Purpose: To determine if gene signatures differentially expressed in head and neck squamous cell carcinomas (HNSCC) are related to alterations in transcription factors nuclear factor-kappaB (NF-kappaB) and TP53 previously associated with decreased cell death, response to therapy, and worse prognosis.
Experimental design: Unique gene signatures expressed by HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. Bioinformatic analysis of the promoters and ontogeny of these clustered genes was done. Expression of proteins encoded by genes of a putative NF-kappaB signature, NF-kappaB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-kappaB genes and cell survival were evaluated by p65 chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) knockdown.
Results: Two groups of HNSCC exhibiting distinct gene signatures were identified: cluster A enriched for histone genes, with a higher prevalence of TP53 promoter binding motifs; and cluster B enriched for injury response genes with NF-kappaB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-kappaB phospho-p65 and weak TP53 staining, and NF-kappaB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-kappaB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA.
Conclusion: NF-kappaB promotes expression of a novel NF-kappaB-related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-type TP53, greater resistance to chemoradiotherapy, and worse prognosis.