Hyun I. Park

Identification and Characterization of Human Endometase (Matrix Metalloproteinase-26) from Endometrial Tumor

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derivedmetalloproteinase. Among more than three million expressed sequence tags sequenced, the endometase gene was only obtained from human endometrial tumor cDNA library. Endometase mRNA was expressed specifically in human uterus, not in other tissues/cells tested, e.g. testis, heart, brain, lungs, liver, thymus, and melanoma G361. Endometase protein has a signal peptide, a propeptide domain, and a catalytic domain with a unique “cysteine switch” propeptide sequence, PHCGVPDGSD, and a zinc-binding motif, VATHEIGHSLGLQH. Endometase is 43, 41, 41, and 39% identical to human metalloelastase, stromelysin, collagenase-3, and matrilysin, respectively. The zymogen was expressed and isolated from Escherichia coli as inclusion bodies with a molecular mass of 28 kDa. The identity and homogeneity of the recombinant protein was confirmed by protein N-terminal sequencing, silver stain, and immunoblot analyses. The pro-enzyme was partially activated during the folding process. Endometase selectively cleaved type I gelatin and α1-proteinase inhibitor; however, it did not digest collagens, laminin, elastin, β-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor. It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-α converting enzyme. Endometase may selectively cleave extracellular matrix proteins, inactivate serpins, and process cytokines.

Endometase/Matrilysin-2 in Human Breast Ductal Carcinoma in Situ and Its Inhibition by Tissue Inhibitors of Metalloproteinases-2 and -4 A Putative Role in the Initiation of Breast Cancer Invasion

Local disruption of the integrity of both the myoepithelial cell layer and the basement membrane is an indispensable prerequisite for the initiation of invasion and the conversion of human breast ductal carcinoma in situ (DCIS) to infiltrating ductal carcinoma (IDC). We previously reported that human endometase/matrilysin-2/matrix metalloproteinase (MMP) 26-mediated pro-gelatinase B (MMP-9) activation promoted invasion of human prostate carcinoma cells by dissolving basement membrane proteins (Y. G. Zhao et al., J. Biol. Chem., 278: 15056–15064, 2003). Here we report that tissue inhibitor of metalloproteinases (TIMP)-2 and TIMP-4 are potent inhibitors of MMP-26, with apparent Ki values of 1.6 and 0.62 nm, respectively. TIMP-2 and TIMP-4 also inhibited the activation of pro-MMP-9 by MMP-26 in vitro. The expression levels of MMP-26, MMP-9, TIMP-2, and TIMP-4 proteins in DCIS were significantly higher than those in IDC, atypical intraductal hyperplasia, and normal breast epithelia adjacent to DCIS and IDC by immunohistochemistry and integrated morphometry analysis. Double immunofluorescence labeling and confocal laser scanning microscopy revealed that MMP-26 was colocalized with MMP-9, TIMP-2, and TIMP-4 in DCIS cells. Higher levels of MMP-26 mRNA were also detected in DCIS cells by in situ hybridization.

Peptide Substrate Specificities and Protein Cleavage Sites of Human Endometase/Matrilysin-2/Matrix Metalloproteinase-26*

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4–P4′ sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1′)-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1′ and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2′, and P3′. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference inK i app values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu49–Thr50 and Ala75–Leu76, which still left the cysteine switch sequence (PHC82GVPD) intact. This suggests that Cys82 may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only ∼5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys82 in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe352–Leu353 and Pro357–Met358 in the reactive loop of α1-proteinase inhibitor and His140–Val141 in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors, and the identification of optimal physiological and pathological substrates of MMP-26 in vivo.

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling.

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1′ pocket in MMPs is a well defined substrate P1′ amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1′ site were selected. The Homephe side chain is designed to probe deep S1′ pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1′ pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1′ pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1′ pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1′ pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1′ pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1′ pockets.

Autolytic processing at Glu586-Ser587 within the cysteine-rich domain of human adamalysin 19/disintegrin-metalloproteinase 19 is necessary for its proteolytic activity.

We investigated the regulation of the proteolytic activity of human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19). It was processed at Glu586(P1)-Ser587(P1′) site in the cysteine-rich domain as shown by protein N-terminal sequencing. This truncation was autolytic as illustrated by its R199A/R200A or E346A mutation that prevented the zymogen activation by furin or abolished the catalytic activity. Reagents that block furin-mediated activation of pro-hADAM19, decRVKR-CMK, A23187, and brefeldin A abrogated this processing. The sizes of the side chains of the P1 and P1′ residues are critical for the processing of hADAM19. The amount of processing product in the E586Q or S587A mutant with a side chain almost the same size as that in the wild type was almost equal. Conversely, very little processing was observed when the size of the side chain was changed significantly, such as in the E586A, E586G, or S587F mutants. Two mutants with presumably subtle structural distinctions from wild type hADAM19, E586D and S587T, displayed rare or little processing and had very low capacities to cleave α2-macroglobulin and a peptide substrate. Therefore, this processing is necessary for hADAM19 to exert its proteolytic activities. Moreover, a new peptide substrate, Ac-RPLE-SNAV, which is identical to the processing site sequence, was cleaved at the E-S bond by soluble hADAM19 containing the catalytic and disintegrin domains. This enzyme cleaved the substrate with K m , k cat, andk cat/K m of 2.0 mm, 2.4/min, and 1200 m −1min−1, respectively, using a fluorescamine assay. Preliminary studies showed that a protein kinase C activator, phorbol 12-myristate 13-acetate, promoted the cellular processing of hADAM19; however, three calmodulin antagonists, trifluoperazine, W7, and calmidazolium, impaired this cleavage, indicating complex signal pathways may be involved in the processing.

Effects of Detergents on Catalytic Activity of Human Endometase/Matrilysin-2, a Putative Cancer Biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 ( approximately 90muM). Their IC(50) values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixons plot; however, the inhibition mechanism of endometase was noncompetitive with a K(i) value of 240muM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a K(d2) (low-affinity calcium-dissociation constant) value of 120 microM, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.

Abstract 2615: Genetic variations in angiogenesis-related genes in prostate cancer recurrence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Prostate cancer is the most common cancer and the second leading cause of cancer death in American men. Among patients who have a radical prostatectomy, ∼30% will have disease recurrence. Currently, the level of prostate specific antigen (PSA), clinical stage (TNM) and tumor grade (Gleason score) are used to estimate prognosis and inform treatment modalities. Although these are extremely useful, additional biomarkers are needed to better predict outcome. Angiogenesis has been associated with Gleason score, tumor stage, progression, metastasis and survival in prostate cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in genes in the angiogenesis pathway may be associated with prostate cancer recurrence. Methods: In a historical cohort of 1061 prostatectomy cases treated at the Moffitt Cancer Center from 1986 to 2003, we identified 311 recurrent cases and 750 non-recurrent cases. A recurrent case was defined by either an elevated PSA level after surgical treatment, clinical metastasis or disease specific death. All other patients were categorized as non-recurrent. We compared genotype frequencies of 2,981 SNPs in 547 angiogenesis genes between the two groups. Genotyping was performed using the Illumina GoldenGate assay and associations between recurrence-free survival and individual SNPs were evaluated using competing Cox regression models to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) under three inheritance models (dominant, recessive and log-additive model). For each SNP, the minimum P-value among all tested inheritance models was selected. We calculated q-values to estimate false discovery rates. Results: The mean age at diagnosis of the study subjects was 59.7 (SD=7.5) years. The recurrence rate was 29.3% with a median recurrence-free survival of 200.8 (range=179.0-241.9) months. A total of 91 SNPs in 56 angiogenesis genes had raw P-values less than 0.01 and q-values less than 3%. We found associations for several families of genes that have been previously associated with aggressiveness and/or survival of prostate cancer, such as cadherins (CDH,11 SNPs), fibroblast growth factor and its receptor (FGF/FGFR, 6 SNPs), insulin-like growth factor and its receptor (IGF/IGFR, 3 SNPs), interleukins (IL, 5 SNPs), metalloproteinases (MMP, 3 SNPs) and thrombospondin (THBS, 4 SNPs). The most significant association was observed for [rs889730][1] in CDH13 (HR= 0.69, 95%CI=0.58-0.83, P=0.000057, additive model). CDH13 contributes to promotion of tumor neovascularization, but down regulation has been associated with a poor outcome in prostate cancer. Conclusion: Data from this comprehensive study of angiogenesis genes and prostate cancer suggests that variants in angiogenesis-related genes may influence prostate cancer recurrence after radical prostatectomy. Further studies are warranted to confirm these preliminary findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2615. doi:1538-7445.AM2012-2615 [1]: /lookup/external-ref?link_type=GEN&access_num=rs889730&atom=%2Fcanres%2F72%2F8_Supplement%2F2615.atom

Abstract 291: Genomewide scale epigenetic profile and prostate cancer recurrence

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Development and progression of prostate cancer reflect the accumulation of genetic and epigenetic alterations. Although genetic changes are involved in the inactivation of genes with important anti-cancer functions, DNA methylation in a promoter region is an important epigenetic mechanism for the down-regulation (silencing) of expression of these genes. Therefore, we conducted a genome-wide profile of prostate tumors to identify epigenetically altered regions that distinguish recurrent from non-recurrent cases. We profiled the epigenetic landscape of 125 prostate tumors using bisulfite treated tumor DNA samples extracted from 65 recurrent and 60 non-recurrent prostate cancer cases. Epigenome-wide methylation analysis was performed at the Molecular Genomic Core Facility of the Moffitt Cancer Center using Illumina Infinium Human Methylation 450K Beadchip, which can quantitatively measure over 485,577 CpG sites with 99% coverage of RefSeq Gene and 96% coverage of CpG islands. For methylation data analysis, intensities were normalized using Illumina GenomeStudio software. Beta values with assigned detection p-values > 0.01 were treated as missing data. We considered the methylation pattern at a larger region instead of methylation at single sites. Thus, in addition to evaluating methylation at single sites, we were interested in the number of significantly differently methylated probes within a gene as a measure of how significant a gene is. We identified 269 differentially methylated CpG sites between recurrent and non-recurrent tumor tissues, with a false discovery rate (FDR) q-value less than 0.05 and a mean methylation difference greater than 0.1 between the two groups (recurrent /non-recurrent cases). Eleven genes where more than 25% of the measured positions were differently methylated (p 0.1 between the medians of the two groups) between recurrent and non-recurrent samples. We further investigated ALEX1 (ARMCX1), which is one of the 11 genes and suggested as a tumor suppressor gene. ALEX1 (Arm protein lost in epithelial cancers, on chromosome X) is involved in a variety of processes such as cell adhesion, embryogenesis and tumorigenesis. Expression of ALEX1 mRNA is significantly reduced in various tumor tissues, including prostate carcinomas and in various cancer cell lines. We investigated expression level of ALEX1 in an additional 392 prostate tumor samples (145 recurrence and 247non-recurrent) using realtime RT-PCR. Based upon average Ct ratios between ALEX1 and β actin, ALEX1 is down regulated 2.67 fold in tumor tissues from recurrent cases as compared with non-recurrent cases (p<0.001). If replicated, these identified biomarkers may be used at the time of diagnosis to predict risk of recurrence and improve clinical treatment decision-making. Citation Format: Hui-Yi Lin, Anders Berglund, Thomas Sellers, Ardeshir Hakam, Hyun Park, Julio Pow-Sang, Jong Y. Park. Genomewide scale epigenetic profile and prostate cancer recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2014-291

Abstract 2558: miRNA-related gene variants and prostate cancer aggressiveness and recurrence.

Introduction: miRNA expression has been correlated with prostate cancer aggressive and recurrence. However, the role of miRNA-related gene variants in prostate cancer aggressiveness and recurrence has not been adequately studied. In this study, we evaluated the association between single nucleotide polymorphisms (SNPs) in miRNA-related genes and prostate cancer aggressiveness and recurrence. Methods: We conducted a two-stage (discovery and replication) analysis of 136 miRNA-related gene variants (in 18 genes) in aggressive and non-aggressive prostate cancer cases. A discovery dataset of 437 aggressive and 603 non-aggressive cases was derived from a historical cohort of prostate cancer cases treated at the Moffitt Cancer Center from 1987 to 2003. A replication set of 659 aggressive and 492 non-aggressive cases was derived from existing genome-wide association data from Cancer Genetic Markers of Susceptibility (CGEMS). In both datasets, patients with a Gleason score > 7 or stage > III were considered to have aggressive disease and all other patients were categorized as non-aggressive. Associations between SNPs and aggressive disease were evaluated using unconditional logistic regression to estimate odds ratios (OR) and 95% confidence intervals (95%CI). We also evaluated the associations between SNPs and recurrence-free survival using Cox regression models to estimate hazard ratios (HR) and 95%CI among 245 recurrent and 979 non-recurrent cases. We calculated q-values to estimate false discovery rates in the analysis of recurrence. Results: For the analysis of aggressiveness, ten SNPs in seven genes (EIF2C2 [rs7009635, rs2280833 and rs13276958], FMR1 [rs28282], GEMIN4 [rs2291779], LIN28B [rs1149280], PIWIL1 [rs1106042], RAN [rs14035] and XPO5 [rs7752811, rs7748977] were significantly associated with aggressive prostate cancer in the discovery dataset. Five of the 10 significant SNPs had data available in the replication dataset, but none of them was statistical significant in this dataset. For the analysis of recurrence, a total of 13 SNPs in seven genes were associated with recurrence-free survival (P-values Conclusion: Findings from this study suggest that variants in miRNA-related genes may influence prostate cancer recurrence, but not aggressiveness. Further studies are warranted to confirm these findings. Citation Format: Ernest K. Amankwah, Hui-Yi Lin, Hyun Park, Selina Radlein, Julio Pow-Sang, Ardeshir Hakam, Ya-Yu Tsai, Jong Y. Park. miRNA-related gene variants and prostate cancer aggressiveness and recurrence. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2558. doi:10.1158/1538-7445.AM2013-2558