Human kallikrein gene 11 (KLK11) mRNA overexpression is associated with poor prognosis in patients with epithelial ovarian cancer.

PURPOSE: The purpose of this study was to examine expression levels of the human tissue kallikrein 11 gene (KLK11) in epithelial ovarian tumors and to identify the relationship between KLK11 expression and patient survival. EXPERIMENTAL DESIGN: KLK11 mRNA expression was examined by semiquantitative PCR in 64 epithelial ovarian tumors (7 adenomas, 6 low malignant potential tumors, and 51 adenocarcinomas) and in 10 normal ovaries. Semiquantitative PCR results were correlated with clinicopathologic variables and overall survival. cDNA from human normal tissues and tumor tissues was also analyzed. RESULTS: KLK11 mRNA expression was detected in various human cancer tissues including breast, lung, colon, prostate, pancreas, and ovarian carcinoma. The mean value of relative KLK11 expression ratio was significantly higher in ovarian tumor samples than in normal ovary samples (compared with normal samples: adenoma, P = 0.0006; low malignant potential tumor, P = 0.0049; and carcinoma, P < 0.0001). No statistically significant associations between KLK11 mRNA expression level and clinical stage, histological type, or histological grade were observed. The log-rank test showed that high KLK11 mRNA expression and advanced clinical stage significantly correlated with poor patient survival (P = 0.0185 and P = 0.0043, respectively). High KLK11 mRNA expression and clinical stage remained significantly associated with overall survival (P = 0.0225 and P = 0.0202, respectively) after multivariate analysis. CONCLUSIONS: KLK11 expression may play an important role in ovarian cancer development and act as an independent prognostic marker in ovarian cancer patients.     

icb-1 Gene counteracts growth of ovarian cancer cell lines

Human gene icb-1 has been originally identified to be involved in differentiation processes of cancer cells. To examine the function of icb-1 in ovarian cancer, we knocked down its expression in three ovarian cancer cell lines and performed microarray-based gene expression profiling with subsequent gene network modeling. Loss of icb-1 expression accelerated proliferation of SK-OV-3, OVCAR-3 and OAW-42 cells and led to upregulation of ovarian cancer biomarkers like KLK10 and CLDN16. Most of the upregulated genes were part of oncogenic pathways regulated by ERα or TNF. Our data suggest that icb-1 gene inhibits growth and progression of ovarian cancer cells.     

KLK6 and KLK13 predict tumor recurrence in epithelial ovarian carcinoma

Background:

The human kallikrein-related peptidase family consists of 15 genes. Twelve of these genes are overexpressed in ovarian cancer and may represent potential markers for diagnosis, prognosis, and/or response to treatment. The aim of this study was to determine the prognostic significance of kallikrein-related peptidase 6 (KLK6) and kallikrein-related peptidase 13 (KLK13) in epithelial ovarian cancer by quantifying gene expression levels with tumour pathology and patient survival data.

Methods:

Total RNA was isolated from 106 patients diagnosed with primary ovarian cancer, as well as 8 normal ovary controls. Samples were analysed by quantitative real-time PCR for KLK6 and KLK13 expression. Correlation between kallikrein gene expression and clinical characteristics was evaluated with the χ²-test. Survival analysis was performed using Kaplan–Meier and Cox proportional hazards regression models.

Results:

Expression levels of both KLK6 and KLK13 mRNA were significantly increased in invasive cancers relative to normal ovaries (P=0.002 and 0.039 respectively). High KLK6 and KLK13 expression was an indicator of poor prognosis, with patients having a shorter recurrence-free survival (P=0.002 and 0.027 respectively). High KLK6 expression was also significantly associated with lower overall survival (P=0.011). When subjected to multivariate analysis, patients with either high KLK6 or KLK13 were 3- and 2.2-fold, respectively, more likely to have a recurrence than patients with low kallikrein expression.

Conclusion:

These data show increased mRNA expression of KLK6 and KLK13 in ovarian cancer compared to normal ovarian tissues. High KLK6 or KLK13 expression in primary ovarian tumours can significantly predict prognosis in terms of recurrence-free survival and overall survival. In all, this study shows KLK6 and KLK13 as potential biomarkers and may be therapeutic targets for treatment of ovarian cancer.     

Downregulation of human kallikrein 10 (KLK10/NES1) by CpG island hypermethylation in breast, ovarian and prostate cancers.

OBJECTIVE: The human kallikrein 10 (KLK10)/normal epithelial cell-specific-1 (NES1) gene is highly expressed in normal mammary, ovary and prostate cells, but its expression is dramatically decreased in cancer cell lines. Recently, it has been shown that CpG island hypermethylation of the KLK10 gene is responsible for the tumor-specific loss of KLK10 gene expression in certain breast cancer cell lines. METHOD: We examined the role of CpG island hypermethylation in the tumor-specific loss of KLK10 expression in breast, ovarian and prostate cancers. We treated cells with the demethylating agent 5-aza-2'-deoxycytidine (dC) and monitored changes in KLK10 mRNA by RT-PCR and secreted hK10 protein expression by ELISA. The following cell lines were used: MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-1, T-47D and BT-474 (breast); BG-1, MDAH-2774, HTB-75, HTB-161, PA-1 and ES-2 (ovary), and LNCaP and PC-3 (prostate). RESULTS: Upregulation of KLK10 mRNA levels, which was accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2'-dC. Genomic sequencing of sodium-bisulfite-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. CONCLUSION: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested.     

Parallel overexpression of seven kallikrein genes in ovarian cancer

Recent evidence suggests that many members of the human kallikrein (KLK) gene family are differentially regulated in ovarian cancer and have potential as diagnostic and/or prognostic markers. We used the serial analysis of gene expression and expressed sequence tag databases of the Cancer Genome Anatomy Project to perform in silico analyses of the expression pattern of the 15 human KLK genes in normal and cancerous ovarian tissues and cell lines. We found that seven KLK genes (KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, and KLK14) are up-regulated in ovarian cancer. Probing 2 normal and 10 ovarian cancer serial analysis of gene expression libraries with gene-specific tags for each KLK indicated that whereas no expression was detected in any normal libraries (with the exception of KLK10 and KLK11), these KLKs were found to be expressed with moderate densities (103-408 tags per million) in 40-60% of the ovarian cancer libraries analyzed. These data were verified by screening the expressed sequence tag databases, where 78 of 79 mRNA clones isolated for these genes were from ovarian cancer libraries. X-profiler comparison of the pools of normal and cancerous ovaries identified a significant difference in expression levels for six of the seven KLKs. We experimentally verified the overexpression of six KLK proteins in cancer versus normal or benign tissues with highly sensitive and specific immunofluorometric assays. A statistically significant stepwise increase in protein levels was found among normal, benign, and cancerous ovarian tissues. The expression of five KLKs showed a strong degree of correlation at the protein level, suggesting the existence of a common mechanism or pathway that controls the expression of this group of adjacent genes during ovarian cancer progression.     

Kallikreins as markers of disseminated tumour cells in ovarian cancer-- a pilot study.

BACKGROUND: Kallikreins are a family of secreted serine proteases, encoded by 15 genes which all localize in tandem on chromosome 19q13.4. Several members of this family have been previously associated with ovarian cancer. Kallikreins 6 (KLK6) and 10 (KLK10) are elevated in tumour cells, serum and ascites fluid of ovarian cancer patients and correlate with disease prognosis. Other kallikreins that have been related to ovarian cancer include KLK4, 5, 7, 8, 9, 11, 13, 14 and 15. We hypothesized that KLK6 and KLK10 can be utilized to monitor dissemination of ovarian cancer cells in blood and ascites fluid of ovarian cancer patients. METHODS: RNA was isolated by immunomagnetic separation of cancer cells and was amplified by RT-PCR. RESULTS: Screening for disseminated cancer cells in blood from 24 ovarian cancer patients, with RT-PCR for KLK6 mRNA, resulted in 75% positivity; however, this was not different from the positivity of normal controls. By utilizing KLK10 as a marker, the positivity of patients was 40% versus 20% of controls. Screening of ascites fluid of ovarian cancer patients revealed 90% positivity for KLK6 and KLK10 mRNA compared with 33% for other cancer types. Significant correlations were identified among mRNA of KLK4, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 in cancer cells isolated from ascites fluid. CONCLUSION: Kallikrein expression by ovarian cancer cells is not specific enough for detecting disseminated disease. Kallikrein expression may have some value for differentiating ovarian cancer from other types of cancer or from non-malignant diseases that lead to ascites accumulation.     

Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer

The tissue kallikrein (KLK) genes are a new source for biomarkers in ovarian cancer. However, there has been no systematic analysis of copy number and structural rearrangements related to their protein expression. Chromosomal rearrangements and copy number changes of the KLK region were studied by FISH with protein levels measured by ELISA. Ovarian cancer and cell lines revealed the KLK region was subject to copy number imbalances or involved in unbalanced translocations and were associated with increased protein expression of KLKs 5, 6, 7, 8, 9, 10 and 11. In this initial study, we introduce the potential for long‐range chromosomal effects and copy number as a mechanism for the previously reported aberrant expression of many KLK genes in ovarian cancers.     

Prognostic value of kallikrein-related peptidase 6 protein expression levels in advanced ovarian cancer evaluated by automated quantitative analysis (AQUA)

Kallikrein-related peptidases, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. In the present study, we sought to determine the prognostic value of kallikrein-related peptidase 6 (KLK6) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. A tissue array composed of 150 advanced stage ovarian cancers, uniformly treated with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For evaluation of KLK6 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Mean follow-up time of the cohort was 34.35 months. One hundred and thirty-five of 150 cases had sufficient tissue for AQUA analysis. In univariate survival analysis, low tumor KLK6 expression was associated with better outcome for overall survival over 3 years (P = 0.019). There was no association between tumor KLK6 expression and progression-free survival (P = 0.128). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor KLK6 expression level was one of the most significant predictor variable for overall survival (95% confidence interval, 1.19-3.50; P = 0.009). High tumor KLK6 protein expression is associated with inferior patient outcome in ovarian cancer. KLK6 may represent a promising disease biomarker and therapeutic target in ovarian cancer.     

In-silico analysis of kallikrein gene expression in pancreatic and colon cancers

Human kallikreins are a cluster of 15 serine protease genes located in the chromosomal band 19q13.4, a non-randomly rearranged region in many solid tumors, including pancreatic cancer. We utilized the SAGE and EST databases of the Cancer Genome Anatomy Project to perform in-silico analysis of kallikrein gene expression in normal and cancerous pancreatic and colon tissues and cell lines using virtual Northern blotting (VNB), digital differential display (DDD) and X-profiler. At least two kallikreins, KLK6 and KLK10, are significantly up-regulated in pancreatic cancer. We probed 2 normal and 6 pancreatic cancer SAGE libraries with gene-specific tags for each of these kallikreins. KLK6 was found to be expressed in 5/6 cancer libraries and showed the most marked (5-fold) increase in average expression levels in cancer vs. normal. These data were verified by screening the EST databases, where all mRNA clones isolated were from cancerous libraries, with no clones detected in normal pancreatic tissues or cell lines. X-profiler comparison of two pools of normal and cancerous pancreatic libraries further verified the significant increase of KLK6 expression levels in pancreatic cancer. DDD data showed a 13-fold increase in KLK10 expression in pancreatic cancer. Three kallikrein genes, KLK6, 8 and 10 are overexpressed in colon cancer compared to normal colon, while one kallikrein, KLK1, is down-regulated. While no expression of KLK6 was detected in normal colon, KLK6-specific tags were detectable in 2 cancer libraries. Similar results were obtained by EST screening; no KLK6 clones were detected in any of the 28 normal libraries examined, while 10 KLK6 EST clones were found in colon adenocarcinoma. KLK10 was not detectable in normal colon. Gene-specific tags were, however, detectable with high density in colon cancer and 7 EST clones were found to be expressed in colon Adenocarcinoma.     

人类组织激肽释放酶6在卵巢上皮性癌中的表达及临床意义

[目的]研究人类组织激肽释放酶（kallikrein,KLK）6蛋白在卵巢上皮性癌组织及腹膜后淋巴结中的表达,探讨其在卵巢上皮性癌中的临床意义。[方法]回顾性分析卵巢肿瘤患者的临床病理资料,采用免疫组化检测72例卵巢癌、16例卵巢交界性肿瘤、20例良性卵巢上皮性肿瘤组织KLK6蛋白的表达;并采用配对设计研究KLK6蛋白在卵巢癌患者腹膜后淋巴结中的表达．探讨KLK6在卵巢上皮性癌发生、发展中的作用。[结果]KLK6在卵巢癌及交界性卵巢肿瘤中的阳性表达分别为52．8％（38／72）、25．O％（4／16）,均显著强于良性上皮性卵巢肿瘤15．0％（3／20）f19〈0．05）。KLK6阳性表达与卵巢癌的临床分期、组织学分级及淋巴结转移有关fP〈0．05）,与组织学类型无相关性（P〉0．05）;KLK6在卵巢癌原发灶与腹膜后转移淋巴结组织中的表达呈正相关（r=8．91,P=0．003）;KLK6阳性与阴性患者的平均生存时间分别为25．9个月和49．2个月（P〈O．051。[结论]KLK6高表达可能在卵巢上皮性癌的发生发展中起潜在作用,KLK6可能与卵巢上皮性癌的浸润、转移有关,KLK6是卵巢癌的不良预后因素之一。     

The promoter and the enhancer region of the KLK 3 (prostate specific antigen) gene is frequently mutated in breast tumours and in breast carcinoma cell lines

KLK3 or prostate specific antigen (PSA) is a serine protease, which is an established tumour marker of prostatic adenocarcinoma. PSA is now used widely for the diagnosis and monitoring of patients with prostate cancer. Recent studies have demonstrated that about 70% of breast cancers produce PSA. In this study, we examined the molecular mechanism underlying the expression of the PSA gene in breast cancer and breast cancer cell lines. We analysed nine breast tumours categorized on the basis of high- or low-PSA expression in tumour cytosols and four breast cancer cell lines. To determine abnormalities associated with PSA expression in breast tumours, genomic DNA was extracted and all five exons of the PSA gene were polymerase chain reaction (PCR) amplified and sequenced on both strands. PCR amplification was also performed for the promoter and enhancer elements of the PSA gene. No mutations were observed in the coding portion of the gene. A polymorphism was observed in exon 2 from three breast tumours. However, sequencing of the promoter and the enhancer elements of the PSA gene reveals several point mutations. Within a 5.8-kb promoter/enhancer region of the PSA gene, we detected 16 different mutational hotspots (appearing more than once in the nine tumours). Among these hotspots, two appeared in seven out of nine tumours. Most importantly, the androgen response element (ARE I) in the proximal promoter was found mutated in four tumours and in the breast carcinoma cell line MCF-7. Mutations associated with the ARE I have been shown previously to result in an 80% decrease in PSA gene expression. The mutations in the core enhancer and promoter region probably contribute to the aberrant expression of the PSA gene in breast tumours, possibly by altering the regulation of the gene by steroid hormones.