Differential Splicing of KLK5 and KLK7 in Epithelial Ovarian Cancer Produces Novel Variants with Potential as Cancer Biomarkers 1 Ying Dong, Aneel Kaushal, Maria Brattsand, Jim Nicklin, and Judith A. Clements 2 The Center of Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4001, Australia [Y. D., A. K., J. A. C.]; Department of Public Health and Clinical Medicine, Dermatology and Venereology, Umeå University, Umeå, Sweden [M. B.]; and Department of Obstetrics and Gynecology, Royal Women’s Hospital, Herston, Brisbane, Queensland, Australia [J. N.] ABSTRACT Purpose: 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 expres- sion of KLK5/hK5 and KLK7/hK7 and their association and to determine whether cancer-related variant transcripts were expressed. Experimental Design: 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 ovar- ian cancer cell lines using semiquantitative RT-PCR; South- ern, Northern, and Western blot analyses; and immunohis- tochemistry techniques. Results: We showed the concordant higher expression of both KLK5/hK5 and KLK7/hK7 in ovarian carcinomas, especially late-stage serous carcinomas, compared with nor- mal 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 West- ern blot and immunohistochemistry analyses showed that these two enzymes are secreted from ovarian carcinoma cells. Conclusions: 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. INTRODUCTION Ovarian carcinoma is the second most common gyneco- logical malignancy and the leading cause of death from gyne- cological malignancy (1). The overall 5-year survival rate of ovarian cancer patients is less than 50% because most of these patients are diagnosed at an advanced stage of the disease, in which the primary tumor has progressed to a highly invasive and metastatic state (2). Thus, development of a reliable early diag- nostic system is very important in the management and treat- ment of ovarian carcinomas. The KLKs 3 are a subgroup of serine proteases that have recently been expanded to 15 members, and the KLK genes localized to chromosome 19q13.4 (3, 4). Many of these genes are highly expressed in ovarian cancer in comparison with normal ovary. We have previously shown high expression of KLK4 (alternatively named prostase, KLK-L1, EMSP-1, and ARM1) and its mRNA variants, as well as the hK4 protein, in ovarian epithelial carcinoma (5). Others have shown that higher KLK4 mRNA levels in ovarian cancer tissues are related to poor prognosis (6). Aberrant expression of KLK6/hK6 [alternatively named protease M, zyme, and neurosin (7–9)], KLK8/hK8 [neu- ropsin, ovasin, tumor-associated differentially expressed gene- 14, and TADG-14 (10, 11)], KLK9 (12), KLK10 [normal epi- thelial-specific 1 gene and NES1 (13)], and KLK11 [trypsin-like serine protease, TLSP, and hippostasin (14)] was also found in ovarian carcinomas. KLK5 [alternatively named stratum cor- neum tryptic enzyme and SCTE (15)] and KLK7 [stratum cor- neum chymotryptic enzyme and SCCE (16, 17)] were originally identified from a keratinocyte library, and their enzymes were purified from stratum corneum of human skin. KLK7 catalyzes the degradation of intercellular cohesive structures in the outer- Received 9/30/02; revised 1/3/03; accepted 1/10/03. 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. 1 Supported by Queensland University of Technology Re-entry Fellow- ship for Women and National Health and Medical Research Council of Australia. 2 To whom requests for reprints should be addressed, at the Center of Molecular Biotechnology, School of Life Sciences, Queensland Univer- sity of Technology, P. O. Box 2434, Brisbane, Queensland 4001, Aus- tralia. Phone: 617-3864-1899; Fax: 617-3864-1534; E-mail: j.clements@ qut.edu.au. 3 The abbreviation used are: KLK, kallikrein; NOE, normal ovarian epithelial; BNG, benign serous adenoma; SER, serous; MUC, muci- nous; END, endometrioid; CCC, clear cell carcinoma; GCT, granulosa cell tumor; nt, nucleotide; aa, amino acid(s); DIG, digoxigenin; RT- PCR, reverse transcription-PCR; UTR, untranslated region; AU, absorb- ance unit(s); EST, expression sequence tag; RACE, rapid amplification of cDNA ends; PI, protease inhibitor; ALP, antileukoprotease. 1710 Vol. 9, 1710 –1720, May 2003 Clinical Cancer Research Cancer Research. on December 9, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from