Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma

Abstract

In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC–specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03–11.11) and 3.45 (95% CI: 1.84–6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA–mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials.

Author Summary

Neck lymph node metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC). To identify genes associated with this critical step of OSCC progression, we compared DNA copy number aberrations and gene expression differences between tumor cells found in metastatic lymph nodes versus those in non-metastatic primary tumors. We identified 95 transcripts (87 genes) with metastasis-specific genome abnormalities and gene expression. Tested in an independent cohort of 133 OSCC patients, the 95 gene signature was an independent risk factor of disease-specific and overall death, suggesting a disease progression phenotype. We knocked down the expression of over-amplified genes in five OSCC cell lines. Knockdown of 18 of the 26 tested genes suppressed the cell growth in at least one cell line. Interestingly, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC. The knockdown of G3BP1 increased programmed cell death in the p53-mutant but not wild-type OSCC cell lines. Taken together, we demonstrate that CNA–associated transcripts differentially expressed in carcinoma cells with an aggressive phenotype (i.e., metastatic to lymph nodes) can be biomarkers with both prognostic information and functional relevance. Moreover, results suggest that G3BP1 is a potential therapeutic target against late-stage p53-negative OSCC.