Oncogene (2007) 26, 3329–3337 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Negative regulation of receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion

SA Mehta1, KW Christopherson2,3,4,6, P Bhat-Nakshatri3,4, RJ Goulet Jr1, HE Broxmeyer2,3,4, L Kopelovich5 and H Nakshatri1,3,4

1Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA; 2Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA; 3Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA; 4Walther Cancer Institute, Indianapolis, IN, USA and 5CADRG, DCP, National Cancer Institute, Bethesda, MD, USA

Chemokine receptor CXCR4 and itsligand CXCL12 are Oncogene (2007) 26, 3329–3337. doi:10.1038/sj.onc.1210120; suggested to be involved in migration, invasion and published online 27 November 2006 metastasis of breast cancer cells. Mutation of the tumor suppressor p53 in breast cancer is associated with Keywords: CXCR4; p53; breast cancer; invasion; metastasis and aggressive clinical phenotype. In this PRIMA-1 report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants(R175H or R280K) of p53 repressCXCR4 expression. Recently described cancer-specific p53 iso- form, D133p53, also failed to repress CXCR4 promoter Introduction activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 CXCR4, a seven transmembrane G-coupled receptor breast cancer cells that contain wild-type p53. Basal originally identified as a coreceptor for CXCR4 promoter activity in HCT116 colon carcinoma line tropic strains of human immunodeficiency virus, is cellsdeleted of p53 [HCT116(p53KO)] was10-fold higher expressed on a wide variety of tissue/organ-specific stem compared to that in parental HCT116 cellswith cells (Kucia et al., 2005). It is activated upon binding its functional wild-type p53. Deletion analysis of CXCR4 ligand CXCL12, also called stromal cell-derived factor promoter identified a seven- p53-repressor (SDF-1a). CXCR4:CXCL12-activated signals cause element homologous to cyclic AMP/AP-1 response calcium flux, matrix metalloproteinase 2 (MMP-2) and (CRE/AP-1) element. Electrophoretic mobility shift and MMP-9 activation, and chemoinvasion chromatin immunoprecipitation assays revealed binding of (Kucia et al., 2005). ATF-1 and cJun to the CRE/AP-1 element. The p53 CXCR4 is overexpressed in cancer and plays a role in rescue drug PRIMA-1 reduced CXCR4 mRNA and cell invasion and metastasis (Balkwill, 2004). Several studies surface expression in MDA-MB-231 cells, which express using animal models have suggested a role for CXCR4 R280K mutant p53. CP-31398, another p53 rescue drug, in metastasis of breast cancer cells (Muller et al., 2001; similarly reduced cell surface levels of CXCR4. PRIMA- Kang et al., 2003; Smith et al., 2004; Liang et al., 2005). 1-mediated decrease in CXCR4 expression correlated CXCR4 is part of the bone and lung metastasis with reduced invasion of MDA-MB-231 cells through signatures for breast cancer (reviewed in Gupta et al., matrigel. These results suggest a mechanism for elevated 2005). Recently, we reported CXCR4-mediated repres- CXCR4 expression and metastasis of breast cancers with sion of major histocompatibility (MHC) class II gene p53 mutations or isoform expression. We propose that p53 expression, which could lead to evasion of antitumor rescue drugs either alone or in combination with immunity (Sheridan et al., 2006). chemotherapeutic drugsmay be effective in reducing CXCR4 expression is regulated by von Hippel– CXCR4-mediated metastasis. Lindau tumor suppressor pVHL, vascular endothelial growth factor, hepatocyte growth factor, USF/c-Myc, Correspondence: Dr H Nakshatri, Department of Surgery, Biochem- YY1, nuclear factor-kappa B (NF-kB) and by promoter istry, and Molecular Biology, Indiana Cancer Research Institute, R4- methylation (Moriuchi et al., 1999; Bachelder et al., 202, 1044 West Walnut Street, R4-268, Indianapolis, IN 46202, USA. 2002; Helbig et al., 2003; Staller et al., 2003; Lee et al., E-mail: [email protected] 2005; Matteucci et al., 2005; Mori et al., 2005). In this 6Current address: Institute of Molecular Medicine. The University of Texas Health Sciences Center, Houston, TX, USA. study, we examined the possibility that CXCR4 is Received 28 March 2006; revised 9 October 2006; accepted 9 October negatively regulated by the tumor suppressor protein 2006; published online 27 November 2006 p53. p53 mutation is a late event in breast cancer and is Regulation of CXCR4 expression in breast cancer SA Mehta et al 3330 associated with poor response to systemic therapy on cell cycle because both luciferase and p53 siRNA- (Berns et al., 2000). Moreover, cancer-specific p53 transfected cells showed similar cell-cycle distribution isoforms with dominant-negative activity have been pattern (Figure 1b). Majority of cells were at G0/G1 identified (Bourdon et al., 2005). p53 is a sequence- phase, because cells had to be maintained in media specific DNA-binding protein that induces expression of lacking estrogen to prevent estrogen receptor from severalgrowth arrest and proapoptotic (Vousden repressing CXCR4 expression (Frasor et al., 2003). and Lu, 2002). It also represses by We performed reciprocalexperiment in MDA-MB- binding to p53-repressor elements or through protein– 231 cells. MDA-MB-231 cells that were isolated after protein interaction (Fridman and Lowe, 2003). Here, we implantation into mammary fat pad of nude mice show that wild-type p53 but not the cancer-specific (TMD-231) were used in these studies, because CXCR4 mutants or D133p53 isoform represses CXCR4 expres- is expressed at much higher levels in these cells sion in breast cancer cells. p53-mediated repression compared to parental cells (Helbig et al., 2003). Wild- requires a cyclic AMP/AP-1 response (CRE/AP-1) like type p53 reduced CXCR4 expression in these cells element in the CXCR4 promoter, which binds to cJun (Figure 1c). and the CRE-building protein (CREB) family transcrip- tion factor ATF-1 (activating transcription factor) (Mayr and Montminy, 2001). We also show that p53 Wild type but not mutants or isoform of p53 represses rescue drug PRIMA-1 at a non-toxic dose reduces CXCR4 promoter activity CXCR4 expression and invasion of breast cancer cells To determine the ability of mutant p53 to repress expressing mutated p53 (Bykov et al., 2002b). CXCR4 and to identify promoter elements involved in p53-mediated repression of CXCR4, we performed cotransfection experiments in MCF-7 breast cancer cells with CXCR4 promoter-reporters and p53 expression Results vectors. Wild-type p53 but not the dominant-negative mutant (p53V143A) reduced the activity of luciferase Transcription factor binding motif search of the reporter controlled by 2.6 kB CXCR4 promoter-enhan- CXCR4 promoter revealed the presence of two sequence cer region (Figure 2a). In TMD-231 cells, wild-type p53 motifs that are 87% (À2249 to À2239) and 85% (À846 but not p53R280K mutant reduced CXCR4 expression to 837) homologous to the consensus p53 binding site À (Figure 2a). (Caruz et al., 1998). To test regulation of CXCR4 To determine whether p53-mediated repression of the expression by p53, we adopted three complementary CXCR4 promoter involves putative p53 response approaches: (1) Analysis of basal CXCR4 expression in elements, we measured the effect of p53 on the activity parental MCF-7 cells and the same cell line transfected of a different CXCR4-promoter-reporter lacking both with siRNA against p53. (2) Co-transfection experi- putative p53 response elements [CXCR4(À827/ þ 7)/ ments in MCF-7 cells with CXCR4 promoter-reporters CAT] (Helbig et al., 2003). CXCR4(À827/ þ 7)/CAT and wild-type or mutant p53 expression vectors. Similar reporter activity was reduced by p53 (Figure 2b). These studies in HCT116 colon carcinoma cells with wild-type results suggest that p53 represses CXCR4 expression p53 and the same cell line with p53 deletion independent of putative p53 response elements. Under [HCT116(p53KO)] (Bunz et al., 2002). (3) Evaluating identicaltransfection conditions, p53 increased the the ability of p53 rescue drugs to alter CXCR4 activity of p21 promoter-enhancer or consensus p53 expression in MDA-MB-231 cells, which expresses response element-driven reporters in both cell types R280K mutant-p53 (Bykov et al., 2002b). (data not shown). We next examined the effect of the dominant-negative MCF-7 cells with reduced p53 levels show elevated (p53V143A) and two cancer-specific mutants of p53 CXCR4 expression compared to parental cells with distinct biological activities (p53R175H or MCF-7 breast cancer cells contain functional wild-type p53R280K) on CXCR4 reporter activity (Scian et al., p53 (Casey et al., 1991) and express low levels of 2004). Neither the dominant-negative mutant nor CXCR4. We investigated whether reducing the levels of cancer-specific mutants repressed CXCR4 reporter p53 in these cells by siRNA leads to elevated CXCR4 activity, suggesting that cancer-specific mutations abro- levels. Northern analysis and flow cytometry analysis gate the ability of p53 to repress CXCR4 expression showed elevated CXCR4 expression in p53 siRNA- (Figure 2b). treated cells compared to control luciferase siRNA- The D133p53 isoform reduces apoptosis mediated by treated cells (Figure 1a and b, respectively). Quantitative wild-type p53, suggesting dominant-negative effect on real-time polymerase chain reaction (RT–PCR) analysis certain p53 functions (Bourdon et al., 2005). This revealed 1.570.15-fold increase in CXCR4 mRNA in isoform failed to repress CXCR4 promoter activity in p53 siRNA-transfected cells compared to luciferase MCF-7 cells (Figure 2b), although it could increase the siRNA-transfected cells (data not shown). In contrast levels of endogenous full-length p53 (Figure 2c). Note to CXCR4, expression of p21, a p53 target gene, was that the wild-type and mutants of p53 are expressed at reduced in p53 siRNA-transfected cells (Figure 1a). similar levels in transfected cells (Figure 2c). Elevated CXCR4 expression in p53 siRNA transfected We compared the CXCR4 promoter activity in cells was not due to the secondary effect of p53 depletion HCT116 and HCT116(p53KO) cells. CXCR4 promoter

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3331

Figure 1 Repression of CXCR4 expression by p53. (a) The effect of siRNA against p53 on CXCR4 expression in MCF-7 cells. CXCR4 and p21 expression was measured by Northern blotting. P53 and p21 protein levels were measured by Western blotting. (b) Cell surface expression of CXCR4 in luciferase or p53 siRNA-transfected MCF-7 cells was measured by flow cytometry. Representative data from two experiments carried out in duplicate is shown. Cell cycle distribution of luciferase or p53 siRNA- transfected cells is also shown. (c) p53 represses endogenous CXCR4 expression in TMD-231 cells. Cells were transfected with 10 mgof the vector pcDNA3 or pcDNA3-p53. RNA was isolated 36 h after transfection and subjected to Northern analysis. The blot was reprobed with 36B4 to ensure integrity of RNA.

activity was more than 10-fold higher in promoter. The fact that CXCR4(À127)/CAT reporter HCT116(p53KO) cells compared to HCT116 cells and activity was not suppressed by p53 indicates that the cotransfected p53 reduced the CXCR4 promoter activity observed repression with other CXCR4 promoter in these cells (Figure 2d). Taken together, these results reporters is not due to nonspecific effect of p53 suggest the presence of p53-repressible element in the overexpression or due to p53-mediated apoptosis of CXCR4 promoter. transfected cells. The À209 to À127 region of the CXCR4 promoter contains sequence elements that bind to SP-1, CRE/AP- CRE/AP-1-like element in the CXCR4 promoter is 1 and Ets-1 family transcription factors (Caruz et al., required for p53-mediated repression 1998) (Figure 3a). p53 has previously been shown to To identify the p53-repressor element within the repress gene expression by interacting with or modulat- CXCR4 promoter, we used additionaldeletion mutants ing the activity of SP-1, AP-1 and Ets-1 (Bargonetti of the CXCR4/CAT reporter: CXCR4(À426)/CAT, et al., 1997; Kim et al., 2003; Sun et al., 2004). We CXCR4(À209)/CAT and CXCR4(À127)/CAT (Helbig analysed the ability of p53 to repress CXCR4/CAT et al., 2003). p53 repressed the activities of reporters with mutations in either SP-1 or Ets-binding CXCR4(À426)/CAT and CXCR4(À209)/CAT repor- sites or a mutant lacking CRE/AP-1 element ters but not the CXCR4(À127)/CAT reporter [CXCR4(À199)/CAT]. The activities of both SP-1 and (Figure 3a), suggesting the presence of p53-repressor Ets-1 mutant CXCR4 promoter-reporters were re- element within À209 to À127 region of the CXCR4 pressed by p53, suggesting that neither SP-1 nor Ets-1

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3332

Figure 2 Wild-type p53 but not cancer-specific mutant/isoform of p53 represses CXCR4 promoter activity. (a) The effect of wild type or the dominant-negative mutant of p53 (p53V143A) on CXCR4 promoter activity in MCF-7 cells (n ¼ 4). *P ¼ 0.0001, pcDNA3 versus p53-transfected cells. The effect of wild-type p53 and MDA-MB-231-specific p53 mutant (p53R280K) on CXCR4 promoter activity in TMD-231 cells is also shown. Results are from two experiments carried out in triplicate. *Po0.0001, pcDNA3 versus p53. (b) The putative p53 response elements are not required for p53-mediated repression of the CXCR4 promoter. MCF-7 cells were transfected with the CXCR4(À897)/CAT reporter lacking both putative p53 response elements and indicated expression vectors. *P ¼ 0.0005, pcDNA3 versus p53. (c) Expression levels of transfected wild type or mutants/isoform of p53 in MCF-7 cells was measured by Western blotting. (d) CXCR4(À897)/CAT activity in HCT116 and HCT116(p53KO) cells. *P ¼ 0.0006; **P ¼ 0.01 pcDNA3 versus p53.

is involved in p53-mediated repression of CXCR4 (data the transcription factor(s) that bind(s) to the CRE/AP-1 not shown). In contrast, p53 failed to repress the activity element is involved in p53-mediated repression of of the CXCR4(À199)/CAT reporter (Figure 3b). Thus, CXCR4.

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3333 p53-repressor element in the CXCR4 promoter binds element of the CXCR4 promoter. Experiments were ATF-1 and c-Jun carried out with extracts from cells that were treated We used electrophoretic mobility shift assay (EMSA) to with 12-O-tetradecanoylphorbol-13-acetate (TPA), a identify transcription factors that bind to the CRE/AP-1 known inducer of AP-1-DNA binding activity (Shaulian and Karin, 2002). Two DNA–protein complexes were detected when the CRE/AP-1 element was incubated with nuclear extracts from HCT116 and HCT116(p53KO) cells with or without TPA treatment (Figure 4a). Also, similar DNA:protein complexes were obtained when extracts from HCT116(p53KO) and same cells transfected with p53 were used in EMSA (data not shown). Similar DNA:protein complexes with nuclear extracts of HCT116, HCT116(p53KO) and HCT116(p53KO) cells transfected with p53 suggests that p53 does not alter protein binding to CRE/AP-1 element. Another complex was detected with nuclear extracts from MCF-7 cells (complex II). Antibody against ATF-1 supershifted complex I. Antibodies against p53, ATF-2, ATF-3, cJun, SP-1 and CREB-1/2 had no effect on DNA:protein complexes. Binding of p53 to CRE/AP-1 element was not observed when nuclear extract from MCF-7 cells overexpressing p53

Figure 4 ATF-1 and cJun bind to the p53-repressible element of the CXCR4 promoter. (a) Binding of transcription factors to CRE/ AP-1 element of the CXCR4 promoter. Nuclear extracts from Figure 3 An CRE/AP-1 like element in the CXCR4 promoter is HCT116, HCT116(p53KO) with or without TPA treatment required for repression by p53. (a) Deletion analysis of the CXCR4 (125 nM for 3 h) or MCF-7 cells were incubated with radiolabelled promoter. Deletion constructs and putative transcription factor CRE/AP-1 probe with or without indicated antibodies and binding sites between À209 and À127 region of the CXCR4 subjected to EMSA. Complex I was supershifted by ATF-1 promoter are depicted in the top. MCF-7 cells were transfected antibody in both HCT116 and MCF-7 cells. ss, supershift; Ab, with indicated CXCR4 promoter deletion mutants and the p53 antibody. Integrity of HCT116 and HCT116(p53KO) extracts was expression vector. *P ¼ 0.0004; **P ¼ 0.0006. (b) Failure of p53 to verified by performing EMSA with SP-1 probe. (b) CHIP assay repress CXCR4 promoter/CAT reporter lacking the CRE/AP-1 identifies binding of ATF-1 and cJun to CXCR4 promoter region element. MCF-7 and HCT116 cells were transfected with indicated encompassing the CRE/AP-1 element. In lane 1, 1/10 of input was CXCR4 promoter/CAT reporter with or without p53. *P ¼ 0.007; used to amplify CXCR4 promoter region. Lane 2 contains negative **P ¼ 0.003 pcDNA3 versus p53-transfected cells. controlwith IgG alone.

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3334 was used (data not shown). As a control, we measured We investigated by flow cytometry and/or Northern the binding of p53 from MCF-7 cells to the consensus blot analysis whether PRIMA-1 or CP31398 can reduce p53 response element (Supplementary Figure S1). CXCR4 expression in TMD-231 cells. Cell surface Sensitive chromatin immunoprecipitation assay (CHIP) expression of CXCR4 in TMD-231 cells was reduced also failed to detect binding of p53 to the CXCR4 to 40 and 20% in cells treated with 10 and 20 mM promoter (data not shown). However, CHIP assay PRIMA-1, respectively (Figure 5a and b). Mean revealed binding of ATF-1 and cJun to the CRE/AP-1 fluorescence intensity of CXCR4 staining was also element of the CXCR4 promoter (Figure 4b). reduced in cells treated with PRIMA-1 (data not shown). These results suggest that mutant p53, when p53 rescue drugs decrease cell-surface expression of rescued by PRIMA-1, can decrease CXCR4 expression. CXCR4 At the concentration of drug used, cells did not undergo PRIMA-1 and CP-31398 have been effective in in vitro and/ apoptosis as determined Trypan blue exclusion assay or in vivo studies in restoring certain function to mutated (data not shown). CP-31398 also reduced cell surface p53 including the R280K mutant p53 present in MDA- expression of CXCR4 in TMD-231 cells (Figure 5c and MB-231 cells (Foster et al., 1999; Bykov et al., 2002b). d). PRIMA-1 reduced CXCR4 mRNA without effecting

Figure 5 PRIMA-1 and CP-31398 reduce cell surface expression of CXCR4 in TMD-231 cells. (a) CXCR4 cell surface expression was measured by flow cytometry. TMD-231 cells were treated with DMSO or PRIMA-1 for 48 h. (b) Effect of PRIMA-1 on CXCR4 expression from three-independent experiments is shown. *P ¼ 0.02 versus control; **P ¼ 0.0001 versus control. (c) The effect of CP- 31398 on CXCR4 expression. (d) Effect of CP-31398 on CXCR4 expression from three experiments. *Po0.0001 versus control; **P ¼ 0.0004 versus control.

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3335

Figure 6 PRIMA-1 reduces invasion of TMD-231 cells through matrigel. Cells were treated with DMSO or PRIMA-1 for 48 h and invasion was measured 24 h after plating on matrigel (see Supplementary Materials and methods). Results from two of three- independent experiments are shown. The P-value shown is for CXCL12-treated cells versus CXCL12 plus PRIMA-1-treated cells.

DNA binding of transcription factors to the CRE/AP-1 compare CXCR4 expression with p53 status. Elevated element (Supplementary Figure S2). CXCR4 expression in clinically aggressive cancers could be due to p53 mutation, both of which are associated with poor clinical outcome (Berns et al., 2000). We have PRIMA-1 reduces invasion of TMD-231 cells through previously shown in a xenograft model that CXCR4- matrigel positive cancer cells are clonally selected in primary CXCR4 has been shown to increase invasive capacity of tumors and at sites of metastasis (Helbig et al., 2003). It cancer cells (Balkwill, 2004). We performed matrigel is possible that a subpopulation of cancer cells with invasion assay to determine whether PRIMA-1 reduces impaired p53 activity has elevated CXCR4 expression the invasive capacity of TMD-231 cells with or without and these cells are selected for growth at sites of CXCL12 treatment. Cells were treated with 20 mM metastasis. PRIMA-1 for 48 h and excess PRIMA-1 was removed A previous study has shown a relationship between by washing. Equalnumbers of viablecontroland CXCR4:CXCL12 activated signals and p53 activity PRIMA-1-treated cells were assayed for invasion with (Khan et al., 2005). Binding of CXCL12 to CXCR4 in or without CXCL12 treatment (Materials and methods neuronal cells leads to p53 acetylation and p21 expres- in Supplementary file). CXCL12 treatment caused a sion suggesting p53 activation. Thus, it is possible that modest increase in invasive capacity of these cells, which activated p53 then reduces CXCR4 expression to was not statistically significant (Figure 6). Invasion of attenuate CXCL12:CXCR4 signaling and this negative CXCL12-treated cells was reduced significantly by regulatory loop is subject to alterations in cancers. PRIMA-1 (Figure 6, P 0.01). Thus, reduced invasion p p53 represses the expression of pro-metastatic and of PRIMA-1 treated cells correlates with inhibition of anti-apoptotic genes, which may involve direct binding CXCR4 expression. of p53 to promoters, interaction of p53 with transcrip- tion factors such as SP-1, AP-1 and Ets-1, or recruit- ment of histone deacetylase complexes containing Discussion mSin3a (Fridman and Lowe, 2003; Kim et al., 2003; Sun et al., 2004). Repression of CXCR4 by p53 appears We present evidence for p53-mediated repression of to be indirect because we did not observe direct binding CXCR4. Because CXCR4 overexpression and p53 of p53 to the CXCR4 promoter region (Figure 4) but mutations are common in cancers, our study suggests requires an element that binds to transcription factors a link between these two major signaling pathways ATF-1 and cJun. Although ATF-1 and cJun are involved in cancer progression (Vousden and Lu, 2002; generally considered as transcription activators, ATF-1 Balkwill, 2004). In breast cancer, CXCR4 expression can also repress transcription (Dong et al., 2002). ATF/ increases progressively with increasing malignant poten- CREB mediate repression of cyclin A promoter by tialand higher expression is associated with lymphnode human T-cell leukemia virus protein Tax (Kibler and positivity, negativity, Her2 over- Jeang, 2001). Crosstalk between AP-1 family members expression and poor survival(Cabioglu et al., 2005; and p53 is well documented, which may involve Salvucci et al., 2005). These studies, however, did not sequestering of the co-activator p300/CBP (Shaulian

Oncogene Regulation of CXCR4 expression in breast cancer SA Mehta et al 3336 and Karin, 2002; Sun et al., 2004). The p53 may Northern analysis similarly sequester p300/CBP away from the CXCR4 RNA was isolated using the Qiagen RNAeasy (Qiagen, promoter and reduces communication between ATF-1/ Valencia, CA, USA) according to the manufacturer’s instruc- cJun and the basaltranscription machinery. tions (see in Supplementary information for additional PRIMA-1 was identified as a p53 rescue drug from details). the library of low-molecular-weight compounds from the NCI using Saos-2-His273 cells carrying tetracycline- EMSA regulated mutant p53 (Bykov et al., 2002b). PRIMA-1 is Nuclear extracts of HCT116, HCT116(p53KO) with or with- the only compound among 45 tested anticancer agents out TPA treatment (125 nM for 3 h), MCF-7 cells and MDA- that selectively induced apoptosis of cell lines with p53 MB-231 cells with or without PRIMA-1 treatment (20 mM for mutation (Bykov et al., 2002a; Li et al., 2005). In our 24 h) were subjected to EMSA with CXCR4-CRE/AP-1, p53 and SP-1 probes as described previously (Helbig et al., 2003). study, PRIMA-1 reduced CXCR4 expression and Details of probes are provided in Supplementary information. invasion of breast cancer cells with mutated p53. These results have important implications because p53 muta- tions are observed in advanced breast cancers that do Cell surface expression of CXCR4 not respond to therapy (Berns et al., 2000). Therefore, a Cell surface expression of CXCR4 was measured by flow combination of PRIMA-1 and chemotherapeutic drugs cytometry as described previously (Helbig et al., 2003) with additional details in Supplementary information. may prove to be effective therapy for metastatic breast cancers. Invasion assay Matrigelinvasion assays were performed using CellInvasion Assay kit from Chemicon International(Temecula,CA, USA) Materialsand methods as per the manufacturer’s protocol. Additional details are in Supplementary information. Plasmid constructs and reagents The human CXCR4 promoter (À897 to þ 7) and deletion mutants were generated by PCR and are described previously Western blot analysis (Helbig et al., 2003). Details of additional constructs can be Details of Western analysis are provided in Supplementary found in Supplementary information. information.

Cancer cell lines, transient transfection assays, CAT assays and CHIP assay luciferase assays CHIP assay was performed using CHIP assay kit (Upstate The human breast cancer cell lines MCF-7 and MDA-MB-231 Biotechnology, Lake Placid, NY, USA) as per instructions were from American Type Tissue Culture Collection. TMD- of the manufacturers. Supplementary Information provides 231, a tumor-derived variant of MDA-MB-231 cells, has been details of the CHIP assay. described previously (Helbig et al., 2003). Details of growth and transient transfection assay conditions of these cell lines and HCT116 cells are in Supplementary information. Acknowledgements

siRNA transfection We thank Drs B Vogelstein, P Staller, M Kaplan, S Boswell siRNA against p53 and luciferase was purchased from and M Smith for various reagents. This work is supported by Dharmacon RNA Technology (Lafayette, CO, USA). MCF- grants R01 CA89153, N01-CN-15133 (to HN), R01 HL67384 7 cells growing in phenol-red free MEM supplemented with and R01 DK53674 (to HB) from the NationalInstitutes of 5% charcoal-dextran treated-serum were transfected with 5 nM Health, American Institute for Cancer Research (03A069- siRNA using TransIT-TKO transfection reagent (Mirus, REN) and MichaelGuest Innovative Research Award from Madison, WI, USA). siRNA-transfected cells were analysed Walther Cancer Institute (to HN). HN is Marian J Morrison 72 h after transfection. Investigator in Breast Cancer Research.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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