Targeting Cancer Stem Cells in HNSCC: Synergic Effect of Cetuximab and ABT-199 in Combination with Photon Radiation

Abstract

Purpose/Objective(s) Concurrent cetuximab based radio-chemotherapy is a validated scheme in head and neck squamous cell carcinoma (HNSCC). Cancer Stem Cells (CSCs) are highly resistant to treatment, have large migratory abilities, and are hypothesized to be responsible for a significant part of recurrences. Apoptotic signaling in CSCs is a major way of treatment escape, through Bcl-2 proteins family. The aim of the present study was to explore the synergic effect between cetuximab and an anti-Bcl-2 antibody (ABT-199), when combined or not with photon radiation. Materials/Methods HNSCC chemo and radio resistant human cell line (SQ20B) and its corresponding stem cell line (SQ20B/CSCs) were used to test the treatment combinations. HaCaT cell line was used to assess toxicity on healthy cell population. SQ20B/CSCs subpopulation was isolated through double cell sorting: side population (SP) (Hoechst exclusion) and CD44 staining: SP/CD44High. SQ20B and SQ20B/CSCs proliferation, invasion/migration, and apoptosis were studied after an exposition to cetuximab 5nM + ABT-199 10mM treatment +/- 10Gy photon irradiation. Invasion and migration were assessed based on scratch wound assay with or without matrigel. Apoptosis was measured using caspases 3/7. 3D spheroid assay was performed to validate the results in a 3D culture approach. EGFR, phospho-EGFR (Tyr1068), Bcl-2 and Bcl-xl protein expression were studied. Results Cetuximab strongly inhibited SQ20B proliferation, migration and invasion whereas it had little effect on SQ20B/CSCs. Conversely, ABT-199 significantly inhibited these properties on SQ20B/CSCs, without showing any effect on SQ20B parental cell line. Cetuximab-ABT-199 combined with radiation had a significant inhibitory effect on both SQ20B and SQ20B/CSCs proliferation, migration and invasion. Although exclusive cetuximab had no pro-apoptotic effect, activation of caspases 3/7 was induced by ABT-199 and enhanced by the cetuximab+ABT-199 combination in both populations. Cetuximab was a strong inhibitor of 3D-spheroid proliferation in SQ20B, whereas ABT-199 strongly decreased spheroid size in CSCs. EGFR was overexpressed in SQ20B, and under-expressed in SQ20B/CSCs. Bcl2 was overexpressed in SQ20B/CSCs. Although cetuximab moderately inhibited HaCaT cell proliferation, the drug combination did not significantly enhanced toxicity. Conclusion Cetuximab+ABT-199 combined with photon radiation significantly inhibited proliferation, invasion and migration of SQ20B HNSCC cell line and its CSCs subpopulation, with an acceptable toxicity profile on healthy cell lines. Apoptotic cell death was enhanced by this drug combination.