Identification of Single Nucleotide Polymorphisms in the Human Kallikrein10 KLK10) Gene and Their Associationwith Prostate,Breast,Testicular, and Ovarian Cancers
Total Page：16
File Type：pdf, Size：1020Kb
The Prostate 51:35 ^ 41 2002) Identification of Single Nucleotide Polymorphisms in the Human Kallikrein10 KLK10) Gene and Their AssociationWith Prostate,Breast,Testicular, and Ovarian Cancers Bhupinder B. Bharaj,1,2 Liu-Ying Luo,1,2 Klaus Jung,3 Carsten Stephan,3 and Eleftherios P. Diamandis1,2* 1Department of Pathologyand Laboratory Medicine, Mount Sinai Hospital,Toronto,Ontario,Canada 2Department of Laboratory Medicine and Pathobiology,University of Toronto,Toronto,Ontario,Canada 3Department of Urology,University Hospital Charite, Humboldt University,Berlin,Germany BACKGROUND. The KLK10 gene $also known as the normal epithelial cell-speci®c 1 gene) is a member of the expanded human kallikrein gene family. Recently, it has been reported that KLK10 is a tumor suppressor gene and that its expression is downregulated in various forms of cancer and cancer cell lines. KLK10 is also upregulated in ovarian cancer. We thus hypo- thesized that the KLK10 gene may be a target for mutations in various cancers. METHODS. We sequenced the ®ve coding exons of the KLK10 gene using genomic DNA from various tumors, normal tissues, and blood, by PCR ampli®cation and automated sequencing. RESULTS. In none of the tumor-derived DNAs, we identi®ed somatic mutations that could inactivate this gene. However, we identi®ed a prevalent germline single nucleotide variation at codon 50 $exon 3) of this gene [GCC $alanine) to TCC $serine)]. The GCC genotype was less prevalent in prostatic cancer patients in comparison to control subjects $P 0.027) but no differences were seen with testicular, ovarian, and breast cancer. We also identi®ed four genetic variations in exon 4, at codons106 [GGC $glycine) to GGA $glycine)], codon 112 [ACG $threonine) to ACC $threonine)], codon 141 [CTA $leucine) to CTG $leucine)], and at codon 149 [CCG $proline) to CTG $leucine)]. None of these variations was signi®cantly different between normal subjects and cancer groups. CONCLUSIONS. We found no evidence for somatic mutations of the KLK10 gene in cancers of the prostate, breast, ovary, and testis. The single nucleotide variation at codon 50 appears to be associated with prostate cancer risk. Prostate 51: 35±41, 2002. # 2002 Wiley-Liss, Inc. KEY WORDS: single nucleotide polymorphisms; normal epithelial cell speci®c-1 gene; kallikreins; human kallikrein 10; cancer biomarkers; serine proteases INTRODUCTION Prostate cancer is one of the leading causes of death in men and accounts for approximately 30% of all newly diagnosed cancers [1]. Molecular genetic studies have revealed a few mutations that seem to be res- Grant sponsor: Natural Sciences and Engineering Research Council ponsible for predisposition to prostate cancer or for of Canada $NSERC); Grant sponsor: Visible Genetics, Inc. early tumor progression [2,3]. With the advent of *Correspondence to: Eleftherios P. Diamandis, Department of Patho- logy and Laboratory Medicine, Mount Sinai Hospital, 600 University DNA ampli®cation and new DNA variation detection Avenue, Toronto, Ontario, M5G 1X5, Canada. technologies, germline polymorphisms have been E-mail: [email protected] identi®ed in many genes and throughout the whole Received 17 August 2001; Accepted 14 December 2001 human genome. These polymorphisms have become DOI 10.1002/pros.10076 ß2002Wiley-Liss,Inc. 36 Bharaj et al. particularly attractive potential markers of cancer risk secreted and found in many biological ¯uids [20]. and/or disease progression. Some polymorphisms can Moreover, we found high levels of hK10 in ovarian alter the protein function or concentration in a bio- tumors; these levels correlate positively with ovarian chemically relevant direction or show a population tumor aggressiveness [21]. Also, serum levels of hK10 distribution that correlates with well-known ethnic may have value for ovarian cancer diagnosis and cancer risk pro®les [4]. Their presence in germline monitoring [22]. However, the mechanism of hK10 up- implies that they have the potential to affect early regulation in ovarian cancer is still obscure. events that initiate carcinogenesis in many tissues, in- A number of tumor suppressor genes are in- cluding the prostate, believed by many to occur at activated by mutations and many single nucleotide puberty or even earlier, during fetal life [5,6]. Also, polymorphisms are associated with malignant dis- they can affect decades-long progression of carcino- eases [23±26]. Given the previous information that genesis, promoted by continued trophic stimulation KLK10 may be a tumor suppressor [19] and that the and division of cells. expression of this gene is altered malignancies [16,19± Serine proteases are a homologous family of protein 22], we hypothesized that KLK10 may be a target for degrading enzymes which are involved in a host of mutations or cancer-predisposing polymorphisms. biological functions, such as coagulation and ®brino- We thus examined the mutational and polymorphic lysis, digestion and activation of the kringle domain status of this gene in DNA isolated from prostate, containing growth factors, like human tissue plasmi- breast, ovarian, and testicular tumors as well as from nogen activator [7,8]. The expression of serine pro- normal tissues. teases, such as plasminogen activator, as well as other classes of proteinases, has been shown to correlate MATERIALS AND METHODS positively with invasiveness and metastatic potential of many tumor cells [9±14]. This is linked to the ability Prostate and Testicular CancerTissues of the tumors to degrade extracellular matrix com- Prostate tissue samples were obtained from 13 ponents, either directly, or indirectly, through the pro- patients who had undergone radical retropubic pros- teolytic activation of other zymogens. tatectomy for prostate adenocarcinoma at the Charite Many serine proteases are secreted into the extra- University Hospital, Berlin, Germany. The patients cellular space in order to function [7]. Some of these did not receive any hormonal therapy before surgery. enzymes can serve as tumor markers. Prostate-speci®c The use of these tissues was approved by the ethics antigen $PSA), a serine protease, is widely used as a CommitteeoftheChariteHospital.Freshtissuesamples tumor marker for the diagnosis and monitoring of were obtained from the cancerous and non-cancerous prostate cancer [15]. parts of the same prostates $matched) that had been The normal epithelial cell-speci®c 1 gene $NES1) removed. Small pieces of the samples were dissected was identi®ed by subtractive hybridization, by virtue immediately and stored in liquid nitrogen until ana- of its downregulation in breast cancer cell lines [16]. lysis. Testicular samples were procured similarly. The gene has been predicted to encode for a secreted serine protease. The NES1 gene, now of®cially known as human kallikrein 10 $KLK10) [17], has high se- Whole Blood FromHealthy Controls quence homology with three protease families, includ- and Prostate Cancer Patients ing the trypsin family, the kringle family, and the Fifty-two healthy males who had no evidence of human kallikrein family [16]. The KLK10 gene spans prostate cancer and total PSA < 2 mg/L and 49 age- 5.5 kb of genomic DNA, contains ®ve coding exons matched patients with histologically con®rmed pros- and one non-coding exon, and resides on chromosome tate cancer, treated with radical prostectomy, were 19q13.4 in the same region where the human kallikrein also included in this study. Blood was collected in family is located [7,18]. This family is now known to heparinized tubes and DNA was extracted as des- contain 15 different genes [7]. cribed below. The KLK10 gene is downregulated in breast and other cancer cell lines [16,19]. Its expression has been Ovarian and Breast Cancer Specimens associated with tumorigenic progression, suggesting that the products of KLK10 proteolytic cleavage are Thirty-four ovarian cancer tissues were obtained likely to be involved in the negative growth regulation from patients who underwent surgery for primary of epithelial cells. Thus, KLK10 may encode for a tumor ovarian carcinoma at Mount Sinai Hospital. Twenty- suppressor protein [19]. seven breast cancer tissues were obtained from Recently, we have shown that the protein encoded women who had undergone surgical treatment for by the KLK10 gene $hK10, human kallikrein 10) is primary breast carcinoma at Mount Sinai Hospital. KLK10 Gene Polymorphisms and Cancer 37 Our protocols have been approved by the Institu- the ampli®ed PCR product on a 1% agarose gel con- tional Review Boards of the Charite University Hospi- taining ethidium bromide. tal, Berlin, Germany and the University of Toronto, Ontario, Canada. DNASequencing DNAExtraction Both strands of the PCR products were sequenced on the Open Gene LR Automated DNA Sequencer DNA was extracted from tissues and whole blood $Visible Genetics Inc., Toronto, Ontario Canada), fol- using the Qiagen QIAmp blood and tissue DNA ex- lowing a general protocol described elsewhere [27]. traction kit $Qiagen, Chatsworth, CA). Approximately The sequencing primers were labeled at the 50 end 25 mg of tissue or 200 mL of whole blood was used for with the ¯uorescent dye Cy 5.5 $Visible Genetics, Inc.). each extraction. Tissues were pulverized into a ®ne The detailed sequences of the nested labeled primers powder on dry ice. Otherwise, we followed the manu- used for sequencing are depicted in Table I. facturer's recommendations. PSAImmunoassay PCR Amplif|cation of the KLK10 Gene The determination of total PSA in all serum samples The primer sequences ¯anking exons 1 through 6 was performed by a time-resolved immuno¯uoro- were designed using Oligo 5.0 software $National Bio- metric assay as described elsewhere [28]. All speci- sciences, Plymouth, MN) according to the KLK10 geno- mens were measured in duplicate. mic DNA sequence deposited in GenBank $accession #AF055481). A detailed description of the primers hK10 Immunofluorometric Assay used to amplify each of the six exons is shown in Table I. PCR ampli®cation was performed in a ®nal The assay for hK10 protein in tumor extracts was volume of 25 mL, containing approximately 100±150 ng performed as previously described [20].