238 ONCOLOGY REPORTS 27: 238-245, 2012 Suppression of the invasion and migration of cancer cells by SERPINB family genes and their derived peptides RUEY-HWANG CHOU1-4, HUI-CHIN WEN1,7, WEI-GUANG LIANG1,5, SHENG-CHIEH LIN1,5, HSIAO-WEI YUAN1, CHENG-WEN WU1,5,6 and WUN-SHAING WAYNE CHANG1 1National Institute of Cancer Research, National Health Research Institutes, Miaoli 35053; 2Center for Molecular Medicine, China Medical University Hospital, Taichung 40402; 3China Medical University, Taichung 40402; 4Department of Biotechnology, Asia University, Taichung 41354; 5College of Life Science, National Tsing Hua University, Hsinchu 30013; 6Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei 11221, Taiwan, R.O.C. Received June 28, 2011; Accepted August 17, 2011 DOI: 10.3892/or.2011.1497 Abstract. Apart from SERPINB2 and SERPINB5, the roles SERPINB RCL-peptides may provide a reasonable strategy of the remaining 13 members of the human SERPINB family against lethal cancer metastasis. in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially Introduction expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of Cancer metastasis is the leading cause of morbidity and each SERPINB gene effectively suppressed the invasiveness mortality in cancer patients. It is a highly complex process, and motility of malignant cancer cells. Among all of the genes, including cell detachment, migration, invasion, circulation in the SERPINB1, SERPINB5 and SERPINB7 genes were more blood vessels, adhesion, colonization at other sites and forma- potent, and the inhibitory effect was further enhanced by tion of secondary tumors (1). Prior to tumor cell detachment co-expression of any two of them. In addition, single treatment from the primary site, which leads to cell migration and inva- of the synthetic peptides corresponding to the P5-P5' sequences sion in the metastasis process, the extracellular matrix (ECM) of the reactive center loop (RCL) of SERPINB1, SERPINB5 microenvironment must be degraded by proteases, such as or SERPINB7 markedly suppressed the invasive and migra- urokinase plasminogen activator (uPA), uPA receptor (uPAR) tory properties of the cancer cells in a dose-dependent manner. and the plasmin network (2,3) and matrix metalloproteinases More significantly, combination treatment of these peptides (MMPs) (4). On the other hand, protease inhibitors negatively in cancer cells further improved the suppressive effect by regulate the proteolysis process in cancer metastasis, e.g. 20-40%. Here, we determined the expression of all SERPINB plasminogen activator inhibitors (PAIs), PAI-1 (SERPINE1) family members in lung and breast cancer patients, and iden- and PAI-2 (SERPINB2) against uPA/uPAR/plasmin network tified those members with potent inhibitory ability toward and the tissue inhibitor of matrix metalloproteinases (TIMPs), invasion and migration, and designed RCL-derived peptides to TIMP-1 to TIMP-4 against MMPs. suppress the malignancy of cancer cells. Forced re-expression Serine protease inhibitors (serpins) regulate many physi- of these anti-invasive SERPINB genes or application of the ological processes, such as blood coagulation, fibrinolysis, inflammation, complement activation and cell migration (5). Based on their phylogenic relationships, the superfamily is divided into 16 different clades (A-P), in which human Correspondence to: Dr W.S.W. Chang, National Institute of Cancer serpins are the first 9 clades (A-I) (6). The clade B serpins Research, National Health Research Institutes, No. 35, Keyan Road, (SERPINB family) is the largest one within the human serpin Zhunan, Miaoli 35053, Taiwan, R.O.C. superfamily. It contains 13 genes located on chromosome E-mail: [email protected] 6p25 (SERPINB1, SERPINB6 and SERPINB9) and 18q21 (the remaining members of the family). Unlike circulating serpins, Dr R.H. Chou, Center for Molecular Medicine, China Medical the SERPINB family genes lack the N and C terminus exten- University Hospital, No. 6, Hsueh-Shih Road, Taichung 40447, Taiwan, R.O.C. sion regions common to other serpins, and lack a secretory E-mail: [email protected] signal peptide; thus they primarily reside within cells (7). Various SERPINB genes have been reported to be cancer- Present address: 7Institute of Cellular and System Medicine, related serpins, but few have been shown to be associated National Health Research Institutes, Taiwan, R.O.C. with cancer invasion and metastasis. Clinical results reveal that a lower level of SERPINB2 expression is associated with Key words: SERPINB family, cancer, migration, invasion poor prognosis and outcome in gastric (8,9), breast (10) and lung cancers (11). Overexpression of SERPINB2 was found CHOU et al: SERPINB GENES AND THEIR DERIVED PEPTIDES SUPPRESS THE MALIGNANCY OF CANCER CELLS 239 Table I. Primers for RT-PCR of SERPINB family genes. Gene symbol Forward primers Reverse primers PCR products (bp) SERPINB1 5'-TCAGCTTGCCCAGGTTCAAACTG-3' 5'-GGATGCTACCTGAGGAATTATGC-3' 300 SERPINB2 5'-GCTGGAGATGTTAGCATGTTCTTG-3' 5'-GGCTTGGTGGAACACTTCAGAAAG-3' 300 SERPINB3 5'-GCGGTCTCGTGCTATCTGG-3' 5'-GTAGGTGATGATCCGAATC-3' 109 SERPINB4 5'-TGGAGCCACGGTCTCTCAG-3' 5'-GGAGATGATAATTCGACTA-3' 115 SERPINB5 5'-CATGGAGGCCACGTTCTGTATG-3' 5'-CCTGGCACCTCTATGGAATCCC-3' 417 SERPINB6 5'-CTCCCGCGGTTTAAACTAGAGG-3' 5'-GCAGAAGAGAATCCCGTTGGTC-3' 300 SERPINB7 5'-GGACCAATCCAAGGCGAATGACC-3' 5'-TAAACAGCGTGGACTGAGGGAGTT-3' 285 SERPINB8 5'-AGCTGGAGGAGAGTTATGACTTG-3' 5'-AGAACCTGCCACAGAACAAGATG-3' 300 SERPINB9 5'-CAAGCCAGACTGTATGAAGAGTAC-3' 5'-AAAGGGTGGTCAGCACAGAACCTG-3' 300 SERPINB10 5'-GCAGACATGATGGAGTTGTATGAAG-3' 5'-GAAGAGGAATGGGTGATTTGCATTG-3' 300 SERPINB11 5'-CGTTTCATGAGTGGACAAGCTCTTC-3' 5'CCTTGAACTGAGCTCTCATTGGTAG-3' 300 SERPINB12 5'-AAGGCACAGATCCTGGAAATGAG-3' 5'-ATTGGGACTTGGAGAGATTCCAG-3' 300 SERPINB13 5'-GCTTCTGCCCAACGACATCG-3' 5'-GCAGCCTCGGTGCCTTCCTC-3' 300 to completely inhibit soluble and cell-surface bound plas- respectively, in RPMI-1640 and MEM α media supplemented minogen activator activity, resulting in the suppression of the with 10% fetal bovine serum (FBS) (Thermo Scientific HyClone, metastasis of melanoma cells in SCID mice (12). SERPINB5 Logan, UT, USA) at 37˚C in a humidified 5% CO2 atmosphere. (MASPIN) has been well studied and was confirmed to be a tumor-suppressor gene, which suppresses tumor growth, Reverse transcription-polymerase chain reaction (RT-PCR). invasion and metastasis in different types of cancers, and has Total RNA was extracted using TRIzol reagent, and cDNA potential therapeutic perspectives (13-15). The squamous- was generated by M-MLV reverse transcriptase according to cell carcinoma antigens (SCCAs), SCCA1 (SERPINB3) and the manufacturer's instructions (Invitrogen Life Technologies, SCCA2 (SERPINB4), have been used as diagnostic markers Carlsbad, CA, USA). The PCR was carried out in a thermocy- for advanced squamous-cell carcinoma. The relative mRNA cler (Biometra, Göttingen, Germany) and conducted in a 25-µl ratio (higher than 0.2) of SERPINB4 over SERPINB3 has reaction volume containing 0.2 µM of each primer, 200 µM been correlated to primary tumor recurrence in head and dNTP, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 1 U Taq neck squamous-cell carcinoma (16). SERPINB13 was found DNA polymerase (Takara, Kyoto, Japan) and 1 µl cDNA under to be down-regulated in squamous-cell carcinoma of the oral the following conditions: 1 cycle at 94˚C for 2 min; 30 cycles cavity and in squamous-cell carcinoma cell lines of head and at 94˚C for 30 sec, 55˚C for 30 sec, 72˚C for 30 sec; and 1 neck cancer (17), but up-regulated in skin carcinoma (18,19). cycle at 72˚C for 10 min. The primers are listed in Table I. For Recently, decreased expression of SERPIN genes including determining the expression patterns of each SERPINB gene in SERPINB2, SERPINB3, SERPINB4, SERPINB7, SERPINB11, normal and tumor tissues, the human lung matched cDNA pair SERPINB12 and SERPINB13 on chromosome 18q21 has been panel and the human breast matched pair total RNA panel (BD demonstrated in oral squamous-cell carcinomas, while the Clontech, Palo Alto, CA, USA) were used. T/N (tumor vs. matched normal tissues) expression ratio of these SERPIN genes was found to have no significant associa- Cloning of the full-length SERPINB genes and stable transfec- tion with clinicodemographic characteristics (20). tion. The full-length SERPINB genes were amplified by PCR Apart from the previously mentioned members of the from human keratinocyte Matchmaker cDNA Library (BD human SERPINB family, however, whether the remaining Clontech), apart from SERPINB4, SERPINB9, SERPINB11 members, such as SERPINB2 and SERPINB5, are associated and SERPINB12, which were amplified from the MGC: 27150 with cancer and whether they are functionally important in cDNA clone (Invitrogen Life Technologies), HEK-293 cell line cancer metastasis, are still unclear. In the present study, we cDNA, normal lung cDNA (Ambion, Austin, TX, USA), and determined the expression patterns of each SERPINB gene in normal brain cDNA (BD Clontech), respectively. The PCR matched normal and tumor tissues from lung and breast cancer program was as followings: 1 cycle at 94˚C for 2 min; 35 cycles patients, constructed individual SERPINB genes, and screened at 94˚C for 30 sec, 55˚C for 30 sec, 72˚C for 2 min and 1 cycle at their effects on the invasive and migratory properties of malig- 72˚C for 10 min. The cloning sites and primer sequences