p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis, and maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We show by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function and loss-of-function experimental systems. Induction of wild-type p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (a) wild-type p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (b) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. [Cancer Res 2009;69(3):753–7]
- breast cancer
p53 is a tumor suppressor gene that is frequently mutated in human cancers, including breast cancer ( 1). The p53 gene encodes a transcription factor that plays a central role in the activation of DNA repair, cell cycle arrest, initiation of apoptosis, and maintenance of genome stability. In normal cells p53 is typically inactive; on activation, the p53 protein is transiently stabilized and accumulates in the nucleus where it functions, in part, to induce or repress the transcription of downstream target genes. Identification of p53-regulated genes has provided critical insights into understanding the biological function of the p53 protein.
Epithelial cell adhesion molecule (EpCAM) is a 40-kDa cell-surface glycoprotein that is expressed at the basolateral membrane of the majority of epithelial tissues. EpCAM is overexpressed in the majority of human epithelial cancers ( 2) and has long been considered to be a candidate for molecular therapy ( 3). Of note, EpCAM expression in primary breast cancers has been associated with poor clinical outcome ( 4– 6). In these studies of primary breast cancer specimens from more than 2,300 patients, EpCAM expression was independently associated with prognosis. Recent in vitro studies confirm the potential functional role of EpCAM in breast cancer; specific ablation of EpCAM expression using RNA interference results in a dramatic decrease in the invasive potential of breast cancer cell lines ( 7).
Despite recent interest in the functional biology of EpCAM, the transcriptional regulation of EpCAM in breast and other epithelial cancers remains to be elucidated. There is experimental evidence to suggest that p53 may regulate the methylation status and amplification of the EpCAM gene ( 8). However, recent analyses of primary breast cancer specimens show no significant correlation between promoter methylation and EpCAM expression, suggesting that alternate mechanisms may be involved in the regulation of EpCAM expression ( 9). Because p53 loss or mutation is a common event in human breast cancer, we investigated the potential role of wild-type p53 in the regulation of EpCAM expression.
Materials and Methods
Cell culture. Human breast epithelial and cancer cell lines MCF10A and MCF10CA1a (CA1a) were obtained from Dr. Fred Miller (Wayne State University, Detroit, MI). MCF-7 cells were obtained from American Type Culture Collection. Wild-type and p53-deficient HCT116 cells were obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD).
Plasmids and luciferase reporter assay. A luciferase reporter corresponding to the p53 response element EpCAM RE-1, EpCAM RE-2 was generated by cloning two copies of EpCAM RE-1, EpCAM RE-2 into pGL3-Basic (Promega). pNeo, p53, p53-V143A, and p53-R273H were obtained from Dr. Simon Powell (Washington University, St. Louis, MO). p53-R280K was cloned from the MDA-MB-231 cell line. These constructs were transiently transfected with 20 ng of TK-Renilla, 400 ng of the EpCAM RE-1, EpCAM RE-2 luciferase reporter, and 200 ng of pNEo or the indicated p53 expression constructs into CA1a or MCF-7 cells. Relative light units reflect the percent mean change in luciferase activity in experimental cells compared with cells cotransfected with empty pGL3-Basic and pcDNA3.1 vectors.
Lentiviral RNA interference. The lentiviral construct pSicoR was obtained from Dr. Tyler Jacks (Massachusetts Institute of Technology, Boston, MA; ref. 10). shRNA target sequences specific for p53 (GACTCCAGTGGTAATCTAC), EpCAM (CTGGCTTTACCAATCTTGA) and a scrambled control (TTCTCCGAACGTGTCACGT) were cloned into the pSicoR vector. shRNA constructs were transfected into HEK293T cells with VSVG and Δ8.9 and viral supernatants were collected to transduce the indicated cell lines at a multiplicity of infection of 10.
Chromatin immunoprecipitation assay. MCF-7 cells were exposed to a minimal dose of UV radiation (10 J/m2) to induce p53 expression and processed for chromatin immunoprecipitation assay at 8 h using the Millipore chromatin immunoprecipitation kit. Briefly, confluent MCF-7 cells were fixed with 1% formaldehyde and lysed. After sonication, p53-bound DNA was immunoprecipitated with proteinA-DO-1 antibody, washed, and eluted in 1% SDS, 0.1 mol/L NaHCO3. Reverse cross-linked DNA was purified using DNA columns and analyzed by PCR analysis using EpCAM- and p21-specific primer sequences.
Immunoblotting. Cells were lysed in radioimmunoprecipitation assay buffer, and the protein concentration was measured with the bicinchoninic acid protein assay (Pierce). Cell extracts were subjected to 12% SDS-PAGE and electrophoretically transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with the indicated primary antibodies and secondary antibodies conjugated with peroxidase (Santa Cruz Biotechnology). Signal was then detected using the enhanced chemiluminescence immunodetection system (Applied Biosystems).
Flow cytometry. EpCAM expression levels were measured by flow cytometry using phycoerythrin-labeled EpCAM antibody using a FACScan flow cytometer (BD Biosciences).
cDNA synthesis and real-time PCR analysis. Total RNA was purified from cell lines using RNeasy (Qiagen). Two micrograms of RNA were reverse transcribed using a cDNA synthesis kit (Ambion). Quantitative mRNA expression was measured using SYBR green chemistry and an ABI Prism 7700 Sequence Detector (Applied Biosystems). Reaction conditions and primer sequences are available on request.
Invasion and adhesion assays. For invasion assays, stably transduced CA1a cells (4 × 104) were added into transwell Matrigel invasion chambers (BD Biosciences) and incubated for 48 to 72 h. Cells invading through the Matrigel membrane were counted by a laboratory technician blinded to the experimental conditions. For the adhesion assay, 1 × 105 stably transduced CA1a cells were plated in triplicate on 96-well fibronectin-coated plates (BD Biosciences). After 30 min of incubation, the plates were placed on a shaker for 2 min, washed twice with PBS, stained with crystal violet, lysed with 2% SDS, and read at 550 nm on an ELISA reader.
Identification of candidate p53 binding sites in the EpCAM gene and in vivo confirmation of wild-type p53 binding to the highest scoring binding site. Candidate p53 binding sites in the EpCAM gene were identified using the p53MH computer algorithm ( 11). The p53MH computer algorithm identified 10 candidate p53 binding sites in the EpCAM genomic sequence ( Fig. 1A ), including two candidate binding sites, RE1 and RE2, located in introns within the EpCAM gene with a score >90%. Chromatin immunoprecipitation assays confirmed p53 binding to the highest scoring candidate binding site, EpCAM-RE1, and to p21, a gene that is bound and regulated by p53 ( Fig. 1B). EpCAM-RE1 and EpCAM-RE2 reporter constructs were then transiently transfected into CA1a cells, and wild-type p53 was induced by transient transfection, UV radiation, or camptothecin treatment. Wild-type p53 expression was associated with a dose-dependent increase in EpCAM-RE1, but not EpCAM-RE2 luciferase activity ( Fig. 1C and D). No luciferase activity was observed following transient transfection with the p53 mutants tested ( Fig. 1C), suggesting that only wild-type p53 binds the EpCAM-RE1 binding site.
Wild-type p53 negatively regulates EpCAM expression in breast cancer cells. In gain-of-function experiments, induction of p53 expression was associated with a dose-dependent decrease in EpCAM expression ( Fig. 2A and B ). No change in EpCAM expression was observed following transient transfection with the mutant p53 constructs ( Fig. 2C). In loss-of-function experiments, specific ablation of p53 expression resulted in a significant reduction (>90%) in p53 mRNA and protein expression ( Fig. 3A and B ), a significant decrease in p21 expression (data not shown), and a significant increase in EpCAM expression ( Fig. 3B and C). We also assessed EpCAM expression in wild-type and p53-deficient HCT116 cells, showing increased EpCAM mRNA and protein expression in p53-deficient HCT116 cells ( Fig. 3D). As an important control, we specifically ablated p53 expression with RNA interference before treatment of MCF-7 breast cancer cells with UV or camptothecin. Under these conditions, no change in EpCAM expression was observed following the UV or camptothecin treatment, confirming that the change in EpCAM expression observed is mediated by p53 (data not shown).
Transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. To determine the functional relevance of p53-mediated transcriptional repression of EpCAM, we assessed the effect of concomitant specific ablation of p53 and EpCAM expression on breast cancer invasion. CA1a cells were stably transduced with lentiviral constructs containing p53, EpCAM, or control shRNA constructs. Transduction with EpCAM shRNA constructs resulted in a significant decrease in EpCAM expression ( Fig. 4A ) and a decrease in breast cancer invasion as measured in a transwell invasion assay ( Fig. 4C). Conversely, transduction with p53 shRNA constructs resulted in a significant increase in EpCAM expression ( Fig. 4A) and an increase in breast cancer invasion ( Fig. 4C). When used in combination with p53 shRNA constructs, EpCAM shRNA constructs were able to successfully abrogate the increase in EpCAM expression and breast cancer invasion observed following specific ablation of p53 expression ( Fig. 4A and C). In parallel adhesion experiments, we observed similar results ( Fig. 4D). These data strongly suggest that transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion.
Identification of p53-responsive genes has provided critical insights into the role of p53 as a tumor suppressor protein. Although genome-wide expression profiling suggests that there may be more than 1,500 p53-responsive genes, the majority of these genes are believed to be indirect targets ( 12). In 2002, it was estimated that there were only 20 genes that had confirmed p53 binding sites, were known to bind p53, and were clearly regulated by p53 ( 11). In this study, we confirmed p53 binding/regulation of the EpCAM gene in vivo and identified a biological function due to p53 binding of the EpCAM gene. Taken together, these studies suggest that EpCAM may be a key transcriptional target of p53.
Regulation of migration and invasion is a recently described tumor suppressor function of p53 ( 13). Although the mechanisms of p53 control of migration and invasion remain to be defined, there is experimental evidence to suggest that p53 is capable of regulating the function of the Rho family of small GTPases, including the prototypic family members RhoA, Rac1, and Cdc42 ( 14, 15). In these studies, phosphoinositide 3-kinase (PI3K) was shown to be a key intermediary between p53 and the Rho GTPases ( 14). In this study, we confirm our previous findings that EpCAM expression is associated with breast cancer invasion ( 7). Although the mechanism(s) of EpCAM control of breast cancer invasion remain to be defined, EpCAM directly interacts with PI3K in normal epithelial cells ( 16), and the introduction of EpCAM in normal epithelial cells is associated with cytoskeletal rearrangements ( 17). These previous studies and the demonstration that EpCAM contributes to p53 control of breast cancer invasion in this study strongly suggest that EpCAM may be an intermediary between p53, PI3K, and the Rho GTPases.
In contrast to the well-studied mechanisms of p53 transcriptional activation, the mechanisms underlying p53-mediated transcriptional repression are less well characterized. Several potential mechanisms for p53-mediated transcriptional repression have been proposed ( 18): (a) interference with DNA-binding transcriptional activators, (b) nonspecific interference with basal transcriptional machinery, (c) alteration of downstream target gene chromatin structure by recruitment of histone deacetylases (HDAC), and (d) recruitment of DNA (cytosine-5) methyltransferase (DNMT) and concomitant promoter methylation. Recruitment of HDAC to the EpCAM gene with concomitant repression of EpCAM expression has recently been described ( 19). In this study, Tai and colleagues also showed that promoter methylation contributes to the repression of EpCAM gene expression in lung cancer cells. It is possible that p53 is actively involved in the recruitment of an HDAC-DNMT1-p53 complex to the EpCAM gene, and that this complex mediates gene repression by promoter methylation as has been recently shown for the survivin gene ( 20).
In the present study, we took a stepwise approach to show that p53 represses the transcription of EpCAM. These data imply that the loss or mutation of wild-type p53 in breast and other epithelial cancers may contribute to EpCAM overexpression. Further study will be necessary to define the mechanisms by which p53 represses EpCAM expression, to determine if evidence of this interaction can be shown in human cancer specimens, and to explore the functional implications in more detail.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Susan G. Komen for the Cure.
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.
We thank Dr. Simon Powell (Memorial Sloan-Kettering Cancer Center, New York, NY) for P53 discussions and advice.
- Received July 15, 2008.
- Revision received October 8, 2008.
- Accepted October 29, 2008.
- ©2009 American Association for Cancer Research.