Document Type: Original Article

Authors

1 Dept. of Oral and Maxillofacial Pathology, School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran.

2 Dept. of Community Medicine and Public Health, Mashhad University of Medical Sciences, Mashhad, Iran.

3 Postgraduate Student of Periodontology, School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran.

Abstract

Statement of the Problem: Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Early diagnosis of OSCC by using biomarkers provides preventive treatment approach to suppress the disease in early stages. CD44 as a cancer stem cell (CSC) marker may be cleaved by MT1-MMP and plays an important role in migration of cancer cells. TGF-B promotes formation of invasive cancer cells phenotype through epithelial mesenchymal transition (EMT) and induces MT1-MMP formation.
Purpose: The aim of this study was to evaluate the expression of TGF-B and CD44 in leukoplakia (premalignant lesion), squamous cell carcinoma (SCC), and normal oral mucosa to determine the role of these markers in the carcinogenesis process of the oral mucosa.
Materials and Method: The expression of TGF-B and CD44 were evaluated in 55 paraffin-embedded specimens (10normal mucosa, 15 non-dysplastic leukoplakia, 15 dysplastic leukoplakia, and 15 OSCC) by immunohistochemistry. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, and Spearman’s rank correlation tests.
Results: Evaluation of CD44 and TGF-B expression in the four studied groups showed statistical significant difference for each marker (p < 0.001). Pairwise comparison of CD44 and TGF-B expression in all groups except normal mucosa and non-dysplastic leukoplakia demonstrated statistical significant difference. In addition, there was positive significant correlation between two markers (r= 0.914, p < 0.001). Diagnostic test’s accuracy for identification of OSCC and dysplastic leukoplakia from non-dysplastic leukoplakia and normal tissues and recognition of OSCC from dysplastic leukoplakia showed optimum sensitivity and specificity.
Conclusion: Increased expression of CD44 as a cancer stem cell marker and TGF-B as an EMT marker from normal mucosa to non-dysplastic leukoplakia, dysplastic leukoplakia, and OSCC and also the significant correlation between these two markers indicated their role in carcinogenesis of oral mucosa.

Keywords

[1]  Neville B, Damm DD, Allen C, Chi A. Oral and maxillofacial pathology. 4th ed. WB Saunders, Elsevier: Missouri; 2016. p.928.

[2]  Oliveira LR, Castilho-Fernandes A, Oliveira-Costa JP, Soares FA, Zucoloto S, Ribeiro-Silva A. CD44+/CD133+ immunophenotype and matrix metalloproteinase-9: Influence on prognosis in early-stage oral squamous cell carcinoma. Head Neck. 2014; 36: 1718-1726.

[3]  Saghravanian N, Anvari K, Ghazi N, Memar B, Shahsavari M, Aghaee MA. Expression of p63 and CD44 in oral squamous cell carcinoma and correlation with clinicopathological parameters. Arch Oral Biol. 2017; 82: 160-165.

[4]  Godge PY, Poonja LS. Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: an immunohistochemical study. Indian J Dent Res. 2011; 22: 493-494.

[5]  Salehinejad J, Sharifi N, Amirchaghmaghi M, Ghazi N, Shakeri MT, Ghazi A. Immunohistochemical expression of p16 protein in oral squamous cell carcinoma and lichen planus. Annals of Diagnostic Pathology. 2014; 18: 210-213.

[6]  Chen C, Zhao S, Karnad A, Freeman JW. The biology and role of CD44 in cancer progression: therapeutic implications. Journal of Hematology & Oncology. 2018; 11: 64.

[7]  Chruscik A, Gopalan V, Lam A. The clinical and biological roles of transforming growth factor beta in colon cancer stem cells: A systematic review. European Journal of Cell Biology. 2017; 97: 15-22.

[8]  Iamaroon A, Pattamapun K, Piboonniyom SO. Aberrant expression of Smad4, a TGF-beta signaling molecule, in oral squamous cell carcinoma. J Oral Sci. 2006; 48: 105-109.

[9]  Richter P, Umbreit C, Franz M, Berndt A, Grimm S, Uecker A, et al. EGF/TGFbeta1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332. J Oral Pathol Med. 2011; 40: 46-54.

[10]          Naor D, Sionov RV, Ish-Shalom D. CD44: structure, function, and association with the malignant process. Advances in Cancer Research. 1997; 71: 241-319.

[11]          Chaiyarit PTK, Satayut S, Dhanuthai K, Piboonratanakit P, Phothipakdee P, Subarnbhesaj A, et al. Alteration of the Expression of CD44 [Corrected] Isoforms in Oral Epithelia and Saliva From Patients With Oral Lichen Planus. J Clin Immunol. 2008; 28: 26-34.

[12]          Karathanasi V, Tosios KI, Nikitakis NG, Piperi E, Koutlas I, Trimis G, et al. TGF-beta1, Smad-2/-3, Smad-1/-5/-8, and Smad-4 signaling factors are expressed in ameloblastomas, adenomatoid odontogenic tumors, and calcifying cystic odontogenic tumors: an immunohistochemical study. J Oral Pathol Med. 2013; 42: 415-423.

[13]          Shimada Y, Ishii G, Nagai K, Atsumi N, Fujii S, Yamada A, et al. Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung. Cancer Sci. 2009; 100: 2054-2059.

[14]          Bocci F, Levine H, Onuchic JN, Jolly MK. Deciphering the Dynamics of Epithelial-Mesenchymal Transition and Cancer Stem Cells in Tumor Progression. Current Stem Cell Reports. 2019; 5: 11-21.

[15]          Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006; 66: 9339-9344.

[16]          Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med. 2011; 17: 313-319.

[17]          Miyazaki H, Takahashi RU, Prieto*Vila M, Kawamura Y, Kondo S, Shirota T, et al. CD44 exerts a functional role during EMT induction in cisplatinresistant head and neck cancer cells. Oncotarget. 2018; 9: 10029–10041.

[18]          Chen L, Li YC, Wu L, Yu GT, Zhang WF, Huang CF, et al. TRAF6 regulates tumour metastasis through EMT and CSC phenotypes in head and neck squamous cell carcinoma. Journal of Cellular and Molecular Medicine. 2018; 22: 1337-1349.

[19]          Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 2009; 28: 15-33.

[20]          Roye GD, Myers RB, Brown D, Poczatek R, Beenken SW, Grizzle WE. CD44 expression in dysplastic epithelium and squamous-cell carcinoma of the esophagus. Int J Cancer. 1996; 69: 254-258.

[21]          Washington K, Gottfried MR, Telen MJ. Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas. Hum Pathol. 1994; 25: 1043-1049.

[22]          Abdulmajeed AA, Dalley AJ, Farah CS. Putative cancer stem cell marker expression in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med. 2013; 42: 755-760.

[23]          Rautava J, Soukka T, Inki P, Leimola-Virtanen R, Saloniemi I, Happonen RP, et al. CD44v6 in developing, dysplastic and malignant oral epithelia. Oral Oncol. 2003; 39: 373-379.

[24]          Bahar R, Kunishi M, Kayada Y, Yoshiga K. CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues. Int J Oral Maxillofac Surg. 1997; 26: 443-446.

[25]          Gonzalez-Moles MA, Bravo M, Ruiz-Avila I, Esteban F, Bascones-Martinez A, Gonzalez-Moles S. Adhesion molecule CD44 expression in non-tumour epithelium adjacent to tongue cancer. Oral Oncol. 2004; 40: 281-286.

[26]          Chang PY, Kuo YB, Wu TL, Liao CT, Sun YC, Yen TC, et al. Association and prognostic value of serum inflammation markers in patients with leukoplakia and oral cavity cancer. Clin Chem Lab Med. 2013; 51: 1291-300.

[27]          Gao Y, Liu J, Pang S. Alteration of CD44v3 and CD44v6 expression in oral premalignant epithelia. Zhonghua Kou Qiang Yi Xue Za Zhi. 1999; 34: 286-288.

[28]          Chen Y, Zhang W, Geng N, Tian K, Jack*Windsor L. MMPs, TIMP-2, and TGF-beta1 in the cancerization of oral lichen planus. Head Neck. 2008; 30: 1237-1245.

[29]          Wagner VP, Cardoso PR, Dos*Santos JN, Meurer L, Vargas PA, Fonseca FP, et al. Immunohistochemical Study of TGF-beta1 in Oral Leukoplakia and Oral Squamous Cell Carcinoma: Correlations Between Clinicopathologic Factors and Overall Survival. Appl Immunohistochem Mol Morphol. 2017; 25: 651-659.

[30]          Mima K, Okabe H, Ishimoto T, Hayashi H, Nakagawa S, Kuroki H, et al. CD44s regulates the TGF-beta-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma. Cancer Res. 2012; 72: 3414-323.

[31]          Ouhtit A, Madani S, Gupta I, Shanmuganathan S, Abdraboh ME, Al-Riyami H, et al. TGF-β2: A Novel Target of CD44-Promoted Breast Cancer Invasion. Journal of Cancer. 2013; 4: 566-572.

[32]          Park NR, Cha JH, Jang JW, Bae SH, Jang B, Kim JH, et al. Synergistic effects of CD44 and TGF-beta1 through AKT/GSK-3beta/beta-catenin signaling during epithelial-mesenchymal transition in liver cancer cells. Biochem Biophys Res Commun. 2016; 477: 568-574.

[33]          Anido J, Saez-Borderias A, Gonzalez-Junca A, Rodon L, Folch G, Carmona MA, et al. TGF-beta Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma. Cancer Cell. 2010; 18: 655-668.

[34]          Bourguignon LY, Singleton PA, Zhu H, Zhou B. Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells. J Biol Chem. 2002; 277: 39703-39712.