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.

Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis.

Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

High Expression of EpCAM and Sox2 is a Positive Prognosticator of Clinical Outcome for Head and Neck Carcinoma.

Locally advanced head and neck squamous cell carcinomas (HNSCC) have limited prognosis due to frequent treatment failure. Currently, TNM-classification and human papillomavirus (HPV) infection are the sole clinical prognosticators of outcome. Tumor heterogeneity and stemness based on epithelial-mesenchymal-transition reportedly associate with therapy resistance. The capacity of epithelial marker EpCAM (EpEX), stemness regulator Sox2 and mesenchymal marker vimentin to predict clinical outcome of HSNCC patients was assessed upon immunohistochemistry staining in two cohorts of HNSCC patients treated with surgery and adjuvant radio (chemo) therapy (n = 94) and primary radio (chemo) therapy (n = 94), respectively. Prognostic values with respect to overall, disease-free and disease-specific survival were assessed in uni- and multivariate cox proportional hazard models to generate integrated risk scores. EpEX, Sox2 and vimentin displayed substantial inter- and intratumoral heterogeneity. EpEXhigh and Sox2high predicted improved clinical outcome in the discovery cohort and in the HPV-negative sub-cohort. EpEXhigh and Sox2high were confirmed as prognosticators of clinical outcome in the validation cohort treated with definitive radio(chemo)therapy. Importantly, EpEXhigh identified patients with improved survival within the HPV-negative subgroup of the validation cohort. Hence, Sox2high and particularly EpEXhigh have potential as tools to predict clinical performance of HNSCC patients, foremost HPV-negative cases, in the frame of molecular-guided treatment decision-making.

EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by outstanding molecular heterogeneity that results in severe therapy resistance and poor clinical outcome. Inter- and intratumoral heterogeneity in epithelial-mesenchymal transition (EMT) was recently revealed as a major parameter of poor clinical outcome. Here, we addressed the expression and function of the therapeutic target epidermal growth factor receptor (EGFR) and of the major determinant of epithelial differentiation epithelial cell adhesion molecule (EpCAM) in clinical samples and in vitro models of HNSCCs. We describe improved survival of EGFRlow/EpCAMhigh HNSCC patients (n = 180) and provide a molecular basis for the observed disparities in clinical outcome. EGF/EGFR have concentration-dependent dual capacities as inducers of proliferation and EMT through differential activation of the central molecular switch phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and EMT transcription factors (EMT-TFs) Snail, zinc finger E-box-binding homeobox 1 (Zeb1), and Slug. Furthermore, soluble ectodomain of EpCAM (EpEX) was identified as a ligand of EGFR that activates pERK1/2 and phosphorylated AKT (pAKT) and induces EGFR-dependent proliferation but represses EGF-mediated EMT, Snail, Zeb1, and Slug activation and cell migration. EMT repression by EpEX is realized through competitive modulation of pERK1/2 activation strength and inhibition of EMT-TFs, which is reflected in levels of pERK1/2 and its target Slug in clinical samples. Accordingly, high expression of pERK1/2 and/or Slug predicted poor outcome of HNSCCs. Hence, EpEX is a ligand of EGFR that induces proliferation but counteracts EMT mediated by the EGF/EGFR/pERK1/2 axis. Therefore, the emerging EGFR/EpCAM molecular cross talk represents a promising target to improve patient-tailored adjuvant treatment of HNSCCs.

Spatiotemporal patterning of EpCAM is important for murine embryonic endo- and mesodermal differentiation.

Epithelial cell adhesion molecule EpCAM is expressed in pluripotent embryonic stem cells (ESC) in vitro, but is repressed in differentiated cells, except epithelia and carcinomas. Molecular functions of EpCAM, possibly imposing such repression, were primarily studied in malignant cells and might not apply to non-pathologic differentiation. Here, we comprehensively describe timing and rationale for EpCAM regulation in early murine gastrulation and ESC differentiation using single cell RNA-sequencing datasets, in vivo and in vitro models including CRISPR-Cas9-engineered ESC-mutants. We demonstrate expression of EpCAM in inner cell mass, epiblast, primitive/visceral endoderm, and strict repression in the most primitive, nascent Flk1+ mesoderm progenitors at E7.0. Selective expression of EpCAM was confirmed at mid-gestation and perinatal stages. The rationale for strict patterning was studied in ESC differentiation. Gain/loss-of-function demonstrated supportive functions of EpCAM in achieving full pluripotency and guided endodermal differentiation, but repressive functions in mesodermal differentiation as exemplified with cardiomyocyte formation. We further identified embryonic Ras (ERas) as novel EpCAM interactor of EpCAM and an EpCAM/ERas/AKT axis that is instrumental in differentiation regulation. Hence, spatiotemporal patterning of EpCAM at the onset of gastrulation, resulting in early segregation of interdependent EpCAM+ endodermal and EpCAM-/vimentin+ mesodermal clusters represents a novel regulatory feature during ESC differentiation.

Confocal laser endomicroscopy in head and neck malignancies using FITC-labelled EpCAM- and EGF-R-antibodies in cell lines and tumor biopsies.

Intraoperative detection of residual malignant cells at tumor margins following excision of primary tumors could help improving surgery and thus patients' outcome. The feasibility of the tumor antigens epidermal growth factor receptor (EGF-R) and epithelial cell adhesion molecule (EpCAM) for antibody-dependent confocal laser scanning endomicroscopy (CLE)-mediated visualization of malignant cells was addressed. Both tumor antigens are highly and frequently expressed in the majority of carcinomas, including head and neck squamous cell carcinomas (HNSCC), and represent prognostic and therapeutic tumor target molecules. FITC-conjugated EGF-R- and EpCAM-specific antibodies served as molecular tools for the detection of antigen-positive cells using the CLE technology. Specificity of both antibodies and their ability to discriminate tumor from non-tumor cells were assessed in vitro with human fibroblasts and PCI-1 HNSCC cell lines, and ex vivo on primary HNSCC samples (n = 11) and healthy mucosa (n = 5). Antigen specificity of the used EpCAM-specific antibody was superior to that of the EGF-R-specific antibody both in vitro and ex vivo (100% vs. 31.25%), and allowed visualization of cellular structures in CLE measurements. These results hold promise for possible future applications in humans.