5th Annual International Symposium on Kallikreins ...

DOI 10.1515/cclm-2014-0340 Clin Chem Lab Med 2014; 52(7): eA21–eA48

Abstract*)

5th Annual International Symposium on Kallikreins and Kallikrein-Related Peptidases Toronto, Canada, 28 September–1 October 2013

ISK 2013 Local Organizing Committee George Yousef, Chair; Maria Pasic, Vice Chair Bharati Bapat; Anastasia Diamandis, Treasurer; Azza Eissa; Heba Khella; Eduardo Martinez-Morillo; Lorna Mirham; Ioannis Prassas; Fabio Rotando; Sara Samaan; Samantha Wala; Nicole White; Peter Yousef

ISK 2013 Program Committee Eleftherios Diamandis, Canada, Chair; Michael Blaber, USA; Judith Clements, Australia; Hans Fritz, Germany; Ulf-Hakan Stenman, Finland; Evi Lianidou, Greece; Hans Lilja, USA; Viktor Magdolen, Germany; Andreas Scorilas, Greece; Georgia Sotiropoulou, Greece; George M. Yousef, Canada

International Scientific Advisory Board Michael Blaber, USA, Chair; Michael Bader, Germany; Dalila Darmoul, France; Maria Brattsand, Sweden; Judith Clements, Australia; Yves Courty, France; Eleftherios Diamandis, Canada; Hans Fritz, Germany; Morley Hollenberg, Canada; Alain Hovnanian, France; Viktor Magdolen, Germany; Mario Plebani, Italy; Manfred Schmitt, Germany; Isobel Scarisbrick, USA

*) These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax and usage are the authors’ responsibility.

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INVITED SPEAKER ABSTRACTS Brattsand, Maria Kallikrein-related peptidases and their inhibitors in the skin In a healthy skin, the production of cells at the basal layer is well coordinated with the shedding of cells at the skin surface, but in different skin disorders this balance is disturbed. In the end of the 1980’s, members of our group discovered that shedding of cells from skin biopsies in vitro was unipolar, and this process could be prevented by adding inhibitors specific for serine proteases. The first enzyme, now named KLK7, was biochemically purified and cloned from stratum corneum in the beginning of the 1990’s. It was characterized to be a serine protease with chymotryptic like activity with ability to degrade the desmosomal protein structures holding the keratinocytes together in the skin. A few years later, we purified and cloned another enzyme, KLK5, with tryptic like activity, and thereafter, also KLK14 could be purified from the same material. All KLKs are produced as inactive pro-enzymes, and KLK5 was shown to be able to activate both its own pro-enzyme as well as proKLK7 and proKLK14 in a pH dependent manner. KLKs may be involved in inflammatory skin disorders through the PAR2 system. KLK5 and KLK14, but neither KLK7 nor KLK8, can induce PAR2 signaling. Patients with Netherton syndrome show the importance of a well regulated protease activity in the skin, and different domains of LEKTI were shown to be efficient towards the KLKs. We have also purified and cloned small, apparently KLK5 specific inhibitor, named SPINK9, which is predominantly expressed in palmo-plantar stratum corneum.

Courty, Yves Kallikrein-related peptidases and lung physiopathology At least 11 kallikrein-related peptidases (KLK1, 3, 5-14) are expressed in the normal or pathological lungs. KLKs were found in the respiratory epithelium of the bronchial tree and in the submucosal glands, but not in the alveolar epithelium. Because these structures discharge their products into the lumen of the airways, KLKs are components of the bronchial secretions. Recent studies using in vitro and in vivo approaches revealed that some KLKs are dysregulated following acute challenges with cigarette smoke and respiratory viruses. These observations suggest that KLKs might be involved in the lung response to external insults. Malignant cells also express and secrete several KLKs. Clinical and preclinical data indicate that KLKs play two roles in lung carcinogenesis. They can be either cancer-promoters (e.g., KLK6, 12-13) or cancer-inhibitors (e.g., KLK8, 10) regulating cell-growth and invasion of malignant cells and angiogenesis. These actions may result from extracellular matrix remodeling, direct activation of membranous receptors or mobilization of growth factors. Although still fragmentary, current data indicate that KLKs may play a role in infectious, inflammatory and malignant lung diseases.

Clements, Judith KLK4 is a key regulator of the tumour microenvironment in prostate cancer Prostate cancer cells reside in a complex stromal microenvironment often referred to as “reactive” stroma which is a critical component of prostate cancer initiation and progression. The mounting evidence for its critical nature has led to increased interest in this niche as a target for new therapeutic approaches. Cancer associated fibroblasts (CAFs) play a key role in this niche regulating the tumour microenvironment. Factors secreted by prostate cancer cells can ‘activate’ non-malignant associated fibroblasts to become CAFs. KLK4 is over-expressed in both localized and bone metastatic prostate cancer and so has the capacity to act as a paracrine factor on the surrounding stroma. To elucidate the role of KLK4 in tumour-stroma cross-talk, its substrates were identified from the prostate cancer lines, LNCaP and PC3 (also derived from a bone metastasis), and the prostate fibroblast line WPMY-1, utilizing the ’PROtein TOpography Migration Analysis Platform’ (Dix et al., Cell, 2008). Gene expression changes following KLK4 treatment were assessed by gene microarray analysis. We identified several novel putative KLK4 substrates and their affected downstream signaling pathways with the TGFβ1 pathway identified as key pathway regulated by KLK4 action. KLK4-treated fibroblasts also expressed elevated levels of a number of genes consistent with a CAF genotype. These findings suggest that KLK4 is a critical regulator of the reactive stromal niche and a potential novel therapeutic target.

Darmoul, Dalila Kallikrein-related peptidase signaling role in the development of human colon cancer Proteolysis, by dysregulated proteases, remains one of the hallmarks of colon cancer progression. However, the traditional view on the role of proteases in tumor growth and progression has significantly changed since it is now admitted that besides their contribution to cancer


5th ISK, Toronto, Canada, 2013 eA23 progression by the degradation of extracellular matrix proteins, some proteases serve as signal molecules controlling tumor growth. Proteases act through specific membrane receptors the protease-activated receptors (PARs), a new class of the GPCR family that are activated by serine proteinase cleavage. By focusing on the role of proteases in colon cancer, we have identified that PARs, are aberrantly expressed in colonic tumors. Further, our studies have implicated proteases signalling via PARs in human colon tumorigenesis. We have demonstrated that trypsin and thrombin behave as very robust new growth factors that can stimulate the migration and proliferation of human colon cancer cells by cleaving PARs. Their mechanism of action involves transactivation of the epidermal growth factor receptor (EGFR) family which are known critical players in tumor growth. Recently, we demonstrated that some members of the tumor-associated serine protease family of kallikreinrelated peptidases (KLKs), primarily known for their clinical use as biomarkers in many cancers, are also aberrantly expressed in malignant colon cancer cell lines and colonic tumours. In addition, some members of KLKs by signaling selectively via different PARs induced ERK1/2 phosphorylation and subsequent cell growth. This may represent an important pool of endogenous PARs ligands. By using recombinant KLKs and generating cells over expressing recombinant KLKs, we showed that KLKs influenced cell-cell adhesion cellular adhesion junctions and subsequent cell migration in vitro. In addition, some KLKs-transfected cells formed larger tumors in nude mice, than vector-transfected colon cancer cells. We suggest that KLKs represent promising new molecular biomarkers which will be useful in establishing clinical prognosis for colon cancer. This KLKs-PAR signaling pathway can be a new therapeutic target for colon tumorigenesis.

Goettig, Peter Activation, catalytic and regulatory mechanisms of kallikrein-related peptidases Physiological activation, catalysis, and regulation of the KLK peptidases depend to various extents on rigidity and flexibility of the protein. In KLK crystal structures exist several states between inflexibility and full disorder, which in some cases explain the respective function. The flexible activation domains of pro-forms become more rigid upon removal of the propeptide. However, activated KLK10 appears to be in a zymogenlike state, which requires additional substrate-induced activation. The KLK active sites comprise rigid specificity pockets, as well as more flexible ones, e.g. in KLK7. Electrostatic surface potentials are connected to specific KLK substrate interactions. Flexibility and modifications of the 99-loop in KLKs 2 and 3 are involved in regulation of the activity. Glycosylation of KLKs 2, 3, and 4 can be another regulatory factor for their enzymatic activity. Ca2+ or Zn2+ binding rigidifies parts of some KLK proteases, as other parts become more flexible, influencing the peptidase activity significantly, e.g. in KLK8. Further regulatory factors of KLKs are natural polypeptidic inhibitors and protein-protein interactions.

Harris, Jonathan Selective inhibition of skin expressed kallikreins The atopic triad comprising atopic dermatitis (eczema), allergic rhinitis, and asthma is experiencing explosive growth amongst the children of the developed world. There is an emerging consensus that these excruciating and disabling conditions develop sequentially with atopic dermatitis as an initiating factor producing the so-called “atopic march” with diabetes and fatty liver disease as identified co-morbidities. Current treatments for atopic dermatitis focus on controlling the disorder’s symptoms using immunomodulators and anti-inflammatory steroids. Both classes of drug have serious side effects and do not provide a long-term cure. The origins of atopic dermatitis are intimately connected to a cycle of skin barrier failure followed by inflammation, sensitization, and infection. There is a growing body of evidence implicating uncontrolled kallikrein proteolytic activity in this process. However, shutting down aberrant proteolysis in the skin is complicated by the plethora of homeostatic pathways in this organ being controlled by proteases, which in turn necessitates very specific modulation. To this end, we have developed a series of inhibitors of skin-expressed kallikreins which combine high selectivity and potency with minimal antigenicity. These exquisitely selectivity reagents are based on the Sunflower Trypsin Inhibitor (SFTI) a plant-derived cyclic inhibitor which we have engineered using substrate-guided and structure-based design techniques to produce inhibitors of KLK4, 5, 7 and 14 with nanomolar potency and the ability to block kallikrein activity in ex vivo assays.

Haun, Randy Effect of suppression of KLK7 on pancreatic tumor development Objective: The aim of this study was to examine the effects of suppressing KLK7 expression on pancreatic tumor development. Methods: KLK7-expressing human pancreatic cancer PK-1 cells were stably transfected with shRNA constructs targeting KLK7 or a scrambled-sequence control. Suppression of KLK7 expression in the transfectants was examined by qPCR and western blot analyses. To observe the functional significance of KLK7 expression on pancreatic tumor growth, KLK7shRNA-transfected and control shRNA-transfected PK-1 cells were used to develop subcutaneous and orthotopic tumor xenografts in immunodeficient mice. Results: Western blot and qPCR analyses demonstrated


eA24 5th ISK, Toronto, Canada, 2013 that KLK7 protein and mRNA, respectively, were effectively suppressed in two independent KLK7-shRNA PK-1 clones compared with two control-shRNA PK-1 clones. Subcutaneous tumors derived from KLK7 shRNA-transfected cells were significantly smaller than those developed from control shRNA-transfected cells. Similarly, no tumor metastases were observed in the orthotopic tumor xenografts in mice inoculated with KLK7 shRNA-transfected cells; whereas, visible liver, lung, and/or spleen metastases were observed in mice receiving control shRNAtransfected cells. Conclusions: Suppressing KLK7 expression in mouse tumor xenografts results in a decrease in tumor growth and metastasis. These findings provide evidence that KLK7 expression plays an important role in pancreatic tumorigenesis and suggests that targeting its aberrant expression may be a useful therapeutic intervention.

Hollenberg, Morley KLKs and their hormone-like signaling actions: a new life for the PSA-KLK family Prostate-specific antigen (PSA-KLK3), one of the most successful biomarkers of prostate disease, was first discovered in 1979 as a unique antigen in human prostatic tissue extracts (Invest Urol. 17:159). Yet, PSA’s proteolytic activity, like that of the serine proteinase in canine seminal plasma, was not appreciated for another 5 years (Bioch. Biophys Res. Comm. 123:482; J Biol Chem. 259: 11520); and its potential physiological role was not known. When proteinase-activated receptor-2 (PAR2) was cloned (PNAS 91:9208,1994), its presence in the prostate along with KLKs suggested that PARs could be a target for KLK-trigged G-protein-coupled signal transduction, as proposed at the first ISK in Lausanne, in 2005 (Biol Chem. 387:677). As will be described, this theme, furthered collaboratively by us along with many others in the KLK field, has generated a new perspective for the role(s) of the KLKs as hormone-like signaling mediators in health and disease.

Hooper, John Kallikreins at the cell surface: transducers of pro-metastatic signals Several kallikrein-related peptidases (KLKs) and members of the related type II transmembrane serine protease family are dysregulated in cancer. Exploring the functional importance of this dysregulation to cancer progression, we provide evidence that several of these proteases interact at the cell surface. In addition, we show that the cell surface receptors Protease Activated Receptor 2 (PAR2) and CDCP1 are activated by these serine proteases. Finally, our recent work indicates that these events can promote cancer in model systems and that evidence of proteolysis of these receptors can be associated with more aggressive disease.

Hovnanian, Alain Of Netherton syndrome and mice: skin and systemic allergy and inflammation cause by defective kallikrein inhibition The epidermis forms a very effective barrier against the external environment and prevents inner fluid loss. Netherton syndrome (NS) is a rare autosomal recessive orphan disease in which skin barrier function is severely impaired secondary to loss-of-function mutations in SPINK5 (serine protease inhibitor of Kazal type 5) encoding LEKTI-1 (lympho-epithelial Kazal type related inhibitor type 5). In vitro and in vivo studies in Spink5 knock-out mice, in transgenic KLK5 and ELA2 murine models have implicated unopposed activity of kallikrein 5, kallikrein 7 and Elastase 2 in the disease mechanism with multiple effects on skin homeostasis, inflammation and allergy. KLK5 cleaves desmoglein 1 resulting in premature stratum corneum detachment and activates PAR2 leading to the activation of the NF-kappa B pathway and the production of pro-allergic (TSLP) and pro-inflammatory (TNF-alpha) cytokines. KLK5 activates KLK7 which has the capacity to contribute to desmosome cleavage and to pro-IL1 beta activation. Danger signals generated by stratum corneum detachment amplify inflammation of the skin. ELA2 aggravates the epidermal defect by impairing profilaggrin and lipid processing. Enhanced TARC and MDC also contribute to skin allergy and inflammation. These features have been confirmed in NS patients and are associated with systemic and cutaneous signs of allergy, with mast cells and eosinophils infiltration in the skin, elevated Th2 cytokines and high serum IgE levels. These results have identified major pathways and therapeutic targets for NS, leading to the development of specific strategies aiming at counteracting unopposed protease activity.

Kundig, Christoph and Patrick Dupuy DM107, an engineered serine inhibitor of the kallikrein proteolytic cascade: Development status and potential to treat skin disorders Several human tissue kallikreins (KLK) were found in the upper stratum granulosum and stratum corneum of human epidermis where they play important roles in skin physiology and pathophysiology. KLKs are regulators of skin barrier functions including desquamation and lipid


5th ISK, Toronto, Canada, 2013 eA25 permeability and are linked to skin inflammation via activation of proteinase-activated receptor (PAR) signaling pathways. Dysregulation of these processes is found in skin disorders and skin KLKs are considered as interesting pharmaceutical targets. The development of kallikreins inhibitors in the field of dermatology has entered a new phase with first compounds in clinical development for Netherton Syndrome and atopic dermatitis. Different approaches which target either single specific kallikreins or interfere more broadly within the KLK proteolytic cascade are tested. At Dermadis we used a serpin backbone to build a multi-specific inhibitor to create a kallikrein cascade blocker. This inhibitor is a biological product, also known as DM107, which displays antitrypsin-like and also antichymotrypsin-like activities. The drug candidate has a protease inhibition profile covering several skin kallikreins believed to be pathologically relevant. Currently, the potential of a topical formulation of the inhibitor to counterbalance the LEKTI deficiency is evaluated. The current development status and clinical development plans will be discussed.

Lilja, Hans KLK3-KLK2 and the natural history of prostate cancer destined to become metastatic Androgens regulate abundant prostate-restricted expression of the active gene products of KLK3 (PSA), and KLK2 (hK2) by androgen receptor (AR) signaling activity, which is critical to normal prostate development and progression of prostate cancer. PSA and hK2 occur in many noncatalytic forms in blood at levels about a million-fold lower than those in seminal fluid, while blood levels can rise thousand-fold at advanced stages of prostate cancer. PSA is well established to stage, monitor disease, treatment efficacy, and detect recurrence of prostate cancer. PSAscreening for prostate cancer reduces cancer deaths but is controversial due to the unclear balance between these benefits versus harms from over-diagnosis and overtreatment. The relationship between the natural history of prostate cancer among a large population of unscreened men and PSA measured at age 40 to 60 suggest that focusing on men at high risk of prostate cancer metastasis and death improves the ratio between benefits and harms of screening. Four kallikrein markers measured in blood contribute increased accuracy in detecting unfavorable prostate cancer among men at higher risk of prostate cancer metastasis or death.

Magdolen, Viktor PC-3 prostate cancer cells overexpressing KLK4: Effects on cell biological processes and mRNA expression KLK4 is overexpressed in malignant versus normal prostate. To elucidate tumor biological functions of KLK4, a prostate cancer PC-3-derived cell line was generated that overexpresses KLK4 and characterized by several cell biological assays versus vector-control (VC) cells. Significant differences regarding proliferation, migration, and cell adhesion were observed. Additionally, we quantitatively analyzed the mRNA expression patterns of selected tumor-associated genes in KLK4 overexpressing and VC cells using low-density micro-arrays. One of the identified potentially differentially expressed genes, COL1A2, encodes the α2(I) chain of collagen type I (ColI). Differential regulation in KLK4 overexpressing cells versus VC cells was validated by an independent QPCR format: COL1A2 mRNA expression was found to be reduced by at least 6-fold in KLK4-overexpressing cells. Expression of COL1A1 mRNA, encoding the α1(I) chain of collagen type I is only marginally affected. KLK4 and COL1A2 expression was additionally analyzed in malignant and adjacent non-malignant prostate cancer tissue. In fact, on average, KLK4 expression was increased in tumor versus normal appearing tissue, whereas COL1A2 expression was found to be distinctly decreased in malignant tissue. The normal isoform of ColI is a heterotrimer of two α1(I) chains and one α2(I) chain. However, carcinomas have been reported to contain α1(I) homotrimers, which are resistant to collagenolytic matrix metalloproteinases and may serve as protease-resistant invasion paths, supporting local proliferation and directed migration of the tumor cells (Makareeva et al. 2010, Cancer Res 70:4366-74). Upon KLK4 overexpression and concomitant dramatic reduction of COL1A2 mRNA expression, the formation of ColI homotrimers may be favored and, by this, contribute to the metastatic potential of prostate cancer cells.

Narain, Niven Validation of potential novel prostate cancer biomarkers identified from a comprehensive pan-omic interrogation of the prostate cancer biology using Bayesian inference approach Prostate Specific Antigen (PSA) test and Digital Rectal Exam (DRE) continue to be the predominant screening tests for prostate cancer with significant limitations in prognosis prediction and disease management. Markers to distinguish i) cancers with low or gray-zone PSA from Benign Prostate Hyperplasia (BPH), ii) indolent from aggressive disease and iii) to identify metastatic disease remain the overarching unmet need in prostate cancer screening. In this study, a comprehensive pan-omic biological interrogation of the human cell model (androgen sensitive LnCAP; androgen refractory PC3 models) was assessed in multiple environmental conditions in presence of bioenergetics perturbants. Pan-omic signatures were captured using mass-spectrometric technologies and overlayed on phenotypic functional assays. An AI based


eA26 5th ISK, Toronto, Canada, 2013 informatics algorithm was utilized to generate molecular interaction networks identifying potential drivers of prostate cancer. Differential expression of four of the potential molecular drivers of prostate cancers (FLNB, LY9 and two others) were characterized and validated in in vitro models and in a small case-control retrospective study in human serum samples from prostate cancer patients and age-matched normal controls. PSA levels in the study samples were used to benchmark patients. Differences in the levels of PSA, and the four markers were detected in the prostate cancer group. Statistical analyses were performed to assess the sensitivity and specificity of the molecular markers by ROC curve analysis. The predictive power of the combination of the four biomarkers out-performed PSA alone. This study demonstrates the power of Interrogative Biology(tm) powered systems based approach as a powerful method for biomarker discovery. Additional validation in a larger cohort in CLIA certified laboratory is ongoing to examine the propensity of these new molecular entities to fulfill the unmet needs in the prostate cancer biomarker industry.

Scarisbrick, Isobel Targeting kallikrein signaling to promote CNS repair Injury to the central nervous system (CNS), including that elicited by trauma, ischemia, infection, neurodegeneration or neoplasia, creates a complex wound that manifests with a cascade of secondary cellular and molecular responses that worsen the initial insult. A growing understanding of these events has resulted in the development of specific clinical management strategies, but there remain few treatments to improve neurological outcomes. An important therapeutic tactic is to minimize secondary injury to prevent pathogenesis and promote an environment that favors endogenous repair and the efficacy of growth promoting interventions and rehabilitation. Among the neurotoxic factors that are now recognized to be deregulated across neurological disorders are serine proteases of the thrombolytic, fibrinolytic and kallikrein families, either as a result of elevations in endogenous cells, secretion by infiltrating immune cells or extravasation. A significant body of evidence is emerging that multiple kallikreins are positioned to contribute to CNS pathology. Recent efforts from our laboratory highlight important roles for kallikrein 6 in pathophysiologic mechanisms impacting the development and progression of spinal cord injury, multiple sclerosis and glioblastoma multiforme. This includes critical roles in inflammation, astrogliosis, oligodendrogliopathy, demyelination, axonopathy and neuron degeneration. In each case, evidence is emerging that the pathophysiological effects of kallikrein 6 are mediated at least in part by selective activation of G-protein coupled Protease Activated Receptors (PARs). Current efforts are focused on testing the hypothesis that kallikrein 6, or the PARs it activates, can be targeted to prevent pathogenesis and promote repair and regeneration in the injured CNS.

Schmitt, Manfred Clinical impact of KLKs in reproductive organ malignancies Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only. Conclusions: Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic drugs to interfere with tumor progression and metastasis.

Scorilas, Andreas Prediction of prostate and bladder cancer risk based on KLK gene targeted microRNAs Prostate cancer (PCa) management is currently hampered by the PSA-driven overtreatment and overdiagnosis. Bladder cancer (BCa) patients often present disease recurrences that cannot be foreseen by the currently used prognostic indicators. The identification of novel biomarkers is, thus, imperative for these major urological malignancies. MicroRNAs (miRNAs) are small regulatory molecules, firmly associated with cancer. They target numerous key tumor-related genes, including KLKs. MiRNAs have recently become renowned as a novel class of cancer biomarkers. They are expected to act as useful clinical tools for predicting accurately and timely tumor aggressiveness and the recurrence risk of cancer patients. The present research project evaluates the prognostic significance of KLK-targeting miRNAs in prostate and bladder malignancies through their quantitative analysis in well-characterized PCa and BCa tissue samples. Our results


5th ISK, Toronto, Canada, 2013 eA27 reveal that miRNA molecules such as miR-224, which targets KLK1, KLK10 and KLK15 genes, and miR-378a, targeting KLK2 and KLK4, can provide significant information regarding the prediction of disease courses of both prostate and bladder cancer patients. KLK-targeting miRNAs (e.g. miR-143, miR-145) display also important discriminatory capacity between malignant and normal tissue and between tumors of differential aggressiveness. The role of KLK-related miRNAs in predicting disease outcome of patients suffering from urological tumors is beginning to unravel.

Simmer, James KLK4: An extracellular matrix enzyme critical for dental enamel formation Matrix metalloproteinase 20 (MMP20) and Kallikrein-related peptidase 4 (KLK4) are two secreted proteases that are critical for proper dental enamel formation. KLK4 is the newest member of the KLK genes clustered on the long arm of human chromosome 19 (19q13.41), having duplicated from KLK5 probably in placental mammals, and seems to be evolving rapidly [Elliott et al., Genomics. 2006;88:591-9]. Mutations in KLK4 cause non-syndromic amelogenesis imperfecta, an inherited condition featuring enamel defects in the absence of non-dental phenotypes. Klk4 knockout/NLS-lacZ knockin mice exhibit hypomaturation type enamel defects (the enamel is of normal thickness, but is soft and undergoes rapid attrition), with no systemic signs. A survey of the NLS-lacZ knockin expression showed strong -galactosidase expression specifically in maturation stage ameloblasts and minor to trace expression in salivary gland ducts and prostate epithelia and no detectable expression in liver, kidney, testis, ovary, oviduct, epididymis, and vas deferens. These observations, together with the scarcity of KLK4 expressed sequence tags in the mouse and human databases strongly support the conclusion that KLK4 functions specifically during enamel maturation. We have generated MMP20/KLK4 double null mice and characterized their enamel phenotype. We conclude that KLK4 cleaves extracellular enamel proteins to facilitate their reabsorption by ameloblasts.

Sotiropoulou, Georgia Insights into KLK functions from novel animal models A number of years elapsed since the identification of novel KLK proteases in mid 90s and the detailed mapping of the extended kallikrein locus in the year 2000. Despite the fact that numerous studies claim the potential utility of KLKs in molecular diagnosis, their specific physiological functions and putative involvement in disease states are largely unexplored. Our current understanding of KLK functions -including the concept of KLK cascades- mostly rely on in vitro observations, which will certainly require validation in vivo. To address this need, we have focused on exploring the (patho)physiological roles of KLK6 and KLK5 by employing novel mouse models. Specifically, we have successfully used SCID mice to investigate the effects of KLK5 and KLK6 in breast cancer, which revealed unexpected pathways affected by these extracellular proteases. More recently, we have generated Klk6 knockout mice with the aim to investigate its hypothesized implication in Parkinson disease. Moreover, for the first time, we have generated the Klk5 knockout, in order to delineate the roles of Klk5 in skin physiology and in overdesquamating and inflammatory skin diseases.

Stenman, Ulf-Hakan Does PSA cause prostate cancer or prevent its growth Prostate cancer is a unique tumor in being very common among elderly men. Histological examination of prostates of men aged over 60 years show that 40 – 80% have at least a microscopic tumor. Early prostate cancers can be detected already in 30-year old men but clinical prostate cancer is very rare in men below 50 year of age. Thus, most prostate cancers develop very slowly and only part of them will threaten the health of the man. Thus, the vast majority of prostate cancers are harmless but 2–3% of all men will die of prostate cancer. Proteases have the potential to drive development of cancer and the prostate produces a large number of proteases, KLK3 or prostate specific antigen (PSA) being the most abundant one. KLK3 has been implicated in the development of prostate cancer by its capacity to release growth factors from their binding proteins. However, other studies indicate that expression of PSA is lower in aggressive than in well differentiated prostate cancers. These apparently contradictory findings may indicate that KLK3 promotes early development of prostate cancer while it slows down the growth of a prostate cancer when the tumor reaches a size of 2-3 mm and requires vascularization. This is in agreement with the finding that KLK3 exerts antiangiogenic activity. The notion that PSA promotes early development of prostate cancer is supported by the finding that men who later develop prostate cancer have higher than average serum concentrations of KLK3 already in their third and fourth decade of life. At this stage, the prostate cancers that can be detected are too small to cause an increase in serum PSA. Therefore it is conceivable that elevated PSA expression at this stage is a risk factor that contributes to the development of prostate cancer.


eA28 5th ISK, Toronto, Canada, 2013 Yousef, George miRNA in kallikrein research: there are more questions than answers miRNAs are small non-coding RNA molecules that regulate the expression of their target genes. Accumulating evidence shows that kallikreins are targeted by miRNAs. Recent evidence shows the presence of miRNA-kallikrein axis of interaction that is actively involved in the pathogenesis of a number of tumors, including kidney, prostate, and ovarian cancers. Data points out to the presence of a divergent network of miRNA action (where a single miRNA targets multiple kallikrein and non-kallikrein genes) and a convergent network, where the same kallikrein is targeted by multiple miRNAs. We have also recently shown that kallikrein overexpression can lead to dysregulation of miRNA expression, and as such, miRNAs can also be downstream effectors of kallikreins. The mechanisms by which kallikreins can control miRNA expression remain to be fully elucidated. Our preliminary results show that kallikreins can affect the expression of essential molecules involved in miRNA biogenesis, including Drosha and Dicer, in addition to others. Preliminary analysis showed that KLK5 overexpression leads to dysregulation of specific miRNAs with a subsequent downstream effect on certain pathways including cell matrix interaction and the MAP-kinase pathways. Taken together, it is now clear that the kallikrein-miRNA interaction is more complex than previously thought. The existence of feedback loops of interaction should be also investigated.

Zlotta, Alexandre Genetic, epigenetic and proteomic analysis of the kallikrein (KLK) family in search for novel diagnostic prognostic and risk susceptibility algorithms for prostate cancer There is a growing need for novel Prostate Cancer (PCa) markers. Our study has focused on the KLK gene family, because of their major role as biomarkers for the diagnosis, prognosis, and monitoring of this disease. We hypothesized that the genetic, epigenetic and proteomic alterations of various KLK related genes may improve the diagnostic specificity of PSA and help identify individuals at risk, thus improving the clinical management of this disease. We used a unique patient population, the Swiss arm of the European Randomized Study of Screening for PCa (ERSPC) as well as a Toronto case-control Cohort. The goal of our work is: (1) To identify genetic profiles (SNPs variants) within the KLK family associated with PCa susceptibility. (2) To identify epigenetic and proteomic profiles improving the detection of prostate tumours and in particular aggressive cancers. (3) Multivariate and cumulative risk analysis combining genetic, epigenetic, proteomic and clinical data to improve PCa diagnosis in terms of genetic susceptibility and aggressive tumour biology. The data presented were integrated with the aid of complex statistical and mathematical models including individualized genetic-epigenetic-proteomic-clinical profiles. In a previous work focusing on the epigenetic regulation of KLKs, we assessed the biological effect of DNA methylation on KLK6 and KLK10 expression. We treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2’deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy. KLK10 DNA methylation was significantly associated with pathological stage in both cohorts.

ORAL PRESENTATION ABSTRACTS Dong, Ying Kallikrein related-peptidases regulate metastasis and chemoresistance in ovarian cancer Ying Dong1, Carson Stephens1, Carina Walpole1, Joakim E. Swedberg1, Loessner Daniela1, Glen M. Boyle2, Peter G. Parsons2, Michael A. McGuckin3, Jonathan M. Harris1 and Judith A. Clements1 1 Cancer Program, Translational Research Institute and Institute of Health and Biomedical Innovation, Faculty of Sciences and Technology, Queensland University of Technology, Woolloongabba, 4102; 2Drug Discovery Group, Division of Cancer and Cell Biology, Queensland Institute of Medical Research, Herston, 4029; 3Mater Medical Research Institute, Woolloongabba, 4102 Queensland, Australia High levels of tumour kallikrein related-peptidase 4 (KLK4) and KLK7 are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. As accumulation of fluid in the peritoneal cavity, ascites, is a common feature of EOC, we wished to determine the role of KLKs in EOC dissemination and chemoresistance in this microenvironment. We examined KLK4- and KLK7-overexpressed SKOV3 EOC cells in 3-dimensional (3D) suspension culture to mimic the ascites fluid. KLK4-SKOV3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV3 cells treated with active KLK4, and KLK4 endogenous expressing OVCA432 cells. MCA formation was reduced by treatment with a KLK4 blocking antibody, or the selective sunflower


5th ISK, Toronto, Canada, 2013 eA29 trypsin KLK4 inhibitor (SFTI-FCQR). SFTI-FCQR also reversed paclitaxel resistance of KLK4-MCAs. KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. In patient samples, a high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumour cells, further supporting a role for KLK4 in the ascitic microenvironment. Interestingly, when recombinant KLK4 enzyme was incubated with serum high molecular weight protein bands were observed and so did the conditioned media of KLK4-SKOV3 cells, indicating the KLK4 enzyme secreted from these cells is enzymatically active. We further found that KLK4-SKOV3 cells expressed high levels of urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in cells from patient ascites. On the other hand, KLK7-transfected SKOV3 cells also formed large compact MCAs and were resistant to paclitaxel, but high levels of α5/β1 integrin were observed, suggesting different signalling pathways induced by these KLKs. Together, our data suggest that KLK4 inhibition in conjunction with paclitaxel treatment may improve the outcome for women with high KLK4 levels in EOC. Key factors in different signalling pathways induced by KLK4 and KLK7 may also be tested for therapeutic potential for this cancer.

Eissa, Azza Kallikrein-related peptidase-8 (KLK8) is induced by TNFα and IL17A resulting in epidermal hyperplasia and elevation of psoriasis-related innate immunity gene expression Azza Eissa, Yijing Yu, Connie Zao, Vinod Chandran, Ulf Meyer-Hoffert and Eleftherios P. Diamandis Kallikrein-8 (KLK8) is an active epidermal protease implicated in normal barrier function and skin diseases. Although aberrant KLK8 expression was reported in the common skin diseases psoriasis and atopic dermatitis (AD), the underlying mechanisms leading to its dysregulation are poorly understood. Lesional psoriatic epidermis is known to lack a functional stratum granulosum, which normally secretes KLK8, and to contain abundant immune T- helper (Th) Th1 and Th17 cells, which are absent in healthy epidermis. Thus, we hypothesized that KLK8 epidermal expression is induced by the aberrant presence of T- helper (Th) cells in psoriatic skin, independent of skin barrier insults. Using HaCat keratinocytes as a differentiation model, we profiled keratinocyte secretion of KLK8 post-treatment with Th1, Th17 and Th2 cytokines alone or in combination, and investigated the effect of KLK8 overexpression on terminal keratinocyte differentiation and innate immunity gene expression in 3D full-thickness human skin equivalents. Our results show that psoriasis-related Th1 and Th17 cytokines, in particular TNFα and IL-17A, synergistically induce KLK8 hypersecretion, while Th2 AD-related cytokines, such as IL4, reduce KLK8 secretion by keratinocytes. KLK8 overexpression, in turn, induces hyperkeratosis and upregulation of innate defense genes expression mimicking psoriatic lesions. Consistent with our in vitro findings, KLK8 expression was reduced in lesional atopic dermatitis skin and in contrast, significantly elevated in lesional skin and sera of psoriatic patients. KLK8 elevation was significantly reduced after effective psoriasis treatment with the TNFα and IL17A-blocker etanercept, correlating positively with psoriasis clearance (p