Cross-talk of TGF-β and Wnt/β-Catenin Pathways Leading to Epithelial Mesenchymal Transition in Oral Submucous Fibrosis in Indian Population

Background: Oral squamous cell carcinoma (OSCC) is a predominant type of cancer in India. It has been observed that OSSC is preceded by premalignant lesions or otherwise reported as potentially malignant disorders (PMD) by WHO during the year 2005. PMD include various oral mucosal diseases such as leukoplakia, erythroplakia, oral submucous fibrosis (OSMF), oral lichen planus, discoid lupus erythematosus and actinic keratosis etc. Literature surveys have highlighted that areca nut–associated oral squamous cell carcinoma is the third most common malignancy in the developing world and OSMF has been reported as the PMD of the oral cavity that has high prevalence rate in India. Since the exact mechanism behind this fibrosis condition is yet to be identified, we propose that, components in areca nut could interfere with certain signaling pathways in the human system that could lead to this precancerous stage. Aim: 1. To investigate the molecular mechanism involved in EMT by cross-talk between TGF-β and Wnt/β-catenin pathways in OSMF patients. 2. To explore the role of HSP90 inhibition in this cross-talk under hypoxic condition. Methods: OSMF tissue samples were obtained via punch biopsy, from patients with areca nut chewing habit and control tissues were collected from healthy individuals. OSMF patients who underwent any local treatment of oral mucosa or those who have any systemic disorder were excluded from the study. Initially, the expression pattern of various TGF-β and Wnt signaling pathway molecules were studied immunohistochemically to confirm the role of these pathways in OSMF. The gene expression of Wnt5a, Wnt3a, GSK-3β, β-catenin, cMyc, TGFβ1, smad3, E-cadherin, snail, α-SMA and Twist were studied by quantitative real time PCR (qRT-PCR). The results of qRT- PCR were further confirmed by Western blot analysis. Human gingival derived fibroblast cells were cultured and the cells were grouped into 2. Group I- normoxia, group II- hypoxia, group III- hypoxia + 17-AAG (an inhibitor of HSP90). mRNA and protein expression patterns were revealed by qRT-PCR and Western blot analysis for HSP90, HIF-1α, Wnt5a, β-catenin, cMyc, TGF-β1, Smad3, p-Smad3, E-cadherin, N-cadherin, Snail and α-SMA. Immunofluorescence analysis of expression of HSP90, HIF-1α, vimentin, snail, TGF-β1, Smad3 were also carried out. Results: The results obtained revealed that, TGF-β and Wnt/β-Catenin signaling molecules are involved in OSMF leading to EMT. The same pattern was observed when the cells were maintained under hypoxic conditions. Conclusion: Overall, this study describes the cross-talk of TGF-β and Wnt/β-Catenin pathways in EMT in tissues collected from OSMF patients and also when the cells were maintained under normoxic and hypoxic conditions. Inhibition of this signaling cross-talk might help to identify a new strategy for therapeutic intervention.