Targeting Cancer Stem Cell Plasticity Through Modulation of Epidermal Growth Factor and Insulin-Like Growth Factor Receptor Signaling in Head and Neck Squamous Cell Cancer

Abstract

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resis- tance. The aim of this studywasto establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the main- tenanceandrepopulation of CSCs.Tumorsphereswereestablished using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher al- dehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to g-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with highand lowALDfractions.ALDHigh clonesshowedhigher expression ofstemcellandepithelial-mesenchymal transition markerscomparedwithALDLow clones. ALDfractions, representingstemcell fractions, fluctuated withserial passaging, equilibrating atalevel specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.