Impact of gene dosage on gene expression, biological processes and survival in cervical cancer: a genome-wide follow-up study.

We investigated the role of tumor copy number (CN)-altered genome (CN-AG) in the carcinogenesis of cervical cancer (CC), especially its effect on gene expression, biological processes, and patient survival. Fifty-nine human papillomavirus 16 (HPV16)-positive CCs were investigated with microarrays-31 for mapping CN-AG and 55 for global gene expression, with 27 CCs in common. Five-year survival was investigated in 55 patients. Deletions and amplifications >2.5 Mb were defined as CN alterations. The %CN-AG varied from 0 to 32.2% (mean = 8.1±8.9). Tumors were classified as low (mean = 0.5±0.6, n = 11), medium (mean = 5.4±2.4, n = 10), or high (mean = 19.2±6.6, n = 10) CN. The highest %CN-AG was found in 3q, which contributed an average of 55% of all CN alterations. Genome-wide, only 5.3% of CN-altered genes were deregulated directly by gene dosage. In contrast, the rate in fully duplicated 3q was twice as high. Amplification of 3q explained 23.2% of deregulated genes in whole tumors (r2 = 0.232, p = 0.006; analysis of variance), including genes located in 3q and other chromosomes. A total of 862 genes were deregulated exclusively in high-CN tumors, but only 22.9% were CN altered. This suggests that the remaining genes are not deregulated directly by gene dosage, but by mechanisms induced in trans by CN-altered genes. Anaphase-promoting complex/cyclosome (APC/C)-dependent proteasome proteolysis, glycolysis, and apoptosis were upregulated, whereas cell adhesion and angiogenesis were downregulated exclusively in high-CN tumors. The high %CN-AG and upregulated gene expression profile of APC/C-dependent proteasome proteolysis were associated with poor patient survival (p<0.05, log-rank test). Along with glycolysis, they were linearly associated with FIGO stage (r>0.38, p<0.01, Spearman test). Therefore, inhibition of APC/C-dependent proteasome proteolysis and glycolysis could be useful for CC treatment. However, whether they are indispensable for tumor growth remains to be demonstrated

DAVID functional annotation cluster analysis in the 2006 genes differentially expressed in cervical cancer.

<p>*The cluster number was obtained when the analysis was run with the whole gene set, including up- and down-regulated genes.</p><p>FC = Fold change is the ratio of the proportion of genes in the tested list versus the Human Gene Reference database.</p><p>NC =  No clustered in up (+) and down (−) regulated genes analyzed separately.</p><p>The clusters in italics were enriched when the functional annotation cluster analysis was run at the highest stringency, and the number inside the parenthesis indicated the order the cluster occupied in the list.</p

Patients followed up on average for 63 months for survival evaluation.

a<p>ACC, Adenocarcinoma. SCC, Squamous Cell Carcinoma. ASCC, Adenosquamous Cell Carcinoma.</p><p>bHT, Radical Hysterectomy. Tele, teletherapy. Brachy, brachytherapy. Chemo, chemotherapy with Cisplatin. i. Means incomplete treatment.</p>c<p>Status alive at the last follow up record and death was caused by primary tumor of cervical cancer, except the case labeled with an asterisk. The cause of death was unknown.</p>d<p>CN indicate the samples analyzed for CN (500 K array), CN/EX indicate the samples analyzed for CN (500 K array) and gene expression (HG 1.0 ST array), and EX indicate the samples analyzed for gene expression (HG 1.0 ST).</p

Survival analysis of women with CC according to International Federation of Gynecology and Obstetrics (FIGO) staging, CN-AG, and gene expression profiles.

<p>The Kaplan-Meier curves for FIGO staging, the whole and 3q %CN-AG, and gene expression profiles of genes involved in glycolysis and APC/C-dependent proteasomal protein catabolic process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097842#pone-0097842-g009" target="_blank">Figure 9</a>) are shown. Patients were followed up an average of 63 months. The p value was calculated by comparing the curves with the log-rank test. Censored patients are labeled with transverse lines.</p

Identification of CN-altered genes in biological processes enriched in high-CN tumors.

<p>Shown are the numbers of 2-copy or CN-altered genes in ≤3 tumors (green bars) and CN-altered genes in ≥4 tumors (blue bars) among the biological processes enriched in the subset of genes deregulated exclusively in high-CN tumors.</p

Segregation of tumors and control samples according to gene expression profile.

<p>Unsupervised hierarchical cluster analysis of 55 CCs and 17 healthy cervical epithelium samples using the expression values of the genes deregulated from glycolysis (panel A) and the anaphase-promoting complex/cyclosome (APC/C)-dependent proteasomal protein catabolic process (panel B) obtained with the HG 1.0 ST microarray. Each row represents a gene and each column represents a sample. Samples name beginning with an “R” are CCs and with a “C” are controls; CCs ending in 1, 2 or 3 belong to low-, medium- or high-CN groups, respectively, whereas those ending with no number were not explored for CN. The length and the subdivision of the branches represent the relationships among the samples based on the intensity of gene expression. The cluster is color-coded using red for upregulation, blue for downregulation, and white for unchanged expression. In panel B, the sub-branches enclosed in squares were considered together in a group with a strong upregulation profile and the remaining upregulated samples were placed in another group with a weak upregulation profile.</p

Amount of copy number (CN)-altered genome and frequency of recurrent CN-altered genes.

<p>Panel A shows box plots with the distribution of tumors (n = 31) according to the percentage of total, deleted, or amplified CN-altered genome (CN-AG). Panel B shows the distribution of tumors, grouped as low (n = 11), medium (n = 10), and high (n = 10) according to the percentage of global and 3q CN-AG. The horizontal lines inside the boxes represent the median (solid) and average (dotted), and the whiskers represent the minimum and maximum values within the 1.56 interquartile range from the end of the box. Values outside this range are represented by black circles. The decline in the accumulated frequency of recurrent CN-altered genes, as increase the number of tumors that shared the same altered gene, is shown for the whole genome (Panel C) or for chromosomes with significant high %CN-AG (Panel D). Combined genes are those that were deleted in some and amplified in other tumors.</p

DAVID functional analysis of genes correlated and non-correlated with 3q amplification in high-CN tumors.

<p>See the legends in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097842#pone-0097842-t003" target="_blank">Table 3</a>.</p

Comparison between CN and gene expression by chromosome arm.

<p>The left side shows the explored genome (Mb) in each chromosome arm that was explored with the 500 K microarray in 31 tumors. The right side shows the number of genes on each arm explored with the Human Gene 1.0 ST microarray in 27 of the tumors in which CN was examined. Each bar represents the percentage of CN-AG (left) or deregulated genes (right) of the chromosomal arm indicated in the middle. Red bars indicate gains or overexpression and blue bars represent losses or subexpression. The dotted line represents the average percentage of the global CN-AG (8.1%; left) or the percentage of deregulated genes (9.5%; right) in the tumor genome. The arms marked with asterisks had an average proportion of CN-AG or percentage of deregulated genes superior and statistically significant compared to the numbers found in the complete tumor genome (p<0.05, chi-square).</p