Expression analysis and study of KLK4 in benign and malignant breast tumours

Georgia Papachristopoulou; Margaritis Avgeris; Andreas Scorilas

doi:10.1160/TH08-01-0037

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2009; 101(02): 381-387
DOI: 10.1160/TH08-01-0037

Cellular Proteolysis and Oncology

Schattauer GmbH

Expression analysis and study of KLK4 in benign and malignant breast tumours

Georgia Papachristopoulou

¹Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece

²Oncological Hospital “G.Gennimatas” IKA-ETAM of Athens, Athens, Greece

,

Margaritis Avgeris

¹Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece

,

Andreas Scorilas

¹Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece

› Institutsangaben

Abstract

Summary

The steroid hormone-regulated gene KLK4 (kallikrein 4) is a new member of the human kallikrein-related peptidase gene family. Up to date, studies report that KLK4 is differentially expressed in many tumours. The purpose of this study was the expression analysis and study of KLK4 in benign and malignant breast tumours. Total RNA was isolated from 16 benign and 45 malignant breast tissue specimens. After testing RNA quality, cDNA was prepared by reverse transcription. Highly sensitive quantitative real-time PCR method for KLK4 mRNA quantification was developed using the SYBR Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative C_T method 2^-ΔΔCT. KLK4 expression was found to vary in both patients’ cohorts; however, a statistically significant elevation of the KLK4 mRNA levels was observed in malignant compared to benign tumour patients. Low KLK4 expression levels were found in well-differentiated tumours (p=0.011) as well as in stage I (p=0.024) patients. Moreover, a statistically significant (r_s=-0.318, p=0.035) negative correlation between the KLK4 expression and progesterone receptor staining was observed. ROC and logistic regression analysis recommended that KLK4 gene expression may be used as a new potential biomarker in breast cancer.

Keywords

KLK4 - kallikreins - breast cancer - biomarker - prognosis

Volltext

Referenzen

References
1 Jemal A, Siegel R, Ward E. et al. Cancer statistics, 2006. CA Cancer J Clin 2006; 56: 106-130.
2 Lopez-Otin C, Diamandis EP. Breast and prostate cancer: an analysis of common epidemiological, genetic, and biochemical features. Endocr Rev 1998; 19: 365-396.
3 Korde LA, Calzone KA, Zujewski J. Assessing breast cancer risk: genetic factors are not the whole story. Postgrad Med 2004; 116: 6-8 11–14, 19-20.
4 DePinho RA. The age of cancer. Nature 2000; 408: 248-254.
5 Hondermarck H, Vercoutter-Edouart AS, Revillion F. et al. Proteomics of breast cancer for marker discovery and signal pathway profiling. Proteomics 2001; 1: 1216-1232.
6 Jemal A, Tiwari RC, Murray T. et al. Cancer statistics, 2004. CA Cancer J Clin 2004; 54: 8-29.
7 Scorilas A. Polyadenylate polymerase (PAP) and 3’ end pre-mRNA processing: function, assays, and association with disease. Crit Rev Clin Lab Sci 2002; 39: 193-224.
8 Scorilas A, Yotis J, Pateras C. et al. Predictive value of c-erbB-2 and cathepsin-D for Greek breast cancer patients using univariate and multivariate analysis. Clin Cancer Res 1999; 5: 815-821.
9 Scorilas A, Trangas T, Yotis J. et al. Determination of c-myc amplification and overexpression in breast cancer patients: evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathological characteristics using univariate and multivariate analysis. Br J Cancer 1999; 81: 1385-1391.
10 Scorilas A, Chiang PM, Katsaros D. et al. Molecular characterization of a new gene, CEAL1, encoding for a carcinoembryonic antigen-like protein with a highly conserved domain of eukaryotic translation initiation factors. Gene 2003; 310: 79-89.
11 Duffy MJ. Biochemical markers in breast cancer: which ones are clinically useful?. Clin Biochem 2001; 34: 347-352.
12 Yousef GM, Chang A, Scorilas A. et al. Genomic organization of the human kallikrein gene family on chromosome 19q13.3-q13.4. Biochem Biophys Res Commun 2000; 276: 125-133.
13 Yousef GM, Diamandis M, Jung K. et al. Molecular cloning of a novel human acid phosphatase gene (ACPT) that is highly expressed in the testis. Genomics 2001; 74: 385-395.
14 Foussias G, Yousef GM, Diamandis EP. Identification and molecular characterization of a novel member of the siglec family (SIGLEC9). Genomics 2000; 67: 171-178.
15 Myers SA, Clements JA. Kallikrein 4 (KLK4), a new member of the human kallikrein gene family is up-regulated by estrogen and progesterone in the human endometrial cancer cell line, KLE. J Clin Endocrinol Metab 2001; 86: 2323-2326.
16 Borgono CA, Michael IP, Diamandis EP. Human tissue kallikreins: physiologic roles and applications in cancer. Mol Cancer Res 2004; 2: 257-280.
17 Yu H, Levesque MA, Clark GM. et al. Prognostic value of prostate-specific antigen for women with breast cancer: a large United States cohort study. Clin Cancer Res 1998; 4: 1489-1497.
18 Yu H, Diamandis EP, Levesque M. et al. Prostate specific antigen in breast cancer, benign breast disease and normal breast tissue. Breast Cancer Res Treat 1996; 40: 171-178.
19 Sauter ER, Daly M, Linahan K. et al. Prostate-specific antigen levels in nipple aspirate fluid correlate with breast cancer risk. Cancer Epidemiol Biomarkers Prev 1996; 5: 967-970.
20 Black MH, Magklara A, Obiezu C. et al. Expression of a prostate-associated protein, human glandular kallikrein (hK2), in breast tumours and in normal breast secretions. Br J Cancer 2000; 82: 361-367.
21 Yousef GM, Scorilas A, Kyriakopoulou LG. et al. Human kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer. Clin Chem 2002; 48: 1241-1250.
22 Anisowicz A, Sotiropoulou G, Stenman G. et al. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med 1996; 2: 624-636.
23 Talieri M, Diamandis EP, Gourgiotis D. et al. Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma. Thromb Haemost 2004; 91: 180-186.
24 Yousef GM, Scorilas A, Nakamura T. et al. The prognostic value of the human kallikrein gene 9 (KLK9) in breast cancer. Breast Cancer Res Treat 2003; 78: 149-158.
25 Yousef GM, Scorilas A, Magklara A. et al. The androgen-regulated gene human kallikrein 15 (KLK15) is an independent and favourable prognostic marker for breast cancer. Br J Cancer 2002; 87: 1294-1300.
26 Sauter ER, Lininger J, Magklara A. et al. Association of kallikrein expression in nipple aspirate fluid with breast cancer risk. Int J Cancer 2004; 108: 588-591.
27 Fritzsche F, Gansukh T, Borgono CA. et al. Expression of human Kallikrein 14 (KLK14) in breast cancer is associated with higher tumour grades and positive nodal status. Br J Cancer 2006; 94: 540-547.
28 Korkmaz KS, Korkmaz CG, Pretlow TG. et al. Distinctly different gene structure of KLK4/KLKL1/prostase/ARM1 compared with other members of the kallikrein family: intracellular localization, alternative cDNA forms, and Regulation by multiple hormones. DNA Cell Biol 2001; 20: 435-445.
29 Hu JC, Zhang C, Sun X. et al. Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors. Gene 2000; 251: 1-8.
30 Yousef GM, Obiezu CV, Luo LY. et al. Prostase/ KLK-L1 is a new member of the human kallikrein gene family, is expressed in prostate and breast tissues, and is hormonally regulated. Cancer Res 1999; 59: 4252-4256.
31 Lobo RA. Androgens in postmenopausal women: production, possible role, and replacement options. Obstet Gynecol Surv 2001; 56: 361-376.
32 Nelson PS, Gan L, Ferguson C. et al. Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression. Proc Natl Acad Sci USA 1999; 96: 3114-3119.
33 Takayama TK, McMullen BA, Nelson PS. et al. Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase. Biochemistry 2001; 40: 15341-15348.
34 Dong Y, Bui LT, Odorico DM. et al. Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms. Endocr Relat Cancer 2005; 12: 875-889.
35 Obiezu CV, Scorilas A, Katsaros D. et al. Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients. Clin Cancer Res 2001; 7: 2380-2386.
36 Obiezu CV, Soosaipillai A, Jung K. et al. Detection of human kallikrein 4 in healthy and cancerous prostatic tissues by immunofluorometry and immunohisto-chemistry. Clin Chem 2002; 48: 1232-1240.
37 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408.
38 Fitzgibbons PL, Page DL, Weaver D. et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000; 124: 966-978.
39 Bundred NJ. Prognostic and predictive factors in breast cancer. Cancer Treat Rev 2001; 27: 137-142.
40 Hamilton A, Piccart M. The contribution of molecular markers to the prediction of response in the treatment of breast cancer: a review of the literature on HER-2, p53 and BCL-2. Ann Oncol 2000; 11: 647-663.
41 Manders P, Tjan-Heijnen VC, Span PN. et al. The complex between urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-I) independently predicts response to first-line endocrine therapy in advanced breast cancer. Thromb Haemost 2004; 91: 514-521.
42 Look M, van Putten W, Duffy M. et al. Pooled analysis of prognostic impact of uPA and PAI-1 in breast cancer patients. Thromb Haemost 2003; 90: 538-548.
43 Harbeck N, Kates RE, Gauger K. et al. Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 2004; 91: 450-456.
44 Key T, Appleby P, Barnes I. et al. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 2002; 94: 606-616.
45 Soreide JA, Lea OA, Varhaug JE. et al. Androgen receptors in operable breast cancer: relation to other steroid hormone receptors, correlations to prognostic factors and predictive value for effect of adjuvant tamoxifen treatment. Eur J Surg Oncol 1992; 18: 112-118.
46 Zhu X, Daffada AA, Chan CM. et al. Identification of an exon 3 deletion splice variant androgen receptor mRNA in human breast cancer. Int J Cancer 1997; 72: 574-580.
47 Brys M. Androgens and androgen receptor: do they play a role in breast cancer?. Med Sci Monit 2000; 6: 433-438.