ABSTRACT: Recurrent/metastatic head and neck squamous cell carcinoma (R/M-HNSCC) is a severe, frequently lethal condition. Targeted therapy includes epidermal growth factor receptor (EGFR) blockade, but overall response rates are low and predictive biomarkers amiss. Deepened understanding of molecular networks governing EGFR-driven progression are required to explore predictive markers and therapeutic targets. We have mapped EGFR-mediated local invasion in a 3D-model using photoconvertible cell-tracers and uncovered invasion-associated, differentially expressed genes (DEGs) correlated with EGFR/MAPK activity and epithelial-to-mesenchymal transition (EMT) in primary malignant cells. A gene regulatory network (GRN) of local invasion was defined, composed of the central hubs inhibin subunit beta alpha (INHBA) and SLUG, and druggable effectors integrin subunit beta 4 (ITGβ4), its ligand laminin 5 (LAMB3/LAMC2), sphingosine kinase 1 (SPHK1), and EGFR ligand amphiregulin (AREG). Blockade of INHBA repressed local invasion, which was rescued by Activin A, Laminin 5, and sphingosine-1-phosphate, proving functional interconnection of the GRN. Functional DEGs (fDEGs) and, most prominently, central hub IHNBA, were associated with tumor buds in TCGA-HNSCC. Newly defined EGFR-activity subtypes, primarily dictated by AREG and epiregulin (EREG), show enhanced fDEGs expression, EMT correlation, and prognostic value. Cell-communication revealed strong interactions of AREG/EREG-governed EGFR-activities with non-malignant cells, potentially impacting on tumor progression. High expression of fDEGs enriched in EGFR-activities (INHBA, ITGA3, ITGΒ4, LAMA3, LAMC2, MT2A, EREG) was associated with response to Cetuximab in PDX models and R/M-HNSCC patients. Thus, we describe a novel GRN of EGFR-mediated local invasion and define druggable effectors with predictive potential regarding the response to Cetuximab.
