Inhibition of Jun NH2-terminal kinases suppresses the growth of experimental head and neck squamous cell carcinoma.

PURPOSE: This study was carried out to investigate whether c-Jun NH2-terminal kinases (JNK) are potential targets for treating head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: JNK activity was first evaluated in 20 paired samples of human HNSCC. The antitumor activity of SP600125, a reversible nonselective ATP-competitive inhibitor of JNKs, was then investigated both in an HNSCC xenograft model and in vitro using immunohistochemistry, immunoblotting, enzyme immunoassay, flow cytometry, and a Matrigel assay of capillary tube formation. Complementary studies were carried out using small interfering RNA to JNK1/2. RESULTS: JNK activity was increased in human HNSCC compared with normal-appearing epithelium. Treatment of mice bearing HNSCC xenografts with SP600125 resulted in >60% inhibition of tumor growth relative to vehicle-treated animals. Inhibition of tumor growth was associated with significant reductions in both cell proliferation and microvessel density. SP600125 inhibited tumor cell proliferation by causing delays in both the S and G2-M phases of the cell cycle. Inhibition of angiogenesis seemed to reflect effects on both tumor and endothelial cells. The JNK inhibitor suppressed the production of vascular endothelial growth factor and interleukin-8 by tumor cells and also inhibited endothelial cell proliferation and capillary tube formation. Reduced amounts and phosphorylation of epidermal growth factor receptor were found in tumor cells after treatment with SP600125. Small interfering RNA-mediated suppression of JNK1/2 led to reduced tumor cell proliferation and decreased levels of epidermal growth factor receptor, vascular endothelial growth factor, and interleukin-8. CONCLUSIONS: JNK activity is commonly increased in HNSCC. Our preclinical results provide a rationale for evaluating JNK inhibition as an approach to treating HNSCC.

Histone deacetylase inhibitors suppress the induction of c-Jun and its target genes including COX-2.

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.