Unknown,Transcriptomics,Genomics,Proteomics

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Mitosis is the main pathway altered in cervical cancer and is a source of potential markers for screening and survival, and therapeutic targets.

ABSTRACT: The effect of preventive vaccination against Human Papillomavirus (HPV) in reducing the burden of cervical cancer (CC) will not be seen before 30 years. It is still necessary to improve the procedures of screening and treatment against CC. Current methods for screening have low sensitivity (Pap test) or specificity (HPV tests) to detect high-grade cervical intraepithelial neoplasias (CIN) and CC (CIN2+). The objective of this study was the identification and characterization of cellular targets present in most CC and absent in normal cervical tissue that can be considered as potential markers for screening or therapeutic targets. Methods and Findings. A pyramidal strategy was used. Initially the expression of 8,638 genes was compared between 43 HPV16-positive CC and 12 healthy cervical epitheliums using the HG-Focus microarray. The expression intensity of 8,370 genes was validated in 24 samples with a second microarray (HG-ST1.0). A total of 997 genes were deregulated in CC and the 21 best ranked were validated and confirmed by qRT-PCR in 67 CC and 25 controls. According to the ROC analysis and the fold change (FC), the best (AUC M-bM-^IM-% 0.97 and FC M-bM-^IM-% 10) six genes (CCNB2, CDC20, PRC1, SYCP2, NUSAP1, CDKN3) belong to the mitosis pathway. They were further explored by qRT-PCR in 29 CIN1 and 21 CIN2/3 lesions to investigate whether they could differentiate CIN2+ from CIN1 and healthy cervical epitheliums (CIN1-). Three of these genes were associated exclusively with invasive tumors (CCNB2, PRC1, SYCP2) and three (CDC20, NUSAP1, CDKN3) also with CIN2/3. The sensitivity and specificity of CDKN3 and NUSAP1, along with CDKN2A, to detect CIN2+ was around 90%. The protein codified by these genes was confirmed by immunohistochemistry in CC. The effect of these markers on the survival was investigated in 40 CC patients followed by 42 months. Only high-expression level of CDKN3 was associated with a poor survival (20%; p = 2 x 10-4, Long-rank test) and it was independent from FIGO staging. Conclusions. CDKN3 and NUSAP1, together with CDKN2A, may be potential targets for development of screening methods. However, further studies are needed from a screening population to define the optimal trade-off between sensitivity and specificity for detection of CIN2+. CDKN3 may be also considered as a survival marker. Inhibition of mitosis is a well-known strategy to combat cancers. Therefore, CDKN3 may be a potential therapeutic target in CC. However, itM-BM-4s still necessary to demonstrate whether itM-BM-4s indispensable for tumor growth. cervical cancer vs. healthy cervix

ORGANISM(S): Homo sapiens

SUBMITTER: Ana MarM-CM--a Espinosa

PROVIDER: E-GEOD-39001 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The effect of preventive vaccination against Human Papillomavirus (HPV) in reducing the burden of cervical cancer (CC) will not be seen before 30 years. It is still necessary to improve the procedures of screening and treatment against CC. Current methods for screening have low sensitivity (Pap test) or specificity (HPV tests) to detect high-grade cervical intraepithelial neoplasias (CIN) and CC (CIN2+). The objective of this study was the identification and characterization of cellular targets present in most CC and absent in normal cervical tissue that can be considered as potential markers for screening or therapeutic targets. Methods and Findings. A pyramidal strategy was used. Initially the expression of 8,638 genes was compared between 43 HPV16-positive CC and 12 healthy cervical epitheliums using the HG-Focus microarray. The expression intensity of 8,370 genes was validated in 24 samples with a second microarray (HG-ST1.0). A total of 997 genes were deregulated in CC and the 21 best ranked were validated and confirmed by qRT-PCR in 67 CC and 25 controls. According to the ROC analysis and the fold change (FC), the best (AUC ≥ 0.97 and FC ≥ 10) six genes (CCNB2, CDC20, PRC1, SYCP2, NUSAP1, CDKN3) belong to the mitosis pathway. They were further explored by qRT-PCR in 29 CIN1 and 21 CIN2/3 lesions to investigate whether they could differentiate CIN2+ from CIN1 and healthy cervical epitheliums (CIN1-). Three of these genes were associated exclusively with invasive tumors (CCNB2, PRC1, SYCP2) and three (CDC20, NUSAP1, CDKN3) also with CIN2/3. The sensitivity and specificity of CDKN3 and NUSAP1, along with CDKN2A, to detect CIN2+ was around 90%. The protein codified by these genes was confirmed by immunohistochemistry in CC. The effect of these markers on the survival was investigated in 40 CC patients followed by 42 months. Only high-expression level of CDKN3 was associated with a poor survival (20%; p = 2 x 10-4, Long-rank test) and it was independent from FIGO staging. Conclusions. CDKN3 and NUSAP1, together with CDKN2A, may be potential targets for development of screening methods. However, further studies are needed from a screening population to define the optimal trade-off between sensitivity and specificity for detection of CIN2+. CDKN3 may be also considered as a survival marker. Inhibition of mitosis is a well-known strategy to combat cancers. Therefore, CDKN3 may be a potential therapeutic target in CC. However, it´s still necessary to demonstrate whether it´s indispensable for tumor growth.

Project description:Cervical cancer develops from pre-cancerous high-grade cervical intraepithelial neoplasia (CIN) lesions harbouring a transforming infection with high-risk human papillomavirus (hrHPV), which is characterised by p16INK4a overexpression. Since it takes one or more decades for these pre-cancerous lesions to progress to invasive squamous cell carcinomas (SCCs), it is obvious that they are heterogeneous in terms of duration of existence and progression risk. We performed array-based comparative genomic hybridisation (array CGH) of 46 p16INK4a immuno-positive CIN2/3 lesions to determine whether this heterogeneity is reflected in their chromosomal profiles. Chromosomal profiles of CIN2/3 lesions were related to those of invasive cervical squamous cell carcinomas (SCCs) and promoter methylation of the CADM1 gene, a tumour suppressor gene known to be functionally involved in the tumorigenic phenotype of cervical cancer cells. Frequent alterations found in CIN2/3 lesions included gains located at chromosome 1, 3, 7 and 20 and losses located at 4, 11, 16, 17 and 19. Unsupervised hierarchical clustering identified two subsets of CIN2/3 lesions, chromosomal profiles of one of which closely resembled invasive SCCs. Gains of 1, 3q and 20 were characteristic for CIN2/3 lesions with chromosomal signatures resembling carcinomas. In addition, dense promoter methylation of the CADM1 gene was significantly more frequent in these CIN2/3 lesions (p=0.004). No chromosomal alterations were detected in one p16INK4a positive and five p16INK4a negative CIN1 lesions. These findings suggest that biomarkers associated with gains at chromosomes 1, 3q and 20 are potential hallmarks of advanced p16INK4a positive CIN2/3 lesions with a high short-term risk of progression. Genomic DNA of 46 high-grade cervical lesions was hybridised to 5K CGH BAC microarrays (5K) produced at the Microarray facility of the VU Medical Center. In total 4632 BAC clones with known chromosomal location were spotted in triplicate, which included the 1Mb resolution Sanger BAC clone set and a subset of clones from the Children’s Hospital Oakland Research Institute (CHORI). All samples were labelled with Cy3 and hybridised together with a pool of normal male reference DNA labelled with Cy5. Hybridisations were essentially performed as described by Snijders et al (Nature Genetics 2001). Both pre-hybridisation and hybridisation were performed in a hybridisation station (HybStation12 – Perkin Elmer Life Sciences). Hybridised arrays were scanned using a G2505B scanner (Agilent, Wilmington, DE, USA). Spots were quantified using ImaGene 5.6.1 software (BioDiscovery Ltd, Marina del Rey, CA, USA) with default settings for the flagging of bad quality spots. Genomic DNA of 6 CIN1 lesions was hybridised on CGH oligo microarrays (44K) produced by Agilent following the manufacturer’s protocol against the same normal reference pool. Hybridised arrays were scanned using the same scanner (G2505B, Agilent). Quantification of these arrays was done using Feature Extraction software version 9.5.1 (Agilent).

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Mitosis is the main pathway altered in cervical cancer and is a source of potential markers for screening and survival, and therapeutic targets.

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