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Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26,p=0.001) and multivariable analysis (hazard ratio: 2.12,p

In serous ovarian cancer, the clinical relevance of tumor cell-expressed plasmin(ogen) (PLG) has not yet been evaluated. Due to its proteolytic activity, plasmin supports tumorigenesis, however, angiostatin(-like) fragments, derived from PLG, can also function as potent anti-tumorigenic factors. In the present study, we assessed PLG protein expression in 103 cases of advanced high-grade serous ovarian cancer (FIGO III/IV) by immunohistochemistry (IHC). In 70/103 cases, positive staining of tumor cells was observed. In univariate Cox regression analysis, PLG staining was positively associated with prolonged overall survival (OS) [hazard ratio (HR)=0.59, p=0.026] of the patients. In multivariable analysis, PLG, together with residual tumor mass, remained a statistically significant independent prognostic marker (HR=0.49, p=0.009). In another small patient cohort (n=29), we assessed mRNA expression levels of PLG by quantitative PCR. Here, elevated PLG mRNA levels were also significantly associated with prolonged OS of patients (Kaplan-Meier analysis; p=0.001). This finding was validated by in silico analysis of a microarray data set (n=398) from The Cancer Genome Atlas (Kaplan-Meier analysis; p=0.031). In summary, these data indicate that elevated PLG expression represents a favorable prognostic biomarker in advanced (FIGO III/IV) high-grade serous ovarian cancer.

Most members of the kallikrein-related peptidase family have been demonstrated to be dysregulated in ovarian cancer and modulate tumor growth, migration, invasion, and resistance to chemotherapy. In the present study, we assessed the mRNA expression levels of KLK6 and KLK8 by quantitative PCR in 100 patients with advanced serous ovarian cancer FIGO stage III/IV. A pronounced correlation between KLK6 and KLK8 mRNA expression (rs = 0.636, p < 0.001) was observed, indicating coordinate expression of both peptidases. No significant associations of clinical parameters with KLK6, KLK8, and a combined score KLK6+KLK8 were found. In univariate Cox regression analysis, elevated mRNA levels of KLK6 were significantly linked with shortened overall survival (OS) (hazard ratio [HR] = 2.07, p = 0.007). While KLK8 values were not associated with patients’ outcome, high KLK6+KLK8 values were significantly associated with shorter progression-free survival (HR = 1.82, p = 0.047) and showed a trend towards significance in the case of OS (HR = 1.82, p = 0.053). Strikingly, in multivariable analysis, elevated KLK6 mRNA values, apart from residual tumor mass, remained an independent predictive marker for poor OS (HR = 2.33, p = 0.005). As KLK6 mRNA and protein levels correlate, KLK6 may represent an attractive therapeutic target for potent and specific inhibitors of its enzymatic activity.

Matrix metalloproteinases (MMPs) are involved in many physiological and pathophysiological processes, including tumor cell invasion and metastasis. For one member of this family, MMP-13 (collagenase-3), a new, highly specific ELISA with a sensitivity of 0.5 ng MMP-13/ml was established. The protein levels of MMP-13 in ascitic fluids of 30 patients with advanced ovarian cancer FIGO stage III (n=19) and IV (n=11) were measured with this ELISA. Using a cutoff value of 0.5 ng MMP-13/mg total protein, two patient subpopulations with short (median 16 months) and long (median 36 months) overall survival were identified. Together with other prognostic markers, determination of MMP-13 in ascitic fluid may help to identify patients at risk for early death and help to individualize adjuvant therapy.

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (NhTIMP-1-chCysWT, N-hTIMP-1- chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.

Misfolded and incorrectly assembled proteins in the secretory pathway are eliminated by ubiquitylation and proteasomal degradation in a process known as ER-associated degradation (ERAD). Retrotranslocation of diverse substrates including misfolded proteins and viruses occurs through channels in the ER membrane, which are also utilized for host cell penetration by A/B class protein toxins such as cholera toxin, ricin or K28. According to the current view, disulfide-bonded proteins must either be reduced or rearranged to ensure translocation competence and entry into the cytosol from the ER. As the underlying mechanisms are still largely mysterious, we here focus on the redox status and disulfide isomerization of ERAD substrates and the role of oxidoreductases in the essential process of ER-to-cytosol retrotranslocation.

Expression of the kallikrein-related peptidase 7 (KLK7) is dysregulated in ovarian cancer. We assessed KLK7 expression by ELISA and quantitative immunohistochemistry and analyzed its association with clinicopathological parameters and patients’ outcome. KLK7 antigen concentrations were determined in tumor tissue extracts of 98 ovarian cancer patients by ELISA. For analysis of KLK7 immunoexpression in ovarian cancer tissue microarrays, a manual quantitative scoring system as well as a software tool for quantitative high-throughput automated image analysis was used. In immunohistochemical analyses, expression levels of KLK7 were not associated with patients’ outcome. However, in multivariate analyses, KLK7 antigen levels in tumor tissue extracts were significantly associated with both overall and progression-free survival: ovarian cancer patients with high KLK7 levels had a significantly, 2-fold lower risk of death [hazard ratio (HR)=0.51, 95% confidence interval (CI)=0.29–0.90, p=0.019] or relapse [HR=0.47, 95% CI=0.25–0.91, p=0.024), as compared with patients who displayed low KLK7 levels. Our results indicate that – in contrast to earlier findings – high KLK7 antigen levels in tumor tissue extracts may be associated with a better prognosis of ovarian cancer patients.

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Human kallikrein-related peptidases (KLKs) are 15 homologous serine proteases involved in several (patho)physiological processes, including cancer. Secreted as precursors, they are activated upon proteolytic release of a short pro-peptide. We searched for interconnection of KLKs within extracellular proteolytic networks leading to activation of protease zymogens and found that (i) pro-KLK activation by other KLKs is scarce, with the exception of pro-KLK11, which is efficiently activated by KLK4 and 5; (ii) pro-KLK4 is activated by matrix metalloproteinase 3; and (iii) trypsin-like KLKs efficiently activate the serine protease urokinase. Our observations provide new insights into the regulation of these important tumor-associated proteases.

Human kallikrein-related peptidases (KLKs) are (chymo)-trypsin-like serine proteinases that are expressed in a variety of tissues such as prostate, ovary, breast, testis, brain, and skin. Although their physiological functions have been only partly elucidated, many of the KLKs appear to be useful prognostic cancer markers, showing distinct correlations between their expression levels and different stages of cancer. Recent advances in the purification of ‘new type’ recombinant KLKs allowed solution of the crystal structures of KLK4, KLK5, KLK6, and KLK7. Along with these data, enzyme kinetic studies and extended substrate specificity profiling have led to an understanding of the non-prime-side substrate preferences of KLK4, 5, 6, and 7. The shape and polarity of the specificity pockets S1–S4 explain well their substrate preferences. KLK4, 5, and 6 exhibit trypsin-like specificity, with a strong preference for Arg at the P1 position of substrates. In contrast, KLK7 displays a unique chymotrypsin-like specificity for Tyr, which is also preferred at P2. All four KLKs show little specificity for P3 residues and have a tendency to accept hydrophobic residues at P4. Interestingly, for KLK4, 5, and 7 extended charged surface regions were observed that most likely serve as exosites for physiological substrates.

The human tissue kallikrein family of serine proteases (hK1–hK15 encoded by the genes KLK1–KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (pin vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the ‘tissue kallikrein overexpressing’ group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; χ2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.

In epithelial ovarian cancer, the high mortality rate is usually ascribed to late diagnosis, since these tumors commonly lack early-warning symptoms, but tumor-associated biomarkers useful for prognosis or therapy response prediction are in short supply. However, members of the tissue kallikrein serine protease family, the serine protease uPA and its inhibitor PAI-1, are associated with tumor progression of ovarian cancer. Therefore, we used ELISA to determine uPA, PAI-1, and tissue kallikreins hK5-8, 10, 11, and 13 in extracts of 142 primary tumor tissue specimens from ovarian cancer patients and studied the strength of association between protein expression levels of these tumor tissue-associated factors. uPA, PAI-1, hk5, and hk8 were related to FIGO stage; hK5 expression was higher in FIGO III/IV than in FIGO I/II patient tissues. PAI-1 and hk5 differed significantly according to nuclear grading; expression of hK5 was higher in G3 than in G1/2 tumors. Associations between uPA, PAI-1, and the tissue kallikreins were weak. There were strong pairwise correlations within the cluster of tissue kallikreins hK5, 6, 7, 8, 10, and 11, but their bivariate distributions depended on nuclear grading. These results support the notion that several tissue kallikreins are co-expressed in ovarian cancer patients, substantiating the existence of a steroid hormone-driven tissue kallikrein cascade in this disease.

The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.

During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin alpha(v)beta3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized alpha(v)beta3 directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of alpha(v)beta3 and was accompanied by rapid and transient tyrosine-phosphorylation of p44(erk-1)/p42(erk-2) mitogen-activated protein kinase. Moreover, overexpression of alpha(v)beta3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon alpha(v)beta3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-alpha (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of human ovarian cancer cells.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor PAI-1, and its cellular receptor uPA-R (CD87) are of crucial importance during cellular invasion and migration, required for a variety of physio- and pathophysiological processes. It has become increasingly evident in recent years that the uPA/uPA-R-system has far more functional properties than plasminogen activation alone. This is reflected by its involvement in cellular events such as proliferation, adhesion, migration, and chemotaxis. Since uPA-R lacks a transmembrane domain and thus on its own is not capable of transmitting signals into cells, association and functional cooperation with other signaling molecules/receptors is needed. In this respect, one group of adhesion and signaling receptors, the integrins, have been identified which constitute, together with the uPA/uPA-R-system, an interdependent biological network by which the uPA/uPA-R-system broadly affects integrin functions and vice versa. Moreover, there is a growing body of evidence that cellular uPA, uPA-R, and PAI-1 expression is under control of specific ECM/integrin interactions and also that integrins are regulated by components of the uPA/uPA-R-system. By this multifaceted crosstalk, cells may modulate their proteolytic, adhesive, and migratory activities and monitor ECM integrity in their microenvironment.

The serpin plasminogen activator inhibitor type-1 (PAI-1), as the primary physiological inhibitor of both urokinase-type (uPA) and tissue-type (tPA) plasminogen activator, plays an important role in the regulation of the fibrinolytic system as well as in extracellular remodeling in both physiological and pathophysiological processes. In plasma as well as in the extracellular matrix PAI-1 binds to vitronectin (Vn), an interaction that affects the function of both proteins. As PAl-1/Vn interaction has a significant regulatory function in fibrinolysis, thrombolysis, and cell adhesion in cancer spread, there is a strong interest in defining the binding sites on PAI-1 and Vn as the basis of a rational design of novel drugs that may modulate PAI-1/Vn-mediated effects. In this minireview, we give an overview on the approaches to define the Vn binding site of PAI-1 and vice versa. Although in the case of PAI-1 the region around alpha-helix E and alpha-helix F of PAI-1 has been demonstrated to be important for its interaction with Vn, the precise location of the Vn-binding region has not completely been resolved. The major high-affinity PAI-1 binding region of Vn is localized within the N-terminal somatomedin B (SMB) domain of Vn. There are indications for at least one other low-affinity PAI-1 binding site in the C-terminal region of Vn, which seems to be involved in the formation of larger PAI-1/Vn complexes.

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA19-31). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo19,31uPA19-31, as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction [Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo19,31uPA19-31 with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of uPA19-31, in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo19,31[D-Cys19]-uPA19-31 as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo19,31[D-Cys19]-uPA19-31 not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo19,31[D-Cys19]-uPA19-31 represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system.

Tissue factor (TF) initiates the extrinsic pathway of blood coagulation via formation of an enzymatic complex with coagulation factor VII/VIIa (FVII/VIIa). Although FVII is the only known ligand for TF, several reports in recent years have shown that the function of TF may not be limited to serving as a trigger of coagulation but that TF could also play a role in cellular signaling, metastasis, adhesion and embryogenesis. To explore the loci of the extracellular domain of TF important for its function, we analyzed the functional and immunological epitopes of TF1-219 by the use of both E. coli expressed TF variants encompassing various portions of the extracellular domain of TF and different anti-TF monoclonal antibodies (mAbs). N- and C-terminally truncated TF variants were analyzed for their VIIa-dependent procoagulant activity (PCA). The results obtained are in agreement with previously performed mutant and structural analyses of the interaction of FVII/FVIIa with the extracellular domain of TF. In addition, we observed that combination of two TF variants, Ec-TF1-122 and Ec-TF120-219, yields a soluble and active two-chain TF molecule with remarkable PCA. The reaction patterns of anti-TF mAbs with truncated TF variants and synthetic TF-derived peptides demonstrated that at least three distinct conformation-dependent epitope areas of TF (residues 1-25, 175-202, and 181 -214, respectively) are detected by these mAbs raised against native TF. In fact, mAbs, which are directed to the same epitope area of TF, behave very similar in various applications including immunohistochemistry and clotting tests. Since mAbs directed to the C-terminal epitope area of TF (residues 181-214) influence TF activity independent of FVIIa-binding, this region may be involved in functions of TF distinct from haemostasis.

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.