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ID: 372 Kallikrein-related (KLK) serine proteases and their roles in prostate cancer: nuclear, cytoplasmic and secreted forms
Abstract
The human tissue kallikrein-related (KLK) proteases are a multigene family of 15 serine proteases, many of which are over-expressed in prostate cancer. Prostate-specific antigen (PSA), a member of this family, is the current serum biomarker for detection and monitoring of prostate cancer. The related prostatic protease, KLK2/KLK2 is also emerging as an additional biomarker for prostate cancer. We have demonstrated that KLK4/KLK4 may also be a useful biomarker for prostate cancer as KLK4/KLK4 expression is higher in the pre-malignant prostatic intra-epithelial neoplasia (PIN) lesion, adenocarcinoma and bone metastasis compared to benign/normal glands. Of interest, there are over 70 KLK splice variants many of which are cancer-specific in their expression and which encode truncated KLK proteins that would not be catalytically active. At least one of these variants, KLK4-205, is nuclear localized which is a novel site for a serine protease-related protein. The functional role of this variant or the native PSA, KLK2 or KLK4 proteases in cancer progression are not fully elucidated, although several biochemical studies suggest a matrix degrading role either directly or indirectly via the activation of urinary plasminogen activator and other factors. In order to further demonstrate a functional role at the cell biology level, we have stably-transfected the prostate cancer PC3 cell line with prepro-PSA, -KLK2 and -KLK4. No change in the proliferative or invasive capacity of these transfected cells was observed, but PSA and KLK4, but not KLK2, over-expression elicited a morphological change and increased migration to various chemo-attractants. We also observed a loss of the cell adhesion protein, E-cadherin and down-regulation of a number of other cell adhesion molecules (desmoplakin, junction plakoglobin, claudin-3 and claudin-7) while expression of the mesenchymal marker, vimentin, was increased. These changes are indicative of an epithelial to mesenchymal transition (EMT) and a more aggressive phenotype. KLK4:PC3 cells showed significantly greater attachment to collagen I and IV and increased migration towards conditioned media from an osteoblastic cell line (Saos-2). PSA and KLK4 expression was also increased in LNCaP:Saos 2 cell co-cultures. These data provide compelling evidence for a role for these kallikrein-related serine proteases in more aggressive disease especially bone metastasis and highlight their potential as biomarkers and therapeutic targets.
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Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity. An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers. Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines. Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death. This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states. Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.
The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding
biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes
preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted
expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an
amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons
and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates
that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate
stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the
porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the
disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing
several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins
hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential
functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and
other proteases.
The human tissue kallikrein (KLK) family of serine proteases, which is important in post-translational processing events, currently consists of just three genes-tissue kallikrein (KLK1), KLK2, and prostate-specific antigen (PSA) (KLK3)-clustered at chromosome 19q13. 3-13.4. We identified an expressed sequence tag from an endometrial carcinoma cDNA library with 50% identity to the three known KLK genes. Primers designed to putative exon 2 and exon 3 regions from this novel kallikrein-related sequence were used to polymerase chain reaction-screen five cosmids spanning 130 kb around the KLK locus on chromosome 19. This new gene, which we have named KLK4, is 25 kb downstream of the KLK2 gene and follows a region that includes two other putative KLK-like gene fragments. KLK4 spans 5.2 kb, has an identical genomic structure-five exons and four introns-to the other KLK genes and is transcribed on the reverse strand, in the same direction as KLK1 but opposite to that of KLK2 and KLK3. It encodes a 254-amino acid prepro-serine protease that is most similar (78% identical) to pig enamel matrix serine protease but is also 37% identical to PSA. These data suggest that the human kallikrein gene family locus on chromosome 19 is larger than previously thought and also indicate a greater sequence divergence within this family compared with the highly conserved rodent kallikrein genes.
Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.
By using the positional candidate gene approach, we were able to identify a novel serine protease gene that maps to chromosome 19q13.3-q13.4. Screening of expressed sequence tags allowed us to establish the expression of the gene and delineate its genomic organization (GenBank accession no. AF135023). We named this gene KLK-L1. Another group, by using a subtraction hybridization method, cloned the same gene and named it prostase (GenBank accession nos. AF113140 and AF113141). Here, we describe the precise mapping and localization of the prostase/KLK-L1 gene between the known genes KLK2 (human glandular kallikrein) and zyme (also known as protease M/neurosin). The direction of transcription of prostase/KLK-L1 is the same as that of zyme but opposite to that of KLK2 and prostate-specific antigen genes. Contrary to the initial impression, prostase/KLK-L1 is expressed at high levels not only in prostate tissue but also in testis, mammary gland, adrenals, uterus, thyroid, and salivary glands. We have further demonstrated with in vitro experiments with the breast carcinoma cell line BT-474 that this gene is expressed and that its expression is up-regulated by androgens and progestins. On the basis of information on other genes that are localized in the same region (prostate-specific antigen, KLK2, zyme, and normal epithelial cell specific-1 gene), we speculate that prostase/KLK-L1 may be involved in the pathogenesis and/or progression of prostate, breast, and possibly other malignancies.
The tissue or glandular kallikreins (KLK) are members of a highly conserved multigene family encoding serine proteases that are central to many biological processes. The rodent KLK families are large, highly conserved and clustered at one locus. The human KLK gene family is clustered on chromosome 19q13.3-13.4, and until recently consisted of just three members. However, recent studies have identified up to 11 new members of the KLK family that are less conserved than their rodent counterparts. Using a Southern blot and sequence analysis of 10 BACs and cosmids spanning approximately 400 kilobases (kb) either side of the original KLK 60-kb locus, we demonstrated that these genes also lie adjacent to this. We have also clarified the position of several microsatellite markers in relation to the extended KLK locus. Moreover, from Southern blot analysis of the cosmids and BACs with a degenerate oligonucleotide probe to the histidine-encoding region of serine proteases, we have shown that there are no other serine protease genes approximately 400 kb centromeric and 220 kb telomeric of the extended locus. We performed an extensive analysis of the expression patterns of these genes by poly(A)(+) RNA dot blot and reverse transcriptase-polymerase chain reaction analysis, and demonstrated a diverse pattern of expression. Of interest are clusters of genes with high prostate (KLK2-4) and pancreatic (KLK6-13) expression suggesting evolutionary conservation of elements conferring tissue specificity. From these findings, it is likely that the human KLK gene family consists of just 14 clustered genes within 300 kb and thus is of a comparable size to the rodent families (13-24 genes within 310 and 480 kb, respectively). In contrast to the rodent families, the newest members of the human KLK family are much less conserved in sequence (23-44% at the protein level) and appear to consist of at least four subfamilies. In addition, like the rat, these genes are expressed at varying levels in a diverse range of tissues although they exhibit quite distinct patterns of expression.
KLK8 (neuropsin/ovasin) is a new member of the human kallikrein gene family, which consists of enzymes with serine protease enzymatic activity. Recent reports have implicated KLK8 in ovarian cancer. KLK8 may have potential clinical value for disease diagnosis or prognosis and it may also be a useful therapeutic target.
We undertook this study to evaluate the prognostic value of KLK8 in ovarian carcinoma by examining its expression in ovarian tumors. Experimental
The KLK8 gene was analyzed by reverse transcription-PCR and direct sequencing in several human normal tissues. Subsequently, its expression was studied in a set of ovarian tumors, and statistical analysis was performed.
We have identified two novel mRNA splice variants of the KLK8 gene, which are abundantly expressed in many tissues. These new variants were named KLK8 type 3 and type 4. Study of the expression of the KLK8 gene and its spliced variants in ovarian tumors indicated that the new variants were expressed very frequently and that full-length KLK8 expression is an independent and favorable prognostic marker for ovarian cancer. Patients with higher KLK8 expression in the tumor have lower grade disease, lower residual tumor left after surgery, live longer, and relapse less frequently. In multivariate analysis, higher KLK8 expression was significantly associated with longer disease-free survival.
These results suggest that KLK8 is a novel, favorable prognostic marker in ovarian cancer. Because KLK8 encodes for a predicted secreted protein, its detection in serum may aid in ovarian cancer diagnosis.
Endometrial cancer is the fourth most common female malignancy in women in developed countries. Estrogen, and to a lesser degree, progesterone, regulate specific target genes that are involved in endometrial tumorigenesis. A family of proteases involved in cellular proliferation, extracellular matrix degradation and thus, implicated in tumorigenesis, and regulated by estrogen and progesterone in a number of systems, are the tissue kallikreins (KLKs). KLK4, a new member of the KLK gene family, was found to be expressed to varying levels in a number of endometrial cancer cell lines- HEC1A, HEC1B, Ishikawa, RL95-2 and KLE- at both the mRNA and protein level. On the addition of 10 nmol/L estradiol, progesterone, or a combination of both over a 48 h period, an increase in the intracellular protein levels of K4 were observed when compared to the control (untreated) cells. We have also identified a novel KLK4 transcript with a complete exon 4 deletion. The significance of this alternative transcript, which would give rise to a truncated protein without a serine residue (which is essential for catalytic activity), is yet to be established. These cell lines now provide a model system to study the role of KLK4 and the molecular mechanisms of KLK4 regulation by estrogen and progesterone, in endometrial tumorigenesis.
Previous studies indicated that a new member of the human kallikrein (KLK) gene family, KLK4, was expressed in prostate, breast, and endometrial carcinoma cell lines and may have potential as a tumor marker. The aim of this study was to examine the expression of KLK4 in the normal ovary and ovarian tumors of different histology, stage, and differentiation and to determine its association with ovarian tumor progression. Using reverse transcription-PCR, Southern blot, and densitometry analyses, we found the level of KLK4 expression was higher in late stage serous (SER) epithelial-derived ovarian carcinomas than in normal ovaries, mucinous epithelial tumors, and granulosa cell tumors. KLK4 was highly expressed in all of the SER ovarian carcinoma cell lines (eight of eight), SER epithelial carcinomas (11 of 11), and two adenomas, whereas it was expressed at a lower level (or not at all) in normal ovaries (four of six), mucinous epithelial tumors (three of four), endometrioid carcinomas (four of five), clear cell carcinomas (two of three), or granulosa cell tumors (three of six). Of particular interest, KLK4 mRNA variants were detected in SER ovarian carcinoma cell lines and primary cultured ovarian tumor cells, but they were not present in normal ovaries. In situ hybridization analysis showed that KLK4 mRNA transcripts are localized to adenocarcinoma cells of ovarian tumor tissues. Similarly, immunohistochemical staining of ovarian carcinoma sections showed immunoreactivity to KLK4 protein product (hK4) antipeptide antibodies. In addition, intracellular hK4 levels, as detected on Western blot analysis, were induced by 100 nM estrogen treatment of the estrogen receptor positive ovarian carcinoma cell line OVCAR-3, >8-24 h. Our results show that the level of KLK4 expression and expression of KLK4 mRNA variants are associated with progression of ovarian cancer, particularly late stage SER adenocarcinomas. Moreover, hK4 may be a candidate marker for the diagnosis and/or monitoring of ovarian epithelial carcinomas.
The tissue kallikreins (KLKs) form a family of serine proteases that are involved in processing of polypeptide precursors and have important roles in a variety of physiologic and pathological processes. Common features of all tissue kallikrein genes identified to date in various species include a similar genomic organization of five exons, a conserved triad of amino acids for serine protease catalytic activity, and a signal peptide sequence encoded in the first exon. Here, we show that KLK4/KLK-L1/prostase/ARM1 (hereafter called KLK4) is the first significantly divergent member of the kallikrein family. The exon predicted to code for a signal peptide is absent in KLK4, which is likely to affect the function of the encoded protein. Green fluorescent protein (GFP)-tagged KLK4 has a distinct perinuclear localization, suggesting that its primary function is inside the cell, in contrast to the other tissue kallikreins characterized so far that have major extracellular functions. There are at least two differentially spliced, truncated variants of KLK4 that are either exclusively or predominantly localized to the nucleus when labeled with GFP. Furthermore, KLK4 expression is regulated by multiple hormones in prostate cancer cells and is deregulated in the androgen-independent phase of prostate cancer. These findings demonstrate that KLK4 is a unique member of the kallikrein family that may have a role in the progression of prostate cancer.
The wild-type or variant mRNAs of several kallikrein (KLK) genes, such as KLK4, are highly expressed in ovarian carcinomas and may have potential as tumor markers. Two of these KLK genes (KLK5 and KLK7) and their proteins (hK5 and hK7) were first identified in the skin epidermis, where hK5 may be the physiological activator of hK7. The purpose of this study was to reexamine the expression of KLK5/hK5 and KLK7/hK7 and their association and to determine whether cancer-related variant transcripts were expressed.
The expression of KLK5/hK5 and KLK7/hK7 was analyzed in the same cohort (n = 37) of benign (n = 4) and malignant ovarian tissue (n = 23) samples and primary cultured cells (n = 21) and in 8 ovarian cancer cell lines using semiquantitative RT-PCR; Southern, Northern, and Western blot analyses; and immunohistochemistry techniques.
We showed the concordant higher expression of both KLK5/hK5 and KLK7/hK7 in ovarian carcinomas, especially late-stage serous carcinomas, compared with normal ovaries and benign adenomas. We also found that one novel KLK5 transcript with a short 5'-untranslated region and a novel KLK7 transcript with a long 3'-untranslated region were highly expressed in the ovarian cancer cell lines OVCAR-3 and PEO1, respectively, but were expressed at very low levels in normal ovarian epithelial cells. Both Western blot and immunohistochemistry analyses showed that these two enzymes are secreted from ovarian carcinoma cells.
Our study demonstrated that hK5 and hK7, or more specifically, the short KLK5 and long KLK7 transcripts, may be useful as tumor markers for epithelial-derived serous carcinomas. However, additional clinical studies assessing serum levels of these putative biomarkers are required to confirm their usefulness in the diagnosis and/or monitoring of these tumors.
The objective is to compare the performance of immunohistochemistry (IHC) with that of reverse transcription (RT)-PCR for detecting clinically significant micrometastases in histopathologically normal archival pelvic lymph nodes (PLN) removed at radical prostatectomy from men with locally advanced nonmetastatic prostate cancer. We stained 1864 fixed, paraffin-embedded PLNs from 199 pT(3)N(0)M(0) prostate cancer patients for prostate-specific antigen (PSA) and cytokeratin. We also assessed human glandular kallikrein (hK2) expression in a subset of 164 patients. In addition, all PLN specimens were assayed for hK2 mRNA using a previously described RT-PCR assay. PSA and cytokeratin were expressed in the same 13 of 199 (7%) cases; hK2 was expressed in 3 of 164 (2%) cases. PSA/cytokeratin and hK2 expression were associated with cancer involvement of seminal vesicles, higher Gleason sum, and a positive RT-PCR-hK2 assay result. In standard postoperative multivariable models, IHC-PSA/IHC-Cytokeratin or IHC-hK2 was associated with prostate cancer progression, development of distant metastases, and prostate cancer-specific survival. However, when RT-PCR-hK2 assay result was added to the models, it was the sole predictor of clinical outcomes. Although IHC-PSA/IHC-Cytokeratin and IHC-hK2 were more specific for identifying patients who would suffer biochemical progression and develop metastases and who would ultimately die of prostate cancer, RT-PCR-hK2 was more sensitive and accurate. Although IHC for PSA, cytokeratin, and hK2 appear to be more specific methods for detecting biologically and clinically significant prostate cancer micrometastases in histopathologically normal PLN, RT-PCR-hK2 appears to be a more sensitive method that maintained a reasonable specificity. In pT(3)N(0) patients, a positive RT-PCR-hK2 assay result when performed on PLN was the strongest predictor of clinical outcomes after radical prostatectomy.
Kallikreins (KLKs) are highly conserved serine proteases that play key roles in a variety of physiological and pathological processes. KLKs are secreted proteins that have extracellular substrates and function. For example, prostate-specific antigen (or KLK3) is a secreted protein that is widely used as a diagnostic marker for prostate cancer. KLK4 is a recently identified member of the kallikrein family that is regulated by androgens and is highly specific to prostate for expression. Here, we show that the gene product of KLK4, hK4, is the first member of the KLK family that is intracellularly localized. We provide strong evidence that the previously assigned first exon that was predicted to code for a signal peptide that would target hK4 for secretion is not part of the physiologically relevant form of KLK4 mRNA. In addition to detailed mapping of the KLK4 mRNA 5' end by RT-PCR, this conclusion is supported by predominantly nuclear localization of the hK4 protein in the cell, documented by both immunofluorescence and cell fractionation experiments. Furthermore, in addition to androgens, hK4 expression is regulated by estrogen and progesterone in prostate cancer cells. Finally, in situ hybridization on normal and hyperplastic prostate samples in tissue microarrays indicate that KLK4 is predominantly expressed in the basal cells of the normal prostate gland and overexpressed in prostate cancer. These data suggest that KLK4 has a unique structure and function compared with other members of the KLK family and may have a role in the biology and characterization of prostate cancer.
Prostate specific antigen (PSA) or human kallikrein 3 (hK3) has long been an effective biomarker for prostate cancer. Now, other members of the tissue kallikrein (KLK) gene family are fast becoming of clinical interest due to their potential as prognostic biomarkers. particularly for hormone dependent cancers. The tissue kallikreins are serine proteases that are encoded by highly conserved multi-gene family clusters in rodents and humans. The rat and mouse loci contain 10 and 25 functional genes, respectively, while the human locus at 19q 13.4 contains 15 genes. The structural organization and size of these genes are similar across species; all genes have 5 coding exons that encode a prepro-enzyme. Although the physiological activators of these zymogens have not been described, in vitro biochemical studies show that some kallikreins can auto-activate and others can activate each other, suggesting that the kallikreins may participate in an enzymatic cascade similar to that of the coagulation cascade. These genes are expressed, to varying degrees, in a wide range of tissues suggesting a functional involvement in a diverse range of physiological and pathophysiological processes. These include roles in normal skin desquamation and psoriatic lesions, tooth development, neural plasticity, and Alzheimer's disease (AD). Of particular interest is the expression of many kallikreins in prostate, ovarian, and breast cancers where they are emerging as useful prognostic indicators of disease progression.
Endometrial cancer is the fourth most common female malignancy in women in developed countries. Estrogen, and to a lesser degree, progesterone, regulate specific target genes that are involved in endometrial tumorigenesis. A family of proteases involved in cellular proliferation, extracellular matrix degradation and thus, implicated in tumorigenesis, and regulated by estrogen and progesterone in a number of systems, are the tissue kallikreins (KLKs). KLK4, a new member of the KLK gene family, was found to be expressed to varying levels in a number of endometrial cancer cell lines- HEC1A, HEC1B, Ishikawa, RL95-2 and KLE- at both the mRNA and protein level. On the addition of 10 nmol/L estradiol, progesterone, or a combination of both over a 48 h period, an increase in the intracellular protein levels of K4 were observed when compared to the control (untreated) cells. We have also identified a novel KLK4 transcript with a complete exon 4 deletion. The significance of this alternative transcript, which would give rise to a truncated protein without a serine residue (which is essential for catalytic activity), is yet to be established. These cell lines now provide a model system to study the role of KLK4 and the molecular mechanisms of KLK4 regulation by estrogen and progesterone, in endometrial tumorigenesis.
Endometrial cancer is the fourth most common female malignancy in women in developed countries. Estrogen, and to a lesser degree, progesterone, regulate specific target genes that are involved in endometrial tumorigenesis. A family of proteases involved in cellular proliferation, extracellular matrix degradation and thus, implicated in tumorigenesis, and regulated by estrogen and progesterone in a number of systems, are the tissue kallikreins (KLKs). KLK4, a new member of the KLK gene family, was found to be expressed to varying levels in a number of endometrial cancer cell lines- HEC1A, HEC1B, Ishikawa, RL95-2 and KLE- at both the mRNA and protein level. On the addition of 10 nmol/L estradiol, progesterone, or a combination of both over a 48 h period, an increase in the intracellular protein levels of K4 were observed when compared to the control (untreated) cells. We have also identified a novel KLK4 transcript with a complete exon 4 deletion. The significance of this alternative transcript, which would give rise to a truncated protein without a serine residue (which is essential for catalytic activity), is yet to be established. These cell lines now provide a model system to study the role of KLK4 and the molecular mechanisms of KLK4 regulation by estrogen and progesterone, in endometrial tumorigenesis.
The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.
Recent studies on human kallikrein 2 (hK2) have revealed striking similarities and significant differences with the closely related kallikrein PSA. Both PSA and hK2 are primarily localized to the prostate and share close structural similarities. Although both kallikreins are produced by the same secretory epithelial cells in the prostate, hK2 is associated more with prostate tumors than PSA and is highly expressed in poorly differentiated cancer cells. The potent trypsin-like activity of hK2 contrasts with the weak chymotrypsin-like activity of PSA. The inactive precursor form of PSA, proPSA, is converted rapidly to active PSA by hK2, suggesting an important in vivo regulatory function by hK2 on PSA activity. The high homology between hK2 and PSA results in significant cross-reactivity to hK2 by polyclonal and some monoclonal antibodies to PSA. Future studies on both PSA and hK2 need to take into account this potential for cross-reactivity. Specific monoclonal antibodies to hK2 have now demonstrated that serum levels of hK2, like PSA, are correlated with prostate cancer. The production of hK2 protein in active protease form and specific monoclonal antibodies to the hK2 antigen will allow extensive future studies delineating the physiological and clinical utility of this new prostate antigen.
The human PRSS17 (serine protease 17) gene, which is located on chromosome 19q in a cluster of genes encoding serine proteases, has been variously designated enamel matrix serine proteinase 1 (EMSP1), prostase, KLK4, and KLK-L1. We have cloned and characterized the mouse and human PRSS17 genes. Both have six exons and five introns. The mouse PRSS17 gene sequence is 10134bp; the human sequence is 7115bp. Computer analysis of the mouse PRSS17 gene sequence upstream of the translation initiation codon identified two potential transcription initiation sites, at nucleotides 2878 and 2336. The first nucleotide of the reported mouse PRSS17 cDNA sequence corresponds to position 2352 on the gene, only 16 bases downstream from one of the putative transcription initiation sites. Repetitive DNA sequences from the MSR1 family are found in both the mouse and human PRSS17 genes. Additionally, the human PRSS17 gene contains Tigger2, MER8, and Alu repetitive sequences. Phylogenetic analyses of human and rodent proteases suggest that the PRSS17 protein is not a member of the kallikrein family of serine proteases but that the PRSS17 gene may have originated prior to the divergence of the kallikrein and trypsin families of proteases. To better characterize the timing of PRSS17 expression in developing teeth, we performed in-situ hybridization on postnatal day 3 developing mouse mandibular incisors. PRSS17 mRNA was not detected in secretory stage ameloblasts but could be detected in odontoblasts, while transition-stage and maturation-stage ameloblasts were strongly positive. This pattern supports a role for the PRSS17 protein in the degradation of enamel proteins.
The tissue kallikreins are serine proteases encoded by highly conserved multi-gene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13-26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5' or 3' untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25-44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.
We have purified, cloned and characterized kallistatin, a tissue kallikrein-binding protein (KBP) in humans and rodents. Kallistatin is a unique serine proteinase inhibitor (serpin) with Phe-Phe residues at the P2 and P1 positions. Structural and functional analysis of kallistatin by site-directed mutagenesis and protein engineering indicate that wild-type kallistatin is selective for tissue kallikrein. Kallistatin is expressed and localized in endothelial and smooth muscle cells of blood vessels and has multiple roles in vascular function independent of the tissue kallikrein-kinin system. First, kallistatin induces vasorelaxation of isolated aortic rings and reduces renal perfusion pressure in isolated rat kidneys. Transgenic mice overexpressing rat kallistatin are hypotensive, and adenovirus-mediated gene delivery of human kallistatin attenuates blood pressure rise in spontaneously hypertensive rats. Second, kallistatin stimulates the proliferation and migration of vascular smooth muscle cells in vitro and neointima formation in balloon-injured rat arteries. Third, kallistatin inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia. These results demonstrate novel roles of kallistatin in blood pressure regulation and vascular remodeling.
The human tissue kallikrein gene family was, until recently, thought to consist of only three genes. Two of these human kallikreins, prostate-specific antigen and human glandular kallikrein 2, are currently used as valuable biomarkers of prostatic carcinoma. More recently, new kallikrein-like genes have been discovered. It is now clear that the human tissue kallikrein gene family contains at least 15 genes. All genes share important similarities, including mapping at the same chromosomal locus (19q13.4), significant homology at both the nucleotide and protein level, and similar genomic organization. All genes encode for putative serine proteases and most of them are regulated by steroid hormones. Recent data suggest that at least a few of these kallikrein genes are connected to malignancy. In this review, we summarize the recently accumulated knowledge on the human tissue kallikrein gene family, including gene and protein structure, predicted enzymatic activities, tissue expression, hormonal regulation, and alternative splicing. We further describe the reported associations of the human kallikreins with various human diseases and identify future avenues for research.
Human kallikrein 4 (gene, KLK4; protein, hK4), a recently discovered member of the kallikrein gene family, shares many characteristics with prostate-specific antigen, the best available marker for prostate cancer. Because the protein has not been detected in any human tissue, we attempted to develop immunologic methods for hK4 analysis and use them to detect hK4 in healthy and cancerous tissue extracts and biological fluids.
We extracted total RNA from 20 pairs of matched (healthy-cancer) prostate tissue samples. KLK4 cDNA was amplified by reverse transcription-PCR (RT-PCR) and cloned in a pPICZalphaA expression vector. We then transformed the construct product into Pichia pastoris yeast strains and induced secreted recombinant protein production by addition of methanol. We purified the recombinant protein by nickel ion-affinity chromatography and used it as an immunogen in rabbits and mice to generate polyclonal anti-hK4 antibodies. These antibodies were used to develop a sandwich-type immunoassay suitable for hK4 quantification in biological fluids and tissue extracts.
The immunoassay had a detection limit of 0.1 microg/L. We detected hK4 in 10 of 21 matched (healthy-cancer) prostate tissues, and hK4 was frequently higher in healthy tissues. In one matched-sample pair, the hK4 content was relatively high in both the healthy [4.62 microg/g of total protein (TP)] and the cancerous (1.22 microg/g of TP) prostate tissue. Among tissue extracts, we found the highest concentrations of hK4 in healthy (0.0-4.62 microg/g of TP) and cancerous (0.0-1.72 microg/g of TP) prostatic extracts and in placental extracts (0.0-0.05 microg/g of TP). We also detected traces of hK4 protein immunoreactivity in amniotic fluid (<0.1-0.6 microg/L), human breast milk (<0.1-0.75 microg/L), and seminal plasma (0.2-0.9 microg/L). Immunohistochemical studies showed cytoplasmic staining for hK4 protein in both malignant and benign epithelial cells of the prostate. However, we did not detect hK4 in cerebrospinal fluid, healthy and cancerous ovarian tissue extracts, and many other human tissue extracts.
hK4 protein is present in some prostatic tissue extracts but at relatively low concentrations, although KLK4 mRNA is readily detectable by RT-PCR. We propose that the protein either is not synthesized efficiently or is degraded very quickly.
Maspin is a noninhibitory member of the serpin family that is down-regulated in breast carcinoma but overexpressed in pancreatic carcinoma. There are no published data regarding the role of maspin in ovarian carcinoma, which is the focus of the present study. Ovarian cell lines (normal and cancer) and tumors (80 invasive, 14 benign, and 10 low malignant potential) were evaluated for maspin expression and localization. Normal ovarian surface epithelial cells had low levels of maspin. Two of four ovarian cancer cell lines (OVCAR3 and SKOV3) expressed maspin, whereas the cell line EG had weak expression, and 222 had no detectable maspin. Subcellular fractionation studies revealed that the two maspin-positive ovarian cancer cell lines contained maspin in both the nuclear and cytosolic compartments. Wild-type maspin was transfected into the aggressive ovarian cancer cell lines SKOV3 and 222. The in vitro invasive activity of the maspin-transfected cell lines was 44-68% lower than respective controls. The histopathology analysis revealed that among the ovarian tumors examined, 57 (71%) were ranked positive for maspin. Thirty (37%) of the invasive tumors overexpressed maspin. Invasive cancers were more likely to have predominantly cytoplasmic staining compared with benign and low-malignant-potential tumors. Maspin overexpression was significantly associated with a high tumor grade (P = 0.004), the presence of ascites (P = 0.02), a lower likelihood of optimal surgical cytoreduction (P = 0.04), and a shorter duration of overall survival (median survival, 6.33 versus 2.67 years; P = 0.003). The Cox proportional hazards multivariate model revealed that maspin overexpression and high stage were independent predictors of survival. Thus, maspin was found to be overexpressed in a substantial proportion of ovarian tumors, which may serve as an adverse prognostic factor; however, its localization may provide new clues as to its activity and function. These paradoxical results may offer new insights regarding the role of maspin in ovarian cancer progression that may also impact diagnosis and treatment strategies.
The ability to identify prostate tumor or prostate tissue specific genes that are expressed at high levels and use their protein products as targets could greatly aid in the diagnosis and treatment of prostate cancer. Using a polymerase chain reaction (PCR)-based subtraction technique, we have recovered the recently described KLK4 (prostase) gene from human prostate cDNA. In this study, KLK4 gene expression in human prostate tumors was further characterized using cDNA quantitative PCR and immunohistochemistry, demonstrating that the gene is specifically expressed at both the mRNA and protein levels in normal human prostate tissue, and in both primary and metastatic prostate tumor samples. Quantitative mRNA analysis also demonstrated low level expression including adrenal gland, salivary gland and thyroid. Finally, it was demonstrated that prostate cancer patient sera contain antibodies that bind specifically to recombinant KLK4 protein. This antibody has been used to detect KLK4-specific peptides in epitope mapping experiments. The relatively specific expression profile and elevated level of KLK4 mRNA and protein in both tumor and normal prostate tissues, in addition to detectable KLK4-specific antibody in cancer patient sera, supports additional efforts to determine if KLK4 can play a role in the diagnosis of prostate cancer, the monitoring of residual disease, or act as a target for immunotherapy.
Hippostasin is a kallikrein-like serine protease, which has two alternatively spliced isoforms, brain-type and prostate-type. We previously reported alternative expression of hippostasin in prostate cancer cell lines.
We studied the expression of a variant-form hippostasin (isoform 3) mRNA by RT-PCR. Localization of the isoform 3 protein was examined by immunohistochemistry. The enzymatic activity of the recombinant protein was measured with synthetic substrates.
A novel isoform of hippostasin contains 25 additional amino acids in the catalytic triad of brain-type hippostasin. Its mRNA was expressed in normal prostate tissue, BPH, and prostate cancer cell lines. The protein was localized in the prostate secretory epithelium. The enzyme activity was similar to that of brain-type hippostasin, which has kallikrein-like activity.
In this study, we have identified a third isoform of hippostasin, which was designated variant-form (isoform 3). Hippostasin isoform 3 may play a role in the prostate, including reproductive and/or tumorigenic functions.
To date, prostate-specific antigen (PSA) has proven to be the most useful tumor marker for prostate cancer (1–3). However, the PSA test does not discriminate well between men with benign disease and early prostate cancer, and it does
not discriminate between aggressive and slow-growing cancers (4,5). Although a useful additional discriminator (5), the advent of free/total PSA assays has not significantly improved this outcome. Thus, an inability to make an early diagnosis
has led to the increased burden of metastatic disease, and many patients are overtreated because clinically aggressive disease
cannot be distinguished from clinically insignificant disease.
The disintegrin metalloprotease ADAM-10 is a multidomain metalloprotease that is potentially significant in tumor progression due to its extracellular matrix-degrading properties. Previously, ADAM-10 mRNA was detected in prostate cancer (PCa) cell lines; however, the presence of ADAM-10 protein and its cellular localization, regulation, and role have yet to be described. We hypothesized that ADAM-10 mRNA and protein may be regulated by growth factors such as 5alpha-dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor, known modulators of PCa cell growth and invasion. Experimental Design: ADAM-10 expression was analyzed by in situ hybridization and immunohistochemistry in prostate tissues obtained from 23 patients with prostate disease. ADAM-10 regulation was assessed using quantitative reverse transcription-PCR and Western blot analysis in the PCa cell line LNCaP.
ADAM-10 expression was localized to the secretory cells of prostate glands, with additional basal cell expression in benign glands. ADAM-10 protein was predominantly membrane bound in benign glands but showed marked nuclear localization in cancer glands. By Western blot, the 100-kDa proform and the 60-kDa active form of ADAM-10 were synergistically up-regulated in LNCaP cells treated with insulin-like growth factor I plus 5alpha-dihydrotestosterone. Epidermal growth factor also up-regulated both ADAM-10 mRNA and protein.
This study describes for the first time the expression, regulation, and cellular localization of ADAM-10 protein in PCa. The regulation and membrane localization of ADAM-10 support our hypothesis that ADAM-10 has a role in extracellular matrix maintenance and cell invasion, although the potential role of nuclear ADAM-10 is not yet known.
To assess the value of the precursor form (-7,5pro) of prostate-specific antigen (PSA) and human kallikrein-2 (hK2) for detecting and grading prostate cancer, as better serum markers with improved specificity are needed in men with lower ranges of total (t)PSA.
tPSA, free PSA (fPSA), the precursor (-7,5)proPSA and hK2 were measured in a subset of participants of the European Randomised Study of Screening of Prostate Cancer. In a pilot study, sera from 143 men biopsied but with no prostate cancer, 142 with BPH, and 146 with prostate cancer were analysed to determine the relative value of serum markers for differentiating between the groups. Then, in 141 men with prostate cancer who had a radical prostatectomy, these serum markers were related to the pathological grading to analyse their value as prognostic variables.
Levels of (-7,5)proPSA, hK2 and fPSA could be used to distinguish between BPH and cancer, but proPSA and hK2, alone or combined, did not improve the specificity of fPSA for discriminating BPH and cancer. There was also no correlation between these serum markers and pathological tumour grade.
The clinical effect of using (-7,5)proPSA or hK2 for detecting and grading prostate cancer remains limited.
The presence of more than one mRNA form is common among kallikrein genes. We identified an mRNA transcript of the human kallikrein gene 5 (KLK5), denoted KLK5 splice variant 1 (KLK5-SV1). This variant has a different 5'-splice site, but encodes the same protein as the classical KLK5 transcript. RT-PCR analysis of this variant transcript expression in 29 human tissues indicated highest expression in the cervix, salivary gland, kidney, mammary gland, and skin. Comparative analysis of the expression levels of KLK5-SV1, another splice variant named KLK5 splice variant 2 (KLK5-SV2), and the classical KLK5 form showed that out of all three mRNA transcripts, the classical form is predominantly expressed (found in more tissues and at higher expression levels) followed by KLK5-SV1. KLK5-SV1 is expressed at high levels in ovarian, pancreatic, breast and prostate cancer cell lines. KLK5-SV1 was also found to be expressed in 9/10 ovarian cancer tissues, but it was not found in one normal ovarian tissue tested. Hormonal regulation experiments suggest that KLK5-SV1 is regulated by steroid hormones in the BT-474 breast cancer cell line. Furthermore, this variant had significantly higher expression in normal prostate tissues compared to their matched cancer tissue counterparts. KLK5-SV1 may have clinical utility in various malignancies and should be further explored as a potential new biomarker for prostate and ovarian cancer.
Human tissue kallikreins (hKs), which are encoded by the largest contiguous cluster of protease genes in the human genome, are secreted serine proteases with diverse expression patterns and physiological roles. Although primarily known for their clinical applicability as cancer biomarkers, recent evidence implicates hKs in many cancer-related processes, including cell-growth regulation, angiogenesis, invasion and metastasis. They have been shown to promote or inhibit neoplastic progression, acting individually and/or in cascades with other hKs and proteases, and might represent attractive targets for therapeutic intervention.
To investigate the clinical utility of the subforms of free prostate-specific antigen (PSA), namely proPSA and "benign" PSA (BPSA), to improve cancer detection when the percent free PSA level is less than 15%. Percent free PSA, while maintaining sensitivity, has greatly improved the specificity of PSA for the early detection of prostate cancer. A low percent free PSA value indicates a greater risk of cancer, but only 30% to 50% of men with percent free PSA levels of less than 15% actually have cancer at biopsy.
Archived sera from 161 consecutive men who were prospectively enrolled in our Early Detection Research Network prostate cancer early detection biomarker program with a percent free PSA value of less than 15% were included in the study. Total PSA, free PSA, proPSA, and BPSA were measured for each sample.
The mean total PSA was 6.1 ng/mL (range 1.8 to 24.0). The mean age of the study group was 62 +/- 7 years. Prostate cancer was detected in 66 (41%) of 161 men. The area under the curve-receiver operating characteristic for total and percent free PSA was 0.51 and 0.54, respectively. BPSA and proPSA/BPSA both improved cancer detection compared with percent free PSA alone; the improvement was statistically significant (P <0.001) . The area under the curve-receiver operating characteristic for proPSA/BPSA was 0.72, giving a sensitivity and specificity of 90% and 46%, respectively.
The results of our preliminary studies have suggested that the ratio of proPSA and BPSA can distinguish cancer with greater accuracy when the percent free PSA value is very low (less than 15%), and may, therefore, provide better clinical utility in this lower range of percent free PSA.
Alternative splicing is prevalent within the human tissue kallikrein gene locus. Aside from being the most important source of protein diversity in eukaryotes, this process plays a significant role in development, physiology and disease. A better understanding of alternative splicing could lead to the use of gene variants as drug targets, therapeutic agents or diagnostic markers. With the rapidly rising number of alternative kallikrein transcripts, classifying new transcripts and piecing together the significance of existing data are becoming increasingly challenging. In this review, we present a systematic analysis of all currently known kallikrein alternative transcripts. By defining a reference form for each of the 15 kallikrein genes (KLK1 to KLK15), we were able to classify alternative splicing patterns. We identified 82 different kallikrein gene transcript forms, including reference forms. Alternative splicing may lead to the synthesis of 56 different protein forms for KLK1-15. In the kallikrein locus, the majority of alternative splicing events occur within the protein-coding region, and to a lesser extent in the 5' untranslated regions (UTRs). The most common alternative splicing event is exon skipping (35%) and the least common events are cryptic exons (3%) and internal exon deletion (3%). Seventy-six percent of kallikrein splice variants that are predicted to encode truncated proteins are the result of frameshifts. Eighty-nine percent of putative proteins encoded by splice variants are predicted to be secreted. Although several reports describe the identification of kallikrein splice variants and their potential clinical utility, this is the first extensive review on this subject. Accumulating evidence suggests that alternative kallikrein forms could be involved in many pathologic conditions or could have practical applications as biomarkers. The organization and analysis of the kallikrein transcripts will facilitate future work in this area and may lead to novel clinical and diagnostic applications.
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