hK PSA F T Prostate Cancer - COnnecting REpositories · related peptidase 2, KLK2) and prostate specific antigen (PSA, KLK3) (Clements 1989; Grauer et al. 1996; Young et al. 1992;

hK2 and PSA: Functions and Targets for Treatment of

Prostate Cancer

Can Hekim

Faculty of MedicineDepartment of Clinical Chemistry

Institute of Clinical MedicineUniversity of Helsinki

Finland

Academic Dissertation

To be publicly discussed with the permission of the Medical Faculty of the University of Helsinki, in Auditorium 2, Biomedicum Helsinki,

on February 24, 2012, at 12 noon

Helsinki 2012Yliopistopaino

2

SUPERVISED BY

Professor Ulf-Håkan Stenman, M.D., Ph.D.Department of Clinical Chemistry

University of Helsinki

Adjunct Professor Hannu Koistinen, Ph.D.Department of Clinical Chemistry


REVIEWED BY

Professor Tero Soukka, Ph.D.Department of Biotechnology

University of Turku

Professor Kimmo Porkka, M.D., Ph.D.Department of Medicine, Division of Hematology


OFFICIAL OPPONENT

Professor Hans Lilja, M.D., Ph.D.Memorial Sloan-Kettering Cancer Center, New York

ISBN 978-952-10-7628-2 (Paperback)ISBN 978-952-10-7629-9 (PDF)

http://ethesis.helsinki.fiHelsinki 2012Yliopistopaino

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The process of scientific research on a subject is like walking towards a mountain, the closer you get, the bigger it grows.

5

List of Original Publications ....................................................................... 7AbBreviations ..................................................................................................... 8Abstract ............................................................................................................... 9Review of the Literature ............................................................................. 101. The Prostate and Prostate Cancer ........................................................................... 10

1.2. Prostate Cancer Diagnosis and Screening .........................................................101.3. Prostate Cancer Treatment................................................................................12

2. The Tissue Kallikrein Family .................................................................................. 142.1. hK2 (human Kallikrein 2) .................................................................................16

2.1.2. hK2 in Tumor Biology ................................................................................182.2. PSA (Prostate Specific Antigen) .........................................................................18

2.2.1. PSA Complexes in Circulation ...................................................................192.2.2. PSA in Tumor Biology ...............................................................................192.2.3. Potential Alternative Treatment Methods for Prostate Cancer Based on PSA ......................................................................................................................21

3. Prostate Cancer Models .......................................................................................... 224. Peptides as Drugs .................................................................................................... 24

4.1. Phage Display Libraries and Peptides ...............................................................264.2. Modification of the Peptides for Improved Stability ..........................................26

Aims of the Study ............................................................................................ 29Materials and Methods ................................................................................ 301. Proteases, Substrates and Antibodies ..................................................................... 302. Immunofluorometric Assays (IFMAs) ................................................................... 30

2.1. IFMA for Free and Total PSA ............................................................................302.2. IFMA for Intact and Total IGFBP-3 ..................................................................302.3. IFMA for Phage expressing hK2-binding Peptides ............................................302.4. Cross-Inhibition Tests ........................................................................................30

3. Determination of Enzymatic Activity ..................................................................... 323.1. hK2 ...................................................................................................................323.2. Other Serine Proteases ......................................................................................32

4. hK2 Inhibition Towards Natural Substrates ........................................................... 324.1. Inhibition of hK2-Mediated Activation of proPSA by Peptides .........................324.2. Inhibition of hK2-Mediated Degradation of IGFBP-3 by Peptides ...................32

5. Enzyme Kinetics Measurements of hK2 Inhibition by Peptides ............................ 326. Phage Display Library Screening ............................................................................ 337. Peptide Synthesis ..................................................................................................... 338. Cyclization of the Peptides ...................................................................................... 339. Similarity Searches .................................................................................................. 3410. Stability Studies ..................................................................................................... 34

Contents

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11. PSA Complexation in Serum ................................................................................ 3412. Chromatographies ................................................................................................ 3413. Immunopurification ............................................................................................. 3514. SDS–PAGE and Western Blotting ......................................................................... 3515. Xenograft Tumor Models ...................................................................................... 3516. Immunohistochemical Staining ........................................................................... 3517. Identification of PSA Complexes and IGFBP-3 Fragments by Mass

Spectrometry ......................................................................................................... 36

Results ................................................................................................................ 371. Identification and Development of hK2-inhibiting Peptides (I) ........................... 372. Effect of hK2-inhibiting Peptides Towards hK2 on Protein Substrates (I, III) ..... 383. Cyclization and Stability Improvements of the Peptides (II) ................................. 404. PSA Forms Complexes in Mouse Plasma and Serum (IV) .................................... 41

Discussion .......................................................................................................... 441. Identification and structure of peptide inhibitors of hK2 (I) ................................ 442. Specificity and Stability of the hK2-inhibiting Peptides (I, II) .............................. 453. Inhibition of hK2 Activity Towards Protein Substrates by hK2-inhibiting Peptides (I, IV) ........................................................................................................................... 464. PSA and hK2 in Prostate Cancer Xenograft Models LNCaP and 22RV1 (IV) ...... 475. Peptide Based Targeting and Therapies for Prostate Cancer (I, III, IV) ............... 47

Conclusions ....................................................................................................... 49Acknowledgments .......................................................................................... 50REFERENCES ......................................................................................................... 52

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This thesis is based on the following published studies that are referred to in the text by their Roman numerals I-IV

I - Hekim, C., Leinonen, J., Närvänen, A., Koistinen, H., Zhu, L., Koivunen, E., Väisänen, V. and Stenman, U-H., Novel peptide inhibitors of human kallikrein 2. Journal of Biological Chemistry 2006, 281(18). 12555-60.

II - Pakkala, M., Hekim, C., Soininen, P., Leinonen, J., Koistinen, H., Weisell, J., Stenman, U-H., Vepsäläinen, J. and Närvänen, A., Activity and stability of human kallikrein-2- specific linear and cyclic peptide inhibitors. Journal of Peptide Science 2007, 13(5): 348-53.

III - Hekim C., Riipi T., Weisell J., Närvänen A., Koistinen R., Stenman U-H., Koistinen H., Identification of IGFBP-3 fragments generated by KLK2 and prevention of frag-mentation by KLK2-inhibiting peptides. Biological Chemistry 2010, 391(4): 475-9.

IV - Hekim, C., Riipi, T., Zhu, L., Laakkonen, P., Stenman, U-H. and Koistinen, H., Complex formation between human prostate-specific antigen and protease inhibitors in mouse plasma. Prostate 2010, 70(5): 482-90.

The publications are reproduced with the permissions of the copyright holders.

List of Original Publications

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%fPSA Percent Free PSA A2M a2-macroglobulin AAT a1-antitrypsin AB Assay Buffer ACT a1-antichymotrypsin AR Androgen Receptor ARA70 Androgen Receptor Associated Protein 70 BPH Benign Prostatic Hyperplasia ECM Extracellular Matrix EMT Endothelial to Mesenchymal Transformation fPSA Free PSA HER2 Human Epidermal Growth Factor Receptor 2 hK2, KLK2 Human Kallikrein 2, Kallikrein-Related Peptidase 2 HRP Horseradish Peroxidase i.v. Intravenous IFMA ImmunofluorometricAssay IGF Insulin-Like Growth Factor IGFBP-3 Insulin-Like Growth Factor-Binding Protein-3 KLK1 Kallikrein 1 KLK2-15 Kallikrein-Related Peptidase 2 to 15 KLKB1 Plasma Kallikrein, Fletcher Factor 1 Mab Monoclonal Antibody MSP ß-Microseminoprotein p2PSA proPSA Isoform Lacking 2 Amino Acids, [-2]proPSA Pab Polyclonal Antibody PAI-1 Plasminogen Activator Inhibitor 1 PAR Protease Activated Receptors PBS Phosphate Buffer Saline PCI Protein C Inhibitor PEG Polyethylene Glycol PHI Prostate Health Index PSA,KLK3 ProstateSpecificAntigen,Kallikrein-RelatedPeptidase3PTHrP Parathyroid Hormone Related Protein RCL Reactive Center Loop s.c. Subcutaneous SNP Single Nucleotide Polymorphism TBS Tris Buffer Saline TGFß Transforming Growth Factor-b TMPRSS-2 Transmembrane Protease, Serine 2 TNM Tumor Node Metastasis tPSA Total PSA uPA Urokinase-Type Plasminogen Activator

AbBreviations

9

AbstractProstate cancer is the most common cancer in males and a major cause of cancer death in industrial-ized countries. It is generally a very slowly growing cancer and potentially curable at early stages by radical prostatectomy or radiotherapy. However, radical therapy is associated with side effects. Therefore, there is need for novel treatments for advanced prostate cancer, and for curing or slowing down the growth of the tumors. Proteases play important roles in the progression of prostate and other cancers. Prostate produces high levels of two kallikreins, human kallikrein 2 (hK2, kallikrein-related peptidase 2, KLK2) and prostate specific antigen (PSA, KLK3). These proteases are secreted into seminal fluid and mediate liquefaction of the seminal clot that forms after ejaculation. Furthermore, enzymatically active PSA has been shown to reduce angiogenesis in vitro and in vivo, which may contribute to the slow growth of prostate cancer. PSA expression is lower in malignant than in normal prostatic epithelium. It is further reduced in poorly differentiated tumors, in which the expression of hK2 is increased. hK2 may mediate tumor growth and invasion by participating in proteolytic cascades degrading extracelullar matrix and thereby promoting tumor spread. Both PSA and hK2 degrade insulin-like growth factor-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 is an important regulator of cell proliferation and survival via IGF-system, and independently. hK2 is more potent than PSA in degrading IGFBP-3. Because its expression is increased in prostate cancer, degradation of IGFBP-3 by hK2 locally in the prostate may promote prostate cancer growth. By using phage display technology we developed biologically active peptides, which specifically inhibit the enzymatic activity of hK2. The peptides were characterized and the motifs required for their inhibitory activity were determined. These may be used to target hK2 for treatment and their binding property be utilized in tumor imaging. However, the peptides were degraded in plasma within minutes and thus, have limited use in vivo. By head-to-tail cyclization we were able to improve plasma stability of the original linear hK2-inhibiting peptide, while the activity towards hK2 was retained. When secreted from the prostate, most of PSA is free and enzymatically active. In circulation major portion of PSA occurs in complexes with protease inhibitors and, thus, is inactive. To justify the use of mouse models for evaluation of the function of PSA and for studies in therapeutic modalities based on modulation of PSA activity it is important to know whether PSA complexation is similar in mouse and man. We characterized the circulating forms of PSA in mouse, by directly adding to mouse serum or using subcutaneous PSA-producing human prostate cancer cell xenograft tumor models. When added to mouse serum, over 70% of PSA forms complexes within 30 min. The complexes contained α2-macroglobulin and serpins of the α1-antitrypsin (AAT) family. Thus, in mouse serum, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than α1-antichymotrypsin (ACT). In mice bearing LNCaP xenograft tumors 70% of immunore-active PSA occurs in complex with AAT. Hence, the metabolism of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the fate of released PSA and would allow the evaluation of treatment modalities based on PSA activity. We studied the degradation of IGFBP-3 by hK2 and identified the cleavage sites by mass spectrometry. Furthermore, we showed that hK2-inhibiting peptides inhibit hK2 activity towards natural protein substrates, including IGFBP-3. As degradation of IGFBP-3 leads to release of IGF-I, which may stimulate cancer growth, these peptides may be useful for treatment of prostate cancer and other diseases associated with increased hK2 activity. The peptides identified in this study have potential therapeutic value for treatment of prostate cancer. They can also be used as lead molecules for the development of peptide analogues suitable for imaging and treatment of prostate cancer.

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1. The Prostate and Prostate Cancer

The main function of the prostate is to produce part of seminal fluid, which carries spermatozoa during ejaculation. Within the prostate, the major fraction of seminal fluid is produced by the luminal cells of glandular acini (Huggins et al. 1942). These epithelial cells, together with the stromal cells, contain cytoplasmic androgen receptors (Chatterjee 2003). Androgens are the major stimulators of prostate growth (Wilson & Foster 1985). They regulate the expression of several prostatic proteins, including proteases such as human kallikrein 2 (hK2, kallikrein-related peptidase 2, KLK2) and prostate specific antigen (PSA, KLK3) (Clements 1989; Grauer et al. 1996; Young et al. 1992; Lawrence et al. 2010). Prostate cancer is the most common non-skin cancer in industrialized countries and the second leading cause of cancer deaths among men (Jemal et al. 2010). In most cases the growth of prostate tumors is a slow process and most tumors remain indolent during the lifetime of a man, and therefore, many tumors do not require clinical attention and treatment (Stenman et al. 1999a; Choo et al. 2002; Klotz 2003). The mechanisms behind the initiation of prostate cancer are largely unknown (Shen & Abate-Shen 2010). Some candidate oncogenes, tumor suppressor genes, and genomic rearrangements associated with the development of prostate cancer have been identified. For example, loss of tumor suppressor gene phosphatase and tensin homolog (PTEN) (Salmena et al. 2008), loss-of-function of homeobox gene NK3 transcription factor related locus 1

(NKX3.1) (Abate-Shen et al. 2008), Serine Protease Inhibitor Kazal-type 1/Tumor-Associated Trypsin Inhibitor (SPINK-1/TATI) overexpression (Paju et al. 2007; Tomlins et al. 2008; Ateeq et al. 2011), gene fusions of Transmembrane Protease, Serine 2 (TMPRSS2) and E-Twenty Six (ETS) family members, especially TMPRSS2-ERG and TMPRSS-ETV1 and -4 (Tomlins et al. 2007; Tomlins et al. 2009), and fusion of KLK2 with ETV4 (Hermans et al. 2008) are associated with prostate cancer. A large scale segregation study on twins shows that hereditary factors could contribute to the development of up to 42% of prostate cancers (Lichtenstein et al. 2000). Genome-wide studies have revealed several loci containing susceptibility genes for hereditary prostate cancer. (Smith et al. 1996; Xu et al. 1998; Wiklund et al. 2003; Schleutker et al. 2003). In addition, several polymorphisms associated with prostate cancer have been found (Thomas et al. 2008). These include single nucleotide pol-ymorphism (SNP) in the genes that encode PSA (KLK3), ß-microseminoprotein (ß-MSP, MSMB) and hK2 (KLK2) (Nam et al. 2003; Severi et al. 2006; Eeles et al. 2008; Liu et al. 2011).

1.2. Prostate Cancer Diagnosis and Screening

Prostatic intraepithelial neoplasia (PIN) may develop already at 20-30 years of age, while the presence of microscopic prostatic tumors can be observed in the next and later decades of life (Sakr et al. 1993). More than 50% of men over 60-80 years of age have an occult prostate cancer (Franks 1954; Sakr et al. 1999). A slowly

Review of the Literature

11


growing prostate cancer has an average doubling time of 2 to 4 years (Schmid et al. 1993). Early stage tumors with a size up to one gram do not usually cause clinical symptoms (Stamey et al. 1987; Stenman et al. 2000). A one gram tumor increases serum PSA on average by 2-3 µg/l. Thus, detection of such cancers is possible by determination of PSA in serum combined with digital rectal examination (DRE) and needle biopsy guided by transrectal ultrasonography (TRUS) (Catalona et al. 1997; Ornstein et al. 1997; Stenman et al. 1999a). Prostate cancer diagnosis is based on examination of tissue obtained by needle biopsy. Tumor aggressiveness is evaluated by a grading system established by Gleason (Gleason & Mellinger 1974). A grade of 1 to 5 is assigned based on the glandular architecture, where lower grades indicate well differentiated and higher grades poorly differentiated glandular structure. The Gleason score is calculated as the sum of the grades of the two most prevalent tumor patterns. The scale is from 2 to 10, and a Gleason score below 6 is classified as a low grade prostate cancer, 7 is an intermediate grade and above 7 is a high grade prostate cancer. The tumor node metastasis (TNM) system is used for staging of prostate cancer. This system, maintained by American Joint Committee on Cancer (AJCC) and International Union for Cancer Control (IUCC), is the most commonly used staging method. The TNM system classifies cancers based on the size and extent of the primary tumor (T), spread into lymph nodes (N) and distant metastases (M) (Table 1). This information can be used to group tumors in 4 stages described by Roman numerals with added letters for defining substages, i.e, stage I, II, III, IV and substages like stage IIA, IIB, etc. Based on this, stage III refers to locally advanced cancer and stage IV to metastatic cancer. If available the TNM information can be combined with Gleason score and serum PSA for tumor staging.

Prostate cancer can be detected at an early stage by screening based on de-termination of serum PSA and biopsy of men with elevated values of PSA (Stamey et al. 1987; Catalona et al. 1991; Brawer et al. 1992; Oesterling et al. 1995; Stenman et al. 2005; Lilja et al. 2008). Trial studies on screening of prostate cancer are ongoing in Europe and USA. The largest ones are “The European randomized study of screening for prostate cancer (ERSPC)” and “the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial”. These trials randomly assign men aged between 50 and 74 for a PSA test (Andriole et al. 2009; Schröder et al. 2009). The European study has shown that PSA based screening reduces mortality. But it also causes over-diagnosis, i.e., it detects prostate cancers in men, who would not develop clinical disease within their lifetime (Draisma et al. 2003). This leads to unnecessary treatments, which may have serious side effects. Therefore, there is a need for strategies that more accurately differentiate between tumors that do and do not need treatment. Traditionally, a cut-off level of 4 µg/l serum PSA has been used to detect prostate cancer. However, moderately elevated PSA levels (4-10 µg/l), so called “grey zone”, in serum do not always indicate malignancy, but may be caused by other prostate related diseases, especially benign prostatic hyperplasia (BPH) (Stamey et al. 1987; Stenman et al. 1999a). Patients with a concentration of serum PSA exceeding 10 µg/l have more than a 50% risk of harboring prostate cancer (Armitage et al. 1988). However, a serum PSA below 4 µg/l does not exclude the presence of malignancy, thus, men with PSA values below 4 µg/l were found to have 15% risk of having a prostate cancer (Thompson et al. 2004). To improve the diagnostic accuracy of PSA, subfractions of PSA, like complexed and free PSA (Stenman et al. 1991; Lilja et al. 1992; Christensson et al. 1993), proPSA, nicked PSA and intact PSA (Mikolajczyk

12

hK2 and PSA: Functions and Targets for Treatment of Postate cancer

et al. 2002; Mikolajczyk et al. 2004; Lilja et al. 2008) can be measured, and parameters like PSA doubling time (Schmid et al. 1993), PSA velocity (Carter et al. 1992), PSA density (Benson et al. 1992) and age specific PSA values can be calculated (Oesterling 1994; Oesterling et al. 1995). The most commonly used method to improve the accuracy of PSA test is to analyze free PSA and calculate the ratio of free to total PSA (percent free PSA, %fPSA) in serum (Finne et al. 2002). %fPSA signifi-cantly improves discrimination between malignant disease and BPH (Stenman et al. 1991; Christensson et al. 1993; Lilja & Stenman 1996). An elevated serum PSA and a low %fPSA indicate increased risk of prostate cancer as compared to elevated PSA alone (Stenman et al. 1994). In serum, the proportion of a 2 amino acid truncated isoform of proPSA, named [-2]proPSA (p2PSA), has been found to be higher in prostate cancer compared with BPH (Mikolajczyk et al. 2001). p2PSA also has been shown to significantly improve prostate cancer detection (Stephan et al. 2009; Sokoll et al. 2008) when used for calculation of the prostate health index (PHI). PHI, calculated using the serum concentrations of p2PSA, fPSA and tPSA by the formula (p2PSA/fPSA) x √(tPSA) (Jansen et al. 2010), has been suggested to be a strong predictor of prostate cancer in men with PSA serum levels of 2-10 µg/l (Guazzoni et al. 2011). The serum levels of hK2 have been shown to add prognostic value for organ confined prostate cancer in men with PSA serum levels of 4-10 ng/ml (Recker et al. 2000; Haese et al. 2001; Raaijmakers et al. 2007). Combining serum concentrations of free and total PSA with hK2 provides further information about the pathologic stage of clinically localized prostate cancer (Steuber et al. 2007; Lilja et al. 2008). A statistical model to predict the results of prostate biopsy has been developed on the basis of blood levels of total PSA, free

PSA, intact PSA and hK2 (Vickers et al. 2008). Here, intact PSA assay measures free PSA that is not cleaved at lysine-145 and lysine-146 (Väisänen et al. 2006). This 4-kallikrein panel is a strong predictor of prostate cancer in men with elevated serum PSA levels (Vickers et al. 2011). Recently, serum/plasma markers have been used also in combination with prostate cancer associated SNP’s in the genes that encode PSA, hK2 and MSP (Klein et al. 2010).

1.3. Prostate Cancer Treatment

Treatment options for prostate cancer depend on the stage of the tumor. Surgery or radiation therapy are mostly curative when the tumor is detected at a localized stage. Surgery permits precise clinical staging and complete removal of the tumor. However, it may cause serious side effects such as incontinence, impotence and complications related to the operation (Begg et al. 2002). Radiation therapy is less strenuous than surgery, but it may also lead to impotence and harm nearby organs like the bladder and rectum (Pollack et al. 2002; Stone & Stock 2007). In men with prostate cancer at stages III or IV, the treatment options include radiation and hormone ablation. Like the normal prostate, development of prostate cancer in the early stages is androgen dependent and can therefore be treated by androgen ablation (Huggins & Hodges 1941). Hormone treatment options include surgical castration, the use of antiandrogens, luteinizing hormone releasing hormone (LHRH) analogues or combinations of these for maximal androgen blockade. These methods either prevent androgen secretion or block the growth stimulating effect of androgens. Most cases initially respond to hormone therapy by stopping tumor growth, but androgen-resistance develops if the patient lives long enough. Several drugs can be used to treat castration resistant prostate cancer, such as MDV3100, an androgen

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Table I : TNM staging system of prostate cancer

Evaluation of the primary tumor (‘T’)

TX Tumor cannot be assessed

T0 Tumor is not present

T1 Tumor present, but not clinically detectable

T1a Tumorincidentalhistologicfindingin≤ 5% of resected tissue; not palpable

T1b Tumorincidentalhistologicfindingin>5%ofresectedtissue

T1c Tumoridentifiedbyneedlebiopsy(duetoelevatedserumPSA)

T2 Tumor is palpable

T2a Tumor is in one-half of one lobe or less

T2b Tumor is in more than one-half of one lobe but not in both lobes

T2c Tumor is in both lobes

T3 Tumor is spread through the prostatic capsule

T3a Unilateral or bilateral extracapsular extension

T3b Tumor invades seminal vesicles

T4 Tumor invades nearby adjacent structures

Evaluation of the regional lymph nodes (‘N’)

NX Nodes cannot be assessed

N0 No regional node metastasis

N1 Node metastasis is present

Evaluation of distant metastasis (‘M’)

MX Metastasis cannot be assessed

M0 No distant metastasis

M1 Distant metastasis is present

M1a Metastasis to distant lymph nodes

M1b Metastasis to bone

M1c Metastasistoothersites(e.g.liver,lung)Adapted from Greene FI, Page DL, Fleming ID, et al.: AJCC Cancer Staging Manual, 6th ed. New York, Springer-Verlag, 2002, p.223

receptor antagonist (Tran et al. 2009), Abiraterone, an inhibitor of the CYP17A1 enzyme that converts testosterone to dihydrotestosterone (de Bono et al. 2011), and docetaxel, a mitosis inhibitor

belonging to the taxane-group (Tannock et al. 2004). These may provide short term effects on survival. Recently, FDA (U.S. Food and Drug Administration) approved immunotherapy based treatment called

14


sipuleucel-T that can be used to treat castration resistant prostate cancer (Kantoff et al. 2010). It enhances the potency of the immune response to prostatic acid phosphatase (PAP), which is expressed in ~95% of prostate cancers (Higano et al. 2010; Patel & Kockler 2008). The vaccine contains immunostimulatory dendritic cells of the patients that have been activated with a recombinant fusion protein of PAP and granulocyte–macrophage colony-stimulating factor (GM-CSF), an immune cell activator (Higano et al. 2010). According to the IMPACT trial study this therapy increased overall survival of the patients from 21.7 to 25.8 months (Kantoff et al. 2010). However, the treatment requires leukapheresis, and it does not eradicate prostate cancer completely.

2. The Tissue Kallikrein Family

The human tissue kallikrein family of serine proteases consists of 15 members. The first kallikrein was discovered in 1930 as a pancreas-derived cardioactive and vasoactive substance (Kraut et al. 1930; Lundwall & Brattsand 2008). It is presently known as tissue kallikrein 1 or kallikrein 1 (KLK1). The effect of kallikrein was attributed to the release of kinin peptides from kininogens (Bhoola et al. 1992). Sub-sequently another enzyme was discovered to have similar function and was named plasma kallikrein (KLKB1) (Chung et al. 1986). Although both of these enzymes are serine proteases that show similar kininogenase activity, they have different expression profiles, protein structure and substrate specificity (Lundwall & Brattsand 2008). The KLKB1 gene is located in chromosomal region 4q34-35, and it is not a member of human tissue kallikrein family. The KLK1 gene that transcribes kallikrein 1 is located together with the 14 other tissue kallikreins, known as kal-likrein-related peptidases 2-15 (KLK2-15), in a gene cluster at chromosomal region

19q13.3-13.4. (Harvey et al. 2000; Gan et al. 2000; Yousef et al. 2000; Lundwall et al. 2006) (Figure 1). The transcription direction of most KLKs is from telomere to centromere, except hK2 (KLK2) and PSA (KLK3), which are transcribed from centromere to telomere. The first identified kallikreins, the so-called classical kallikreins, KLK1, hK2 and PSA contain a unique 11 amino acid long kallikrein loop and share a higher degree of amino acid sequence similarity of 62-79% than KLK4-15, which share 25-49% sequence similarity (Villoutreix et al. 1994; Harvey et al. 2000). Within the kallikrein locus, the genes for hK2 and PSA are located in close proximity and their active forms have 79% sequence similarity. In contrast to humans, mouse or rat kallikrein loci do not contain functional KLK2 and KLK3 genes, but only KLK2 pseudogenes (Pavlopoulou et al. 2010). All KLK genes express single chain serine proteases containing a catalytic triad consisting of aspartate-57, histidine-102 and serine-195 (numbering of amino acids according to bovine chymotrypsin). All KLKs have either trypsin- or chy-motrypsin-like substrate specificity. The kallikreins are transcribed as proenzymes with a signal peptide, which is cleaved off prior to the transportation from the endoplasmic reticulum into secretory granules (Lawrence et al. 2010). They contain 3 to 37 amino acid long activation peptides or pro-peptides ending with lysine or arginine, which suggests that the zymogens are activated by enzymes with trypsin-like specificity (Paliouras & Diamandis 2006). Kallikreins have been shown to activate each other and are also activated by plasmin, urokinase, thrombin, factor Xa, plasma kallikrein and trypsin (Takayama et al. 1997; Paju et al. 2000; Yoon et al. 2008). hK2 cleaves the activation peptides of both prohK2 and proPSA, thereby activating itself and PSA (Mikolajczyk et al. 1997; Lövgren et al.

15


1997; Denmeade et al. 2001a; Williams et al. 2010). PSA has also been shown to be activated by trypsin, prostasin and several other kallikreins (Takayama et al. 1997; Paju et al. 2000; Takayama et al. 2001; Yoon et al. 2008). The fact that several kallikreins are substrates for other kallikreins and

they are co-expressed in some tissues, like prostate and skin, suggests the existence of so-called kallikrein activation cascade (KLK activome) (Figure 2) (Paliouras & Diamandis 2006; Pampalakis & Sotiropou-lou 2007). Several KLKs are expressed in the

Figure 1. KLK locus, gene and protein form. a) The KLK gene is located in the long arm of chromosome 19 in region 13.4. The direction of gene transcription is from telemore to centromere except KLK2 and KLK3. The “classical” KLK genes (KLK1, -2 and -3) are shown in black, whereas KLK4-15 shown in grey and KLK1 pseudogene in light grey. b) KLK genes consist of 5 coding exons and 4 intervening introns with a conserved intron phase of I, II, I, 0. The codons for the residues of catalytic triad are conserved, histidine (H) near the end of coding-exon 2, aspartic acid (D) in the middle of coding-exon 3, and serine (S) near the start of coding-exon 5. Most KLK genes include also one or two non-coding exons in the 5´ and 3´ untranslated regions (UTR). c) KLK proteins are translated as pre-proenzymes, of which the signal sequence (Pre) is cleaved of prior to transportation from endoplasmic reticulum for secretion. propeptide (Pro) remains until the activation of the KLKs. Modified and adapted from Borgoño and Diamandis 2004 Nat Rev Cancer 4(11), pp.876–890.

prostate and are present in seminal fluid at various concentrations. Among these, hK2 and PSA are most abundantly produced by prostatic epithelial cells (Wang et al. 1981; Schedlich et al. 1987). Within the prostate, the expression of hK2 and PSA is regulated by androgens through the androgen receptor (AR) (Lawrence et al. 2010). AR elements (ARE) are located in

the PSA promoter region (Riegman et al. 1991a; Riegman et al. 1991b; Cleutjens et al. 1997). The highest concentrations of hK2 and PSA are found in seminal plasma and prostatic extracellular fluid, but they are also present at lower concentrations in breast milk, breast cytosol, breast cyst fluid, saliva, urine and plasma (Clements 1989; Denmeade et al. 2001b; Shaw & Diamandis

KLK locus (19q13.4)

KLK gene

KLK protein

a

b

c

5ʼ UTR 3ʼ UTR

Chromosome 19

1 15 3 2 1 4 5 6 7 8 9 10 11 12 13 14

Coding exons

p13.3

p13.2

p13.1

p12 q12 q13.1

q13.2

q13.3

q13.4

Pre

H57 D102 S195

Serine-protease domain

1 2I II I 03 4 5

H D S

Pro CN

5ʼ 3ʼ

16


2007; Emami et al. 2009). hK2, PSA, KLK5 and -14 are directly or indirectly involved in the degradation of semeno-gelins, which are the main gel-forming proteins of seminal plasma. Proteolysis of the gel is mainly carried out by PSA, but also by hK2, and this leads to liquefaction of the seminal clot (Lilja 1985; Deperthes et al. 1996; Michael et al. 2006; Emami et al. 2008). This process is essential for the spermatozoa to gain proper motility and to fertilize the oocyte. KLK1, -4 to -11 and -14 have been isolated from skin, and several possible functions have been suggested including degradation of desmosomes by KLK5, -7 and -8, leading to desquamation (Kishibe et al. 2007; Lundwall & Brattsand 2008). Besides having functions in seminal fluid and skin, KLK4 is involved in the formation of teeth enamel (Simmer et al. 1998). In the brain, KLK8, also known as neuropsin, degrades and rearranges extra-cellular matrix (ECM) proteins and may play a role in synaptic plasticity associated with learning and memory (Shimizu et al. 1998; Tamura et al. 2006). KLK6 and -8 may also be involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis (Iwata et al. 2003; Terayama et al. 2007; Lundwall & Brattsand 2008). One of the hallmarks of cancer is the increased expression/activity of proteases associated with matrix-degrad-ing proteolytic pathways, contributing to the invasive properties of cancer cells (Hanahan & Weinberg 1999; Hanahan & Weinberg 2011). All KLKs except KLK12 have been found to be differentially expressed in several cancers [reviewed in (Borgoño & Diamandis 2004; Paliouras & Diamandis 2006; Sardana et al. 2008)]. In prostate cancer, hK2, KLK4, -11, -14 and -15 have been found to be overexpressed and have been suggested to serve as tumor markers (Darson et al. 1997; Obiezu et

al. 2002; Yousef et al. 2003; Paliouras et al. 2007). However, with the exception of hK2 the utility of these as tumor markers remains to be demonstrated.

2.1. hK2 (human Kallikrein 2)

hK2 is a 28 kDa serine protease produced by prostatic epithelial cells and secreted into prostatic lumen and seminal fluid (Schedlich et al. 1987). Despite the high structural similarity with PSA, hK2 has a trypsin-like specificity with 20,000-fold higher enzymatic activity than PSA towards synthetic substrates in vitro (Mikolajczyk et al. 1997; Mikolajczyk et al. 1998). The proteolytic efficiency of hK2 against physi-ological substrates varies widely (Clements et al. 2004). Activation of proPSA by hK2 is probably physiologically important, since both proteases are produced by the prostate and secreted into seminal fluid. Thus, hK2 may contribute to liquefaction of seminal clot by activating PSA and also by digesting semenogelins, although the proteolytic efficiency of hK2 towards semenogelins is much lower than that of PSA (Deperthes et al. 1996). When the architecture of prostate is disrupted, hK2, like PSA, leaks into circulation (Kwiatkowski et al. 1998). In prostate cancer, tissue expression of hK2 increases with increasing grade and high serum concentrations of hK2 are associated with poor prognosis (Darson et al. 1997; Lintula et al. 2005). Therefore, serum hK2 combined with serum PSA can improve diagnosis of prostate cancer (Saedi et al. 1998; Partin et al. 1999; Becker et al. 2000). Furthermore, measurement of serum hK2 can also help in discriminating organ confined from non-organ confined prostate cancer (Haese et al. 2000; Recker et al. 2000). In circulation, hK2 forms complexes with A2M, C1-inactivator, protein C inhibitor (PCI), α2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1)

17


Figure 2. KLK activome and functions of active KLKs in tumorigenesis, skin desquamation, innate immunity, neurodegeneration, hypertension and semen liquefaction. Sotiropoulou et al. Functional Roles of Human Kallikrein-related Peptidases J Biol Chem 2009, 284(48), pp.32989-32994. © the American Society for Biochemistry and Molecular Biology. Reprinted with permission.

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and an unidentified serpin with a molecular weight of 63 KDa (Grauer et al. 1998; Heeb & España 1998; Mikolajczyk et al. 1998; Mikolajczyk et al. 1999b). In seminal plasma, the most abundant hK2 complex is that with PCI (Deperthes et al. 1995).

2.1.2. hK2 in Tumor Biology

Insulin-like growth factor-binding protein 3 (IGFBP-3), a common substrate for hK2 and PSA, is cleaved by hK2 with much higher potency than by PSA (Cohen et al. 1992; Réhault et al. 2001; Koistinen et al. 2002). Proteolysis of IGFBP-3 leads to increased bioavailability of IGF-I, which is the main ligand of IGFBP-3 (Jones & Clemmons 1995). IGF-I has promitotic and antiapoptotic effects in normal and differentiated cells (Bol et al. 1997). In the prostate it acts in a paracrine fashion (Meinbach & Lokeshwar 2006). IGF-I is produced by stromal cells and exerts its activity on epithelial cells carrying IGF-receptors (Kaplan et al. 1999). IGFBP-3 is expressed by prostatic epithelial cells (Chan et al. 1998). Increased IGF-I and decreased IGFBP-3 serum concentrations have been associated with an increased risk of developing prostate cancer in some (Chan et al. 1998), but not in all studies (Finne et al. 2000a; Chan et al. 2002). IGFBP-3 or its fragments also have IGF-in-dependent effects on cell growth, i.e., they induce apoptosis and inhibit the growth of prostate cancer cell lines (Rajah et al. 1997; Boyle et al. 2001; Silha et al. 2006). hK2, like KLK4, -5, -6 and -14, have been shown to activate protease activated receptors (PAR), thereby exerting direct stimulus on the cells (Oikonomopoulou et al. 2006). PARs are G-protein coupled transmembrane receptors, which are activated through proteolytic cleavage of the N-terminus of the extracellular domain of the protein (Vu et al. 1991; Coughlin 1999; Coughlin 2000). In prostate cancer cells, hK2 activates PAR2 leading to phos-

phorylation and downstream activation of ERK1/2 signaling pathways and stimulation of cell proliferation (Mize et al. 2008). PAR1, -2 and -4 are highly expressed in prostate cancer and benign tissue, and also in the prostate cancer cell line LNCaP (Black et al. 2007). hK2 may act as a promotor of tumor invasion and metastasis by degrading components of ECM and by activating pathways related to the degradation of connective tissues (Clements et al. 2004; Lawrence et al. 2010). Another common physiological substrate of PSA and hK2 is the ECM component fibronectin (Lilja 1985). Like with IGFBP-3, hK2 cleaves fibronectin with much higher efficiency than PSA (Lövgren et al. 1999; Deperthes et al. 1996). hK2 activates urokinase-type plasminogen activator (uPA) (Frenette et al. 1997) and inactivates plasminogen activator inhibitor-1 (PAI-1) (Mikolajczyk et al. 1999a). As increased uPA activity is associated with prostate cancer (Achbarou et al. 1994; Lyon et al. 1995), these findings support the role of hK2 in extracellular proteolytic cascades in prostate cancer. Thus, hK2 may play an important role in prostate cancer growth and invasion (Figure 3) (Rittenhouse et al. 1998; Koistinen et al. 2008).

2.2. PSA (Prostate Specific Antigen)

PSA is a 28 kDa single chain serine protease with a restricted chymotrypsin-like substrate specificity, cleaving preferentially after tyrosine residues (Coombs et al. 1998). PSA is produced by prostatic epithelial cells and released into the lumen of the prostatic ducts as an essential component of seminal fluid (Wang et al. 1981). Its concentration in seminal plasma is 0.39-3 g/l (Wang et al. 1998). The main physiological function of PSA is to digest the major gel-forming proteins semenogelin-I and -II after ejaculation (Lilja 1985). In seminal fluid PSA occurs in at least 5 isoforms named

19


PSA-A to -E based on their order of elution in anion exchange chromatography (Zhang et al. 1995). Among these isoforms, PSA-A and -B have different isoelectric points due to differences in glycosylation, but have similar enzymatic activity, whereas PSA-C, -D and -E are partially degraded or “nicked” isoforms of PSA (Zhang et al. 1995; Mattsson et al. 2008). The major nicks in PSA occur between arginine-85 and phenylalanine-86, lysine-145 and lysine-146, and lysine-182 and serine-183. (Watt et al. 1986; Lundwall & Lilja 1987; Zhang et al. 1995; Mattsson et al. 2008). The content of PSA forms cleaved after lysine-145 and lysine-182 are increased in BPH tissues and are therefore termed benign PSA (BPSA) (Mikolajczyk et al. 2000). In seminal plasma, about 35% of PSA is cleaved (Zhang et al. 1995; Mattsson et al. 2008). The intact isoforms of PSA, i.e., PSA-A and -B, are fully active and form complexes with α1-antichymotrypsin (ACT), α1-protease inhibitor (API, also known as α1-antitrypsin, AAT) and A2M in vitro (Christensson et al. 1990; Chris-tensson & Lilja 1994; Leinonen et al. 1996). From the normal prostate, only a minor part of PSA escapes into circulation. In prostatic diseases the barriers that prevent PSA leaking into circulation are disrupted, which results in elevation of the PSA concentration in blood (Stenman et al. 1999a). However, poorly differentiated prostate cancers produce less PSA than well differentiated ones (Abrahamsson et al. 1988; Stege et al. 2000; Paju et al. 2007). Thus, an increased PSA concentration in circulation is an indication of prostatic disease (Stamey et al. 1987; Armitage et al. 1988). As mentioned before, this makes PSA a very useful marker for prostate cancer, although a high serum PSA may also be caused by BPH and prostatitis (Stamey et al. 1987). Increased tumor volume correlates with PSA concentration in plasma (Stamey et al. 1987). In prostate cancer metastases, expression of PSA is

generally maintained, but metastatic sites are heterogeneous and PSA expression varies (Stein et al. 1984; Roudier et al. 2003).

2.2.1. PSA Complexes in Circulation

When enzymatically active PSA reaches circulation, it rapidly forms complexes with serine protease inhibitors (serpins) and A2M (Christensson et al. 1990; Stenman et al. 1991; Christensson & Lilja 1994; España et al. 1996). In plasma 5-40% of the immunoreactive PSA occurs in free form, while 60-95% is in complex with ACT and a minor part with API. These PSA forms are detected by assays for total PSA (Stenman et al. 1991; Lilja et al. 1992; Zhang et al. 1997). In addition, 1-10% of PSA in plasma is complexed with A2M, which is not recognized by conventional immunoassays. It can be detected with PSA antibodies after denaturation by SDS or high pH (Zhang et al. 1998). As mentioned earlier, the proportion of free/total PSA (%fPSA) gives additional information for discriminating between BPH and prostate cancer (Stenman et al. 1991).

2.2.2. PSA in Tumor Biology

Besides having a physiological role in re-production, PSA may also be involved in tumor progression (Fortier et al. 1999; Clements et al. 2004; Koistinen et al. 2008). Like hK2, PSA degrades IGFBP-3 causing release of IGF-I, which may stimulate tumor development (Cohen et al. 1992; Sutkowski et al. 1999; Koistinen et al. 2002). PSA may play a role in the development of bone metastases by cleaving and inactivat-ing parathyroid hormone related protein (PTHrP) (Cramer et al. 1996; Iwamura et al. 1996) and by promoting osteoblast pro-liferation, inducing apoptosis in osteoclast precursors and promoting prostate cancer cell adhesion to bone marrow endothelial cells (Yonou et al. 2007; Goya et al. 2006; Romanov et al. 2004). PSA has been shown

20


to activate the latent form of the soluble transforming growth factor-β2 (TGF-β2), which has both tumor suppressor and promoter functions (Dallas et al. 2005; Killian et al. 1993). TGF-β activates cy-toskeleton modulators, thereby rearranging cell cytoskeleton, inducing cell migration (Bhowmick et al. 2001), and causing loss of the epithelial phenotype via downregu-lation of E-cadherin (Thiery 2002). Trans-fection of non-PSA producing prostate cancer cell lines with PSA causes the cells to undergo epithelial to mesenchymal transformation (EMT) (Veveris-Lowe et al. 2005). This type of phenotypical change is generally associated with aggressive cancer (Thiery 2002). EMT-like processes include regulation of the cytoskeleton, cellular adhesion and migration (Thiery et al. 2009; Yilmaz & Christofori 2009). Other studies have shown that PSA may be associated with prostate cancer progression by degrading ECM components such as fibronectin and by activating matrix metal-loproteinase 2 (MMP2) (Lilja 1985; Pezzato et al. 2004; Ishii et al. 2004; Webber et al. 1995). PSA has also been found to cleave galectin-3 dimers and releasing functional-ly active monovalent lectin, which may be involved in progression of prostate tumors (Saraswati et al. 2011). Inhibition of PSA expression by small interfering RNA (siRNA) has shown that PSA may promote growth of prostate cancer cells, via enhancing ARA70 induced AR transactivation, and increase subcutaneous growth of LNCaP prostate cancer cells in mice (Niu et al. 2008; Williams et al. 2011). Furthermore, transfection of enzymatically active PSA into a non-PSA producing cell line has been found to accelerate tumor growth (Williams et al. 2011). Ligation of prostate cancer cell surface GRP78 with PSA-A2M complex has been shown to activate a proliferative and antiapoptotic feedback loop that promotes tumor growth (Misra et al. 2011). While the observations described

above indicate a tumor promoting effect of PSA, other studies show that PSA actually inhibit tumor growth. When PSA is added to the culture medium, prostate cancer cell lines show various changes in their gene expression at the mRNA and protein levels (Bindukumar et al. 2005). Several of these differentially expressed genes are related to regulation of proan-giogenic and antiangiogenic factors. This also showed that in vivo growth of prostate tumor xenografts is suppressed when PSA is injected in the vicinity of the tumor area. PSA exhibits antiangiogenic properties on endothelial cells in culture and in animal models (Fortier et al. 1999; Fortier et al. 2003; Mattsson et al. 2009). This implies that PSA may act as an antiangiogenic agent in prostate cancer. Therefore, the slow growth of prostate cancer, at the stage when the tumor needs blood vessels, could be attributed to PSA. The antiangiogenic activity of PSA has been ascribed to the ability of PSA to generate angiostatin-like fragments by cleaving plasminogen (Heidtmann et al. 1999). However, this has not been confirmed in other studies, and thus, other antiangiogenic mechanisms cannot be ruled out. Clinical observa-tions suggest that PSA inhibits rather than promotes tumor growth. The expression of PSA is actually lower in malignant tissues than in the benign prostate (Abrahamsson et al. 1988; Paju et al. 2007). Tumors with high PSA expression have been associated with low microvessel density and antiangi-ogenic activity (Papadopoulos et al. 2001; Ben Jemaa et al. 2010). Furthermore, low PSA concentrations in tumor tissue are associated with adverse outcome (Stege et al. 2000). The role of PSA in prostate cancer progression requires further elucidation. The studies cited above indicate that PSA may both stimulate and inhibit tumor growth (Figure 3). While this seems con-troversial, it is conceivable that PSA in the early phase of development promotes

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PSAhK2PAI-1

scuPA

uPA

Plasminogen

Fibrinogen

TGFß

FibronectinLaminin

Extracellular Matrix Degradation

Angiogenesis

Cell Proliferation & Differentiation

Fibrin

Plasmin

PAR2

PTHrP

ROS

latent TGFß

IGFBP-3

proPSA

IGF ApoptosisEMT

CleavageConversion/ActivationInhibition

AbolitionStimulation

Figure 3. Possible roles for hK2 and PSA during tumor progression.

tumor growth, but when the tumor reaches a size that it requires new blood vessels for further growth, PSA inhibits angiogenesis and thereby slows down tumor growth.

2.2.3. Potential Alternative Treatment Methods for Prostate Cancer Based on PSA

As mentioned earlier, PSA has been shown to exert both properties that may promote

and inhibit tumor growth. Inhibition of PSA expression by shRNA in LNCaP cells reduced the growth rates of these cells and transfection of PSA into DU-145 cells increased cell proliferation (Williams et al. 2011). This underlines the importance of the enzymatic activity of PSA. Treatment of PC-3M prostate cancer cell line with enzymatically active PSA results in up-

and down-regulation of several proteins (Bindukumar et al. 2008), some of which are related to prostate cancer progression. Most significant changes like upregula-tion of laminin receptor (Ménard et al. 1998), DJ-1 (Tillman et al. 2007) and Glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) (Rondinelli et al. 1997) have been shown to favor tumor inhibition. In another study, PSA has been shown, at

concentrations found in a healthy prostate gland, indirectly to enhance natural killer (NK) cells to secrete interferon-γ, which has antitumor and immunoregulatory activities (Fang et al. 2008). Antiangio-genic property of PSA is suggested to be mediated by its ability to generate active angiostatin fragments from lys-plasmi-nogen (Heidtmann et al. 1999). These recent findings of PSA suggesting a role in

22


maintaining homeostasis in the prostate cancer indicate that downregulation of PSA expression may act on predisposition of the prostate tissue to the development of cancer and it may be linked to a more aggressive disease. PSA may exert some of its antitumor effects via enzymatic activity, such as angiostatin generation, however, some of its actions may not require its enzymatic activity (Bindukumar et al. 2005). Considering that decreased amounts of PSA in prostate tumor tissue is associated with poorly differentiated cells and late stages of malignancy (Stege et al. 2000), it may be feasible to maintain PSA activity for treatment of prostate cancer. Furthermore, enhancing the PSA activity may even be more beneficial to regain the physiological homeostasis within the prostate tissue. Recently PSA-specific binding peptides have been identified by phage display library screening, which enhance the enzymatic activity of PSA (Wu et al. 2000). These peptides, as they are or their peptidomimetic derivatives, are potential drugs for prostate cancer. As these peptides specifically target PSA, they also have potential use for imaging of prostate cancer metastases. This may improve prostate cancer diagnosis by providing more information on the progress of prostate tumor growth. Another alternative approach for treatment of prostate cancer is utilizing cytotoxic agents, such as doxorubicin or thapsigargin prodrugs, coupled to PSA substrates so that they are activated in the vicinity of prostate tumor where the PSA concentration is highest (DeFeo-Jones et al. 2000; Denmeade et al. 2003). This type of therapeutical method could reduce the side effects of a cytotoxic drug as it is engineered to be tumor tissue specific.

3. Prostate Cancer Models

In cancer research, in vitro and in vivo studies are carried out with various models that try to recapitulate the characteristics

of the particular cancer type of interest. For prostate cancer these include various cell lines, some of which form xenograft tumors when introduced in animals, transgenic animal models, genetically engineered models and spontaneously tumor forming animals. Most commonly used prostate cancer cell lines in the literature are DU-145 (Stone et al. 1978), PC-3 (Kaighn et al. 1979) and LNCaP (Horoszewicz et al. 1983), which were established from bone, brain and lymph node metastases of human prostatic adenocarcinoma, respectively. LNCaP cells express mutant AR (Veldscholte et al. 1990), whereas PC-3 and DU-145 express very little or none (Sobel & Sadar 2005). The proliferation of LNCaP cells can be stimulated by treating them with androgens (Horoszewicz et al. 1983), and due its mutant AR also with several steroid hormones and anti-androgens (Veldscholte et al. 1992). Androgen treatment of LNCaP cells also increases the production of PSA and hK2 (Montgomery et al. 1992; Young et al. 1992; Grauer et al. 1996). As most of the prostate cancer cell lines express either very little or no AR, LNCaP is perhaps the most relevant cell line for studying the biology of early prostate cancer. Late stage tumors generally develop androgen resistance as a response to hormone therapy (Koivisto et al. 1998). In this respect, progression of prostate cancer development has been mimicked by coinoculating LNCaP cells with non-tumorigenic MS fibroblasts into athymic nude mice and in vivo passaging several sublines (Thalmann et al. 1994; Hyytinen et al. 1997). By maintaining the LNCaP tumors in castrated mice, also an-drogen-independent LNCaP sublines have been established (Thalmann et al. 1994). Another androgen-dependent prostate cancer cell line that expresses PSA is 22RV1 (Sramkoski et al. 1999). The cell line has been established by in vitro propagation from a xenograft model for primary human prostate carcinoma CWR22,

23


which represents relapsed primary cancer (Wainstein et al. 1994; Sramkoski et al. 1999). Another cell line derived from the same xenograft tumors is CWR22Pc, which grows androgen dependently and expresses high levels of PSA (Dagvadorj et al. 2008). When injected subcutaneously into nude mice, CWR22Pc grows androgen dependently and releases PSA into circulation. The PSA levels in the serum of this model were shown to correlate with tumor volume. In addition, MDA PCa 2a and -b cell lines, established from bone metastasis of a patient with advanced prostate cancer, are androgen sensitive, and produce hK2 and PSA (Navone et al. 1997; Lawrence et al. 2010). Also VCaP cells, derived from vertebral metastatic lesions, express large quantities of PSA (Korenchuk et al. 2001). Subcutaneous or orthotopical xenograft tumor models for prostate cancer are either derived from in vitro cultured cells like those mentioned above (van Weerden & Romijn 2000; Sobel & Sadar 2005) or propagated in vivo such as CWR22, PC-82, LAPC-4 and -9, and LuCaP23 (Craft et al. 1999; Klein et al. 1997; Ellis et al. 1996; Hoehn et al. 1980; van Weerden & Romijn 2000). Most commonly used mice strain for this purpose is nude mice (Foxn1nu), which have a reduced immune system due to defect in development of thymus gland (Flanagan 1966; Pantelouris 1968; Segre et al. 1995; Kaestner et al. 2000). LNCaP xenograft tumors can be established by injecting the cells together with basement matrix support under the skin of nude mice (Papsidero et al. 1981; Lim et al. 1993). For studies of certain pathways or oncogenes in prostate cancer development, transgenic mouse models have been established. The main strategy is to manipulate the target genes for gain- or loss-of-function. Transgenic adenocar-cinoma of the mouse prostate (TRAMP) is a mouse model developed using rat probasin promoter to express the SV40

early gene (T/t antigen:Tag), which was targeted to the differentiated columnar epithelial cells of the mouse prostate (Greenberg et al. 1994). This model mimics human disease histologically and patho-logically with metastatic spread to distant sites. Transgenic Lady model, which is developed using prostate-specific probasin promoter, is less aggressive reflecting the early stages of prostate cancer. Also PSA and hK2 transgenic mouse have been described (Williams et al. 2010; Zhang et al. 2000). However, the levels of PSA produced by this transgenic model were found to be 1000-fold lower than in human prostate and PSA was not detectable in the serum of these models. Another strategy to mimic the natural initiation and progression of cancer is to use mouse prostate reconstitution model (Thompson 1996). In this model, by introducing the ras and myc, or both oncogenes in recombinant retrovirus, different phenotypes of prostatic pathology can be established, like dysplasia, hyperplasia and frank carcinomas, respec-tively (Thompson et al. 1991). Spontaneous prostate tumors develop in some strains of rats, such as, the Dunning R-3327, Lobund Wistar and ACI/Seg (Pollard 1998; Dunning 1963). Spontaneous prostate ad-enocarcinoma occurs most commonly in dogs resembling closely to the human disease (Lamb & Zhang 2005). Most of the cell lines suitable for prostate cancer studies are derived from metastatic lesions and unfortunately, do not mimic early stages of prostate cancer development very well (Peehl 2005). Primary cultures of prostate cancer and their gene expression profile comparison to healthy prostate tissue provide useful tools for prostate cancer research (Peehl 2005). However, several challenges remain including isolation and characterization of these cultures with diverse genomic profile. Coordinated collection for large number of fresh prostate tumor tissues focused

24


on primary cultures of prostate cancer is possible by biobanking (Dhir 2008). Similar as it is with the in vitro culture of the cell lines, prostate tumor animal models currently in use can recapitulate certain aspects of the disease, but none of these can truly represent the complexity of human prostate cancer.

4. Peptides as Drugs

Peptides are 3 to 50 amino acid long single chained amino acid polymers connected by peptide bonds (Jones 2006). In biological systems, peptides have several roles such as acting as hormones, growth factors and neurotransmitters. Most of the naturally occurring regulatory peptides exert their activities by binding to specific target proteins or receptors, and activate or inhibit downstream pathways that regulate biological processes of cells including proliferation and apoptosis (Reubi 2003). Analogues of these naturally occurring peptides and active domains of proteins can be chemically synthesized to develop peptide drugs (Watt 2006). Bioactive peptides, that are native for the human body, are mostly highly stable in vivo and not immunogenic (Sato et al. 2006). Many bioactive peptides are already in use as drugs. These include insulin to treat diabetes, adrenocorticotropic hormone (ACTH) to treat secondary adrenal in-sufficiency, oxytocin for inducing labor, ghrelin to treat obesity, and glucagon-like peptide 1 (GLP-1) for the control of diabetes (Dhillo & Bloom 2001). Another approach to peptide drug development is discovery of novel peptides by scanning peptide libraries against proteins. These libraries can be generated chemically or genetically (Latham 1999; Landon et al. 2004; Laakkonen & Vuorinen 2010). For example, by using phage display libraries peptide inhibitors for proteases, such as gelatinases, have been developed (Koivunen et al. 1999).

Currently, a majority of drugs in clinical use are small molecule compounds that have favorable pharmaceutical properties such as small size, straightfor-ward synthesis, low price, oral availability and ability to cross biological membranes (Lipinski et al. 2001). As drugs, peptides have several disadvantages over these traditional small molecule drugs such as larger size, poor in vivo stability, fast clearance from circulation, and their production can be challenging and costly. Despite their drawbacks, peptides have unique properties that may be beneficial (Table 2). Peptides can bind to their targets with higher affinity and specificity. Furthermore, they can be used to target mechanisms that small molecules cannot (Sato et al. 2006). Peptides are mostly non-toxic, have low immunogenicity and do not accumulate in the internal organs like liver or kidney. Peptides can be chemically synthesized in mass amounts with high yield and purity, although production in large quantities can be expensive. Peptides are also considered to be alternative to antibody-based drugs that have high affinity and specificity (Ladner et al. 2004). Although antibodies are highly stable in circulation and mostly non-immunogenic, low tissue penetration properties makes them less suitable for treatment of solid tumors. Compared to antibodies, peptides have higher activity per mass. Manufac-turing of peptides is usually cheaper than that of antibodies and they are more stable during long time storage (Ladner et al. 2004). Monoclonal antibodies (Mabs) have been used in treatment of cancer (Mehren et al. 2003). Anti-HER2 targeting Mabs are used as a therapy for metastatic breast cancer that overexpresses HER2 (Le et al. 2005). Rituximab, a monoclonal antibody that targets B-lymphocyte antigen CD20 and stimulates cytotoxic mechanisms, has been used for treatment of non-Hodg-kins lymphoma and chronic lymphocytic lymphoma (CLL) (Mavromatis & Cheson

25


2003; Plosker & Figgitt 2003). However, most of the patients treated with rituximab experience infusion-related or hemato-logical adverse effects (Plosker & Figgitt 2003). Introducing non-human Mabs into human body also can trigger immunogenic reactions towards these Mabs (White et al. 2001). Furthermore, accumulation of Mabs in kidneys and liver may cause damage to these organs (Abdel-Nabi et al. 1992). These problems with Mab treatment can be partially overcome by converting the Mabs to a single chain Fv domain, which is the active part of the antibody (White et al. 2001; Mehren et al. 2003). Recently, peptibody technology, which fuses the highly stable Fc part of an antibody with a peptide, has been emerging (Wang et al. 2004). Peptibody, peptide-antibody hybrid, combines high stability of antibodies with powerful ligand selection for targets property of peptide libraries (Molineux & Newland 2010). Romiplostim, a peptibody that mimics thrombopoietin (TPO) and acts as TPO receptor agonist, has been used to treat immune thrombocytopenia (ITP), an autoimmune disorder characterized by decreased number of circulating platelets. In 2010, over 60 therapeutic peptide drugs were on market and many more were under development (Vlieghe et al. 2010). An example of recently approved peptide drug is enfuvirtide (Fuzeon, T-20), a 36-amino acid long therapeutic peptide that is used

to treat HIV-1 infection (Matthews et al. 2004). The peptide is a replicate of residues 127-162 of the transmembrane viral gly-coprotein gp41 (Wild et al. 1994), which inhibits the fusion of viral and plasma membranes, thereby preventing entry of the virus into host cells (Kilby et al. 1998). The recommended dosage for adults is 90 mg per day, which is a large amount considering the production costs (Fung & Guo 2004). Despite the high production price, which is due to the complexity of the peptide, the unique properties of this peptide makes it a most effective treatment method for HIV-1 infection (Badia et al. 2007). Besides being therapeutic tools, certain peptides have the ability to penetrate into cell cytoplasm by transloca-tion accross cell membranes (Wagstaff & Jans 2006). These peptides can be linked to “cargo” molecules for intracellular delivery, which makes them efficient drug-carrier molecules for targeting cytoplasmic pathways. Although peptide drugs mostly have some disadvantages such as low oral bio-availability and tendency for quick me-tabolization, the interest of pharmaceu-tical companies in therapeutic peptides is increasing. This is mostly because of few side effects, high specificity and easy mimicry of peptides or proteins that are part of physiological mechanisms.

Table 2: Advantages and challenges of peptides as drugs

Advantages Challenges

Highspecificity Low oral bioavailability

High activity Strenuousdelivery(injection)

Littlenon-specificbindingtomolecularstructures other than desired target

Poor transportation across membranes

Minimization of drug-drug interactions Challenging & costly synthesis

Low toxicity Solubility

Often very potent Risk of immunological effects

Biological & chemical diversity for target Rapid clearance from bodyModified and adapted from Vivien 2005 Chem Eng News 83(11), pp.17-24

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4.1. Phage Display Libraries and Peptides

One commonly used method to identify novel ligands for target proteins is phage display technology (Smith 1985; Smith & Petrenko 1997). The method is based on random expression of peptides with predefined structures as part of the coating proteins on the phage (Scott & Smith 1990). Collections of phage that are engineered to express these certain predefined structures with random amino acid sequence are called phage libraries (Smith 1985). Phage display is mainly used for identification of peptides or antibody recognition sites, but it can also be applied to epitope discovery and mimicry, and substrate identifica-tion for enzymes (Smith & Petrenko 1997; Ruoslahti & Rajotte 2000; Cloutier et al. 2002). Peptides conjugated to the phage coating protein generally have fixed number of amino acids and may have fixed positions for certain amino acids (Scott & Smith 1990). Based on their ability to form disulphide bridges, cysteine residues can be used to create variations in the tertiary structures. Most of the ligands found by phage display have high specificity towards the target (Parmley & Smith 1988). When applied for enzymes, it is possible to identify peptides that modulate their enzymatic activity (Koivunen et al. 1999). In screening, a phage library is incubated with the target protein followed by selection and enrichment of bound phage (Smith 1985). After several successive rounds of selection and enrichment (Figure 4), the DNA of the phage is sequenced to identify the peptide ligand (Sidhu et al. 2000). After the iden-tification of the peptide for the target, it can be synthesized chemically. If the target is an enzyme, the effect of the peptide on the enzymatic activity can be studied, as binding may modulate, i.e., inhibit or enhance the activity. Characterization of the peptides can be carried out by determining the amino acids necessary for the binding

or for the modulatory effect. This can be achieved by performing step-by-step single modifications of the peptides, such as alanine scanning, deletion of the residues and trimming from C- and N- termini. Other methods to screen and identify ligands for target receptors or proteins include peptide arrays (Frank 2002), peptide bead libraries (Lowe 1994) and positional scanning of synthetic combi-natorial libraries (PS-SCL) (Barrios & Craik 2002; Rubio-Godoy et al. 2002), all of which uses solid-phase combinatorial chemistry to generate diversity.

4.2. Modification of the Peptides for Improved Stability

When developing therapeutic peptides, probably the biggest challenge is their poor stability. After oral administration they are rapidly degraded by the enzymes in gastrointestinal tract (Veber et al. 2002). Therefore, peptides need to be delivered by alternative routes, such as subcutane-ous (s.c.) or intravenous (i.v.) injection (Weber et al. 1992). However, after i.v. injection peptides may also be degraded by peptidases in the circulation (Witt et al. 2001). And even if they withstand the proteolytic attack, they are often excreted via kidneys into urine because of their small size. Blood contains several active exo-peptidases which proteolyze peptides from N- or C- termini. Therefore, especially open-ended linear peptides are often vulnerable in circulation and rapidly degraded into single amino acids by exo-peptidases. Addition of one or several polyethylene glycol (PEG) groups to the N- or C- termini or both ends of the peptides prevents peptides being degraded by the exopeptidases (Harris & Chess 2003). Conjugation of PEG, termed as PEGylation, to peptides may change the charge of the peptide resulting in improved solubility and activity, but it may also

27


CC

C CC

C C C C C C CCC

C CC

CC

C CC

CC

C CC

CC

C CC

CC

C CC

CC

C CC

AA A A A

BB B B B D

D D D D

EE E E E

C C C C C C CCC

C CC

A library of phage displaying random peptide sequences on their pIII coat protein, is exposed to a target antibody captured protein in micro-titer wells.

Unbound phage are washed away

Specifically-bound phage are eluted

After 3 to 4 rounds, individual clones are isolated and their DNA sequenced for the identification of the peptide

Specifically-bound phage are amplified by infecting host cells

Panning and selection steps are repeated 3 or 4 times

result in loss of function. PEGylation of a peptide increases its size according to the number of added residues. This reduces clearance from circulation via kidneys, thereby prolonging its half-life (Veronese & Pasut 2005). Similarly, peptides can

be conjugated to albumin, which as well prevents excretion via kidneys. Also as mentioned earlier, coupling peptides to Fc parts of antibodies is an efficient method to increase the half-life of peptides (Wang et al. 2004).

Figure 4. Short schematic description of phage display method for identification of peptide ligands against proteins that are captured by an antibody.

28


Another method to prevent exo-peptidase degradation and to improve the stability is cyclization. Linear peptides can be cyclized either by adding cysteine residues to both ends to form disulphide bonds or simply by engineering a peptide bond from head-to-tail (Adessi & Soto 2002; Clark et al. 2005). However, cyclization of a bioactive peptide may impair its functions. Cyclization and PEGylation may prevent degradation by exopeptidases, but not by another class of peptidases called endopeptidases that cleave peptide bonds within the peptide. Contrary to exopeptidases, most endopeptidases in circulation are inhibited by serpins, A2M and other protease inhibitors (Huntington 2003). However, active endopeptidas-es are abundant in the small intestines making oral administration challenging for therapeutic peptides. Also the targeted tissues may contain endopeptidases. Several other strategies can be used to improve the stability of peptides. These include modification of the chemical structure of the peptide, engineering of pseudopeptides and peptidomimetics (Adessi & Soto 2002). Modifications of the chemical structure of peptides include synthesis of the peptides as D-isomers with reversed amino acid sequence (Milton et al. 1992; Guichard et al. 1994), N- or C- termini modifications, replacement of natural amino acids with non-coded amino acids and alkylation of N-amide nitrogen (Heavner et al. 1986). Pseudopeptides can be made by replacement or alteration of atoms that are part of the peptide backbone (Benovitz & Spatola 1985). These include amide bond surrogates, translation of the peptides into peptoids or β-peptides,

both of which are based on modified amino acids with side-chain substitu-ents (Zuckermann et al. 1992; Seebach et al. 1996), and conversion to azapeptides or azatides, where one or more α-carbon atoms are replaced by nitrogen atoms (Magrath 1992). Peptidomimetics, where peptides are mimicked by using chemical methods, may be another efficient way of combining the desired properties of both peptides and chemical compounds (Perez et al. 2002). By definition (IUPAC, In-ternational Union of Pure and Applied Chemistry) peptidomimetics do not have classical peptide characteristics such as en-zymatically scissile peptide bonds. Peptides provide promising molecular scaffolds for drug discovery, i.e, they can serve as lead molecules in drug discovery (Mergener 1998). This way, peptide traits such as high specificity and potency could be combined with drug properties, which are cheap production, oral administration and high stability (Lipinski et al. 2001; Hummel et al. 2006). Peptidomimetics or conversion to small drug compounds may significant-ly reduce production costs and increase stability in the digestive tract so that they could be administered orally. However, as for peptides, rather quick degradation and renal clearance are not always undesirable properties as short half-life of peptides may make them less toxic and easily manageable. After their task is complete, disappearance of the peptides may ease the burden on the liver and decrease potential side effects due to tissue accumulation. As a drug, the ideal peptide would be stable enough to penetrate the target tissue, but then be cleared from the system in a suitable time period.

29

hK2 and PSA are nearly exclusively produced by the healthy human prostate and by prostate tumors, which makes these proteases potential targets for prostate cancer therapy. This work aims to find hK2-specific novel peptide ligands that can be used to study the role of hK2 in prostate tumors and as lead molecules for development of peptide-based drugs for prostate cancer treatment. It also intends to further characterize the behavior of PSA in animal models.

The main goals in this study are

I. To identify and characterize specific peptides modulating the activity of hK2.

II. To improve the stability of hK2-inhibiting peptides for in vivo studies.

III. To study the inhibition of hK2 activity towards cancer-related substrates by hK2-inhibiting peptides.

IV. To characterize PSA complexes in mouse serum.

Aims of the Study

30

1. Proteases, Substrates and Antibodies

The proteases, substrates and antibodies used are described in Table 3.

2. Immunofluorometric Assays (IFMAs)

2.1. IFMA for Free and Total PSA

Free and total PSA were quantified simulta-neously using a dual-label IFMA (DELFIA Prostatus free/total PSA kit, Perkin-Elmer, Turku, Finland) according to the manu-facturer’s instructions. The assay for total PSA recognizes PSA-ACT and PSA-API, but not PSA-A2M complexes (Zhang et al. 1998).

2.2. IFMA for Intact and Total IGFBP-3

To measure concentrations of intact and total IGFBP-3 we have used two in-house built assays (Koistinen et al. 1994; Koistinen et al. 2002). For the intact IGFBP-3 assay, 25 µl of standards and samples were pipetted into the 1B6 antibody coated wells followed by addition of 200 µl assay buffer (AB) [TBS (0.05 M Tris buffer, pH 7.7, containing 0.154 M NaCl and 8 mM NaN3) containing 2.3 g/l bovine serum albumin and 0.15 g/l bovine globulin]. After overnight incubation at +4 °C wells were washed and europium-labeled tracer antibody 5C11 in DTPA containing AB was added for 1 h incubation at room temperature followed by another washing step. Enhancement solution (PerkinElmer Life Sciences) in 200 µl volume was added and incubated 5 min. Then fluorescence was measured with a Victor 1420 fluorometer (PerkinElmer Life Sciences) in time-resolved mode. To detect total IGFBP-3, i.e., both cleaved and intact proteins, similar protocol was followed except the catcher antibody was 5C11 and the tracer 7F8.

2.3. IFMA for Phage expressing hK2-binding Peptides

The phage IFMA was performed essentially as described (Wu et al. 2000). The solid phase antibody (5E4) was coated onto mi-crotitration wells at a concentration of 5 mg/l in TBS for 16 h at 22 °C, the solution was discarded, and the wells were saturated with 10 g/l BSA in TBS for 3h at 22 °C. In the assay 50 ng of hK2 in 200 µl of AB was captured onto wells coated with mAb 5E4. Following a 1 h incubation the wells were washed and 2.5 µl of phage (108-109 infectious particles) in 200 µl of AB was added. After incubation for another hour, the wells were washed and filled with 200 µl AB containing 100 ng of europium-labeled (Hemmilä et al. 1984) polyclonal anti-phage antibody recognizing the M13 coat protein of the phage. After incubation for 1 h the wells were washed and enhancement solution was added. The flu-orescence was measured with a Victor 1420 fluorometer (PerkinElmer Life Sciences) in time-resolved mode.

2.4. Cross-Inhibition Tests

The relative binding sites of the peptides on hK2 were mapped by competition experiments. The protocol was based on the IFMA method described above, except that various cloned phage and various synthetic peptides were reacted together with hK2. Briefly, each of the synthetic peptides at a concentration of 5 µM was added into wells in which hK2 had been captured by mAb 5E4. After incubation for 30 min, hK2-binding peptide expressing phage (1010-1011 units) were added, and after 1 h incubation the wells were washed, and bound phage were detected as described above for phage IFMA.

Materials and Methods

31


Table 3: Proteases, substrates and antibodies used in this study.

Protease Remarks Study Source

Enzymatically active PSA (PSA-B)

purifiedfromhumanseminalplasma I, IIIZhang et al. 1995, Wu et

al. 2004

proPSApurifiedfromLNCaPcellculturemedium

I Wu et al. 2004

hK2 recombinant produced in insect cells I, III, IV Lövgren et al. 1999

IGFBP-3 recombinant human III Peprotech(London,UK)

Bovine trypsin, α-chymotrypsin

IAthens Research and

Technology(Athens,GA)

Human plasmin, thrombin,plasma kallikrein

I Sigma-Aldrich

Trypsin 1, 2, 3 and C produced in-house I Koistinen et al. 2009

Substrate Protease specificity (sequence)

Study Source

Chromogenic substrates

S-2586PSA and chymotrypsin(MeO-Suc-Arg-Pro-Tyr-pNA)

IChromogenix In. Lab.

(Milano,Italy)

S-2222Trypsin(CO-Ile-Glu-(OR)-Gly-Arg-pNA)

I Chromogenix In. Lab.

S-2238Thrombin(H-D-Phe-pipecolicacid-Arg-pNA)


S-2403Plasmin (pyroGlu-Phe-Lys-pNA)


S-2302hK2 and plasma kallikrein(H-D-Pro-Phe-Arg-pNA)

I, II Chromogenix In. Lab.

Fluorogenic substrates

FluorogenicPSAsubstrate(4-morpholinecarbonyl-HSSKLQ-amidomethylcoumarin)basedonDenmeadeetal.1997

IIIJPT Peptide Technologies

(Berlin,Germany)

Antibody Description Study Source

5E4anti-PSA and -hK2 monoclonal antibody

I, IV Leinonen et al. 2002

ab6188 anti-M13 bacteriophage coat protein I Abcam(Cambridge,UK)

5C11, 1B6, 7F8 anti-IGFBP-3 monoclonal antibodies III Koistinen et al. 1994

P0260 rabbit anti-mouse IgG III Dako, Glostrup, Denmark

A0562 rabbit anti-PSA polyclonal antibody IV Dako

P0217swine anti-rabbit polyclonal IgG conjugated with HRP

IV Dako

J1105(Vectastain) control rabbit IgG IVVector Labs, Burlingame,

CA

I5381 non-specificmouseantibody IV Sigma-Aldrich

32


3. Determination of Enzymatic Activity

3.1. hK2

hK2 (0.17 µM) was incubated with a 100 –1000-fold molar excess of hK2-specific synthetic peptides in 20 mM Tris buffer, pH 8.0, containing 1 g/l BSA, for 30 min at room temperature. After addition of the S-2302 substrate to a final concentra-tion of 0.2 mM, the absorbance at 405 nm was monitored at 5-10 min intervals for 1 h with a Victor 1420 Multilabel fluorometer. Student’s t test was used to evaluate the significance of the hK2 inhibition by the peptides.

3.2. Other Serine Proteases

The specificity of the peptides was analyzed by studying their effect on the activity of other serine proteases including α-chymotrypsin, bovine trypsin, human trypsin 1, 2, 3, and C, urokinase, thrombin, plasmin, plasma kallikrein, and PSA. All of the substrates for these serine proteases were chromogenic (see table 3). The con-centrations suggested by the manufac-turer were used. As in hK2 enzymatic activity measurement, the activities of the proteases were monitored with a Victor 1420 Multilabel fluorometer at 405 nm absorbance in 5-10 min intervals.

4. hK2 Inhibition Towards Natural Substrates

4.1. Inhibition of hK2-Mediated Activation of proPSA by Peptides

hK2 is known to activate proPSA (Lövgren et al. 1997). To determine whether the peptides are able to inhibit this activity, the hK2-binding peptides, hK2p01, hK2p02, and hK2p03, were incubated with hK2 for 50 min at room temperature in TBS buffer containing 1 g/l BSA before the addition of proPSA. Concentrations of peptides, hK2, and proPSA were 0-100 µM, 7.5 nM, and 150 nM, respectively, in a final volume of 50 µl. After incubation for 2 h, 10 µl of 2.4

mM fluorogenic PSA substrate was added. Fluorescence (excitation at 355 nm and emission at 460 nm) was measured after 17.5 h of incubation in a Victor 1420.

4.2. Inhibition of hK2-Mediated Degradation of IGFBP-3 by Peptides

hK2 (5 nM final concentration) was preincubated with hK2-inhibiting peptides hK2p01, hK2p01b, hK2p02 and hK2p03 (1, 10 and 100 µM) for 30 min at room temperature in TBS buffer containing 1 g/l BSA before addition of IGFBP-3 (1.5 nM final concentration). After incubation for 24 h at 37 °C, the concentrations of IGFBP-3 were measured using two sandwich-type immunofluorometric assays, one recognizing only intact IGFBP-3 and the other both cleaved and intact (i.e., total) IGFBP-3 (section 2.2).

5. Enzyme Kinetics Measurements of hK2 Inhibition by Peptides

The inhibitory capacity of the peptides was characterized by determining IC50 value for hK2 inhibition on the basis of the velocity of S-2302 cleavage at 200 µM concentration by 0.17 µM hK2 in the presence of 0-400 µM peptide. In addition, the Ki value for the most potent inhibitory peptide was determined by a Lineweaver-Burk plot after determining the velocity of the cleavage of 0-400 µM S-2302 substrate by 0.2 µM hK2 in the presence of 4 µM of peptide. Ki values were also determined for peptides inhibiting hK2 towards IGFBP-3. hK2 (5 nM final concentration) was pre-incubated with hK2-inhibiting peptides hK2p01, hK2p01b, hK2p02 and hK2p03 (1, 10 and 100 µM) for 30 min at room temperature in TBS buffer containing 1 g/l BSA before addition of IGFBP-3 (1.5 nM final concentration). The final volume in the 96-well microtiter wells was 120 µl. After incubation for 24 h at 37 °C, the concentrations of intact and total IGFBP-3 were measured using sandwich-type im-

33


munofluorometric assays (section 2.2). Ki values were determined using 0.2 nM hK2, 3-50 nM IGFBP-3 and 0-10 µM peptides and the SigmaPlot 11.1 program with Enzyme Kinetics Module 1.3 (both from Systat Software Inc., San Jose, CA, USA).

6. Phage Display Library Screening

Construction of the phage display libraries, CX7C, CX8C, X9, X10, X11, CX3CX3CX3C (C=cysteine, X=any of the 20 randomly occurring natural amino acids) in fUSE-5-phage and screening of these libraries were done as previously described (Koivunen et al. 1994b; Koivunen et al. 1994a). Each peptide library was screened against hK2, which was captured by 5E4 coated onto microtiter wells (Wu et al. 2000). mAb 5E4 binds PSA and hK2 in an equimolar manner and reacts with an epitope, which is remote from the active site of these enzymes (Leinonen et al. 2002; Stenman et al. 1999b). hK2 in the amount of 24 ng was incubated in antibody-coated wells for 30 min at 37 °C in 100 µl of TBS followed by washing with TBS to remove unbound hK2. An aliquot of each phage library containing 1010-1011 transducing units was added into the microtiter wells in 100 µl of TBS containing 10 g/l BSA. Incubation took place at 4 °C with gentle shaking for 16-24 h in the first round and for 1 h for the following three rounds. The wells were washed 10 times with TBS containing 0.5% Tween 20. To elute bound phages, 100 µl of 0.1 M glycine-HCl, pH 2.2, containing 1 g/l BSA was added. The eluted phage suspension was neutralized with 1 M Tris buffer, pH 9.0, amplified by infection of kanamycin-resistant Escherichia coli K91 cells and purified by polyethylene glycol precipitation (Wu et al. 2000). After four rounds of selection and amplification, single-stranded DNA from individual phage clones was prepared. The relevant part of the viral DNA, after amplification by PCR (Koivunen et al. 2001), was sequenced

with an ABI 310 Genetic analyzer and the Dye Terminator Cycle Sequencing Core kit (PE Applied Biosystems, Foster City, CA, USA) using as a primer 5´-TAA TAC GAC TCA CTA TAG GGC AAG CTG ATA AAC CGA TAC AAT-3´.

7. Peptide Synthesis

Peptides hK2p01-hK2p06 (Table 4) were synthesized with an Apex 396 DC multiple peptide synthesizer (Advanced ChemTech, KY, USA) with Fmoc [N-(9-fluorenyl)methoxycarbonyl] strategy and TBTU/diisopropylethanolamine as coupling reagents. All peptides were synthesized on Gly-Wang resin (Advanced ChemTech). The side-chain protecting groups used in synthesis were t-butyloxycarbonyl for tryptophan and lysine, tert-butyl for serine and theonine, 2,2,4,6,7-pentame-thyldihydrobenzofurane-5-sulfonyl for arginine, and trityl for glutamine. For cysteine both acetamidomethyl and trityl protection groups were used. The peptides were purified by HPLC (Shimadzu, Kyoto, Japan) using a C18 reversed-phase column with acetonitrile as eluent [0.1% trifluoroacetic acid (TFA), 0-60% acetoni-trile (ACN) gradient for 60 min]. Alanine replacement experiments were done for peptides hK2p01-hK2p03 by changing each of the amino acids to alanine indi-vidually.

8. Cyclization of the Peptides

Iodination method was used to cyclize peptides containing cysteines with Acm (Veber et al. 1972). Into the peptide solution with 2 mg/ml concentration in 50% acetic acid (AcOH) were added 1 M HCl (0.1 ml/mg of peptide), which was immediately followed by the addition of 0.1 M iodine solution in 50% AcOH in H2O (5 equiv./Acm). After vigorous mixing for 40 min at room temperature, the reaction was stopped by adding 0.1 M sodium thiosulfate to a final concentra-

34


tion. The solution was filtered (0.45 μm) and the peptides were purified by HPLC as described above. Head-to-tail cyclization was performed on the resin. The allyl ester deprotection was done manually with Pd(0) under argon. Mixture of Fmoc – peptidyl(resin) – OAll and PhSiH3 containing the catalyst in a small amount of DCM was stirred for 2.5 h at room temperature under argon. After washing the resin with the catalyst-dissolving mixture, 0.5% DIEA in DMF, 0.5% sodium diethyl dithiocarbamate in DMF and DCM, and drying, the N-terminal Fmoc group was removed using 20% piperidine in DMF. Head-to-tail cyclization process was done twice by the addition of HBTU/DIEA (8 equiv., 1 : 1, mol/mol) in DMF for 2 times for 3 h at room temperature. Peptides were released from the Rink resin using 95% TFA.

9. Similarity Searches

BLAST searches (www.ncbi.nih.gov/BLAST) (Altschul et al. 1997) of the hK2 inhibiting sequences were performed using the Swiss-Prot database.

10. Stability Studies

Initially, stability was studied by using modified trypsin (sequencing-grade modified trypsin V5113, Promega, Madison). Each peptide (400 µg) dissolved in 400 µl of 200 mM NH4HCO3 buffer (pH 8.0) was mixed with 1 μl of trypsin solution (1 mg/ml in NH4HCO3, pH 8.0). The peptides were incubated at 37 °C and 30 μl aliquots were sampled every 30 min. Thirty microliters of 2% TFA and 5% ACN in water was added, and the samples were analyzed by HPLC and by mass spectrome-try using an ABI QSTAR XL with a MALDI interface (AB SCIEX, Framingham, MA, USA). The stability of the peptides in human plasma was studied by mixing 400 μl of

human male plasma collected into EDTA coated vacutainer tubes (BD Biosciences) and 400 μl of each peptide (1 mg/ml) in PBS (phosphate-buffered saline). The peptides were incubated at 37 °C and 100 μl aliquots were taken after 30 min and then every hour up to 4 h. The head-to-tail cyclic peptides were incubated for 24 h. The peptide was separated from plasma proteins on a centrifugal filter device (Microcon YM-10, cut off 10 kDa, Millipore, Bedford, MA, USA) by centrifu-gation at 14,000 rpm using an Eppendorf 5415 D centrifuge (Eppendorf, Hamburg, Germany) for 10 min. Remaining plasma protein on the filter was washed with 50 μl of 0.1% TFA and 0.1% Tween 20 in H2O and centrifuged again for 10 min. The filtrates were analyzed by analytical HPLC and mass spectrometry.

11. PSA Complexation in Serum

To estimate the rate of the PSA complex formation and to identify the complexes in serum, 200 ng of PSA in 1 ml of TBS was added to 100 µl of mouse serum and 10 µl aliquots, diluted with 90 µl of AB of the dual-label IFMA kit (DELFIA Prostatus free/total PSA kit), were frozen at time points between 0 and 24 h. For further purification and identification, PSA (7-24 µg) in 7-24 µl TBS was added to 300 µl of mouse serum. Female human serum was used as a control. After incubation for 48 h, free and total PSA were quantified by IFMA.

12. Chromatographies

Free and complexed PSA in serum (250 µl) were separated by gel filtration on a HiLoadTM 16/60 Superdex 200 column (Amersham Biosciences, Arlington Heights, IL, USA) and eluted with TBS. Flow rate was 15 ml/h, and 1-ml fractions were collected. Fractions containing complexed PSA were identified by IFMA and further fractionated by anion-exchange

35


chromatography on a 1-ml Resource Q column (Amersham Biosciences) equili-brated with 10 mM Tris-HCl buffer, pH 8.0, containing 8 mM NaN3 (buffer A). Bound proteins were eluted with a linear gradient composed of 60 ml of buffer A and 60 ml buffer A containing 300 mM NaCl. PSA-containing fractions of 2 ml volume were identified by IFMA, pooled and concen-trated to 1 ml using a Centriplus YM-10 filtration device (Amicon, Beverly, MA, USA).

13. Immunopurification

PSA-complexes in mouse and human serum and in chromatographic fractions were purified by immunoadsorption using Mab 5E4 coupled to Sepharose beads (Leinonen et al. 2002; Wu et al. 2004). To reduce non-specific binding to the column, the fractions were first preincubated for 2 h at room temperature with Sepharose beads coupled with non-specific mouse antibody (I5381, Sigma-Aldrich). The samples were then incubated with 30 mg of 5E4-beads for 2 h at room temperature on a shaker. After washing the beads three times by addition of PBS and centrifugation at 8,000 rpm for 3 min, bound proteins were eluted with 24 µl of 0.1% trifluoroacetic acid and neutralized with 1 µl of 1 M Tris-HCl.

14. SDS–PAGE and Western Blotting

Immunopurified PSA was boiled in reducing SDS-sample buffer and separated by electrophoresis on 4-12% NuPAGE Bis-Tris gels run in MES buffer (Invitrogen, Carlsbad, CA, USA), followed by silver staining or transfer of proteins to Immobilon-P Transfer Membrane (Millipore, Bedford, MA). Non-specific binding to the membrane was blocked by incubating the filter for 2 h at room temperature with 1% bovine serum albumin in PBS. The membrane was incubated overnight at +4 °C with rabbit anti-PSA polyclonal antibody, followed

by detection with horseradish peroxidase-conjugated swine anti-rabbit immuno-globulin G, using enhanced chemilumi-nescence (ECL) Western blotting detection reagents (GE Healthcare, Amersham, UK).

15. Xenograft Tumor Models

The human prostate carcinoma cell lines LNCaP and 22RV1 (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium (Cambrex, Verviers, Belgium) containing 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, 1% non-essential amino acids, penicillin (100 U/ml), and streptomycin (100 mg/ml) (all from Sigma-Aldrich). The cells were kept in a humidified incubator at 37 °C with 5% CO2. Athymic nude mice (NMRI-nu/nu) and SCID (severe combined immuno-deficiency) mice were purchased from Taconic (Bomholt, Denmark) and from Charles River Laboratories (Wilmington, MA, USA). To produce prostate cancer xenografts in mice, LNCaP or 22RV1 cells (1.5 x 106 per mouse) in 50 µl of 6.7 mM phosphate-buffered saline (PBS) without Ca and Mg (Cambrex) were mixed with 50 µl Matrigel basement membrane preparation (BD Biosciences, Franklin Lanes, NJ, USA). The mixture was injected subcutaneously into the flank in the abdominal region of 4- to 6-week-old mice. The mice were sacrificed before the tumors reached a threshold volume of 1,500 mm3, usually 6-8 weeks after injection of the cells. Tumor volume was measured with a caliper and calculated according to the formula V = width x height x depth / 2. Animal Experiment Board of State Provincial Office of Southern Finland has approved the study protocols in this work.

16. Immunohistochemical Staining

Mouse tumor tissues were fixed in 10%

36


neutral buffered formalin, embedded in paraffin and cut in 5-µm sections. Sections were deparaffinized and incubated with rabbit anti-PSA polyclonal antibody or control rabbit IgG as a negative control. For detection HRP-conjugated antirabbit polymer (Envision system, Dako) was used with 3-amino-9-ethyl-carbazole (AEC). The sections were counterstained with Mayer’s hemalum solution (Merck, Darmstadt, Germany) and mounted with Gurr Aquamount (BDH, Poole, UK).

17. Identification of PSA Complexes and IGFBP-3 Fragments by Mass Spectrometry

IGFBP-3 (1.75 µM) was incubated with hK2 (0.25 µM) for 24 h at 37 °C in 20 µl of TBS containing 0.06% BSA. Protein bands were cut from the silver stained gel, alkylated and treated with modified trypsin (Promega, Madison, WI, USA) for mass spectrometry (Shevchenko et al. 1996). IGFBP-3 fragments were con-centrated using ZipTip pipette tips (µC18

ZipTip®, Millipore). For LC-MS analysis the peptides were separated on a CapLC instrument (Waters, Milford, MA, USA) with a 0.075 mm x 150 mm C18 column (Atlantis dC18, 100 Å, 3 µm, Waters) and eluted with a linear gradient of acetonitrile (5-50% in 30 min) in 0.1% formic acid. Flow rate was 0.3 µl/min and the eluent was directly injected into a quadrupole/time-of-flight hybrid mass spectrometer (Q-TOF Micro, Waters) equipped with an electrospray ionization (ESI) source. Fragmentation spectra of the peptides were acquired by colliding doubly or triply charged precursor ions with argon collision gas at accelerating voltages of 20-70 V. Protein and peptide identifications were performed automatically with the MASCOT search engine (Matrix Science, Inc., Boston, MA, USA) using primary sequence databases. Spectra were also interpreted manually using Biolynx peptide sequencing software (Waters) and FASTA sequence comparison software (Lipman & Pearson 1985).

37

1. Identification and Development of hK2-inhibiting Peptides (I)

Several rounds of phage display library panning with different libraries resulted in identification of 6 peptides binding to hK2 from libraries X10 and X11 (Table 4). Three of these were 10 amino acid long linear peptides from X10 library and the other three were 11 amino acid long cyclic peptides from X11 library. Arginine was present in all six peptides. Some common motifs can be found between the peptides. The peptides hK2p02 and hK2p03 share

the amino acid sequence of ARRPFP. In the other peptides from X11 library arginine is located two residues after the first cysteine. hK2-binding phage-peptide IFMA, where antibody-captured hK2 bound phage was traced with europium-labeled anti-phage polyclonal antibodies, confirmed binding of the synthetic peptides to hK2. One of the 6 identified peptides (hK2p05) showed weak binding and it was excluded from further studies. The synthetic peptides competed with the phage that display same peptides.

Table 4: hK2-binding peptides identified by phage display.

Code Library Peptide sequenceNo. of isolates

IC501 IC50

2 Ki3

hK2p01 X10SRFKVWWAAG 1 3.4 2.6 1.05

hK2p02 X10AARRPFPAPS 19 30.5 6.5 1.87

hK2p03 X10PARRPFPVTA 1 162.0 18.6 3.22

hK2p04 X11CFRQGCWVIT 3

hK2p05 X11FRTCLRSWACM 6

hK2p06 X11CYRMPTCMQRD 4

Results

IC50 and Ki values are in µM and refer to inhibition of hK2 enzymatic activity1with synthetic peptide substrates2with proPSA3with IGFBP-3

Furthermore, the cross-inhibition assay also showed that peptides hK2p01, hK2p02 and hK2p03 compete with each other. These three peptides were found to inhibit the enzymatic activity of hK2 towards a synthetic chromogenic substrate (Figure 5). The specificity was also confirmed by studying the effect of peptides on the enzymatic activity of several serine proteases with similar structure or catalytic activity than of hK2. Inhibition of hK2 by the peptides was dose dependent and

Lineweaver-burke analyses suggested that the peptides are competitive inhibitors. Half maximal inhibitory concentrations (IC50) for the peptides towards the synthetic substrate were 3.4 µM for hK2p01, 30.5 µM for hK2p02 and 162.0 µM for hK2p03 (Table 4). The residues that are essential for inhibition of hK2 were determined by making structural modifications and testing the effect on inhibition activity towards hK2. Alanine replacement and

38


0

0.075

0.150

0.225

0.300

0 5 10 15 20 25 30

no peptidehK2p03hK2p02hK2p01hK2p01b

Abs

orba

nce

(A40

5)

Time (min)

sequence trimming of the amino acid residues showed that the motif required for the inhibitory effect of hK2p01 peptide is RFKXWW, while for hK2p02 and hK2p03 it is ARRPXP. In the alanine replacement experiments, changing the first amino acid at the N-terminus of the peptide hK2p01 from serine to alanine actually increased

the inhibitory efficacy and decreased the IC50-value from 3.4 to 2.2 µM. Deletion of the N-terminal serine reduced the inhibitory activity. The modified hK2p01 peptide with alanine in the N-terminus (hK2p01b) showed the strongest inhibition potency towards synthetic peptide substrates with a Ki value of 1.41 µM.

Figure 5. Inhibition of hK2 (0.17 µM) by 1000-fold molar excess of hK2-inhibiting peptides. Absorbance at 405 was measured to observe cleavage of the chromogenic substrate S-2302 at 5 min intervals for 30 min (n=12, mean±S.E). *, p<0.001 (Student’s t-test).

2. Effect of hK2-inhibiting Peptides Towards hK2 on Protein Substrates (I, III)

To confirm that the peptides also inhibit activity towards protein substrates, we studied inhibition of proPSA activation by hK2. In our experiments, proPSA was activated by hK2, and hK2-inhibiting peptides inhibited activation of proPSA by hK2 in a dose-dependent fashion. The

IC50 values for the peptides were 2.6 µM for hK2p01, 6.5 µM for hK2p02 and 18.6 µM for hK2p03 (mean, n=8, p<0.001 between all, Student’s t test). To inhibit hK2 activity towards IGFBP-3, the same three hK2-inhibiting peptides and one modified peptide (hK2p01b) were studied. When preincubated at three different concentra-tions, peptides inhibited the enzymatic activity of hK2 towards IGFBP-3 in a

*

*

*

**

39

Results

KLK2Peptide (µM) 100 10 1

+- + + +- + + +- + + +- + + +- + + +-100 10 1 100 10 1 100 10 1 100100

100

80

60

40

20

0100 100 100 100 10 1

hK2p01 hK2p02 hK2p03hK2p01b Controlpeptide

Rel

ativ

e pe

rcen

tage

(%)

10 20 30 (G) 40 50 60 MQRARPTLWA AALTLLVLLR GPPVARAGAS SAGLGPVVRC EPCDARALAQ CAPPPAVCAE

70 80 90 100 110 120 LVREPGCGCC LTCALSEGQP CGIYTERCGS GLRCQPSPDE ARPLQALLDG RGLCVNASAV

130 140 150 160 170 180 SRLRAYLLPA PPAPGNASES EEDRSAGSVE SPSVSSTHRV SDPKFHPLHS KIIIIKKGHA

190 200 210 220 230 240 KDSQRYKVDY ESQSTDTQNF SSESKRETEY GPCRREMEDT LNHLKFLNVL SPRGVHIPNC

250 260 270 280 290 DKKGFYKKKQ CRPSKGRKRG FCWCVDKYGQ PLPGYTTKGK EDVHCYSMQS K

dose-dependent fashion as shown by im-munoassays that measure the intact and cleaved IGFBP-3 (Figure 6). The order of the inhibitory efficiencies of the peptides

was the same than those found with the synthetic peptide substrates, hK2p01b being the most efficient with a Ki value of 0.28 µM (Table 4).

Figure 6. The relative percentage of cleaved vs. intact IGFBP-3. Control peptide is previously identified 12 amino acid long cyclic PSA-stimulating peptide that does not bind to hK2.

Figure 7. hK2 cleavage sites of IGFBP-3 and fragments generated by hK2.

We identified the cleavage sites of IGFBP-3 generated by hK2 (Figure 7). After the digestion by hK2, the IGFBP-3 fragments were analyzed by mass spec-trometry and 9 to 35 amino acid long eight fragments of IGFBP-3 were found (Figure 7). The peptide fragments were

mostly ending with an arginine, but also with lysine and methionine. Cleavage after arginine and lysine are in accordance with trypsin-like substrate specificity of hK2 (Deperthes et al. 1996). One fragment identified with low confidence score was observed to be cleaved after methionine.

40


3. Cyclization and Stability Improvements of the Peptides (II)

Linear peptides are highly susceptible to proteolytic attacks in circulation. We found that this was also the case with hK2 inhibiting peptides, the hK2p01b peptide being degraded within minutes. To increase stability, the linear peptides were cyclized in two different ways; via addition of cysteine to both ends to form a disulphide bridge, and by cyclization with a peptide bond, which is referred as head-to-tail cyclization. For the cyclization process, the amino acid sequences of hK2-inhibiting peptides that are required for inhibition were used as scaffolds. The rest of the amino acids were replaced by alanine. Several derivatives were synthesized by addition of alanine residues to both ends to modify sizes of the peptides before cyclization. Most ARRPAP motif containing peptide variants (referred as KLK2a in publication III) did not inhibit hK2 after cyclization with a disulphide bridge, but one showed slight (23%) inhibition. The head-to-tail cyclized variants of RFKVWW containing peptide (hk2p01b and hK2p01-ht, in publication III referred as KLK2b and KLK2b-ht, respectively) inhibited hK2 activity with similar or even higher activity than the parent compounds. The stability of the hK2 inhibiting peptides towards proteolytic degradation was studied by incubation with trypsin (0.1 µmol/l) and human plasma followed by HPLC analyses. Trypsin cleaves peptide bonds after lysine and arginine, and human plasma is a rich source for proteases. The linear hK2p01b was completely degraded by trypsin in 30 min, whereas cleavage of the cyclic peptides was much slower and after 4 h of incubation 43% of hK2p01b-ht peptide was intact. In human plasma, the linear and the cysteine cyclized forms of the peptides were degraded within 30 min while head-to-tail forms of hK2p01b remained intact for 24 h (Figure 8).

Arginine residue in the amino acid sequence of the peptides may indicate that the peptides could also be substrates for hK2 itself. However, we did not observe degradation of the peptides by hK2 (data not shown).

Search of the proteins/peptides that contain similar sequences to those essential for hK2 inhibition was performed by using BLAST. The sequences ARRPXP and RFKXWW were searched in the UniprotKB/Swiss-Prot database of human proteins. Several proteins contained the sequence ARRPXP of which most were intracellu-lar proteins or hypothetical proteins with evidence at the transcript level. Among the most relevant matches were Stromal cell-derived factor 1 (UniProt accession code P48061), Spasmolytic polypeptide (Q03403), serine/threonine protein kinase p21-activated kinase 4 (PAK4) (O96013), cytokine receptor-like factor 1 precursor (O75462), and substance P chain of pro-tachykinin 1 precursor (P20366). No

Figure 8. HPLC analyses of hK2p01b-ht peptide A) without and with trypsin after 4 h and B) with human plasma after 30 min and 24 h of incubation times.

A

B 30 min 24 h

no trypsin 4 h

Plas

ma

Tryp

sin

41

Results

proteins containing the RFKXWW motif were found.

4. PSA Forms Complexes in Mouse Plasma and Serum (IV)

When subcutaneously transplanted into nude mice, LNCaP cells and 22RV1 cells grow tumors, which produce PSA that is released into circulation. The tumor volumes and the PSA plasma concentrations correlated (Figure 9). When

LNCaP xenograft tumor size reached 1000 mm3 PSA levels in mouse plasma were 100-300 µg/l and that in mice bearing 22RV1 xenograft tumors were 5-8 µg/l. hK2 serum levels in these LNCaP xenografts were up to 2 µg/l (unpublished results). During tumor growth, the proportion between free and total PSA remained fairly constant, 68±7.5% (mean±SD) of PSA being complexed. Tumor size or total PSA concentrations were not

Tumor volume (mm3)Tumor volume (mm3)

R 2 = 0.8286

R 2 = 0.845

0

2

4

6

8

10

0 500 1000 1500

22RV1 xenografts

Total PSAFree PSA

LNCaP xenografts

R2 = 0.7344

R 2 = 0.8079

0

50

100

150

200

250

300

350

0 500 1000 1500

Total PSAFree PSA

PSA

(ng

/ml)

related to the proportion of complexed PSA, which was similar in LNCaP and 22RV1 xenografts. The complexation rate was calculated by adding pure PSA to mouse serum followed by incubation at 37 °C and measurement of free and total PSA levels in aliquots collected at several time intervals. A decrease in free and total PSA concentrations were observed within 30 min by immunoassay. After 24 h of incubation, 27% of PSA remained free while 36% was in immunoreactive complexes and 37% was undetectable, most likely in complex with A2M, which is not detectable by the immunoassays used. Western blotting using anti-PSA polyclonal antibody showed that PSA

formed several complexes in mouse serum. The observed bands corresponded to 33, 85, 160 and 200 kDa. A weak band was observed at 65 kDa (Figure 10). To further characterize the complexes, PSA added to serum was immunopurified, followed by SDS-PAGE. Western blot analysis showed that there were no visible complexes immediately after addition of PSA to the mouse and human serum. After 18 h PSA was in complexes in both sera. PSA complexes in mouse serum were seen in the region between 75-230 kDa, while in human a strong band at 120 kDa and a diffuse band at 200-500 kDa region were found. Similar bands were seen at the silver staining. To enrich PSA and its complexes

Figure 9. Total and free PSA values measured from the serum of LNCaP and 22RV1 xenografts. Modified and adapted from Hekim et al., Complex formation between human prostate-specific antigen and protease inhibitors in mouse plasma Prostate 2010, 70(5), pp.482–490. Reprinted with permission.

42


mouse serum + PSA

Immunopurification

PSA -+ +Serum +- +

a1a2

250 -150 -100 -

75 -

50 -

37 -

25 -

250 -150 -100 -

75 -

50 -

37 -

25 -

20 -

mouseserum human

serum

+ - + - + - + - +

IncubationTime (h)

0 0 0 18 18 18 180 0PSAIncubationtime (h)

----0 0 0 0018 18

+++++1818

mouseserum

humanserum

250 -150 -100 -

75 -

50 -

37 -

25 -

PSA Western Blotting

α2-macroglobulin 1

α1-antitrypsin

Prostate specific antigen 3

α1-antitrypsin 1-2

Prostate specific antigen

Prostate specific antigen

α1-antitrypsin 1-4α1-antitrypsin 1-5α1-antitrypsin 2

α1-antitrypsin 2, 4

α1-antitrypsin 2, 4, 5

α1-antitrypsin 1-5α1-antitrypsin 4

2

Gel filtration chromatography

Fraction no.

fPSAtPSA

0

1000

2000

3000

50 60 70 80 90 100 110

Ion exchange chromatography

Fraction no.

PSA

(µg

/L)

PSA

(µg

/L)

fPSAtPSA

0

400

800

1200

0 10 20 30 40 50

E b1

b2

b3b4

97 -

66 -

55 -

40 -

Silver staining

Immunopurification

Silver staining

Figure 10. Flow chart for identification of PSA complexes in mouse serum. The collected fractions from chromatography steps are shown in boxes with a dashed line. Except for human PSA, all proteins are of mouse origin

1 The peptides identified by MS belong to the region between N-terminus and bait region.2 The identified peptides could be derived from members of the α1-antitrypsin family.3 Roughly equal amounts of PSA and α1-antitrypsin 1-5 were found by MS.4 Minor amounts as compared to PSA.5 One of the identified peptides is found in the C-terminal part, which is cleaved upon the activation of the serpin

43

Results

before immunopurification, gel filtration and ion exchange chromatography were performed. After immunopurification and SDS-PAGE, silver staining revealed 4 bands of sizes between 37-93 kDa. These bands together with the two other bands from the silver staining without

immunopurification were in-gel trypsin digested and identified by MS/MS. The analyses showed that the bands at the sizes of 58 and 93 kDa contained mostly serpins from α1-antitrypsin family, and smaller bands of 37 and 40 contained both members of α1-antitrypsin family and PSA.

44

Among proteases produced by the prostate, hK2 and PSA are the most organ specific and most abundant (Wang et al. 1981; Schedlich et al. 1987; Shaw & Diamandis 2007). Both enzymes have been suggested to play roles in tumor growth in prostate cancer (Borgoño & Diamandis 2004). PSA exhibits antiangiogenic properties and may thus reduce tumor growth, whereas hK2 may favor tumor growth by cleaving or activating growth factor regulators, such as IGFBP-3 and PAR2, and increase metastasis by degrading ECM (Koistinen et al. 2008). In a previous (Wu et al. 2000) and present study (I) we have identified peptides that bind specifically to PSA and hK2, and enhance the enzymatic activity of PSA or inhibit hK2, respectively. These peptides are potential drugs for therapy of prostate cancer. Inhibition of hK2 by specific peptides may also slow down tumor growth. However, in order to study the effect of the peptides in vivo the stability of the peptides usually have to be improved. Moreover, to develop medical interventions based on modulation of the activity of PSA or hK2 it is necessary to learn about the behavior of these proteases in the tumor microenvironment and in the circulation using animal models of prostate cancer.

1. Identification and structure of peptide inhibitors of hK2 (I)

In this study, novel hK2-binding peptides were identified using phage display technology. Three linear peptides that inhibit the enzymatic activity of hK2 were found. By using same method, specific peptides for PSA have previously been identified in our laboratory (Wu et al. 2000). The PSA-specific peptides were

identified in cyclic libraries and they enhanced the enzymatic activity of PSA. No peptides were identified when PSA or hK2 were directly adsorbed to the plastic wells. A critical factor in identification of both PSA- and hK2-specific peptides was to capture the target protein using antibodies for the screening process. With this strategy we were able to expose the active site of the enzymes to the peptides displayed by phage and to identify peptides that bind to this region. Despite the high structural similarity between PSA and hK2 and the same antibody used to capture, the peptides identified with these enzymes did not contain any common motifs. In the case of enzymatic activity modulating peptides, this is apparently related to the different substrate specificity of these enzymes. Moreover, the peptides binding to PSA stimulated its enzymatic activity, while those binding to hK2 inhibited. All of the hK2 binding peptides contained arginine residue. This is not surprising as arginine is located at the P1 position of hK2 substrates (Cloutier et al. 2002). hK2 probably requires arginine with an amino acid before for initial binding to the catalytic cleft. When arginine is replaced or deleted, the peptides completely lost their inhibitory effect towards hK2 confirming the necessity of arginine at a certain position. Replacement or deletion of the other residues reduced this inhibitory effect or some residues could be completely replaced or discarded without significant effect on activity. These studies revealed the motif required for the inhibitory activity. Two inhibiting peptides contained almost identical motif ARRPXP. The motif of the more potent peptide inhibitor was RFKXWW, but for the maximal effect

Discussion

45

Discussion

the motif requires at least one amino acid before arginine. In the original peptide, the first amino acid was serine, but alanine-scanning studies showed that alanine in this position actually increases the potency. Phage screening was performed using linear and constraint cyclic peptide libraries, but peptides binding to hK2 were only found with linear phage libraries. Interestingly, three of the peptides that were identified with the X11 library contained two cysteine residues and they are likely to be cyclic. In these peptides, like in the other ones, arginine was the 2nd amino acid after cysteine and the peptides contained the CXR motif. These three cysteine containing peptides, with the exception of hK2p06, did not show any effect on the enzymatic activity of hK2. No peptides were identified in cyclic libraries used for screening. The reason for this may be because the constraint libraries contained 3, 7, 8 or 11 amino acids between the cysteine residues, whereas there were 4 or 5 amino acids in the cyclic peptides identified. Amino acid sequence similarity search of the hK2-inhibitory peptides was performed by using BLAST and found several hits for the peptide motif ARRPXP. However, some of these proteins are not expressed by prostate and the rest are intracellular, therefore, their interaction with hK2 is unlikely. No proteins containing the motif RFKXWW were found.

2. Specificity and Stability of the hK2-inhibiting Peptides (I, II)

In our study, hK2-inhibitory peptides were specific for hK2 and did not show any effect on several other proteases. Although active forms of PSA and hK2 share 79% identical amino acid sequence, the peptides did not either bind to or affect the enzymatic activity of PSA. This was not surprising, as PSA has a different substrate specificity. The assay where the phage peptides

compete with synthetic peptides showed that the hK2-inhibitory peptides bind to the same or adjacent site. The peptides are competitive inhibitors and inhibit hK2 in a dose-dependent fashion. The identified hK2-inhibitory peptides are linear, which makes them highly susceptible towards proteolytic attacks and especially to exopeptidases that are active in human plasma (Sato et al. 2006). Furthermore, the peptides contain arginine, which makes them potential substrates for trypsin and trypsin-like proteases including hK2 itself. Indeed, trypsin cleaved the peptides very efficiently although hK2 did not. We found that the hK2-inhibitory peptides were rapidly degraded in plasma. For the peptides to be used for in vivo studies, the stability must be improved so that they can withstand proteolytic attack at least until they bind to their target and inhibit it. The stability of peptides can be increased by several methods including PEGylation (using PEG chains as a carrier), retro-inversion and cyclization. Conjugation of PEG molecules to N- or C- terminus or to both termini of the peptide can protect the peptide from exopeptidases (Veronese & Pasut 2005). By addition of multiple PEG molecules (e.g., 5 kDa) to the peptide, it is possible to make it big enough (>30 kDa) to reduce renal clearance from the circulation (Sato et al. 2006). As a carrier molecule PEG also has been shown to have favorable properties such as high water solubility, high mobility and low immunogenicity (Werle & Bernkop-Schnürch 2006). Retro-inversion, i.e., synthesizing the peptide with D-amino acids instead of natural L-amino acids, can also improve stability towards proteases. For example, octreotide, a 14 amino acid long synthetic peptide drug based on somatostatin used to treat gastrointestinal tumors, has half life of few minutes. This has been extended up to 1.5 h by shortening the sequence to 8 amino acids

46


and replacing them with D-amino acids (Harris 1994; Werle & Bernkop-Schnürch 2006). Modification for improving the half-life of peptides require optimization. We used cyclization and retro-inversion methods to increase the stability of the peptides. Retro-inversion of the peptides abolished their inhibitory activity towards hK2 (unpublished). Cyclization of the peptides was done by adding cysteine residues to form disulfide bridges or head-to-tail cyclization via a peptide bond. In both types of cyclization the peptides kept their inhibitory properties, furthermore, head-to-tail cyclized peptide was relatively stable against tryptic digestion and proteolytic degradation in human plasma. Diffusion from circulation into tissues may take time. Therefore, more stable peptide-drug may have better therapeutic activity. However, it is also important that the peptide-drug clears out from the human body afterwards. Accumulation in the liver or kidney may cause organ damage. Thus, the half-life of therapeutic peptides need to be optimized. In our study the size of the hK2-inhibiting peptide is small enough to be cleared through the kidneys. However, the excretion can also be too rapid, i.e., before reaching the target tissue. In these cases, PEGylation or conjugation with a larger molecule, such as albumin, may be a solution for both stability and clearance.

3. Inhibition of hK2 Activity Towards Protein Substrates by hK2-inhibiting Peptides (I, IV)

We studied the inhibitory effect of the peptides towards proPSA activation and IGFBP-3 cleavage. For both of these protein substrates the peptides inhibited hK2 in a dose-dependent fashion. The order of inhibitory efficacies of the peptides towards hK2 were the same than with synthetic substrates. This is important as active PSA has been suggested to take part in the early development of prostate cancer and

also play a role in degradation of the ECM (Webber et al. 1995). When overexpressed in prostate tumors, hK2 may promote tumor growth by digesting IGFBP-3, which regulates cell proliferation via IGF-system (Jones & Clemmons 1995). It may also exert an apoptotic effect independent of IGF-system (Rajah et al. 1997; Boyle et al. 2001; Silha et al. 2006). Therefore, inhibiting hK2 activity towards proPSA and IGFBP-3 may be favorable in treating or slowing down prostate tumor growth. However, it should also be noted that hK2 is not the only enzyme that can cleave IGFBP-3. Trypsin and PSA, which are both expressed in prostate cancer, also cleave IGFBP-3 (Cohen et al. 1992; Koistinen et al. 2002). PSA cleaves IGFBP-3 with less efficiency compared with trypsin and hK2 (Koistinen et al. 2002). In addition to the confirmation of IGFBP-3 cleavage by hK2, we identified the IGFBP-3 fragments generated by hK2. Identification of the hK2 cleaved IGFBP-3 fragments in serum may facilitate prostate cancer diagnosis. Because of their different substrate specificities, the fragments generated by PSA and hK2 are different (Okabe et al. 1999). Since in advanced malignant prostate tissue expression of hK2 is increased and PSA is reduced, there may be increased amounts of hK2-generated IGFBP-3 fragments that has tryptic-cleavage sites rather than chymotryptic-cleavage sites. The natural substrates of hK2 suggest a role in prostate tumor biology. Based on substrate specificity, hK2 may act on proteolytic cascade increasing proliferation and tissue invasion (Rittenhouse et al. 1998; Koistinen et al. 2008). hK2 is also involved in other tumor promoting mechanisms. It can stimulate cell proliferation through ERK1/2 pathway by activating PAR2 (Mize et al. 2008). Therefore, studies on the effect of the hK2-inhibiting peptides also on the PAR2 activation pathways are justified.

47

Discussion

4. PSA and hK2 in Prostate Cancer Xenograft Models LNCaP and 22RV1 (IV)

Several PSA-expressing tumor cell lines can be transplanted into mice to establish animal models of prostate cancer (van Weerden & Romijn 2000). We produced LNCaP and 22RV1 xenografts and showed that blood PSA levels correlate with the volume of the tumor, which confirms PSA as a valid surrogate marker for the followup of tumor growth. In the serum of LNCaP xenografts, by using immunoassay, we were able to detect hK2, suggesting this model is suitable also for hK2-targeting studies. When using these xenograft models it is important to know the fate of PSA in circulation, and especially whether it is enzymatically active or inactivated by complexation with serpins and other inhibitors. This information is also useful for the development of PSA-based medical interventions, in which PSA is targeted or the enzymatic activity of PSA is utilized (Williams et al. 2007; Denmeade et al. 1998). In this study, we showed that PSA behaves similarly in the circulation of human and mice. Like in human plasma, active PSA released into the circulation from the xenograft tumors rapidly forms complexes with protease inhibitors in mouse plasma. After addition of PSA to mouse serum, complexation occurred within 30 min, which is also similar to the rate in human plasma (Zhang et al. 1995; Christensson et al. 1990). Immunoassay of total and free PSA showed 70% to be complexed and 30% free. Around 30% of the added PSA was undetectable indicating complexation with A2M. These proportions are similar to those found in human plasma of prostate cancer patients (Finne et al. 2000b; Lilja 1993). When we identified the composition of these PSA complexes by tryptic digestion and mass spectrometry, we found that the major

PSA complex was with A2M and serpins of the AAT family. In humans, the major PSA complexes are PSA-ACT and PSA-A2M (Christensson et al. 1990; Lilja et al. 1991; Stenman et al. 1991; Zhou et al. 1993). PSA also forms complexes with AAT in human serum (Stenman et al. 1991; Zhang et al. 1999), but the proportion is low compared to that with ACT. Serpins contain a reactive center loop (RCL) with a recognition site that is specifically cleaved by various serine proteases (Forsyth et al. 2003). Upon cleavage of the RCL, serpins change conformation and form a covalent bond with the enzyme. This mechanism is called “suicide mechanism” due to the irreversible structural change of the serpin after complex formation. Mouse AATs and ACT orthologue serpin A3N contain different recognition sites in their RCL than human AATs and ACT. This explains why PSA in mouse serum forms complexes with mouse AATs, but not with mouse ACT.

5. Peptide Based Targeting and Therapies for Prostate Cancer (I, III, IV)

Therapeutic peptide drugs currently in the pharmaceutical markets are used to treat various diseases (Kelley 1996; Loffet 2002; Stevenson 2009). Furthermore, there are hundreds of peptides in the drug development pipeline and in clinical trials (Vlieghe et al. 2010). The increasing utility of peptides is related to their high specificity towards their targets (Lien & Lowman 2003). However, the short half-life of peptides is a main challenge for their therapeutic use, but modified peptides can be very useful drugs (McGregor 2008). hK2-inhibiting peptides may have significant therapeutic value as increased expression levels of hK2 is associated with aggressive stages of prostate cancer (Darson et al. 1997; Lintula et al. 2005). Studies have suggested that enzymatic activity of hK2 plays a role in the tumor progression

48


by degrading components of the ECM and proteins involved in regulation of cell growth (Clements et al. 2004). Therefore, inhibition of hK2 by these peptides may slow down the growth of prostate cancer and prevent formation of metastases. We have shown that hK2-inhibitory peptides inhibit IGFBP-3 degradation and proPSA activation. However, these findings need to be validated in cell and animal model studies. In order to use in animal studies, the peptides need to be stable in circulation and have an acceptable clearance rate. As part of this study, we have improved the stability of the peptides by cyclizing them, but for studies on the effect on tumor growth, they probably need to be modified to have a longer half-life. Making a drug out of a peptide is a long and challenging process. Although we have made a major advancement by making them stable in plasma, there still remain

many steps before they can be tested for prostate cancer therapy. In prostate cancer detection these hK2-inhibiting peptides also have a potential use in tumor imaging, which permits the direct visualization of targets. To a certain extend prostate cancer metastases express hK2 (Darson et al. 1999) and PSA (Stein et al. 1984). Thus, the peptides for hK2 or PSA can be used to target and image these prostate originated tumor cells. For example; when radiolabeled, the peptides can be tracked by positron emission tomography (PET) scan and the tumor or metastatic site can be observed. The advantages of hK2-inhibiting peptides would be not only binding, but also have a therapeutic effect on the target tumor tissue. Although secreted target proteins are not the best choice for imaging, high concentrations of hK2 or PSA around the source tissue might make the imaging possible.

49

By using phage display we identified several inhibitory peptides that are specific for hK2, a protease produced abundantly by prostate. The peptides were found to inhibit hK2 activity against synthetic peptide substrate and physiological protein substrates. Relevant to prostate cancer progression, the peptides inhibited activation of proPSA and proteolysis of IGFBP-3 by hK2. The required motifs for their inhibitory activity were determined by chemically synthesizing structural variants of the peptides and testing their effect on the enzymatic activity of hK2. As a result of these studies we increased the potency of the most efficient inhibitory peptide. We could also reduce the size of the peptide from 10 to 8 residues by keeping only the amino acids necessary for the inhibitory effect. The stability of this peptide in plasma was improved by

cyclization in a head-to-tail fashion. Hence, the stability of the peptide in plasma was extended significantly. This will allow us to study the effect of the peptide on tumor growth in xenograft models, like LNCaP model that was found to produce both PSA and hK2, and was validated regarding the complexation of PSA in circulation. As hK2 promotes tumor growth by degrading ECM and IGFBP-3, and by activating PAR2 and uPA, specific inhibition of the enzymatic activity of hK2 by these peptides may inhibit or slow down the tumor growth. Furthermore, the expression of hK2 is limited to prostate, therefore, its inhibition does not need to be associated with adverse side effects. Thus, hK2-inhibiting peptides may be potentially useful for targeting and treating prostate cancer.

Conclusions

50

Acknowledgments

This study was carried out at the Department of Clinical Chemistry under professor Ulf-Håkan Stenman and more recently head professor Pirkko Vihko. To them I am much thankful for providing me with excellent working facilities and resources that made the study workflow very smooth. Although this book was written under one single name, the work would not have been realized without the contribution of many, to which I wish to express my deepest gratitude. Ulf-Håkan Stenman (Uffen), to whom I am most grateful for being a true mentor and great scientific inspiration. Also indebted to him for taking care of every detailed necessary thing with assurance. His endless vision and creativity continually produced fruitful ideas, which always led the project in the proper direction. A great privilege is that I have been doing scientific research under Uffe’s supervision. Hannu Koistinen, to whom I am truly thankful for guiding me very patiently throughout my whole PhD studies. Also appreciate very much his useful skill of solving every problem regardless of being small or big. Galvanizing scientific discussions with Hannu always raised enthusiasm in me. Tero Soukka and Kimmo Porkka, the official pre-examiners of this thesis, for their careful evaluation, constructive criticism and valuable comments. Katja Junttu, for handling the thesis paperwork with care and attention. Jari Leinonen and Ping Wu, for introducing and letting me to inherit the project. Ale Närvänen, for peptide col-laboration that opened a whole different

perspective to the project within the lab and out at the pub. Miikka Pakkala and Janne Weisell, for continually supplying with peptides of every kinds. Leena Valmu and Tero Riipi for looking out for peptides and protein fragments with that miracle machine and spending time to analyze those. Eric Wallén and Kristian Meinander for being creative acrobats on mimicking peptides. Erkki Koivunen, for teaching phage display technology and providing necessary tools for identifying something novel and unique. Kim Petterson, Timo Lövgren and Ville Väisänen for providing us hK2, which is one of the main tools in this study. Zhu Lei, for being specialist on purification of complexed proteins and using his expertise in this project. Pirjo Laakkonen, for being such a fun person to work with and introducing me to animal experiments, which instantly give meaning to a study. And work is nothing without motivating fun colleagues and labmates, Patrik Finne, Jakob Stenman, Krisse Hotakainen, Annukka Paju, Susanna Lintula, Heini Lassus, Piia Vuorela, Johanna Mattsson, Laura Hautala, Suvi Ravela, Anna Lempiäinen, Anne Kaukinen, Tuomas Heikkinen, Outi Kilpivaara, Sari Tuupanen, Reetta Vainionpää, Anna-Kaisa Herrala, Ileana Quintero, César Araujo, Wan-Ming Zhang, Liisa Airas, Gynel Arifsdhan, Emilia Korhonen, Annikki Löfhjelm, Riitta Koistinen, Ralf Bützow, Anne Ahmanhaimo, Sanna Kihlberg, Oso Rissanen, Laura Sarantaus, Erik Mandelin and Jari Leinonen. Thank you for creating such fantastic work atmosphere. Hannu, Erik and Jari with whom I had the most intimate times around coffee and various drink tables inland and outland (outland

51

across the sea includes Oso and Ali). Those special moments influenced and also led this work in the right direction. Since the beginning of the work those who helped me uncountable times with everything practical in the lab Henrik Alfthan, Taina Grönhom, Maarit Leinimaa and Marianne Niemelä. To them I cannot thank enough for taking care of me so well. Ansa Karlberg also for taking care of so many bureaucratic things and making everything easier. Helena Taskinen for being extra great hands on my experiments. Krisse Nokelainen for being so dexterous in so many experiments and sharing the lab-bench with me. Friends in science but outside the lab for parties and joyful moments, especially Mihalis Stefanidakis, Rabeh Soliymani, Marc Baumann, Aki Koivistoinen, Katja Helenius, Tuomas Tammela, Johanna Partanen, Alexandra Gylfe, Iulia Diaconu, Roxana Ola, Tea Blom, Heli Lehtonen, Saara Nuutinen, Nora Pöntynen. And Demir, for introducing me to the scientific research world of Finland and teaching me how to survive in once outland and now home. Friends outside of the lab who have been morally supportive and giving cheerful times during the study times. Among the recent gang Ersen Fırat, Milja Sirviö, Zeynep and Nico von Flittner, Laura and Juan Martin, Hirono and Marc Magistrali, Cetin Gürsel, Yonca and Murat Ermutlu, Özlem Filiz, Anna and Çağrı Akgül, Tina Schubarth, Melis Arı with special thanks to Laura Murto for language advice and Halil Gürhanlı for proof-read-ing the thesis . Life gets wiser with beautiful tunes, very much indebted to my bandmates Andy, Ali, Kirill Shapochkin, Mikko Uola, Sami Typpö and Teppo Meriläinen in “the RAN” for letting me phase into another world of creativity that is music. Andy (Andrea Ansidei), for being a great friend and companion, and living through great deal of moments that created

a bond closer than a brother with an extraordinarily common sense. Ali Pekcan, my brother-in-love, with whom we also lived a great deal of surreal days&nights, for being a true friend ready to help me with anything in the blink of an eye and a great buddy to share all of my futuristic ideas and acts in science and art. Most special acknowledgment goes to Jari Leinonen, who took my hand in the lab, shared the best and worst days, listened to my confessions and guided me through all my problems inside and outside the lab. My parents-in-law Ülkü and Önder Pekcan, for endless support and patience. My parents Latife and Nezih Hekim, for being so generous and understand-ing when I left home for a special cause. Especially my mother Latife for the positive attitude in her motivations and my father Nezih for being a true scientist, and giving inspiration and great support. My beloved sisters Şirin and Zeynep Hekim, for helping me to widen my vision, encouraging me and having the great escape fun times. My love, my life and my best friend Zeynep who has inspired, supported and motivated me in every step of this long and challenging process. Every work needs to contain love so it becomes something notable. Our common passion for science and hunger for knowledge made the two of us start this adventure. Together we faced and crossed toughest barriers. To her I am deeply thankful for believing in me and always being there with me. Finally, my son Aral, for giving more joy to this life and for showing me the effort of learning and doing research has a solid purpose. Also for reminding me once again that kids are scientists, or vice versa.

Helsinki, 2012

52

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Documents

hK PSA F T Prostate Cancer - COnnecting REpositories · related peptidase 2, KLK2) and prostate specific antigen (PSA, KLK3) (Clements 1989; Grauer et al. 1996; Young et al. 1992;