Epithelial-mesenchymal transition (EMT) is a critical process that drives tumor invasion and metastasis in oral squamous cell carcinoma (OSCC). Although transforming growth factor beta 1 (TGF-β1) and epidermal growth factor (EGF) are well-established inducers of EMT, the signaling crosstalk underlying their combined effects during the initial phase remains poorly defined. This study investigated the impact of combined TGF-β1 and EGF stimulation on EMTrelated phenotypic and molecular changes in HSC-3 OSCC cells. The cells were treated with TGF-β1 (10 ng/mL) and EGF (100 ng/mL), either individually or in combination. Cell proliferation, morphological changes, EMT marker expression, signaling pathway activation, and migratory capacity were assessed using cell counting kit-8 assays, quantitative morphological analysis, RT-qPCR, immunoblotting, and wound healing assays, respectively. Although TGF-β1 or EGF alone induced partial EMT-like changes, combined stimulation produced a pronounced spindleshaped morphology, an increased cell aspect ratio, and loss of epithelial cell-cell contact. Consistently, cotreatment led to greater repression of E-cadherin and enhanced upregulation of N-cadherin and Vimentin compared with single treatments. Notably, dual exposure markedly increased Smad2 and extracellular signal-regulated kinase (ERK) phosphorylation while accelerating cell migration. These findings indicate that TGF-β1 and EGF cooperatively promote EMT and migratory behavior through the coordinated activation of Smad2 and MAPK/ERK signaling pathways, highlighting this crosstalk as a potential therapeutic target for OSCC metastasis.

Introduction
Materials and Methods
    1. Reagent
    2. Cell culture
    3. Cell morphology assay
    4. Cell proliferation
    5. Real-time quantitative polymerase chain reaction
    6. Immunoblotting
    7. Wound healing assay
    8. Statistical analysis
Results
    1. Effects of TGF-β1 and EGF co-stimulation on cellproliferation and morphology
    2. Regulation of EMT marker expression by TGF-β1and EGF co-stimulation
    3. Enhanced cell migration induced by TGF-β1 andEGF
    4. Activation of EMT-related signaling pathways byTGF-β1 and EGF
Discussion
Funding
Conflicts of Interest
References

• Bo Ram Keum(Department of Oral Physiology, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea)
• Hyung Joon Kim(Department of Oral Physiology, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea) Corresponding author
• Soon Chul Heo(Department of Oral Physiology, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea, Institute of Tissue Regeneration Engineering (ITREN), Mechanobiology Dental Medicine Research Center, Dankook University, Cheonan 31116, Republic of Korea)