Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). KLK5 was shown to be differentially expressed at the mRNA level in breast and ovarian cancer. Until now, detection of hK5 protein in either biological fluids or tissues has not been described due to lack of suitable reagents and methods. The aim of this study was to develop immunological reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and biological fluids, and to study the possible clinical utility of hK5 as a biomarker for endocrine-related malignancies. Recombinant hK5 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5 antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various biological fluids, tissue extracts, and serum samples from normal individuals and patients with various malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%) cancer. High levels were also detected in ascites fluid from metastatic ovarian cancer patients and in ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in biological fluids and tissue extracts. Our preliminary data indicate that hK5 is a potential biomarker in patients with ovarian and breast cancer.     

Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma.

Human kallikrein 11 (hK11) is a putative serine protease of the human kallikrein gene family. Currently, no methods are available for measuring hK11 in biological fluids and tissues. Our aim was to develop immunological reagents and assays for measuring hK11 and examine if the concentration of this kallikrein is altered in disease states. We produced recombinant hK11 protein in a baculovirus system and used it to develop monoclonal and polyclonal antibodies against hK11. We then developed an immunofluorometric procedure for measuring hK11 in biological fluids and tissue extracts with high sensitivity and specificity. We further quantified hK11 in various biological fluids and in serum of patients with various cancers. The hK11 immunofluorometric assay is highly sensitive (detection limit, 0.1 microg/l) and specific (no detectable cross-reactivity for other homologous kallikreins). We established the tissue expression pattern of hK11 at the protein level and found the highest levels in the prostate, followed by stomach, trachea, skin, and colon. We have immunohistochemically localized hK11 in epithelial cells of various organs. We further detected hK11 in amniotic fluid, milk of lactating women, cerebrospinal fluid, follicular fluid, and breast cancer cytosols. However, highest levels were seen in prostatic tissue extracts and seminal plasma. hK11 in seminal plasma and prostatic extracts is present at approximately 300-fold lower levels than prostate-specific antigen and at approximately the same levels as hK2. hK11 expression in breast cancer cell lines is up-regulated by estradiol. Elevated serum levels of hK11 were found in 70% of women with ovarian cancer and in 60% of men with prostate cancer. This is the first reported immunological assay for hK11. Analysis of this biomarker in serum may aid in the diagnosis and monitoring of ovarian and prostatic carcinoma.     

Human tissue kallikrein gene family: applications in cancer

Human tissue kallikrein genes, located on the long arm of chromosome 19, are a subgroup of the serine protease family of proteolytic enzymes. Initially thought to consist of three members, the human kallikrein locus has now been extended and includes 15 tandemly located genes. These genes, and their protein products, share a high degree of homology and are expressed in a wide array of tissues, mainly those that are under steroid hormone control. PSA (hK3) is one of the human kallikreins, and is the most useful tumor marker for prostate cancer screening, diagnosis, prognosis and monitoring. hK2, another prostate-specific kallikrein, has also been proposed as a complementary prostate cancer biomarker. In the past 5 years, the newly discovered kallikreins (KLK4-KLK15) have been associated with several types of cancer. For example, hK4, hK5, hK6, hK7, hK8, hK10, hK11, hK13 and hK14 are emerging biomarkers for ovarian, breast, prostate and testicular cancer. New evidence raises the possibility that some kallikreins are directly involved with cancer progression. We here review the evidence linking kallikreins and cancer and their applicability as novel biomarkers for cancer diagnosis and management.     

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.     

Coordinated steroid hormone-dependent and independent expression of multiple kallikreins in breast cancer cell lines

The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well in the pathogenesis of endocrine-related cancers. Previous experiments have shown that many kallikrein genes are under steroid hormone regulation in breast cancer cell lines. We here examine the coordinated expression of multiple kallikrein genes in several breast cancer cell lines after steroid hormone stimulation. Breast cancer cell lines were treated with various steroid hormones and kallikrein (KLK/hK) expression of hK3 (prostate-specific antigen, PSA), hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 was analyzed at the RNA level via RT-PCR and at the protein level by immunofluorometric ELISA assays. We identified several distinct hK hormone-dependent and hormone-independent expression patterns. Hormone-specific modulation of expression was seen for several kallikreins in BT-474, MCF-7, and T-47D cell lines. hK6 was specifically up-regulated upon estradiol treatment in all three cell lines whereas PSA expression was induced by dihydrotestosterone (DHT) and norgestrel stimulation in BT-474 and T-47D. hK10, hK11, hK13, and hK14 were specifically up-regulated by DHT in T-47D and by estradiol in BT-474 cells. Bioinformatic analysis of upstream proximal promoter sequences for these hKs did not identify any recognizable hormone-response elements (HREs), suggesting that the coordinated activation of these four hKs represents a unique expression “cassette”, utilizing a common hormone-dependent mechanism. We conclude that groups of human hKs are coordinately expressed in a steroid hormone-dependent manner. Our data supports clinical observations linking expression of multiple hKs with breast cancer prognosis.     

Human kallikrein 13 protein in ovarian cancer cytosols: a new favorable prognostic marker.

PURPOSE: Human kallikrein 13 (hK13; encoded by the KLK13 gene) is a secreted serine protease expressed in endocrine tissues, including the prostate, testis, breast, and ovary. We have previously reported steroid hormone regulation of the KLK13 gene and its clinical value as a marker of favorable prognosis in breast cancer at the mRNA level. We hypothesized that hK13 may represent a potential biomarker for ovarian carcinomas. PATIENTS AND METHODS: Using a newly developed enzyme-linked immunosorbent assay (ELISA), hK13 levels were quantified in 131 ovarian tumor extracts and correlated with various clinicopathological variables and outcome (progression-free survival [PFS], overall survival [OS]), over a median follow-up period of 42 months. RESULTS: hK13 concentration in ovarian tumor cytosols ranged from 0 to 18.4 ng/mg of total protein. An optimal cutoff value of 0.13 ng/mg (67(th) percentile) was selected, based on the ability of hK13 values to predict the PFS of the study population, to categorize tumors as hK13-positive or negative. Women with hK13-positive tumors most often had early stage (stage I/II) disease, no residual tumor after surgery and optimal debulking success (P <.05). Univariate and multivariate Cox regression analyses revealed that patients with hK13-positive tumors had a significantly longer PFS and OS than hK13-negative patients (P <.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK13-positive tumors (P =.007 and P =.002, respectively). CONCLUSION: These results indicate that hK13 is an independent marker of favorable prognosis in ovarian cancer.     

Kallikrein 11 expressed in human breast cancer cells releases insulin-like growth factor through degradation of IGFBP-3

Expression of human kallikrein 11 (hK11), secreted as a trypsin-like serine protease, is related to the prognosis of some human cancers, but its physiological functions in the steps of cancer progression are still unknown. In order to elucidate the enzymatic function of hK11 we investigated the substrate of hK11 and expression of KLK11, the gene encoding hK11. Among 20 human cancer cell lines tested, two estrogen receptor possessing [ER(+)] breast cancers showed the highest expression of KLK11. The investigation of the hK11 substrate showed that hK11 could degrade itself and insulin-like growth factor (IGF) binding protein 3 (IGFBP-3). Expression of KLK11 was detected in both human breast cancer tissue and in non-cancerous mammary glands, and significantly higher KLK11 expression was observed in histological grade I/II than in grade III breast cancers. The above results indicate that the hK11 expressed in ER(+) breast cancer cells may play a crucial role in breast cancer progression by increasing the bioavailability of IGFs via degradation of IGFBP-3.     

Human tissue kallikreins: the cancer biomarker family

Human tissue kallikreins (KLKs) are attracting increased attention due to their role as biomarkers for the screening, diagnosis, prognosis, and monitoring of various cancers including those of the prostate, ovarian, breast, testicular, and lung. Human tissue kallikrein genes represent the largest contiguous group of proteases within the human genome. Originally thought to consist of three genes, the identification of the human kallikrein locus has expanded this number to fifteen. These genes, and their encoded proteins, share a high degree of homology and are expressed in different tissues. Prostate-specific antigen (PSA), the most commonly known kallikrein, is a useful biomarker for prostate cancer. Several other kallikreins, including kallikreins 2 (KLK2) and 11 (KLK11) are emerging as complementary prostate cancer biomarkers. Along with these kallikreins, several others have been implicated in the other cancers. For example, KLK5, 6, 7, 10, 11, and 14 are emerging biomarkers for ovarian cancer. The identification of kallikrein substrates and the development of proteolytic cascade models implicate kallikrein proteins in cancer progression. This review describes the current status of kallikreins as cancer biomarkers.     

Human kallikrein 8 (hK8/TADG-14) expression is associated with an early clinical stage and favorable prognosis in ovarian cancer

The purpose of this study was to examine human kallikrein 8 (hK8/TADG-14) expression in epithelial ovarian tumors and to investigate the association of hK8 expression levels with patient survival. Human kallikrein 8 protein (hK8) expression was examined by immunohistochemistry in 74 ovarian adenocarcinomas and 6 normal ovaries. Results of immunostaining were correlated with clinicopathological variables and overall survival of the patients. Human kallikrein 8 gene (KLK8) mRNA expression was examined by semi-quantitative PCR in 35 ovarian tumors and 7 normal ovaries. Expression of hK8 was not detected on the surface epithelium of normal ovaries. In contrast, hK8 expression was detected in 51.4% (38/74) of carcinomas with a significantly higher detection rate of hK8 expression being observed in early stage disease compared to advanced stage disease (p=0.0192). Data analysis using the log-rank test showed hK8 expression correlated significantly with favorable patient survival (p=0.0328). Younger age (p=0.0008), early clinical stage (p<0.0001), and low histological grades of the tumors (p=0.0018) were also associated significantly with a favorable prognosis. In a multivariate model, age (p=0.0186) and clinical stage (p<0.0001) remained associated significantly with overall survival, whereas hK8 expression and histological grade lost their significance. There was significant relationship between the hK8 expression status and KLK8 mRNA expression levels (p=0.0304). Expression of hK8 is increased during the development of ovarian cancer and down-regulated during ovarian cancer progression. Expression of hK8 is a favorable prognostic marker in patients with ovarian cancer.     

Human kallikrein 8, a novel biomarker for ovarian carcinoma

Human kallikrein 8 (hK8; neuropsin) is a serine protease and new member of the hK family. The aim of this study was to examine if hK8 may serve as a novel cancer biomarker. An hk8-ELISA, developed in-house, was used to study the distribution of hK8 in various biological fluids and tissue extracts from healthy individuals and ovarian cancer patients of different stages of the disease (International Federation of Obstetrics and Gynecology II-IV). For ovarian cancer patients, very high levels in ascites fluid were observed (相似文献   

Immunofluorometric quantification of human kallikrein 5 expression in ovarian cancer cytosols and its association with unfavorable patient prognosis.

Human kallikrein 5 (hK5; encoded by the KLK5 gene) is a secreted serine protease expressed in hormonally regulated tissues, including the breast and ovary. We have previously reported regulation of the KLK5 gene by estrogens and progestins and its clinical value as a marker of poor prognosis in breast and ovarian cancers. We thus hypothesized that hK5 may represent a potential biomarker for ovarian carcinomas, at the protein level. Using a newly developed ELISA, hK5 levels were quantified (nanograms per milligram of total protein) in 22 low malignant potential (LMP) and 132 epithelial ovarian tumors and correlated with various clinicopathological variables and outcome [progression-free survival (PFS), overall survival (OS)]. hK5 concentration in LMP tumors ranged from 0 to 2.3 ng/mg (mean = 0.24) and from 0 to 220 ng/mg (mean = 3.35) in ovarian tumor cytosols (p = 0.002). Using a cutoff value of 0.15 ng/mg, 60% of ovarian tumors were categorized as hK5 positive. We found a strong correlation between patients with hK5-positive tumors and disease stages III/IV and grade 3 tumors (all p < 0.05). Univariate survival analysis revealed that hK5-positive patients had a significantly shorter PFS and OS (p < 0.05). Kaplan-Meier survival curves further confirmed an increased risk of relapse and death in women with hK5-positive tumors (p = 0.015 and p = 0.019, respectively). Multivariate analysis indicated that the prognostic value of hK5 was not independent from other parameters in the entire group of patients. When stratified by tumor grade (G1/2 vs. G3) and debulking success (optimal vs. suboptimal), univariate and multivariate analyses demonstrated that hK5 was an independent indicator of poor prognosis for patients with grade 3 tumors and with optimal debulking (p < 0.05). In patients with disease stage I/II versus III/IV, hK5 positivity was independently associated with a shorter PFS (p = 0.046) and marginally decreased OS (p = 0.08), in multivariate analysis. Lastly, we observed a fairly weak, positive, but statistically significant correlation between the expression levels of tissue hK5 and tissue CA125 (r(s) = 0.297; p < 0.001). Our findings provide evidence for an association between hK5 and more aggressive forms of epithelial ovarian cancer.     

Human kallikrein 8 protein is a favorable prognostic marker in ovarian cancer.

Human kallikrein 8 (hK8/neuropsin/ovasin; encoded by KLK8) is a steroid hormone-regulated secreted serine protease differentially expressed in ovarian carcinoma. KLK8 mRNA levels are associated with a favorable patient prognosis and hK8 protein levels are elevated in the sera of 62% ovarian cancer patients, suggesting that KLK8/hK8 is a prospective biomarker. Given the above, the aim of the present study was to determine if tissue hK8 bears any prognostic significance in ovarian cancer. Using a newly developed ELISA, hK8 was quantified in 136 ovarian tumor extracts and correlated with clinicopathologic variables and outcome [progression-free survival (PFS); overall survival (OS)] over a median follow-up period of 42 months. hK8 levels in ovarian tumor cytosols ranged from 0 to 478 ng/mg total protein, with a median of 30 ng/mg. An optimal cutoff value of 25.8 ng/mg total protein (74th percentile) was selected based on the ability of hK8 values to predict the PFS of the study population and to categorize tumors as hK8 positive or negative. Women with hK8-positive tumors most often had lower-grade tumors (G1), no residual tumor after surgery, and optimal debulking success (P < 0.05). Univariate and multivariate analyses revealed that patients with hK8-positive tumors had a significantly longer PFS and OS than hK8-negative patients (P < 0.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK8-positive tumors (P = 0.001 and P = 0.014, respectively). These results indicate that hK8 is an independent marker of favorable prognosis in ovarian cancer.     

The serum concentration of human kallikrein 10 represents a novel biomarker for ovarian cancer diagnosis and prognosis

Human kallikrein 10 (hK10) is a secreted serine protease that is highly expressed in ovarian tissue. We hypothesized that hK10 might represent a novel serological marker for ovarian cancer. We quantified by immunoassay, hK10 in sera from 97 normal women (controls), 141 patients with benign gynecologic diseases, and 146 patients with ovarian cancer. We then examined the diagnostic and prognostic value of this measurement in ovarian cancer. We found that normal serum hK10 ranged from 50 to 1040 ng/liter (mean = 439 ng/liter). hK10 concentration is significantly elevated in serum of presurgical ovarian cancer patients (range: 106-11,746 ng/liter; mean = 1067 ng/liter) but not in serum of patients with benign gynecologic diseases (range: 120-1200 ng/liter; mean = 447 ng/liter). When a cutoff of 700 ng/liter was selected (diagnostic specificity = 90%), the diagnostic sensitivity for ovarian cancer is 54%. About 35% of CA125-negative ovarian cancer patients (CA125 < 23 kU/liter) were hK10 positive at 90% specificity. In patients with stage I/II ovarian cancer, use of these two markers in combination results in a 21% increase in sensitivity, at 90% specificity, compared with CA125 alone. High serum hK10 was strongly associated with serous epithelial type, late-stage, advanced grade, large residual tumor (>1 cm), suboptimal debulking, and no response to chemotherapy (all Ps < 0.001). In univariate Cox survival analysis, high serum hK10 is associated with increased risk for relapse and death (hazard ratio = 2.59 and 3.15, respectively, P 相似文献   

Expanded human tissue kallikrein family--a novel panel of cancer biomarkers.

The full characterization of the human kallikrein gene locus has allowed identification of all members of this gene family on chromosome 19q13.4 and the establishment of common structural criteria, at both the mRNA and protein level. The human kallikrein gene family now consists of 15 members; their mRNA and protein structure, tissue expression and hormonal regulation patterns have been delineated. In addition to prostate-specific antigen (PSA, hK3), which is an established tumor marker for prostate cancer diagnosis and follow-up, and human glandular kallikrein (hK2), an emerging prostate cancer biomarker, accumulating evidence indicates that many other members of the human kallikrein gene family are also implicated in endocrine-related malignancies. Many kallikreins are differentially regulated in breast, prostate, ovarian and testicular cancers. In addition, preliminary reports indicate that three newly identified kallikreins (hK6, hK10 and hK11) are serum biomarkers for diagnosis and monitoring of ovarian and prostate cancer. The mechanism by which kallikreins might be involved in the pathogenesis and/or progression of cancer is not as yet fully understood. Preliminary reports indicate a possible role of kallikreins in controlling vital processes, like apoptosis, angiogenesis and tumor metastasis by cleavage of critical substrates such as growth factors, hormones or extracellular matrix. In this review, we present data on the differential expression of kallikreins in cancer at both the mRNA and protein levels, and propose future directions of research towards our understanding of the involvement of kallikreins in cancer and their possible diagnostic, prognostic and therapeutic applications.     

Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma.

PURPOSE: Human kallikrein 10 (hK10; also known as the normal epithelial cell-specific 1 gene and protein) is a secreted serine protease, which belongs to the human kallikrein family. It has been reported that hK10 is down-regulated in breast and prostate cancer cell lines and that it may function as a tumor suppressor. Recently, we developed a highly sensitive and specific immunoassay for hK10 and found that this protein is abundantly expressed in ovarian tissue. In this study, we measured quantitatively hK10 levels in ovarian cancer cytosolic extracts and evaluated the prognostic value of this biomarker in ovarian cancer. EXPERIMENTAL DESIGN: Specimens from eight normal ovarian tissues, eight ovarian tissues with benign disease, and 182 ovarian tumors were investigated. RESULTS: hK10 concentration in ovarian tumor cytosols ranged from 0 to 84 ng/mg of total protein, with a median of 2.6. This median was highly elevated in comparison with normal and benign ovarian tissues (P < 0.001). A cutoff of 1.35 ng/mg was selected to categorize tumors as hK10 high and hK10 low. With chi(2) test and Fisher's exact test, high concentration hK10 was found to be associated with advanced disease stage, serous histological type, suboptimal debulking, and large residual tumor (>1 cm; all P < 0.05). hK10 status was additionally correlated with clinical outcome, including progression-free (PFS) and overall survival (OS) using the Cox model. In univariate analysis, we found that patients with hK10 high tumors were more likely to die and relapse, in comparison with patients with hK10 low tumors (hazards ratios for PFS and OS were 1.93 and 2.42, respectively; P < 0.05). Although this correlation disappeared after the entire patient population was subjected to multivariate analysis, it remained significant in the subgroup of patients with stage III/IV ovarian cancer (hazards ratios for PFS and OS were 1.98 and 2.12, respectively; P < 0.05). CONCLUSIONS: Our results indicate that hK10 is a new, independent, unfavorable prognostic marker, especially for late-stage ovarian cancer.