Title Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p 53 , nuclear factor-κ B and other signal transcription factors in head and neck squamous cell carcinoma

Background: Differentially expressed gene profiles have previously been observed among pathologically defined cancers by microarray technologies, including head and neck squamous cell carcinomas (HNSCCs). However, the molecular expression signatures and transcriptional regulatory controls that underlie the heterogeneity in HNSCCs are not well defined. Results: Genome-wide cDNA microarray profiling of ten HNSCC cell lines revealed novel gene expression signatures that distinguished cancer cell subsets associated with p53 status. Three major clusters of over-expressed genes (A to C) were defined through hierarchical clustering, Gene Ontology, and statistical modeling. The promoters of genes in these clusters exhibited different patterns and prevalence of transcription factor binding sites for p53, nuclear factor-κB (NF-κB), activator protein (AP)-1, signal transducer and activator of transcription (STAT)3 and early growth response (EGR)1, as compared with the frequency in vertebrate promoters. Cluster A genes involved in chromatin structure and function exhibited enrichment for p53 and decreased AP-1 binding sites, whereas clusters B and C, containing cytokine and antiapoptotic genes, exhibited a significant increase in prevalence of NF-κB binding sites. An increase in STAT3 and EGR1 binding sites was distributed among the over-expressed clusters. Novel regulatory modules containing p53 or NF-κB concomitant with other transcription factor binding motifs were identified, and experimental data supported the predicted transcriptional regulation and binding activity. Conclusion: The transcription factors p53, NF-κB, and AP-1 may be important determinants of the heterogeneous pattern of gene expression, whereas STAT3 and EGR1 may broadly enhance gene expression in HNSCCs. Defining these novel gene signatures and regulatory mechanisms will be important for establishing new molecular classifications and subtyping, which in turn will promote development of targeted therapeutics for HNSCC. Published: 11 May 2007 Genome Biology 2007, 8:R78 (doi:10.1186/gb-2007-8-5-r78) Received: 17 October 2006 Revised: 7 February 2007 Accepted: 11 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R78 Genome Biology 2007, 8:R78 R78.2 Genome Biology 2007, Volume 8, Issue 5, Article R78 Yan et al. http://genomebiology.com/2007/8/5/R78 Background Numerous basic and clinical studies suggest that development and malignant progression of cancer is rarely due to a defect in a single gene or pathway. Multiple genetic alterations accumulate during carcinogenesis, potentially leading to aberrant activation or suppression of multiple pathways and downstream genes that have important functions in determining the malignant phenotypes of cancer. Microarray technology has enabled us to study global gene expression profiles of cancers and identify gene programs or 'signatures' that are critical to the heterogeneous characteristics and malignant phenotypes of cancers, even of the same pathologic type [1-3]. In head and neck squamous cell carcinomas (HNSCCs), gene expression profiling has been used in attempts to identify biomarkers for diagnosis [4], differential sensitivity to chemotherapy [5], risk for recurrence [6], survival [7], malignant phenotype [8], and metastasis [9]. Although considerable variability in the composition of gene signatures was observed in these studies, they provided evidence for subsets within HNSCCs, which are possibly due to differences in molecular pathogenesis that affect malignant potential. However, the transcriptional regulatory mechanisms that control the heterogeneous and shared patterns of gene expression profiles observed, and their relationship to malignant phenotypes, are not well defined. The transcriptional regulation of gene expression is mainly dependent on the composition of transcription factor binding site (TFBSs), and complex interactions among transcription factors and regulatory proteins that bind to gene promoters [10]. In murine and human squamous cell carcinoma (SCC), we and others have identified transcription factors that are inactivated or mutated (for instance, the tumor suppressor p53), or are constitutively activated (such as nuclear factorκB [NF-κB], activator protein [AP]-1, signal transducer and activator of transcription [STAT]-3, and early growth response [EGR]1). These transcription factors have been independently implicated as tumor suppressor or oncogenic transcription factors that regulate the expression of individual genes related to phenotypic characteristics that are important in cancer development. Among these transcription factors, p53 has been implicated as a master regulator of genomic stability, cell cycle, apoptosis, and DNA repair [11,12]. Mutation or silencing of the p53 gene is an important molecular event in tumorigenesis, which has been associated with nearly 50% incidence among all cancers [13-15], including HNSCC [16-20]. NF-κB is a nuclear transcription factor that is activated in HNSCCs and other cancers. We and others have shown that constitutive activation of NF-κB1/RelA is among the important factors that control expression of genes that regulate cellular proliferation, apoptosis, angiogenesis, immune and proinflammatory responses, and therapeutic resistance in HNSCCs [21-26] and other cancers [27-29]. AP-1, STAT3, and EGR1 are considered important transcription factors that are involved in regulating gene expression in human cancers, including HNSCCs. Constitutive activation of AP-1 and STAT3 appear to be important factors for tumor cell proliferation, survival, and angiogenesis in vitro or in vivo [21,24,29-34]. EGR1 is a zincfinger transcription factor that is rapidly and transiently induced in response to a number of stimuli, including growth factors, cytokines, and mechanical stresses [35,36]. The study of regulatory controls involving multiple transcription factors for clustered gene expression obtained from microarray data meets with many experimental challenges. In a previous study of step-wise progression of murine SCCs, we combined gene expression profiling data with a bioinformatic analysis of promoter TFBSs and ontology limited to NF-κB regulated genes, and provided evidence that this transcription factor is one of the critical regulatory determinants of expression of multiple genes and malignant phenotype [37,38]. However, this approach involving analysis of a single pathway and this TFBS appears far from providing a complete explanation for the heterogeneity and multiplicity of genes expressed in clusters in HNSCCs and other cancers, or associated differences in phenotypic and biologic behavior observed. Identification of common TFBSs in gene clusters through in silico analysis can provide a framework for further elucidating the network and complex interactions of regulatory mechanisms that are involved in gene expression in cancer [39,40]. In the present study, microarray combined with computational prediction was utilized to define gene expression patterns and putative TFBSs for genes that are differentially expressed among ten HNSCC cell lines and nonmalignant keratinocytes. The differentially expressed microarray profiles classified subsets of HNSCC cells related to differences in p53 genotype, protein expression, and unique gene signatures. The potential relationship of novel gene expression signatures and prevalence of TFBSs for p53, NF-κB, AP-1, STAT3, and EGR1 were identified, and novel transcription regulatory modules for specific gene clusters were predicted. The predicted results were then validated by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assay. Our study suggests that integration of genome-wide microarray profiling and computational analyses is a powerful way to identify gene signatures as determinants for cancer heterogenicity and malignant phenotypes, and their underlying regulatory control mechanisms. Results Identification of novel gene clusters in University of Michigan SCC cells with different p53 status by cDNA microarray expression profiling cDNA microarray analysis was performed using a panel of ten HNSCC cell line series from the University of Michigan (UMSCC), derived from eight patients with aggressive HNSCC Genome Biology 2007, 8:R78 http://genomebiology.com/2007/8/5/R78 Genome Biology 2007, Volume 8, Issue 5, Article R78 Yan et al. R78.3