P. T. White1, C. Subramanian1, P. T. Grogan1, E. Brandes1, H. Zhang2, R. Gallagher2, B. N. Timmermann2, M. S. Cohen1 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Kansas,Department Of Medicinal Chemistry,Lawrence, KS, USA
Introduction: Head and neck squamous cell carcinoma (HNSCC) survival rates have been stagnant for the last four decades highlighting the need for novel therapeutic approaches and a better understanding of the disease biology. Recent advances indicate that cancer stem cells (CSCs) in the tumor are responsible for recurrence and metastasis, and that standard cisplatin treatment enriches CSC numbers via BMI-1 upregulation. Withanolides are 28-carbon steroidal lactones that have been shown by our group and others to have potent anticancer activity through inhibition of HSP90-chaperoned kinases. These selectively target key proliferative pathways in cancers (including notch, β-catenin, and NF-κB) that also play a critical role in CSC maintenance. We hypothesize that a novel withanolide, withalongolide A (WGA) and its triacetate derivative (WGA-TA) will prevent tumor growth and invasion through inhibition of CSC epithelial to mesenchymal transition (EMT) and cell migration.
Methods: Validated human HNSCC cell lines (JMAR, MDA1986,UMSCC-11B) were grown in 2D culture and treated with 0.1 to 5μM WGA or WGA-TA for 24h. Proteins involved in the maintenance of CSCs and EMT were analyzed by Western blot (WB) and matrigel invasion assays were performed post treatment to evaluate migration. Orosphere formation assay was conducted to determine self-renewal after treatment. The percentage of apoptotic CSCs was determined by flow cytometry (FC) and confirmed by WB.
Results: Treatment of HNSCC cells with WGA-TA demonstrated a dose dependent increase in inhibition of CSC markers compared to WGA, including BMI-1, CD44, EZH2, notch1 and other proteins involved in CSC maintenance. 5 µM WGA-TA treatment had the highest inhibition on CSC regulatory proteins and markers (80% for EZH2, 45%BMI-1, 60% for CD44) which was significant (p<0.01)vs. 5 µM WGA (30% inhibition for EZH2, 35% for CD44, and none for BMI-1) and vs. controls (p<0.01). Similarly 5 µM WGA-TA significantly inhibited levels of Akt (95%), p-Akt (80%), and p-GSK3β (90%) compared to WGA (95%, 80% and 30%, respectively, p<0.001 vs controls). 5 µM WGA-TA treatment also decreased the EMT protein vimentin by 60%. In the migration assay, treatment with 5μM WGA-TA showed greater than 80% suppression of migration compared to control (p<0.001), as opposed to 20-30% for 5 µM WGA treatment (p<0.01 vs WGA-TA). An increase in apoptotic CSCs by >50% vs controls, and decrease in orosphere formation occured in a dose-dependent manner with WGA-TA.
Conclusions: WGA-TA represents a novel therapy for HNSCC targeting key pathways and kinases implicated in the maintenance of CSCs, EMT, and invasion. These withanolides target BMI-1, EZH2 and its downstream effectors in HNSCC CSCs resulting in decreased EMT and tumor migration. Further in vivo translation is needed to define the role of this CSC inhibition on tumor growth kinetics, invasion, and metastatic spread.