Role In Ovarian Carcinogenesis Research Articles

Polymorphisms of the Interleukin-1 Gene Cluster and Ovarian Cancer

Interleukin-1 (IL-1) is known to be critically involved in ovarian carcinogenesis. We investigated a panel of polymorphisms of the IL-1 and the IL-1 receptor antagonist (IL-1 RA) genes in patients with ovarian cancer. One hundred thirty-four patients with surgically staged ovarian cancer, 27 patients with borderline ovarian cancer, and 134 healthy controls were genotyped for three polymorphisms of the IL-1 gene (-889 C/T polymorphism of the IL-1alpha gene [IL1A], -511 C/T polymorphism of the IL-1beta promoter [IL1B promoter], a polymorphism of IL-1beta exon 5 [IL1B exon 5]), and an 86-base pair repeat in intron 2 of the IL-1 RA gene [IL1RN]) using polymerase chain reaction and pyrosequencing. Allelic frequencies did not differ between patients with ovarian cancer and controls. In the ovarian cancer group, polymorphism did not correlate with any of the investigated clinicopathologic variables, including tumor stage, lymph node, and histologic grade. In univariate and multivariate models, there was no correlation between any polymorphism and patients' overall and disease-free survival. We investigated interleukin polymorphisms in ovarian cancer but did not find any association between common polymorphisms of IL1A, IL1B, and IL1RN and the occurrence of ovarian cancer. Allelic variation within the IL-1 gene clusters does not seem to play a role in ovarian carcinogenesis and does not appear to be a useful tool for possible screening and risk evaluation.

Inactivation of the p16INK4A gene by methylation is not a frequent event in sporadic ovarian carcinoma.

The p16INK4A tumour suppressor gene (p16) encodes for a cyclin-dependant kinase inhibitor which plays a role in the phosphorylation of the retinoblastoma protein (pRb) during regulation of the G1-S phase of the cell cycle. Loss of heterozygosity at 9p21, the chromosomal location of the p16 gene, has been reported in a broad range of tumours and this is usually indicative of the presence of a tumour suppressor gene, the second allele being frequently inactivated by mutation or deletion. The p16 gene, however, is often found not be mutated or deleted and it has been suggested that hypermethylation of the CpG islands of the gene may be an alternative mechanism of gene inactivation. We sought to determine the levels of p16 abnormality in a series of epithelial ovarian carcinomas in an attempt to clarify the presently conflicting evidence of whether or not hypermethylation of the p16 gene plays a role in ovarian carcinogenesis. We analysed 57 primary ovarian tumours and their corresponding blood DNA using SSCP analysis, sequencing and the methylation specific PCR (MSP) technique. We found low levels of mutation (6.7% of malignant tumours) and no evidence of methylation in any of our samples, suggesting that neither mutation or hypermethylation of the CpG islands of the p16 gene play an important role in ovarian carcinogenesis.

Frequent Loss of PTEN Expression Is Linked to Elevated Phosphorylated Akt Levels, but Not Associated with p27 and Cyclin D1 Expression, in Primary Epithelial Ovarian Carcinomas

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.

Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.

Focal adhesion kinase (FAK) is a tyrosine kinase that is important to such key functions such as cell adhesion, motility, and invasion. A MEDLINE search of the years 1980-1998 found no previous reports of FAK expression in human ovarian carcinoma. The authors performed experiments to determine whether FAK expression is elevated in this disease. Ten normal human ovarian tissue samples and 26 cancer samples from patients with Stage I-IV ovarian carcinoma were obtained. Two ovarian carcinoma cell lines were also analyzed. FAK expression was determined by Western blot analysis with the V39 anti-human FAK polyclonal antibody. The level of FAK protein expression was determined using densitometric scanning of the 125 kD band on autoradiographs of Western immunoblots. Serous cancers expressed fourfold-increased values of FAK relative to normal ovarian tissue (P < 0.0001), and nonserous adenocarcinomas expressed threefold- to fourfold-increased values of FAK (P < 0. 0006). Ovarian carcinoma cell lines also expressed increased values of FAK. With a cutoff of 40, an elevated FAK level was associated with a sensitivity of 93% and specificity of 100%. There was no significant difference in FAK expression with regard to grade or stage of tumor. FAK is significantly overexpressed in ovarian carcinoma, implying that FAK may play an important role in ovarian carcinogenesis. FAK expression may be useful as a screening tool to identify newly developed disease or as a tumor marker in confirmed cases of epithelial ovarian carcinoma. FAK may also serve as a potential target for therapeutic disruption of ovarian carcinoma progression.

Altered expression of transforming growth factor-beta ligands and receptors in primary and recurrent ovarian carcinoma.

Resistance to the potent growth inhibitory effects of transforming growth factor-beta (TGF-beta) is a characteristic of many malignancies. TGF-beta insensitivity has been attributed to alterations in the number and function of the TGF-beta receptors as well as disturbances of downstream signal transduction. Paradoxically, increased levels of TGF-beta ligand have been demonstrated in several types of malignant tumors. TGF-beta also may play a role in ovarian carcinogenesis; however, the nature of this interaction has yet to be defined completely. To explore the potential role of TGF-beta-mediated autocrine and paracrine influences in epithelial ovarian carcinoma, mRNA expression levels of the three TGF-beta ligand isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and the three TGF-beta receptors (TbetaR-I, T/betaR-II, and TbetaR-III) were examined by Northern blot analysis in both primary and recurrent ovarian carcinoma specimens. Immunohistochemical analysis was performed to localize expression of TbetaR-I and TbetaR-II, whereas the presence of genetic alterations in TbetaR-1 was examined through Southern blot analysis. Compared with normal ovarian tissue, both primary and recurrent ovarian carcinomas demonstrated significant overexpression of the TGF-beta1 and TGF-beta3 mRNA transcripts. TGF-beta2 expression was detectable in 75% of primary and only 53% of recurrent tumor specimens. Alterations also were detected in TbetaR mRNA expression. Expression levels of TbetaR-III were significantly reduced in both primary and recurrent ovarian carcinomas. Furthermore, detectable levels of TbetaR-I and TbetaR-III mRNA transcripts were present in only 47% and 50% of recurrent ovarian tumors, respectively. Immunohistochemical staining demonstrated that TbetaR-I and TbetaR-II expression localized to tumor cells; however, receptor staining in stromal tissue also was detected. Southern blot analysis of TbetaR-I did not reveal any major genetic changes to account for the absence of TbetaR-I expression. Alterations in expression of TGF-beta ligands and receptors consistently were greater in recurrent ovarian carcinomas compared with primary tumors, and may reflect a phenotype that promotes tumor recurrence or chemoresistance. Together, these data suggest that enhanced expression of TGF-betaI and TGF-beta3, as well as the loss of expression of TbetaR-I and TbetaR-III, contribute to ovarian carcinogenesis and/or tumor progression.

DOC-2, a candidate tumor suppressor gene in human epithelial ovarian cancer.

Using RNA fingerprinting (RAP) strategy and Northern blot analysis, we identified a differentially expressed sequence DOC-2 which is detectable in all normal human ovarian surface epithelial (HOSE) cell cultures but not in ovarian cancer cell lines and tissues. Subsequent cloning of DOC-2 from a cDNA library generated from the HOSE cells was carried out using the 3' and 5' RACE approach. A 3268 base pair full length cDNA of DOC-2 was isolated and sequenced. The predicted protein has a length of 770 amino acids. Homology search of all NCBI sequences indicated that the amino acid sequence of DOC-2 shares 93% homology with the mouse p96/mDab2 phosphoprotein and has a phosphotyrosine interacting domain (PID) and multiple SH3 binding motifs. Chromosomal localization by FISH showed that the DOC-2 gene is located on 5p13. Western blot analysis showed that the 105 kDa DOC-2 protein was down-regulated in all the carcinoma cell lines. In-situ immunohistochemistry performed on normal ovaries, and benign, borderline and invasive ovarian tumor tissues showed down regulation of DOC-2 protein particularly in serous ovarian tumor tissues. When DOC-2 was transfected into the ovarian carcinoma cell line SKOV3, the stable transfectants showed significantly reduced growth rate and ability to form tumors in nude mice. These data suggest that down-regulation of DOC-2 may play an important role in ovarian carcinogenesis.

No Evidence Exists for Methylation Inactivation of thep16Tumor Suppressor Gene in Ovarian Carcinogenesis

Thep16ink4/CDKN2/MTS1tumor suppressor gene encodes a cyclin-dependent kinase inhibitor which plays an important role in regulation of the G1/S phase cell cycle checkpoint. Loss of heterozygosity (LOH) at thep16locus, 9p21, has been documented in a wide variety of tumors including ovarian carcinoma. However, inactivating mutations of the remaining allele and homozygous deletions are relatively infrequent events in primary tumors, even in cases where expression of p16 at the mRNA and protein level is clearly absent. These findings initially cast doubt on the role ofp16as a tumor suppressor genein vivo. Recently, an alternative mechanism of p16 inactivation involving methylation of the CpG island in the 5′ region of the gene has been demonstrated in a number of malignancies and cell lines. In this study we have analyzed the methylation status of four CpG dinucleotides in a panel of 23 ovarian tumors using a multiplex PCR approach to correlate our findings with the LOH data in this region. Using the microsatellite markers D9S171 and D9S1679 LOH was demonstrated in 4/22 (18%) informative cases. All 23 tumors showed no evidence of methylation at thep16locus including the 4 tumors demonstrating LOH at 9p21. These results suggest that methylation inactivation of thep16gene does not play an important role in ovarian carcinogenesis.

Human ovarian cancer of the surface epithelium

Epidemiologic studies have shown that the risk of cancer in the ovarian surface epithelium is decreased by factors that suppress ovulation, whereas uninterrupted ovulation has been associated with increased risk. This suggests that ovulation may play a critical role in ovarian carcinogenesis. More recently, molecular studies have demonstrated alterations in specific oncogenes and tumor suppressor genes in ovarian cancers. Overexpression of the HER-2/ neu oncogene occurs in approximately 30% of ovarian cancers and correlates with poor survival. Although mutation of the K- ras oncogene has been found in some mucinous ovarian cancers, mutations in this gene appear to be more common in borderline ovarian tumors. Amplification of c- myc occurs in approximately 30% of ovarian cancers and is more frequently seen in serous cancers. Mutation of the p53 tumor suppressor gene, with resultant overexpression of mutant p53 protein, occurs in 50% of stage III/IV and 15% of stage 1/11 ovarian cancers. Most p53 mutations in ovarian cancers are transitions, which suggests that they arise spontaneously rather than due to exogenous carcinogens. In contrast to the acquired genetic alterations described above that are a feature of sporadic ovarian cancers, 5–10% of ovarian cancers probably arise due to inherited genetic defects. Recently, the BRCA1 tumor suppressor gene has been identified and shown to be responsible for most cases of hereditary ovarian cancer. Further studies are needed to augment our understanding of the molecular pathogenesis of ovarian cancer.

Absence of Prohibitin Gene Mutations in Human Epithelial Ovarian Tumors

Multiple loss of heterozygosity (LOH) studies of ovarian cancers have found a high incidence of chromosome 17 loss in these tumors. Several authors have suggested that the region commonly deleted encompasses 17q12-21. In addition, this region has recently been reported to be linked to the familial breast/ovarian cancer syndrome. Recently the human prohibitin gene was mapped to region 17q12-22. Prohibitin causes arrest of DNA synthesis by fibroblast and HeLa cells and prohibitin shows significant homology to a gene (Cc) thought to be important for the regulation of development of Drosophila melanogaster. These findings have led many to consider the prohibitin gene a potential tumor suppressor gene. In addition, sequence analysis of exon 4 of human prohibitin gene revealed mutations in 4 of 23 sporadic breast carcinomas. Because of the proposed function for prohibitin, its alterations in breast cancers, and the fact that its location on 17q falls within a commonly deleted region in ovarian cancers, we have undertaken an analysis of the sequence of prohibitin in epithelial ovarian cancers. Using several polymorphic DNA probes, we identified 20 epithelial ovarian tumors which demonstrated LOH for the region that contains the prohibitin gene. To evaluate whether mutations of prohibitin may be important in ovarian carcinogenesis, we have sequenced exons 4 and 5 of this gene using the technique of genomic amplification with transcript sequencing. Only normal exon 4 and 5 sequence was observed among the 20 tumors screened. These results demonstrate that this region of the prohibitin gene is not mutated in epithelial ovarian cancers and suggest that the prohibitin gene does not play a role in ovarian carcinogenesis. Sequencing of further exons and introns are needed to confirm this latter hypothesis.