Members of the EGFR/ErbB family of tyrosine kinases are found to be highly expressed and deregulated in many cancers, including head and neck squamous cell carcinoma (HNSCC). The ErbB family, including EGFR, has been demonstrated to play key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSCs) which are believed to be responsible for tumor initiation and maintenance. In this study, we investigated the possible role of EGFR as a regulator of “stemness” in HNSCC cells. Activation of EGFR by the addition of EGF ligand or ectopic expression of EGFR in two established HNSCC cell lines (UMSCC-22B and HN-1) resulted in the induction of CD44, BMI-1, Oct-4, NANOG, CXCR4, and SDF-1. Activation of EGFR also resulted in increased tumorsphere formation, a characteristic ability of cancer stem cells. Conversely, treatment with the EGFR kinase inhibitor, Gefinitib (Iressa), resulted in decreased expression of the aforementioned genes, and loss of tumorsphere-forming ability. Similar trends were observed in a 99.9% CD44 positive stem cell culture derived from a fresh HNSCC tumor, confirming our findings for the cell lines. Additionally, we found that these putative cancer stem cells, when treated with Gefitinib, possessed a lower capacity to invade and became more sensitive to cisplatin-induced death in vitro. These results suggest that EGFR plays critical roles in the survival, maintenance, and function of cancer stem cells. Drugs that target EGFR, perhaps administered in combination with conventional chemotherapy, might be an effective treatment for HNSCC.

In established head and neck squamous cell carcinoma cell lines, SP cells were found using methods that determine expression and function of the drug transporter BCRP1/ABCG2. Activation of EGFR, a gene implicated in tumorigenesis in HNSCC leads to increased SP, and conversely, inhibition of EGFR leads to decrease in SP. This finding could help explain the role of EGFR in regulating cancer stem cells and thus tumorigenesis in HNSCC.

BackgroundCancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells.MethodsMTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose.ResultsIn putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR.ConclusionsThese results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and Akt.

Protein tyrosine kinases (TKs) are overexpressed in many carcinomas and sarcomas. We studied the expression of the following TKs in head and neck squamous cell carcinoma (HNSCC): platelet-derived growth factor receptor (PDGFR), c-kit, epidermal growth factor receptor (EGFR), and a serine-threonine kinase, Akt. Formalin-fixed, paraffin-embedded tumor blocks from 44 consecutive patients with primary HNSCC and 5 specimens of benign pharyngeal and laryngeal mucosa were retrieved for immunohistochemical analysis. Of the specimens, 38 had enough material to stain for all 4 antibodies. The study included 21 pharyngeal (base of tongue, 14; tonsil, 6; soft palate, 1), 16 laryngeal, and 1 floor of the mouth carcinoma. All 4 kinases in the tumor samples were expressed highly (PDGFR, 95%-100%; EGFR, 38%-43%; c-kit, 50%-86%; p-Akt, 57%-81%), with EGFR, c-kit, and p-Akt significantly higher than in benign samples. None of the kinase expressions correlated with disease-free survival. The expression of the kinases raises the possibility of treatment of HNSCC by tyrosine and serine-threonine kinase inhibitors.

Protein tyrosine kinases (TKs) are overexpressed in many carcinomas and sarcomas. We studied the expression of the following TKs in head and neck squamous cell carcinoma (HNSCC): platelet-derived growth factor receptor (PDGFR), c-kit, epidermal growth factor receptor (EGFR), and a serine-threonine kinase, Akt. Formalin-fixed, paraffin-embedded tumor blocks from 44 consecutive patients with primary HNSCC and 5 specimens of benign pharyngeal and laryngeal mucosa were retrieved for immunohistochemical analysis. Of the specimens, 38 had enough material to stain for all 4 antibodies. The study included 21 pharyngeal (base of tongue, 14; tonsil, 6; soft palate, 1), 16 laryngeal, and 1 floor of the mouth carcinoma. All 4 kinases in the tumor samples were expressed highly (PDGFR, 95%-100%; EGFR, 38%-43%; c-kit, 50%-86%; p-Akt, 57%-81%), with EGFR, c-kit, and p-Akt significantly higher than in benign samples. None of the kinase expressions correlated with disease-free survival. The expression of the kinases raises the possibility of treatment of HNSCC by tyrosine and serine-threonine kinase inhibitors.