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

Validation of gene expression of 9 genetic markers by qRT-PCR.

<p>The intensity of gene expression, expressed in Log2 values, is shown in box plots. Expression of the 6 genes validated in this study (<i>CCNB2</i>, <i>PRC1</i>, <i>SYCP2</i>, <i>CDKN3</i>, <i>CDC20</i>, and <i>NUSAP1</i>) and the 3 well-known genes (<i>CDKN2A</i>, <i>MKI67</i>, and <i>PCNA</i>) associated with CC are compared among the 4 groups, including healthy cervical epitheliums (Normal, n = 25), low-grade CIN (CIN1, n = 29), high-grade CIN (CIN2/3, n = 21), and invasive CC (cancer, n = 44). The upper and lower boundaries of the boxes represent the 75^th and 25^th percentiles, respectively. The black line within the box represents the median value, and the whiskers represent the minimum and maximum values that lie within 1.5× the interquartile range from the end of box. Values outside this range are represented by black circles. The fold change (FC) was calculated by dividing the median of each pathological group by the median of the control group.</p

Survival analysis of women with cervical cancer according to FIGO staging and <i>CDKN3</i> expression.

<p>The Kaplan-Meier curves for FIGO staging and <i>CDKN3</i> are shown. Patients were followed-up for at least 42 months. For gene expression, cancer patients with higher (red line) and lower (blue line) fold change values were compared (see material and methods). The p value was calculated by comparing the curves with the log-rank test. Censored patients are labeled with black dots, but only four of them were censored before the minimal period of follow-up (42 months).</p

Segregation of tumor and control samples according to the expression of deregulated genes.

<p>Unsupervised hierarchical cluster analysis of 43 CCs and 12 healthy cervical epitheliums using the expression values obtained with the HG-Focus microarray of all 997 deregulated genes (panel A) or the 23 top ranked genes selected for validation (panel B). Each row represents a gene and each column represents a sample. 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, green for downregulation, and black for unchanged expression. Panel C shows the principal components analysis (PCA) using the values in panel B; blue circles represent the CCs (n = 43) and yellow circles represent the controls (n = 12). Both sets of genes clearly separated the samples into the 2 main groups using both types of analysis.</p

DAVID functional annotation cluster analysis at the highest stringency of the 100 genes most deregulated in cervical cancer compared with normal cervical epithelium^*.

*<p>Enrichment Score is the -log₁₀ of the average p-value of the terms in the cluster. Fold change is the ratio of the proportion of genes in the tested list versus the Human Gene Reference database.</p

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

Histological analysis of marker genes.

<p>Protein expression was determined by immunohistochemistry using sections from formalin-fixed, paraffin-embedded tissue. Proteins explored were CDKN3A, SYCP2, PRC1, CDC20, CCNB2, PCNA, CDKN2A, MKI67, and CDC2. Representative experiments in adeno cell carcinomas (panel A) and squamous cell carcinomas (panel B) are shown. The specific signals are shown as brown staining (counterstained with hematoxylin; original magnification, ×400; bars, 10 µm).</p