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Publicly Available Published by De Gruyter May 10, 2014

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

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 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 kallikrein-related 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 zymogen-like 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 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 shRNA-transfected 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 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 non-catalytic 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. PSA-screening 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 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 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.

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

1Cancer 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 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<0.001). Together, our findings highlight the differential regulation of epidermal KLK8 by the distinct immune T-helper cells implicated in psoriasis and AD and corroborate KLK8 as a protease regulator of keratinocytes’ differentiation and innate immunity. Since KLK8 skin surface activity is central to the interlinked skin barrier and immune dysfunction of psoriatic skin, KLK8-specific inhibitors may have future potential as topical barrier-enhancing agents in psoriasis.

Kontos, Christos

Kallikrein-related peptidase 11 (KLK11) mRNA expression predicts short-term relapse and poor overall survival in colorectal cancer patients

D.K. Alexopoulou1, C.K. Kontos1, S. Christodoulou2, I.N. Papadopoulos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2Fourth Surgery Department, University General Hospital “Attikon”, University of Athens, Athens, Greece

Objective: We analyzed the mRNA expression of KLK11 in colonic tumors and non-cancerous counterparts, and examined its prognostic potential in colorectal cancer (CRC). Methods: Total RNA was isolated from 120 primary tumors and 41 paired non-cancerous mucosa from CRC patients. 2 μg of total RNA were reversely transcribed into cDNA. A highly sensitive real-time PCR methodology, based on SYBR Green chemistry, was developed for KLK11 mRNA quantification, and KLK11 expression was analyzed in tissue specimens. Data and results: A significant upregulation of KLK11 mRNA was found in malignant colorectal tumors, compared to their adjacent non-cancerous mucosae (P<0.001). KLK11 expression was significantly associated with the tumor invasion (P=0.048) and histological grade (P=0.004). Survival analysis showed that KLK11 mRNA expression constitutes an unfavorable prognostic biomarker in CRC, predicting poor disease-free and overall survival (P=0.045 and P=0.040, respectively), independently of the tumor size and histological grade. Conclusion:KLK11 mRNA expression can be considered as a novel, molecular prognostic biomarker predicting short-term relapse and poor overall survival in CRC, with additional prognostic value for CRC patients with highly invasive tumors and/or positive lymph nodes.

Acknowledgements: This work has been financially supported by the Hellenic Society of Medical Oncology.

Kryza, Thomas

KLK12 stimulates angiogenesis through a PDGF-B dependent paracrine pathway

T. Kryza 1, C. Achard3, C. Parent1, S. Marchand-Adam1,2, A. Guillon-Munos1,*, S. Iochmann1, B. Korkmaz1, R. Respaud2, Y. Courty1 and N. Heuz-Vourc’h1

1CEPR, UMR 1100/EA6305, Tours, France; 2 CHRU de Tours, Tours, France; 3 UMR892, Nantes, France

KLK12 was recently suspected to participate to angiogenesis. To gain insight into the molecular mechanisms implicated, we analyzed KLK12 effect on endothelial cells (ECs) secretome using a protein array and found that KLK12 cleaves ECM-bound PDGF-B through processing of its retention motif, thereby releasing active and soluble PDGF-B. Both PDGF-B and VEGF-A are involved in KLK12 mediated angiogenesis, as shown using an in vitro 3D angiogenesis model and neutralizing antibody. Analysis of molecular interplay between KLK12, PDGF-B and VEGF-A revealed that the release of PDGF-B from EC ECM by KLK12 induced VEGF-A secretion by stromal cells, which in turn acted on ECs favoring differentiation and capillary-like formation. Altogether our results show for the first time that PDGF-B is a new substrate for KLK12. Actually, KLK12 is the first physiologically-relevant protease candidate to process PDGF-B retention motif and liberate soluble active PDGF-B. Changes in PDGF-B bioavailability modulates VEGF secretion by stromal cells, through a paracrine mechanism, ultimately leading to angiogenesis. Because both KLK12 and PDGF-B are up-regulated in cancer cells, such as non-small cell lung cancer, this protease might be associated with tumor angiogenesis.

Meyer-Hoffert, Ulf

Characterization of SPINK6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury

Jan Fischer1, Zhihong Wu1, Tomasz Kantyka2, Maria Sperrhacke1, Olga Dimitrieva1, Yulia Koblyakova1, Kerstin Ahrens1, Nina Graumann1, Hansjrg Baurecht1, Karina Reiss1, Jens-Michael Schrder1, Ehrhardt Proksch1, Ulf Meyer-Hoffert1

1Department of Dermatology, University Clinic Schleswig-Holstein, Campus Kiel, Kiel, Germany 2Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Cracow, Poland

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLK)s is crucial for epidermal barrier function and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal-domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5 KLK6, KLK7, KLK12, KLK13 and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis in the stratum granulosum and stratum corneum, in sebaceous glands and the inner root sheath of hair follicles. Stimulation with flagellin, TNFα/IFNγ, epidermal growth factor and IL-1β significantly induced Spink6 mRNA expression, whereas stimulation with all-trans retinoic acid resulted in a significant downregulation of Spink6 mRNA expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and contributes to skin barrier function. Differences in KLK inhibition by human and mouse Spink6 may pave the way for specific inhibitors of tissue kallikreins.

Morizane, Shin

Inflammatory cytokines regulate expression of lympho-epithelial Kazal-type-related inhibitor in epidermal keratinocytes

S. Morizane, K. Iwatsuki.

Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Okayama, 700-8558, Japan

Objective: To investigate a link between expression of lympho-epithelial Kazal-type-related inhibitor (LEKTI), a specific kallikrein inhibitor, and inflammatory cytokines in keratinocytes. Method: Cultured normal human epidermal keratinocytes (NHEKs) were stimulated with inflammatory cytokines and LEKTI protein in cultured media was measured by using ELISA. Immunohistochemical staining for LEKTI was performed using skin samples from normal skin (NS) and the lesions of psoriasis (PS, a Th17 disease) or atopic dermatitis (AD, a Th2 disease). Results: TNF-alpha and a Th17 cytokine, IL-17, significantly up-regulated LEKTI protein in NHEK-cultured media, but Th2 cytokines, IL-14 or IL-13, did not. Immunohistochemical analysis confirmed LEKTI expression in AD lesions was almost same as in NS, but it was significantly higher in PS lesions than in AD lesions. Conclusions: TNF-alpha and IL-17 induce LEKTI expression in keratinocytes, which might account for a difference of LEKTI expression between PS and AD.

Olkhov-Mitsel, Ekaterina

Epigenetic regulation of kallikrein related peptidases as a novel panel of prostate cancer biomarkers

E. Olkhov-Mitsel1,2, T. Van der Kwast2,3, K. Kron3, H. Ozcelik1, L. Briollais1, N. Fleshner3, E.P. Diamandis1,2,3,4, A. Zlotta4,5, B. Bapat1,2,3

1Lunenfeld-Tanenbaum Research Institute, Toronto, ON; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, ON; 3University Health Network, Toronto, ON; 4Mount Sinai Hospital, Toronto, ON; 5Princess Margaret Hospital, Toronto, ON

Kallikrein related peptidases (KLKs) are dysregulated in a wide range of malignancies and have been shown to have broad range of applications as cancer biomarkers. The most widely recognized KLK biomarker is the prostate-specific antigen (PSA), encoded by KLK3, which is used for prostate cancer (PCa) diagnosis. However, the utility of PSA is controversial and improved biomarkers are needed for PCa, the most commonly diagnosed cancer in North American men. Other members of the KLK gene family are likely candidates since their expression is altered in PCa. Epigenetic mechanisms such as DNA methylation and aberrant microRNA (miRNA) expression have been shown to regulate KLKs in carcinogenesis. Previously, we established tumor-specific hypermethylation of KLK10 in PCa. To better understand KLK10 regulation, and potentially PCa pathogenesis, we have identified a panel of miRNAs predicted to target this gene. Notably, miR-137 was predicted to target KLK10 and was hypomethylated on CpG Island microarrays. This suggests a combinatorial regulation of KLK10 and a miRNA that possibly targets it, miR-137, by DNA methylation. Further, we found that high miR-137 expression was significantly associated with Gleason Score (P-values=0.035). Next, we examined the methylation status of other KLK targeting miRNAs, identified in silico, using genome-wide DNA methylation profiling. We found differential methylation of 47 KLK targeting miRNAs to be significantly associated with high-grade PCa and methylation of 24 such miRNAs to be associated with recurrence. This further characterizes molecular subtypes of PCa, which will allow for potentially establishing a diagnostic and/or prognostic model of clinical significance for the disease.

Pampalakis, Georgios

Extracellular α-synuclein is resistant to direct KLK6 proteolysis

Methodios Ximerakis1, Georgios Pampalakis2, Theodoros Roumeliotis3, Vasia S. Sykioti1, Spiros D. Garbis3, Leonidas Stefanis1,4, Georgia Sotiropoulou2 and KostasVekrellis1

1Division of Basic Neurosciences, Biomedical Research Foundation, Academy of Athens, Athens and 2Department of Pharmacy, University of Patras, Rion-Patras, Greece; 3Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; 4Second Department of Neurology, University of Athens Medical School, Athens, Greece

A plethora of evidence supports that α-synuclein plays a central role in the pathogenesis of Parkinson’s disease (PD). Although the physiological roles of both intracellular and extracellular α-synuclein remain elusive, the levels of the protein appear to be critical in PD pathogenesis. In this respect, oligomeric intermediates of the protein are considered to be the toxic species. In addition, secreted α-synuclein has been demonstrated to propagate via a prion-like mechanism. A key determinant of disease onset and progression is the balance between α-synuclein production and clearance. It is therefore possible that deregulation in the normal degradation and clearance of extracellular α-synuclein could underlie some or many cases of the disease. The degradation/clearance mechanisms of secreted α-synuclein have received very little attention and the enzymes involved are not well-studied. As a result it has not yet been possible to enhance catabolism by pharmacological manipulation. Here, we sought to investigate factors and mechanisms that regulate the extracellular α-synuclein turnover. As a source of secreted α-synuclein we used conditioned media from SH-SY5Y cells that were modified to overexpress and secrete this protein under biologically relevant conditions and concentrations. We provide evidence for a differential susceptibility of recombinant and naturally secreted α-synuclein to KLK6 proteolysis. Our data strongly demonstrate that unlike recombinant α-synuclein that is efficiently cleaved at the N- and C-termini by KLK6, secreted α-synuclein forms are resistant to direct proteolysis by KLK6. We further provide evidence that secreted α-synuclein can be cleaved by KLK6 only through activation of a secreted metallopeptidase, thus leading to the novel hypothesis that a proteolytic cascade may be engaged in the processing/turnover of secreted α-synuclein.

Sidiropoulos, Konstantinos

miRNA-mediated anti-oncogenic pathway induction by kallikrein 5 over-expression in breast cancer cell lines

K.G. Sidiropoulos1,3, N.M White1, A. Bui1, P. Boulos1, Q.C Ding1, G. Sotiropoulou2, G. Pampalakis2, H. Khella1, G.M. Yousef1,3

1Department of Lab Medicine and Pathobiology, University of Toronto, Canada; 2 Department of Pharmacy, School of Health Sciences, University of Patras, Rion-Patras, Greece, 3Department of Pathology, St. Michael’s Hospital, University of Toronto, Canada

Kallikrein 5 (KLK-5) has been demonstrated to be down-regulated in breast and other cancers. In order to understand the role of KLK5 in breast cancer pathogenesis, stable breast cell lines (MDA-MB-231) were created over-expressing KLK5 and an examination of miRNA expression changes were analyzed using a miRNA microarray approach. We identified 25 up-regulated and 60 downregulated miRNAs with KLK5 expression. These included up-regulated miRNAs miR-183-5p, miR-206; and down-regulated miRNAs: miR-935, mir-519a-5p, and miR-23b-5p. Pathway analysis of the gene targets of these miRNAs showed that the most enriched pathways were related cell-cell interaction, MAPK signaling, apoptosis and p53 pathways. KLK5 expression caused up-regulation of a set of miRNAs determined to be enriched in ECM-receptor interaction pathways. These gene targets were confirmed to have altered expression in KLK5 expressing stable as compared to mock stable lines and included a reduction in cell to matrix adhesion molecules including integrins such as beta-1 (ITGB1), reduced ECM protease expression, and reduced basement membrane constituents. An analysis of the miRNA-ECM interactions in breast cancer was carried out. Our data shows a significant overexpression of miR-183-5p levels upon KLK5 transfection, while integrin beta 1 (ITGB1) levels were downregulated up to 3-fold. Transfection with an inhibitor of miR-183-5p resulted in reversal of the inhibition of ITGB1. To further explore the mechanisms of KLK5’s effect on miRNAs, we examined the expression miRNA biogenesis which demonstrated a significant reduction in Drosha, Dicer, Exportin5, Ago 1 and Ago2 expression. From this data, KLK5 likely potentiates down-regulation of multiple miRNA biogenesis pathways. To examine the effects of KLK5 on the biological characteristics of breast cancer, we analyzed the TCGA database and a sub-set of 16 patients were recovered which had a change in KLK5, miR-183-5p and ITGB1 expression of at least 1 z-score. The data demonstrated a trend in which high expression of KLK5 and miR-183-5p was correlated with low ITGB1 expression and a basal-like breast cancer subtype with no metastatic patients. Conversely, lower expression of KLK5 and miR-183-5p was correlated with higher ITGB1 expression and mainly a luminal A or B breast cancer subtype. Interestingly, patients with the lowest level of KLK5 mir-183-5p and highest ITGB1 expression were correlated with confirmed breast cancer metastasis independent of breast molecular subtype.

Skala, Wolfgang

Crystal structures of human kallikrein-related peptidase 2 guide the search for its zinc inhibition mechanism

W. Skala1, S. Guo1, V. Magdolen2, H. Brandstetter1, P. Goettig1

1Division of Structural Biology, Department of Molecular Biology, Universitt Salzburg, Billrothstrae 11, 5020 Salzburg, Austria; 2Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der TU Mnchen, Ismaninger Strae 22, 81675 Mnchen, Germany

Human kallikrein-related peptidase (KLK) 2 is predominantly expressed in prostatic tissue and secreted into seminal fluid, where it most likely contributes to sperm liquefaction by activating KLK3/PSA. KLK2 belongs to the closely related "classical" KLKs 1-3, which share an extended 99- or kallikrein loop near their non-primed substrate binding side. In order to elucidate the structure of KLK2, we expressed a hexahistidine-tagged zymogen form as inclusion bodies in E. coli. In vitro folding followed by a three-step purification protocol, including autoactivation, yielded tens of milligrams of active, monomeric protease. Crystals diffracting up to 1.9 resolution allowed us to solve two structures of KLK2 by using initial phases from molecular replacement employing KLK3 as search model. The refined KLK2 crystal structures resemble KLK3, while they exhibit some notable differences. Their active sites contain the inhibitors benzamidine or d-Phe-Pro-Arg chloromethyl ketone, respectively. Interestingly, about ten residues in the 99-loop lack electron density, indicating that this loop is disordered in the crystal, possibly due to cleavage after Lys95E. Although Zn2+ ions are potent inhibitors of KLK2 (IC50=21.51.4 μM), no metal ion binding sites are evident from the structure. Nevertheless, kinetic analyses of several mutants (H25A, H91A, H95fA and H101A) pinpoint the Zn2+ binding site to the 99-loop/active site interface. In conclusion, our novel structure completes those of the classical human KLKs and represents the first structure of a classical KLK expressed in prokaryotes.

Talieri, Maroulio

Silencing of kallikrein-related peptidase 7 (KLK7) in HCT116 colon cancer cells: invasiveness and proteomic analysis

M. Devetzi1,2,3, M. Talieri3, T. Trangas2, M. Samiotaki1, G. Panayotou1

1Biomedical Sciences Research Center Alexander Fleming, Vari, 16672, Greece; 2Department of Biological Applications & Technology, University of Ioannina, Ioannina, 45110, Greece; 3Department of Cellular Physiology, G. Papanicolaou Research Center of Oncology, Saint Savvas Oncologic Hospital, Athens, 11522, Greece

It has been shown that KLK7 is up-regulated in colon cancer on the mRNA and protein levels, but its function in the progression of this type of cancer remains unclear. Small interfering RNA (siRNA) targeting KLK7 was transfected into HCT116 colon cancer cells, which highly express KLK7. KLK7 mRNA and protein levels were examined by real time qPCR and Western blot, respectively. The Matrigel(tm) invasion assay was implemented to assess cell invasion. LC-MS/MS proteomic analysis was applied in order to find differentially expressed proteins between KLK7-silenced and non-silenced colon cancer cells. Statistical analysis was performed to evaluate the significance of the findings. KLK7 expression was significantly downregulated (65-80% knockdown) at the mRNA and protein levels in HCT116 cells transfected with KLK7 siRNA, and the invasive capacity of cells was significantly decreased (p=0.02). Proteomic analysis identified 1895 proteins out of which 257 were significantly differentially expressed between KLK7-silenced and non-silenced colon cancer cells. Proteins involved in cell migration as well as in intracellular protein transport were differentially expressed between the two groups. KLK7 peptidase may play an important role in colon cancer invasion and could be a potential target for anticancer therapy.

This research has been co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)-Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.

Yoon, Hyesook

Kallikrein 6 promotes neurotoxicity by activating protease activated receptors

Hyesook Yoon1,2, Maja Radulovic1, Jianmin Wu2, Sachiko I. Blaber3, Michael Blaber3, Michael G. Fehlings4, Isobel A. Scarisbrick1,2,5.

1Neurobiology of Disease Program; 2Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester MN; 3Department of Biomedical Science, Florida State University, Tallahassee, FL; 4Department of Surgery, Toronto Western Research Institute, Toronto, ON, Canada; 5Department of Neurology, Mayo Clinic Rochester, MN

Trauma to the CNS generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. KLK6 is a serine protease recently implicated in neurodegeneration and in these studies we investigate the role of protease activated receptors 1 (PAR1) and PAR2 in mediating these effects. First we demonstrate KLK6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and -2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant KLK6 triggered ERK1/2 signaling in primary cerebellar granule neurons and in the NSC34 motoneuron cell line, in a PI3K and MEK-dependent fashion. Novel lipopeptide inhibitors specific for PAR1 or PAR2, in addition to neurons derived from PAR1 deficient mice were utilized to demonstrate both receptors play a role in KLK6-elicited signaling, including activation of ERK1/2 and the pro-apoptotic protein Bim. Both KLK6 and thrombin promoted degeneration of cerebellar neurons and potentiated glutamate neurotoxicity in vitro. Importantly, neurotoxicity in each case was reduced or blocked in neurons derived from PAR1 deficient mice. Taken together these data point to a novel KLK6-PAR1 and -PAR2 mediated signaling axis in CNS neurons that is positioned to mediate neurotoxicity occurring in cases of SCI.

Supported by NIH R01 NS052741, NMSS PP2009 and CA1060A11.

Yu, Yijing

Investigation of the biological substrates of skin kallikrein-related peptidases

Yijing Yu1,2, Azza Eissa1,2, Hari Kosanam2, Eleftherios P. Diamandis1,2,3

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada, M5T 3L9; 3Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, Canada, M5G 2C4

Kallikrein-related peptidases (KLKs) are a family of serine proteases expressed in various tissues. In the skin, it has been reported that KLKs contribute to its physiology (e.g. desquamation), the pathogenesis of skin cancers (e.g. melanoma) and inflammatory diseases (e.g. psoriasis and atopic dermatitis) by activating or inactivating certain substrates. Biological substrates for most of kallikreins still remain to be discovered. Recombinant mature-form of KLK7and KLK8 proteins were produced in the P. pastoris expression system. The purified KLKs were confirmed by western blotting and mass spectrometry (MS). The enzymatic activities of recombinant KLKs were determined by flurogenic substrate and zymography assays. To screen the pathobiological substrates for KLKs, three melanoma cell lines WM35, WM 902 and WM9 were cultured, treated with (without) active-form KLK 7, and analyzed by MS. Intracellular proteins were removed. Peptide sequences only found in KLK7-treated samples which contain phenylalanine or tyrosine cleavage site were selected as candidate sequences. Sixteen candidates for KLK7 in melanoma studies had been detected, including tenascin, Cysteine-rich angiogenic inducer 61(CYR61), and connective tissue growth factor. Selected reaction monitoring (SRM) assays will be designed for detected peptide sequences, and performed with more than 3 biological samples. In addition, the immortal human keratinocyte cell line HaCaT conditioned medium is being tested using TAILS and PROTOMAP experiments, to discover more physiological substrates of KLKs.

POSTER PRESENTATION ABSTRACTS

Adeli, Kourosh

Systematic analysis of kallikrein expression in cancer cell lines and tissues

Kourosh Adeli, Ioannis Prassas, Niron Sukumar, Apostolos Dimitromanolakis, Eleftherios P. Diamandis

Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto M5T 3L9, ON, Canada

Objective: Evaluate mRNA co-expression profiles in the kallikrein family and provide insights into the regulation of kallikreins at the gene level. Methods: mRNA expression levels of all the genes in the kallikrein family were evaluated in 4708 tissue samples and 917 cell lines. The microarray gene profiles were collected across the NCBI GEO repository and the Cancer Cell Line Encyclopedia. All samples have been assayed in the same microarray platform (HG U133 Plus 2.0) to ensure consistency of expression levels. Raw data were processed according to the typical Affymetrix data analysis pipeline. Scatter plots and Pearson correlation were used to assess the level of co-expression between all pairs of kallikrein genes. Results: Four distinct patterns of kallikrein co-expression were observed: (Subgroup 1): KLK 2,3,4 and 15, (Subgroup 2): KLK 5,6,7,8,9,10 and 11, (Subgroup 3): KLK 11and 12, and (Subgroup 4): KLK 10,11 and 13. These four subgroups were found to be tightly co-regulated along many cancer cell lines and tissues. Distinct KLK expressing profiles have also been identified between normal and cancer tissues. Conclusion/Future Plans: Despite the well-established involvement of KLKs in various physiological and pathophysiological conditions, little is known about their coordinated genomic regulation. Identification of certain co-expressing KLK subgroups will eventually allow us to pinpoint the critical underlying regulatory elements and further elucidate the cascade-like regulation of these genes.

Batra, Jyotsna

A genetic variation affecting miRNA binding to KLK3 is associated with the risk of prostate cancer

S. Stegeman1, F. Lose2, A. Spurdle2, J. Clements1, J. Batra1

1Australian Prostate Cancer Research Centre-Queensland and Institute of Health and Biomedical Innovation, QUT, Brisbane, Queensland, Australia - 4102; 2Molecular Cancer Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia - 4006

Objectives: Kallikreins (KLKs), encoded by a gene cluster on chromosome 19q13.4, are secreted serine proteases with diverse expression patterns and physiological roles. We hypothesised that genetic factors influencing KLK3 expression may have implications in prostate cancer risk prediction, and interpretation of PSA (KLK3) screening might therefore need correction for these risk associated functional KLK3 genetic variants. Thus, we aim to identify functional causal polymorphism within the KLK locus. Results: We genotyped approximately 1,200 prostate cancer cases and 1,300 controls determining that a single nucleotide polymorphism (SNP) within the 3’ untranslated region of KLK3 is associated with prostate cancer risk (OR=0.74 95% CI=0.63-0.88, p=0.001). Using a range of online tools, miRNA 3162-5p was predicted to have greater binding affinity for the T allele of the rs1058205 SNP versus the C allele (C allele frequency 0.36). Reporter vector assays with over-expression of miR-3162-5p resulted in a 20% decrease in luciferase signal (p=0.04) for the KLK3 rs1058205 SNP T allele vector construct whilst no significant change was observed for the C allele suggesting that miR-3162-5p has specific affinity for the T allele. Further validating these results, we observed a 36% reduction in cellular KLK3 protein expression following miR-3162-5p over-expression in the LNCaP cell line (TT rs1058205 SNP). Conclusion: Results to date show a prostate cancer associated SNP rs1058205 in the KLK3 3’ UTR affects miR-3162-5p targeting in prostate cancer. Thus this SNP may be better suited for incorporation into risk modeling of prostate cancer in comparison to the original SNP identified by genome-wide association studies at this locus.

Clements, Judith

1- Kallikrein 4 proteolysis of the EphB4 receptor tyrosine kinase initiates novel paracrine and nuclear actions of EphB4 in prostate cancer cells

Jessica Lisle1,2, Inga Mertens-Walker1,2, Carson Stephens1, Scott Stansfield1, Judith Clements1,2, Adrian C Herington1,2 and Sally-Anne Stephenson1,2

The Institute of Health and Biomedical Innovation, Faculty of Health1, and the Australian Prostate Cancer Research Centre-Queensland2, Queensland University of Technology, 37 Kent St, Woolloongabba, Queensland, Australia 4102

The EphB4 receptor tyrosine kinase is over-expressed in >60% of prostate cancers (PCa) and increases cancer cell survival, invasion and migration. Through interaction with its cognate ligand, ephrin-B2, expressed on adjacent endothelial cells, EphB4 also promotes tumour angiogenesis. In PCa cells we have identified two sequential protease-mediated cleavages of EphB4, generating both a soluble extracellular and an intracellular fragment that have potentially major impact on the known mechanisms of action of EphB4. We have shown that the PCa-associated serine protease kallikrein-like peptidase 4 (KLK4) cleaves EphB4 at arginine 508 to generate a soluble extracellular ligand-binding domain of 70kDa which can be released from the cell, providing a novel mechanism for interaction with ephrin-B2 expressed on other cell types. This has implications for angiogenic outcomes - inhibition if the fragments exist in a monomeric form, but stimulation if the fragments exist in a dimeric/clustered form. Following the initial cleavage, the residual 50kDa membrane-embedded fragment is then subsequently cleaved by the protease γ-secretase to generate an intracellular C-terminal fragment of 47 kDa. This 47kDa fragment can then translocate to the nucleus of the PCa cell line LNCaP, translocation being mediated via a classical nuclear localization signal and interaction with α-importin. We have recently demonstrated via ChIP experiments that nuclear EphB4 binds to DNA in regions that harbour genes and/or transcription factors already implicated in PCa. This study provides the first evidence of proteolytic regulation of EphB4 in PCa. The sequential generation of both N- and C-terminal fragments suggests unique mechanisms of action which may provide novel insights into how EphB4 contributes to PCa.

2- Proteomic screening of kallikrein-related peptidase 7 substrates in ovarian cancer

Silva, Lakmali1; Stoll, Thomas2; Stansfield, Scott1; Stephens, Carson1; Hastie, Marcus2; Gorman, Jeff2; Dong, Ying1; Irving-Rodgers, Helen1; Clements, Judith1

1Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4102; 2Queensland Institute of Medical Research, University of Queensland, Brisbane, QLD 4006

Kallikrein-related peptidase 7 (KLK7) is a serine protease that is over-expressed in ovarian cancer cells, compared to normal ovarian tissues. KLK7 over production in ovarian cancer cells has been predicted to enhance cell migration, invasion and resistance to chemotherapy, three of the hallmarks of cancer progression. To target KLK7 action in ovarian cancer as a potential therapy, its functional role must be elucidated. As KLK7 exerts its function through cleaving protein targets, we sought to identify substrates of KLK7 in ovarian cancer. To screen for protein cleavage sites of KLK7, trypsin generated peptides of proteins produced by human cells were treated with KLK7, or a catalytically inactive mutant KLK7, as a control, and analysed by liquid chromatography-tandem mass spectrometry to identify the peptide fragments. Protein databases (MatrixDB) were screened for potential proteins with these peptide sequences and proteins were further interrogated using three-dimensional (3-D) conformation data in the Protein Data Bank (PDB) database to check the accessibility of these predicted cleavage sites. Through both bioinformatics screening and biochemically, we demonstrated that KLK7 cleave Integrin Beta 6 (ITGB6) that plays a role in cell adhesion and cell-extracellular matrix interactions. ITGB6 expression has shown to increase in late stage ovarian tumour tissues and is associated with increased invasiveness of cancer cells. Identification of substrates of KLK7 in the ovarian cancer microenvironment will help demonstrate the diverse role played by KLK7 in the progression of ovarian cancer, by re-modelling the extracellular matrix and enhancing cell invasiveness.

Courty, Yves

1- Kallikrein-related peptidase 13 is dysregulated in the lungs following influenza A virus infection

F. Gueugnon1,2, A. Guillon1,2, V. Thibault1,2, J. Kearley3, A. Petit-Courty 1,2, M. Si-Tahar 1,2, A. Humbles3 and Y. Courty 1,2

1INSERM U1100, CEPR, Facult de Mdecine, 37 000 Tours, France; 2 Universit Franois Rabelais, Tours, France; 3 MedImmune, Gaithersburg, MD, USA

Several kallikrein-related peptidases (KLK) are expressed in the lungs but their functions remain elusive. This study aimed at providing a better understanding of the involvement of KLKs in the local host response to influenza A virus (IAV) infection. Balb/c mice were challenged by IAV and mRNA expression of mKlk13 was quantified by real-time qRT-PCR in lung tissue 24h to 6-days post-infection. Klk13 expression was significantly decreased in infected mice compared with control mice. Previous acute exposure to cigarette smoke did not change the response. Human lung cell lines (BEAS-2B, NCI-H292) and primary airway epithelial cells cultured at air-liquid interface were also infected by IAV. Immortalized cell lines responded to IAV infection by an increase in KLK13 expression. In contrast, KLK13 was down-regulated in human primary cells, especially in airway epithelial cells collected from patients suffering COPD (Chronic Obstructive Pulmonary Disease). Our results suggest that KLK13 may be involved in the airway epithelial cell response to influenza infection.

2- Acute cigarette smoke exposure differentially regulates the pulmonary expression of kallikrein-related peptidases

F. Gueugnon1,2, A. Vallet1,2, A. Guillon1,2, J. Kearley3, A. Petit-Courty1,2, M. Si-Tahar 1,2, A. Humbles3 and Y. Courty 1,2

1 INSERM U1100, CEPR, Facult de Mdecine, 37 000 Tours, France; 2 Universit Franois Rabelais, Tours, France; 3MedImmune, Gaithersburg, MD, USA

Cigarette smoking is a recognized causative agent or precipitant of various lung diseases including cancer and Chronic Obstructive Pulmonary Disease (COPD). Airways inflammation and remodeling both occur in the lungs of patients with COPD, and it is likely that inflammation contributes to the structural abnormalities. Airway epithelial cells are the main expression site for kallikrein-related proteases (KLKs) and this study aimed to determine whether KLKs are involved in the early inflammatory response induced by cigarette smoke (CS). Cell cultures from airway-derivate cell lines (BEAS-2B, NCI-H292) were incubated with CS extract for 6h and total RNA was prepared 16 h after CS exposure. Gene expression profiles (KLK 1, 4, 5, 7-11, 13-14) were then determined using real-time qRT-PCR. Treatment with CS extract did not modulate expression of the KLK genes in the epithelial cell lines. Balb/c mice were exposed to air or whole mainstream CS for 2 h during 4 days in a purpose-designed chamber. Three genes (mKlk 7, 8 and 13) were found significantly dysregulated following acute CS exposure. mKlk 7 and 8 were down-regulated while mKlk13 was up-regulated. These results suggest that some KLKs may be involved in the early response of the lung to CS injury probably through interplays between epithelial cells and other cell types.

3- Kallikrein-related peptidase 6 regulates the growth and survival of lung cancer cells through activation of proteinase-activated receptor 2 and epidermal growth factor receptor

N. Michel1,2, C. Parent1,2, E.S. Lavergne3,4, M-L Jourdan2,5, A. Vallet1,2, S. Iochmann1,2, P. Reverdiau1,2, O. Musso 3,4, N. Heuz-Vourc’h 1,2, and Y. Courty 1,2

1INSERM U1100, CEPR, Facult de Mdecine, 37 000 Tours, France; 2Universit Franois Rabelais, Tours, France; 3INSERM, UMR991, Rennes, France; 4Universit de Rennes 1, Rennes, France; 5INSERM UMR 1069, Tours, France

We have previously revealed that elevated expression of kallikrein-related peptidase 6 (KLK6) is an indicator of poor prognosis for patients suffering non-small cell lung cancer (NSCLC). Utilizing an isogenic NSCLC cell model system, we demonstrated that ectopic KLK6 expression enhanced cell growth through an acceleration of the cell cycle, between the G1 and S phases. However, the mechanism that sustains KLK6 signaling remained unknown. We show here that KLK6 promotes proliferation and restrains apoptosis of lung tumoral cells in vitro via a ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2).

da Motta, Guacyara

Involvement of proteoglycans in human plasma prekallikrein interaction with cell surface

C. L. Veronez, F. D. Nascimento1, H. B. Nader, I. L. S. Tersariol, G. Motta

Departamento de Bioqumica, Escola Paulista de Medicina/UNIFESP, Sao Paulo and 1Biomaterials and Biotecnology Research Group-UNIBAN, Sao Paulo, SP, Brazil

Human plasma prekallikrein (PK) is the zymogen of a protease related to hemostasis, inflammation, fibrinolysis, adipogenesis, tissue repair and angiogenesis. High molecular weight kininogen (HK) may function as PK putative receptor on cell surface and HK endocytosis is mediated by proteoglycans (PG). In this work the aim is to evaluate PK assembly and processing on cells and whether PG along with HK could influence this interaction. We used endothelial cells lines, ECV304 and RAEC; epithelial cells CHO-K1 (wild-type) and CHO-745 (deficient in PG); PK endocytosis was studied using confocal microscopy and PK cleavage was determined by immunoblotting using antibody directed to sequence C364 TTKTSTR371 on PK. At 37C PK was determined as pixels/cell, in absence or presence of added HK, as 1,418.4 0.010 or 1,070.3 0.001 for ECV304; 440.0 0.000 or 642.8 0.001 for RAEC; 1,319.1 0.003 or 631.3 0.001 for CHO-K1. None PK internalization was determined for CHO-745 in both conditions. PK colocalized with LysoTracker respectively, in absence and presence of added HK, 76.0% and 88.5% for ECV304; 82.2% and 62.2% for RAEC, 40.7% and 57.0% for CHO-K1. After assembly on cell surface PK fragments (53kDa, 48kDa, 32kDa) were detected in fluid phase of all cell lines studied, independent of added HK. In conclusion, PK interaction with cell surface is temperature-dependent, independent of added HK, which results in PK endocytosis promoted by PG and proteolysis probably by different proteases, as a control mechanism of plasma kallikrein activity.

Domanskyy, Mykola

Inhibition of KLK2 by SPINK1

Mykola Domanskyy, Ulf-Hkan Stenman and Hannu Koistinen

Department of Clinical Chemistry, Hartman Institute, University of Helsinki, Helsinki, 00014, Finland

Serine protease inhibitor Kazal type-1 (SPINK1), also known as TATI (tumour-associated trypsin inhibitor), is a 56 amino acid trypsin inhibitor, whose levels are elevated in several cancers, including prostate cancer. Some mutations, such as N34S, are associated with pancreatitis. Furthermore, SPINK1 was recently found to be associated with ETS rearrangement-negative prostate cancers and to stimulate the invasion of prostate cancer cells, perhaps by activating the EGF-receptor. Recently, the mouse ortholog of SPINK1, SPINK3, was identified as a potent inhibitor of human KLK2, KLK3, and KLK5. We studied the effect of SPINK1on KLK2 and KLK3. Synthetic cDNA encoding SPINK1 56 amino acids without signal peptide was optimized for expression in E. coli. The SUMOstar E. coli Expression System was used for expression and purification of recombinant SPINK1. Final purification was done using an antibody affinity column and HPLC. Enzyme activity was measured using a chromogenic substrate and recombinant human KLK2. SPINK1 was found to inhibit KLK2, but not KLK3. It is possible that SPINK1 has a dual role in prostate cancer, i.e., it induces tumor growth by stimulating the EGF receptor. SPINK1 on the other hand inhibits the activity of trypsin and KLK2 and thus suppress activation of protease-activate receptors (PARs), which also may affect tumour growth.

Drucker, Kristen

Kallikrein 6 mediates therapy resistance in glioblastoma

Kristen L. Drucker1,2, Alex R. Paulsen2, Caterina Giannini3, Paul A. Decker4, Sachiko I. Blaber5, Michael Blaber5, Robert B. Jenkins3, Isobel A. Scarisbrick6

1Department of Physical Medicine and Rehabilitation; 2Neurobiology of Disease Program; 3Department of Laboratory Medicine and Pathology; 4Biomedical Statistics, Mayo Clinic Rochester, MN; 5Department of Biomedical Science, Florida State University; 6Department of Neurology; Mayo Clinic Rochester, MN

Kallikreins (KLKs) have prognostic value in specific malignancies, but few studies have addressed their clinical significance to glioblastoma (GBM). KLK6 is up regulated at sites of CNS pathology and recently linked to reactive astrogliosis and therefore of potential high relevance to GBM. To determine the clinical significance of KLK6 to GBM, we investigated the association between patient survival and levels of KLK6-immunoreactivity (IR) in 60 grade IV astrocytoma patient tumor specimens. Also, we evaluated the effects of elevated KLK6 on the response of glioblastoma cell lines (U251, SF767) to cytotoxic agents. A range of KLK6 expression was observed across grade IV tumors with higher levels a poor prognostic indicator of patient survival (univariable analysis, P=0.02). GBM patients with KLK6-IR scores below 10 had a median survival of 408d and patients with scores at 10 or above had a median survival of 276d (HR=2.36, 95% CI=1.19-4.68, univariable analysis P=0.01). The mechanism by which KLK6 contributes to reduced patient survival is suggested by results demonstrating KLK6 promotes resistance of GBM cell lines to apoptosis inducing agents, including radiation and/or temozolomide. The ability of KLK6 to elicit pro-survival effects was shown to depend at least in part on activation of the thrombin receptor, protease activated receptor 1 (PAR1). These results suggest KLK6 and PAR1 represent targets for the development of therapies to enhance the sensitivity of GBM to cell death-inducing agents.

Supported by R01 NS052741, P50 CA108961 and T32 NS07494.

Furio, Laetitia

Skin-specific kallikrein-5 transgenic mice reproduce cutaneous and systemic hallmarks of Netherton syndrome: a viable model for therapeutic development

L. Furio1,2, S. de Veer1,2, C. Deraison2, A. Briot2, C. Bonnart2, A. Robin2 and A. Hovnanian1,2,31University Paris Descartes Sorbonne Cite, France; 2INSERM U781 and Imagine Institute of Genetic Diseases, Paris, France; 3Department of Genetics, Necker hospital, Paris, France

Netherton syndrome (NS) is a severe genetic skin disease caused by mutations in SPINK5 encoding the lymphoepithelial Kazal-type-related serine protease inhibitor (LEKTI). Of the proteases which show overactivity in NS (kallikrein-related peptidase (KLK) 5, KLK7 and elastase 2), KLK5 is a potential key initiator of the proteolytic cascade. To address the role of KLK5 in the disease, we have generated a transgenic murine model overexpressing human KLK5 in the skin (TgKLK5). The transgene was highly expressed in the granular layer of TgKLK5 epidermis and was associated with increased proteolytic activity. This proteolytic activity was further characterized by gel zymography from skin protein extracts: KLK5 activity was elevated, as well as KLK7, KLK14 and elastase 2 activities, three proteases which have been proposed to be activated by KLK5. TgKLK5 mice displayed low weight at birth with a growth delay. Skin barrier was defective with increased transepidermal water loss. The stratum corneum detached from the underlying epidermis as a result of desmosomal cleavage. Whiskers and hairs were abnormal and reduced. Additionally, TgKLK5 mice displayed hallmarks of cutaneous inflammation and allergy including high expression of IL-1β, TNF-α and the pro-Th2 cytokine TSLP with mast cells and eosinophils infiltration. Overtime, persistent scratching led to alopecic, erosive and crusty skin lesions with lymph nodes hyperplasia. Inflammation was also detected at the systemic level, with high serum IgE and TSLP levels. CD4 T cells from lymph nodes showed increased secretion of Th2 and Th17 cytokines. These results help deciphering the role of KLK5 in NS phenotype and provide a useful viable model for the development of new therapeutic strategies.

Guo, Shihui

Eukaryotic expression and biochemical analysis of prostatic KLKs

S. Guo1, W. Skala, V. Magdolen2, H. Brandstetter1, P. Goettig1

1Department of Molecular Biology, University of Salzburg, Salzburg, Austria; 2Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany

Currently, structure-function studies of prostatic KLK peptidases rely mostly on proteins from E. coli or inhomogeneous natural samples. In order to study pure glycosylated variants resembling the natural KLKs, we used Leishmania (LEXSY) and HEK293 cells for secretory expression. Both systems allowed successful expression of soluble prostate KLK zymogens, which could be activated by enterokinase. KLK2 expression is more efficient in LEXSY cells, resulting in a glycosylated variant that is active against the tryptic substrate Bz-PFR-pNA. Expression of glycosylated KLK3 (PSA) and KLK4 was more efficient in HEK cells. All three KLKs were homogeneous in SDS-PAGE analysis and glycosylated as evidenced by glycan staining (Periodic Acid-Schiff stain). After treatment with Peptide-N-Glycosidase F, they exhibited lower molecular weights as expected by the amino acid sequence. For example, these findings are consistent with single potential N-linked glycosylation site of KLK4, which appears to have an influence on the activity. KLKs 3 and 4 exhibited activity against chymotryptic (Suc-AAF-AMC) or tryptic substrates, respectively. We further confirmed the known effects of stimulating and inhibitory ions, such as citrate and Zn2+ on KLK3 and the formation of a covalent complex with the natural inhibitor alpha-1-antichymotrypsin (ACT). Using a known crystallization condition, glycosylated KLK4 crystals were obtained, which require optimization for diffraction experiments. This study aims to clarify the effect of glycosylation on catalysis by comparison with non-glycosylated prostatic kallikreins from E. coli, in particular with the processing of natural substrates.

Jessa, Fatima

Establishing reference intervals for kallikrein 5, 6, 7, and 8 in healthy females

Fatima Jessa1,2, Felix Leung1,3, Eleftherios P. Diamandis1,2,3, and Vathany Kulasingam1,2

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; 2Department of Clinical Biochemistry, University Health Network, Toronto, ON, Canada; 3Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada

Introduction: Kallikreins are a family of serine proteases that are upregulated in many cancers. Certain kallikrein proteins (KLK 5, 6, 7, 8, 10, 11, and 14) are upregulated in ovarian cancer and documented to be potential diagnostic and/or prognostic biomarkers. However, before levels of these kallikreins can be measured for clinical purposes, a reference interval for each kallikrein needs to be determined. The reference range for a particular analyte is the prediction interval in which 95% of a reference group fall into, and generally denotes the “healthy” individuals in a population. Objective: The objective of this study was to establish reference intervals for kallikreins 5, 6, 7 and 8 among a population of apparently healthy, metabolically stable females. Methods: Approximately 150 serum samples from a population of healthy females were collected. A sandwich ELISA was used to measure the levels of the kallikreins of specific interest, in the serum of the test subjects. Reference intervals were established after outlier exclusion using both the non-parametric and Robust statistical methods to obtain the 2.5th and 97.5th percentiles, as per Clinical Laboratory Standards Institute guidelines. Results: Both the non-parametric and Robust statistical methods yielded similar reference intervals for all 4 kallikreins examined. The reference interval for Kallikrein 5 was established to be 0.2-0.8 μg/L using the nonparametric method. The reference interval for Kallikrein 6 was 0.6-5.6 μg/L, for Kallikrein 7 was 5.1-33.9 μg/L and for Kallikrein 8 was 0.9-5.0 μg/L using the nonparametric method. There was no association between kallikrein values and age. Conclusion: Our established reference intervals provide an important role when it comes to analyzing the levels of specific kallikrein proteins in ovarian cancer patients, since these proteins have potential diagnostic and/or prognostic value.

Koistinen, Hannu

Prostate-specific antigen cleaves nidogen-1

Mattsson JM, Ravela S, Stenman UH and Koistinen H

Department of Clinical Chemistry, Haartman Institute, University of Helsinki, Finland

Prostate-specific antigen (PSA or KLK3) exerts anti-angiogenic activity, which we have recently shown to be related to the enzymatic activity of PSA. PSA cleaves components of extracellular matrix (ECM), e.g., laminin and fibronectin, as well as unidentified proteins in Matrigel™ basement membrane preparation. Proteolytic products of some ECM proteins are known to be anti-angiogenic. We studied the PSA-generated protein fragments derived from Matrigel™ and aimed to characterize their anti-angiogenic activity. PSA was purified from seminal fluid and incubated with Matrigel™. Analysis of the resulting proteolytic fragments by SDS-PAGE and mass spectrometry revealed that PSA cleaves nidogen-1. This was confirmed by using recombinant nidogen-1. The nidogen-1 fragments did not have any anti-angiogenic effect on HUVECs in the tube formation model, where the cells were grown on Matrigel™ basement membrane preparation. Nidogen-1 is a novel substrate for PSA. The cleavage if nidogen-1may lead to disruption of basement membrane structure and, thus, may facilitate cancer cell invasion. However, the nidogen-1 fragments per se do not mediate the anti-angiogenic function of PSA.

Kolin, David

Prognostic significance of human tissue kallikrein-related peptidase 6 (KLK6) in gastric cancer

D.L. Kolin1, K. Sy2, F. Rotondo1, M.N. Bassily3, K. Kovacs1, C. Streutker1, G.M. Yousef1

1St. Michael’s Hospital, Toronto, ON M5B 1W8; 2Credit Valley Hospital, Mississauga, ON L5M 2N1; 3Department of Community Medicine and Public Health, Menoufiya University, Egypt

The prognosis of patients following surgery for gastric cancer is often poor and is estimated using traditional clinicopathological parameters which can be inaccurate predictors of future survival. Kallikreins are a group of serine proteases which are differentially expressed in many human tumours and are being investigated as potential cancer biomarkers. The aim of this study was to correlate human tissue kallikrein 6 (KLK6) expression with histopathological and clinical parameters in gastric cancer. We constructed a gastric tumour tissue microarray (TMA) from 113 gastrectomy specimens, and used immunohistochemistry (IHC) to measure KLK6 expression. A whole-slide scanned image of the TMA was analyzed using an automated algorithm by first segmenting the images based on stroma and epithelium, then separating the KLK6 IHC stain from the counterstain, and finally quantifying the staining intensity. KLK6 expression was significantly positively correlated with nodal involvement (p=0.002), and was predictive of advanced stage disease (p<0.05). Kaplan-Meier survival curves revealed KLK6 positive tumours were significantly associated with lower overall survival (p=0.04). Interestingly, there was no significant difference in KLK6 expression in stromal vs. epithelial tissue, or Lauren classification. KLK6 expression shows promise as a novel prognostic marker in gastric cancer. Automated analysis of TMA IHC staining has the potential to reduce both inter- and intra-observer variability and to reveal differences in staining not detected by conventional manual scoring.

Kontos, Christos

1- Enhanced miR224 transcription predicts short-term relapse and poor overall survival in colorectal adenocarcinoma patients

P. Adamopoulos1, C.K. Kontos1, S.M. Rapti1, S. Christodoulou2, I.N. Papadopoulos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2Fourth Surgery Department, University General Hospital “Attikon”, University of Athens, Athens, Greece

Objective: We developed a highly sensitive quantitative real-time PCR methodology and used it to quantify miR-224, which is known to target KLK15, in colorectal tumors and paired non-cancerous colorectal mucosa, aiming at examining the prognostic potential of miR-224 expression and its putative clinical application in colorectal adenocarcinoma prognosis. Methods: Total RNA was isolated from 119 malignant colorectal adenocarcinomas and 70 paired non-cancerous mucosa. 2 μg of RNA were polyadenylated and reversely transcribed. A quantitative real-time PCR (qPCR) based on SYBR Green chemistry was developed and applied for the quantification of miR-224 levels, followed by extensive biostatistical analysis. Data and results: miR-224 levels in malignant colorectal tumors ranged between 0.002 and 0.796 a.u. (MIR224 copies / RNU48 copies) with a median of 0.035, and were significantly elevated, compared to miR-224 levels in non-cancerous counterparts (P<0.001). Enhanced miR-224 expression appears as a powerful prognosticator in colorectal adenocarcinoma, predicting short-term relapse and poor overall survival (OS) in these patients (P=0.008 and P=0.002, respectively), independently from established clinicopathological parameters. Conclusion: miR-224 constitutes probably a novel molecular biomarker with independent prognostic value in colorectal adenocarcinoma.

Acknowledgements: This research was partially funded by the University of Athens, Special Account of Research Grants, no 10812.

2- Kallikrein related peptidase 4 (KLK4) mRNA expression: an independent favorable prognosticator of head and neck squamous cell carcinoma patients’ disease-free survival

E. Foteinou1, C.K. Kontos1, A.I. Yiotakis2, I. Yiotakis2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2First Ear, Nose & Throat Clinics, Athens General Hospital “Hippokration”, University of Athens, Athens, Greece

Objective: We sought to analyze KLK4 mRNA expression in head and neck squamous carcinoma (HNSCC) tissue specimens and to examine its prognostic value and potential clinical application as a novel molecular tissue biomarker in HNSCC. Methods: Total RNA was isolated from 116 primary tumors and 74 paired non-cancerous adjacent tissues of HNSCC patients. 2 μg of total RNA were reversely transcribed into cDNA. A highly sensitive real-time PCR methodology, based on SYBR Green chemistry, was developed for KLK4 mRNA quantification (detection limit: 10 mRNA copies), and KLK4 expression analysis was performed, followed by extensive biostatistical analysis. Data and results:KLK4 mRNA expression was considerably downregulated in HNSCC specimens, compared to non-cancerous paired head and neck tissues (P=0.030). Additionally, KLK4 mRNA levels are correlated with small tumor size (P=0.028). KLK4 mRNA expression status constitutes an independent favorable prognosticator of patients’ disease-free survival (DFS), as it predicts longer DFS regardless of patient’s nodal status, tumor invasion, size, and histological grade (P=0.022). Conclusion:KLK4 mRNA expression can be considered as a novel potential tissue biomarker predicting short-term relapse of HNSCC patients.

Acknowledgements: This work has been financially supported by the Hellenic Society of Medical Oncology.

3- Kallikrein-related peptidase 6 (KLK6) mRNA expression: an independent unfavorable prognosticator in colon cancer

C.K. Kontos1, D.K. Alexopoulou1, S. Christodoulou2, I.N. Papadopoulos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2Fourth Surgery Department, University General Hospital “Attikon”, University of Athens, Athens, Greece

Objective: We analyzed the mRNA expression of KLK6 in colonic tumors and paired non-cancerous colonic mucosae, and examined its prognostic potential in colon cancer. Methods: Total RNA was extracted from 110 primary colon adenocarcinomas and 39 adjacent non-cancerous mucosae from colon cancer patients. 2 μg of total RNA were reversely transcribed into cDNA. A highly sensitive real-time PCR methodology was developed for KLK6 mRNA quantification, and KLK6 expression was analyzed in tissue specimens. Data and results: An eminent upregulation of KLK6 mRNA expression was observed in malignant tumors, compared to their normal counterparts (P<0.001). KLK6 expression was significantly associated with the tumor invasion (P=0.024) and presence of distant metastasis (P=0.013). Kaplan-Meier analysis showed that KLK6 mRNA expression is likely to constitute an unfavorable prognostic biomarker in colon cancer. Multivariate Cox regression analysis revealed also that the unfavorable prognostic value of KLK6 mRNA does not depend on the tumor size, histological grade, and TNM stage of colon cancer patients. Conclusion:KLK6 mRNA expression can be considered as a novel, molecular prognostic biomarker predicting poor disease-free and overall survival in colon cancer. Undoubtedly, its study as a molecular biomarker in this malignancy merits further investigation.

Acknowledgements: This work has been financially supported by the Hellenic Society of Medical Oncology.

4- miR-34a overexpression is a strong indicator of unfavorable survival outcome in colon cancer patients

C.K. Kontos1, S.M. Rapti1, S. Christodoulou2, I.N. Papadopoulos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2Fourth Surgery Department, University General Hospital “Attikon”, University of Athens, Athens, Greece

Objective: We quantified miR-34a levels in colonic tumors and non-cancerous tissue counterparts, to examine their prognostic potential and putative clinical application. Methods: Total RNA was extracted from 113 malignant colonic tissue specimens and 61 paired non-cancerous mucosa resected from colon cancer patients. 2 μg of total RNA were polyadenylated using poly(A)-polymerase and reversely transcribed into cDNA using MMLV. A novel real-time PCR methodology, based on SYBR Green chemistry, was developed and used for the quantification of miR-34a levels, followed by advanced biostatistical analysis. Data and results: miR-34a levels in colonic tumors ranged between 1.88 and 105.26 a.u. (MIR34A copies / 1,000 RNU48 copies) with a meanSE of 29.512.23. Comparison of miR-34a levels in paired (cancerous vs. non-cancerous) specimens did not reveal any significant differences. miR-34a seems to constitute an unfavorable predictor of disease-free (DFS) and overall survival (OS), predicting poor outcome in colon cancer patients (P=0.015 and P=0.019, respectively), independently from established clinicopathological tumor features. Conclusion: miR-34, which is predicted to target KLK13, can be considered as a novel biomarker predicting poor DFS and OS of colon cancer patients, independently from tumor invasion, nodal status, presence of distant metastases, tumor size and histological grade.

Acknowledgements: This work has been supported by the Commission of the European Community through the INsPiRE project (EU-FP7-REGPOT-2011-1, Proposal no: 284460).

5- miR-96: a novel molecular tissue biomarker predicting unfavorable outcome in colorectal cancer patients

S.M. Rapti1, C.K. Kontos1, S. Christodoulou2, I.N. Papadopoulos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece; 2Fourth Surgery Department, University General Hospital “Attikon”, University of Athens, Athens, Greece

Objective: We analyzed the levels of miR-96 in colorectal cancer specimens and paired non-cancerous mucosa, in order to examine the prognostic value and potential clinical application of this miRNA as a novel molecular biomarker in colorectal cancer. Methods: Total RNA was extracted from 116 primary tumors and 61 paired non-cancerous mucosa of colorectal cancer patients. 2 μg of total RNA were polyadenylated using poly(A)-polymerase and then cDNA was prepared by reverse transcription using an oligo-dT-adaptor primer. A highly sensitive real-time PCR methodology, based on SYBR Green chemistry, was developed for miRNA quantification and expression analysis of miR-96 was performed. Data and results: miR-96 was significantly upregulated in colorectal tumors, compared to non-cancerous paired mucosa (P<0.001). miR-96 overexpression is associated with positive nodal status and advanced TNM stage (P=0.044 and P=0.029, respectively), and is likely to constitute an unfavorable predictor of overall survival (OS), predicting poor OS independently from tumor invasion, nodal status, presence of distant metastases, tumor size and histological grade (P=0.038). Its overexpression can also predict an increased risk for relapse (P=0.041). Conclusion: miR-96, which is predicted to target KLK10, can be considered as a novel molecular biomarker predicting poor DFS and OS of colorectal cancer patients.

Acknowledgements: This research was partially funded by the University of Athens, Special Account of Research Grants, no 10812.

Mavridis, Konstantinos

MicroRNA 224: a KLK-targeting miRNA with dregulated expression and promising prognostic value in prostate cancer

K. Mavridis1, K. Stravodimos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Panepistimiopolis, 15701, Athens, Greece; 2First Urology Department, Medical School, University of Athens, “Laiko” General Hospital, 17 Ag Thoma St., 11527, Athens, Greece

Hsa-miR-224-5p (miR-224) is a tumor-related miRNA, which targets the expression of several Kallikrein (KLK) genes. The present study aims to analyze miR-224 expression in prostate cancer (CaP) cancer and to evaluate its prognostic significance. Snap-frozen tissue samples were obtained from a well-characterized cohort of CaP and benign prostatic hyperplasia (BPH) patients. MiR-224 expression levels were measured using qPCR after the isolation, polyadenylation and reverse transcription of total RNA via a poly(T) adapter. MiR-224 was found to be downregulated in CaP compared to BPH patients (p<0.001). The expression levels of miR-224 were also gradually decreased in advanced pathological stage and high Gleason score CaP tumors (all p values <0.05). MiR-224 expression was associated with improved biochemical progression-free survival of CaP patients (HR=0.314, p=0.013). Our data reveal that the kallikrein-targeting miR-224 can be viewed as a potential prognostic biomarker for prostate cancer patients.

Acknowledgements: This research has been co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)-Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. (UoA -BIOPROMO, MIS 377046).

Meinander, Kristian

Hydrocarbon isosteres of disulfide bridges in peptides that stimulate the proteolytic activity of KLK3

Kristian Meinandera, Janne Weisellb, Miikka Pakkalab, Roope Kallionpa, Can Hekimc, Ulf-Hkan Stenmanc, Hannu Koistinenc, Jouko Vepslinenb, Ale Nrvnenb, Kristina Luthmand, and Erik A.A. Wallna

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bSchool of Pharmacy, University of Eastern Finland, Kuopio, Finland; cDepartment of Clinical Chemistry, University of Helsinki, Finland; dMedicinal Chemistry, Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden

Stimulation of the enzymatic activity of kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) may be beneficial for patients with prostate cancer due to the antiangiogenic activity of KLK3. So far, only a few peptides have been reported to stimulate KLK3. The most potent ones are denoted C4 and B2, both of which increase the proteolytic activity of KLK3 several-fold at micromolar concentrations. However, no small molecule compounds with comparable activity have been reported.

The in vivo use of natural peptides is highly challenging. With this in mind we set out to construct synthetic protocols for replacing both internal and terminal disulfide bridges with more stable hydrocarbon isosteres. We have synthesized several pseudopeptide analogs based on both the C4 and B2 peptides. All of shese pseudopeptides show a significant stimulating effect on KLK3, the most potent ones shoving a more than two-fold activation at a concentration of 20 μg/ml (13-14 μM).

Oliva, Maria Luiza

In vitro and in vivo studies of the plant kallikrein inhibitor as an antithrombotic agent

Marlon V. Brito1, Cleide de Oliveira1, Bruno R. Salu1, Sonia Andrade Chudzinski2, Paula M. Diniz1, Ana C. Sato1, Cristina P. Vicente3, Misako U. Sampaio1, Francisco H. A. Maffei4 and Maria Luiza V. Oliva1

1Departamento de Bioqumica, Universidade Federal de Sao Paulo, Sao Paulo, SP, Brazil; 2Laboratrio de Bioqumica e Biofsica, Instituto Butantan, Sao Paulo, SP, Brazil; 3Universidade Estadual de Campinas, Instituto de Biologia, Campinas, SP, Brazil; 4Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu, Botucatu, SP, Brazil

Bauhinia bauhinioides kallikrein inhibitor (BbKI), a plant Kunitz-type serine peptidase inhibitor, shows specific action impairing the viability of some tumor cells. The potent inhibitory activity of BbKI against human and rat plasma kallikrein (Kiapp 2.6 nM and 5.4 nM, respectively) may be responsible for its efficacy in prolongation of activated partial thromboplastin time (aPTT) of rat and human plasma. As the association between cancer and thrombosis is well established, and studies indicate that the increased risk of tumor in patients with have thromboembolic events may be due to the activation of the coagulation system by tumor cells, or by therapeutic strategies. The antithrombotic action of BbKI, in a model of venous and arterial thrombosis, was studied in Wistar rats and Black 6 C57 mice, respectively. BbKI reduced the thrombus weight of the rat by 65% in comparison to that of NaCl group in a concentration of 5.5 μM. In model arterial thrombosis photochemically induced, in mouse carotid artery, in the group treated with BbKI prior to the thrombus induction the time of total occlusion (87.27 14.94 min) was prolonged in relation to that of the control group (51.97 10.52 min). These data indicate the BbKI in preventing thrombosis.

Financial support: FAPESP, CNPq, CAPES.

Pampalakis, Georgios

Generation of the Klk5-/-mouse to investigate the roles of KLK5 in skin

Georgios Pampalakis1#, Laetitia Furio2,3,4#, Georgia Sotiropoulou1*, Alain Hovnanian2,3,4*

1Department of Pharmacy, University of Patras, Rion-Patras, 26500, Greece; 2University Paris Descartes Sorbonne Paris Cit, Imagine Institute, 3INSERM U781, and 4Department of Genetics, Necker Hospital, Paris, France. #, equal contributions

Current in vitro studies suggest that KLK5/Klk5 plays a central executor role in the regulation of the epidermal desquamation proteolytic cascade, mainly due to its activity to autoactivate. Also, the aberrant activity of KLK5/Klk5 has been correlated with skin inflammation due to the generation of pro-inflammatory peptides (i.e. LL-37) derived from abnormal proteolytic processing of cathelicidin. Recently, it was shown that in Netherton syndrome KLK5 drives the inflammatory manifestations through Par2 signaling. In order to investigate in vivo the importance of KLK5 in skin homeostasis and to answer the question “what will be the effects of Klk5 inactivation on normal desquamation?” we have generated, for the first time, the Klk5 knockout mouse. The inactivation cassette was inserted into the first intron separating the first and the second coding exons of the Klk5 gene. Heterozygous Klk5+/- mice were crossed to derive Klk5-/- mice, which appeared macroscopically normal, indistinguishable from their wild-type controls (C57BL/6 background) and fertile. A detailed description of the Klk5-/- phenotype will be presented.

Radulovic, Maja

Critical role for protease activated receptors in kallikrein 6-mediated astrogliosis

Maja Radulovic1, Hyesook Yoon1,2, Jianmin Wu2, Sachiko I. Blaber3, Michael Blaber3, Isobel A. Scarisbrick1,2,4

1Neurobiology of Disease Program; 2Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, MN; 3Department of Biomedical Science, Florida State University, Tallahassee, FL; 4Department of Neurology, Mayo Clinic, Rochester, MN

A remarkable feature of the expression of kallikrein 6 (KLK6) in the central nervous system (CNS) is its abundance in oligodendroglia and neurons, but relative lack of expression in astrocytes. We have shown in cases of CNS injury however, that KLK6 is induced in reactive astrocytes and exhibits prolonged expression in astroglial scar tissue in cases of spinal cord injury, multiple sclerosis and glioblastoma multiforme. In this study, we address the hypothesis that KLK6 plays a role in the promotion of astrogliosis by selective activation of protease activated receptors (PARs). We demonstrate that KLK6 promotes rapid transformation of primary murine astrocytes from an epithelioid to a stellate morphology in vitro by a mechanism that depends on the presence of PAR1 or PAR2. In addition, KLK6-induced secretion of interleukin 6 and ERK1/2 signaling were also shown to be dependent on activation of PAR1 or PAR2. Together, these results define fundamental roles for the KLK6-PAR signaling axis in the promotion of astrogliosis. These studies also position KLK6 and PARs as novel therapeutic targets to prevent astrogliosis and promote neural recovery in cases of CNS injury and disease.

Supported by NIH R01 NS052741, NMSS PP2009 and CA1060A11.

Rotondo, Fabio

Immunoexpression of human kallikrein 10 in human pituitary adenomas

F. Rotondo1, A. Di Ieva2, K. Kovacs1, M.D. Cusimano2, G. Yousef1

1Department of Laboratory Medicine, Division of Pathology and 2Division of Neurosurgery, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada

Human kallikrein 10 (hK10), a serine protease, plays an important role in the regulation of cell proliferation and tumor growth. In the present work, we investigated hK10 immunoexpression in various types of surgically removed human pituitary adenomas. Specimens were fixed in formalin, embedded in paraffin. Staining was performed by the streptavidin-biotin-peroxidase complex protocol using the LSAB+ Kit (DAKO, Carpenteria, CA) and an hK10-specific rabbit polyclonal antibody (1:150). Results showed that 6 lactotroph adenomas removed from patients not receiving dopamine agonist medication were strongly immunopositive for hK10. Immunopositivity was localized in the cytoplasm and was clearly visible in many adenoma cells. In 44 other adenomas (somatotroph, gonadotroph, subtype 1, subtype 3, oncocytic and null cell adenomas), immunopositivity was very mild, seen only in few unevenly distributed adenoma cells. Previous studies have shown immunopositivity in nontumourous human lactotrophs and corticotrophs (Petraki el at, J Histochem Cytochem, 2002). Since others have shown that hK10 possesses tumor suppressing properties and is decreased in rapidly growing tumor cells could explore hK10 immunoexpression in lactotroph adenomas exposed to dopamine agonist therapy which inhibits tumor growth.

This work was supported by the generous donations from the Jarislowsky and Lloyd Carr-Harris Foundations.

Samaan, Sara

A miRNA-kallikrein network that affects prostate cancer progression

Sara Samaan1, Zsuzsanna Lichner1, Qiang Ding1, Carol Saleh1, Joseph Samuel1, George M Yousef1,2

1Departmentof Laboratory Medicine and the Keenan Research Centre in the Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Toronto, Canada, M5B 1W8; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada, M5S 1A8

miRNAs are short RNA nucleotides which negatively control the expression of their targets. Recent work shows that miRNAs are dysregulated in prostate cancer. Kallikreins (KLKs) are genes that encode serine proteases and have been found to be useful cancer biomarkers. The overall objective of this study is to investigate the interaction of miRNAs and KLKs in prostate cancer pathogenesis. We analyzed miRNAs that are predicted to target 5 KLKs implicated in prostate cancer pathogenesis: KLK1, KLK2, KLK3, KLK4, and KLK15. We identified 23 miRNAs that are dysregulated between high risk and low risk biochemical failure patients and are predicted to target these kallikreins. This demonstrated that kallikreins as potential downstream effector molecules through which miRNAs can affect tumor progression. Furthermore, we identified 14 significantly dysregulated miRNAs between different Gleason grades that are predicted to target the prostate cancer-related kallikreins. A polymorphic minisatellite repeat sequence was then identified in the kallikrein locus between KLK4 and KLK14. This minisatellite sequence consists of a number of microsatellite repeat sequences, which are shared by a number of prostate-cancer related genes on Chromosome 19 and are predicted miRNA targets. Based on gene target prediction analysis, miR-422a was selected to be transfected into LNCaP prostate cancer cells to measure its effect on the expression levels of KLK4, KLK14, and other targeted genes. Our results show the presence of a miRNA-KLK network in prostate cancer progression, which may have therapeutic implications.

Scarisbrick, Isobel

Mechanism of kallikrein 6-mediated oligodendrogliopathy

Joshua E. Burda1, Maja Radulovic1, Hyesook Yoon1,2, Jianmin Wu2, Sachiko I. Blaber3, Michael Blaber3, Isobel A. Scarisbrick1,2,4

1Neurobiology of Disease Program; 2Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, MN; 3Department of Biomedical Science, Florida State University, Tallahassee, FL; 4Department of Neurology, Mayo Clinic, Rochester, MN

Kallikrein 6 (KLK6) is among the most densely expressed serine proteases in the adult central nervous system (CNS), yet little is known regarding its physiological roles. In prior studies we have shown that KLK6 is preferentially expressed by the myelin producing oligodendroglia. Moreover, levels of KLK6 are elevated at sites of CNS injury and disease, including actively demyelinating lesions in multiple sclerosis patients, as well as at sites of injury following spinal cord trauma and in stroke. Taken with studies demonstrating that myelin proteins are rapidly hydrolyzed by KLK6, we hypothesized that this secreted serine protease plays key roles in oligodendrocyte biology and pathophysiology. Here we demonstrate that KLK6 is a key regulator of oligodendrocyte process outgrowth and myelin gene expression. In addition, elevated levels of KLK6 promoted rapid oligodendrogliopathy in the case of primary murine oligodendroglia in vitro or after microinjection into the spinal cord white matter of adult C57BL6 mice. Importantly, KLK6-mediated oligotoxic effects were blocked or significantly reduced in cells and tissues derived from PAR1 genetically deficient mice. These studies suggest that the KLK6-PAR1 signaling axis plays critical roles in oligodendrocyte differentiation and pathobiology and may represent a target for the development of myelin regeneration therapies in cases of CNS white matter injury or disease.

Supported by NIH R01 NS052741, NMSS PP2009 and CA1060A11.

Scorilas, Andreas

1- Downregulation of the KLK2- & KLK4-targeting miR-378a predicts the short-term relapse of prostate cancer patients

M. Avgeris1, K. Mavridis1, K. Stravodimos2, A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Panepistimiopolis, 15701, Athens, Greece, 2First Urology Department, Medical School, University of Athens,Laiko General Hospital, 17 Ag Thoma St., 11527, Athens, Greece

The deregulation of several miRNAs in prostate cancer (PCa) provides a novel pool of candidate tumor markers. Using in silico approaches, we have identified hsa-miR-378a-3p (miR-378a) as a potential post-transcriptional regulator of KLK2 and KLK4. The aim of the study is the evaluation of the clinical significance of miR-378a for the prediction of PCa patients’ outcome. Prostate tissue specimens obtained from 73 PCa and 64 BPH patients. Following extraction, total RNA was polyadenylated and reverse transcribed using a poly(T) adapter. A SYBR-Green based qPCR assay was applied thereafter for the quantification of miR-378a levels. Although miR-378a levels were not significantly altered between PCa and BPH, a statistically strong downregulation of miR-378a was observed in higher Gleason score (p=0.018) and larger diameter (rs=-0.402; p=0.034) tumors, as well as in patients with elevated PSA serum levels (rs=-0.365; p<0.001). Moreover, Kaplan-Meier survival curves pointed out the significantly shorter disease-free survival (DFS) of the PCa patients with reduced miR-378a levels (p=0.034). Finally, in high-recurrence risk patients, the reduced miR-378a levels revealed to be an independent predictor of poor DFS (p=0.030). Our data highlight that miR-378a loss increases the risk for short-term relapse of the PCa patients.

Acknowledgements: This research has been co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)-Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. (UoA -BIOPROMO, MIS 377046).

2- KLK8 mRNA expression analysis in breast cancer: downregulation in cancerous tissues and in metastatic disease

K. Michaelidou1, A. Kladi-Skandali1, C. Haritos3, S. Stamatopoulou2, I. Misitzis3, A. Ardavanis2 and A. Scorilas1

1Department of Biochemistry and Molecular Biology, University of Athens, Greece; 2First Department of Medical Oncology and 3The Breast Clinic,Saint Savvas, Anticancer Hospital, Athens, Greece

KLK8, like other members of the KLK family, may prove to be a promising clinical tool for breast cancer (BC). The objective of this study was the quantification of KLK8 mRNA expression in BC tissues, and the initial determination of its clinical utility as a potential BC biomarker. Therefore, total RNA was isolated from 50 breast tumors and their matched normal adjacent tissues, cDNA was prepared and KLK8 mRNA expression analysis was performed by comparative Ct method. The mRNA levels of KLK8 were found to be significantly downregulated in BC specimens compared to non-cancerous counterparts (p=0.002). Furthermore, patients with metastatic BC showed significantly lower KLK8 expression compared to those with organ confined tumors (p=0.031). Moreover, KLK8 mRNA levels were found to be significantly increased in HER2-positive compared to HER2-negative BC (p=0.017). Additionally, a negative correlation was observed between KLK8 and PgR expression levels (rs= -0.352; p=0.048). On the contrary, no significant relationship was observed between KLK8 mRNA levels and histological tumor grade, TNM stage, or ER status. Nevertheless, our preliminary analysis, which will be extended in a larger patients’ cohort, proposes a possible tumor suppressor role of KLK8 in BC and demonstrate its biomarker potential for this malignancy.

Acknowledgements: This work has been financially supported by the Hellenic Society of Medical Oncology. This research was also partially funded by the University of Athens Special Account of Research Grants no 10812.

Scott, Erika

Mining databases for SNPs in the kallikrein genes

E. Scott*, N. Sukumar*, A. Konvalinka, E. P. Diamandis

Department of Laboratory Medicine and Pathobiology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario M5T 3L9. *equal contributions

The kallikrein (KLK) proteins are a group of 15 serine proteases encoded sequentially by the KLK locus on chromosome 19. Various single nucleotide polymorphisms (SNPs) found within the KLK locus have been identified and shown to be associated with many forms of cancer. The purpose of this study was to mine databases to update the KLK SNP information and to discover novel, potentially pathogenic SNPs. Two databases, Exome Variant Server and Ensembl, were mined for SNPs found in each of the KLK genes and the information was combined, analyzed, and compared to previous studies. As part of the analysis, the PolyPhen algorithm was used to predict damaging SNPs. Many of these SNPs were plotted on both the primary protein sequence and the structure of each KLK. The total number of SNPs obtained in this study, 23 656, far exceeded the total number of SNPs described in previous studies. Specifically, we found 1741 non-synonymous SNPs while previous studies reported much fewer. Furthermore, 35 nonsense SNPs were present in all 15 KLKs, many of which were in KLK13. In conclusion, we have greatly expanded the knowledge of SNPs in the KLK genes. Nonsense SNPs are particularly useful because natural KLK knockouts can be studied for association with certain forms of cancer. In addition, intronic and intergenic SNPs need to be more closely examined to determine possible associations with disease due to their effect on regulation. Our study represents an expansive mine of information that is valuable to both scientists and clinicians studying KLKs and KLK-related diseases.

Shaninian, Jasmin (Hasmik)

Secretome and degradome profiling shows that KLK 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells

H. Shahinian1, D. Loessner2, M. L. Biniossek3, J. A. Clements2, V. Magdolen4, O. Schilling3,5

1University Heart Center Freiburg-Bad Krozingen, Germany; 2School of Biomedical Sciences, Queensland University of Technology, Australia; 3Institute of Molecular Medicine and Cell Research, Univ. Freiburg, Germany; 4Dept. of Obstetrics and Gynecology, Technical University of Munich, Germany; 5BIOSS Centre for Biological Signaling Studies, Univ. Freiburg, Germany

Kallikrein-related peptidases, in particular KLK4, 5, 6 and 7 (4-7), often have elevated expression levels in ovarian cancer. In OV-MZ-6 ovarian cancer cells, combined expression of KLK4-7 reduces cell adhesion and increases cell invasion and resistance to paclitaxel. We investigated how KLK4-7 shapes the secreted proteome (“secretome”) and proteolytic profile (“degradome”) of ovarian cancer cells. The secretome comparison consistently identified > 900 proteins in three replicate analyses. Expression of KLK4-7 predominantly affected the abundance of proteins involved in cell-cell communication, including increased levels of transforming growth factor β-1 (TGFβ-1). KLK4-7 co-transfected OV-MZ-6 cells share prominent features of elevated TGFβ-1 signaling, including increased abundance neural cell adhesion molecule L1 (L1CAM). Augmented levels of TGFβ-1 and L1CAM upon expression of KLK4-7 were corroborated in vivo by an ovarian cancer xenograft model. The degradomic analysis showed that KLK4-7 expression mostly affected cleavage sites C-terminal to arginine, corresponding to the preference of kallikreins 4, 5 and 6. Putative kallikrein substrates include chemokines, such as growth differentiation factor 15 (GDF 15) and macrophage migration inhibitory factor (MIF). Proteolytic maturation of TGFβ-1 was elevated. KLK4-7 have a pronounced, yet non-degrading impact on the secreted proteome, implying a novel link between these proteases and TGFβ-1 signaling in tumor biology.

Sotiropoulou, Georgia

1- Insights into the molecular pathways underlying the dual roles of KLK6 in breast cancer

Georgia Sotiropoulou1*, Georgios Pampalakis1, Chris Smith2, Olga Papadodima3, Aristotelis Chatziioannou3, Vassileios Zoumpourlis3, Eleftherios P. Diamandis2

1University of Patras, School of Health Sciences, Department of Pharmacy, Rion-Patras, Greece; 2Mount Sinai Hospital, Department of Pathology & Laboratory Medicine, Toronto, ON, Canada; 3National Hellenic Research Foundation, Athens, Greece

Previously, we suggested that KLK6 is an epigenetically silenced tumor suppressor in human breast cancer and that inhibition of tumor growth by KLK6 is observed only when KLK6 is expressed at physiological concentrations (Pampalakis et al. Cancer Res 2009). Namely, KLK6 re-expression at physiological levels in KLK6-negative MDA-MB-231 breast cancer cells resulted in remarkable inhibition of their tumorigenicity in vivo and in vitro. Aberrant overexpression of KLK6 at levels by >50-100 fold higher than normal mammary epithelial cells or mammary tissues (and comparable to those observed in a small subgroup of breast cancers) is associated with hyperproliferation of MDA-MB-231 and very efficient tumor formation in SCID mice. Here, we aimed to shed light to the pathways underlying the concentration-dependent effects of KLK6 in breast cancer by integrating phenotypic assays with transcriptomic profiling (Illumina platform) and HT proteomics. For proteomic profiling, cell lysates were fractionated by SDS-PAGE, gel bands were cut, in gel digested with trypsin, and the resulting peptides were analysed by LC-ESI-MS/MS. The profiles of MDA-MB-231 parental cells were compared in parallel with three different MDA-MB-231 clones stably transfected to synthesize: (a) wild-type KLK6 at normal levels (C28WT), (b) wild-type KLK6 at abnormally increased levels (C5WT), and (c) a mutant form of KLK6 carrying a point substitution at the active site Ser that completely inhibits its protease activity (C22MS). We identified a total of 3,779 proteins of which 2,487 were represented by ≥2 MS/MS peptides and distributed as following: 1,776 in parental, 2,020 in C28WT, 1,228 in C5WT, and 1,593 in C22MS. Ingenuity Pathway Analysis (IPA) was used to construct potential networks. We identified novel pathways that are altered upon reactivated expression of KLK6. These involved the transcription factor NF-kB as well as genes/proteins with established roles in apoptosis. For example, expression of the apoptosis-related proteins CASP8 and BCL10 is associated with physiological KLK6 levels but they were found inactivated in KLK6 overexpressors. Based on gene expression signatures and differential protein profiles we suggest putative pathways to explain the dual roles of KLK6 in breast cancer.

2- Development and characterization of a new polyclonal specific for mouse Klk6

Lenitsa Zingou, Georgios Pampalakis, Georgia Sotiropoulou

Department of Pharmacy, University of Patras, Rion-Patras 26500, Greece

The kallikrein-related peptidase 6 (KLK6 for human; Klk6 for mouse) has been implicated in neurodegenerative diseases including multiple sclerosis and Parkinson’s disease and in various types of cancer. Therefore, the investigation of the role(s) of Klk6 in mouse models used for the study of the aforementioned human diseases becomes a necessity. In this sense, the availability of new anti-KLK6 antibodies is of great importance. Recently, we developed a highly specific IgYs polyclonal antibody against human KLK6 [Sotiropoulou G et al. Chem Cent J 2012; Licensed to Merck/Millipore: Cat#: ABN357 Anti-Neurosin (KLK6)]. Nonetheless, no antibody is currently available that is highly specific for mouse Klk6. Here, we generated a novel rabbit anti-peptide polyclonal for mouse Klk6. Peptides corresponding to two non-overlapping Klk6 sequences of high hydrophilicity and predicted antigenicity were synthesized, conjugated to keyhole limpet hemocyanin (KLH) and used for immunization. Immune antiserum was purified on affinity columns on which we immobilized the peptides used for immunization. The specificity and affinity of the purified polyclonal was assessed against active recombinant mouse Klk6 protein, which we have produced in Pichia pastoris KM71, and the detection limit was determined. Further, we tested the crossreactivity of our antibody against recombinant and endogenous KLK6. Finally, we tested whether the purified α-Klk6 polyclonal recognizes the endogenous Klk6 protein with high sensitivity/specificity in mouse tissues and cell lines. Using the generated polyclonal, we analyzed the expression profile of Klk6 in an established in vitro model of mouse skin carcinogenesis.

Srinivasan, Srilakshmi

Functional validation of a KLK3 variant, rs17632542, associated with prostate cancer risk

S. Srinivasan1; C. Stephens1; A. Spurdle2; J. Clements1; J. Batra1, 2

1Australian Prostate Cancer Research Centre-Queensland and Institute of Health and Biomedical Innovation, Queensland University of Technology at the Translational Research Institute, Brisbane, Queensland, Australia – 4102; 2Molecular Cancer Epidemiology Laboratory, Genetics and Population Health Division, Queensland Institute of Medical Research, Brisbane, Queensland, Australia – 4006

Background and Aims: KLK3 or PSA is the current biomarker for prostate cancer (PrCa) and has a significant role in the proteolytic cascades involved in seminal clot dissolution and prostate tumor biology. Fine-mapping studies identified a missense single nucleotide polymorphism (SNP) rs17632542 (Ile178Thr) in exon-4 of KLK3 to be associated with PrCa risk. We aim to delineate the molecular consequences of this missense rs17632542 SNP. Methods:In-silico tools were employed to analyse the effect of the SNP on splicing, protein stability and glycosylation. Mini-gene assays verifying the differential splicing effect for the SNP were performed in LNCaP and PC3 cell lines. Recombinant KLK3 (261 aa) protein harbouring the variant (Thr) and wild-type (Ile) at amino-acid position 179, were made in insect cell lines. Protein stability analysis by differential scanning fluorimetry (DSF) and substrate activity assays using a fluorescent peptide were performed. Results and Conclusions:In-silico analysis for the rs17632542 SNP suggests an alteration in a KLK3 splice site, KLK3 protein stability and glycosylation for the risk allele. Mini-gene assay herein provide evidence for differential allele specific splicing. DSF and activity assays using peptide substrates suggested the SNP affects the stability and activity of the KLK3 protease. These results indicate the missense rs17632542 SNP to have biological effect on the expression and/or function of the KLK3 protein which has role in PrCa prognosis.

Talieri, Maroulio

1- Immunohistochemical localisation and clinical evaluation of kallikrein-related peptidase 14 (KLK14) in colon cancer

M. Devetzi1, G. Papachristopoulou2,3, V. Petrou2, N. Arnogiannaki2, A. Scorilas3, D. Kypraios4, D. Xynopoulos4, M. Talieri1

1Department of Cellular Physiology, G. Papanicolaou Research Center of Oncology, Saint Savvas Oncologic Hospital, Athens 11522, Greece; 2Department of Pathology Saint Savvas Oncologic Hospital, Athens 11522, Greece; 3Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens 15701, Greece; 4Department of Gastroenterology, Saint Savvas Oncologic Hospital, Athens 11522, Greece

Human kallikrein-related peptidase 14 (KLK14) is a steroid hormone-regulated gene encoding for a trypsin-like protease, expressed mainly in the central nervous system and endocrine tissues. KLK14 has been reported to have possible utility as tumor biomarker for various types of cancer. In this study, the KLK14 antigen expression was assessed in 75 colon cancer tissues, using the indirect immunoperoxidase method for immunostaining. Statistical analysis evaluated the results. KLK14 is intensely expressed on the epithelium of colon tissue either carcinoma or adenoma, but not on the normal colon tissue. KLK14 is mainly localised on epithelial cells and less on the stroma. KLK14 demonstrated cytoplasmic staining, seen on the colon lumen and mucus. Neoplastic cells show cytoplasmic staining on the lumen surface. Colon cancer shows granular cytoplasmic staining, whereas colon adenomas show a cytoplasmic homogeneous staining which is present only on the lumen surface. Less differentiated tumors of more advanced stage showed more intense KLK14 immunostaining. Follow-up analysis revealed that KLK14 was significantly associated with shorter overall survival (p=0.02) and disease-free survival (p=0.01). Present data suggest that KLK14 protein is up-regulated in colon cancer and its expression predicts poor prognosis for colon cancer patients.

2- The expression of kallikrein-related peptidase 5 (KLK5) in colon cancer and its clinical relevance

G. Papachristopoulou1,3, A. Malachias2, N. Arnogiannaki1, A. Scorilas3, M. Devetzi4, D. Xynopoulos2, M. Talieri4

1Department of Pathology, Saint Savvas Oncologic Hospital, Athens 11522, Greece; 2Department of Gastroenterology, Saint Savvas Oncologic Hospital, Athens 11522, Greece; 3Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens 15701, Greece; 4Department of Cellular Physiology, G. Papanicolaou Research Center of Oncology, Saint Savvas Oncologic Hospital, Athens 11522, Greece

Most members of the Kallikrein-related peptidase family have been extensively studied as potential biomarkers for several carcinomas and other diseases. KLK5 (HSCTE) was originally identified from a keratinocyte library, and its enzyme was purified from stratum corneum of human skin. KLK5 has been shown to be differentially expressed in a variety of endocrine tumors, but is not as yet examined in colon cancer. In this study, we assessed the expression status of KLK5 in 170 colon tissues as well as in 70 adenomas and their paired normal colonic mucosa, by real time qPCR using TaqMan probes. It was shown that KLK5 positive expression is associated with patients nodal status (p=0.022) and with tumor histological grade (p=0.033). Cox univariate analysis revealed that KLK5 positive expression is associated with disease-free survival (p=0.028) and overall survival of patients (p=0.048). Kaplan-Meyer survival models showed that patients with positive KLK5 have lower disease-free survival (p=0.009) and overall survival (p=0.019). ROC analysis demonstrated that KLK5 expression had significant discriminatory value between cancer and adenoma tissues (AUC 0.77; 95% CI=0.69-0.85, p=0.03). Present data suggest that KLK5 gene expression has discriminatory power (p<0.001) between colon cancer and adenoma tissue. KLK5 gene expression predicts poor prognosis for colon cancer patients.

3- Expression and clinical evaluation of kallikrein-related protease 6 (KLK6) in colon cancer

A. Vakrakou1, G. Papachristopoulou2, M. Devetzi1, A. Scorilas3, N. Arnogiannaki2, M. Nikaki4, D. Xynopoulos4, M. Talieri1

1Department of Cellular Physiology, G. Papanicolaou Research Center of Oncology, Saint Savvas Oncologic Hospital, Athens 11522, Greece; 2Department of Pathology, Saint Savvas Oncologic Hospital, Athens 11522, Greece; 3Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece; 4Department of Gastroenterology, Saint Savvas Oncologic Hospital, Athens 11522, Greece

Human tissue kallikrein-related peptidase 6 (KLK6) belongs to the human Kallikrein family of genes and is a serine protease. It is deregulated in human malignancies and plays important role in cell growth regulation, angiogenesis, invasion and metastasis. A small clinical study suggests that KLK6 mRNA expression might correlate to poor prognosis of colon cancer. In this study we investigated the potential clinical significance of KLK6 gene in colon tissues. We assessed the expression status of KLK6 in 170 colon cancer tissues and 70 adenomas as well as in their paired normal colonic mucosa, by real time qPCR using TaqMan probes. Cancer specimens showed higher levels of KLK6 mRNA expression than non cancer tissues (p=0.01). ROC analysis demonstrated that KLK6 expression had significant discriminatory value between cancer and non cancer tissues (AUC 0.64; p=0.03). KLK6 mRNA expression is associated with TNM stage (p=0.01) and grade (p=0.002) of the tumour in a statistically significant manner by Mann-Whitney test, but not with the size of the tumour (p=0.24). Present data suggest that KLK6 gene expression has discriminatory power (p<0.001) between colon cancer and adenoma tissue. KLK6 gene expression predicts poor prognosis for colon cancer patients.

Veronez, Camila

Genetic profile of plasma prekallikrein in hereditary angioedema type 1 Brazilian patients

C.L. Veronez1, N. Cagini1, M. Buzolin2, L.A. Velloso3, E. Mansour3, J.B. Pesquero1

1Department of Biophysics, Federal University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil, CEP 04039-032; 2Department of Pediatrics, State University of Campinas, Campinas, Brazil; 3Department of Internal Medicine, State University of Campinas, Campinas, Brazil

Genetic C1-inhibitor (C1-INH) deficiency leads to hereditary angioedema (HAE) types I and II that result from unregulated plasma prekallikrein (PK) activation and overproduction of bradykinin. Since PK inhibition has been shown to be a reliable therapeutic alternative for HAE attacks, we aimed to analyze its genetic features in patients with diagnosis of HAE. Therefore the coding region and flanking regions of the PK gene of patients from 12 different Brazilian families were analyzed by sequencing. Clinical history, enzymatic assays and molecular analysis of C1-INH gene were considered in diagnosis. We found the missense alterations p.Ser143Asn and p.Ala178Thr, both lying in the protein domain A2 around high-molecular weight kininogen (HMWK) binding site; the mutation p.Ser381Ala affecting the last amino acid of A4, close to the disulfide bond linking the low and high chain and the mutation p.Arg560Gln in the low chain. In addition we also detected two small insertions in introns 1 and 7, one small deletion in intron 13 and one silent mutation in exon 15 and 23 base changes in intronic regions. This initial genetic characterization suggests a large number of alterations in PK gene that may influence protein interaction with factor XII, HMWK, C1-INH and other inhibitors, and can help to explain symptoms and drug response variability in HAE patients, mainly in treatments with PK inhibitors.

Wang, Xiaolu

Development of orally active bradykinin B1 antagonists

X. Wang, J. Madden, A. Parkes, D. Hallett, B. Ellenbroek, M. Gemkow, J. Kemp and A. Davenport

Evotec AG, Essener Bogen 7, 22419 Hamburg, Germany

The Bradykinin B1 and B2 receptors are GPCRs that play a role in pain and inflammation. The B2 receptor is constitutively expressed under normal physiological conditions and its activation in response to inflammatory or noxious insult is believed to account for acute pain responses. In contrast, the B1 receptor is not expressed in healthy tissue but is upregulated following tissue injury. Activation of B1 receptors is believed to play a significant role in the chronic phase of inflammatory pain responses. Therefore, identifying an orally active small molecule B1 antagonist has been the goal of a number of drug discovery companies. Evotec’s approach to finding small molecule B1 antagonists was to identify leads that, contrary to most B1 antagonists, were active at rat or mouse B1 receptors, thus enabling them to be profiled in rodent models of pain and inflammation. This has been achieved and more than one series identified with in vivo activity. This poster describes one such series, which was derived from an existing compound in the B1 receptor antagonist literature. The lead compound from this new series is orally bioavailable in rats, has excellent selectivity over other drug targets and lacks potential liabilities such as hERG channel blockade. Notably however, despite having a good overall profile and exposure in vivo, this B1 antagonist was inactive in a number of in vivo models. An explanation for the observed pharmacokinetic/ pharmacodynamic discrepancy would be the PGP liability of the compound.

White, Nicole

Kallikrein-related peptidase 6 in breast cancer: effect on miRNA expression and the MAPK pathway

N.M.A. White1,2, P. Boulos1, K.G. Sidiropoulos 1, G. Pampalakis3, A. Bui1, Q. Ding1, G. Sotiropoulou3, G.M. Yousef1,2

1Dept of Laboratory Medicine, St. Michael’s Hospital, Toronto, Canada, M5B 1W8; 2Dept of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada, M5S 1A8; 3Department of Pharmacy, University of Patras, 26500, Rion-Patras, Greece

Objective: This study aimed to examine the effect of KLK6 on miRNA expression and their downstream pathways in breast cancer cell line. Methods: We stably transfected the KLK6 negative breast cancer cell line MDA-MB-231 with KLK6 cDNA resulting in cells that highly overexpress KLK6. We preformed miRNAprofiling and compared KLK6 transfected vs. non-transfected cells. Results: We found 59 miRNAsupregulated and 72 downregulated inKLK6-expressing vs. control cells. Although the number of miRNAs dysregulated up or down was similar, the degree of dysregulation was very different. Upregulated miRNAsshowed an average increase of 2.16 (log fold change), while the average decrease was only -0.7. We hypothesized this may be due to changes in miRNA biogenesis and examined genes involved in miRNA processing. We found increased expression in Dicer, Drosha, Exportin-5, Ago1 and Ago2 in KLK6-expressing cells. We also examined the effect of increased KLK6 and miRNA expression on the MAPK pathway and found there was a general increase in MAPK related genes in cells with high KLK6. We correlated dysregulated gene expression with miRNAs and found 16 genes that were upregulated were potential targets of downregulatedmiRNAs. In particular, miR-34a was predicted to target 4 genes. We validated the miRNA-target interaction in our cell line model and found that upon miRNA transfection, there was a significant decrease in MAPK13, MAP2K1 and CDKN1C as well as the transcription factor SMAD4. Conclusions: These results suggest that KLK6 may contribute to the regulation of miRNAs, either directly or indirectly; and those same miRNAs can have an effect the MAPK pathway.

Yousef, George

Human tissue kallikrein-related peptidase 6 and 10 are highly expressed in early gastroesophageal junction adenocarcinoma

A. Grin1,2, M. Tripathi1,2, F. Rotondo1,2, M.N. Bassily3, G. M. Yousef1,2

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada; 2Department of Laboratory Medicine, and the Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael’s Hospital Toronto, Canada; 3Department of Community Medicine and Public Health, Menoufiya University, Egypt

Gastroesophageal junction (GEJ) adenocarcinoma and its precursor, Barrett’s esophagus (BE), are increasing in incidence. The diagnosis of dysplasia and early GEJ cancer can be challenging and while early tumors are generally associated with a good prognosis, some behave aggressively. Members of the kallikrein gene family represent potential diagnostic and prognostic biomarkers. The aim of this study was to investigate the expression of human tissue kallikrein-related peptidase 6 (KLK6) and human tissue kallikrein-related peptidase 10 (KLK10) by immunohistochemistry in early GEJ adenocarcinoma and in BE with and without dysplasia. KLK6 and KLK10 immunohistochemistry was performed on a series of 30 early GEJ adenocarcinomas and an expression score was given for the tumor and surrounding dysplastic and normal Barrett’s mucosa, if present. KLK6 and KLK10 showed significantly increased expression in tumor compared to both dysplastic (p=0.009; p=0.05) and non-dysplastic BE (p=0.0002; p=0.0001). Increased KLK6 staining intensity was seen in tumor at the invasive front (p=0.006). There was no significant correlation between KLK 6 or KLK10 and histopathologic markers of poor prognosis. KLK6 and KLK10 are highly co-expressed in early GEJ adenocarcinomas. KLK6 and KLK10 expression may be useful diagnostic markers in GEJ tumors and KLK6 may be a marker of disease aggressiveness and invasiveness.


*)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.


Published Online: 2014-5-10
Published in Print: 2014-7-1

©2014 by Walter de Gruyter Berlin/Boston