting its vital role in cellular migration and invasion. In the present study, we evaluated and compared the CaD expression in metastatic and non-metastatic OSCC immunohistochemically in an attempt to explain its possible role in tumour cell progression and migration.


  Materials and Methods Top


A total of 50 OSCC samples were collected from the archival blocks of the Department of Oral Maxillofacial Pathology and Oral Microbiology, Vinayaka Missions Sankarachariyar Dental College, Salem. Out of 50 study blocks 25 are metastatic and 25 from non-metastatic oral squamous cell carcinoma (OSCC) cases. Conventional OSCC occurring in any area of the oral cavity with or without regional lymph node metastasis was included whereas variants of OSCC were not included in the study. The ethical committee of Vinayaka Mission’s Sankarachariyar Dental College, Salem (VMDC/IEC/Approval No 80) approved this study on 13/10/2017.

Study Design

The archival blocks collected were subjected to immunohistochemical staining. The sections are treated with ready-to-use rabbit monoclonal anti-human CaD antibody (PathnSitu, CA-94551 USA), counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. CaD expression was scored using Klein et al.[13] scoring system for both staining intensity and the percentage of cells stained. Accordingly scores 0, 1, 2, and 3 of staining intensity denoted negative, weak, mild, and strong, respectively. The percentage of cells taken from the stain were scored as 0—negative, grade 1-—<30% positive cells, grade 2—30–60 % positive cells, and grade 3—>60% positive cells. The final score for each case was calculated by adding the scores of proportion of positivity score and intensity score. The final score 0–1 was considered negative, score 1–2 was considered mild expression, score 2–4 was moderate expression, and score 4–6 was severe expression.

Statistical Analysis

The final data was analyzed using SPSS software version 11.5 (SPSS Inc., Chicago, IL). The significant difference between the groups are analyzed using Chi square test. P value less than 0.05 was considered as statistically significant.


  Results Top


In immunohistochemical analysis of metastatic and non-metastatic OSCC, CaD expression was observed in cell membrane and cytoplasm of tumour cells with more intensity in the periphery of tumour islands and adjacent stroma. In adjacent dysplastic surface epithelium of the tumour showed positivity in the basal cells. In few areas of tumor island, CaD expression was noted in newly formed blood vessels [Figure 1].
Figure 1 Caldesmon expression in newly formed blood vessels in well differentiated squamous cell carcinoma

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In non-metastatic OSCC, the overall staining is graded as follows; grade 1 expression of CaD was observed in 90% of well-differentiated cases, 60% of grade 2 expression and 40% of grade 1 expression were noted in moderately differentiated OSCC. In poorly differentiated OSCC grade 3 expression was observed in 60% cases and grade 2 in 40% cases. The overall staining expression of CaD in non-metastatic OSCC shows a significant difference among the grades of OSCC with a P value of 0.001 [Table 1]. The staining intensity was scored as weak, mild, and strong with the significant P value of 0.002 in different grades of non-metastatic OSCC [Table 2], [Figure 2].
Table 1 Overall staining expression of CaD in different grades of non-metastatic oral squamous cell carcinoma with significant P value

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Table 2 Intensity of staining of CaD in different grades of non-metastatic oral squamous cell carcinoma with significant P value

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Figure 2 Caldesmon expression with mild staining intensity was observed in moderate differentiated squamous cell carcinoma

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In metastatic OSCC overall staining was observed in different grades are as follows; 90% grade 1 expression was observed in well-differentiated cases, 90% of grade 2 expression in moderately differentiated cases, and 100% of grade 3 expression observed in poorly differentiated cases of OSCC with a significant P value of 0.001 [Table 2]. Strong positivity and staining intensity of tumour cells were observed in poorly differentiated OSCC with high significant at P value of (0.001) [Table 3] and [Table 4], [Figure 3]. When comparing the CaD expression in metastatic and non-metastatic OSCC, the overall staining and intensity of staining were not statistically significant.
Table 3 Overall staining expression of CaD in different grades of metastatic oral squamous cell carcinoma with significant P value

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Table 4 Intensity of staining of CaD in different grades of metastatic oral squamous cell carcinoma with significant P value

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Figure 3 Caldesmon expression with Strong staining intensity was observed in poorly differentiated squamous cell carcinoma

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  Discussion Top


Stabilization of actin filaments, remodelling of cytoskeleton and cellular motility are regulated by the actin-binding protein, CaD. Role of CaD in the cytoskeletal rearrangement of non-smooth muscle is unclear.[14] In non-smooth muscle cells, CaD is involved in the formation and maintenance of stress fibres, which is responsible for formation of adhesion sites in the tumour cell periphery.[15] Tamara et al.[16] in 2005 investigated that CaD plays a vital role in the microfilament remodeling and cell motility of endothelial cells which is the early step in angiogenesis and thereby promotes invasiveness. The aggressiveness of a tumour chiefly depends upon the ability of cancer cells to invade adjacent tissue and undergo metastasis. Epithelial-mesenchymal transition is a complex process which is categorized into three types; including type 1 in embryogenesis, type 2 in wound healing, organ fibrosis, tissue regeneration, and type 3 where the cancer cells are involved in invasion and metastasis.[17] Previous studies highlighted the role of several growth factors and cytokines like TGF-β, EGF, insulin-like growth factor, hepatocyte growth factor, fibroblast growth factor, interleukin, and transcription factors that modulates the plasticity of tumour cells and promotes invasiveness.[18],[19],[20],[21],[22] Chang et al.[7] in 2013 noted increased CaD expression in metastatic OSCC with a poor prognosis. In our study CaD expression increased with increasing grade of OSCC which is similar to the previous studies. Based on our results, CaD expression is high in poorly differentiated metastatic and non-metastatic OSCC significantly. Also, positive expression was noted in new blood vessels and surrounding stroma which denotes its increased potential for tumour progression and metastasis. When comparing the CaD expression in metastatic and non-metastatic OSCC, significant results were not obtained.

EMT is the first step for tumour progression and migration which leads to metastasis and influences the prognosis of the tumour. Few other markers like e-cadherin, IL-8, TGF-β, etc., have potential roles in epithelial-mesenchymal transition and migration of tumour cells. In summary, though there was significant overexpression of CaD from differentiated tumors to poorly differentiated tumors, metastatic potential of the tumor didn’t seem to have any relation. However, more samples are required to validate these predictions and confirm the role of CaD in epithelial mesenchymal transition, tumor progression, and invasion.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Koontongkaew S. The tumor microenvironment contribution to development, growth, invasion and metastasis of head and neck squamous cell carcinomas. Journal of Cancer 2013;4:66-83.  Back to cited text no. 1
    
2.
Tao L, Huang G, Song H, Chen Y, Chen L. Cancer associated fibroblasts: an essential role in the tumor microenvironment (Review). Oncology Letters 2017;14:2611-20.  Back to cited text no. 2
    
3.
Hanahan H, Weinberg RH. Hallmarks of cancer: the next generation. Cell 2011;144:646-74.  Back to cited text no. 3
    
4.
Kalluri R, Zeisberg M. Fibroblasts in cancer. Nature Reviews in Cancer 2006;6:392-401.  Back to cited text no. 4
    
5.
Sobue K, Sellers JR. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. The Journal of Biological Chemistry 1991;266:12115-8.  Back to cited text no. 5
    
6.
Sobue K, Morimoto K, Inui K, Kanda K, Kakiuchi S. Control of actin-myosin interaction of gizzard smooth muscle by calmodulin- and caldesmon linked flip-flop mechanism. Biomedical Research 1982;3:188-96.  Back to cited text no. 6
    
7.
Chang KP, Wang CLA, Kao HK, Liang Y, Liu SC, Huang LL, Hseuh C, Hsieh YJ, Chien KY, Chang YS, Yu JS, Chi LM. Overexpression of caldesmon is associated with lymph node metastasis and poorer prognosis in patients with oral cavity squamous cell carcinoma. Cancer 2013;4003-11.  Back to cited text no. 7
    
8.
Mayanagi T, Sobue K. Diversification of caldesmon-linked actin cytoskeleton in cell motility. Cell Adhesion & mMigration 2011;5:150-9.  Back to cited text no. 8
    
9.
Lúcio PSC, Cavalcanti AL, Alves PM, Godoy GP, Nonaka CFW. Myofibroblasts and their relationship with oral squamous cell carcinoma. Braz J Otorhinolaryngol 2013;79:112-8.  Back to cited text no. 9
    
10.
Tomasek J, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano regulation of connective tissue remodelling. Molecular Cell Biology 2002;3:349-63.  Back to cited text no. 10
    
11.
Davis FM, Stewart TA, Thompson EW, Monteith GR. Targeting EMT in cancer: opportunities for pharmacological intervention. Trends in Pharmacological Sciences 2014;35:479-88.  Back to cited text no. 11
    
12.
Zhang J, Zheng G, Zhou L, Li P, Yun M, Shi Q, Wang T, Wu X. Notch signalling induces epithelial‑mesenchymal transition to promote metastasis in oral squamous cell carcinoma. International Journal of Molecular Medicine 2018;42:2276-84.  Back to cited text no. 12
    
13.
Mirzapoiazova T, Kolosova IA, Romer L, Garcia JGN, Verin AD. The role of caldesmon in the regulation of endothelial cytoskeleton and migration. Journal of Cellular Physiology 2005;203:520-8.  Back to cited text no. 13
    
14.
Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue − a review. Fedchenko and Reifenrath Diagnostic Pathology 2014;9:1-12.  Back to cited text no. 14
    
15.
Mayanagi T, Morita T, Hayashi K, Fukumoto K, Sobue K. Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via there organization of the actin cytoskeleton. The journal of biological chemistry 2008;28:31183-96.  Back to cited text no. 15
    
16.
Mirzapoiazova T, Kolosova IA, Romer L, Garcia JGN, Verin AD. The role of caldesmon in the regulation of endothelial cytoskeleton and migration. Journal of Cellular Physiology 2005;203:520-8.  Back to cited text no. 16
    
17.
Acloque JPTH, Huang RYJ, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;871-90.  Back to cited text no. 17
    
18.
Mimeault M, Batra SK. Interplay of distinct growth factors during epithelial-mesenchymal transition of cancer progenitor cells and molecular targeting as novel cancer therapies. Annals of Oncology 2007;18:1605-19.  Back to cited text no. 18
    
19.
Zavadil J, Bottinger EP. TGF-b and epithelial-to-mesenchymal transitions. Oncogene 2005;24:5764-74.  Back to cited text no. 19
    
20.
Lee CL, Chang JSM, Syu SH, Wong TS, Chan JYW, Tang YC, Yang ZP, Yang WC, Chen CT, Lu SC, Tang PH, Yang TZ, Chu PY, Hsiao JR, Liu KJ. IL-1b promotes malignant transformation and tumor aggressiveness in oral cancer. J Cell Physiol 2015;230:875-84.  Back to cited text no. 20
    
21.
de Lima TB, Klein IP, de Oliveira MG, Rados PV, Filho MS, Visioli F. Analysis of the epithelium mesenchymal transition process on oral squamous cell carcinomas. Brazilian Dental Journal 2017;28:543-7.  Back to cited text no. 21
    
22.
Krisanaprakornkit S, Iamaroon A. Review article epithelial-mesenchymal transition in oral squamous cell carcinoma. International Scholarly Research Network ISRN Oncology 2012;1-10.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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



 

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