# The Role of TGF-β1 and Mutant SMAD4 on Epithelial-Mesenchymal Transition Features in Head and Neck Squamous Cell Carcinoma Cell Lines

## TL;DR

This study explores how TGF-β1 and mutant SMAD4 influence epithelial-mesenchymal transition in head and neck cancer cells, linking these factors to metastasis potential.

## Contribution

The study reveals that epithelial HNSCC cells respond more strongly to TGF-β1 and identifies mutant SMAD4 as a driver of mesenchymal features in HNSCC.

## Key findings

- Epithelial HNSCC cells and HaCaT cells showed stronger responses to TGF-β1 than mesenchymal HNSCC cells.
- Mutant SMAD4 was detected in the most mesenchymal HNSCC cell line and contributes to mesenchymal features.
- Overexpression of mutant SMAD4 in HaCaT cells increased TGF-β1 and vimentin expression.

## Abstract

Head and neck squamous cell carcinomas (HNSCCs) represent more than 90% of all malignancies in the upper aero-digestive tract. Their ability to metastasize is tightly associated with the patient’s survival. The process of epithelial–mesenchymal transition (EMT) is thought to be a central mechanism for invasion and metastasis. Here, we investigated the responsiveness of HNSCC cell lines and the HaCaT control cell line to the EMT master regulator TGF-β1 and observed major differences in the level of sensitivity to this cytokine. The more epithelial HNSCC cells and HaCaT control cells responded more extensively to TGF-β1 than the less epithelial HNSCC cells. Mutant SMAD4 was detected in the most mesenchymal HNSCC cell line and appears to contribute to mesenchymal features in HNSCC cells. These observations could help to explain the different histomorphological phenotypes of HNSCC tumors, which could be linked to the risk of metastatic spread.

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.