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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored <i>CCND1</i>, <i>EGFR</i>, SOX2, and <i>ERBB2</i>, respectively. Analysis of survival data and RNAi screening data suggested that <i>GRB7</i>, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of <i>GRB7</i> reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting <i>GRB7</i> had a synergistic inhibitory effect when combined with trastuzumab, an anti-<i>ERBB2</i> antibody. Survival analysis of the independent cohort also showed that high <i>GRB7</i> expression was associated with poor prognosis in ESCC.</p><p>Conclusion</p><p>Our integrative analysis provided important insights into ESCC pathogenesis. We identified <i>GRB7</i> as a novel ESCC driver gene and potential new therapeutic target.</p></div

Results of analysis on RNA interference screening data of Project Achilles.

<p>Results of analysis on RNA interference screening data of Project Achilles.</p

Knockdown of <i>GRB7</i> expression in ESCC cell lines.

<p>(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) <i>GRB7</i> 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 <i>GRB7</i>-knockdown cells. Each bar represents the average of 3 measurements. (d) Inhibitory effects of siRNA targeting <i>GRB7</i> in combination with trastuzumab. Cells were transfected with siRNA targeting <i>GRB7</i> 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.</p

GISTIC-defined peaks associated with altered expression in ESCC.

<p>Q value indicated the significance of copy number alteration in each region.</p><p>The % aberration included any gain over 0.1 or loss under −0.1 (log2 ratio).</p><p>Candidate targets were genes that have actual correlations between expression and copy number alterations in 'genes in peak'.</p><p>GISTIC-defined peaks associated with altered expression in ESCC.</p

Clinical significance of <i>GRB7</i> mRNA expression in ESCC is validated in the validation set.

<p>(a) Analysis of <i>GRB7</i> 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 <i>GRB7</i> mRNA expression.</p

Results of univariate and multivariate analysis of clinicopathlogical factors for 5-year overall survival in the validation set.

<p>CI: confidence interval, well: well differentiated squamous cell carcinoma, mod: moderately differentiated squamous cell carcinoma, poor: poorly differentiated squamous cell carcinoma.</p><p>Results of univariate and multivariate analysis of clinicopathlogical factors for 5-year overall survival in the validation set.</p