A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. METHODS: Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. RESULTS: An EGFR-mediated EMT gene signature composed of n = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. CONCLUSIONS: EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.

SLUG-related partial epithelial-to-mesenchymal transition is a transcriptomic prognosticator of head and neck cancer survival.

Partial epithelial-to-mesenchymal transition (pEMT) contributes to cellular heterogeneity that is associated with nodal metastases and unfavorable clinical parameters in head and neck squamous cell carcinomas (HNSCCs). We developed a single-cell RNA sequencing signature-based pEMT quantification through cell type-dependent deconvolution of bulk RNA sequencing and microarray data combined with single-sample scoring of molecular phenotypes (Singscoring). Clinical pEMT-Singscores served as molecular classifiers in multivariable Cox proportional hazard models and high scores prognosticated poor overall survival and reduced response to irradiation as independent parameters in large HNSCC cohorts [The Cancer Genome Atlas (TCGA), MD Anderson Cancer Centre (MDACC), Fred Hutchinson Cancer Research Center (FHCRC)]. Differentially expressed genes confirmed enhanced cell motility and reduced oxidative phosphorylation and epithelial differentiation in pEMThigh patients. In patients and cell lines, the EMT transcription factor SLUG correlated most strongly with pEMT-Singscores and promoted pEMT, enhanced invasion, and resistance to irradiation in vitro. SLUG protein levels in HNSCC predicted disease-free survival, and its peripheral expression at the interphase to the tumor microenvironment was significantly increased in relapsing patients. Hence, pEMT-Singscores represent a novel risk predictor for HNSCC stratification regarding clinical outcome and therapy response that is partly controlled by SLUG.

Interactome analysis reveals endocytosis and membrane recycling of EpCAM during differentiation of embryonic stem cells and carcinoma cells.

Transmembrane epithelial cell adhesion molecule (EpCAM) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESCs). EpCAM displays spatiotemporal patterning during embryogenesis, tissue morphogenesis, cell differentiation, and epithelial-to-mesenchymal transition (EMT) in carcinomas. Potential interactors of EpCAM were identified in murine F9 teratoma cells using a stable isotope labeling with amino acids in cell culture-based proteomic approach (n = 77, enrichment factor >3, p value ≤ 0.05). Kyoto Encyclopedia of Genes and Genomes and gene ontology terms revealed interactions with regulators of endosomal trafficking and membrane recycling, which were further validated for Rab5, Rab7, and Rab11. Endocytosis and membrane recycling of EpCAM were confirmed in mF9 cells, E14TG2α ESC, and Kyse30 carcinoma cells. Reduction of EpCAM during mesodermal differentiation and TGFß-induced EMT correlated with enhanced endocytosis and block or reduction of recycling in ESCs and esophageal carcinoma cells. Hence, endocytosis and membrane recycling are means of regulation of EpCAM protein levels during differentiation of ESC and EMT induction in carcinoma cells.