Methods: Three human HNSCC lines (UMSCC-17B, SCC-58, TR146), representing malignant squamous cells of the larynx, tongue, and cheek, were cultured either as monolayers or free floating spheres in serum-free medium under low oxygen (5% O2) to enrich for CSCs. Monolayer and sphere cells were treated with 10 uM of Phenamil for 48 hours, and expression of CSC markers (CD44 and BMI1), SMURF1, and downstream target genes (ID1, SMAD6) was analyzed using qRT-PCR.
Results: Sphere cells showed increased expression of CD44, BMI1, and SMURF1, as well as an overall decrease in ID1 and SMAD6 expression when compared to matched monolayer controls. Phenamil treatment of sphere cells markedly reduced expression of CSC markers and SMURF1. Furthermore, ID1 and SMAD6 expression increased after phenamil treatment when compared to expression levels observed in untreated spheres.
Conclusion: Our results indicate that HNSCC CSCs upregulate SMURF1 expression to modulate BMP-signaling. This has important implications since non-CSC tumor cells, which display active BMP-signaling, are responsive to current treatments, unlike CSCs which may be more resistant to current therapies and contribute to poor treatment responses. We propose that reactivating BMP-signaling will sensitize CSCs to current treatments and improve patient outcomes.