Oncogene (2002) 21, 7114 – 7120 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc SHORT REPORTS Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera Craig H Day*,1, Gary R Fanger1, Marc W Retter1, Bonnie L Hylander2, Remedios B Penetrante2, Raymond L Houghton1, Xinqun Zhang1, Patricia D McNeill1, Aristides Maltez Filho3, Marcos Nolasco3, Roberto Badaro3, Martin A Cheever1, Steven G Reed1,4,5, Davin C Dillon1 and Yoshihiro Watanabe1 1Corixa Corporation, 1124 Columbia Street, Suite 200, Seattle, Washington, WA 98104, USA; 2Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Street, Buffalo, New York, NY 14263, USA; 3Hospital Aristides Maltez, Bahia, Brazil; 4Department of Pathobiology, University of Washington, Box 357238, Seattle, Washington, WA 98195, USA; 5Infectious Disease Research Institute, 1124 Columbia Street, Suite 600, Seattle, Washington, WA 98104, USA The ability to identify prostate tumor or prostate tissue radiation therapy. Either technique can fail to eliminate specific genes that are expressed at high levels and use their all tumor cells, resulting in recurrence of disease. This protein products as targets could greatly aid in the possibility is increased if diagnosis of prostate cancer is diagnosis and treatment of prostate cancer. Using a made late, with extracapsular involvement. polymerase chain reaction (PCR)-based subtraction A number of human prostate-specific antigens have technique, we have recovered the recently described previously been identified, including prostate-specific KLK4 (prostase) gene from human prostate cDNA. In antigen (PSA), prostatic acid phosphatase and pros- this study, KLK4 gene expression in human prostate tate-specific membrane antigen. PSA is currently a tumors was further characterized using cDNA quantitative well-established clinical marker as an indicator for PCR and immunohistochemistry, demonstrating that the prostate cancer (Daher and Beaini, 1998), with the gene is specifically expressed at both the mRNA and advantages that it is secreted and enters the circulatory protein levels in normal human prostate tissue, and in both system, and that virtually all primary prostate tumors primary and metastatic prostate tumor samples. Quanti- maintain PSA expression. Increasing levels of PSA in tative mRNA analysis also demonstrated low level peripheral blood has been shown to be an effective expression including adrenal gland, salivary gland and clinical marker useful in detecting the presence of thyroid. Finally, it was demonstrated that prostate cancer prostate cancer, either in the initial diagnosis or in the patient sera contain antibodies that bind specifically to detection of disease progression following procedures recombinant KLK4 protein. This antibody has been used to such as radical prostatectomy (Abi-Aad et al., 1992). detect KLK4-specific peptides in epitope mapping experi- However, two problematic issues remain with utilizing ments. The relatively specific expression profile and PSA as a marker for the diagnosis of prostate cancer elevated level of KLK4 mRNA and protein in both tumor and the detection of residual disease. First, there is a and normal prostate tissues, in addition to detectable high level of false positive tests due to PSA expression KLK4-specific antibody in cancer patient sera, supports in benign prostatic hyperplasia that result in unneces- additional efforts to determine if KLK4 can play a role in sary biopsies. Second, PSA can fail as a marker for the diagnosis of prostate cancer, the monitoring of residual residual disease since not all metastases maintain disease, or act as a target for immunotherapy. expression of PSA (Daher and Beaini, 1998; Sissons Oncogene (2002) 21, 7114 – 7120. doi:10.1038/sj.onc. et al., 1992), and the presence of the PSA gene by 1205786 reverse transcription (RT) – PCR from blood does not unequivocally indicate specific prostatic metastases Keywords: antibody; KLK4; prostase; prostate cancer; (Thiounn et al., 1997). Due to these limitations, protein expression additional markers for prostate cancer are needed. Recently, an additional prostate-specific androgen- regulated serine protease was identified, termed Prostate cancer is the most common cancer among men, prostase (Nelson et al., 1999), KLK-L1 (kallikrein- and is the second leading cause of male cancer deaths in like1) (Yousef et al., 1999) and also KLK4 (Stephenson the US (Grenlee et al., 2000). The current treatment et al., 1999). The initial characterization of this gene options include prostatectomy or prostate ablation by (Nelson et al., 1999) demonstrated that prostase mRNA expression is restricted to normal prostate tissue and the prostate tumor cell line LNCaP. The *Correspondence: C Day; E-mail: [email protected] prostase cDNA was shown to encode a putative 254 Received 22 March 2002; revised 12 June 2002; accepted 18 June amino acid polypeptide, and was radiation-hybrid- 2002 panel mapped to chromosome 19q13, a region contain- Prostase characterization and expression in prostate CH Day et al 7115 ing several other serine proteases including three expression was detected in normal lung or colon tissue known human kallikreins (hK1, hK2 and hK3 samples. [PSA]). A genomic analysis of this region more Quantitative PCR was then performed to determine precisely mapped and localized the prostase gene and mRNA levels of KLK4 in prostate tumors, normal the related positions of other known genes in the area prostates and other normal human tissues. As shown in including PSA, KLK1, KLK2, zyme and neuropsin Figure 1b, increased expression of KLK4 mRNA is (Yousef et al., 1999). An additional study mapped the evident in all 22 prostate tumors and all three normal kallikrein-related prostase gene to the KLK locus, and prostate samples analysed. Elevated expression of based upon sequence similarities and like-character- KLK4 is evident in one metastatic prostate tumor istics amongst the three human kallikrein genes, sample (met-lymph node), and low but detectable proposed prostase be designated the fourth human expression is apparent in a second metastatic prostate kallikrein (KLK4) (Stephenson et al., 1999). Fluores- tumor sample (met-ascites). Elevated expression of cence in situ hybridization analysis confirmed the KLK4 is also evident in all three of the benign localization of KLK4, or PRSS17, to the long arm of prostatic hyperplasia (BPH)-positive samples examined human chromosome 19, and more specifically as compared to non-prostate samples. In addition, low narrowed the gene’s location to 19q13.3?q13.4 but detectable levels of expression were observed in the (DuPont et al., 1999). normal adrenal, salivary and thyroid gland samples, In this study we quantitatively characterize KLK4 while the remaining 29 normal tissues tested yielded expression, extending the mRNA expression profile of exceedingly low or no detectable quantities of amplified KLK4 by examining primary and metastatic human KLK4 mRNA. prostate tumors as well as mRNA expression char- To characterize KLK4 expression at the protein acteristics of a large panel of human cell lines. In level, both rabbit polyclonal antisera and monoclonal addition, anti-KLK4 antibodies were generated and antibodies were produced. Recombinant KLK4 was used to demonstrate that KLK4 is a secreted protein. generated by expression of a six histidine-tagged, C- Using immunohistochemical analysis, the prostate- terminal 159 amino acid fragment in E. coli and the specific expression profile was confirmed at the protein product was purified using nickel-nitrilotriacetic acid level. Lastly, we demonstrate that sera from patients (NTA) column chromatography for subsequent use as with prostate cancer contain antibody specific to an immunogen in rabbits. Polyclonal antisera was recombinant KLK4 protein. Collectively, these findings tested for KLK4 specificity and then affinity purified suggest further evaluation of KLK4 as a diagnostic for use in Western blot and IHC analysis. marker and as a potential therapeutic target. To determine if the KLK4 gene encoded a secreted To date, characterization of the expression of this protein, it was cloned into the pcDNA3.1 mammalian new serine protease family member has focused on expression vector and used to transfect HEK293 cells. mRNA levels in human normal tissues and prostate Analysis of transiently transfected HEK293 cell lysates tumor cell lines (Nelson et al., 1999; Yousef et al., revealed the expression of an *22 kD protein that is 1999), hormonal regulation of a human endometrial recognized by the anti-KLK4 affinity purified rabbit cancer cell line (Myers and Clements, 2001), serous sera (data not shown). Analysis of supernatants ovarian carcinomas (Dong et al., 2001) and on recovered from the same transfection revealed the expression levels as an indicator of ovarian cancer expression of an *25 kD protein due to glycosylation. patient prognoses (Obiezu et al., 2001). In order to Furthermore, N-terminal amino-acid sequence analysis extend the characterization of the tissue-specific revealed the cleavage of the putative signal sequence expression of KLK4, cDNA quantitative PCR was (residues 1 – 26) (Nelson et al., 1999), retaining the used to analyse the expression of KLK4 in a large presence of the four amino acid propeptide (SCSQ) panel of samples, including normal prostate, primary indicating incomplete cleavage of the protein into its and metastatic prostate tumors, human cell lines and a predicted