Overexpression of kallikrein 10 in epithelial ovarian carcinomas.

OBJECTIVE: We wanted to identify genes up-regulated in ovarian tumors that might serve as early markers for ovarian cancer. One promising focus is the family of genes that encode secreted proteases, which play essential roles in tumor invasion and metastasis. Kallikrein 10 (KLK10) is a member of the kallikrein family of serine proteases which include 15 proteins, including the prostate-specific antigen (PSA), also known as hK3. We investigated whether KLK10 and its related protein, hK10, might serve as equally accurate markers for ovarian carcinoma. METHODS: Transcriptional profiling was performed using RNA isolated from normal ovarian epithelium, ovarian cancer cell lines, and primary ovarian tumors. Microarray data were confirmed by Northern blot analysis of 66 ovarian tumor samples and 6 ovarian cancer cell lines. In situ hybridization and Western blot analysis confirmed the Northern blot findings. RESULTS: KLK10 was more highly expressed by primary ovarian tumors than by NOE. Thirty-two of 35 primary serous ovarian carcinoma samples (91.4%) expressed higher levels of KLK10 than NOE did. Eleven of 15 nonserous epithelial ovarian carcinoma samples (73.3%) and 8 of 11 primary peritoneal carcinoma samples (72.7%) also expressed KLK10. Overall, 84.8% of all epithelial ovarian and peritoneal carcinoma tumor samples showed elevated expression of KLK10. Similarly, Western blot analysis demonstrated that levels of the KLK10-related protein, designated hK10, are elevated in primary ovarian tissue lysates, but the protein is undetectable in immortalized ovarian epithelium. Finally, in situ hybridization established that KLK10 mRNA is much more highly expressed by tumor tissue than by normal epithelium and stromal tissues. CONCLUSIONS: Our data support recent immunoassay findings of elevated levels of hK10 in the tumor tissue and serum of ovarian cancer patients. Thus, it is likely that KLK10 and other kallikreins will serve as useful diagnostic and prognostic markers in patients with ovarian carcinoma.

Transcriptional profiling reveals that several common fragile-site genes are downregulated in ovarian cancer.

Previous transcriptional profiling analysis of 14 primary ovarian tumors identified approximately 12,000 genes as decreased in expression by at least twofold in one or more of the tumors sampled. Among those genes were several known to be mapped to common fragile sites (CFSs), some of which had previously been shown to exhibit a loss of expression in ovarian carcinoma. Therefore, we selected a subset of genes to determine whether they localized within CFSs. Of the 262 genes that were downregulated at least twofold in 13 of 14 tumors, 10 genes were selected based on the following criteria: localization to a CFS band; documented aberrations in at least one malignancy; and feasibility of scoring breakage at the specific CFS. Fluorescence in situ hybridization analysis was performed using bacterial artificial chromosome clones encompassing portions of the genes to determine the position of the genes relative to their corresponding CFSs. Nine genes were determined to localize within seven previously uncloned CFSs. Semiquantitative reverse-transcription/polymerase chain reaction analysis of the cell lines and primary ovarian tumors validated the downregulation of seven of the 10 genes. We identified portions of seven uncloned CFSs and provide data to suggest that several of the genes mapping within CFSs may be inactivated in ovarian cancer.

Identification of underexpressed genes in early- and late-stage primary ovarian tumors by suppression subtraction hybridization.

To identify novel tumor suppressor genes involved in ovarian carcinogenesis, we generated four down-regulated suppression subtraction cDNA libraries from two early-stage (stage I/II) and two late-stage (stage III) primary ovarian tumors, each subtracted against cDNAs derived from normal ovarian epithelial cell brushings. Approximately 600-700 distinct clones were sequenced from each library. Comparison of down-regulated clones obtained from early- and late-stage tumors revealed genes that were unique to each library which suggested tumor-specific differences. We found 45 down-regulated genes that were common in all four libraries. We also identified several genes, the role of which in tumor development has yet to be elucidated, in addition to several under expressed genes, the potential role of which in carcinogenesis has been described previously (Bagnoli et al., Oncogene, 19: 4754-4763, 2000; Yu et al., Proc. Natl. Acad. Sci. USA, 96: 214-219, 1999; Mok et al., Oncogene, 12: 1895-1901, 1996). The differential expression of a subset of these genes was confirmed by semiquantitative reverse transcription-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control in a panel of 15 stage I and 15 stage III tumors of mixed histological subtypes. Chromosomal sorting of library sequences revealed that several of the genes mapped to known regions of deletion in ovarian cancer. Loss of heterozygosity (LOH) analysis revealed multiple genomic regions with a high frequency of loss in both early- and late-stage tumors. To determine whether loss of expression of some of the genes corresponds to loss of an allele by LOH, we used a microsatellite marker for one of the novel genes on 8q and have shown that loss of expression of this novel gene correlates with loss of an allele by LOH. In conclusion, our analysis has identified down-regulated genes, which map to known as well as novel regions of deletions and may represent potential candidate tumor suppressor genes involved in ovarian cancer.