Growth inhibition and activation of the oxidative stress signaling pathway by 17-AAG in head and neck squamous cell carcinoma

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INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with differences in clinical behavior and treatment outcome among patients with comparable stages of disease. Our recently published data has shown that a molecular profile could identify a group of HNSCC with a high risk of recurrence. Within this high-risk profile, we have found a pattern of gene expression consistent with activation of the Nuclear Factor-kappa B (NF-\#312;B) signaling pathway. 17-AAG, a heat shock protein 90 (Hsp90) inhibitor, inhibits TNF-induced NF-\#312;B activation in lung cancer cell lines. The effects of Hsp90 inhibition on the TNF-induced NF-\#312;B activation in HNSCC was investigated in this study.
 EXPERIMENTAL PROCEDURES: The cellular effects of 17-AAG on HNSCC cell lines SQ20B and FaDu were determined by MTS assay. The cells were treated with 17-AAG and placebo, with and without TNF stimulation, to assess the TNF-induced NF-\#312;B activation. Total and phospho-IKK and NF-\#312;B protein levels were determined by western blots. Global gene expression changes were determined by DNA microarrays after 4- and 8-hours of 17-AAG treatments. The expression data was mined using significance analysis of microarrays (SAM) and Ingenuity Pathways Analysis (IPA).
 RESULTS: 17-AAG demonstrated a significant inhibition of cell growth in both cell lines (SQ20B IC₅₀ 100nM , FaDu IC₅₀ 50nM) while there was no significant changes in the protein levels and phosphorylated states of IKK and NF-\#312;B. The gene expression data analysis by SAM comparing the control and 4-hours of 17-AAG treatment samples showed upregulation of members of Hsp70 and Hsp40 family proteins. The analysis by IPA suggested the activation of an oxidative stress response-like state with upregulation of ER resident kinase, PERK (EIF2AK3), and its associated protein, BiP (HSPA5).
 CONCLUSIONS: Inhibition of Hsp90 appears to decrease cell proliferation in a dose dependent manner in HNSCC cell lines, through an oxidative stress signaling pathway, rather than through the TNF-induced NF-KB pathway. This suggests that 17-AAG may have a role as a radiation sensitizer and further investigation is warranted in HNSCC.