|Ahead of print publication
Potentiated action on the progression of OSMF by hypoxia mediated signaling pathway by the epithelial mesenchymal transition and angiogenic apparatus
Sanjana Gupta, Devi Charan Shetty, Nikita Gulati, Saurabh Juneja, Anshi Jain
Department of Oral and Maxillofacial Pathology and Microbiology, I.T.S. Center for Dental Studies and Research, Ghaziabad, Uttar Pradesh, India
|Date of Submission||27-Mar-2021|
|Date of Decision||01-Jul-2021|
|Date of Acceptance||30-May-2021|
|Date of Web Publication||25-Apr-2022|
Department of Oral and Maxillofacial Pathology and Microbiology, I.T.S. Center for Dental Studies and Research, Muradnagar, Ghaziabad - 201 206, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Epithelial–mesenchymal transition (EMT) is a complex process, in which epithelial cells acquire the characteristics of invasive mesenchymal cells. EMT has been implicated in cancer progression and metastasis as well as the formation of many tissues and organs during development.
Aim: The aim of the study was to ascertain the role of hypoxia-mediated signaling pathways influencing EMT and angiogenesis in progression of oral submucous fibrosis (OSMF).
Materials and Methods: Evaluation of the immunoexpression of alpha-smooth muscle actin (α-SMA), E-cadherin, vimentin, and factor VIII receptor antigen in OSMF and oral squamous cell carcinoma (OSCC) arising from OSMF was done. Differences between the different variables were analyzed using ANOVA test and Pearson's Chi-square test, and Mann–Whitney test was also calculated.
Results: The mean α-SMA positive myofibroblasts increased from Group 1 (OSMF) to Group 2 (OSCC), especially those in the deeper connective tissue stroma. The mean labeling index of vimentin and mean vessel density immunoexpression was more in Group 2 (OSCC) as compared to Group 1 (OSMF). Mean α-SMA correlated negatively with E-cadherin expression and positively with vimentin and factor VIII immunoexpression. E-cadherin expression correlated negatively with factor VIII and positively with Vimentin expression.
Conclusions: The molecular mechanisms responsible for the development of OSCC in patients with OSMF require unification of multiple progressive pathogenetic mechanisms involved in the progression of the disease.
Keywords: Angiogenic apparatus, epithelial–mesenchymal transition, oral submucous fibrosis
|How to cite this URL:|
Gupta S, Shetty DC, Gulati N, Juneja S, Jain A. Potentiated action on the progression of OSMF by hypoxia mediated signaling pathway by the epithelial mesenchymal transition and angiogenic apparatus. J Can Res Ther [Epub ahead of print] [cited 2022 Dec 8]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=343920
| > Introduction|| |
Oral cancers arising in oral submucous fibrosis (OSMF) are said to constitute a clinicopathologically distinct disease. The morphological and histological differences underlying in OSMF transforming to oral squamous cell carcinoma (OSCC) are attributed to the differential mechanisms of the carcinogenesis of areca nut. OSMF is associated with chewing of areca nut which has been declared as Group 1 carcinogen by the International Agency for Research on Cancer. On comparing the risk ratio, it has been estimated that people with OSF are 19.1 times more likely to develop oral cancer than those without it.
Identification of signature genes influencing epithelial–mesenchymal transition (EMT) may unravel novel pathways, which are critical to the progression of oral cancer. Hypoxia-inducible factors are responsible for the regulation of hypoxia that mediates the adaptive responses to oxygen depletion. They are involved in carcinogenesis and tumor growth through the regulation of genes involved in angiogenesis, glycolytic metabolism, and other biological mechanisms such as oxygen transport, iron metabolism, glycolysis, glucose uptake, growth factor signaling, apoptosis, invasion, and metastasis.
Whereas, myofibroblasts are a unique group of cells with smooth muscle properties and can be identified by the expression of alpha-smooth muscle actin (α-SMA), which are alleged to be primary producers of extracellular matrix after injury. Alteration in quantity and functioning myofibroblasts has been implicated in various fibrotic diseases. Various cytokines have been linked with tumor stroma in OSCC such as transforming growth factor beta-1 from cancerous cells that are accountable for differentiation of fibroblasts into myofibroblasts, neo-angiogenesis, and increase in the inflammatory cells and overexpression of mesenchymal markers such as Vimentin.
Angiogenesis is a noteworthy process that occurs in a number of physiological events such as embryonic growth, chronic inflammation, wound healing, and in pathological conditions such as progression of a tumor. The quantification of microvasculature can be done by the assessment of microvessel density.
Oral epithelium shows adaption to different mechanical demands and maintains its structure by a process of continuous cell renewal where a lot of factors are involved in maintaining the balance between proliferation and differentiation. E-cadherin is localized on the surface of epithelial cells and is also involved in transduction of signals controlling various cellular events including polarity, differentiation, growth, and cellular migration. Studies have shown that collagen formation has been induced by arecoline and also responsible for demonstrating cytotoxicity and stimulating double-stranded nucleic acid synthesis and cell morphology change. This cell morphological change implicates arecoline in cytoskeletal disturbance related with interference in cell mitosis and intracellular transport mechanisms. The Vimentin antibody recognizes a 57 kDa intermediate filament (IF) and labels a variety of mesenchymal cells including melanocytes, lymph cells, endothelial cells, and fibroblasts. The expression of Vimentin is appreciably enhanced in cell growth, cell cycling, tumor differentiation, and during the process of tumorigenesis. There has been little research done for the possible effects of arecoline on the cytoskeleton components. EMT is a process, in which there is a reduced expression of epithelial genes (E-cadherin) and an increase in the expression of mesenchymal genes (N-cadherin and Vimentin) and EMT transcription factors. Together with an altered localization of the catenin, the epithelial cells lose their phenotype and intercellular adhesions. Transcription factors act synergistically to bring about the epithelial cell reprogramming. EMT has detrimental role in the progression of fibrosis and cancer metastasis. Therefore, this study was conducted to assess hypoxia-mediated signaling pathways influencing EMT and angiogenesis in progression of OSMF.
| > Materials and Methods|| |
Patients and tissue samples
The study was conducted in the Department of Oral and Maxillofacial Pathology and Microbiology, I. T. S Dental College, Muradnagar, Ghaziabad, on archival tissue samples which were submitted for histopathological evaluation. The samples were fixed in 10% neutral-buffered formalin and embedded in paraffin wax to obtain two sections of 3 μm thin were taken for immunohistochemistry procedure. Study sample was composed of total ten cases of histopathologically confirmed cases of OSMF and five cases of OSMF transforming into OSCC. Clinical data such as age, gender, habit duration, and type were obtained for each case.
Grading of oral submucous fibrosis
Tissue specimens were grouped based on Khanna JN and Andrade NN (1995) follows as Group I: very early cases (Fine fibrillar collagen network interspersed with mark edema, blood vessels dilated and congested, large aggregate of plump young fibroblasts present with abundant cytoplasm, inflammatory cells mainly consist of polymorphonuclear leukocytes with few eosinophils. The epithelium is normal, Group II: Early cases (juxta-epithelial hyalinization present, collagen present as thickened but separate bundles, blood vessels dilated and congested, young fibroblasts seen in moderate number, inflammatory cells mainly consist of polymorphonuclear leukocytes with few eosinophils and occasional plasma cells, flattening, or shortening of epithelial rete-pegs evident with varying degree of keratinization.), Group III: Moderately advanced cases (juxtaepithelial hyalinization present, thickened collagen bundles, residual edema, constricted blood vessels, mature fibroblasts with scanty cytoplasm and spindle-shaped nuclei, inflammatory exudates which consists of lymphocytes and plasma cells, epithelium markedly atrophic with loss of rete pegs, muscle fibers seen with thickened, and dense collagen fibers, and Group IVA: Advanced cases, Group IVB: Advanced cases (Collagen hyalinized smooth sheet, extensive fibrosis, obliterated the mucosal blood vessels, and eliminated melanocytes, absent fibroblasts within the hyalinized zones, total loss of epithelial rete pegs, presence of mild to moderate atypia, and extensive degeneration of muscle fibers.
Immunohistochemistry with alpha-smooth muscle actin, Vimentin, factor VIII and E-cadherin
Three-micrometer-thick sections from archival formalin-fixed paraffin-embedded tissues were placed on poly-L-lysine-coated slides for immunohistochemistry α-SMA, Vimentin, factor VIII, and E-cadherin expressions were analyzed by immunohistochemical examination with antibodies. For each E-cadherin, α-SMA, and Vimentin antibody, the deparaffinized sections were heated in antigen retrieval machine in Tris EDTA buffer (pH 9.1). The slides were allowed to cool in Tris EDTA buffer and washed in phosphate-buffered saline (PBS). For factor VIII, the deparaffinized tissue sections were placed on slide tray and hydrated to water. Excess water was removed from the glass slide without letting the tissue dry. Freshly prepared pepsin enzyme solution was applied to the specimen to cover it completely. Slides with specimen were incubated for approximately 30 min at 37°C. The slides were then placed in PBS buffer rinse for three changes × 5 min each. Immunohistochemical staining was performed by the avidin-biotin complex procedure with a streptavidin-biotin complex peroxidase kit. Primary antibody-Monoclonal anti-E-cadherin antibody (Pathn Situ, Livemore, CA, USA Clone no. EP6, Catalog no. HAR039-6ML RTU), primary antibody–Monoclonal anti-α-SMA antibody (BiogenexInd Ltd Pvt Ltd, Clone number-Am 128-5M, Catalogue number-AM1281117), factor VIII-related antigen monoclonal antibody (Biogenex Pvt Ltd. BGX016A) and primary antibody–Monoclonal anti-Vimentin antibody (Biogenex Ind. Pvt. Ltd. Catalog no: Clone no-Am074-5M, Catalogue no-AM0740616) along with secondary antibody-poly-HRP secondary detection system (Biogenex Ind Pvt ltd) were used. For E-cadherin-Normal Oral mucosa (Epithelium), for Vimentin--Normal Oral mucosa (Fibroblasts), for factor VIII--Normal Oral mucosa (Blood Vessels), and for α-SMA-normal salivary gland tissue was taken as positive control. Positive staining E-cadherin and factor VIII were identified as membranous, whereas positive staining of α-SMA and Vimentin were identified as cytoplasmic brown staining.
Assessment of immunoscoring
Expression of all four molecules (E-cadherin, Vimentin, α-SMA, and factor VIII) were assessed semi-quantitatively, qualitatively, and quantitatively. The IHC expression of E-cadherin was assessed semi-quantitatively according to (Cortesina and Martone.) (0 = no positive cells, + = 0%–25% positive cells, ++ =26%–50% positive cells, +++ = 50%–75% positive cells, ++++ = more than 75% cells), and qualitative scoring was done according to Sharada et al. (0 = absent, 1= <25%, 2 = 25%–50%, 3 = >50%). Extent was assessed according to Martano et al. (0 = negative, 1 = central, 2 = peripheral, and 3 = both) and localization was assessed according Kaur et al. (0 = negative, 1 = membranous, 2 = cytoplasmic, and 3 = both). Quantitative assessment of factor VIIIR was done where three fields per lesion sections were counted in the areas that appeared to contain the greatest number of blood vessels on scanning at low magnification. Large blood vessels as well as any single brown staining endothelial cell which were clearly separate from other blood vessels was included in the blood vessel count. Branching structures were counted as one, unless there was a break in the continuity of the vessels, in which case it was counted as two distinct vessels. From the three fields counted for the antibody, the mean of these three fields was then used for subsequent analysis. Quantitative assessment of Vimentin was also done. The positive cells were counted in five high power fields at 40X. Labeling index was calculated for immunopositive cells in basal layer of epithelium. Furthermore, for α-SMA, the mean number of myofibroblast was counted in five high fields 40X in juxtaepithelial area and five high power field 40X in deep connective tissue area in all grades of OSMF and comparison of mean number of myofibroblast was done between the juxtaepithelial and deep connective tissue area in all grades of OSMF.
| > Results|| |
The study groups had a mean age range of 34.8 ± 2.147 with the majority of male patients. The majority of participants had a habit of both smoking and chewing tobacco (53.5%) with a duration of <10 years (33.3%).
Immunoexpression of alpha-smooth muscle actin, Vimentin, E-cadherin, and factor VIII in study cases
The results depict the mean α-SMA positive myofibroblasts increased from Group 1 (OSMF) to Group 2 (OSCC). The mean number of α-SMA positive myofibroblasts in superficial stroma of OSMF cases was 23.48 ± 14.03 and in OSCC were 16.86 ± 1.82, whereas in the deep stroma, the mean number of α-SMA-positive myofibroblasts OSMF cases was 14.4 ± 6.91 and in OSCC cases was 24.62 ± 3.63. Intensity of α-SMA in myofibroblasts increased from mild to strong in both Group 1 (OSMF) and Group 2 (OSCC). Majority of cases (50% in Group 1 and 60% in Group 2) had distribution of myofibroblasts in juxtaepithelial and deep connective tissue stroma. The labeling index of Vimentin in Group 1 (OSMF) was 18.0 ± 7.93 and Group 2 (OSCC) was 22.2 ± 3.96. The intensity in Group 1 showed strong intensity in four cases, moderate intensity in five cases, and one case showed mild intensity. The intensity in Group 2 showed strong intensity in three cases and moderate intensity in two cases. In Group 1, the maximum expression of ≥50% was observed in four cases (40%) and in Group 2 60% showed 25%–50% positivity. In Group 1, the intensity increased from mild-to-moderate in majority of cases, whereas in Group 2, 40% of cases showed moderate intensity. The immunoexpression was localized to the membrane in majority of cases (80%) in Group 1, whereas it showed both membranous (40%) and cytoplasmic (40%) localization in Group 2 (OSCC). The mean vessel density increased from 13.74 ± 3.26 in Group 1 (OSMF) to 17.03 ± 2.02 in Group 2 (OSCC) [Table 1].
|Table 1: Assessment of alpha-smooth muscle actin, Vimentin, E-cadherin, and factor VIII immunoexpression in study groups|
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On correlating immunoexpression of α-SMA, Vimentin, and factor VIII in varying histopathological grades of study cases, the α-SMA deep connective tissue count increased from moderately advanced OSMF (16.240 ± 7.3857) to advanced OSMF (12.560 ± 6.6864) to OSCC (24.620 ± 3.6335), whereas α-SMA superficial connective tissue count decreased from moderately advanced OSMF (26.240 ± 16.1248) to advanced OSMF (20.720 ± 12.8137) to OSCC (16.860 ± 1.8243). The Vimentin and factor VIII immunoexpression also increased from moderately advanced OSMF to advanced OSMF to OSCC, but the results were statistically significant for factor VIII quantitative immunoexpression where the mean vessel density increased from 13.196 ± 3.19971 in moderately advanced OSMF to 14.292 ± 3.60615 in advanced OSMF to 17.030 ± 2.02856 in OSCC [Graph 1] and [Figure 1], [Figure 2].
|Figure 1: Moderately advanced OSMF showing a) strong immunopositivity of Alpha SMA positive myofibroblasts in both juxtaepithelial and deep connective tissue (IHC, 10x magnification, inset 40x magnification) b)strong immunopositivity of Vimentin in basal cells of epithelium IHC, 10x magnification, inset 40x magnification) c)strong immunopositivity of Factor VIIIRAg in the blood vessels (IHC, 10x magnification, inset 40x magnification) d) strong immunopositivity of Alpha SMA positive myofibroblasts in both juxtaepithelial and deep connective tissue (IHC, 10x magnification, inset 40x magnification)|
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|Figure 2: Moderately differentiated OSCC showing a)strong membranous immunopositivity of E-cadherin in peripheral tumour cells and weak to absent immunoexpression in central tumour cells (IHC 10x magnification, inset 40x) b) strong immunopositivity ofVimentin in peripheral cells of tumour islands IHC, 10x magnification, inset 40x magnification) c) strong immunopositivity of Factor VIII in the blood vessels at the invasive front(IHC, 10x magnification, inset 40x magnification) d) strong immunopositivity of Alpha SMA in periphery of tumour islands (IHC, 10x magnification, inset 40x magnification)|
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Correlation coefficient of alpha-smooth muscle actin, Vimentin, E-cadherin, and factor VIII in study cases
The spearman correlation coefficients of qualitative immunohistochemical parameters of α-SMA, E-cadherin, Vimentin, and factor VIII in various histopathological grades were calculated where the E-cadherin intensity negatively correlated to Vimentin intensity (−0.538) [Table 2].
|Table 2: Spearman correlation coefficients of qualitative immunohistochemical parameters of alpha-smooth muscle actin, E-cadherin, vimentin and factor VIII in various histopathological grades|
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The Pearson correlation coefficients of quantitative immunohistochemical parameters of α-SMA, E-cadherin, Vimentin, and factor VIII in various histopathological grades were also calculated. The mean α-SMA myofibroblasts in deep connective tissue correlated mean vessel density counted by factor VIII immunoexpression (0.495), whereas α-SMA positive myofibroblasts in superficial connective tissue correlated positively to Vimentin positive cells showing EMT (0.508). E-cadherin semi-quantitave immunoexpression correlated negatively with mean α-SMA-positive myofibroblasts (−0.158). The correlation coefficients were statistically significant (P < 0.05) [Table 3].
|Table 3: Pearson correlation coefficients of quantitative immunohistochemical parameters of alpha-smooth muscle actin, E-cadherin, Vimentin, and factor VIII in various histopathological grades|
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| > Discussion|| |
The local and systemic upregulation of inflammatory and fibrogenic cytokines and downregulation of antifibrotic cytokines are described to be fundamental to the pathogenesis of OSMF. One of the biggest complications associated with OSF is the higher risk of transforming to OSCC. The concept of epithelial and mesenchymal transition (EMT) was first proposed by Greenburg and Hay EMT may play a key role in tumor invasion and metastasis as well as transformation of precancerous state to cancer. They are a persuasive regulator of many cellular functions and phenomena including cell proliferation, differentiation, survival, adhesion, migration, motility, and apoptosis. Myofibroblasts are phenotypically intermediate between smooth muscle cells and fibroblast. They help in extracellular matrix (ECM) reorganization by the production of numerous inflammatory mediators, growth factors, and proteins of the ECM. Apoptosis has been accountable for its disappearance after completion of the repair process, but their persistent presence stimulates dysfunctional repair mechanisms, causing excess contraction, ECM secretion, and thus, fibrosis.
EMT consists of certain elements such as cytoskeleton proteins, cytokines, and nuclear proteins which are also commonly related with OSMF, lead to the possibility of EMT in the process of fibrosis in oral mucous, triggered by betel quid chewing habit in OSMF. In OSMF, increased expression of myofibroblasts may suggest that myofibroblasts production and persistence could be one of the mechanisms which has been utilized by transforming growth factor-beta to induce a fibrotic response in OSMF.
In the present study, the mean number of α-SMA-positive myofibroblasts in superficial stroma and deep stroma of OSMF was comparable to that in the connective tissue stroma of OSCC. Our results were similar to the previous study conducted by Angadi et al., 2011 and Reddy et al., 2018, who reported an increase in the mean number of myofibroblasts in OSMF. Myofibroblasts are responsible for the initiation of ECM changes in the connective tissue. Activation of buccal mucosa fibroblast into myofibroblasts could be due to arecoline-induced ZEB 1 pathway in OSMF. Buccal mucosal fibroblasts transdifferentiate into myofibroblasts by ZEB1-driven α-SMA expression. These myofibroblasts further lead to the activation of ECM in buccal mucosa causing tissue contraction and finally progressing to OSMF. Some authors have also suggested a decrease in number of total myofibroblasts with disease progression because of the shift in fibrosis by transdifferentiation of myogenic cells to myofibroblasts. Our results were in contradiction to Reddy et al., who explained the decrease because of apoptosis or dedifferentiation of these cells [Table 1]. On the other hand, Kale et al. suggested distribution of myofibroblasts in all layers of connective tissue, because as the disease progresses the myogenic differentiation of myofibroblasts predominate around the blood vessels.
IF plays a supporting or general structural role. Vimentin which is a Class 2 IF is the most abundant IF and expressed principally in the mesenchymal cell. Proposed function of Vimentin has been suggested as a sign of dedifferentiation demonstrating cytotoxicity and also stimulates double-stranded nucleic acid synthesis and cell morphology changes. Certain change that occurs in cell morphology implicates arecoline in cytoskeletal disturbance associated with interference in cell mitosis and intracellular transport mechanisms. The effect of arecoline on the Vimentin in normal human buccal mucosal fibroblasts has been studied previously by very few authors. Changes in the intensity of staining in the arecoline-induced OSF are not evidently recorded and localized according to the individual tissue or the cell staining. In the present study, labeling index of Vimentin in Group 1 (OSMF) was 18.0 ± 7.93 and Group 2 (OSCC) was 22.2 ± 3.96. The intensity in Group 1 showed strong intensity in four cases, moderate intensity in five cases, and one case showed mild intensity. The intensity in Group 2 showed strong intensity in three cases and moderate intensity in two cases [Table 1]. A significant increase has been noted in Vimentin expression in the epithelium from normal to dysplastic tissue to OSCC. From a biological point of view, the reduction in E-cadherin expression and upregulation of Vimentin expression in dysplastic and cancerous tissues propose that EMT is occurring and may be playing a role in OSCC development. Upregulation of Vimentin at the tumor invasive front was also observed in OSCC tissues. This is consistent with the role of Vimentin in cell migration and supports findings from previous studies showing a core.
E-cadherin is a calcium-dependent transmembrane glycoprotein located in the epithelial tissue, which is an essential cell adhesion molecule and signal transduction factor, can direct the formation of protein complexes has been attached to the actin cytoskeletons along with beta-catenin formation which can prevent and reduce tumor cell adhesion. E-cadherin is responsible for the loss of EMT. The dysplastic layer shows reduction in dysplastic layer. This observation of our study was consistent with the studies conducted by Yogesh et al., Kaur et al. (2011, 2013) who stated that atypical features of dysplasia are strongly correlated to the loss of expression of E-cadherin. Yogesh et al. in his study also explained that there is a variation in the expression of E-cadherin in the dysplastic epithelium with varying degrees of dysplasia or severity of dysplasia and location of tissue would suggest that these alterations are the result of the progression of dysplasia and could be a late event, which is suggestive of change toward a cell phenotype with the ability to invade. In our study, the expression of E-cadherin was in the form of membranous to cytoplasmic OSCC which was in accordance with many other studies. Higher cytoplasmic expression of E-cadherin in OSCC suggested a redistribution of the E-cadherin complex out of tight junctions and increase in its degradation by cytoplasmic endocytosis. Miyazawa found that change expression of E-cadherin/β-catenin, Vimentin was also observed in the front infiltration of OSCC; Nguyen et al. using immunohistochemical method confirmed the high expression of N-cadherin and the metastasis and invasion of cancer cells are associated with HNSCC patients, Nijkamp et al. also reported low expression of E-cadherin in head-and-neck squamous cell carcinoma more prone to transfer than high expression group.
Angiogenesis is a critical event in the tumor growth and metastasis, mediated by several growth factors released by the tumor cells in the local environment. In the present study, the mean vessel density increased from 13.74 ± 3.26 in Group 1 (OSMF) to 17.03 ± 2.02 in Group 2 (OSCC) [Table 1]. There has been a triggering of angiogenic switch in moderately advanced OSF due to the increased demand for blood to provide nutrition for the proliferating abnormal cells, and this will account for the increased Mean vessel density (MVD). Tumor neovascularization promotes growth because cell population needs new vessels to allow the exchange of nutrients, oxygen, and waste products for simple diffusion. Factor VIII does not help in distinguishing between angiogenesis occurring due to ischemia/hypoxia. Debnath et al. found increase in MVD in early stages and decrease in advanced stages of OSMF. They also found that MVLD and MVAP (Mean vascular Area percentage and Mean vascular Luminal diameter) increased with increasing stage of the OSMF. Murgod et al. found increase in MVD in early stages and decrease in advanced stages of OSMF and exponentially rises in OSCC. This is due to neoangiogenesis in the mucosa to compensate for the hypoxia created by fibrosis. In the present study, MVD increased progressively from normal mucosa to OSMF and to OSCC [Table 1], thus confirming the findings of Sabarinath et al., who showed that the mean MVD had a tendency to increase as the disease progressed, although the increase among the various stages of OSMF was not statistically significant. Singh et al. and Fang et al. found a significant increase of MVD in the early stage of OSMF in comparison with the other stages. Our present results suggest that the rise in MVD noted in the early stage of OSMF is probably due to neoangiogenesis in the mucosa to compensate for the hypoxia created by fibrosis. As the disease advances and the stroma becomes more and more hyalinized, the mediators of angiogenesis seem to diminish, which could explain the decrease in MVD observed in the late stage of OSMF. Following further the pathological mechanism, the tissue tries to cope up with hypoxia by actively promoting neovascularization as an adaptive response on the part of the mucosa in survival of the atrophic epithelium in OSF. Tilakratne et al. have hypothesized that dense fibrosis and less vascularity of the epithelium, in the presence of an altered cytokine activity creates a unique environment for carcinogens from both tobacco and areca nut to act on the epithelium. They assumed that carcinogens from areca nut accumulate over a long period of time either on or immediately below the epithelium allowing the carcinogens to act for a longer duration before it diffuses into deeper tissue. According to them, less vascularity may deny the quick absorption of carcinogens into the systemic circulation.
In our study, E-cadherin was expressed throughout the epithelial layers of oral mucosa cultures but lost by the individual invasive cells, whereas Vimentin expression was not conspicuous in the epithelial layers but was abundant for individual invasive cells within the connective tissue. It is implied that deregulation of the canonical pathway may be a contributor to OSCC development. Orsulic et al. found that the binding of E-cadherin to b-catenin at the epithelial cell membrane prevents b-catenin accumulation in the cytoplasm, thereby inhibiting nuclear migration of b-catenin and the formation of a functional transcription factor. It also prevents the activation of certain oncogenes, one of which is the gene for Vimentin.
The inflammatory reaction antecedent to fibrosis in the onset of oral submucous fibrosis (OSF), and the role of EMT indicates a previously unexplored pathogenesis for progression of OSMF to OSCC. Identification of signature genes influencing EMT may unravel novel pathways, which are critical to the headway of oral cancer. These markers of EMT may prove to be efficient targets to control the further spread and improve the prognosis of OSCC. The inflammatory reaction antecedent to fibrosis and the role of EMT in fibrogenesis and malignant transformation in other organs points to the involvement of EMT in the pathogenesis of OSF and its malignant transformation. The inflammatory cytokines produced in response to the inflammation may mediate the progression of OSF through various EMT conduits. The membranous loss of E-cadherin, β-catenin, with an overwhelming expression of Vimentin, N-cadherin, and α-SMA seen in OSF further confirms the role of EMT in OSF.
| > Conclusions|| |
The molecular mechanisms responsible for the development of OSCC in patients with OSMF requires unification of multiple progressive pathogenetic mechanisms involved in progression of the disease. The presence of areca nut habit in these patients promotes the property of stemness and acquisition of mesenchymal phenotype in epithelial cells with predisposition to develop into an epithelial malignancy with increased invasive potential. The results of the present study highlight the role of molecular markers in understanding the disease progression at various fronts including epithelial–mesenchymal transformation in a hypoxic microenvironment. Such changes also favor transformation of myofibroblasts into an altered myofibroblasts with protumorigenic phenotype. These molecular pathways also alter the response of extracellular matrix and angiogenic response in favor of disease progression and malignant transformation. Interplay of these molecules may provide insights into identifying cases with higher risk of malignant transformation to malignancy.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]