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Eleftherios P Diamandis - One of the best experts on this subject based on the ideXlab platform.
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an integrated cell line based discovery strategy identified follistatin and Kallikrein 6 as serum biomarker candidates of breast carcinoma
Journal of Proteomics, 2016Co-Authors: Alain Mange, Lampros Dimitrakopoulos, Antoninus Soosaipillai, Peter Coopman, Eleftherios P Diamandis, Jerome SolassolAbstract:Abstract Secreted proteins constitute a relevant source of putative cancer biomarkers. Here, we compared the secretome of a series of four genetically-related breast cancer cell lines as a model of aggressiveness using quantitative mass spectrometry. 537 proteins (59.5% of the total identified proteins) predicted to be released or shed from cells were identified. Using a scoring system based on i) iTRAQ value, ii) breast cancer tissue mRNA expression levels, and iii) immunohistochemical staining (public database), a short list of 10 candidate proteins was selected. Using specific ELISA assays, the expression level of the top five proteins was measured in a verification set of 56 patients. The four significantly differentially expressed proteins were then validated in a second independent set of 353 patients. Finally, follistatin (FST) and Kallikrein 6 (KLK6) in serum were significantly higher (p-value Biological significance Discovery of new serum biomarkers that exhibit increased sensitivity and specificity compared to current biomarkers appears to be an essential field of research in cancer. Most biological markers show insufficient diagnostic sensitivity for early breast cancer detection and, for the majority of them, their concentrations are elevated only in metastatic forms of the disease. It is therefore essential to identify clinically reliable biomarkers and develop effective approaches for cancer diagnosis. One promising approach in this field is the study of secreted proteins through proteomic analysis of in vitro progression breast cancer models. Here we have shown that FST and KLK6 are elevated in breast cancer patient serum compared to healthy controls. We expect that our discovery strategy will help to identify cancer-specific and body-fluid-accessible biomarkers.
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prognostic significance of multiple Kallikreins in high grade astrocytoma
BMC Cancer, 2015Co-Authors: Kristen L Drucker, Eleftherios P Diamandis, Caterina Gianinni, Paul A Decker, Isobel A. ScarisbrickAbstract:Background: Kallikreins have clinical value as prognostic markers in a subset of malignancies examined to date, including Kallikrein 3 (prostate specific antigen) in prostate cancer. We previously demonstrated that Kallikrein 6 is expressed at higher levels in grade IV compared to grade III astrocytoma and is associated with reduced survival of GBM patients. Methods: In this study we determined KLK1, KLK6, KLK7, KLK8, KLK9 and KLK10 protein expression in two independent tissue microarrays containing 60 grade IV and 8 grade III astrocytoma samples. Scores for staining intensity, percent of tumor stained and immunoreactivity scores (IR, product of intensity and percent) were determined and analyzed for correlation with patient survival. Results: Grade IV glioma was associated with higher levels of Kallikrein-immunostaining compared to grade III specimens. Univariable Cox proportional hazards regression analysis demonstrated that elevated KLK6- or KLK7-IR was associated with poor patient prognosis. In addition, an increased percent of tumor immunoreactive for KLK6 or KLK9 was associated with decreased survival in grade IV patients. Kaplan-Meier survival analysis indicated that patients with KLK6-IR < 10, KLK6 percent tumor core stained < 3, or KLK7-IR < 9 had a significantly improved survival. Multivariable analysis indicated that the significance of these parameters was maintained even after adjusting for gender and performance score. Conclusions: These data suggest that elevations in glioblastoma KLK6, KLK7 and KLK9 protein have utility as prognostic markers of patient survival.
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separation of Kallikrein 6 glycoprotein subpopulations in biological fluids by anion exchange chromatography coupled to elisa and identification by mass spectrometry
Proteomics, 2012Co-Authors: Antoninus Soosaipillai, Eleftherios P Diamandis, Uros Kuzmanov, Christopher R Smith, Ihor Batruch, Anastasia DiamandisAbstract:Kallikrein 6 (KLK6) has been shown to be aberrantly glycosylated in ovarian cancer. Here, we report a novel HPLC anion exchange method, coupled to a KLK6-specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids. Biological fluids were fractionated using anion exchange and resulting fractions were analyzed for KLK6 content by ELISA producing a four-peak elution profile. Using this assay, the KLK6 elution profile and distribution across peaks of a set (n = 7) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and cerebrospinal fluid (CSF) of normal individuals (n = 7). Glycosylation patterns of recombinant KLK6 (rKLK6) were characterized using tandem mass spectrometry (MS/MS), and found to consist of a highly heterogeneous KLK6 population. This protein was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. The rKLK6 glycoform composition of each peak was assessed by lectin affinity and MS/MS based glycopeptide quantification by product ion monitoring. The combined results showed an increase in terminal alpha 2-6 linked sialic acid in the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals.
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combinatorial peptide libraries facilitate development of multiple reaction monitoring assays for low abundance proteins
Journal of Proteome Research, 2010Co-Authors: Andrei P Drabovich, Eleftherios P DiamandisAbstract:Low-abundance proteins present in biological fluids are often considered an attractive source of new disease biomarkers. Since such proteins are poorly observed in proteome-scale discovery experiments due to an overwhelming mass of high-abundance proteins, the development of quantitative multiple reaction monitoring (MRM) assays for low-abundance proteins is a challenging task. Here, we present a strategy that facilitates the development of MRM assays for large numbers of unpurified lowabundance proteins. Our discovery strategy is based on the reduction of the dynamic range of protein concentrations in biological fluids by means of one-bead one-compound combinatorial peptide libraries (CPL). Our 2D-LC-MS/MS approach allowed us to identify a total of 484 unique proteins in ovarian cancer ascites, and 216 proteins were assigned as low-abundance ones. Interestingly, 74 of those proteins have never been previously described in ascites fluid. Treatment with CPL allowed identification of a significantly higher number of unique peptides for low-abundance proteins and provided important empirical fragmentation information for development of MRM assays. Finally, we confirmed that MRM assays worked for 30 low-abundance proteins in the unfractionated ascites digest. Using a multiplexed MRM method, relative amounts of five proteins (Kallikrein 6, metalloproteinase inhibitor 1, macrophage migration inhibitory factor, follistatin-related protein, and mesothelin) were determined in a set of ovarian cancer ascites. Multiplexed MRM assays targeting large numbers of proteins can be used to develop comprehensive panels of biomarkers with high sensitivity and selectivity, and to study complex protein networks.
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prognostic impact of cd68 and Kallikrein 6 in human glioma
Anticancer Research, 2009Co-Authors: Tadej Strojnik, Eleftherios P Diamandis, Rajko Kavalar, Irena Zajc, Katerina Oikonomopoulou, Tamara T LahAbstract:Aims: To evaluate the expression of CD68 and Kallikrein 6 in human gliomas, and investigate their prognostic significance for survival of brain cancer patients in comparison to some known prognostic markers. Patients and Methods: Histological sections of 51 primary astrocytic tumours (11 benign, 40 malignant) were immunohisto- chemically stained for CD68, cathepsin B, Kallikrein 6 and Ki-67. CD68 and Kallikrein 6 expressions were also analyzed by real-time PCR in nine brain tumour biopsies. Results: Both microglia and tumour cells expressed CD68. High CD68 and cathepsin B staining scores were significantly, more frequent in the malignant than in the benign tumours (p=0.036 and p=0.014, respectively). In contrast, the benign group presented a stronger immunoreactivity for Kallikrein 6 compared with the malignant tumours (p=0.013). A CD68 staining score of tumour cells higher than 3 was a significant predictor of shorter overall survival (p<0.01) in all patients and of borderline significance in the malignant group (p=0.057). Strong CD68 staining was of greater predictive value in the subgroup of anaplastic astrocytomas (p=0.021). Furthermore, as expected on the basis of our previous studies, prognostic significance was confirmed for cathepsin B, but not for any of the other markers under evaluation. Conclusion: Kallikrein 6 was down-regulated in malignant glioma, but this differential expression did not have an impact on patient prognosis. In contrast, immunostaining of glioma tissue for CD68 and for cathepsin B may be used for prognosis of survival in these patients. This finding suggests that besides the known role of cathepsin B in invasion and angiogenesis, CD68 may be also associated with glioma progression.
Niv Papo - One of the best experts on this subject based on the ideXlab platform.
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pre equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases
Biochemical Journal, 2018Co-Authors: Itay Cohen, Si Naftaly, Efrat Benzeev, Alexandra Hockla, Evette S Radisky, Niv PapoAbstract:High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially towards proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and Kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6,500- to 230,000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by interaction of the mutated APPI residues with nonconserved enzyme residues located in or near binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.
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pre equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases
Biochemical Journal, 2018Co-Authors: Itay Cohen, Si Naftaly, Efrat Benzeev, Alexandra Hockla, Evette S Radisky, Niv PapoAbstract:High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and Kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.
Natalia A Ignatenko - One of the best experts on this subject based on the ideXlab platform.
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specific microrna mrna regulatory network of colon cancer invasion mediated by tissue Kallikrein related peptidase 6
Neoplasia, 2017Co-Authors: Earlphia Sells, Ritu Pandey, Hwudaurw Chen, Bethany A Skovan, Haiyan Cui, Natalia A IgnatenkoAbstract:Abstract Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of Kallikrein 6 (KLK6), a member of a Kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates ( P =.005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.
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Specific microRNA–mRNA Regulatory Network of Colon Cancer Invasion Mediated by Tissue Kallikrein–Related Peptidase 6
Elsevier, 2017Co-Authors: Earlphia Sells, Ritu Pandey, Hwudaurw Chen, Bethany A Skovan, Haiyan Cui, Natalia A IgnatenkoAbstract:Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of Kallikrein 6 (KLK6), a member of a Kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P = .005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors
Antoninus Soosaipillai - One of the best experts on this subject based on the ideXlab platform.
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an integrated cell line based discovery strategy identified follistatin and Kallikrein 6 as serum biomarker candidates of breast carcinoma
Journal of Proteomics, 2016Co-Authors: Alain Mange, Lampros Dimitrakopoulos, Antoninus Soosaipillai, Peter Coopman, Eleftherios P Diamandis, Jerome SolassolAbstract:Abstract Secreted proteins constitute a relevant source of putative cancer biomarkers. Here, we compared the secretome of a series of four genetically-related breast cancer cell lines as a model of aggressiveness using quantitative mass spectrometry. 537 proteins (59.5% of the total identified proteins) predicted to be released or shed from cells were identified. Using a scoring system based on i) iTRAQ value, ii) breast cancer tissue mRNA expression levels, and iii) immunohistochemical staining (public database), a short list of 10 candidate proteins was selected. Using specific ELISA assays, the expression level of the top five proteins was measured in a verification set of 56 patients. The four significantly differentially expressed proteins were then validated in a second independent set of 353 patients. Finally, follistatin (FST) and Kallikrein 6 (KLK6) in serum were significantly higher (p-value Biological significance Discovery of new serum biomarkers that exhibit increased sensitivity and specificity compared to current biomarkers appears to be an essential field of research in cancer. Most biological markers show insufficient diagnostic sensitivity for early breast cancer detection and, for the majority of them, their concentrations are elevated only in metastatic forms of the disease. It is therefore essential to identify clinically reliable biomarkers and develop effective approaches for cancer diagnosis. One promising approach in this field is the study of secreted proteins through proteomic analysis of in vitro progression breast cancer models. Here we have shown that FST and KLK6 are elevated in breast cancer patient serum compared to healthy controls. We expect that our discovery strategy will help to identify cancer-specific and body-fluid-accessible biomarkers.
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separation of Kallikrein 6 glycoprotein subpopulations in biological fluids by anion exchange chromatography coupled to elisa and identification by mass spectrometry
Proteomics, 2012Co-Authors: Antoninus Soosaipillai, Eleftherios P Diamandis, Uros Kuzmanov, Christopher R Smith, Ihor Batruch, Anastasia DiamandisAbstract:Kallikrein 6 (KLK6) has been shown to be aberrantly glycosylated in ovarian cancer. Here, we report a novel HPLC anion exchange method, coupled to a KLK6-specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids. Biological fluids were fractionated using anion exchange and resulting fractions were analyzed for KLK6 content by ELISA producing a four-peak elution profile. Using this assay, the KLK6 elution profile and distribution across peaks of a set (n = 7) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and cerebrospinal fluid (CSF) of normal individuals (n = 7). Glycosylation patterns of recombinant KLK6 (rKLK6) were characterized using tandem mass spectrometry (MS/MS), and found to consist of a highly heterogeneous KLK6 population. This protein was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. The rKLK6 glycoform composition of each peak was assessed by lectin affinity and MS/MS based glycopeptide quantification by product ion monitoring. The combined results showed an increase in terminal alpha 2-6 linked sialic acid in the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals.
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differential n glycosylation of Kallikrein 6 derived from ovarian cancer cells or the central nervous system
Molecular & Cellular Proteomics, 2009Co-Authors: Uros Kuzmanov, Antoninus Soosaipillai, Christopher R Smith, Nianxin Jiang, Eleftherios P DiamandisAbstract:Disturbed glycosylation patterns have been observed in the majority of human cancers. Over the past 40 years, a number of physiologically expressed proteins containing abnormal glycan structures have been shown to be tumor-associated antigens (1). For example, prostate-specific antigen (PSA)1 and ribonuclease 1 were found to be differentially glycosylated in prostate and pancreatic cancers, respectively (2, 3). It has been suggested that the disturbed glycosylation of proteins is an early event of oncogenic transformation, aiding in the invasion and metastasis of tumor cells (1, 4–11). As such, a selective advantage might be conferred on tumor cells with increased glycan structures, allowing them to evade immune response during the invasion and metastasis processes (12). In ovarian cancer, a number of proteins are found to be aberrantly glycosylated, including CA125 (13), α1-proteinase inhibitor (14), haptoglobin (14), other acute phase proteins (15), and IgGs (15). In particular, there is mounting evidence of increased sialylation of proteins and deregulated sialylation pathways in ovarian cancer (16). Altered sialylation of proteins in this disease is indicated by increased levels of the sialyl LewisX and sialyl-Tn antigens in ovarian carcinoma, even at early stages of progression (15, 17, 18). This coincides with the findings showing disrupted sialyltransferase protein expression (19–21) and altered mRNA expression of several sialyltransferases in ovarian cancer cells (22). Human tissue Kallikreins are a family of 15 secreted serine proteases with trypsin or chymotrypsin-like activities. Through the use of RT-PCR, ELISA, immunohistochemical, and bioinformatic techniques, most Kallikreins have been shown to be deregulated in a number of malignancies including breast, ovarian, prostate, and testicular cancer (23–25). Elevated levels of Kallikrein 6 (KLK6), a trypsin-like protease, in serum and tissue extracts have been shown to forecast for poor prognosis in ovarian cancer (26–32). KLK6 has a wide expression pattern at both the mRNA and protein levels. However, immunohistochemical and ELISA studies have shown that the major site of KLK6 expression is the central nervous system (CNS), with very high (mg/liter) levels of the protein detected in cerebrospinal fluid (CSF) (33–35). As such, the major source of KLK6 in the circulation of normal individuals is the CNS. The up-regulation of KLK6 in ovarian cancer and its unfavorable prognostic value have been well-established (33, 36, 37). It has been previously shown that virtually all ovarian tumors express KLK6, some of them at extremely high levels (33, 37). During ovarian cancer development and progression, tumor-derived KLK6 diffuses into the general circulation (33, 36). Despite these highly favorable characteristics of KLK6 as an ovarian cancer biomarker, the sensitivity of the test performed in serum (for both early and late stage disease) has been shown not to exceed that of the classical ovarian cancer biomarker, CA125 (36). The combination of KLK6 and CA125 resulted in modest increases in sensitivity (10–30% over and above CA125 alone) for both early and late stage disease (36). At early stages, the increase of serum KLK6 contributed by ovarian cancer cells is usually not sufficient to raise KLK6 above the normal serum levels. Therefore, the ability to differentiate KLK6 originating from the CNS (normally found in the serum of healthy individuals) and KLK6 originating from ovarian tumors could potentially increase the diagnostic value of KLK6 as an ovarian cancer biomarker. Toward this purpose, the differential N-glycosylation patterns of KLK6 from ascites fluid of ovarian cancer patients and CSF of healthy individuals were examined. Initially, anion-exchange chromatography with the two biological fluids resulted in different elution patterns, indicative of differential post-translational modifications or processing. Different N-glycosylation patterns of the two isoforms of KLK6 were confirmed by glycosidase digestion followed by gel shift mobility assays. Additionally, the presence of sialylation on the two isoforms was determined by lectin-antibody sandwich ELISA methodology. The composition and structure of the glycans present on the two subpopulations of KLK6 were elucidated by monitoring KLK6 glycopeptides by electrospray ionization-Orbitrap tandem mass spectrometry (MS/MS). Our main finding is that KLK6 from ovarian cancer ascites (but not CSF) is extensively sialylated. This difference in sialylation may be exploited in the future for developing a specific biomarker for ovarian carcinoma.
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human Kallikrein 6 hk6 a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma
Journal of Clinical Oncology, 2003Co-Authors: Eleftherios P Diamandis, Antoninus Soosaipillai, Andreas Scorilas, S Fracchioli, Marleen Van Gramberen, Henk W A De Bruijn, Alfthan Henrik, Linda Grass, George M Yousef, Ulfhakan StenmanAbstract:Purpose: The discovery of new ovarian cancer biomarkers that are suitable for early disease diagnosis and prognosis may ultimately lead to improved patient management and outcomes. Patients and Methods: We measured, by immunoassay, human Kallikrein 6 (hK6) concentration in serum of 97 apparently healthy women, 141 women with benign abdominal diseases, and 146 women with histologically proven primary ovarian carcinoma. We then calculated the diagnostic sensitivity and specificity of this test and examined the association of serum hK6 concentration with various clinicopathologic variables and patient survival. Results: Serum hK6 concentration between normal and benign disease patients was not different (mean, 2.9 and 3.1 μg/L, respectively). However, hK6 in presurgical serum of ovarian cancer patients was highly elevated (mean, 6.8 μg/L; P < .001). Serum hK6 decreased after surgery (to a mean of 3.9 μg/L) in 68% of patients. The diagnostic sensitivity of serum hK6 at 90% and 95% specificity is 52% and 47%, ...
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higher expression of human Kallikrein 10 in breast cancer tissue predicts tamoxifen resistance
British Journal of Cancer, 2002Co-Authors: Antoninus Soosaipillai, Eleftherios P Diamandis, Liuying Luo, Maxime P Look, John A FoekensAbstract:The human tissue Kallikreins are secreted serine proteases, encoded by a group of homologous genes clustered in tandem on chromosome 19q13.3-4. Human Kallikrein 6 and human Kallikrein 10 are two new members of this family. Recently, we developed highly sensitive and specific immunofluorometric assays for human Kallikrein 6 and human Kallikrein 10, which allow for their quantification in tissue extracts and biological fluids. Both human Kallikrein 6 and human Kallikrein 10 are found to be down-regulated in breast cancer cell lines, suggesting that they may be involved in breast cancer pathogenesis and progression. In this study, we investigated the potential value of human Kallikrein 6 and human Kallikrein 10 as prognostic and predictive factors in breast cancer. We quantified human Kallikrein 6 and human Kallikrein 10 protein levels in 749 breast tumour cytosolic extracts and correlated this data with various clinicopathological variables and patient outcomes. Human Kallikrein 6 and human Kallikrein 10 are positively correlated with each other. Higher human Kallikrein 6 and human Kallikrein 10 protein levels are associated with younger age, pre-menopausal, status and tumours which are negative for oestrogen and progesterone receptors. No correlation was found between human Kallikrein 6 and human Kallikrein 10 levels and tumour size, grade, and nodal status. Survival analysis showed that neither human Kallikrein 6 nor human Kallikrein 10 are related to the rate of relapse-free and overall survival. In the analysis with respect to response to tamoxifen therapy, although human Kallikrein 6 levels were not associated with tamoxifen responsiveness, higher levels of human Kallikrein 10 were significantly associated with a poor response rate. This association remained significant in the multivariate analysis. Furthermore, higher human Kallikrein 10 levels were significantly related with a short progression-free and post-relapse overall survival after start of tamoxifen treatment for advanced disease. Taken together, our results suggest that although human Kallikrein 6 and human Kallikrein 10 are not prognostic markers for breast cancer, human Kallikrein 10 is an independent predictive marker for response of tamoxifen therapy.
Isobel A. Scarisbrick - One of the best experts on this subject based on the ideXlab platform.
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Clinical significance and novel mechanism of action of Kallikrein 6 in glioblastoma
2016Co-Authors: Kristen L Drucker, Sachiko I Blaber, Michael Blaber, Paul A Decker, Alex R. Paulsen, Caterina Giannini, Joon H. Uhm, Brian P. O’neill, Robert B. Jenkins, Isobel A. ScarisbrickAbstract:Background. Kallikreins have prognostic value in spe-cific malignancies, but few studies have addressed their clinical significance to glioblastoma multiforme (GBM). Kallikrein 6 (KLK6) is of potential high rele-vance to GBM, since it is upregulated at sites of CNS pathology and linked to reactive astrogliosis. Here we examine the clinical value of KLK6 as a prognostic indi-cator of GBM patient survival and its activity in promot-ing resistance to cytotoxic agents. Methods. The association between patient survival and levels of KLK6 immunoreactivity were investigated in 60 grade IV astrocytoma tumor specimens. Levels of KLK6 RNA were also evaluated in a separate set of GBM patient tumors (n 23). Recombinant KLK6 or en-forced KLK6 overexpression in GBM cell lines was used to evaluate effects on astrocytoma cell survival. Results. A range of KLK6 expression was observed across grade IV tumors, with higher levels a poor prog-nostic indicator of patient survival (P .02) even after adjusting for gender and Eastern Cooperative Oncology Group performance scores (P .01). KLK6 reduced the sensitivity of GBM cell lines to cytotoxic agents, including staurosporine and cisplatin, and to the current standard of patient care: radiotherapy or temozolomide alone or in combination. The ability of KLK6 to promote resistance to apoptosis was dependent on activation of the thrombin receptor, protease activat-ed receptor 1. Conclusions. Taken together, these results indicate that elevated levels of KLK6 in GBM are likely to promote the resistance of tumor cells to cytotoxic agents and are an indicator of reduced patient postsurgical survival times
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genetic targeting of protease activated receptor 2 reduces inflammatory astrogliosis and improves recovery of function after spinal cord injury
Neurobiology of Disease, 2015Co-Authors: Maja Radulovic, Hyesook Yoon, Karim Mustafa, Michael G Fehlings, Isobel A. ScarisbrickAbstract:Inflammatory-astrogliosis exacerbates damage in the injured spinal cord and limits repair. Here we identify Protease Activated Receptor 2 (PAR2) as an essential regulator of these events with mice lacking the PAR2 gene showing greater improvements in motor coordination and strength after compression-spinal cord injury (SCI) compared to wild type littermates. Molecular profiling of the injury epicenter, and spinal segments above and below, demonstrated that mice lacking PAR2 had significantly attenuated elevations in key hallmarks of astrogliosis (glial fibrillary acidic protein (GFAP), vimentin and neurocan) and in expression of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor (TNF) and interleukin-1 beta (IL-1β)). SCI in PAR2-/- mice was also accompanied by improved preservation of protein kinase C gamma (PKCγ)-immunopositive corticospinal axons and reductions in GFAP-immunoreactivity, expression of the pro-apoptotic marker BCL2-interacting mediator of cell death (BIM), and in signal transducer and activator of transcription 3 (STAT3). The potential mechanistic link between PAR2, STAT3 and astrogliosis was further investigated in primary astrocytes to reveal that the SCI-related serine protease, neurosin (Kallikrein 6) promotes IL-6 secretion in a PAR2 and STAT3-dependent manner. Data point to a signaling circuit in primary astrocytes in which neurosin signaling at PAR2 promotes IL-6 secretion and canonical STAT3 signaling. IL-6 promotes expression of GFAP, vimentin, additional IL-6 and robust increases in both neurosin and PAR2, thereby driving the PAR2-signaling circuit forward. Given the significant reductions in astrogliosis and inflammation as well as superior neuromotor recovery observed in PAR2 knockout mice after SCI, we suggest that this receptor and its agonists represent new drug targets to foster neuromotor recovery.
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prognostic significance of multiple Kallikreins in high grade astrocytoma
BMC Cancer, 2015Co-Authors: Kristen L Drucker, Eleftherios P Diamandis, Caterina Gianinni, Paul A Decker, Isobel A. ScarisbrickAbstract:Background: Kallikreins have clinical value as prognostic markers in a subset of malignancies examined to date, including Kallikrein 3 (prostate specific antigen) in prostate cancer. We previously demonstrated that Kallikrein 6 is expressed at higher levels in grade IV compared to grade III astrocytoma and is associated with reduced survival of GBM patients. Methods: In this study we determined KLK1, KLK6, KLK7, KLK8, KLK9 and KLK10 protein expression in two independent tissue microarrays containing 60 grade IV and 8 grade III astrocytoma samples. Scores for staining intensity, percent of tumor stained and immunoreactivity scores (IR, product of intensity and percent) were determined and analyzed for correlation with patient survival. Results: Grade IV glioma was associated with higher levels of Kallikrein-immunostaining compared to grade III specimens. Univariable Cox proportional hazards regression analysis demonstrated that elevated KLK6- or KLK7-IR was associated with poor patient prognosis. In addition, an increased percent of tumor immunoreactive for KLK6 or KLK9 was associated with decreased survival in grade IV patients. Kaplan-Meier survival analysis indicated that patients with KLK6-IR < 10, KLK6 percent tumor core stained < 3, or KLK7-IR < 9 had a significantly improved survival. Multivariable analysis indicated that the significance of these parameters was maintained even after adjusting for gender and performance score. Conclusions: These data suggest that elevations in glioblastoma KLK6, KLK7 and KLK9 protein have utility as prognostic markers of patient survival.
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differential expression of multiple Kallikreins in a viral model of multiple sclerosis points to unique roles in the innate and adaptive immune response
Biological Chemistry, 2014Co-Authors: Michael Panos, George P Christophi, Moses Rodriguez, Isobel A. ScarisbrickAbstract:Recent studies provide a functional link between Kallikrein 6 (Klk6) and the development and progression of disease in patients with multiple sclerosis (MS) and in its murine models. To evaluate the involvement of additional Kallikrein family members, we compared Klk6 expression with four other Kallikreins (Klk1, Klk7, Klk8, and Klk10) in the brain and spinal cord of mice infected with Theiler's murine encephalomyelitis virus, an experimental model of progressive MS. The robust upregulation of Klk6 and Klk8 in the brain during the acute phase of viral encephalitis and in the spinal cord during disease development and progression points to their participation in inflammation, demyelination, and progressive axon degeneration. More limited changes in Klk1, Klk7, and Klk10 were also observed. In addition, Klk1, Klk6, and Klk10 were dynamically regulated in T cells in vitro as a recall response to viral antigen and in activated monocytes, pointing to their activities in the development of adaptive and innate immune function. Together, these results point to overlapping and unique roles for multiple Kallikreins in the development and progression of virus-mediated central nervous system inflammatory demyelinating disease, including activities in the development of the adaptive and innate immune response, in demyelination, and in progressive axon degeneration.
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distinct promoters regulate tissue specific and differential expression of Kallikrein 6 in cns demyelinating disease
Journal of Neurochemistry, 2004Co-Authors: George P Christophi, Sachiko I Blaber, Michael Blaber, Moses Rodriguez, Paul J Isackson, Isobel A. ScarisbrickAbstract:Kallikrein 6 is a serine protease expressed abundantly in normal adult human and rodent CNS, and therein is regulated by injury. In the case of CNS demyelinating disease, K6 expression in CNS occurs additionally in perivascular and parenchymal inflammatory cells suggesting a role in pathogenesis. Herein we describe two unique transcripts that occur within the human and mouse K6 genes that differ in their 5'-untranslated regions. These transcripts have identical translation initiation sites in exon 3, are expressed in a tissue-specific fashion and are differentially regulated in response to CNS injury. While the human and mouse 5'-transcripts differ in sequence they are identical in genomic organization and tissue-specific expression. The most 5'-transcript, designated transcript 1, includes exon 1-7, and was detectable in all CNS regions, but not in any non-CNS tissues examined (spleen, thymus, liver, kidney, pancreas, submandibular gland and peripheral nerve). In contrast, transcript 2 lacks exon 1, but contains a unique sequence at the 5'-end of exon 2, designated exon 2A. Transcript 2 was expressed both in CNS and in each peripheral tissue. In a murine model of human CNS demyelinating inflammatory disease induced by Theiler's picornovirus, mouse K6 transcript 1 was up-regulated in brain and spinal cord at acute and more chronic phases of CNS inflammation and demyelination, while overall transcript 2 expression was not significantly altered. However, in isolated splenocyte cultures, transcript 2 was up-regulated two-fold by cellular activation. Tissue-specific expression patterns and differential regulation in CNS disease indicates that each K6 5'-transcript is probably regulated by unique promoter elements and may serve as a molecular target to treat inflammatory demyelinating disease.
