(CANCER RESEARCH 58. 4782-4786. November I. I998| Advances in Brief The Role for NES1 Serine Protease as a Novel Tumor Suppressor1 Jaya Goyal, Karen M. Smith, Janet M. Cowan, David E. Wazer, Sam W. Lee, and Vimla Band2 Dt'iHiniiit'in* of Radiation Oncology ¡J.G.. D.E. W., V. B.j. und Pediatrics ¡J.M.C.I. New England Medical Center, tinti Department of Hiochemistry ¡V.B.f. unti Genetics Program ¡K.M.S.. V. BJ. Tufts University School of Medicine. Boston. Massachusetts 02111. anil Department t>f Medicine. Beth Israel Deaconess Medical Center. Harvard Medical School. Boston. Massachusetts 02115 ¡S.W. L.¡ Abstract Previously (Liu et al, Cancer Res., 56: 3371-3379, 1996), we isolated a novel serine protease-like gene—Normal Epithelial Cell Specific-1 I.V/-..S/)—that is expressed in normal mammary epithelial cells but is down-regulated in most breast cancer cell lines. Here, we demonstrate that stable expression of NES1 in the NESl-negative MDA-MB-231 breast cancer cell line suppressed the oncogenicity as revealed by inhibition of the anchorage-independent growth and tumor formation in nude mice. Fluorescence in situ hybridization localized the V/..S7 gene to chromosome 19ql3.3, a region that contains genes for related proteases (including the prostate-specific antigen) and is rearranged in human cancers. Similar to breast cancer cell lines, prostate cancer cell lines also lacked NES1 niKN.V and protein expression. Together, these results strongly suggest a tumor- suppressor role for NES1 in breast and prostate cancer. Introduction Carcinomas, the malignant tumors arising from epithelial cells, constitute the majority of human cancers. In nearly all cases, the etiology of these cancers is unknown. Malignant transformation rep resents a complex multistep process in which genetic changes and environmental factors including radiation, viruses, carcinogens, and dietary components are considered to play a role ( 1). To gain insight into biochemical pathways involved in epithelial cell oncogenesis. we and others have used in vitro models of epithelial cell transformation. In one such model, we exposed the normal mammary epithelial cell strain 76N to fractionated doses of y-irradi- ation in vitro, which was similar to a therapeutic regimen used for cancer treatment and which resulted in the tumorigenic conversion of the cells (2). To isolate genes the products of which contributed toward oncogenic transformation in this system and to identify dif ferentially expressed mRNAs, we carried out subtractive hybridiza tion between the normal parental cell strain 76N and its radiation- transformed cell line 76R-30. This strategy resulted in the isolation of a novel putative serine protease. NES I,3 the mRNA expression of which was observed in 76N cells but was down-regulated in 76R-30 cells (2). Using a panel of normal and tumor mammary epithelial cell lines, we showed that the expression of NES 1 mRNA and protein was absent in a majority of breast tumor cell lines (2). The predicted NES1 polypeptide showed high homology to a number of serine proteases (3), in particular the members of the trypsin family, the kallikrein family, and the family of proteases that activate the kringle domain-containing growth factors (4-6). The Received 7/23/98; accepted 9/17/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Department of Defense Breast Cancer Program. - To whom requests for reprints should be addressed, at Department of Radiation Oncology. Box 824. New England Medical Center. 750 Washington Street. Boston. MA 02111. Phone: (617) 636-4776: Fax:1617»636-6205: E-mail: VBAND@opal.Tufts.Edu. 'The abbreviations used are: NESI. normal epithelial specific-1: PSA. prostate- specific antigen: FISH, fluorescence in silit hybridization: IGF. insulin-like growth factor: IGFBP-3. insulin-like growth factor-binding protein 3: DAPI. 4'.6-diamidino-2-phenylin- dole: DAPI-II. DAPI with /'-phenylenediamine in PBS and glycerol. kallikrein family includes the PSA that is increased in the serum of prostate cancer patients and serves as a prognostic marker (7, 8). The kringle domain-containing growth factors include human tissue plas- minogen activator and human hepatocyte growth factor activator, which have been linked to oncogenesis (5, 6. 9, 10). The former is increased during tumorigenic progression of cells, whereas the latter is a mitogenic growth factor for a known proto-oncogene, c-met (hepatocyte growth factor receptor; Ref. 11). The involvement of close homologues of NESI in oncogenic transformation suggests a potential function for NESI in cell growth. Here, we show that the transfection-mediated reconstitution of NESI expression in a NESl-negative breast tumor cell line, MDA- MB-231, results in suppression of the tumorigenic phenotype both in vitro and in vivo. The NESI gene localized to chromosome 19ql3.3 within the same region that contains PSA (8). Similar to mammary epithelial cells, NESI mRNA and protein were expressed in normal and immortal prostatic epithelial cells but not in tumorigenic prostate epithelial cell lines. Together, these data suggest that NESI plays a tumor suppressor role in breast, prostate, and possibly other epithelial cells. Materials and Methods Tissue Sample and Cell lines. Normal prostate tissue was from a prostac- tomy specimen obtained from the Beth Israel Deaconess Medical Center (Boston). Normal prostate epithelial cells immortalized with the human pap- illoma virus type 18 (designated as HPV18-I cells) or SV40 large T antigen (SV40-1; obtained from Dr. J. S. Rhim. National Cancer Institute, Frederick. MD; Refs. 12. 13), the ND1 prostate cancer cell line (obtained from Dr. P. Narayan. University of Florida. Gainesville. FL: Ref. 14). and the MDA-MB- 231 breast cancer cell line (obtained from Dr. Ruth Lupu. Lawrence Berkeley National Laboratory. Berkeley, CA) have been described (15). PC3, DU-145, and LNCaP prostate cancer cell lines were obtained from American Tissue Type Collection (Manassas. VA). All of the cell lines used in this study were grown in a-MEM with 10% PCS (16). Transfection. NES! cDNA was cloned into pCMV-neo vector and 8 ¿igof Hindlll lineari/ed plasmid was used for transfection into MDA-MB-231 cells using the calcium phosphate coprecipitation method as described earlier (16). After G418 ( 1 mg/ml) selection for 2 weeks, single colonies were isolated and subcultured at 1:3 split ratio. Northern Blotting. Total cellular RNA was isolated from subconfluent monolayer cells using the guanidinium-isothiocynate method. Ten ¿tgof each RNA was resolved on formaldehyde-1.2% agarose gel and was transferred onto a nylon membrane (Hybond-N: Amersham. Arlington Heights, IL). Hybridi/ation was carried out with <2P-labeled NESI cDNA probe (nucleotide 651-1072) as described previously (2). Western Blotting. Culture supernatants from vector- and /VESy-transfected cells were collected for 24 h after serum deprivation. Fifty ^g (breast cells) or 300 /xg (prostate cells) of each supernatant [protein quantitation done using a Bicinchoninic acid protein assay reagent kit (Pierce Chemical Co.. Rockford. IL)] were separated on a 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-P Millipore. Marlborough. MA). The mem branes were blocked with TBST (25 mM Tris. 0.15 M NaCI, and 0.1% Tween 20) containing 5% each of nonfat dry milk and BSA. incubated with either rabbit anti-NESl antibody or monoclonal antibody against PSA (Ab-2. Neo- markers. Fremont. CA). followed by either goat antirabbit IgG or goat anti- 4782 Research. on November 10, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from