Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target

Abstract

Background: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease.

Methodology and principal findings: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines.

Conclusions: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Figure 1. Percentage of called gains and losses and their effect on gene expression in 32 NSCLC patients.

Summary plot for called gains and losses in 32 resected NSCLC patients with DNA copy number changes indicated in grey. Positive values indicate the percentage of samples found with a gain. Negative values indicate the percentage of samples harboring a loss at the specified chromosome location. Genes in specified regions affected by copy number gain are indicated in green and genes affected by copy number loss are indicated in red. A selection of affected genes is indicated. The full list of 359 affected transcripts can be found in supplementary table S1.

Figure 2. Loss of 14q32.2-32.33 chromosomal region and HSP90 expression in relation with survival.

(A) Kaplan-Meier curves for overall survival are shown for 29 patients in relation to gene dosage in chromosome region 14q32.33. (B) Overall survival for 29 patients in relation to HSP90 expression. Low expression was defined as expression lower than the median of the total 32 samples, “normal” expression was defined as higher than the median of 32 samples. Three of 32 patients included in the analysis of gene dosage and expression were excluded from the survival analysis because of a survival time of less than 30 days.

Figure 3. HSP90 expression and survival in three validation sets of NSCLC patients.

Overall survival for (A) 140 patients with NSCLC, validation set 1 (B) 111 NSCLC patients, validation set 2 and (C) 54 patients with NSCLC, validation set 3. The cut off for distinction between low and “normal” expression was based on the 33-percentile of expression values.

Figure 4. Sensitivity of a panel of wild type EGFR-expressing NSCLC cell lines to the Hsp90 inhibitor 17-AAG.

(A–D) time- and dose-dependent inhibition of the in vitro growth of H460, H157, H441, and A549 NSCLC cell lines following exposure to 17-AAG. Cells were seeded at 105/well, and viable cell number was determined on subsequent days as described in Methods. 17-AAG concentrations at (H441) or above (A549, H460 & H157) 30 nM uniformly resulted in time-dependent loss of cell viability. The IC50 value of 17-AAG (continuous exposure for 72 h) for each cell line is as follows: H460 = 30 nM, H157 = 15 nM, H441 = 8 nM, and A549 = 20 nM.