Immunohistochemical Expression of the Epithelial to Mesenchymal Transition Proteins E-cadherin and ß-catenin in Grades of Oral Squamous Cell Carcinoma.

BACKGROUND: E-Cadherin/β-Catenin protein complexes play a major role in epithelial to mesenchymal transition (EMT) and vice versa. Such types of EMT are implicated physiologically during embryonic development and pathologically in tissue fibrosis and tumorigenesis. AIMS: The aim was the evaluation of E-Cadherin and β-Catenin immunoreactivity in various grades of oral squamous cell carcinoma (OSCC) and to correlate their pattern of expression.
MATERIALS AND METHODS: Immunohistochemical expression of E-Cadherin/β-Catenin was evaluated in a total n = 30 tissue samples comprising of n = 10 well-differentiated squamous cell carcinoma (WDSCC), n = 10 moderately differentiated squamous cell carcinoma (MDSCC), and n = 10 poorly differentiated squamous cell carcinoma (PDSCC). Based on the intensity of staining, an immunoreactivity scoring was calculated. STATISTICAL ANALYSIS: The scorings obtained were subjected to independent t-test, paired t-test, Chi-square test, and ANOVA test using SPSS version 20.0 statistical analysis software. P < 0.05 was considered statistically significant.
RESULTS: A significant difference was observed in the expression of β-Catenin between normal mucosa and WDSCC; normal mucosa and MDSCC. A gradual decrease in the immunoreactivity score of E-Cadherin is seen in WDSCC, MDSCC, and PDSCC.
CONCLUSION: Therefore, dysregulation of these proteins can lead to tumor progression, invasion, and metastasis. Further studies are warranted to specify the role of these EMT proteins as prognostic/therapeutic markers in patients suffering from OSCC. Copyright:

INTRODUCTION

Head and neck cancer accounts for >650,000 cases and 330,000 deaths annually.[1] Among these, oral squamous cell carcinoma (OSCC) is most common in the Indian subcontinent.[23] The most accepted etiology for the high incidence of tumorigenesis are the habits of chewing tobacco, areca nut, and other allied products coupled with alcohol consumption and poor oral hygiene.[45] The inability to repair damaged DNA due to mutation in proto-oncogene and Tumor Suppressor Gene results in genetic alteration causing cancer.[67] More specifically, mutation in gene and protein controlling the cell cycle, apoptosis, angiogenesis, and cell adhesion aid in carcinogenesis.[8] Replacement of epithelial phenotype and downregulation of markers like E-Cadherin, desmoplakin, cytokeratins, claudins, and β-Catenin of tight intercellular junction by a mesenchymal phenotype that results in cancer progression, invasion and metastasis.[910]

Cell to cell junction is crucial for maintaining the integrity and morphology of the squamous epithelium facilitated by a wide family of trans-membrane glycoproteins called cadherins amongst which the epithelial cadherin (E-Cadherin) plays a vital role in epithelial cell to cell adhesion.[1112] It is a 120 KDa, calcium-dependent transmembrane glycoprotein, the intracellular domain of which is linked to the actin and the catenins like α, β, γ -Catenins. B-Catenin is believed to be playing a major role in cell to cell junction by controlling the cadherin-mediated cell adhesion. The disruption of intercellular adhesion is a principal component in order to elicit invasive properties in malignant epithelial cells. In this regard, alteration in the E-Cadherin and β-Catenin complex is implicated in the oncogenesis.[11]

Segregation of β-Catenin from E-Cadherin in the cell membrane decreases cell–cell adhesion and increases cell migration and invasive properties of malignant cells.[13] E-Cadherin and β-Catenin protein complexes also participate actively in epithelial-mesenchymal transition (EMT) which is instrumental in cancer development.[14151617]

This has persuaded us to evaluate the expression pattern of E-Cadherin and β-Catenin in different grades of OSCC.

MATERIALS AND METHODS

Sample size

A total of 30 formalin-fixed paraffin-embedded (FFPE) tissue blocks previously diagnosed histologically as various grades of OSCC were retrieved from the department archives. For control, 10 normal oral mucosa samples were obtained from various patients who provided their consent for biopsy.

Sections of 4 μm were obtained from all the samples and were subjected to hematoxylin and eosin staining and were evaluated by three independent oral pathologists using Bryne's grading system, and afterward categorized into four groups with 10 cases each, namely Group 1 – well-differentiated squamous cell carcinoma (WDSCC), Group 2 – moderately differentiated squamous cell carcinoma (MDSCC), Group 3 – poorly differentiated squamous cell carcinoma (PDSCC), and Group 4 – normal oral mucosa.[18]

Immunohistochemical staining

4 μm thick sections were cut from the 40 FFPE blocks and were mounted on poly-L-lysin-coated positively charged slides. The slides were deparaffinized, rehydrated, and subjected to antigen retrieval using TRIS–EDTA buffer at pH 9.0. The slides were then incubated with primary anti-β-Catenin and anti-E-Cadherin rabbit monoclonal antibody (PathNSitu Biotechnologies Pvt. Ltd. California, USA). Afterward, the sections were treated by PolyExcel HRP/DAB detection systems (PathNSitu Biotechnologies Pvt. Ltd. California, USA). Human colon carcinoma and normal oral epithelium served, respectively as external and internal positive controls.

Immunohistochemical evaluation

The presence of brown-colored end products at the site of target antigen was suggestive of positive immunoreactivity [Figures 1–3]. E-Cadherin expression was evaluated in accordance with the presence or absence of membrane staining on the cell membrane. β-Catenin expression evaluated on the basis of the membrane as well as cytoplasmic staining. Immunoreactivity was semiquantitatively assessed in conformity with the staining intensity and distribution applying the immune reactive score as adapted from Balasundaram et al.,[19] the immunoreactivity score (IRS) was a multiplication product of intensity score and proportion score. The intensity score was defined as; 0: negative, 1: weak, 2: moderate, and 3: strong. The proportion score was defined as; 0: negative, 1: <10% positive cells, 2: 10%–50% positive cells, 3: 50%–80% positive cells, and 4: >80% positive cells. The total score ranged from 0 to 12. Immunoreactivity was divided into three groups based on the final score. Negative immunoreactivity grade (IRG) was interpreted as a total score of 0. A total score of 1–4 was defined as low IRG. Moderate IRG was determined as a total sum of 5–8 and high IRG was specified as a total score of 9–12. Cytoplasmic staining of β-Catenin was performed based on counting the number of positive cells per 50 membrane positive cells. 5 representative fields were counted for all the above assessments and arranged and tabulated.

Figure 1

E-Cadherin expression (a-d) and β-Catenin expression (a1-d1) in well-differentiated squamous cell carcinoma. (a and a1) Negative expression, (b and b1) weak expression, (c and c1) moderate expression, (d and d1) strong expression (×100)

Figure 3

E-Cadherin expression (a-c) and β-Catenin expression (a1-c1) in poorly differentiated squamous cell carcinoma. (a and a1) weak expression, (b and b1) moderate expression, (c and c1) strong expression (×100)

E-Cadherin expression (a-c) and β-Catenin expression (a1-c1,) in moderately differentiated squamous cell carcinoma. (a and a1) weak expression, (b and b1) moderate expression, (c and c1) strong expression (×100)

Statistical analysis

The results obtained were subjected to independent t-test, paired t-test, Chi-square test, and ANOVA test. All the statistical tests were carried out using IBM corp. SPSS software version 20.0, Armonk, New York for Windows. A P = 0.05 was considered as statistically significant.

RESULTS

A total of 40 samples were considered for the study, among which 26 were male and 14 were female, with a mean age of 47.23 + 12.01 and 57.71 ± 12.97, respectively. There was a definite decrease in the E-Cadherin IRS with a mean of 6.40 ± 4.006 in WDSCC, 6.30 ± 3.773 in MDSCC, and 5.30 ± 4.138 in PDSCC when compared with normal oral mucosa which had a mean score of 11.20 ± 1.687. This difference was statistically significant [Table 1].

Table 1

Comparison of expression of E-Cadherin and β-Catenin in normal epithelium with various grades of squamous cell carcinoma based on immunoreactivity score

Sample type	n	E-Cadherin		β-Catenin
		Mean±SD	P	Mean±SD	P
Normal	10	11.20±1.687	0.003	9.20±1.932	0.002
WDSCC	10	6.40±4.006		4.10±3.872
Normal	10	11.20±1.687	0.001	9.20±1.932	0.001
MDSCC	10	6.30±3.773		4.80±3.155
Normal	10	11.20±1.687	0.001	9.20±1.932	0.879
PDSCC	10	5.30±4.138		9.00±3.590

Independent t-test (P < 0.05 is significant). SD: Standard deviation, SCC: Squamous cell carcinoma, WDSCC: Well-differentiated SCC, MDSCC: Moderately differentiated SCC, PDSCC: Poorly differentiated SCC

The β-Catenin IRS for normal mucosa was 9.20 ± 1.932. There was an obvious decrease in β-Catenin IRS in WDSCC and MDSCC with a mean of 4.10 ± 3.872 and 4.80 ± 3.155, respectively. There was a significant difference observed between normal mucosa and WDSCC as well as between normal mucosa and MDSCC. No significant difference in β-catenin expression was seen between PDSCC and normal mucosa as evident in Table 1.

Chi-square test showed no statistically significant difference established between various grades of SCC and IRG [Table 2]. When the E-Cadherin IRS across various grades of SCC was analyzed using ANOVA, it failed to establish any significant variance. However, the IRS what is it of β–Catenin showed a statistically significant increase in score with P = 0.009 from WDSCC to MDSCC to PDSCC [Table 3].

Table 2

E-Cadherin and β-Catenin expression across various grades of squamous cell carcinoma

Marker	IRG	SCC type			Total	P
		WDSCC	MDSCC	PDSCC
E-Cadherin	High	3	2	2	7	0.772
	Moderate	4	4	4	12
	Low	2	4	2	8
	Negative	1	0	2	3
Total		10	10	10	30
β-Catenin	High	1	1	6	8	0.081
	Moderate	3	2	2	7
	Low	5	7	2	14
	Negative	1	0	0	1
Total		10	10	10	30

Chi-square test (P < 0.05 is significant). SCC: Squamous cell carcinoma, WDSCC: Well-differentiated SCC, MDSCC: Moderately differentiated SCC, PDSCC: Poorly differentiated SCC, IRG: Immunoreactivity grade

Table 3

Variation of E-Cadherin and β-Catenin expression across various grades of squamous cell carcinoma

Type	n	E-Cadherin		β-Catenin
		Mean±SD	P	Mean±SD	P
WDSCC	10	6.40±4.006	0.793	4.10±3.872	0.009
MDSCC	10	6.30±3.773		4.80±3.155
PDSCC	10	5.30±4.138		9.00±3.590
Total	30	6.00±3.869		5.97±4.072

ANOVA test (P < 0.05 is significant). SD: Standard deviation, SCC: Squamous cell carcinoma, WDSCC: Well-differentiated SCC, MDSCC: Moderately differentiated SCC, PDSCC: Poorly differentiated SCC

When E-Cadherin expression was compared with β-Catenin expression across various grades of OSCC, no significant difference was noticed in WDSCC and MDSCC, whereas in PDSCC, there was a significant increase in β-Catenin expression noticed when compared with E-Cadherin expression with a mean difference of − 3.7 (P = 0.00) [Table 4].

Table 4

Comparison between E-Cadherin expression and β-Catenin expression across grades of squamous cell carcinoma

Type	n	Mean difference±SD	P
WDSCC	10	2.3±4.95	0.175
MDSCC	10	1.5±2.79	0.124
PDSCC	10	-3.7±1.89	0.00

Paired t-test (P < 0.05 is significant). SD: Standard deviation, SCC: Squamous cell carcinoma, WDSCC: Well-differentiated SCC, MDSCC: Moderately differentiated SCC, PDSCC: Poorly differentiated SCC

ANOVA test shows a significant increase in number of cytoplasmic positivity per 50 β Catenin positive cells across grades of OSCC (p=0.001). The mean of cytoplasmic expression in normal mucosa was 3.70 ± 2.584, but in WDSCC, it was 7.10 ± 5.915; in MDSCC, it was 15.0 ± 14.438; and in PDSCC, it was 21.7 ± 11.729 [Table 5].

Table 5

Analysis of number of β-Catenin cytoplasmic positivity per 50β-Catenin positive cells across grades of squamous cell carcinoma

Type	n	Mean±SD	Significant
Normal	10	3.70±2.584	0.001
WDSCC	10	7.10±5.915
MDSCC	10	15.00±14.438
PDSCC	10	21.70±11.729
Total	40	11.88±11.820

ANOVA test (P < 0.05 is significant). SD: Standard deviation, SCC: Squamous cell carcinoma, WDSCC: Well-differentiated SCC, MDSCC: Moderately differentiated SCC, PDSCC: Poorly differentiated SCC

DISCUSSION

Dysregulation of E-Cadherin and β-Catenin is the turning point in tumor progression, invasion, and metastasis.

With this in the background, immunohistochemical staining for E-Cadherin and β-Catenin was performed and the slides were evaluated for positive immunoreactivity. In normal oral mucosa, there was a distinct and crisp E-Cadherin expression observed in the cell membrane of the basal layer, suprabasal layer, and spinous layer. The superficial layers were devoid of expression. Similar findings were also noted by Kaur et al. and Liu et al.[1220] β-Catenin also had a similar kind of expression in normal oral mucosa with a crisp and distinct immunostaining evident in the cell membrane of basal suprabasal and spinous layer. Many cells also showed cytoplasmic positivity. However, Liu et al. noticed β-Catenin expression only in the basal and suprabasal layers.[20]

When E-Cadherin expression was evaluated in normal oral mucosa and various grades of OSCC, it was found that there was a significant decrease in E-Cadherin expression in various grades of SCC. The reduced expression was found to be statistically significant and there was a downward progress in the expression pattern noticed. Similar findings were noticed by Mayer et al. who reported that 92% of the primary OSCC showed reduced expression of E-Cadherin.[21] Balasundaram et al. and Mehendiratta et al. also reported decreased E-Cadherin expression in OSCC.[1922] Many researchers also found a decreased β-Catenin expression in OSCC.[1920212223242526272829303132] When we evaluated β-Catenin expression in normal mucosa and various grades of SCC, there was an obvious decrease in expression noticed in WDSCC and MDSCC. But in PDSCC, the β-Catenin expression was quite comparable with normal oral mucosa without any statistically significant difference.

When the IRS of β-Catenin across various grades of squamous cell carcinoma was evaluated, a statistically significant increase in score was noticed. This was against the findings of Zaid, who noticed a gradual decrease in β-Catenin expression with decrease in histopathological grades.[11] Our finding was also against the findings of Zhao et al. who noticed a significant reduction in the expression of β-Catenin expression across decreasing grades of SCC.[33] Tanaka et al. fail to elicit any difference in β-Catenin expression when histopathological grades were considered.[34]

When E-Cadherin expression was compared with β-Catenin expression in different grades of OSCC, no significant difference was noticed in WDSCC and MDSCC. But in PDSCC, β-Catenin shows an increase in expression which was inversely proportional in relation to E-Cadherin expression.

Various authors have outlined the positive cytoplasmic expression of β-Catenin in SCC.[113134333536] Zaid reported an increase in cytoplasmic expression from WDSCC to MDSCC to PDSCC.[11] We also evaluated β-Catenin expression in the cytoplasm by counting the number of cells showing cytoplasmic expression among 50 membrane positive cells. Increased cytoplasmic expression of β-catenin was noticed from normal mucosa to PDSCC and this was in accord with the findings of Freitas et al and lopes et al.[3135] Hence, it can be said that the membranous β-Catenin moves to the cytoplasmic compartment and increases its potential in activating the tumoral gene transcription.[1136] Zhao et al. in their study suggested that loss in adhesion of E-Cadherin may cause a redistribution of β-Catenin from the cell membrane to the cytoplasm.[33] This may explain our findings as well.

In a stable epithelial cell, if β-catenin detaches from E- Cadherin, it becomes soluble leading to various cell signaling pathways.[11] The present study outlines a decreased expression of E-Cadherin in OSCC which explains the malignant and invasive nature of the neoplasm. But a decreasing trend in expression was not established in decreasing grades of histological differentiation. β-Catenin on the other hand showed an increasing trend in expression in decreasing grade of histodifferentiation. PDSCC showed higher β-Catenin expression both in the membrane and cytoplasm. There was a high level of cytoplasmic expression noticed in PDSCC. Release of β-Catenin from E-Cadherin causes decreased cell adhesion, increased cell migration, and invasiveness.[13] In EMT, there is a loss of epithelial marker proteins such as E-Cadherin, zonula occludens-1, and cytokeratin and gain of a mesenchymal phenotype with expression of mesenchymal proteins, namely vimentin, α-smooth muscle actin, and fibroblast-specific protein-1, along with the production of interstitial matrix components type I collagen and fibronectin.[1415] Hence, the loss of cell-cell adhesion promotes EMT and is linked with the etiopathogenesis of diseases involving EMT.

In the present study, we can conclude that there is a decreased expression of E-Cadherin in OSCC which proves that E-Cadherin downregulation may lead to loss of cellular adhesion resulting in noncohesive tumor cells ideal for deeper infiltration and invasion. But strangely, we found an increased cytoplasmic β-Catenin expression. Hence, it can be inferred that β-Catenin is breaking away from the membrane E-Cadherin complex and overexpressing in cytoplasm facilitating cellular dedifferentiation, tumor progression, invasion, and metastasis at the molecular signaling level.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.