Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4442-4446, May 1995 Cell Biology ETS1 suppresses tumorigenicity of human colon cancer cells (transcription factor) HIROAKI SUZUKI*t, VINCENZO ROMANO-SPICA*, TAKIS S. PAPASt, AND NARAYAN K. BHAT*§ *Laboratory of Molecular Oncology, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201; *Center for Molecular and Structural Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425-2213; and §Program Resources, Inc./DynCorp, Frederick Cancer Research and Development Center, P.O. Box B, Frederick, MD 21702-1201 Communicated by Robert C. Gallo, National Cancer Institute, Bethesda, MD, January 3, 1995 (received for review June 24, 1994) ABSTRACT We have ectopically expressed transcription 202 (Glu202 to Gly202) and 212 (Asp212 to His212) is capable of factor ETS1 in two different highly tumorigenic human colon binding to ETS1-binding sequence (EBS) motifs in DNA but cancer cell lines, DLD-1 and HCT116, that do not express lacks transcriptional activity. Overexpression of mutant ETS1 endogenous ETS1 protein and have obtained several indepen- protein in DLD-1 cells did not reduce their tumorigenic dent clones. The expression ofwild-type ETS1 protein in these potential. Since DLD-1 cells express copious amounts of other colon cancer cells reverses the transformed phenotype and ETS-related proteins (9), results presented in this paper tumorigenicity in a dose-dependent manner. By contrast, demonstrate that reduction in tumorigenicity appears to be expression in DLD-1 cells of a variant form of ETS1, lacking due to overexpression of wild-type ETS1. transcriptional activity, did not alter the tumorigenic prop- erties of the cells, suggesting that the reduction in tumorige- nicity in these clones was specific for the wild-type ETSI gene MATERIALS AND METHODS products. Since these colon cancer cells have multiple genetic Plasmid DNAs. A 1.95-kb full-length human ETS1 cDNA alterations, the system described in this paper could be a good (ref. 18; gift ofJ. M. Leiden, University of Chicago) was cloned model to study the suppression of tumorigenicity at a tran- into the HindIII site of pcDM7 and pcDNAI (Invitrogen). scriptional level, which could lead to the design and develop- pEBSCAT plasmid DNA has been described (25). ment of novel drugs for cancer treatment. Isolation of cDNA Coding for Mutant ETS1. Total genomic DNA from DE1-2-3 cells (1.2 ,ug) was used for polymerase ETS1 is a member of the ets gene family and is a cellular chain reaction (PCR) with sense (5'-ggattcCACCATGAAG- counterpart of the v-ets oncogene of the avian erythroblastosis GCGGCCGT-3'; positions -4 to 14) and antisense (5'- virus E26'(1-3). The ETS1 gene is expressed at high levels in caagcttgTCAGTGCCATCACTC-3'; positions 1321-1335) lymphoid cells (4-9) and in astrocytes and endothelial cells ETS1 primer pairs to clone integrated ETS1 cDNA as de- (10-12). ETS1 is a nuclear phosphoprotein (13-15) that binds scribed (26). to purine-rich DNA sequences and functions as a transcription Cell Culture and Transfection. The human colon carcinoma factor (1-3, 16-19). The DNA-binding domain is localized at cell lines DLD-1 (27) and HCT116 (28) were transfected with the carboxyl-terminal region (20-22), and the transactivation human ETS1 cDNA expression vectors along with pSV2neo by domain is localized in the protein domain encoded by exons 5 use of Lipofectin reagent (GIBCO/BRL). Transfectants were and 6 ofETS1 (23). The ETS1 protein binds to its target DNA selected with G418 (400 ,ug/ml). DE-1-1, DE1-1-7, and sequences present in the transcriptional regulatory regions of DE1-3-2 are DLD-1 transfectants obtained with pcDM7 ETS1 a number of "housekeeping" genes, as well as certain types of expression vector, whereas DE1-2-3, DE1-2-4, and DE1-2-8 tissue-specific genes, and regulates their transcription (1-3). are DLD-1 transfectants obtained with pcDNAI ETS1 expres- Thus, it is possible that the aberrant expression of the ETS1 sion vector. DME1-1, DME1-9, DME1-12, DME1-21, and gene may result in the alteration of growth. The expression of DME1-26 are DLD-1 transfectants obtained with PRC cyto- the ETS1 gene is induced at late stages of thymocyte differ- megalovirus mutant ETS1 expression vector. HE1-2 and entiation (7). In resting T cells, the ETS1 gene is expressed at HE1-3 are HCT116 transfectants obtained with PRC cyto- high levels, and upon activation of T cells, the ETS1 gene megalovirus ETS1 expression vector. product is decreased to low levels (8). ETS1 gene expression Cell Labeling, Immunoprecipitation, Polyacrylamide Gel is also induced during differentiation of P19 murine embryonal Electrophoresis, and Electrophoretic Mobility-Shift Assay. carcinoma cells (24). These results suggest that ETS1 plays a These were done as described (7, 9, 29-31). role in induction of cellular differentiation and suppression of cell growth. To identify whether ETS1 has tumor-suppressive activities, RESULTS AND DISCUSSION we have ectopically expressed ETS1 in two types of human Generation of DLD-1 Cells Expressing ETS1 Proteins. We colon cancer cell lines, DLD-1 and HCT116, that do not expressed the full-length ETS1 protein in DLD-1 and HCT116 express endogenous ETS1 protein. We have obtained several cells under the control of the cytomegalovirus promoter and independent clones expressing different amounts offull-length obtained several independent cell lines. These cell lines were ETS1 protein. Overexpression of ETS1 in these colon cancer derived from single cell clones. DNA blot analyses of trans- cells did not change their anchorage-dependent cell growth. fectants digested with multiple restriction enzymes confirmed However, rates of anchorage-independent growth were re- that these transfectants truly represented independent clones duced in a dose-dependent manner. Cells expressing the (data not shown). ETS1 protein formation was examined by highest levels of ETS1 made smaller and fewer colonies in soft radioimmunoprecipitation using ETS1-specific monoclonal agar and showed reduced tumor incidence in nude mice. A In DLD-1 transfectants mutant form of ETS1 with amino acid substitution at codon antibody (13, 31). (DE1-1-1, DE1-1-7, Abbreviations: CAT, chloramphenicol acetyltransferase; EBS, ETS1- The publication costs of this article were defrayed in part by page charge binding sequence. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Department of Pathology, Hokkaido University, accordance with 18 U.S.C. §1734 solely to indicate this fact. School of Medicine, Sapporo 060, Japan. 4442 Downloaded by guest on September 26, 2021 Cell Biology: Suzuki et at Proc. Natl. Acad. Sci. USA 92 (1995) 4443 (-) neo-1 0 DLD-1 (-) e' rt't'n o'onGo o ,n ...I...I bo D S Q0 O a n a c (- r r I I .: . :' I'- '' '''"' - - - - "':;.-.''.. Peptide + + ++ + + ; r *;, ,,I,':-.. .. .... , ''':; -1 *. ''s E.p51-,,..:,~ i:,:'. ;_*.: ...*l _..ETS1 p42 -.- 1-1 (+I 2-4 (+) 2-3 (+ + +,*Mut) FIG. 1. ETS1 protein in DLD-1 transfectants. Cells were labeled with [35S]methionine (100 ,uCi/ml) for 1 hr and were immunoprecipi- tated with the human ETS1-specific monoclonal antibody E44 in the absence (-) or presence (+) of cognate peptide (31). In T cells, the two ETS-1 isoforms detected (p51 and p42) are the products of full-length and alternatively spliced forms of ETS1 mRNA (13). DE1-2-3, DE1-2-4, DE1-2-8, and DE1-3-2), ETS1 protein 1-7 (+ + + + +) 2-8 (+ + +) 3-2(+ +) (p51) is encoded by exogenous ETS1 (Fig. 1), because (i) it is undetectable in parental cells (DLD-1) as well as in cells transfected with pSV2neo alone (DLD-1-Neol, -Neo2, and -Neo3), (ii) it is immunoprecipitated by two different ETS1 antibodies and the immunoprecipitation can be blocked by cognate peptides (13, 30, 31), (iii) the levels of p51 correlated well with the levels of ETS1 mRNA initiated from the cyto- megalovirus promoter (data not shown), and (iv) p51 is similar in size to the endogenous full-length proteins detected in FIG. 2. Soft-agar colony formation assays of the ETS1 transfec- T-lymphoma cells (Fig. 1) (8, 13, 30). Also, we have previously tants. Cells were plated in 0.33% noble agar (Difco) in RPMI 1640 in DE1-1-7 with 15% fetal bovine serum at 104 cells per 60-mm plate and stained shown that the exogenous ETS1 protein expressed after 10 of in the nucleus and binds to the with p-iodonitrotetrazolium violet (1 mg/ml; Sigma) days cells is localized purine-rich incubation. ETS-1 protein expression: (-), negative; (+)-(+ + + + +), DNA sequences; further, this product is similar in biochemical relative levels of ETS1 in various DLD-1 ETS1 transfectants in cells expressed properties to the ETS1 protein expressed lymphoid as shown in Fig. 1. Wild-type and mutant (*Mut) ETS1 are expressed (29). However, we observed that ETS1 migrated as a doublet at similar levels. (p51/p50) in extracts derived from DE1-2-3 cells but as a single species (p51) in all other ETS1 transfectants examined (Fig. 1). ETS1. We found no substantial differences in their growth Formation of p50 ETS1 protein appears to be due to mutation rates (Table 1), suggesting that ETS1 expression did not affect in ETS1 (for details, see Fig. 4). The significance of p50 ETS1 the anchorage-dependent growth rates of the DLD-1 trans- protein is discussed below. fectants and was not toxic to these cells. On the other hand, Overexpression of ETS1 in DLD-1 Cells Reduces Their anchorage-independent growth rates, indicated by the colony- Ability to Form Colonies in Soft Agar and to Form Tumors in forming ability in soft agar, were reduced in ETS1 transfec- Nude Mice. To determine whether overexpression of ETS1 has tants.