Tumor and Microenvironment

ORIGINAL ARTICLE
Year : 2018  |  Volume : 1  |  Issue : 1  |  Page : 16--24

Significance of phosphorylated epidermal growth factor receptor, matrix metalloproteinases, and E-cadherin in oral cancer

Bhairavi N Vajaria1, Kinjal R Patel1, Rasheedunnisa Begum2, Jayendra B Patel1, Franky D Shah1, Prabhudas S Patel1,  
1 Biochemistry Research Division, Gujarat Cancer Research Institute, Ahmedabad, India
2 Department of Biochemistry, Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India

Correspondence Address:
Dr. Prabhudas S Patel
Biochemistry Research Division, Gujarat Cancer Research Institute, Ahmedabad, Gujarat
India

Abstract

Objective: The most challenging problem in oral cancer is late monitoring and disease spread (metastasis). The study aimed to simultaneously evaluate phosphorylated epidermal growth factor receptor (pEGFR), truncated E-cadherin protein, and matrix metalloproteinases (MMPs) in patients with oral cancer. Methodology: pEGFR and truncated E-cadherin protein were measured from 25 paired tissues by ELISA and Western blot, respectively. Plasma MMPs levels were studied by gelatin zymography from 100 controls and 100 patients with oral cancer. The results revealed significant higher expression of pEGFR and truncated E-cadherin protein in malignant oral cancer tissues as compared to adjacent normal. Plasma MMPs were significantly elevated in patients with oral cancer as compared to the controls. An increase in the levels of pEGFR and truncated E-cadherin protein was observed in advanced and metastatic disease as compared to early and nonmetastatic disease. The levels of MMPs were increased in advanced disease as compared to early disease. Kaplan–Meier's survival analysis indicated that elevated expression of pEGFR, truncated E-cadherin protein, active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 has reduced the overall survival. Conclusion: Simultaneous elevation of pEGFR, truncated E-cadherin protein, and MMPs indicated its role in oral carcinogenesis. Further combination therapies targeting these markers might help in combating oral cancer.


How to cite this article:
Vajaria BN, Patel KR, Begum R, Patel JB, Shah FD, Patel PS. Significance of phosphorylated epidermal growth factor receptor, matrix metalloproteinases, and E-cadherin in oral cancer.Tumor Microenviron 2018;1:16-24


How to cite this URL:
Vajaria BN, Patel KR, Begum R, Patel JB, Shah FD, Patel PS. Significance of phosphorylated epidermal growth factor receptor, matrix metalloproteinases, and E-cadherin in oral cancer. Tumor Microenviron [serial online] 2018 [cited 2023 Jun 4 ];1:16-24
Available from: http://www.TMEResearch.org/text.asp?2018/1/1/16/203050


Full Text



 Introduction



Recently, there is an upsurge in the incidence of oral cancer, especially in India.[1],[2] Metastasis is a serious problem in patients with oral cancer and is a major cause of increasing mortality. Hence, understanding the molecular mechanisms that trigger tumor progression and metastases is one of the great confronts in cancer research. Several steps in the metastatic cascade depend on cell surface molecules such as E-cadherin which play a critical role in cell–cell adhesion, along with molecules such as matrix metalloproteinases (MMPs) and epidermal growth factor receptor (EGFR). MMPs are the enzymes involved in the degradation of extracellular matrix (ECM). MMPs and EGFR are responsible for increased invasion and cell proliferation.

E-cadherin, a transmembrane glycoprotein, is involved in homophilic adhesion in epithelial cells. Earlier studies have documented reduced expression of E-cadherin in oral squamous cell carcinoma (OSCC), which is associated with a poor prognosis.[3],[4] Oral cancer has also been characterized by diminished E-cadherin levels.[4],[5],[6],[7],[8] It has been reported that ectodomain shedding of E-cadherin might play an important role in invasion process associated with tumor progression.[9],[10] Truncation of E-cadherin by MMPs causes decreased cell–cell adhesion.[11]

Tissue invasion and metastasis require extensive remodeling of ECM, which requires a number of extracellular enzymes. MMPs are the enzymes which play an important role in metastasis and invasion by proteolytic degradation of ECM, disruption of cell–cell adhesion, and cell matrix adhesion, migration, and angiogenesis.[12],[13] Earlier studies from our laboratory have documented increased levels of MMP-2 and MMP-9 in oral cancer and have demonstrated its association with metastasis.[14] The importance of growth factors in tissue remodeling and their implication in cancer progression and metastasis are well recognized. EGFR is a tyrosine kinase receptor of the ErbB family which plays an important role in proliferation, invasion, and metastasis.[15] Abnormal amplification of EGFR gene has been observed in various human tumors including OSCC.[15],[16],[17],[18],[19],[20] It has been earlier suggested that phosphorylated EGFR (pEGFR) is the active form of EGFR and hence its expression is necessary to be evaluated. pEGFR expression has been shown to be correlated with the evaluation of prognosis and disease progression in patients with breast cancer and lung cancer.[21],[22] Studies have suggested that increased expression of EGFR is responsible for membrane to cytoplasmic localization of E-cadherin in OSCC and the consequent increase of E-cadherin cointernalization with EGFR.[23] Earlier results have suggested that reduction in E-cadherin results in the upregulation of EGFR transcriptionally.[24] Furthermore, in vitro studies have indicated that the activation of EGFR downregulates E-cadherin.[25]

It is known that MMPs are essential for tissue invasion and metastasis and E-cadherin disruption plays a key role in metastasis. Moreover, pEGFR, which is the active form of EGFR, is known to be involved in cellular proliferation. Therefore, it is necessary to evaluate these markers simultaneously to understand the mechanism of cancer progression and metastasis. However, to the best of our knowledge, there are no earlier reports on simultaneous evaluation of E-cadherin, MMPs, and pEGFR in patients with oral cancer. Hence, the present study evaluated E-cadherin, MMPs, and pEGFR simultaneously in patients with oral cancer to study the molecular basis of metastasis in oral cancer.

 Methodology



Participants

The study was approved by Institutional Review Board of Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India. Due consent was obtained from all the participants who participated in the study. The participants included in the study comprised 100 healthy individuals (as controls), who had no major illness in the recent past, and 100 untreated patients with oral cavity cancer. The participants were enrolled from Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India. Pathological tumor, node, and metastasis staging of malignant disease was performed as per the American Joint Committee on Cancer norms.[26] The clinicopathological details of patients with oral cancer including disease site, metastasis, stage, and differentiation are mentioned in [Table 1]. The numbers in [Table 1] represent the number of patients with oral cancer analyzed with each clinical characteristic.{Table 1}

Sample collection

Blood samples were collected by venous puncture and plasma was separated from heparinized Vacuettes of 100 patients with oral cancer and 100 healthy controls. Tissue samples from patients with oral cancer (25 paired tissues) were collected on ice from operation theater immediately after surgical resection of the tumors. Adjacent normal tissue samples were selected from the tumor-free margins at least 2–3 cm away from the tumor as defined by the pathologist. The tissue specimens were washed with ice-cold phosphate-buffered saline (pH 7.4) and were stored at −80°C until analyzed.

Estimation of phosphorylated epidermal growth factor receptor

pEGFR expression was estimated using DuoSet IC human phospho-EGFR ELISA kit (R and D systems Cat No: DYC1095B-2), which was specific for the detection of tyrosine pEGFR only.

The optical density was determined immediately using a microplate reader set to 450 nm and 540 nm. The readings at 540 nm were subtracted from the readings at 450 nm. This subtraction was performed to correct for optical imperfections in the plate. The final readings were subtracted from the blank optical density after wavelength correction at 540 nm. The standard curve was prepared using pEGFR control at a concentration range of 10–40 ng/ml.

Total proteins

Total protein levels from plasma euglobulin fraction/tissue lysates were determined using Lowry's method.[27] In brief, 10 μl of plasma euglobulin samples was mixed with 490 μl of distilled water and 2.25 ml of reagent C was added and the tubes were incubated for 10 min at room temperature. Thereafter, 0.25 ml of Folin–Ciocalteau reagent was added and the tubes were incubated for 30 min at room temperature in dark. The tubes were read spectrophotometrically at 750 nm. The standard curve using bovine serum albumin (Sigma, USA) as standard was linear from 10 to 60 μg.

Estimation of matrix metalloproteinases by Gelatin zymography

Gelatin zymography was performed using methodology as described by Lorenzo et al.[28] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; containing 0.5 mg/ml gelatin) using 7.5% polyacrylamide gel was performed using 100 μg of plasma euglobulin fraction with sample buffer dye without reducing agent. Gels were washed and kept in 50 mM Tris HCl pH 7.5; containing 10 mL CaCl2, 1 μM ZnCl2, 0.02% (w/v) NaN3, and 1% (V/V) Triton X-100, overnight. The gels were then stained with 0.1% (W/V) Coomassie Brilliant Blue R-250 and destaining was performed in 7% (V/V) acetic acid. These zymograms were analyzed using gel documentation system (Alpha Innotech, USA). The integrated density value (IDV), or the sum of all the pixel values after background correction, was evaluated for each proteinase activity. To check for reproducibility, the samples were run in the same gels as well as in different gels.

Estimation of truncated E-cadherin protein by Western blot

To perform Western blot, total tissue lysate was prepared from 50 mg of tissue homogenized in lysis buffer (0.05 M Tris pH 7.5 containing 2 mM ethylenediaminetetraacetic acid [EDTA], 5 mM sodium fluoride, 5 mM EDTA, 150 mM sodium chloride, 1.0% nonidet P-40, 1 mM phenyl methyl sulfonyl fluoride, 10 mM aprotinin, 1 mM sodium orthovanadate, and 0.3 mM leupeptin). After centrifugation, the lysate supernatant was taken in another fresh microcentrifuge tube and its total protein content was estimated.[27] The running gel of 10% was prepared for E-cadherin protein. The lysate supernatant equivalent to 100 μg proteins was mixed with 6X sample loading buffer, and denatured by boiling for 3 min. The samples were loaded after 5 min. The proteins were separated using denaturing and reducing SDS-PAGE at a constant voltage (100 V) until the dye front reached the bottom of the gel. After electro-transfer, the membrane was incubated in 5% w/v blocking reagent in Tris buffer saline tween 20 (TBS-T) for 1 h at room temperature, After three washes with TBS-T (1 min × 15 min and 2 min × 5 min), the membrane was incubated overnight with primary antibody, i.e., HECD 1 mouse monoclonal antibody (Calbiochem, USA) with gentle shaking. The membrane was rinsed three times with TBS-T (1 min × 15 min and 2 min × 5 min), and Horse radish peroxidase-conjugated anti-mouse secondary antibody (1:1000 dilution in TBS-T) was incubated for 2 h with gentle shaking. The detection was performed by autoradiography (chemiluminescence method) using ECL Western blotting detection kit (GE Healthcare, UK) by capturing luminescence on light-sensitive hyperfilm.

The densitometric analysis of protein band was performed using densitometer (Alpha Innotech Inc, USA) and IDV, i.e., sum of pixel values after background correction was evaluated.

Statistical analysis

Statistical analysis of the data was performed using Statistical Package for Social Science software version 17.0. (SPSS Inc., Chicago, USA) Student's paired t-test was used to compare the levels of pEGFR and truncated E-cadherin protein between malignant and adjacent normal oral cancer tissues. Student's independent t-test was carried out to assess the levels of significance of pEGFR, truncated E-cadherin protein, and various forms of MMPs with various clinico-pathological parameters such as stage, metastasis, differentiation, and infiltration. Pearson's correlation analysis was used to evaluate the correlation between pEGFR, truncated E-cadherin protein, and various forms of MMPs. To obtain optimal cutoff point for survival analysis, receiver operating characteristic (ROC) curve analysis was performed using MedCalc software (Medcalc Software, Ostend, Belgium). ROC curve analysis was also performed to evaluate the discriminatory efficacy of various forms of MMPs between controls and patients with oral cancer, as well as for pEGFR and truncated E-cadherin expression in distinguishing malignant and adjacent normal tissues. Kaplan–Meier survival analysis was performed to analyze correlation of the markers with overall survival, and the significance of differences in survival rates was analyzed by Log-rank test. The values are expressed as the mean ± standard error of mean. P ≤ 0.05 was considered statistically significant. pEGFR and truncated E-cadherin protein were estimated from 25 paired malignant and adjacent normal oral cancer tissues. Plasma MMPs were estimated from 100 controls and 100 patients with oral cancer.

 Results



Expression of phosphorylated epidermal growth factor receptor

[Figure 1] depicts the levels of pEGFR expression between malignant and adjacent normal tissues. As depicted in [Figure 1], the levels were significantly higher (P = 0.019) in malignant tissues as compared to adjacent normal tissues by paired t-test.{Figure 1}

Levels of truncated 97 kDa truncated E-cadherin protein

[Figure 2]a represents the Western blot analysis of truncated E-cadherin protein in malignant and adjacent normal tissues of patients with oral cancer. As illustrated in [Figure 2]b, the bar chart reveals a significant increase (P = 0.025) of truncated 97 kDa E-cadherin protein in malignant tissues as compared to adjacent normal tissues by paired t-test.{Figure 2}

Comparison of plasma pro, active, and total matrix metalloproteinases-2 and matrix metalloproteinases-9 in controls and patients with oral cancer

[Figure 3]a depicts the gelatin zymography pattern in controls and patients with oral cancer. The representative pattern reveals pro and active forms of MMP-2 and MMP-9. Activation ratio was defined as the ratio of active MMP/total MMP. [Figure 3]b and c depict the levels of different forms of MMPs in controls and patients with oral cancer. As depicted in [Figure 3]b and c, active MMP-2, pro and active MMP-9, total MMP-2, and MMP-9 were significantly higher in patients with oral cancer (P < 0.0001, P < 0.0001, P = 0.051, and P < 0.0001, respectively) as compared to the controls by student's independent t-test.{Figure 3}

Receiver operating characteristic curve analysis of phosphorylated epidermal growth factor receptor, truncated E-cadherin protein, and matrix metalloproteinases

To evaluate the distinguishing capacity of the pEGFR expression and truncated E-cadherin protein in discriminating malignant and adjacent normal tissues, ROC curves were constructed. [Table 2] indicates the ROC curve analysis which depicts that pEGFR and truncated E-cadherin protein could distinguish adjacent normal and malignant tissues with AUC of 0.674 (P = 0.0620) and 0.673 (P = 0.2602), respectively. ROC curves were also constructed for plasma pro, active, and total MMP-2 and MMP-9. The results depicted in [Table 2] indicated that Pro MMP-2, active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 could significantly discriminate controls and patients with oral cancer (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, and P = 0.006, respectively).{Table 2}

Levels of markers with various clinicopathological parameters

The levels of pEGFR were compared between early and advanced stages of disease. The pEGFR expression was found to be higher in advanced stage as compared to early stage (P = 0.723), however the levels were nonsignificant. The expression of pEGFR was found to be higher in metastatic tumors as compared to nonmetastatic tumors (P = 0.416), however nonsignificant.

The levels of truncated E-cadherin protein (97 kDa) were found to be elevated in advanced stage of disease as compared to early stage (P = 0.313) and were found to be increased in infiltrative tumors (lymphocytic infiltration) as compared to noninfiltrative tumors (P = 0.121). The levels were significantly higher in metastatic as compared to nonmetastatic tumors (P < 0.0001). The levels of pro MMP-2, active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 were observed to be higher in advanced stage as compared to early stage, and the levels were significant for pro MMP-2 (P = 0.003). An increasing trend of plasma active MMP-2, pro MMP-9, active MMP-9, total MMP-2, and total MMP-9 was observed from well to moderate to poorly differentiated tumors. However, no significant difference was observed between well versus moderate (P = 0.152, P = 0.521, P = 0.081, P = 0.066, P = 0.414, and P = 0.075, respectively), well versus poor (P = 0.981, P = 0.450, P = 0.472, P = 0.451, P = 0.48, and P = 0.467, respectively), and moderate versus poorly differentiated tumors (P = 0.525, P = 0.735, P = 0.686, P = 0.632, P = 0.803, and P = 0.674, respectively).

Kaplan–Meier's survival analysis

Kaplan–Meier's survival analysis was performed to analyze the association of pEGFR expression, truncated E-cadherin protein, and different forms of plasma MMPs with overall survival. As ROC curve analysis is used to analyze diagnostic utility, ROC cutoff was taken to determine its prognostic utility as well by survival analysis. The significance of differences in survival rates was analyzed by Log-rank test. Kaplan–Meier's overall survival analysis is depicted in [Table 3], which shows the optimal ROC cutoff, Log-rank Chi-square, and level of significance.{Table 3}

Kaplan–Meier's survival analysis of pEGFR [Table 3] indicated that values above ROC cutoff were associated with lower overall survival, however the levels were nonsignificant (P = 0.739, χ2 = 0.111). The mean statistics could not be computed as all the cases were censored. Kaplan–Meier's survival analysis depicted that patients with oral cancer with values above ROC cutoff of active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 [Table 3] had lower overall survival as compared to those with values below ROC cutoff. Kaplan–Meier's survival analysis of E-cadherin [Table 3] depicted that levels of truncated E-cadherin protein above ROC cutoff had lower overall survival as compared to values below the cutoff (χ2 = 0.400, P = 0.527).

Correlation between matrix metalloproteinases, E-cadherin, and phosphorylated epidermal growth factor receptor

The correlation of pEGFR, matrix metalloproteinases, and truncated E-cadherin protein was evaluated by Pearson's correlation analysis. [Table 4] depicts that truncated E-cadherin protein showed a significant positive correlation with plasma pro MMP-2 (r = 0.948, P < 0.0001), plasma active MMP-2 (r = 0.748, P = 0.008), plasma active MMP-9 (r = 0.873, P < 0.0001), plasma total MMP-2 (r = 0.778, P = 0.005), and activation ratio MMP-2 (r = 0.062, P = 0.857). As depicted in [Table 4], pEGFR was positively correlated with truncated E-cadherin protein (r = 0.729, P = 0.163). However, no significant correlation of pEGFR was observed with various forms of plasma MMPs. Moreover, pro MMP-2, pro MMP-9, active MMP-2, and MMP-9 were observed to be significantly (P < 0.0001) positively intercorrelated.{Table 4}

 Discussion



Metastasis is a major clinical problem and is responsible for a majority of oral cancer-related deaths. The loss of cellular adhesion, detachment of the tumor cells, migration, and spread to distant organs are important steps in metastasis. The role played by MMPs, EGFR, and E-cadherin in progression of oral cancer could be a valuable approach to understand the molecular basis of oral cancer and for the management of these patients.

The present study simultaneously evaluated pEGFR, truncated E-cadherin, and MMPs as markers of metastasis in patients with oral cancer. It was observed that the expression of pEGFR was significantly higher in malignant tissues as compared to adjacent normal tissues. Increased expression of EGFR gene has been observed in various human tumors including OSCC.[15],[16],[17],18],[19],[20],[29] EGFR expression in premalignant lesion has been documented to be useful for predicting the neoplastic potential of dysplastic tissues. Mechanisms leading to constitutive activation of EGFR include increased production of ligands, elevated levels of EGFR protein, EGFR mutations, and defective downregulation of EGFR.[30]

In the present study, Kaplan–Meier's survival analysis indicated that higher values of pEGFR were associated with lower overall survival. Earlier studies have indicated increased EGFR expression to be correlated with poor survival.[20],[31],[32] In the present study, elevated expression of pEGFR was observed in advanced stage as compared to early stage, and levels were also found to be higher in metastatic tumors as compared to nonmetastatic tumors. The results were supported by previous studies that have reported a significant association of EGFR expression with the stage of disease.[31] Studies have also documented that EGFR-positive immunoreactivity in oral cancer was not correlated with clinicopathological parameters.[16] A study by Myers et al. showed that targeted molecular therapy of EGFR arrests the growth of oral cancer in vitro and reduces its proliferation in an experimental xenograft animal model.[33] Since overexpression of pEGFR was also observed in malignant oral cancer tissues in the present study, targeting EGFR may be a promising approach to combat the disease.

In the present study, pEGFR expression was observed to be positively correlated with truncated E-cadherin protein. It has been reported that decreased expression of E-cadherin resulted in an overexpression of EGFR in keratinocytes, whereas E-cadherin transfection reversed this effect.[34] Several studies have observed decreased E-cadherin expression in oral cancer.[4],[6],[7],[8] Previous studies have reported that loss of E-cadherin results in the upregulation of EGFR mRNA in head and neck cancer.[24] Supporting the data, we hypothesize that increase in truncation of E-cadherin might be upregulating pEGFR expression, as observed in the present study. Earlier studies have also observed that mutation in E-cadherin results in increased EGFR activation and decreased E-cadherin-EGFR association.[35],[36] In contrast, formation of E-cadherin-mediated cell–cell adhesion has been shown to activate EGFR in various experimental settings.[37],[38] It has been suggested that during epithelial-mesenchymal transition, several classes of receptor tyrosine kinases, including EGFR, Her2-neu, insulin-like growth factor 1 receptor (IGF-1R), and c-Met, can inhibit E-cadherin-dependent adhesion.[39],[40],[41] The regulation is bidirectional as E-cadherin can also inhibit activation of EGFR, Her2-neu, IGF-1R, and c-Met.[42]

Loss of E-cadherin plays a key role in metastasis with involvement of proteinases. In the present study, truncated E-cadherin protein showed a significant positive correlation with plasma pro MMP-2, plasma active MMP-2, plasma active MMP-9, plasma total MMP-2, and activation ratio MMP-2. Hence, the results suggested that elevated expression of MMPs causes loss of cell–cell adhesion by increased truncation of E-cadherin protein. An overexpression of E-cadherin has been reported in invasive bronchial tumor cell lines which was responsible for decreased invasiveness and reduced expression of MMP-1, MMP-3, MMP-9, and MT1-MMP.[43]

The levels of pro MMP-2, active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 were observed to be higher in advanced stage as compared to early stage and the levels were significant for pro MMP-2. An increasing trend of plasma active MMP-2, pro MMP-9, active MMP-9, total MMP-2, and total MMP-9 was observed from well to moderate to poorly differentiated tumors. Earlier studies have observed that MMP-9 expression was associated with tumor metastasis of OSCC.[44],[45] Moreover, high tumor and stromal MMP-2 and MMP-9 expression was significantly associated with positive lymph node metastasis and was linked to poor clinical outcomes.[44],[46]

In the present study, no significant association of pEGFR was observed with plasma MMPs. Earlier reports have shown that EGFR activation leads to decreased E-cadherin, and MMP inhibition improves EGF-stimulated junctional disruption and loss of E-cadherin protein.[41] Activation of EGFR promotes SCC cell migration and invasion through inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin through ERK1/2 and PI3-K signaling pathway.[47],[48] In the present study, simultaneous elevation of truncated E-cadherin protein, EGFR, MMP-2, and MMP-9 was observed. Similarly, in human ovarian tumors and paired peritoneal metastases, the immunohistochemical staining for activated pEGFR and MMP-9 was colocalized with regions of reduced E-cadherin.[49] However, there are lack of reports on simultaneous evaluation of pEGFR, truncated E-cadherin protein, and MMPs in oral cancer. Studies have depicted that loss of E-cadherin activates EGFR-MEK/ERK signaling which promotes invasion through the ZEB1/MMP-2 axis in non-small cell lung cancer.[48] Thus, based on these studies, we hypothesize that decrease in E-cadherin is involved in the upregulation of pEGFR which is further involved in upregulating MMPs. Moreover, increase in MMPs further causes decrease in E-cadherin levels by disruption of cell–cell adhesion [Figure 4].{Figure 4}

 Conclusion



The results revealed significant increase of different forms of plasma MMPs in patients with oral cancer as compared to controls as well as significant elevated expression of pEGFR and truncated E-cadherin protein in malignant oral cancer tissues. The results signified the role of MMPs, pEGFR, and truncated E-cadherin protein in oral cancer progression and metastasis. Overall, the study outcome represented that increased expression of pEGFR might cause decrease in E-cadherin levels by upregulating MMPs. Moreover, decrease in E-cadherin might be involved in EGFR activation. Nowadays, much advancement in EGFR-based inhibitors and novel strategies in targeting EGFR have also been documented.[50],[51] MMP inhibitors might be effective in controlling metastasis.[50],[52] Several combination therapies targeting different molecular markers such as EGFR and MMPs of oral cancer might be effective in controlling the disease. The present study also opened the way for the development of combination therapies to combat oral cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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