Amplified Genes May Be Overexpressed, Unchanged, or Downregulated in Cervical Cancer Cell Lines

Several copy number-altered regions (CNAs) have been identified in the genome of cervical cancer, notably, amplifications of 3q and 5p. However, the contribution of copy-number alterations to cervical carcinogenesis is unresolved because genome-wide there exists a lack of correlation between copy-number alterations and gene expression. In this study, we investigated whether CNAs in the cell lines CaLo, CaSki, HeLa, and SiHa were associated with changes in gene expression. On average, 19.2% of the cell-line genomes had CNAs. However, only 2.4% comprised minimal recurrent regions (MRRs) common to all the cell lines. Whereas 3q had limited common gains (13%), 5p was entirely duplicated recurrently. Genome-wide, only 15.6% of genes located in CNAs changed gene expression; in contrast, the rate in MRRs was up to 3 times this. Chr 5p was confirmed entirely amplified by FISH; however, maximum 33.5% of the explored genes in 5p were deregulated. In 3q, this rate was 13.4%. Even in 3q26, which had 5 MRRs and 38.7% recurrently gained SNPs, the rate was only 15.1%. Interestingly, up to 19% of deregulated genes in 5p and 73% in 3q26 were downregulated, suggesting additional factors were involved in gene repression. The deregulated genes in 3q and 5p occurred in clusters, suggesting local chromatin factors may also influence gene expression. In regions amplified discontinuously, downregulated genes increased steadily as the number of amplified SNPs increased (p<0.01, Spearman's correlation). Therefore, partial gene amplification may function in silencing gene expression. Additional genes in 1q, 3q and 5p could be involved in cervical carcinogenesis, specifically in apoptosis. These include PARP1 in 1q, TNFSF10 and ECT2 in 3q and CLPTM1L, AHRR, PDCD6, and DAP in 5p. Overall, gene expression and copy-number profiles reveal factors other than gene dosage, like epigenetic or chromatin domains, may influence gene expression within the entirely amplified genome segments

Mitosis Is a Source of Potential Markers for Screening and Survival and Therapeutic Targets in Cervical Cancer

<div><p>The effect of preventive human papillomavirus (HPV) vaccination on the reduction of the cervical cancer (CC) burden will not be known for 30 years. Therefore, it’s still necessary to improve the procedures for CC screening and treatment. The objective of this study was to identify and characterize cellular targets that could be considered potential markers for screening or therapeutic targets. A pyramidal strategy was used. Initially the expression of 8,638 genes was compared between 43 HPV16-positive CCs and 12 healthy cervical epitheliums using microarrays. A total of 997 genes were deregulated, and 21 genes that showed the greatest deregulation were validated using qRT-PCR. The 6 most upregulated genes (<em>CCNB2, CDC20, PRC1, SYCP2, NUSAP1</em>, <em>CDKN3</em>) belong to the mitosis pathway. They were further explored in 29 low-grade cervical intraepithelial neoplasias (CIN1) and 21 high-grade CIN (CIN2/3) to investigate whether they could differentiate CC and CIN2/3 (CIN2+) from CIN1 and controls. <em>CCNB2</em>, <em>PRC1</em>, and <em>SYCP2</em> were mostly associated with CC and <em>CDC20</em>, <em>NUSAP1</em>, and <em>CDKN3</em> were also associated with CIN2/3. The sensitivity and specificity of <em>CDKN3</em> and <em>NUSAP1</em> to detect CIN2+ was approximately 90%. The proteins encoded by all 6 genes were shown upregulated in CC by immunohistochemistry. The association of these markers with survival was investigated in 42 CC patients followed up for at least 42 months. Only <em>CDKN3</em> was associated with poor survival and it was independent from clinical stage (HR = 5.9, 95%CI = 1.4–23.8, p = 0.01). <em>CDKN3</em> and <em>NUSAP1</em> may be potential targets for the development of screening methods. Nevertheless, further studies with larger samples are needed to define the optimal sensitivity and specificity. Inhibition of mitosis is a well-known strategy to combat cancers. Therefore, <em>CDKN3</em> may be not only a screening and survival marker but a potential therapeutic target in CC. However, whether it’s indispensable for tumor growth remains to be demonstrated.</p> </div

ROC analysis of 4 gene markers selected for detection of CIN2/3 and CC.

<p>See legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055975#pone-0055975-t003" target="_blank">Table 3</a>.</p><p>The last 4 rows included the combined analysis of CDKN3, NUSAP1, CDC20 and CDKN2A as indicated. Samples were considered positive when at least 2 of the 3 markers were positive.</p>a<p>All comparisons gave a p-value <1×10⁻⁹, chi square.</p

Genes explored by qRT-PCR.

a<p>Genes in bold were selected to be explored in pre-invasive samples.</p>b<p>The analysis was performed with 44 HPV16-positive CC, 22 CC positive for other HPVs and 25 cervical controls. Fold change (FC) was calculated with the median values as follows: tumor/control for upregulated genes and control/tumor for downregulated genes (see Materials and Methods). The difference between the groups was statistically significant (p<1×10⁻¹⁵; Mann-Whitney Rank Sum Test) for all but 2 genes (NDN, SLC18A2). NDN and SLC18A2 had a p>0.05.</p>c<p>Included carcinomas positives for HPV-18 (5), -31 (5), -33 (2), -45 (5), -51 (2), -58 (2) and -59 (1).</p

Patients followed up for at least 42 months for survival evaluation.

a<p>ACC, Adenocarcinoma. SCC, Squamous Cell Carcinoma. ASCC, Adenosquamous Cell Carcinoma.</p>b<p>HT, Radical Hysterectomy. Tele, teletherapy. Brachy, brachytherapy. Chemo, chemotherapy with Cisplatin.</p>c<p>Status alive was registered at the last follow up, death was caused by primary tumor of cervical cancer, except the case labeled with an asterisk, and unknown cases were lost during the follow up study. The cause of death of case labeled with an asterisk was unknown.</p

Correlation of expression intensity of 23 genes examined by HG-Focus and HG-ST1.0 microarrays.

<p>The Log₂ values of the standardized intensity signals (RMA values) of 23 genes examined by the 2 microarrays in 19 CC and 5 normal cervical epithelium were plotted. The linear trend (black line) is included, which was calculated with Person’s correlation test. r =  correlation coefficient, p =  p-value.</p

Distribution of deregulated genes according to the fold change (FC) and Δ-score values.

<p>All 997 genes (circles) that were deregulated in cervical cancer (CC) tumors compared to the control samples by the SAM method are graphed in a Volcano plot. The x-axis represents the FC in gene expression (cancer sample/control sample) expressed in Log2 and the y-axis display the absolute Δ-score, a modified t-test calculated with the SAM method, the higher the Δ-score values, the higher the statistical significance. The Log2 (FC) values are positive for upregulated genes and negative for downregulated genes. Circles colored in red and orange represent the genes involved in M-phase of the cell cycle and those colored in blue and orange are the genes that were validated by qRT-PCR.</p