An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma

Abstract

Background: Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.

Methods: We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.

Results: We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.

Conclusion: Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Fig 1. Integrative analysis and candidate genes.

(a) Schema of the analysis flow in this study. (b) Plots of recurring high-level amplifications and deletions in 62 ESCCs from GISTIC analysis. The X-axis shows the G score (top) and false discovery rate (q value; bottom) for recurrent peaks across the genome, with the green line indicating an arbitrary false discovery rate (FDR) cutoff of 0.25. Labels on the right denote the positions of peaks in significantly altered regions. (c) Results of integrative analysis and G scores obtained from GISTIC analysis in chromosome 17. The y-axis on left side represents the G score, while the y-axis on the right side represents the p-value from Wilcoxon tests using the edira algorithm. The G score of the positive value indicates amplification, while that of the negative value indicates deletion. The red and blue lines show G scores of amplifications and deletions, respectively. The dotted red line indicates the threshold of amplification, and the dotted blue line indicates the threshold of deletion. Dot plots indicate p-values from Wilcoxon tests in each position. Dot plots in light blue represent deletions, and dot plots in orange represent amplifications. The regions in yellow indicate significant correlations between expression and copy number alterations. (d) Forest plot of hazard ratios of candidate genes for survival. The table on the left side of the Forest plot shows the p-value from univariate and multivariate survival analysis for each gene.

Fig 2. Knockdown of GRB7 expression in ESCC cell lines.

(a) Reductions in mRNA and protein levels of GRB7 at 48 hours after siRNA transfection in KYSE410 and TE4 cells. The results are the mean ± SD from 3 replicates of a single experiment. (b) GRB7 inactivation reduced proliferation of KYSE410 and TE4 cells. Cell growth was measured on days 2, 3, and 4 by MTT assay. Absorbance at day 0 was assigned a value of 1. The results are the mean ± SD from 6 replicates of a single experiment. (c) Migration and invasion assays using GRB7-knockdown cells. Each bar represents the average of 3 measurements. (d) Inhibitory effects of siRNA targeting GRB7 in combination with trastuzumab. Cells were transfected with siRNA targeting GRB7 or negative control siRNA and treated with or without trastuzumab (0.1 and 1.0 μg/mL). Cells were then seeded in 96-well plates, and cell growth was monitored every 24 hours using MTT assays. Absorbance at day 0 was assigned a value of 1. The results are the mean ± SD from 6 replicates of a single experiment.

Fig 3. Clinical significance of GRB7 mRNA expression in ESCC is validated in the validation set.

(a) Analysis of GRB7 mRNA expression in tumor tissues and the corresponding normal mucosa by real-time RT-PCR. (b) Kaplan-Meier survival curves for ESCC patients according to GRB7 mRNA expression.