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.

Figure 1. Identification of DNA copy number–associated differentially expressed genes in metastatic OSCC.

A) Integrative genomic analysis workflow; B) Heat-map of the 1,988 transcripts with differential expression between non-metastatic primary and nodal metastatic OSCC. Specimen code: Red = metastatic OSCC; Yellow: primary OSCC; C) Top panel: the z-score of the DNA copy number difference between primary and metastatic OSCC. Each bar represents one of the differentially expressed genes. Genes with significant Z-score differences are highlighted in red. Bottom panel: Manhattan plot showing the -Log10 transformed p-value of the correlation coefficients of CNA and gene expression. Each dot represents one of the differentially expressed genes. Genes with different copy number in primary and metastatic OSCC are highlighted in red. The 4 regions with cluster of CNA-associated differentially expressed genes are indicated.

Figure 2. Association between DNA copy number–associated differentially expressed genes and survival.

A) Hierarchical clustering of an independent dataset of 133 patients using the 95 CNA-associated differentially-expressed gene signature. Expression variances of the genes were summarized by principal components analysis. A hierarchical clustering analysis was performed using the first three principal component (PC) scores; B) Overall survival of the two patient clusters stratified by the 95-CNA-associated differentially expressed signature; C) Cumulative incidence of OSCC-specific death for the two patient clusters.

Figure 3. Results of siRNA knockdown screen.

(A–E) Cell viability after the siRNA-mediated gene knock-down (KD) screen in UM-SCC-14A, UM-SCC-14C, PCI-15A, PCI-15B, and JHU-019. Cell viability was measured 4 days after the siRNA transfection. Results were presented as relative to control cells treated with transfection reagent only. Error bar represent the standard deviation of 9 repeats; (F) Average viability in cell lines derived from primary OSCC (x-axis) and from metastatic OSCC (y-axis). KD of Kiff11 and transfection reagent only was used as positive and negative control. Arrow indicates the three repeats of KD of G3BP1.

Figure 4. Effects of G3BP1 knockdown in OSCC.

Cell viability, cytotoxicity, and apoptosis in OSCC cell lines PCI-15B (A), UM-SCC-17A (B), UM-SCC-47 (C) after the KD of G3BP1. The results are presented as relative to the reading in control wells that are transfected with negative control siRNA. Error bar indicates standard deviation of the readings in three repeat wells; (D) Confirmation of the KD of G3BP1 in these cell lines with western blot. Beta-actin (ACTB) is used as a loading control.