KLK4T2 Is a Hormonally Regulated Transcript from the KLK4 Locus

The Prostate, 2008

BACKGROUND. The kallikrein-related (KLK) serine protease, prostate specific antigen is the current marker for prostate cancer (PCa). Other members of the KLK family are also emerging as potential adjunct biomarkers for this disease. Our aim was to identify and characterize novel KLK-related genes with potential as PCa bio-markers. METHODS. Low stringency DNA screening was coupled with amplification techniques to identify novel sequences. Transcripts were examined by Northern blot, RT-PCR, and in situ hybridization analysis and in silico bioinformatics approaches. Protein characterization was performed by Western blot and confocal microscopy analysis. Gene regulation studies were performed by quantitative PCR and promoter reporter assays. RESULTS. We identified a novel kallikrein-related mRNA designated KRIP1 (kallikreinrelated, expressed in prostate 1) which, together with the recently reported CKLK1 and KLK31P transcripts, is transcribed from KLKP1; a gene evolved from, and located within, the KLK locus. Significantly, in contrast to these other non-coding KLKP1 transcripts, the KRIP1 mRNA generates an $18 kDa intracellular protein-the first non-serine protease identified from the KLK locus. KRIP1 mRNA is abundant only in normal prostate and is restricted to cells of epithelial origin in normal and diseased glands. Ligand binding of the androgen receptor increases transcription from the KLKP1 gene. Consistently, KRIP1 mRNA levels are lower in PCa samples compared to benign prostatic hyperplasia. CONCLUSIONS. Transcription from KLKP1 is reduced as cells de-differentiate on the pathway to malignancy. KLKP1/KRIP1 has potential as a marker of both PCa progression and recent evolutionary events within the KLK locus.

medicallaboratory journal, 2019

Background and Objectives: Prostate cancer is a highly prevalent malignancy with a high mortality rate in men. Many studies have investigated the diagnostic value of various genes involved in prostate cancer, but there is no data for Kallikrein 2 (KLK2) and Kallikrein 3 (KLK3) expression patterns among Iranian patients. Therefore, we aimed to evaluate the expression of these two genes in Iranian patients with prostate cancer. Methods: In this case-control study, three groups consisting of healthy individuals, patients with benign prostatic hyperplasia (BPH) and patients with prostate cancer were studied. Peripheral blood samples were collected from all subjects, mRNA was extracted after cell lysis, and cDNA was synthesized. Real-time PCR was performed to assess gene expression levels relative to a reference gene (18s rRNA gene). Results: The KLK2 gene was overexpressed in patients with prostate cancer. KLK2 expression differed significantly between the cancer patients and controls. Relative expression of the KLK3 gene in the BPH group was higher than that in the control and cancer groups. However, we observed no significant difference in the expression of KLK3 between the control and cancer subjects. Conclusion: We demonstrate that analysis of KLK2 expression is a favorable method of diagnosing prostate cancer and discriminating normal individuals from those with BPH or prostate cancer. We also found that the KLK3 gene is highly overexpressed in individuals with BPH, which might indicate that this gene is not cancer-specific.

Genetics and Molecular Biology, 2006

We used the multiplex semi-quantitative reverse-transcriptase PCR (RT-PCR) to investigate kallikrein 2 and 3 (KLK2 and KLK3) mRNA levels in prostate tissue from 42 prostate cancer patients, 33 of whom were also assessed for peripheral blood KLK2 expression by qualitative semi-nested RT-PCR. We found that KLK2 was an important tissue biomarker for distinguishing between prostate cancer patients and those with benign prostatic hyperplasia, particularly when KLK2 expression was > 60% of that of the β2-microglobulin constitutive gene. Patients with an average relative expression value ≥ 0.6 (cutoff value) had an eleven-fold higher chance of having prostate cancer. When one or two tissues samples were evaluated for KLK2 expression using the cutoff value the estimated chance of having prostate cancer was increased by seven times for one positive sample and 45 times for two positive samples. There was no significant correlation between KLK3 gene expression and prostate cancer diagnosis. Logistic regression for blood and tissue KLK2 expression successfully detected 92% of the prostate cancer cases. The detection of KLK2 in blood showed a sensitivity of 59% and a specificity of 82%. This study indicates that the KLK2 gene may be a useful molecular marker for the diagnosis of prostate cancer and that analysis of KLK2 expression in blood and tissues could provide a novel approach for the clinical investigation of this type of cancer.

The Prostate, 2006

BACKGROUND. Fifteen human tissue kallikrein (KLK) genes have been identified as a cluster on chromosome 19. KLK expression is associated with various human diseases including cancers. Noncoding RNAs such as PCA3/DD3 and PCGEM1 have been identified in prostate cancer cells. METHODS. Using massively parallel signature sequencing (MPSS) technology, RT-PCR, and 5 0 rapid amplification of cDNA ends (RACE), we identified and cloned a novel gene that maps to the KLK locus. RESULTS. We have characterized this gene, named as KLK31P by the HUGO Gene Nomenclature Committee, as an unprocessed KLK pseudogene. It contains five exons, two of which are KLK-derived while the rest are ''exonized'' interspersed repeats. KLK31P is expressed abundantly in prostate tissues and is androgen regulated. KLK31P is expressed at lower levels in localized and metastatic prostate cancer cells than in normal prostate cells. CONCLUSIONS. KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that may play a role in prostate carcinogenesis or maintenance.

The Prostate, 2003

BACKGROUND. Many members of the human kallikrein gene family are differentially expressed in cancer and a few have potential as diagnostic/prognostic markers. KLK14 is a newly discovered human kallikrein gene that is mainly expressed in the central nervous system and endocrine tissues. Since KLK14 was found to be regulated by steroid hormones in prostate cancer cell lines, we hypothesized that it will be differentially expressed in prostate cancer tissues compared to their normal counterparts. METHODS. Matched prostate tissue samples from the cancerous and non-cancerous parts of the same prostates were obtained from 100 patients who underwent radical prostatectomy. Quantitative analysis of KLK14 expression levels were performed by real-time RT-PCR using SYBR Green I dye on the LightCycler TM system. Associations with clinico-pathological parameters were analyzed. RESULTS. KLK14 overexpression in the cancerous compared to non-cancerous tissue was found in 74% of patients (P < 0.001). Mean level of expression was 154 arbitrary units (Au) in cancerous tissues and 14.2 Au in the non-cancerous tissues. The ratio of the cancerous to noncancerous KLK14 expression values was higher in patients with late stage (stage III) compared to stage II (P ¼ 0.002), and in grade 3 compared to grade 1/2 tumors (P ¼ 0.001). A statistically significant increase was also observed in patients with higher in Gleason score (>6) compared to Gleason score ¼ 6 tumors (P ¼ 0.027). No correlation was found between KLK14 tissue expression levels and serum prostate-specific antigen. CONCLUSIONS. KLK14 expression is significantly higher in cancerous compared to noncancerous prostatic tissue. The up-regulation of the KLK14 gene in advanced and more aggressive tumors may indicate a possible role for the hK14 protein in tumor spread and opens the possibility of hK14 being a candidate new marker for prostate cancer diagnosis and prognosis.

Journal of Biological Chemistry, 2002

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and human kallikrein 2 are produced and secreted in the prostate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3 genes that result from inclusion of intronic sequences adjacent to the first exon. The novel proteins encoded by these transcripts, named PSA-linked molecule (PSA-LM) and hK2-linked molecule (K-LM), share only the signal peptide with the original protein product of the respective gene. The mature proteins are entirely different and bear no similarity to the kallikrein family or to other proteins in the databases. As is the case with PSA, PSALM is expressed in the secretory epithelial cells of the prostate and is up-regulated in response to androgenic stimulation. A similar pattern of expression is suggested for K-LM. Prostate-specific antigen (PSA) 1 is considered the best cancer biomarker currently available and is widely used for screening, diagnosis, and monitoring of prostate cancer (1, 2). PSA (also named human kallikrein 3, hK3) is encoded by the KLK3 gene (3), a member of the human kallikrein gene family (4). These genes are clustered on chromosome 19q13.3-13.4 and have a highly conserved structural organization (4, 5). Another member of the kallikrein gene family, KLK2, displays 80% homology with KLK3 (3, 6) and is located adjacent to it (4). The KLK2 protein product, human kallikrein 2 (hK2; previously known as human glandular kallikrein 1, or hGK1), is emerging as an additional prostatic tumor marker (7). The kallikrein genes code for a group of serine proteases in-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Journal of Thrombosis and Haemostasis, 2006

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5 0 -truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclearlocalized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles. 12 875-889

DNA and Cell Biology, 2001

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. 435

Thrombosis and Haemostasis, 2013

The kallikreins are a family of serine proteases with a range of tissue-specific and essential proteolytic functions. Among the most well-studied are the prostate tissue specific KLK2 and KLK3 genes and their secreted protease products, hk2 and PSA. Members of the so-called classic kallikreins, these highly active trypsin-like serine proteases play established roles in human reproduction. Both hK2 and PSA expression is regulated by the androgen receptor, whose activity has a fundamental role in prostate tissue development and progression of disease. This feature, combined with the ability to sensitively detect different forms of these proteins in blood and biopsies, result in a crucially important biomarker for the presence and recurrence of cancer. Emerging evidence has begun to suggest a role for these kallikreins in critical vascular events. This review discusses the established and developing biological roles of hK2 and PSA, as well as the historical and advanced use of their detection to accurately and non-invasively detect and guide treatment of prostatic disease.

Biological Chemistry, 2010

KLK4 is a member of the human kallikrein-related peptidase family of (chymo)trypsin-like serine proteases. The aim of the present study was to generate polyclonal antibodies (pAb) directed against KLK4 for the analysis of KLK4 by immunohistochemistry in human tissues. Recombinantly expressed human mature KLK4 was used for immunization of chickens. pAb 617A is an affinity-purified monospecific pAb fraction reacting with a linear epitope within a flexible surface-exposed loop of KLK4. pAb 617C is the KLK-directed pAb fraction completely depleted from pAb 617A. In healthy adult tissues, KLK4 was immunodetected by both antibody fractions in kidney, liver, and prostate, but not in other organs such as colon and lung. To evaluate protein expression of KLK4 in prostate cancer, samples of tumor tissue plus corresponding tumor-free areas of 44 prostate cancer patients, represented on a tissue microarray, were investigated. Distinct KLK4 immunostaining was observed with both antibodies in can...