Identification of differentially expressed genes in HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas

Abstract

Human papillomaviruses (HPVs) have been implicated in the pathogenesis of a subset of squamous cell carcinoma of the head and neck (SCCHN). The goal of this study was to compare the cellular gene expression profiles of HPV-positive and HPV-negative oropharyngeal carcinomas with those of the normal oral epithelium. Using Affymetrix Human U133A GeneChip, our results showed that 397 genes were differentially expressed in HPV-positive SCCHN compared to the normal oral epithelium. The upregulated genes included those involved in cell cycle regulation (CDKN2A), cell differentiation (SFRP4) and DNA repair (RAD51AP1), while the downregulated genes included those involved in proteolysis (PRSS3). We also found 162 differentially expressed genes in HPV-negative SCCHN compared to the normal oral mucosa. The upregulated genes included those involved in cell proliferation (AKR1C3) and transcription regulation (SNAPC1), while downregulated genes included those involved in apoptosis (CLU) and RNA processing (RBM3). Our studies also identified a subgroup of 59 differentially expressed genes in HPV-positive SCCHN as compared to both HPV-negative SCCHN and normal oral tissues. Such upregulated genes included those involved in nuclear structure and meiosis (SYCP2), DNA repair (RFC5), and transcription regulation (ZNF238). Genes involved in proteolysis (KLK8) and signal transduction (CRABP2) were found to be downregulated in HPV-positive SCCHN. The results of GeneChip experiments were validated by quantitative real-time RT-PCR analysis of a few representative genes. Our results reveal specific gene expression patterns in HPV-positive and HPV-negative oropharyngeal squamous carcinomas that may serve as potential biomarkers for the development of SCCHN.

Fig. 1

Analysis of HPV16 E6 and E7 gene expression in oropharyngeal tissue samples by RT-PCR. CaSki (HPV-16 positive) and C-33A (HPV-negative) cervical cell lines were used as controls. Expression of E6 splicing variants (E6, 477 bp, E6*I, 293 bp, E6*II, 176 bp) and E7 (137 bp) genes was found only in SK20, SK30 and SK31 SCCHN samples. The integrity of all the RNA samples was validated by amplification of the house-keeping gene GAPDH (452 bp).

Fig. 2

Summary of differentially expressed genes identified by microarray analysis using two different statistical analyses. The ovals represent the 3 groups of samples used in our study. The overlapping regions indicate the number of genes found to be up- or down-regulated between two different groups of samples.

Fig. 3

Unsupervised hierarchical cluster analysis in oropharyngeal tissue samples. From a total of 22,215 transcripts on the microarray, 8,286 genes showed variations in expression across all the samples. From this group of filter genes, we identified 7 clusters of genes that show differential expression between the 3 groups of samples (HPV-positive SCCHN, HPV-negative SCCHN and normal oral mucosa). Genes also found in the supervised statistical analysis are underlined.

Fig. 4

Chromosomal location of genes found to be differentially expressed in the hierarchical cluster analysis. Squares to the right of the chromosomes indicate genes with increased expression in HPV-positive oropharyngeal squamous carcinomas as compared to HPV-negative carcinomas or the normal oral epithelium (clusters 1, 2 and 3). Circles to the left of the chromosomes indicate genes with reduced expression in HPV-positive oropharyngeal squamous carcinomas as compared to the HPV-negative and normal samples (clusters 4 and 5). The chromosome map was obtained and modified from the Department of Pathology, University of Washington web page http://www.pathology.washington.edu/research/cytopages/idiograms/human/ (Idiogram Album: Human copyright © 1994 David Adler).

Fig. 5

Validation of microarray data in oropharyngeal tissue samples by quantitative RT-PCR. Total RNA from tissue samples was used to verify four upregulated genes (cyclin-dependent kinase inhibitor 2A [CDKN2A], synaptonemal complex protein 2 [SYCP2], zinc finger protein 238 [ZNF238] and replication factor C 5 [RFC5]), and two downregulated genes (cellular retinoic acid binding protein 2 [CRABP2], kallikrein 8 [KLK8]) that were differentially expressed based on the microarray data. All reactions were performed in triplicates and the error bar represents the standard deviation. Relative expression of the target gene was calculated using the 2 delta CT method, where GAPDH was used as the endogenous control gene.