Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M511962200 on May 1, 2006

J. Biol. Chem., Vol. 281, Issue 28, 18973-18982, July 14, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/28/18973    most recent
M511962200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wai, P. Y.
Right arrow Articles by Kuo, P. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wai, P. Y.
Right arrow Articles by Kuo, P. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Ets-1 and Runx2 Regulate Transcription of a Metastatic Gene, Osteopontin, in Murine Colorectal Cancer Cells*

Philip Y. Wai1, Zhiyong Mi, Chengjiang Gao, Hongtao Guo, Carlos Marroquin, and Paul C. Kuo

From the Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, November 7, 2005 , and in revised form, April 4, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) –107 to –174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt –101 to –123 and nt –121 to –145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt –115 to –118 and nt –129 to –134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer continues to be the second leading cause of cancer-related deaths in the United States (1, 2). Many patients with advanced or recurrent disease develop distant metastases and fail locoregional therapy. The molecular mechanisms underlying tumor metastasis are not completely understood, but recent evidence implicates osteopontin (OPN)2 as a key regulator of cancer cell metastasis.

OPN, an extracellular matrix protein secreted by a wide variety of cancers, functionally enables tumor progression (3–8). The secreted phosphoprotein binds the {alpha}vbeta integrin and CD44 families of receptors to propagate cellular signals (8–14). In colorectal cancer, gene profiling studies have identified a positive correlation between advanced or metastatic colon tumors and abundant OPN expression (15, 16). We have previously shown that stable, RNAi-mediated down-regulation of OPN expression in CT26 colon adenocarcinoma cells results in suppression of matrix metalloproteinase-2 (MMP-2) expression, decreased in vitro tumor cell motility and invasiveness, and attenuation of functional in vivo hepatic metastasis (17). Together, these data suggest that OPN is necessary for CT26 colorectal metastasis. Subsequent studies in our laboratory have confirmed that OPN protein expression is highly up-regulated in metastatic CT26 but absent in nonmetastatic cell lines such as YAMC.

In this study, we investigated the molecular determinants that regulate OPN expression in CT26 colorectal tumor cells. CT26, an undifferentiated murine colon adenocarcinoma cell line, produces aggressive pulmonary and hepatic metastases in murine models (18, 19), and constitutively expresses OPN at high levels. We hypothesized that specific cis-regulatory domains and trans-factors control OPN expression in CT26 colorectal cancer. Using transient transfection and clonal deletion analysis of OPN promoter-luciferase constructs, we identified cis-regulatory regions within the OPN promoter in CT26 cells versus YAMC control. Gel-shift assays sublocalized 20–30-base pair regions in this cis-domain that could form DNA-nuclear protein complexes. Competition with unlabeled mutant probes characterized the nucleotide sequence of the essential nuclear factor binding sites. A TRANSFAC data base query and antibody-supershift assays identified the corresponding trans-regulatory nuclear proteins as Ets-1 and Runx2. Chromatin immunoprecipitation (ChIP) assays evaluated the in vivo binding capacity of these targets to the OPN promoter. Consensus binding site mutations in subcloned OPN promoter-luciferase constructs demonstrated a significant decrease in transcriptional activity. Antisense oligonucleotides to Ets-1 and Runx2 determined the functional effect of decreased expression of these trans-regulatory nuclear proteins in CT26 cells. Together, these data suggest that expression of the tumor metastasis protein, OPN, requires Ets-1 and Runx2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—CT26 murine colon carcinoma cells were grown as monolayer cultures in DMEM-10% fetal bovine serum (FBS) (Invitrogen) supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were maintained in a 37 °C incubator with 5% CO2-humidified air. YAMC cells were provided as a gift from Dr. Robert Whitehead (Vanderbilt University Medical Center, Nashville, TN). YAMC is a conditionally immortalized murine colonic epithelial cell line that expresses SV40-large-T-antigen when stimulated by interferon-{gamma} at 33 °C. SV40-large-T-antigen is heat labile and degraded at 37 °C. YAMC cells were grown in monolayer in RPMI 1640 (Invitrogen) supplemented with 5% FBS, 1 µg/ml insulin, 5 IU/ml mouse interferon-{lambda}, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a 33 °C incubator with 5% CO2 in humidified air. The utilization by established investigators of the metastatic and nonmetastatic companion cell lines CT26 and YAMC in studying colon cancer is well described in the literature (20, 21).

Western Blot Analysis—Total cell lysates were prepared and analyzed by SDS-PAGE as described previously (22). Briefly, 35 µg of protein/lane was resolved on 4–20% polyacrylamide gels (Gradipore Inc., Hawthorne, NY) and transferred to polyvinylidene membranes (Amersham Biosciences). The primary antibodies used were: anti-OPN (R&D Systems, Minneapolis, MN), anti-glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX), anti-PEBP2{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), anti-aml-3 (Calbiochem). Secondary antibodies used were: donkey anti-goat IgG-horseradish peroxidase (Santa Cruz Biotechnology), donkey anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology), or goat anti-rabbit (Santa Cruz Biotechnology). Immunodetection was performed using horseradish peroxidase-based SuperSignal chemiluminescent substrate (Pierce). For quantification, the bands were scanned into an AlphaImager 3400 (Alpha Innotech, San Leandro, CA) and normalized by dividing the measured density of protein bands by the density of glyceraldehyde-3-phosphate dehydrogenase control bands from corresponding cell lysates.

Plasmid Constructs—5'-Deletion fragments of the OPN promoter subcloned into pXP2 plasmid encoding luciferase were gifts from Dr. D. Denhardt (Rutgers University). Deletion constructs were constructed by PCR from the following primers, and the fragments were subsequently cloned into pGL3-basic luciferase reporter plasmid: OPN –69 (–69 to +79), OPN –88 (–88 to +79), OPN –107 (–107 to +79), OPN –120 (–120 to +79), OPN –134 (–134 to +79), OPN –160 (–160 to +79), OPN –174 (–174 to +79), OPN –209 (–209 to +79), OPN –258 (–258 to +79), OPN –472 (–472 to +79), OPN –512 (–512 to +79), OPN –670 (–670 to +79), OPN –777 (–777 to +79), and OPN –1467 (–1467 to +79). Consensus binding site mutations corresponding to Ets-1 (nt –120 to –114; 5'-GAGGAAG-3' to 5'-ACTTTTA-3'), Runx2 (nt –136 to –129; 5'-AACCACAA-3' to 5'-GGACAATTT-3') and an ETS-1/Runx2 double mutant were constructed using QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, CA), with the OPN –512 wild type fragment as template, and used in transient transfection assays. Mutational accuracy was verified using DNA sequencing and restriction enzyme digestion.

Transient Transfection and Activity Assay—Preliminary experiments using pGL3-beta-galactosidase were performed to determine that transfection efficiency was comparable between the two cell lines, CT26 and YAMC. DNA transfections of CT26 and YAMC cells were carried out in 12-well plates using Lipofectamine 2000 (Invitrogen). 24 h prior to transfection, cells were harvested using trypsin and plated at a density of 2 x 105 cells/well in 12-well plates in DMEM-10% FBS without antibiotics. 2 µg of plasmid DNA diluted in Opti-MEM I (Invitrogen) and 2 µl of Lipofectamine 2000 diluted in Opti-MEM I were combined and incubated for 20 min at room temperature. These reagents were incubated with the previously plated cells for 4 h at 37°C in a CO2 incubator. After 4 h, the transfection medium was then replaced with DMEM-10% FBS. 0.1 µg of pRL-SV40-Renilla, which contains a full-length Renilla reniformi luciferase gene under the control of a constitutive promoter was added to each well as a control for transfection efficiency between different wells. 24 h after transfection, the cells were harvested in 150 µl of reporter lysis buffer (Promega), and dual luciferase reporter assays were performed by following the manufacturer's directions. 20 µl of lysate was used for measurement in a luminometer (Turner Designs TD-20/20, Sunnyvale, CA). The results were reported as relative luciferase activity, which represents a ratio of luciferase firefly activity/Renilla activity. The data represent the mean ± S.D. of three different experiments repeated in triplicate.

Nuclear Extract Preparation—Monolayers of CT26 cells were washed with phosphate-buffered saline and harvested by scraping into cold phosphate-buffered saline. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride; then 10% Nonidet P-40 was added and vortexed briefly. The nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES, pH 7.9, 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM dithiothreitol, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14,000 rpm, and the supernatant containing the nuclear proteins was stored at –80 C until use.

Gel-shift and Supershift Assays—The assays were performed using CT26 and YAMC nuclear extracts. In competitive binding assays, unlabeled oligonucleotides were added at 100 M excess. Supershift assays were performed by the addition of 1 µl of polyclonal antibody directed against mouse Ets-1 (sc-111 X, Santa Cruz Biotechnology) and Runx2 (S-19 X, Santa Cruz Biotechnology; AML-3, Calbiochem). The oligonucleotides used in gel-shift assays were as follows (underlined for emphasis): probe A, nt –123 to –101 (5'-CCA GAG GAG GAA GTG TAG GAG CAG GT-3'); probe B, nt –145 to –121 (5'-TTT TTT TTT AAC CAC AAA ACC AGA G-3'); and probe C, nt –165 to –140 (5'-TGT TTC CTT TTC TTC CTT TTT TTT TT-3'). Each probe was prepared by end-labeling the double-stranded oligonucleotides with [32P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega, Madison, WI), followed by G-50 column purification (Biomax, Odenton, MD). The reactions were resolved on a 6% nondenaturing acrylamide gel in 1x Tris borate-EDTA buffer. All olignonucleotides used in the gel-shift assays were HPLC (high pressure liquid chromatography) grade. 20-bp oligonucleotides were synthesized to contain sequential 2–3-nt mutations (Fig. 3, C and D) and used as competitors in gel-shift assays to identify essential protein binding sites. Probe A mutants containing the nt –118 to –115 (5-GGAA-3' to 5'-TTTT-3') binding site mutation or the Ets-1 consensus binding site mutation (5'-gagACTTTTAtgtaggagcaggt-3') were constructed (Integrated DNA Technologies, Coralville, IA), labeled using [32P]ATP as described above, and used in gel-shift assays. Probe B mutants containing the nt –134 to –129 (5-CCACAA-3' to 5'-ACATTT-3') binding site mutation or the Runx2 consensus binding site mutation (5'-ttttGGACATTTaaccagaggaggaagtgta-3') were constructed (Integrated DNA Technologies), labeled using [32P]ATP as described above, and used in gel-shift assays.

Transcription Factor Data Base Analysis—The nucleotide sequence corresponding to the cis-regulatory domain in the OPN promoter was cross-referenced with the TRANSFAC transcription factor data base to identify consensus binding sites of known transcription factors. Candidate transcription factors were screened for using antibody-supershift analysis in the gel-shift assays, as described above, to confirm the presence of the factor in our oligonucleotide-protein complexes.

Chromatin Immunoprecipitation Assay—Chromatin isolated from CT26 and YAMC cells were fixed and immunoprecipitated using the ChIP assay kit (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. The purified chromatin was immunoprecipitated using 10 µg of anti-AML-3, anti-PEBP2{alpha}, anti-Ets-1, or IgG isotype. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The PCR product was 265 bp in length. The PCR program was: 94 °C for 4 min followed by 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s, for a total of 28 cycles, and then 72 °C for 7 min. PCR products were resolved in 10% acrylamide gels. The average size of the sonicated DNA fragments subjected to immunoprecipitation was 300 bp as determined by ethidium bromide gel electrophoresis. The ChIP assay utilized PCR primers 5'-CCTTTCATCCCCACTGATGT-3' and 5'-TGAGGTTTTTGCCACTACCC-3'.

Antisense Oligonucleotide Design and Assay—Sense and antisense oligonucleotides were designed according to GenBankTM sequences X53953 [GenBank] (Ets-1: sense, 5'-AGCCAACCCTACCTACCCAG-3'; antisense, 5'-TGGGTAGGTAGGGTTGGCT-3') and NM 009820 (Runx2: sense, 5'-GCCACCACTCACTACCACAC-3'; antisense, 5'-GTGTGGTAGTGAGTGGTGGC-3') to inhibit the expression of Ets-1 and Runx2. Transfection of sense or antisense to Ets-1 or Runx2 and cotransfection of Ets-1/Runx2 sense or antisense into CT26 cells was performed using Lipofectamine 2000, as described above. After 48 h, cells were collected for analysis of OPN protein expression by Western blotting.

Ets-1 and Runx2 siRNA and Assays—We utilized an RNAi-mediated approach to develop cell lines that could be utilized in an in vivo model of metastasis. Ets-1 and Runx2 siRNA (sc-35346, sc-37146; Santa Cruz Biotechnology) were used at a concentration of 10 µM for transient transfection of CT26 cells as described above. CT26 were transfected using mismatch siRNA or Ets-1/Runx2 siRNA. After 24 h, cells were transfected again using the corresponding siRNA. After 72 h from the initial transfection, cell lysates were collected and analyzed using Western blotting to confirm the extent of Ets-1, Runx2, and OPN protein expression. Subsequently, cells were assayed for viability by trypan blue exclusion. Stably down-regulated cell lines would then be developed for use in a previously established model of in vivo experimental metastasis (17).


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1.
Deletional analysis of OPN promoter-luciferase constructs in CT26 murine colon cancer cells. A, the histograms are representations of luciferase activity from OPN promoter constructs subcloned into pGL-3 and normalized to Renilla-luciferase activity from co-transfected pRL-SV40-Renilla plasmids. The lengths of the OPN promoter tested were OPN –88 (–88 to +79), OPN –258 (–258 to +79), OPN –472 (–472 to +79), OPN –670 (–670 to +79), and OPN –777 (–777 to +79). The values are expressed as the mean ± S.D. of three triplicate experiments. *, p < 0.05 versus OPN –88 and versus YAMC control. B, the histograms are representations of luciferase activity from OPN promoter constructs subcloned into pGL-3 and normalized to Renilla-luciferase activity from co-transfected pRL-SV40-Renilla plasmids. The length of the OPN promoter –88 to –258 was further analyzed using OPN –69 (–69 to +79), OPN –107 (–107 to +79), OPN –120 (–120 to +79), OPN –134 (–134 to +79), OPN –160 (–160 to +79), OPN –174 (–174 to +79), OPN –209 (–209 to +79), and OPN –258 (–258 to +79). The values are expressed as the mean ± S.D. of three triplicate experiments. *, p < 0.05 versus OPN –69.

 
Statistical Analysis—Data are expressed as mean ± S.D. Statistical analysis was performed using SigmaStat, version 3 (Systat Software, Point Richmond, CA). Individual comparisons were made with Student's t test. The criterion for significance was p < 0.05 for all comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transient Transfection Analysis of OPN Promoter Deletion Constructs—To localize potential cis-acting elements in the OPN promoter, deletion constructs were analyzed in CT26 colon cancer cells using transient transfection (Fig. 1). Serial deletion constructs demonstrated a significant 3-fold increase in luciferase activity between nt –88 and –258 in CT26 cells (Fig. 1A). Transient transfection of these deletion constructs in YAMC, a nonmetastatic colonic epithelial cell line, did not demonstrate any increased luciferase activity (Fig. 1A). CT26 demonstrated an ~8-fold increased luciferase activity in comparison with YAMC. Deletion fragments containing nucleotides upstream from –258 did not confer any additional, significant increase in luciferase activity in CT26 cells (Fig. 1A). Using further serial deletion constructs, this area of increased OPN promoter activity was further localized to the length of the promoter from nt –107 to –160 (Fig. 1B). Fragment –120 and –134 demonstrated an incremental 3.8-fold and a 6.4-fold increase in luciferase activity in comparison with control. These results suggest that more than one enhancer may reside in the nt –107 to –160 cis-regulatory domain of the OPN promoter.


Figure 2
View larger version (84K):
[in this window]
[in a new window]
  		FIGURE 2.
Gel-shift analysis of the nt –107 to –160 cis-regulatory domain. [32P]ATP-labeled oligonucleotides corresponding to probe A (nt –101 to –125), probe B (nt –121 to –145), and probe C (nt –140 to –165) were used to screen the nt –107 to –160 domain for protein binding regions (lanes 4–6). Three complexes (E1, E2, and E3) were formed in the presence of probe A. Two complexes (R1 and R2) were formed with probe B. Unlabeled specific competitor oligonucleotides (SC) corresponding to probes A, B, and C were used in >50-fold excess to evaluate the specificity of binding to the nuclear proteins (lanes 7–9). A nonspecific unlabeled competitor oligonucleotide (NC) corresponding to nt –174 to –202 was used as control (lanes 10–12). Free probe in the absence of nuclear extract is also shown (lanes 1–3). The blot is representative of three separate experiments. Comp, competitor.

 
Gel-shift Analysis—To determine whether trans-activating nuclear factors may regulate the nt –107 to –160 region of the OPN promoter, gel-shift analysis was performed using [32P]ATP-labeled fragments (~20 to 30 nt) that span this region of interest (Fig. 2). Nuclear proteins were isolated from CT26 cells and incubated with three overlapping, labeled oligonucleotide probes: probe A (nt –101 to –123); probe B (nt –121 to –145); and probe C (nt –140 to –165), as described under "Experimental Procedures." Nuclear proteins bound to probe A (Fig. 2, lane 4) and probe B (Fig. 2, lane 5) but not to probe C. Three gel-shift complexes (E1, E2, and E3) were present with probe A, whereas two distinct complexes (R1 and R2) were seen with probe B. Gel-shift complexes E1, E2, and E3 and R1 were extinguished in the presence of 50-fold excess unlabeled probe A or B, respectively (Fig. 2, lanes 7 and 8), but persisted in the presence of a 50-fold excess of nonspecific unlabeled competitor (Fig. 2, lanes 10 and 11). These gel-shift complexes corresponding to E1, E2, E3, and R1 suggest that potential trans-regulatory nuclear proteins are bound to the OPN promoter in the region of nt –101 to –145 in CT26 cells.

The binding sites were further characterized by serial mutations of the OPN promoter between nt –101 to –123 in probe A (Fig. 3A) and between nt –121 to –145 in probe B (Fig. 3C). These mutated sequences were then used as excess unlabeled competitors in gel-shift assays (Fig. 3, B and D). Mutation of nucleotides –115 to –118 in probe A resulted in persistence of complexes E1, E2, and E3 (Fig. 3B, lanes 8 and 9), whereas mutations of nt –101 to –114 and nt –119 to –123 resulted in ablation of complexes E1, E2, and E3 in competition gel-shift assays (Fig. 3B, lanes 1–7, 10, and 11). These data suggest that nt –115 to –118 (AAGG) represents an essential binding site for the nuclear proteins corresponding to the E1, E2, and E3 complexes. Mutation of nt –129 to –134 in probe B resulted in persistence of complexes R1 and R2 (Fig. 3D, lanes 5–7), whereas mutations of nt –121 to –128 and –135 to –145 resulted in ablation of R1 in competition gel-shift assays (Fig. 3D, lanes 1–4 and 8–12). These data suggest that nt –129 to –134 (AACACC) represents an essential binding site for the nuclear proteins corresponding to the R1 complex. Together, these data from the probe A and B serial deletions indicate that two potential enhancer binding sites (nt –115 to –118 and nt –129 to –134) reside within the cis-regulatory region nt –101 to –145.

Identification of trans-Regulatory Proteins and Confirmation of DNA-Protein Binding—To identify the nuclear factors that bind our cis-element of interest, we searched the TRANSFAC data base for consensus binding sites of known transcription factors within the nt –101 to –145 segment (Fig. 4). The region nt –115 to –118 matched with the known consensus binding site of Ets (Fig. 4A), whereas the region nt –129 to –134 matched with Runx2, SBF-1 (spliceosome-binding factor-1), and C/EBP (Fig. 4B). We selected these nuclear proteins as candidates for specific binding in supershift and ChIP assays. Gelshift assays were repeated in the presence of Ets-1 antibody (Fig. 5A, lanes 7 and 8) and Runx2 antibodies (Fig. 5B, lanes 7–8 and 10–11) with the previously described DNA probes A and B. In the presence of antibody against Ets-1, complexes E1, E2, and E3 were diminished, and a specific supershift band was formed (Fig. 5A, lanes 7 and 8). In the presence of antibody against Runx2 (S-19 X), complex R1 was diminished with formation of a supershift band (Fig. 5B, lanes 7 and 8). This effect was more pronounced with the use of {alpha}-aml-3, a different antibody raised against Runx2, with the formation of an intense supershift band (Fig. 5B, lanes 10 and 11). No shift was detected using IgG isotype, and no complexes were formed between the antibodies and DNA probe in the absence of nuclear extract (Fig. 5, A, lane 5, and B, lanes 6 and 9). No supershift complexes were formed using antibodies to SBF-1 and C/EBP (data not shown). ChIP assays were then performed to confirm the in vivo binding of Ets-1 and Runx2 to this portion of the OPN promoter (Fig. 5C). Control reactions using no antibody or IgG isotype did not exhibit specific immunoprecipitation, whereas reactions containing antibodies to Ets-1 or Runx2 demonstrated specific binding to this region of the OPN promoter. Chromatin from YAMC cells did not result in specific Ets-1 or Runx2 antibody-mediated immunoprecipitation (Fig. 5D). Together, these data suggest that Ets-1 and Runx2 bind specifically to enhancer domains in the cis-regulatory region nt –101 to –145 of the OPN promoter in CT26 cells.


Figure 3
View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 3.
Characterization of the nuclear binding sites using competitive gel-shift analysis. A, to sublocalize binding sites within nt –101 to –123, 11 mutant oligonucleotides based on the probe A template were synthesized to contain sequential 2–3-bp mutations. WT, wild type. B, these 11 mutant unlabeled oligonucleotides were used in >50-fold excess in competitive gel-shift assays against 32P-labeled probe A (nt –101 to –123). Differential ablation of nuclear protein-probe A binding is shown for mutants 1–11. Control reactions showing NE + probe A, NE + probe A + specific competitor (SC), and NE + probe A + nonspecific competitor (NC) are shown for comparison. Three specific complexes (E1, E2, and E3) are distinguishable in the blot. The blot is representative of three separate experiments. C, to sublocalize binding sites within nt –120 to –145, 12 mutant oligonucleotides based on the probe B template were synthesized to contain sequential 2–3-bp mutations. D, these 12 mutant unlabeled oligonucleotides were used in >50-fold excess in competitive gel-shift assays against 32P-labeled probe B (nt –120 to nt –145). Differential ablation of nuclear protein-probe B binding is shown for mutants 1–12. Control reactions showing NE + probe B, NE + probe B + specific competitor, and NE + probe B + nonspecific competitor are shown for comparison. Two specific complexes (R1 and R2) are distinguishable in the blot. The blot is representative of three separate experiments.

 


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4.
Consensus binding sites of known transcription factors in the cis-regulatory region nt –101 to –145. The TRANSFAC data base was queried for consensus binding sites of known transcription factors for the sequence nt –101 to –145. A, binding sites are shown for Ets, upstream stimulating factor (USF), and alcohol dehydrogenase regulator-1 (ADR1) in the domain –101 to –123. B, binding sites are shown for C/EBP, Runx2, and spliceosome-binding factor-1 (SBF-1).

 
Functional Analysis of ETS-1 and Runx2 on DNA Binding, OPN Transcription, and Protein Expression—To determine whether trans-activation of Ets-1 and Runx2 is critical to the transcription of OPN in CT26 cells, we constructed OPN promoter sequences containing mutant binding sites for Ets-1 and Runx2 and analyzed them in gel-shift and luciferase-promoter assays. Mutation of nt –115 to –118 from AAGG to TTTT resulted in ablation of the E1 and E3 complexes with significant reduction in the binding of E2 in gel-shift assays (Fig. 6, lane 2). This effect was duplicated by mutating the consensus binding site for Ets-1 with complete ablation of complexes E1, E2 and E3 (Fig. 6, lane 3). Both mutation of nt –129 to –134 from AACACC to TTTACA and mutation of the consensus binding site for Runx2 resulted in ablation of complexes R1 and R2 (Fig. 6, lanes 5 and 6).


Figure 5
View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 5.
Identification of the trans-regulatory protein and confirmation of binding. A, gel-shift assays using probe A were repeated in the presence of antibody to Ets-1. Supershift bands (SS) were detected with increasing concentration of anti-Ets-1 in lane 7 (µg/µl) and lane 8 (µg/µl). Probe A + NE + IgG isotype (lane 6), probe A + anti-Ets-1 in the absence of NE (lane 5), free probe A (lane 1), probe A + NE (lane 2), probe A + NE + specific competitor (SC)(lane 3), and probe A + NE + nonspecific competitor (NC) (lane 4) were used as controls. This blot is representative of three experiments. B, gel-shift assays using probe B were repeated in the presence of antibodies to Runx2 (S-19 X and {alpha}-aml-3). Supershift bands were detected with increasing concentration of S-19 X and {alpha}-aml-3 in lanes 7 and 8 and lanes 10 and 11, respectively. Probe B + NE + IgG isotype (lane 5), probe B + S-19 X in the absence of NE (lane 6), probe B + {alpha}-aml-3 in the absence of NE (lane 9), free probe B (lane 1), probe B + NE (lane 2), probe B + NE + specific competitor (lane 3), and probe B + NE + nonspecific competitor (lane 4) were used as controls. This blot is representative of three experiments. C, ChIP analysis of Ets-1 and Runx2 binding. Chromatin from CT26 cells was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer (Upstate Biotechnology). The purified chromatin was immunoprecipitated using 10 µg of anti-Ets-1, SC-19 X, anti-aml-3, corresponding IgG isotype, or no antibody (no Ab) as control. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of three experiments. D, chromatin from YAMC cells was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer (Upstate Biotechnology). The purified chromatin was immunoprecipitated using 10 µg of {alpha}-Ets-1, SC-19 X, {alpha}-aml-3, the corresponding IgG isotype, or no antibody as control. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of three experiments.

 
The OPN promoter fragment –512 demonstrated promoter activity similar to full-length OPN promoter in CT26 cells (data not shown) and was used as a template for constructing consensus mutants. OPN –512 wild type, OPN –512{Delta}Ets, OPN –{Delta}Runx, and OPN –{Delta}Ets/Runx double mutant were subsequently transfected into CT26 cells (Fig. 7A). Luciferase activity was reduced by ~40 and ~52% in OPN –512{Delta}Ets and OPN –512{Delta}Runx, respectively, in comparison with OPN –512 wild type (Fig. 7A). In OPN –{Delta}Ets/Runx double mutant, luciferase activity was reduced by ~63% in comparison to OPN –512 wild type.

Transient transfection of antisense oligonucleotides against Ets-1 and Runx2 resulted in a significant >3-fold and >4-fold decrease in OPN protein expression, respectively (Fig. 7B). Co-transfection of Ets-1 and Runx2 antisense resulted in a >7-fold decrease in OPN protein expression in comparison with control (Fig. 7B). Together, these data suggest that ETS-1 and Runx2 trans-activation are critical for OPN expression in CT26 cells.

Functional Analysis of ETS-1 and Runx2 Down-regulation on in Vivo Metastasis—We aimed to develop stable Ets-1/Runx2-down-regulated cell lines that could be used in in vivo assays of tumor metastasis. Initially, Ets-1 and Runx2 siRNA were used to evaluate for efficiency of OPN down-regulation. Co-transfection of Ets-1 and Runx2 siRNA resulted in decreased levels of OPN protein comparable with that achieved with antisense (data not shown). However, analysis of tumor cell viability at 72 h after transfection demonstrated that >76% of cells were nonviable compared with control in triplicate experiments (Fig. 8). No viable cells were seen in culture for Ets-1/Runx2 siRNA transfectants at >4 days in culture (data not shown). Cell lines that stably expressed down-regulated levels of Ets-1/Runx2 could not be generated for use in in vivo murine metastasis models.


Figure 6
View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 6.
Gel-shift analysis of Ets-1 and Runx2 interactions with bindingsite mutations. Gel-shift assays were repeated using mutant probes containing either the –118 to –115 (GGAA to TTTT) mutation (lane 2: probe A-{Delta}GGAA), the Ets-1 consensus binding site mutation (lane 3), the –134 to –129 (CCACAA to ACATTT) mutation (lane 5: probe B-{Delta}CCACAA)), or the Runx2 consensus binding site mutation (lane 6). Control reactions with probe A + NE and probe B + NE are shown (lanes 1 and 4). The blot is representative of three experiments. WT, wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
OPN is a ~298-amino acid secreted phosphoprotein that is expressed at high levels by T-lymphocytes, epidermal cells, bone cells, macrophages, endothelial cells, and tumor cells in remodeling processes such as inflammation, ischemia-reperfusion, bone resorption, arteriosclerosis, and tumor progression (3). Increased OPN expression is associated with tumor invasion or metastasis in cancers of the breast (23–25), stomach (26), lung (27, 28), prostate (29), liver (30, 31), and colon (15). In previous studies, we have shown that up-regulated expression of OPN is a critical determinant of the metastatic phenotype of CT26 colon cancer cells. Using RNA interference, stable down-regulation of OPN protein levels by ~3.0-fold compared with CT26 wild type resulted in a >3.0-fold decrease in MMP-2 expression, a 3.6-fold decrease in tumor cell motility in vitro, a 4.1-fold reduction in tumor invasiveness in vitro, and a 2.0-fold attenuation of in vivo hepatic metastasis (17). This demonstration of OPN function in colorectal metastasis supports similar data from various cancer models (32–34). Although the target genes activated by OPN in metastasis are being actively investigated, the molecular mechanisms that regulate this increased OPN expression in colorectal cancer remain unknown. Analysis of the OPN promoter has uncovered multiple consensus binding sites for known transcription factors (3–8, 35). Several signaling pathways/transcription factors regulate OPN expression in models of breast cancer, melanoma, and leukemia; these include AP-1, Myc, Oct-1, upstream stimulating factor (USF), v-Src, TGF-beta/BMPs/Smad/Hox, Wnt/beta-catenin/APC/GSK-3beta/Tcf-4, Ras/RRF, and TP53 (3–8). To date, the regulatory factors that control OPN expression in colorectal cancer are not known.


Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 7.
Transient transfection analysis of Ets-1 and Runx2 consensus binding site mutations on OPN promoter activity. A, the histograms are representations of luciferase activity from OPN promoter constructs containing Ets-1 consensus binding site mutations (–512{Delta}Ets), Runx2 consensus binding site mutations (–512{Delta}Runx), or combined Ets-1/Runx2 binding site mutations (–512{Delta}Dm) subcloned into pGL-3 and normalized to Renilla-luciferase activity from co-transfected pRL-SV40-Renilla plasmids. Deletion fragments corresponding to OPN –69 to +79, –120 to +79, and –512 to +79 were included as controls. The values are expressed as the mean ± S.D. of three triplicate experiments. *, p < 0.05 versus –512 wild type (wt). B, transient transfection analysis of antisense oligonucleotides to Ets-1 and Runx2 and their effect on OPN protein expression. Sense and antisense oligonucleotides to Ets-1 (lanes 2 and 4), Runx2 (lanes 3 and 5), or Ets-1/Runx2 (lanes 6 and 7) were transiently transfected into CT26 cells. Cell lysates were collected after 48 h, and OPN protein levels were measured in Western blots. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels are shown as an internal control for loading. The blot is representative of three separate experiments.

 


Figure 8
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 8.
Transient transfection analysis of Ets-1 and Runx2 down-regulation on CT26 proliferation. The histograms are representations of viable cells 72 h after transfection with mismatch siRNA or Ets-1/Runx2 siRNA. Cells were cultured in 12-well plates, transfected with siRNA at a concentration of 10 µM, and retransfected 24 h later. Cell viability was determined using trypan blue exclusion at 72 h after initial transfection. The blot is representative of the mean ± S.D. of three separate experiments.

 
In this present study, we have identified a specific OPN promoter region, nt –107 to –160, which significantly increases transcriptional activity in CT26 colorectal cancer cells. Using gel-shift assays, two cis-regulatory enhancer domains were localized to nt –115 to –118 and nt –129 to –134. These regulatory domains were demonstrated to specifically bind the nuclear proteins Ets-1 and Runx2. Loss-of-trans-activation of these nuclear factors through mutation of the consensus binding sites or through antisense inhibition resulted in decreased OPN promoter activity and protein expression. These data suggest that Ets-1 and Runx2 are critical transcription factors that regulate OPN expression in CT26 colorectal cancer cells. A direct analysis of Ets-1/Runx2(–/–) CT26 mutants in an in vivo metastasis model was not feasible, but we have previously demonstrated that OPN expression is integral to the CT26 metastatic phenotype in a syngeneic BALB/c murine model (17). In the context of this previously published data, our current study demonstrating in vitro down-regulation of OPN expression by Ets-1/Runx2 antisense and in vivo binding of Ets-1/Runx2 to CT26 DNA in our ChIP assays strongly suggest that Ets-1 and Runx2 function as regulators of OPN-mediated metastasis in CT26 cells.

Ets-1 and Runx2 are critical to normal physiologic development, and their activity is also enhanced during tumorigenesis. Ets-1 is expressed in lymphoid cells, developing T and B cells, endothelial cells, and vascular smooth muscle cells (36–39), and it belongs to a family of ~30 mammalian nuclear transcription factors. These proteins contain multiple conserved domains including a winged-helix-loop-helix domain that mediates binding to purine-rich DNA sequences with a central GGA(A/T) core, a Pointed domain that regulates protein-protein interactions, a Ras-responsive phosphorylation site at threonine 38, an autoinhibitory module on exon VII that is activated by calcium-dependent phosphorylation on Ser-25, Ser-257, Ser-282, and Ser-285, and a C-terminal domain that mediates trans-activation (40–43). The Ets nuclear proteins cooperate with other transcription factors to activate or repress transcription in a variety of processes including cell proliferation, apoptosis, development, differentiation, angiogenesis, and oncogenic transformation (41–43). Augmentation of Ets-mediated gene transcription results from coactivation with AP-1, AML-1, CBP (CREB-binding protein)/p300, Lef-1 Sp1, NF-{kappa}B, Stat-5, and Maf-B, whereas suppression of Ets function occurs with Erg, Daxx, and Ubc9 (41–43). The Runx gene family encodes the DNA binding {alpha}-subunit of a heterodimeric transcription complex. The Runx genes have several aliases including acute myeloid leukemia (AML), core-binding factor-{alpha} (CBF{alpha}), and polyomavirus enhancer-binding factor-2 (PEBP2) (44). The Runx transcription factors play a pivotal role during normal development and tumorigenesis (45–47). The mammalian Runx genes (Runx1, Runx2, Runx3) encode three different isoforms of the {alpha}-subunit and share a highly conserved 128-amino acid Runt homology domain that mediates DNA binding. Dimerization with the PEBP2beta/CBFbeta-subunit enhances DNA binding of the {alpha}-subunit (47). Despite their structural similarity, the Runx genes have divergent biological roles in mammalian development with Runx1-, Runx2-, and Runx3-null mice showing major defects in hematopoietic, osteoblastic and neuronal development, respectively (48).

Ets-1 and Runx2 protein expression are up-regulated in tumor progression, and their activation is intimately linked with cellular migration, invasion, and tumor metastasis. Ets-1 transforms NIH3T3 fibroblasts (41), regulates MMP-1, MMP-3, MMP-7, and MMP-9, and urokinase-type plasminogen activator during tumor invasion and metastasis, and is in turn up-regulated by VEGF, basic fibroblast growth factor (bFGF), and hypoxia-inducible factor-1 (HIF-1) during tumor angiogenesis (41–43, 49). In the context of colorectal cancer, immunohistochemical studies have shown that Ets-1 expression is significantly increased in adenomas, carcinomas in situ, and colonic adenocarcinomas but is absent from normal or hyperplastic polyps (50). In other colon cancer models, Ets-1 and Ets-2 expression is directly linked to lymph node metastasis (50), depth of invasion (51), VEGF expression, and lowered survival rates (52). In contrast, there have been no published studies describing a similar role for Runx2 in colorectal carcinoma. However, the Runx genes have been shown to function in tumor suppression of gastric cancer (53), granulocytic differentiation of myeloid precursor cells and leukemias (54), and osteoblast differentiation and cleidocranial dysplasia (55–57). Both Runx enhancer and repressor activity have been demonstrated previously in molecular models (45, 58).

Together, these previous studies provide a limited understanding of how Ets-1 and Runx2 mediate functional colorectal metastasis. In this context, our study represents the first report describing a mechanistic role for Ets-1 and Runx2 in the trans-activation of OPN expression in a colon cancer cell line. There are previous reports using osteogeneic (59) and mammary cell lines (60) that describe a regulatory role for these factors in OPN expression. Runx2 was shown to induce OPN expression in NIH3T3 fibroblasts with Ets-1 acting as a synergistic co-activator (61). More recently, Runx2 activation of OPN expression has been reported in metastatic mammary epithelial cells (55, 62). Inman and Shore (55) confirm that Runx2 functions as a transcriptional activator of the murine OPN promoter in HC11 mouse mammary epithelial cells and that targeted down-regulation of Runx2 results in >50% reduction of OPN promoter activity in HC11 cells (55). In other studies, El-Tanani et al. (60) demonstrate that a cooperative interaction between Ets, PEA3, beta-catenin/Lef-1, and AP-1 activates OPN transcription in RAMA 37 cells (60). In the context of these data, our results add support to the role of Ets-1 and Runx2 as significant activators of cancer metastasis.

Our attempts to develop stable Ets-1/Runx2 down-regulated cell lines for use in functional, in vivo metastatic assays were unsuccessful, as our Ets-1/Runx2-deficient cell lines were not viable. These results are consistent with data from other investigators who have demonstrated that Ets-1 knock-out results in increased T-cell apoptosis (63), abnormal development and differentiation in NK cells and B-cells (64, 65), reduced cell adhesiveness in HeLa cells (66), and diminished cell proliferation in glioma cells (67). In a similar fashion, Runx2 gene knock-out in mice causes a lethal phenotype (57) and RNAi-mediated down-regulation of the Runx homologue, run, in Caenorhabditis elegans results in an early larval lethal phenotype (69). Recent studies from Passaniti and colleagues (70) demonstrate that RNAi-mediated knock-out of Runx2 inhibits cell proliferation and delays cell cycle progression in human bone marrow endothelial cells. This inhibition of cell proliferation occurred over a similar time course in comparison with our data. Together, these data suggest that Ets-1 and Runx2 regulate essential, basic processes during mesenchymal cell differentiation and also during tumor progression.

An investigation into the molecular interactions between Ets-1 and Runx2 in our cell model will be the focus of future studies. We suspect that Ets-1 and Runx2 cooperate to activate OPN transcription in a synergistic fashion in CT26. Previous studies in other cell models have shown that Ets-1 can bind the Runx2-related AML-1. Ets-1 stimulates DNA binding activity of AML-1 by associating with its negative regulatory domain for DNA binding (NRDB) (71, 72). In turn, AML-1 binds to the exon VII domain and blocks the inhibitory module of Ets-1 (71, 68). This cooperative synergism is supported by the findings of Sato et al. (61), who demonstrate that Ets-1 and PEBP2{alpha} cooperate to regulate OPN transcription in skeletal tissues (61). The precise mechanisms that govern the regulated activity of Ets-1 and Runx2 are complex and unknown. Further insight into the study of these paired transcriptional activators of OPN may identify potential therapeutic targets in the regulation of cancer metastasis.


   FOOTNOTES
 
* This work was supported by a Clowes faculty development award (to P. C. K.) from the American College of Surgeons, National Institutes of Health Grants R01AI44629 and R01GM65113 (to P. C. K.), and an Ethicon-Society of University Surgeons fellowship award (to P. Y. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Surgery, 330 Cedar St., FMB102, Yale University School of Medicine, New Haven, CT 06510. Tel.: 203-785-2697; Fax: 203-737-2116; E-mail: philip.wai{at}yale.edu.

2 The abbreviations used are: OPN, osteopontin; YAMC, young adult mouse colon cells; Ets, E26 transformation-specific sequence; RNAi, RNA interference; C/EBP, CCAATT/enhancer-binding protein; VEGF, vascular endothelial growth factor; nt, nucleotide(s); NE, nuclear extract; MMP, matrix metalloproteinase; ChIP, chromatin immunoprecipitation; siRNA, short interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AML, acute myeloid leukemia. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R. C., Ghafoor, A., Feuer, E. J., and Thun, M. J. (2005) CA-Cancer J. Clin. 55, 10–30[Abstract/Free Full Text]
  2. Parker, S. L., Tong, T., Bolden, S., and Wingo, P. A. (1996) CA-Cancer J. Clin. 46, 5–27[Abstract]
  3. Wai, P. Y., and Kuo, P. C. (2004) J. Surg. Res. 121, 228–241[CrossRef][Medline] [Order article via Infotrieve]
  4. Denhardt, D. T., and Guo, X. (1993) FASEB J. 7, 1475–1482[Abstract]
  5. Craig, A. M., and Denhardt, D. T. (1991) Gene 100, 163–171[CrossRef][Medline] [Order article via Infotrieve]
  6. Denhardt, D. T., and Noda, M. (1998) J. Cell. Biochem. Suppl. 30–31, 92–102
  7. O'Regan, A., and Berman, J. S. (2000) Int. J. Exp. Pathol. 81, 373–390[CrossRef][Medline] [Order article via Infotrieve]
  8. Weber, G. F. (2001) Biochim. Biophys. Acta 1552, 61–85[Medline] [Order article via Infotrieve]
  9. Goodison, S., Urquidi, V., and Tarin, D. (1999) Mol. Pathol. 52, 189–196[Abstract]
  10. Denhardt, D. T., Noda, M., O'Regan, A. W., Pavlin, D., and Berman, J. S. (2001) J. Clin. Investig. 107, 1055–1061[Medline] [Order article via Infotrieve]
  11. Weber, G. F., Ashkar, S., Glimcher, M. J., and Cantor, H. (1996) Science 271, 509–512[Abstract]
  12. Katagiri, Y. U., Sleeman, J., Fujii, H., Herrlich, P., Hotta, H., Tanaka, K., Chikuma, S., Yagita, H., Okumura, K., Murakami, M., Saiki, I., Chambers, A. F., and Uede, T. (1999) Cancer Res. 59, 219–226[Abstract/Free Full Text]
  13. Ashkar, S., Weber, G. F., Panoutsakopoulou, V., Sanchirico, M. E., Jansson, M., Zawaideh, S., Rittling, S. R., Denhardt, D. T., Glimcher, M. J., and Cantor, H. (2000) Science 287, 860–864[Abstract/Free Full Text]
  14. Gao, C., Guo, H., Downey, L., Marroquin, C., Wei, J., and Kuo, P. C. (2003) Carcinogenesis 24, 1871–1878[Abstract/Free Full Text]
  15. Agrawal, D., Chen, T., Irby, R., Quackenbush, J., Chambers, A. F., Szabo, M., Cantor, A., Coppola, D., and Yeatman, T. J. (2002) J. Natl. Cancer Inst. 94, 513–521[Abstract/Free Full Text]
  16. Yeatman, T. J., and Chambers, A. F. (2003) Clin. Exp. Metastasis 20, 85–90[CrossRef][Medline] [Order article via Infotrieve]
  17. Wai, P. Y., Mi, Z., Guo, H., Sarraf-Yazdi, S., Gao, C., Wei, J., Marroquin, C. E., Clary, B., and Kuo, P. C. (2005) Carcinogenesis 26, 741–751[Abstract/Free Full Text]
  18. Corbett, T. H., Griswold, D. P., Jr., Roberts, B. J., Peckham, J. C., and Schabel, F. M., Jr. (1975) Cancer Res. 35, 2434–2439[Abstract/Free Full Text]
  19. Ikubo, A., Aoki, Y., Nagai, E., and Suzuki, T. (1999) Clin. Exp. Metastasis 17, 849–855[CrossRef][Medline] [Order article via Infotrieve]
  20. Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P., and Jat, P. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 587–591[Abstract/Free Full Text]
  21. Pozzi, A., Yan, X., Macias-Perez, I., Wei, S., Hata, A. N., Breyer, R. M., Morrow, J. D., and Capdevila, J. H. (2004) J. Biol. Chem. 279, 29797–29804[Abstract/Free Full Text]
  22. Gao, C., Guo, H., Wei, J., Mi, Z., Wai, P., and Kuo, P. C. (2004) J. Biol. Chem. 279, 11236–11243[Abstract/Free Full Text]
  23. Tuck, A. B., O'Malley, F. P., Singhal, H., Harris, J. F., Tonkin, K. S., Kerkvliet, N., Saad, Z., Doig, G. S., and Chambers, A. F. (1998) Int. J. Cancer 79, 502–508[CrossRef][Medline] [Order article via Infotrieve]
  24. Tuck, A. B., O'Malley, F. P., Singhal, H., Tonkin, K. S., Harris, J. F., Bautista, D., and Chambers, A. F. (1997) Arch. Pathol. Lab. Med. 121, 578–584[Medline] [Order article via Infotrieve]
  25. Tuck, A. B., Arsenault, D. M., O'Malley, F. P., Hota, C., Ling, M. C., Wilson, S. M., and Chambers, A. F. (1999) Oncogene 18, 4237–4246[CrossRef][Medline] [Order article via Infotrieve]
  26. Ue, T., Yokozaki, H., Kitadai, Y., Yamamoto, S., Yasui, W., Ishikawa, T., and Tahara, E. (1998) Int. J. Cancer 79, 127–132[CrossRef][Medline] [Order article via Infotrieve]
  27. Shijubo, N., Uede, T., Kon, S., Maeda, M., Segawa, T., Imada, A., Hirasawa, M., and Abe, S. (1999) Am. J. Respir. Crit. Care Med. 160, 1269–1273[Abstract/Free Full Text]
  28. Chambers, A. F., Wilson, S. M., Kerkvliet, N., O'Malley, F. P., Harris, J. F., and Casson, A. G. (1996) Lung Cancer 15, 311–323[CrossRef][Medline] [Order article via Infotrieve]
  29. Thalmann, G. N., Sikes, R. A., Devoll, R. E., Kiefer, J. A., Markwalder, R., Klima, I., Farach-Carson, C. M., Studer, U. E., and Chung, L. W. (1999) Clin. Cancer Res. 5, 2271–2277[Abstract/Free Full Text]
  30. Gotoh, M., Sakamoto, M., Kanetaka, K., Chuuma, M., and Hirohashi, S. (2002) Pathol. Int. 52, 19–24[CrossRef][Medline] [Order article via Infotrieve]
  31. Pan, H. W., Ou, Y. H., Peng, S. Y., Liu, S. H., Lai, P. L., Lee, P. H., Sheu, J. C., Chen, C. L., and Hsu, H. C. (2003) Cancer 98, 119–127[CrossRef][Medline] [Order article via Infotrieve]
  32. Irby, R. B., McCarthy, S. M., and Yeatman, T. J. (2004) Clin. Exp. Metastasis 21, 515–523[CrossRef][Medline] [Order article via Infotrieve]
  33. Mi, Z., Guo, H., Wai, P. Y., Gao, C., Wei, J., and Kuo, P. C. (2004) J. Biol. Chem. 279, 46659–46667[Abstract/Free Full Text]
  34. Rangaswami, H., Bulbule, A., and Kundu, G. C. (2005) J. Biol. Chem. 280, 19381–19392[Abstract/Free Full Text]
  35. Hijiya, N., Setoguchi, M., Matsuura, K., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1994) Biochem. J. 303, 255–262[Medline] [Order article via Infotrieve]
  36. Ghysdael, J., Gegonne, A., Pognonec, P., Dernis, D., Leprince, D., and Stehelin, D. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1714–1718[Abstract/Free Full Text]
  37. Anderson, M. K., Hernandez-Hoyos, G., Diamond, R. A., and Rothenberg, E. V. (1999) Development (Camb.) 126, 3131–3148[Abstract]
  38. Tanaka, K., Oda, N., Iwasaka, C., Abe, M., and Sato, Y. (1998) J. Cell. Physiol. 176, 235–244[CrossRef][Medline] [Order article via Infotrieve]
  39. Naito, S., Shimizu, S., Maeda, S., Wang, J., Paul, R., and Fagin, J. A. (1998) Am. J. Physiol. 274, C472–C480 Ref 39: Is Vol. 274 correct?[Medline] [Order article via Infotrieve]
  40. Petersen, J. M., Skalicky, J. J., Donaldson, L. W., McIntosh, L. P., Alber, T., and Graves, B. J. (1995) Science 269, 1866–1869[Abstract/Free Full Text]
  41. Dittmer, J. (2003) Mol. Cancer 2003, 2: 29[CrossRef][Medline] [Order article via Infotrieve]
  42. Oikawa, T., and Yamada, T. (2003) Gene 303, 11–34[CrossRef][Medline] [Order article via Infotrieve]
  43. Hsu, T., Trojanowska, M., and Watson, D. K. (2004) J. Cell. Biochem. 91, 896–903[CrossRef][Medline] [Order article via Infotrieve]
  44. van Wijnen, A. J., Stein, G. S., Gergen, J. P., Groner, Y., Hiebert, S. W., Ito, Y., Liu, P., Neil, J. C., Ohki, M., and Speck, N. (2004) Oncogene 23, 4209–4210[CrossRef][Medline] [Order article via Infotrieve]
  45. Lund, A. H., and Lohuizen, M. (2002) Cancer Cell 1, 213–215[CrossRef][Medline] [Order article via Infotrieve]
  46. Coffman, J. A. (2003) Cell Biol. Int. 27, 315–324