We previously showed using microarray profiling biotechnology that stepwise transformation and metastatic progression of SCC in a murine model results in the expression of gene programs related to the signal transcription factor NF-kappaB. Inhibition of NF-kappaB modulated over half the genes differentially expressed and attenuated the malignant phenotype, indicating it may be a critical target for prevention or therapy of head and neck cancer (reviewed in 1). Recent gene expression profiling and bioinformatic analysis of the promoters of gene clusters differentially expressed in human HNSCC provided evidence for increased prevalence of binding motifs for NF-kappaB as well as other signal transcription factors, such as p53, AP-1, STAT3 and EGR-1 (Yan et al, Genome Biology, 2007). NF-kappaB, AP-1, STAT3 and EGR-1 activation has previously been associated with pathogenesis and therapeutic resistance, and the subsets expressing wt or mt 53 have been reported to differ in response to chemotherapy. These observations suggested the hypothesis that key alterations in a network of signal transcription factors can interact in determining gene expression and development of HNSCC of differing malignant potential and sensitivity or resistance to therapy.? ? ? During the present period, we reported on the identification of a novel NF-kappaB gene signature that is differentially expressed in HNSCC in association with TP53 status (2). 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. We identified two groups of HNSCC exhibiting distinct gene signatures: 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. We conclude that 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. Further, these findings suggested that a common mechanism may be responsible activating NF-kappaB and inactivating p53 in this subset. In another project (Z01-DC-000073, Friedman et al, Clin Cancer Res., 2007), we demonstrated that the anti-malarial and inflammatory drug quinacrine can restore p53 expression, function and cisplatin sensitivity of HNSCC. Quinacrine may hold potential alone and in combination with DNA damaging agents for investigational therapy in clinical researcf trials.? ? In a second study, we took a systems biology approach to define NF-kappaB regulons, interacting signal pathways and networks in the malignant phenotype of head and neck cancer cell lines differing in p53 status (3). In collaboration with colleagues at University of Pennsylvania, we used their newly developed computational strategy, COGRIM (Clustering Of Gene Regulons using Integrated Modeling), to identify NF-kappaB regulons (a set of genes under regulation of the same transcription factor) for 1,265 genes differentially expressed by head and neck cancer cell lines differing in p53 status. There were 748 NF-kappaB targets predicted and individually annotated for RELA, NFkappaB1 or cREL regulation, and a prevalence of RELA related genes was observed in over-expressed clusters in a tumor subset. Using Ingenuity Pathway Analysis, the NF-kappaB targets were reverse-engineered into annotated signature networks and pathways, revealing relationships broadly altered in cancer lines (activated proinflammatory and down-regulated Wnt/beta-catenin and transforming growth factor-beta pathways), or specifically defective in cancer subsets (growth factors, cytokines, integrins, receptors and intermediate kinases). Representative predicted NF-kappaB target genes were experimentally validated through modulation by tumor necrosis factor-alpha or small interfering RNA for RELA or NFkappaB1. These studies provide evidence that NF-kappaB globally regulates diverse gene programs that are organized in signal networks and pathways differing in cancer subsets with distinct p53 status. The concerted alterations in gene expression patterns reflect cross-talk among NF-kappaB and other pathways, which may provide a basis for molecular classifications and targeted therapeutics for heterogeneous subsets of head and neck or other cancers.? ? In another collaborative study, we contributed evidence that an anti-oxidant, Tempol, protects against oxidative DNA damage in cells from patients, and development of epithelial cancers in mice deficient in genes that cause Fanconi Anemia (4). Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and marked cancer susceptibility. FA patients have an elevated risk of developing hematologic malignancies and solid tumors, especially SCC, as a result of a defect in DNA repair. Free radical formation and oxidative damage of DNA occurs continuously, but is normally repaired. Tempol is a nitroxide antioxidant and a superoxide dismutase mimetic. The reduction in oxidative DNA damage in tempol-treated FA fibroblasts suggested that Tempol could reduce the rate of accumulation of alterations in key genes and delay tumor development. Using Fancd2(-/-) knockout mice as a model of FA, the potential of tempol as a tumor-delaying agent for solid tumors was studied. Dietary tempol increased the mean tumor-free survival time of Fancd2(-/-) Trp53(+/-) mice by 27% (P < 0.01), from 308 to 390 days, without changing the overall tumor spectrum. More strikingly, tempol delayed the onset of epithelial tumors and increased the mean epithelial tumor-free survival time by 38% (P < 0.0001), from 312 to 432 days, in Fancd2(-/-) Trp53(+/-) mice. These results show that tempol can significantly delay tumor formation in Fancd2(-/-) Trp53(+/-) mice. Furthermore, tempol treatment did not adversely affect the repopulating ability of FA hematopoietic stem cells. The reduction in oxidative DNA damage in tempol-treated FA fibroblasts and mice suggests that its tumor-delaying function may be attributed to its antioxidant activity. These findings suggest that Tempol and anti-oxidants may warrant clinical study for prevention in cancer prone hereditary disorders such as FA in which oxidative damage contributes to cumulative DNA damage and cancer.
