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 EGFR^low/EpCAM^high 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. Head and neck squamous cell carcinomas (HNSCCs) display poor survival, with death rates above 55%. Major factors affecting survival are metastases’ formation and therapy resistance. Phenotypic changes during partial epithelial-mesenchymal transition (EMT) provide tumor cells with increased migration, invasion, and therapy resistance. Understanding molecular mechanisms of EMT, as a central process of the metastatic cascade and the development of therapy resistance, is therefore important. In the present work, we identified molecular cross talk between epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) as a novel determinant of clinical outcome in HNSCCs. Low levels of EGFR but high levels of EpCAM (EGFR^low/EpCAM^high) were associated with favorable prognosis, with survival rates above 90%, whereas EGFR^high/EpCAM^low correlated with poor survival, below 10%. EGFR was shown to have a concentration-dependent capacity to induce proliferation and EMT. Proteolytic cleavage of the extracellular domain of EpCAM (EpEX) produces a ligand of EGFR that induces EGFR-dependent proliferation but counteracts EGF-induced EMT. We delineate an EGFR/extracellular signal–regulated kinase 1/2 (ERK1/2)/EpCAM signaling axis that may be a promising therapeutic target for HNSCCs.

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

EpCAM, a carcinoma cell-surface marker protein and a therapeutic target, has been primarily addressed as a cell adhesion molecule. With regard to recent discoveries of its role in signaling with implications in cell proliferation and differentiation, and findings contradicting a direct role in mediating adhesion contacts, we provide a comprehensive and updated overview on the available structural data on EpCAM and interpret it in the light of recent reports on its function. First, we describe the structure of extracellular part of EpCAM, both as a subunit and part of a cis-dimer which, according to several experimental observations, represents a biologically relevant oligomeric state. Next, we provide a thorough evaluation of reports on EpCAM as a homophilic cell adhesion molecule with a structure-based explanation why direct EpCAM participation in cell–cell contacts is highly unlikely. Finally, we review the signaling aspect of EpCAM with focus on accessibility of signaling-associated cleavage sites.

Protein homo-oligomerization is a very common phenomenon, and approximately half of proteins form homo-oligomeric assemblies composed of identical subunits. The vast majority of such assemblies possess internal symmetry which can be either exploited to help or poses challenges during structure determination. Moreover, aspects of symmetry are critical in the modeling of protein homo-oligomers either by docking or by homology-based approaches. Here, we first provide a brief overview of the nature of protein homo-oligomerization. Next, we describe how the symmetry of homo-oligomers is addressed by crystallographic and non-crystallographic symmetry operations, and how biologically relevant intermolecular interactions can be deciphered from the ordered array of molecules within protein crystals. Additionally, we describe the most important aspects of protein homo-oligomerization in structure determination by NMR. Finally, we give an overview of approaches aimed at modeling homo-oligomers using computational methods that specifically address their internal symmetry and allow the incorporation of other experimental data as spatial restraints to achieve higher model reliability.

Recombinant protein expression is central to biotechnology's application. However, not all proteins can be expressed in all organisms, and, given the vast experimental space, it can be challenging to identify the conditions that will yield successful protein expression. The field lacks a predictive model of soluble protein expression that could replace laborious experimental trial and error. Here, we discuss the state of the field and identify the lack of large, high-fidelity datasets as the primary bottleneck to progress. We outline a proposed path toward an extensible experimental platform for collecting soluble overexpression data across organisms. We suggest that the resulting data should be used to train predictive models of protein expression toward answering the question: can protein expression be solved?