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Hepsin, a Cell Surface Serine Protease Identified in Hepatoma Cells, Is Overexpressed in Ovarian Cancer

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Hepsin, a Cell Surface Serine Protease Identified in Hepatoma Cells, Is Overexpressed in Ovarian Cancer CANCER RESEARCH 57. 2554-2887. July 5. 9971 Advances in Brief Hepsin, a Cell Surface Serine Protease Identified in Hepatoma Cells, Is Overexpressed in Ovarian Cancer HirotoshiTanimoto,Yan Yan,John Clarke,SoheliaKorourian,KazushiShigemasa,Tim H. Parmley, Groesbeck P. Parham, and Timothy J. O'Brien' Departments of Obstetrics and Gynecology [H. T.. Y. Y.. J. C.. T. H. P.. G. P. P.. T. J. 0.], Pathology (S. K.]. and Biochemistry and Molecular Biology IT. J. 0.1, Unisersily of Arkansas for Medical Sciences, Little Rock. Arkansas 72205-7/99. and Department of Obstetrics and Gynecology. Hiroshima University School of Medicine, Hiroshima, Japan 737 fK. 5.1 Abstract collagenase (3, 10—13).A significant correlation with tumor progres sion and the immunoreactivity for Mr 72,000 type IV collagenase is Extracellular proteases mediate the digestion of neighboring extracel observed in colorectal cancer (3), and the decrease in the Mr 72,000 lular matrix componentsin initial tumor growth, allow sheddingor des type IV collagenase by pharmacological treatment causes loss of the quamation of tumor cells into the surrounding environment, provide the basis for invasion of basement membranes in target metastatic organs, invasive capacity in vitro (10). Here we investigated the expression of and are required for release and activation of many growth and anglo serine proteases in ovarian tumors by selecting conserved domains in genie factors. We identified overexpression of the serine protease hepsin the immediate vicinity of the catalytic triad of histine-aspartic acid gene in ovarian carcinomas and investigated the expression of this gene in and serine. These sentinel amino acids are surrounded by relatively 44 ovarian tumors (12 low malignant potential tumors and 32 carcinomas) well-conserved domains separated by approximately 50—!00 amino and 10 normal ovaries. Quantitative PCR was used to determine the acids of less-conserved areas. Using redundant primers (14) to these relative expression of hepsin compared to that of fl-tubulin. The mRNA conserved domains, we have examined the expression of serine pro expression levels of hepsin were significantly elevated in 7 of 12 low teases in ovarian carcinoma tissue compared to that of normal ovary. malignant potential tumors and in 27 of 32 carcinomas. On Northern blot These candidate serine proteases have been identified by subcloning analysis, the hepsin transcript was abundant in carcinoma but was almost and sequencing the appropriate PCR products. In this initial report, we never expressed in normal adult tissue, including normal ovary. Our results suggest that hepsin is frequently overexpressed in ovarian tumors describe the overexpression of the extracellular serine protease hepsin and therefore may be a candidate protease in the invasive process and in ovarian carcinoma tissues. Hepsin is located in the plasma mem growth capacity of ovarian tumor cells. brane (anchored by a transmembrane amino-terminal domain; Refs. 15 and 16), allowing its protease catalytic domain access to the Introduction extracellular space. Cell surface proteases such as hepsin could both function as an activator of other extracellular proteases or act directly Proteases have been known to play essential roles in many physi by degrading the extracellular matrix of surrounding cells. Previously, ological functions. They mediate specific proteolysis and contribute to extracellular proteases have been reported to play an important role in biological processes such as tissue rearrangement, complement acti tumor cell growth (17). It has been shown that hepsin is required for vation, and blood coagulation (I). It is a well-established concept that cell growth and maintenance of normal cell morphology (18), and it the proteolytic degradation of the extracellular matrix is an indispen also activates the extrinsic blood coagulation pathway on the cell sable step in tumor invasion and metastasis (2). In this process, surface, leading to thrombin formation (19); however, it is not known proteases have a key role in paving the way for spreading tumor cells. if such activities directly correlate with metastatic capacity. A large number of reports have demonstrated an increased production Proteases have been classified into four families: (a) serine pro of several classes of proteases, including MMPs2 such as Mr 72,000 teases; (b) metalloproteases; (c) aspertic proteases; and (d) cysteine type IV collagenase (gelatinase A, MMP-2, Ref. 3) and matrilysin proteases (20). Hepsin, an extracellular serine protease, was originally (pump- 1, MMP-7, Ref. 4), cysteine proteases such as cathepsin B (5), isolated from cDNA libraries prepared from human liver and hepa aspartic proteases such as cathepsin D (6), and serine proteases such toma cell lines (15). The total length of the cDNA is approximately as plasminogen activator (7) in tumor cells. The proteolysis of the 1.8 kb, and the nucleotidesequenceincludesa single open reading extracellular matrix in tumor invasion and metastasis seems to be a frame for a protein of 417 amino acids. The amino acid sequence is highly complicated process and probably involves a cascade of events similar to that of other serine proteases, and it contains His, Asp, and requiring a variety of proteases (2). In this cascade, the integrated Ser residues at positions 203, 257, and 353, respectively, as the capacity for extracellular matrix digestion, tumor cell invasion, and catalytic triad for enzymatic activity ( 15, 16). The specific role and metastatic growth may be mediated by proteases with unique speci behavior of hepsin on the surface of malignant cells is still not well fications. This hypothesis is supported by the findings that some understood. agents that specifically inhibit one of these proteases reduce tumor cell invasion (8, 9). Among the known proteases associated with the Materials and Methods tumor cell growth and metastasis process is the Mr 72,000 type IV Tissue Samples. Fresh surgical specimens of ovarian tumors were obtained from 44 patients (I 2 LMP tumors and 32 carcinomas). Clinical staging was Received 5/8/97; accepted 5/29/97. The costs of publication of this article were defrayed in part by the payment of page determined according to the criteria of the International Federation of Gyne charges. This article must therefore be hereby marked advertisement in accordance with cologists and Obstetricians. Normal ovaries were obtained from 10 patients l8 U.S.C. Section 1734 solely to indicate this fact. who underwent surgery for benign gynecological disease. The materials were I To whom requests for reprints should be addressed, at Department of Obstetrics and obtained immediately after the surgical procedure, frozen in liquid nitrogen, Gynecology. University of Arkansas for Medical Sciences, 4301 West Markham Slot #718. Little Rock. AR 72205-7199. and stored at —80°Cbefore mRNA isolation. 2 The abbreviations used are: MMP, matrix metalloprotease; LMP, low malignant mRNA Extraction and eDNA Synthesis. mRNA extraction from tissue potential. and cDNA synthesis were carried out by the methods described previously 2884 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. HEPSIN OVEREXPRESSION IN OVARIAN CANCER (21). mRNA was isolatedusing a RiboSepmRNA isolation kit (Becton genesCaseTable 1 Patientcharacteristics andexpression ofhepsin Dickinson Labware). in this procedure, polyadenylated mRNA was isolated expression directly from the tissue lysate using an affinity chromatography media oh Histological type― Stage/gradeLN5mRNA of hepsin' godeoxythymidylic acid cellulose. cDNA was synthesized from 5.0 @xgof I Normal ovary n mRNA by random hexamer priming using a first-strand cDNA synthesis kit 2 Normal ovary n (Clontech). Using PCR amplification of genomic free cDNA, we identified 3 Normal ovary n products of the correct bp size. These PCR products were subcloned into a 4 Normal ovary n 5 Normal ovary n Promega T vector and grown in Escherichia coli for subsequent sequencing. 6 Normal ovary n The primary PCR product identified indicated a high homology to the trypsin 7 Normal ovary n like serine protease hepsin, previously identified and cloned from hepatoma 8 Normal ovary n cells (15). 9 Normal ovary n 10 Normal ovary n Quantitative PCR. The mRNA overexpression of hepsin was determined I I S adenoma (LMP) I/I N 4+ using quantitative PCR. Quantitative PCR was performed according to the 12 S adenoma (LMP) I/I NE 4+ method of Noonan et a!. (22) with some modification as reported previously 13 S adenoma (LMP) 1/I NE (21). Oligonucleotideprimers used were: hepsin, 5'-TGTCCCGATGGC 14 S adenoma (LMP) 1/1 N 2+ 15 S adenoma (LMP) 3/I P 4+ GAGTG1Tr-3' (forward) and 5'-CCTGTFGGCCATAGTACTGC-3' (re 16 S adenoma (LMP) 3/1 P 4+ verse); and @3-tubu1in,5'-TGCATfGACAACGAGGC-3' (forward) and 5'- 17 S adenoma (LMP) 3/1 P 4+ CTGTCTrGACATrGUG-3' (reverse). @3-tubuhin was used as an internal 18 M adenoma (LMP) 1/I NE 4+ control. The predicted sizes of the amplified genes were 282 bp for hepsin and 19 M adenoma (LMP) 1/I N n 20 M adenoma (LMP) 1/I N n 454 bp for 3-tubulin. The primer sequences used in this study were designed 21 M adenoma (LMP) 1/1 NE n according to the cDNA sequences described by Leytus et a!. (hepsin; Ref. 15) 22 M adenoma (LMP) I/l NE n and Hall et a!. (/3-tubuhin;Ref. 23). The PCR reaction mixture consists of 23 5 carcinoma 1/2 N 4+ cDNA derived from 50 ng of mRNA, 5 pmol of sense and antisense primers 24 5 carcinoma 1/3 N 4+ 25 S carcinoma 3/1 NE 2+ for both the hepsin gene and the (3-tubulingene, 200 ,xmolof deoxynucleotide 26 S carcinoma 3/2 NE 4+ triphosphates, 5 pCi of [a-32P]dCTP, and 0.25 unit of Taq DNA polymerase 27 S carcinoma 3/2 P 4+ with reaction buffer (Promega) in a final volume of 25 pi. The target sequences 28 5 carcinoma 3/2 NE 2+ were amplified in parallel with the @3-tubuIingene.Thirty cycles of PCR were 29 5 carcinoma 3/3 NE 2+ 30 5 carcinoma 3/3 NE 4+ carried out in a thermal cycler (Perkin-Elmer).
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