Tanta Dental Journal

: 2017  |  Volume : 14  |  Issue : 2  |  Page : 62--67

Role of CD44 and cortactin in metastasis of oral squamous cell carcinoma

Yomna S Abd El-Aziz1, Eman M. E. Megahed1, Wafaa A. E. Mostafa1, Iman M Ezzo2,  
1 Department of Oral Pathology, Faculty of Dentistry, Tanta University, Tanta, Egypt
2 Department of Pathology, Tanta Cancer Center, Tanta, Egypt

Correspondence Address:
Yomna S Abd El-Aziz
Department of Oral Pathology, Faculty of Dentistry, Tanta University, Tanta


Context: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer, and over the last decade its incidence has increased by 50%. The tendency of OSCC for local and regional metastasis is high and this is thought to be the greatest contributor to the morbidity and mortality associated with OSCC. Aim: The aim of this study was directed to evaluate the role of both cluster of differentiation 44 (CD44) and cortactin (CTTN) in lymph node (LN) metastasis of OSCC. Materials and methods: The study groups comprised blocks of OSCC with LN metastasis (n = 20) and blocks of OSCC without LN metastasis (n = 20). The blocks were prepared for immunohistochemical staining for CD44 and CTTN. Results: The result of this study revealed significant correlation between expression of both CD44 and CTTN and LN metastasis. Conclusion: From the present study, it can be concluded that assessment of CD44 and CTTN in OSCC represent an important tool for prediction of risk of metastasis for each case and the significant correlation between CD44 and CTTN could provide insights of their critical role in metastasis.

How to cite this article:
Abd El-Aziz YS, Megahed EM, Mostafa WA, Ezzo IM. Role of CD44 and cortactin in metastasis of oral squamous cell carcinoma.Tanta Dent J 2017;14:62-67

How to cite this URL:
Abd El-Aziz YS, Megahed EM, Mostafa WA, Ezzo IM. Role of CD44 and cortactin in metastasis of oral squamous cell carcinoma. Tanta Dent J [serial online] 2017 [cited 2018 May 24 ];14:62-67
Available from: http://www.tmj.eg.net/text.asp?2017/14/2/62/207305

Full Text


Oral cancer is the sixth most common cancer in the world. The mortality associated with recurrent disease is more than 50% [1]. More than 95% of oral cancers are classified histologically as oral squamous cell carcinoma (OSCC) [2].

Many OSCC are thought to be associated and perhaps derived from oral epithelial dysplaia which appears clinically as leukoplakia, erythroplakia, and speckled leukoplakia [3]. The most important risk factors for OSCC are use of tobacco or betel quid and the regular drinking of alcoholic beverages. Their effect are dependent upon dose, frequency and duration of use, and are accelerated and exaggerated by the concurrent use of two or more of these agents [4].

The most important prognostic factor of OSCC is the presence of lymph node (LN) metastasis which is the main culprit for tumor recurrence. Evaluation for LN metastasis is the key point to determine proper therapy planning. The ability to predict LN metastasis with immunohistochemical (IHC) parameters can improve patient outcome [5].

Cortactin (CTTN) is an actin-associated scaffolding protein that binds and activates the actin-related protein 2/3 complex, and regulates branched actin networks in the formation of dynamic cortical actin-associated structures [6]. Its overexpression has been reported in many types of malignant tumors and has been postulated to increases tumor aggressiveness, possibly through promotion of tumor invasion and metastasis [7]. Some studies have documented a role for CTTN in promoting cell motility and invasion, including a critical role in invadopodia, actin rich-subcellular protrusions associated with degradation of the extracellular matrix (ECM) by cancer cells [8].

The establishment of invasive and metastatic behaviors requires that tumor cells acquire novel adhesion and migration properties to detach from their original sites and to localize to distant organs [9].

Human cluster of differentiation 44 (CD44) is a transmembrane hyaluronan binding glycoprotein that can bind to hyaluronic acid (HA), an ECM, and regulate a variety of cellular functions, such as cell migration, proliferation, cell–cell interaction, and apoptosis. These cellular functions of CD44 imply that a disorder of CD44 expression plays a crucial rule in the behavior of a malignant tumor [10]. Recently, CD44 has been identified as a biomarker of cancer stem cells in many malignancies [11]. However, CD44 clinical significance is currently disputed due to conflicting results within literature.

So, it is now clear that both CTTN and CD44 may play pivotal role in metastasis, therefore, this study will be carried out to shed light on the pathological significance of both cortactin and CD44 in invasion and metastasis of OSCC and this could provide us promising tools for prediction of metastasis in oral cancer.

 Materials and Methods

A total of 40 paraffin embedded archival tissue blocks of OSCC were used in this study. The specimens were retrieved from archives of Department of Oral Pathology, Faculty of Dentistry, Tanta University and Department of Pathology, Tanta Cancer Center. They were obtained from untreated OSCC patients and consisted of material obtained from surgically resected primary tumor. The study groups comprised blocks of OSCC with LN metastasis (n = 20) and blocks of OSCC without LN metastasis (n = 20).

To confirm the diagnosis of the samples in the study, first, 5 μm sections were prepared and were stained using the hematoxylin and eosin staining protocol. The diagnosis was then confirmed by two pathologists. Afterward, the blocks of OSCC were prepared for IHC staining for CD44 and CTTN.


For IHC staining, 4 μm sections were prepared from each paraffin block and were deparaffinized in the xylene solution and then dehydrated in a graded alcohol series. To block the internal peroxidase activity, hydrogen peroxide (3%) in a PBS was used. Then, antigen retrieval was carried out in a microwave for 10 min with appropriate buffer solutions as follows; 10 mmol/l citrate buffer, pH 6 for CD44 and 1 mmol/l EDTA, pH 8 for CTTN. Further incubations using primary mouse monoclonal antibody anti-CD44 (clone 156–3C11; Lab Vision, Fremont, California, USA), dilution 1:50 and anti-CTTN (clone 30; Santa Cruz Biotechnology, Santa Cruz, California, USA), dilution 1:50 were used as the primary antibodies for 30 min and were incubated in a moist chamber in room temperature for 1 day, followed by the application of secondary antibody (for 15 min), 3,3'-diaminobenzidine (producing brown staining), and Meyer's hematoxylin (for background staining). The samples were placed in PBS immediately after each of the above-mentioned steps. Breast carcinoma specimens were positive and serve as positive control according to the manufacturer's instructions. The negative control was prepared by the replacement of primary antibody with nonimmune normal rabbit or mouse sera.

Assessment of immunohistochemistry stained sections

Presence of brown-colored reaction membranous (CD44) or cytoplasmic (CTTN), was considered as positive reaction. The evaluation of immunostaining was carried out by using the Image J software, version 4.10.03 (Nikon, Tokyo, Japan). Assessment of CD44 and CTTN expression was done using IHC Profiler according to Varghese et al.[12] and a score for each case was assigned as high positive(+3), positive (+2), low positive (+1) and negative (0).

Statistical analysis

The data obtained were then subjected to statistical analysis by using SPSS statistics for Windows, version 17.0 (SPSS Inc., Chicago, Illinois, USA). The χ2-test and nonparametric Kendall's-τ were applied. Also, Tukey's test of significance was conducted to look for any significant associations between CD44 and CTTN expressions and LN metastasis. P values of at least 0.05 were considered statistically significant.


Immunoprofile of oral squamous cell carcinoma specimens with positive lymph node metastasis


CD44 was expressed in all OSCC specimens as membranous staining of all cellular borders. CD44 expression was found to be directly proportional to LN metastasis as its expression was upregulated in OSCC specimens with positive LN metastasis. Overall, 14 (70%) out of 20 OSCC specimens with positive LN metastasis showed positive (+2) CD44 expression (Figure 1] and [Figure 2] and the remaining specimens (30%) exhibited low positive expression [Table 1] and [Figure 3] with mean value of 1.7 ± 0.46.{Table 1}{Figure 1}{Figure 2}{Figure 3}


All OSCC specimens with positive LN metastasis showed cytoplasmic CTTN expression with mean value 1.6 ± 0.49. CTTN expression was found to be upregulated with LN metastasis. Overall, 12 (60%) out of 20 specimens revealed positive (+2) CTTN expression [Figure 2], whereas the remaining specimens (40%) showed low positive (+1) expression [Figure 1] and [Figure 4] and [Table 2]).{Table 2}{Figure 4}

Immunoprofile of oral squamous cell carcinoma specimens with negative lymph node metastasis


CD44 expression in OSCC specimens with negative LN metastasis ranged between low positive (+1) and positive (+2) with mean value 1.35 ± 0.48. In 13 (65%) out of 20 specimens, CD44 expression was low positive (+1) [Figure 5]. The remaining specimens (35%) showed positive (+2) expression [Figure 3] and [Figure 6], [Table 1]).{Figure 5}{Figure 6}


A total of 15 (75%) out of 20 OSCC specimens with negative LN metastasis showed low positive (+1) expression [Figure 6] and the remaining specimens (25%) exhibited positive (+2) expression [Figure 4] and [Figure 5], [Table 2]) with mean value of 1.25 ± 0.43.

Correlation between CD44 and CTTN expression

In the present study, it was found that CD44 expression in squamous cell carcinoma specimens was highly significantly correlated to CTTN expression with increasing malignant criteria of tumor cells and LN metastasis (P < 0.01) [Table 3].{Table 3}


Despite advances in diagnosis and treatment of OSCC, the prognosis of this malignant tumor has remained relatively poor [13]. Cervical LN and distant metastasis are the major causes of mortality in OSCC patients. So, tumor markers reflecting molecular alterations of the primary tumor and surrounding stroma may provide valuable information about the metastatic potential of a tumor [14].

Metastasis is very sophisticated, multistep process consisting of cell detachment from the primary tumor, invasion to the ECM, entry into the blood or lymph vessels, dispersal through the circulation and proliferation following extravasation in the target organ [15]. Tumor cells must alter cell–cell and cell–matrix adhesion to invade and metastasize. CD44 is a cell membrane adhesion molecule that interacts in cell–cell and cell–matrix adhesions [5].

In the present study, CD44 expression was significantly upregulated with regional LN metastasis (P < 0.05). Similar observations were reported by previous studies [16],[17],[18],[19]. Analysis of these data suggests that CD44 activated some proteinases enzymes, for example, matrix metalloproteinase (MMP)-9, which responsible for digesting ECM, thereby promoted the degradation of tumor cell-mediated matrix, resulting in the enhancement of invasion and metastasis.

Conversely, Mostan et al.[20] postulated that reduced CD44 expression has significant correlation with cervical LN metastasis. Even more, Kawano et al.[21] and Liu et al.[22] showed no correlation between CD44 expression and LN metastasis in nasopharyngeal carcinoma and laryngeal carcinoma respectively and explained that 'this inconsistency may be due to different standards for evaluating staining or variation of antibody specificity or sensitivity'.

Another step in the metastasis process is the migration of malignant epithelial cells after degradation of the basement membrane. After penetrating the basement membrane, tumor cells migrate through the stroma. Therefore, degradation of ECM and basement membrane components is a crucial step for cancers to progress from carcinoma in situ to invasive carcinoma and the development of metastasis [23]. Cortactin is one of the molecules that are related to cell motility and degradation of the basement membrane and ECM components.

The present study revealed a significant correlation between CTTN expression and regional LN metastasis (P < 0.05) which goes along with previous studies on head and neck squamous cell carcinoma [24], OSCC [25], hepatocellular carcinoma [26], and lung cancer [27]. In a like manner, Luo et al.[7] reported that in experimental metastasis assay, intravenous injection of in-vitro cultured esophageal cancer cells transfected with cortactin silence RNA (synthetic RNA degrade mRNA after transcription) in nude mice decreased lung metastasis and prolonged survival time compared with control group which is injected by cancer cells not transfected by CTTN silence RNA.

These findings could be explained as the major function of CTTN is to regulate and stabilize actin assembly which is important for formation and proper function of invadopodia. Also, secretion of MMPs by invadopodia and degradation of ECM are regulated by CTTN. In the same way, Li et al.[28] postulated that the overexpression of CTTN potentiates bone metastasis of breast cancer by increasing the adhesive affinity of tumor cells for bone marrow endothelial cells. These findings assume that CTTN overexpression endows cancer cells with various capabilities for metastasis.

Based on previous data, the current study revealed a highly significant correlation (P < 0.01) between CD44 and CTTN expression. This correlation may be explained by data reported by Wehrel-Haller et al.[29]who postulated that cell motility is a complex event depend on the coordinated remodeling of the actin cytoskeleton, on regulated assembly of actin network, and on turnover of focal adhesion. CD44 has been demonstrated to serve as a docking molecule to retain MMP-9 activity at the cell surface [30]. MMP-9 proteolytically cleaves CD44 into extracellular domain (CD44ECD) and intracellular domain (CD44ICD). CD44ECD induces cell crawling at the leading edge on a HA matrix, along with invadopodia extension which induces mechanical stretching of cells. CTTN is required for formation and proper function of invadopodia. This process rapidly activates metalloproteases followed by CD44 cleavage and facilitates cell detachment from the HA matrix at the rear of the cells. CD44ICD translocates to the nucleus and induces high levels of CD44 mRNA, promoting attachment of the newly synthesized CD44 [31]. Thus, the rapid turnover of CD44, actin cytoskeleton remodeling and regulated actin assembly enable efficient cell migration.

Consequently, CD44 and CTTN act in synergistic manner to enhance cancer cell motility, invasion and metastasis. Altogether, the results of this study suggest that CD44 and CTTN can be used as important tools in prediction of metastasis in oral cancer.


From the present study, it can be concluded that assessment of CD44 and CTTN in OSCC represent an important tool for prediction of risk of metastasis for each case and the significant correlation between CD44 and CTTN could provide insights of their critical role in metastasis.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics. CA Cancer J Clin 2008; 58:71–96.
2Sargeran K, Murtomaa H, Safavi SM, Vehkalahti MM, Teronen O. Survival after diagnosis of cancer of the oral cavity. Br J Oral Maxillofac Surg 2008; 46:187–191.
3Napier SS, Speight PM. Natural history of potentially malignant oral lesions and conditions: an overview of the literature. J Oral Pathol 2008; 37:1–10.
4Petti S. Lifestyle risk factors for oral cancer. Oral Oncol 2009; 45:340–350.
5Kosunen A, Pirinen R, Ropponen K, Pukkila M, Kellokoski J, Virtaniemi J, et al. CD44 expression and its relationship with MMP-9, clinicopathological factors and survival in oral squamous cell carcinoma. Oral Oncol 2007; 43:51–59.
6Daly RJ. Cortactin signaling and dynamic actin networks. Biochem J 2004; 382:13–25.
7Luo ML, Shen XM, Zhang Y, Wei F, Xu X, Cai Y, et al. Amplification and overexpression of CTTN (EMS1) contribute to the metastasis of esophageal squamous cell carcinoma by promoting cell migration and anoikis resistance. Cancer Res 2006; 66:11690–11699.
8Weaver AM. Cortactin in tumor invasiveness. Cancer Lett 2008; 265:157–166.
9Misra S, Heldin P, Hascall VC, Karamanos NK, Skandalis SS, Markwald RR, et al. Hyaluronan-CD44 interactions as potential targets for cancer therapy. FEBS J 2011; 278:1429–1443.
10Takamune Y, Ikebe T, Nagano O, Nakayama H, Ota K, Obayashi T, et al. ADAM-17 associated with CD44 cleavage and metastasis in oral squamous cell carcinoma. Virchows Arch 2007; 450:169–177.
11Curley MD, Garrett LA, Schorge JO, Foster R, Rueda BR. Evidence for cancer stem cells contributing to the pathogenesis of ovarian cancer. Front Biosci 2011; 16:368–392.
12Varghese F, Bukhari AB, Malhotra R, De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One 2014; 9:e96801.
13Marur S, Forastiere AA. Challenges of integrating chemotherapy and targeted therapy with radiation in locally advanced head and neck squamous cell carcinoma. Curr Opin Oncol 2010; 22:206–211.
14Hagen BEK, Simon JR. Comparative study of the expression of p53, Ki67, Ecadherin and MMP-1 in verrucous hyperplasia and verrucous carcinoma of the oral cavity. Head Neck Pathol 2007; 1:118–122.
15Jarvinen A, Autio R, Kilpinen S. High-resolution copy number and gene expression microarray analyses of head and neck squamous cell carcinoma cell lines of tongue and larynx. Genes Chromosomes Cancer 2008; 47:500–509.
16Simionescu C, Mărgăritescu C, Surpăţeanu M, Mogoantă L, Zăvoi R, Ciurea R, et al. The study of E-cadherin and CD44 immunoexpression in oral squamous cell carcinoma. Rom J Morphol Embryol 2008; 49:189–193.
17Okamoto M, Nishimine M, Kishi M, Kirita T, Sugimura M, Nakamura M, et al. Prediction of delayed neck metastasis in patients with stage I/II squamous cell carcinoma of the tongue. J Oral Pathol Med 2002; 31:227–233.
18Yüce I, Bayram A, Cağlı S, Canöz O, Bayram S, Güney E. The role of CD44 and matrix metalloproteinase-9 expression in predicting neck metastasis of supraglottic laryngeal carcinoma. Am J Otolaryngol 2011; 32:141–146.
19Wu XJ, Li XD, Zhang H, Zhang X, Ning ZH, Yin YM, et al. Clinical significance of CD44s, CD44v3 and CD44v6 in breast cancer. J Int Med Res 2015; 43:173–179.
20Mostaan LV, Khorsandi MT, Sharifian SM, Shandiz FH, Mirashrafi F, Sabzari H, et al. Correlation between E-cadherin and CD44 adhesion molecules expression and cervical lymph node metastasis in oral tongue SCC: predictive significance or not. Pathol Res Pract 2011; 207:448–451.
21Kawano T, Nakamura Y, Yanoma S, Kubota A, Furukawa M, Miyagi Y, et al. Expression of E-cadherin, and CD44s and CD44v6 and its association with prognosis in head and neck cancer. Auris Nasus Laryn×2004; 31:35–41.
22Liu WW, Zeng ZY, Wu QL, Hou JH, Chen YY. Overexpression of MMP-2 in laryngeal squamous cell carcinoma: a potential indicator for poor prognosis. Otolaryngol Head Neck Surg 2005; 132:395–400.
23De Vicente JC, Fresno MF, Villalain L, Vega JA, Hernández Vallejo G. Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma. Oral Oncol 2005; 41:283–293.
24Rodrigo JP, García-Carracedo D, García LA, Menéndez S, Allonca E, González MV, et al. Distinctive clinicopathological associations of amplification of the cortactin gene at 11q13 in head and neck squamous cell carcinomas. J Pathol 2009; 217:516–523.
25Liu HS, Lu HH, Lui MT, Yu EH, Shen W, Chen YP, et al. Detection of copy number amplification of cyclin D1 (CCND1) and cortactin (CTTN) in oral carcinoma and oral brushed samples from areca chewers. Oral Oncol 2009; 45:1032–1036.
26Chuma M, Sakamoto M, Yasuda J, Fujii G, Nakanishi K, Tsuchiya A, et al. Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma. J Hepatol 2004; 41:629–636.
27Noh SJ, Baek HA, Park HS, Jang KY, Moon WS, Kang MJ, et al. Expression of SIRT1 and cortactin is associated with progression of non-small cell lung cancer. Pathol Res Pract 2013; 209:365–370.
28Li Y, Tondravi M, Liu J, Smith E, Haudenschild CC, Kaczmarek M, et al. Cortactin potentiates bone metastasis of breast cancer cells. Cancer Res 2001; 61:6906–6911.
29Wehrle-Haller B, Imhof BA. Actin, microtubules and focal adhesion dynamics during cell migration. Int J Biochem Cell Biol 2003; 35:39–50.
30Peng ST, Su CH, Kuo CC, Shaw CF, Wang HS. CD44 crosslinking-mediated matrix metalloproteinase-9 relocation in breast tumor cells leads to enhanced metastasis. Int J Oncol 2007; 31:1119–1126.
31Chetty C, Vanamala SK, Gondi CS, Dinh DH, Gujrati M, Rao JS. MMP-9 induces CD44 cleavage and CD44 mediated cell migration in glioblastoma xenograft cells. Cell Signal 2012; 24:549–559.