model to study the suppression of tumorigenicity at a tran- scriptional level, which ... expression vector, whereas DE1-2-3, DE1-2-4, and DE1-2-8 are DLD-1 ...
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) 202 (Glu202 to Gly202) and 212 (Asp212 to His212) is capable of binding to ETS1-binding sequence (EBS) motifs in DNA but lacks transcriptional activity. Overexpression of mutant ETS1 protein in DLD-1 cells did not reduce their tumorigenic potential. Since DLD-1 cells express copious amounts of other ETS-related proteins (9), results presented in this paper demonstrate that reduction in tumorigenicity appears to be due to overexpression of wild-type ETS1.
ABSTRACT We have ectopically expressed transcription factor ETS1 in two different highly tumorigenic human colon cancer cell lines, DLD-1 and HCT116, that do not express endogenous ETS1 protein and have obtained several independent clones. The expression of wild-type ETS1 protein in these colon cancer cells reverses the transformed phenotype and tumorigenicity in a dose-dependent manner. By contrast, expression in DLD-1 cells of a variant form of ETS1, lacking transcriptional activity, did not alter the tumorigenic properties of the cells, suggesting that the reduction in tumorigenicity in these clones was specific for the wild-type ETSI gene products. Since these colon cancer cells have multiple genetic alterations, the system described in this paper could be a good model to study the suppression of tumorigenicity at a transcriptional level, which could lead to the design and development of novel drugs for cancer treatment.
MATERIALS AND METHODS Plasmid DNAs. A 1.95-kb
full-length human ETS1 cDNA
(ref. 18; gift of J. M. Leiden, University of Chicago) was cloned into the HindIII site of pcDM7 and pcDNAI (Invitrogen). pEBSCAT plasmid DNA has been described (25). Isolation of cDNA Coding for Mutant ETS1. Total genomic DNA from DE1-2-3 cells (1.2 ,ug) was used for polymerase chain reaction (PCR) with sense (5'-ggattcCACCATGAAGGCGGCCGT-3'; positions -4 to 14) and antisense (5'caagcttgTCAGTGCCATCACTC-3'; positions 1321-1335) ETS1 primer pairs to clone integrated ETS1 cDNA as described (26). Cell Culture and Transfection. The human colon carcinoma cell lines DLD-1 (27) and HCT116 (28) were transfected with human ETS1 cDNA expression vectors along with pSV2neo by use of Lipofectin reagent (GIBCO/BRL). Transfectants were selected with G418 (400 ,ug/ml). DE-1-1, DE1-1-7, and DE1-3-2 are DLD-1 transfectants obtained with pcDM7 ETS1 expression vector, whereas DE1-2-3, DE1-2-4, and DE1-2-8
ETS1 is a member of the ets gene family and is a cellular counterpart of the v-ets oncogene of the avian erythroblastosis virus E26'(1-3). The ETS1 gene is expressed at high levels in lymphoid cells (4-9) and in astrocytes and endothelial cells (10-12). ETS1 is a nuclear phosphoprotein (13-15) that binds to purine-rich DNA sequences and functions as a transcription factor (1-3, 16-19). The DNA-binding domain is localized at the carboxyl-terminal region (20-22), and the transactivation domain is localized in the protein domain encoded by exons 5 and 6 of ETS1 (23). The ETS1 protein binds to its target DNA sequences present in the transcriptional regulatory regions of a number of "housekeeping" genes, as well as certain types of tissue-specific genes, and regulates their transcription (1-3). Thus, it is possible that the aberrant expression of the ETS1 gene may result in the alteration of growth. The expression of the ETS1 gene is induced at late stages of thymocyte differentiation (7). In resting T cells, the ETS1 gene is expressed at high levels, and upon activation of T cells, the ETS1 gene product is decreased to low levels (8). ETS1 gene expression is also induced during differentiation of P19 murine embryonal carcinoma cells (24). These results suggest that ETS1 plays a role in induction of cellular differentiation and suppression of cell growth. To identify whether ETS1 has tumor-suppressive activities, we have ectopically expressed ETS1 in two types of human colon cancer cell lines, DLD-1 and HCT116, that do not express endogenous ETS1 protein. We have obtained several independent clones expressing different amounts of full-length ETS1 protein. Overexpression of ETS1 in these colon cancer cells did not change their anchorage-dependent cell growth. However, rates of anchorage-independent growth were reduced in a dose-dependent manner. Cells expressing the highest levels of ETS1 made smaller and fewer colonies in soft agar and showed reduced tumor incidence in nude mice. A mutant form of ETS1 with amino acid substitution at codon
are DLD-1 transfectants obtained with pcDNAI ETS1 expression vector. DME1-1, DME1-9, DME1-12, DME1-21, and DME1-26 are DLD-1 transfectants obtained with PRC cytomegalovirus mutant ETS1 expression vector. HE1-2 and HE1-3 are HCT116 transfectants obtained with PRC cytomegalovirus ETS1 expression vector.
Cell Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, and Electrophoretic Mobility-Shift Assay.
These were done as described
(7, 9, 29-31).
RESULTS AND DISCUSSION Generation of DLD-1 Cells Expressing ETS1 Proteins. We expressed the full-length ETS1 protein in DLD-1 and HCT116 cells under the control of the cytomegalovirus promoter and obtained several independent cell lines. These cell lines were derived from single cell clones. DNA blot analyses of transfectants digested with multiple restriction enzymes confirmed that these transfectants truly represented independent clones (data not shown). ETS1 protein formation was examined by radioimmunoprecipitation using ETS1-specific monoclonal antibody (13, 31). In DLD-1 transfectants (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 payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
binding sequence.
tPresent address: Department of Pathology, Hokkaido University, School of Medicine, Sapporo 060, Japan.
4442
Proc. Natl. Acad. Sci. USA 92
Cell Biology: Suzuki et at ,n
S
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FIG. 1. ETS1 protein in DLD-1 transfectants. Cells were labeled with [35S]methionine (100 ,uCi/ml) for 1 hr and were immunoprecipitated 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
3-2(+ +)
transfecSoft-agar colony formation assays of the ETS1 (Difco) in RPMI 1640 plated in 0.33% noble agar60-mm with 15% fetal bovine serum at 104 cells per plate and stained with p-iodonitrotetrazolium violet (1 mg/ml; Sigma) after 10 days of incubation. ETS-1 protein expression: (-), negative; (+)-(+ + + + +), relative levels of ETS1 expressed in various DLD-1 ETS1 transfectants as shown in Fig. 1. Wild-type and mutant (*Mut) ETS1 are expressed FIG. 2.
tants. Cells were
at similar levels.
no substantial differences in their growth did not affect 1), suggesting that ETS1 expression the anchorage-dependent growth rates of the DLD-1 transfectants and was not toxic to these cells. On the other hand, rates, indicated by the colonyanchorage-independent growth were reduced in ETS1 transfecin soft agar, ability forming tants. The ability to form colonies in soft agar was dependent on the level of wild-type ETS1 expression (Table 1 and Fig. 2). Not only the efficiencies but also the sizes of soft agar colonies
ETS1. We found rates (Table
Overexpression of ETS1 Ability to Form Colonies in Soft Agar and to Form Tumors in
Nude Mice. To determine whether overexpression of ETS1 has any effect on cell proliferation, we studied the growth properties of the DLD-1 transfectants expressing different levels of Table 1. Growth and
2-8 (+ + +)
1-7 (+ + + + +)
(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 different ETS1 -Neo3), (ii) it is immunoprecipitated by two 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 cytois similar megalovirus promoter (data not shown), and (iv) p51 in size to the endogenous full-length proteins detected in T-lymphoma cells (Fig. 1) (8, 13, 30). Also, we haveinpreviously shown that the exogenous ETS1 protein expressed DE1-1-7 cells is localized in the nucleus and binds to the purine-rich DNA sequences; further, this product is similar in biochemical in lymphoid cells properties to the ETS1 protein expressed that ETS1 migrated as a doublet (29). However, we observed but as a single (p51/p50) in extracts derived from DE1-2-3 cells examined (Fig. 1). species (p51) in all other ETS1 transfectants Formation of p50 ETS1 protein appears to be due to mutation in ETS1 (for details, see Fig. 4). The significance of p50 ETS1 protein is discussed below. in DLD-1 Cells Reduces Their
tumorigenicity of ETS1 transfectants ETS1
Cell line Controls DLD-1 DLD-1 neo-1 DLD-1 neo-2 DLD-1 neo-3 ETS1 transfectants
Wild-type DE-1-1
DE1-2-4 DE1-3-2 DE1-2-8 DE1-1-7 Wild-type and mutant
expression* -
-
+ + ++ +++ +++++
Tumor incidence Tumor i nude mice 4 wk 3 wk 2 wk
Soft-agar
diameter,$
Doubling time,§
mm
hr
colonies, %
Tumor
3/3 5/6 5/6 4/5
3/3 5/6 5/6 5/5
3/3 6/6 5/6 5/5
13.2 9.4 10.0 11.0
21.5 19.0 18.0 24.2
30.5 20.9 24.0 ND
5/7 2/5 2/6 0/4 0/9
7/7 3/5 4/6 1/4 0/9
6/7 5/5 4/6 2/4 3/9
12.8 5.3 4.4 3.8 3.7
22.0 24.5 39.0 21.0 19.5
13.2 17.5 3.4 10.4 7.4
31.4 23.2 7.1 4/5 3/5 1/5 *-, Undetectable; +, low; + + + + +, high (comparable to the level in lymphoid cells as shown in Fig. 1). tCells (2 x 106) of each cell line were injected subcutaneously into the right anterior flank area of 6- to 8-week-old female nude mice. tMean diameter of the tumors in nude mice 4 weeks after injection. §Cells were plated at 2 x 105 in RPMI 1640 medium containing 15% fetal bovine serum. soft-agar colonies per 104 cells plated, scored after 10 days of incubation. ND, not done. ¶Percent IIWild-type and mutant ETS1 proteins are expressed at similar levels. DE1-2-3
++++ll
4444
Cell Biology: Suzuki et at.
Proc. Natl. Acad Sci USA 92
suppressed by transfection of ETS1 cDNA (Fig. 2); i.e., cells expressing higher levels of ETS1 (DE1-1-7 and DE1-2-8) showed a reduction in the number of colonies in soft agar, as well as in their colony size. The reduction in soft-agar colony number was noticeable only when ETS1 expression occurred at moderate levels, suggesting that the ETS1 gene product(s) may be competing with other gene products which are involved in anchorage-independent growth. The DE1-3-2 cells showed less colony-forming ability in soft agar than the DE1-2-8 cells, which express higher levels of ETS1 protein; this observation is consistent with the longer doubling time exhibited by the DE1-3-2 cells. The parental and neo transfectants (controls) all formed tumors in nude mice within 3 weeks. In contrast, DE1-1-7 cells, expressing high levels of the ETS1 protein, produced no
(1995)
tumors in all nine nude mice studied within the same observation time (Table 1). However, in transfectants expressing abundant ETS1, the tumor incidence was delayed by 3 weeks and the tumors were smaller (Table 1). Three other ETS1 transfectants (DE1-2-8, -3-2, and -2-4) also showed reduced tumor incidences, but at different levels. However, DE1-2-3 cells, expressing the p51/p50 ETS1 doublet, did not show a decrease in the number of colonies formed in soft agar (Fig. 2) or in the incidence of tumor formation in nude mice
were
(Table 1).
Characterization of Mutant ETS1. In DE1-2-3 cells, in addition to p51, a faster migrating 50-kDa ETS1 protein (p50) was specifically recognized by three different ETS1 antibodies raised against the amino-terminal (13, 31), middle (30), and carboxyl-terminal regions (15) of ETS1 in both radioimmu-
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FIG. 3. (A) (Left) Characterization of the mutant p50 ETS-1 protein. Cells were labeled and lysates were immunoprecipitated with anti-ETS-1 monoclonal antibody E44. Bound antigens were released as described (30) and were used for immunoprecipitation with either E44 (lanes 1, 3, and 5) or G19E (ref. 15) (lanes 2, 4, and 6) antibody. Lanes: 1 and 2, DLD-1; 3 and 4, DE1-1-7; 5 and 6, DE1-2-3. (Right) Clone 0-15 encodes mutant ETS1 protein. Plasmid DNAs encoding mutant or wild-type ETS1 were used to generate proteins in TNT reticulocyte lysate (Promega). Total 35S-labeled translation product with no added DNA (lane 10) or with wild-type (lane 11) or mutant ETS1 (lane 12) cDNA and the ETS1 immunoprecipitates from DE1-2-3 cell extract (lane 7) or from mutant (lane 8) or wild-type (lane 9) translation products were analyzed. (B) Schematic representation of wild-type (ETS1) and mutant (mETS1) proteins and designated motifs: RVPS (Arg-Val-Pro-Ser), phosphorylation domain; TA domain, transactivation domain; DNA BD, DNA-binding domain. The amino acid substitutions from Glu (E) to Gly (G) and Asp (D) to His (H) in mutant ETS1 protein and their location are indicated. (C) Mutant ETS1 protein binds to DNA. Sequences of an ETS1-binding oligonucleotide (ETS2 1-8) and a mutant form (ETS2 1-8M) to which ETS1 does not bind (29) are shown at the top. Aliquots representing equal amounts of wild-type (lanes 2) and mutant (lanes 3) ETS1 proteins were mixed with 32P-labeled ETS2 1-8M oligonucleotide and the protein-DNA complexes were analyzed. Lanes 1, translation product without any added plasmid DNA. For competition, a 50-fold molar excess of nonradioactive ETS2 1-8 or ETS2 1-8M oligonucleotide was used. Positions of ETS1-DNA complex and free probe (FP) are indicated at right. (D) Wild-type ETS1, but not mutant ETS1, activates transcription through the EBS motif. DLD-1 cells were transfected with an optimum amount of wild-type and mutant ETS1 expression vectors along with pEBSCAT plasmid DNA. Forty-eight hours after transfection, chloramphenicol acetyltransferase (CAT) activity was measured by diffusion assay (NEN kit). (Inset) Expression of ETS1 protein was measured by radioimmunoprecipitation 48 hr after transfection of DLD-1 cells without any added DNA (lane 1) or with mutant (lane 2) or wild-type (lane 3) ETS1 expression plasmid.
Proc. Natl. Acad. Sci. USA 92 (1995)
Cell Biology: Suzuki et al.
noprecipitation and Western blot analyses. As shown in Figs. 1 and 3A both p50 and p51 ETS1 were expressed at similar levels and recognized by antibodies raised against peptides derived from epitope regions encoded by exons 6, 7, 8, and 9 and from the carboxyl terminus of the human ETS1 protein (13, 15, 30, 31), indicating that p50 formation was not due to truncation of the protein but appeared to be due to some other mutation(s) in ETS1. Mutant ETS1 Protein Has Amino Acid Substitutions. To characterize the mutation, the ETS1 DNA integrated in the genome of DE1-2-3 cells (following transfection of exogenous ETS1 cDNA) was isolated from genomic DNA by use of PCR and characterized according to the size of the ETS1 protein produced in an in vitro transcription-coupled translation system. The protein immunoprecipitated from the in vitro translation products generated from clone 0-15 was very similar to the p50 ETS1 protein seen in DE1-2-3 cell extracts (Fig. 3A), suggesting that clone 0-15 encodes p50 ETS1. DNA sequence analyses of clone 0-15 revealed that it was identical to the ETSI sequence (26), except for four nucleotide substitutions (at codons 202, 208, 212, and 296) in its entire open reading frame. Two substitutions in codons 208 (GTC to GTT) and 296 (CCC to CCT) did not effect any change in its amino acid residues. However, substitutions in codons 202 (GAG to GGG) and 212 (GAC to CAC) did change Glu202 to Gly202 and Asp212 to His212, respectively (Fig. 3B). Substitution of neutral amino acid residues for acidic residues could account for the observed faster migration of this mutant ETS1 protein (p50), similar to the mutation (Glu6 to Val6) in sickle cell hemoglobin (32). Mutant ETS1 Protein Binds to DNA But Lacks Transcriptional Activity. Structure function analysis of ETS1 has shown that the minimal DNA-binding domain consists of 85 aa and is localized at the carboxyl-terminal region (21, 22). The transactivation domain maps to the protein domain encoded by exons 5 and 6 of the ETS1 gene (23), a region not well conserved among other members of the ets family of genes. Exon 6 of human ETS1 encodes aa 177-243 (33); therefore, the mutations observed in p50 appear to be clustered around the putative transactivation domain of ETS1 (Fig. 3B). Both mutant and wild-type ETS1 were capable of binding to DNA (Fig. 3C). However, mutant (p50) ETS1 was unable to transactivate CAT gene expression through EBS, whereas wild-type (p51) ETS1 transactivated the EBS-CAT gene by 2- to 3-fold (Fig. 3D). The failure to transactivate EBS-CAT gene expression by mutant ETS1 was not due to transfection efficiency or expression of p50 ETS1, because similar levels of p50 and p51 ETS1 were expressed in DLD-1 cells (Fig. 3D). These results are consistent with the finding that there is no amino acid substitution in the DNA-binding domain and suggest that the mutation in the putative transactivation domain affects transcriptional activation. Mutant ETS1 Protein Has Lost the Ability to Reduce Tumorigenicity of DLD-1 Cells. We expressed mutant ETS1 protein in DLD-1 cells and obtained several independent transfectants expressing different amounts of mutant ETS1 protein (Fig. 4A). DME-1, DME1-9, and DME1-21 cells express mutant ETS1 comparable to wild-type ETS1 expressed in DE1-2-8 transfectants. Transfectants expressing mutant ETS1 proteins were more tumorigenic than wild-type ETS1 expressing transfectants (Table 2). These results further confirm that the reduction in tumorigenicity of colon cancer cells appears to be due to overexpression of wild-type ETS1. Overexpression of Wild-Type ETS-1 in HCT116 Cells Also Reduces its Tumorigenicity. We have also obtained HCT116 transfectants expressing various amounts of ETS1 protein (Fig. 4B) and found that the transfectants expressing higher levelsin of wild-type ETS1 protein exhibited reduced tumorigenicity nude mice (Table 2). Colon tumor formation is associated with multiple genetic changes, including inactivation of the tumor-suppressor genes
,
,
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4445
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FIG. 4. Expression of ETS1 protein in DLD-1 and HCT116 colon cancer cell lines. (A) DLD-1 cells were transfected with an expression vector carrying mutant ETS1 cDNA and independent clones expressing mutant ETS1 proteins were selected for further analysis. The ETS1 transfectants and control cells were metabolically labeled with [35S]methionine for 1 hr and ETS1 proteins were detected by monoclonal antibody E44. Transfectants expressing wild-type ETS1 (DE11-7 and DE1-2-8) and mutant ETS1 (DME1-1, DME1-9, DME1-12, DME1-21, and DME1-26) are shown. (B) HCT116 cells were transfected with an expression vector carrying wild-type ETS1 cDNA and independent clones expressing wild-type ETS1 were selected for further analysis. The ETS1 transfectants and parental HCT116 cells were metabolically labeled with [35S]methionine and ETS1 proteins were detected by immunoprecipitation as in A. ETS1 proteins expressed in DLD-1 (DE1-1-7) and HCT transfectants (HE1-2 and HE1-3) are shown. Size markers (68 and 43 kDa) are at left.
p53, DCC, MCC/APC, as well as activation of the oncogenes one mutant Ki-ras allele (Gly13 to Asp13), as well as point mutations in p53 (Ser241 to Phe241) (36). We observed no changes in the level of wild-type or mutant p53 proteins between the DLD-1-ETS-1 transfectants and control cells by radioimmunoprecipitation analysis (data not shown). Furthermore, tumorigenicity of DLD-1 cells could be suppressed by knocking out the mutant Ki-ras allele (36), suggesting that the activated Ki-ras gene product plays a dominant role in tumor formation. Activated Ras proteins have been shown to activate the MAP kinase signal transduction pathway, thereby inducing
myc and Ki-ras (34, 35). DLD-1 cells contain one normal and
Table 2. Tumorigenicity of DLD-1 and HCT116 ETS1 transfectants Tumor incidence in nude mice ETS1 4 wk 2 wk 3 wk Cell line expression ETS1 Wild-type +++++ DE1 1-7 6/25 4/25 0/25 +++ DE1 2-8 6/10 3/10 0/10 Mutant ETS1
DME1-1 DME1-9 DME1-12 DME1-21 DME1-26 Parental HCT116
++ ++ + +++ + -
Wild-type ETS1
HCT-HE1-2 HCT-HE1-3
+ +++
6/9 8/9 9/9 8/10
1/9 2/9 6/9 2/10 2/5 7/17
4/9 7/9 9/9 8/10 5/5 15/17
17/17
8/18 0/18
16/17 9/18
16/17 13/18
5/5
Tumor growth was assayed as described in Table 1. Cells (2 x 106) were injected subcutaneously into the right anterior flank area of 6- to 8-week-old female nude mice (three to five animals per experiment) and tumor formation was examined on a weekly basis.
4446
Proc. Natl. Acad. Sci USA 92
Cell Biology: Suzuki et ail
many target genes, including those encoding Ets family of transcription factors (1-3, 37). In fact, overexpression of truncated ETS2 protein containing the DNA-binding domain has been shown to block the myc gene expression mediated through the Ras signal transduction pathway (38). It is also interesting that DLD-1 wild-type ETS1 transfectants express the same levels of ETS2 proteins, which indicates that the exogenous expressed ETS1 protein does not target the ETS2 promoter. Though DLD-1 and HCT116 cells have multiple genetic alterations leading to tumor formation, the expression of wild-type ETS1 may be able to overcome these changes and suppress tumorigenicity by either blocking the downstream targets for ETS2 gene products or by inducing new gene have products required for tumor suppression. In fact, inweDE1-7 shown before that a 54.5-kDa protein is enhanced cells compared with parental or neo-transfected cell lines (29). It is also possible that the ETS1 gene products may be able to modify the preexisting proteins of a specific signal transduction pathway involved in colon tumor cell formation. More direct evidence is needed to substantiate these points. The approach that we have taken to perturb the transcription process in colon cancer cells could pave the way for development of innovative therapeutic agents to suppress colon cancer. T.S.P. was supported partially by grants from the Swint Foundation, the John I. Smith Foundation, and the Scientific and Environmental Affairs Division of the North Atlantic Treaty Organization. 1. Bhat, N. K. &
2. 3. 4.
5. 6. 7. 8. 9. 10. 11.
Papas,
T. S.
(1994)
in
Challenges of Modem
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