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CCR5 Expression Influences the Progression of Human Breast Cancer in a p53-dependent Manner

CCR5 Expression Influences the Progression of Human Breast Cancer in a p53-dependent Manner Chemokines are implicated in tumor pathogenesis, although it is unclear whether they affect human cancer progression positively or negatively. We found that activation of the chemokine receptor CCR5 regulates p53 transcriptional activity in breast cancer cells through pertussis toxin–, JAK2-, and p38 mitogen–activated protein kinase–dependent mechanisms. CCR5 blockade significantly enhanced proliferation of xenografts from tumor cells bearing wild-type p53, but did not affect proliferation of tumor xenografts bearing a p53 mutation. In parallel, data obtained in a primary breast cancer clinical series showed that disease-free survival was shorter in individuals bearing the CCR532 allele than in CCR5 wild-type patients, but only for those whose tumors expressed wild-type p53. These findings suggest that CCR5 activity influences human breast cancer progression in a p53-dependent manner. Key words: chemokine receptor • breast cancer • p53 • CCR5 polymorphism • p38 Introduction Many cancers express an extensive network of chemo- Current evidence does not establish whether these che- kines and chemokine receptors (1, 2). Studies in animal mokine biological activities in the tumor microenviron- models suggest that chemokines may act on tumor cells ment contribute to cancer growth and spread, or to host and/or tumor-infiltrating leukocytes (3–6). Tumor-pro- antitumor response and cancer regression. Studies using the duced chemokines are thought to have distinct roles in CC chemokine CCL5 (RANTES) as a model reported that the biology of primary and metastatic disease, including elevated CCL5 levels in the tumor environment contribute (a) directing leukocyte infiltration into the tumor, (b) reg- to improving the immune response against breast carcino- ulating the antitumor immune response, (c) controlling mas (7), but also correlate with poor prognosis in breast can- tumor angiogenesis, (d) functioning as autocrine or para- cers (8, 9). CCL5 also has a role in the chemotaxis and me- crine growth and survival factors, and (e) controlling tumor tastasis of breast cancer cell lines (10–12). Selected cell lines cell movement. with an enhanced chemotactic response to CCL5 neverthe- less showed decreased growth potential in nude mice, sug- gesting an inverse correlation between CCL5-induced The online version of this article contains supplemental material. chemotactic and proliferation signals (12). Compatible with Address correspondence to Santos Mañes, Department of Immunology this, basic fibroblast growth factor–induced proliferation of and Oncology, Centro Nacional de Biotecnología, Campus de Canto- endothelial cells was inhibited by distinct chemokines, in- blanco, Universidad Autonoma de Madrid, E-28049 Madrid, Spain. Phone: 34-91-585-4660; Fax: 34-91-372-0493; e-mail: [email protected] cluding CCL5, via a noncompetitive mechanism (13). R. Colomer’s present address is Instituto Catalan de Oncologia, Medical These results indicate that the role of chemokines in tumor Oncology Division, Hospital Josep Truet, Fança s/n, E-17007 Gerona, progression is complex and poorly understood. Spain. S. Montero’s present address is Pharma Mar S.A., Avda de los Reyes 1, Colmenar Viejo, E-28770 Madrid, Spain. Abbreviations used in this paper: BrdU, bromodeoxyuridine; DFS, disease- K. Harshman’s present address is Center for Integrative Genomics, free survival; MAPK, mitogen-activated protein kinase; siRNA, small University of Lausanne, CH-1015 Lausanne, Switzerland. interfering RNA. 1381 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2003/11/1381/9 $8.00 Volume 198, Number 9, November 3, 2003 1381–1389 http://www.jem.org/cgi/doi/10.1084/jem.20030580 The Journal of Experimental Medicine Here we studied the role of CCR5 in breast tumor pro- two intraperitoneal injections of bromodeoxyuridine (BrdU; 10 mg/ml, 50 l/mouse) 6 h apart, and primary tumors were re- gression by analyzing proliferation of xenografts derived moved 4 h after the last injection. Some tumors were fixed in from tumor cells expressing wild-type or mutated p53 in 70% ethanol, paraffin embedded, and analyzed for BrdU incorpo- combination with wild-type or mutated CCR5. We also ration using anti-BrdU antibody (Becton Dickinson) or active took advantage of the fact that 1% of the Caucasian pop- p38 MAPK using anti–phospho-p38 antibody (no. 9216; New ulation is homozygous for the CCR532 polymorphism, England BioLabs, Inc.). Other tumors were snap frozen in Tissue which renders a nonfunctional CCR5 receptor, to study Freezing Medium (Sakura Finetek) and cryosections were used the effect of this mutation in a clinical series of breast can- for in situ apoptosis determination by the TUNEL method cer patients. We describe a new mechanism by which (MEBSTAIN Apoptosis kit II; Immunotech) or p21 detection. CCR5 influences human breast cancer progression, de- Finally, some tumors were mechanically disrupted. Cell lysates pending on the status of the p53 tumor suppressor. were prepared with RIPA buffer and used both to measure hu- man CCL5 (Cytoscreen; Biosource International) and for p53, p21, and Mdm2 analysis in Western blot. CCL5 levels, BrdU in- Materials and Methods corporation, and TUNEL data were compared using the two- tailed Mann-Whitney test. Cell Lines and Stimulation. MCF-7 and MDA-MB-231 cells 32 Genotyping of the Breast Cancer Cohort. The breast can- (American Type Culture Collection) were transduced with retro- cer cohort comprised primary (nonmetastatic) breast cancer pa- viral pLZ-KDEL32-IRES-gfp or pLZ-IRES-gfp (mock) super- tients diagnosed at the Hospital Universitario 12 de Octubre, natants, and green fluorescent protein–expressing cells were se- Madrid, Spain, from January 1992 to December 1995. Stage IV lected by fluorescence-activated cell sorting (11). The percentage patients at diagnosis were excluded from this series. DNA was of CCR5 cells observed after subtracting the control value was isolated from biopsies of 547 patients and the CCR532 allele multiplied by average fluorescence intensity to calculate surface determined with an automated method based on real-time PCR CCR5 expression in mock and KDEL32-expressing cells (14). 175H in a Lightcycler system (16). Patients included in this study were MCF-7-p53 cells were obtained by transfection with pWZL- 175H disease free after surgery and showed no initial metastases or me- Hygro-p53 (provided by M. Serrano, Centro Nacional de tastases within 3 mo after surgery. Biotechnologia, Madrid, Spain; reference 15), followed by hy- Chemotherapy was administered to premenopausal patients gromycin selection. p53 silencing by small interfering RNA with tumors larger than 1 cm, postmenopausal patients with neg- (siRNA) was performed by transfecting MCF-7 cells with a pool ative estrogen receptor expression, and patients showing affected of p53-specific siRNA (SMARTpool kit; Dharmacon) at 100 lymph nodes. Patients with conservative surgery and those show- nM using Oligofectamine (GIBCO BRL). Transfection effi- ing four or more axillary lymph nodes affected were treated with ciency was 75  5.1% as estimated using a fluorescent-labeled radiotherapy. Patients whose tumors expressed hormone recep- siRNA. As a control, MCF-7 cells were transfected with nonspe- tors were treated with tamoxifen for 5 yr. Clinical follow-up was cific pooled siRNA duplexes (Dharmacon). Finally, MCF-7 cells performed according to institutional protocols by physical exami- were transfected with Flag-tagged dominant negative mutants for nation every 3 mo in the first 2 yr after surgery, every 6 mo in the MAP kinase kinases 3 and 6 (dnMKK3 and dnMKK6; provided next 3 yr, and every year after the sixth year. Mammography was by R.J. Davis, Howard Hughes Medical Institute, University of performed yearly and other diagnostic tests were performed when Massachusetts, Worcester, MA). 175H relapse was suspected. Disease-free survival (DFS) was defined as Serum-depleted mock, KDEL32-, p53 -, siRNA-, the time (in months) from diagnosis of disease, usually coinciding dnMKK3-, dnMKK6-, or dnMKK3 plus dnMKK6–expressing with surgery, to first relapse or to the last clinical revision for pa- MCF-7 cells were stimulated with 100 nM CCL5 (PeproTech), tients with no recurrence. A total of 133 out of 541 patients re- irradiated (30 Gy) or UV irradiated with UVC (254 nm, 30 2 lapsed (24.5%). J/m ), and then incubated at 37C for the times indicated. Equal Allelic frequencies between patient groups where compared protein amounts from cell lysates prepared with RIPA buffer (50 using standard tests with Yates correction, the Mantel-Haens- mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxy- zel test, or Fisher’s exact test where necessary, using Statcalc soft- cholate, 1% NP-40) were analyzed by Western blot with anti- ware (EpiInfo 5.1). For statistical analysis of genotypes, 32/ p53 (Oncogene Research Products), anti-p21 and anti-Mdm2 and 32/32 individuals were grouped to avoid values of 7. (Santa Cruz Biotechnology, Inc.), anti-p38 and anti–phospho- DFS was estimated using the Kaplan-Meier method and com- p38 mitogen-activated protein kinase (MAPK; no. 9211; New pared by the log-rank test. England BioLabs, Inc.), and anti-Flag (Sigma-Aldrich) antibodies. Online Supplemental Material. Fig. S1 shows DFS curves in Protein loading was confirmed with anti-tubulin antibody breast cancer patients grouped according to CCR5 status. Fig. (Sigma-Aldrich). For some experiments, cells were pretreated for S1 is available at http://www.jem.org/cgi/content/full/jem. 14 h with 100 M tyrphostin AG 490, 10 M LY 294002, 10 20030580/DC1. M PD 98059, 10 M SB 203580 (all from Calbiochem), 0.75 g/ml pertussis toxin (Sigma-Aldrich), or DMSO before CCL5 stimulation. Densitometric analysis of Western blots was per- formed with NIH Image software. Results Breast Tumor Xenograft Models and Immunohistochemistry. Eight CCR5 Signaling Increases p53 Transcriptional Activity. female BALB/c-SCID mice were injected subcutaneously in CCR5-mediated signals may regulate transcription of sev- both flanks with 3  10 mock or KDEL32-expressing MDA- 6 eral p53 target genes in certain cell types (17). To study the MB-231 or 5  10 MCF-7 cells. In the case of MCF-7, mice association between CCR5 and p53 in cancer cells, we an- were pretreated with 1 g/ml 17 -ethynyl estradiol (Sigma- alyzed CCR5-dependent signaling in the MCF-7 breast Aldrich) in drinking water for 1 wk before cell injection and during the experimental period. 4 wk after cell injection, mice received carcinoma line, which expresses CCR5 and wild-type p53. CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine Figure 1. CCR5 regulates p53 transcrip- tional activity. (A) Time course induction of WAF1 p53 (dotted line, ), p21 (solid line, ), and Mdm2 (dashed line, ) in CCL5-stimulated MCF-7 cells. Western blots from four indepen- dent experiments were quantified by densitometry and the values were normalized using the tubulin loading control. Data points are plotted relative to mean values obtained before chemokine addi- tion (n 5). (B) CCL5-induced p53 targets in 175H MCF-7 cells expressing the p53 mutant. One representative experiment of three is shown. (C and D) MCF-7 cells transfected with control or p53-specific siRNA oligonucleotides were stimulated with CCL5 (C) and incubated at 37C for the times indicated or  irradiated (30 Gy; D) and incubated for 3 h at 37C. Cell lysates were analyzed by Western blot. One representative experiment of three is shown. (E) CCR5 was detected in live mock- or KDEL32-expressing MCF-7 cells by FACS . Surface CCR5 expression in mock- and KDEL32-expressing cells was estimated by multiplying average fluorescence intensity by the percentage of CCR5 cells (n 3). (F and G) Mock- and KDEL32-expressing cells were stimulated with CCL5 (F) or  irradiated (30 Gy; G) and incubated at 37C for the times indicated. Cell lysates were analyzed by Western blot. One representative experiment of three is shown. Stimulation of MCF-7 cells with CCL5, a ligand for surface (18). KDEL32 expression reduced CCR5 levels by WAF1 CCR5, increased levels of well-established p53-regulated 70% (Fig. 1 E). CCL5 did not induce p21 or Mdm2 WAF1/CIP1 genes, such as p21 and Mdm2 (Fig. 1 A). p53 pro- in KDEL32-expressing MCF-7 cells (Fig. 1 F), indicating tein levels were essentially unaltered after CCL5 stimula- that CCR5 is the specific receptor by which CCL5 induces WAF1 tion, although a slight increase was observed 60 min after these genes. p21 and Mdm2 up-regulation were similar, stimulation. To determine whether CCL5-mediated however, in mock- and KDEL32-expressing MCF-7 cells WAF1 p21 and Mdm2 up-regulation is dependent on p53 after  irradiation, suggesting that KDEL32 expression did transcriptional activity, MCF-7 cells stably expressing a not affect the p53 pathway in these cells (Fig. 1 G). 175H dominant negative p53 mutant (15) were stimulated p53 Transcriptional Activity Requires CCR5-mediated p38 WAF1 with CCL5. CCL5 did not increase p21 or Mdm2 Activation. To study the mechanism by which CCR5 ac- 175H protein levels in MCF-7-p53 cells (Fig. 1 B). Concur- tivates p53, we used chemical inhibitors to block CCR5- rently, transfection of MCF-7 cells with p53-specific mediated signaling. Binding of chemokines to their recep- WAF1 siRNA prevented CCL5-induced p21 and Mdm2 up- tors leads to activation of heterotrimeric G proteins and regulation (Fig. 1 C). As a control, p53-specific siRNA also JAK family tyrosine kinases. These in turn initiate multiple inhibited  irradiation–induced up-regulation of p53 activ- signaling cascades, including phosphatidylinositol-3 kinase ity (Fig. 1 D). These results suggest that CCL5-induced as well as p38 and p42/p44 MAPK pathways (19). Incuba- transcriptional activation of p53 target genes, such as tion of MCF-7 cells with the inhibitors LY 294002 and PD WAF1 p21 and Mdm2, requires functional p53 protein. 98059, which block phosphatidylinositol-3 kinase and To analyze whether CCL5-induced p53 activation re- p42/p44 MAPK pathways, has only a marginal effect on WAF1 quires CCR5-mediated signaling at the cell surface, we spe- CCR5 induction of p21 and Mdm2 (Fig. 2 A). In con- WAF1 cifically blocked CCR5 in MCF-7 cells with a CCR5 trast, CCR5-mediated induction of p21 and Mdm2 dominant negative mutant. We previously showed that was inhibited by treatment of MCF-7 cells with pertussis overexpression of a CCR5 deletion mutant (KDEL32), toxin, AG 490, and SB 203580 (Fig. 2 A), which impede which is phenotypically similar to the natural CCR532 G , JAK-2, and p38 MAPK activation, respectively. In ad- (32) mutant, abrogates CCR5 function in MCF-7 cells dition, CCL5 induced p38 phosphorylation in mock-trans- (11). This effect is probably due to a trans inhibitory effect fected MCF-7 cells, but not in KDEL32-expressing cells of the KDEL32 mutant on CCR5 transport to the cell (Fig. 2 B), indicating that CCR5 mediates p38 MAPK acti- Mañes et al. The Journal of Experimental Medicine per se (Fig. 2 C). Finally, we used dominant negative mu- tants for MKK3 and MKK6 to block p38 MAPK activation (20). Overexpression of dnMKK3 or dnMKK6 mutants produced only modest inhibition of the CCL5-induced in- WAF1 crease in p21 and Mdm2 levels. Maximum inhibition was observed when dnMKK3 and dnMKK6 were co- expressed (Fig. 2 D). Likewise, UV-induced p38 phos- phorylation was maximally inhibited in cells coexpressing dnMKK3 and dnMKK6 (Fig. 2 E). The results suggest that specific CCR5 activation at the cell surface enhances p53 transcriptional activity in MCF-7 breast cancer cells by a G -, JAK-, and p38-dependent mechanism(s). The CCR5-p53 Pathway Regulates In Vivo Proliferation of Human Breast Tumor Xenografts. To study the CCR5-p53 pathway in tumor growth, we compared in vivo prolifera- tion of mock- and KDEL32-expressing MCF-7 (wild- type p53) or MDA-MB-231 (mutated p53) cells implanted into SCID mice. As for MCF-7, KDEL32 mutant ex- pression in MDA-MB-231 cells reduced surface CCR5 levels by 62  4.2%. We measured the amount of human CCL5 in extracts from mock and KDEL32 xenografts from both cell lines. CCL5 levels were comparable in mock and KDEL32 tumors (MCF-7-mock: 35.1  7.5 pg/100 g, n 8; MCF-7-32: 41.7  10.6 pg/100 g, n 8, P 0.5; MDA-MB-231-mock: 33.06  5.34 pg/ 100 g, n 5; MDA-MB-231-32: 50.21  17 pg/100 g, n 7, P 0.15, Mann-Whitney test), suggesting that all tumor types were equally exposed to CCL5. BrdU incorporation experiments using the MCF-7 xe- nografts showed that the percentage of BrdU cells was significantly higher in KDEL32 than in mock tumors (Fig. 3 A; P 0.05, Mann-Whitney test). It should be noted, however, that subcutaneous MCF-7 xenograft im- plantation and growth is dependent on estradiol supple- mentation during the experimentation period, and that estradiol induces equal proliferation of mock and KDEL32- expressing cells in vitro (unpublished data). This may at- Figure 2. CCR5 regulates p53 activity through in a G -, JAK2-, and tenuate the differences observed in BrdU incorporation WAF1 and Mdm2 p38 MAPK–dependent manner. (A) Measurement of p21 between mock- and KDEL32-expressing MCF-7 xe- induction analyzed by Western blot after CCL5 stimulation of MCF-7 nografts. Similar BrdU chase experiments using MDA- cells pretreated with the indicated chemical inhibitors. Data points represent MB-231 tumors showed no BrdU incorporation differ- the mean  SD of densitometric values obtained in two independent experiments for each inhibitor. Data are plotted relative to those obtained ences when mock- and KDEL32-expressing xenografts in DMSO-treated cells before CCL5 addition. (B and C) Mock- and were compared (Fig. 3 B). TUNEL assays of the xenografts KDEL32-expressing cells were stimulated with CCL5 (B) or UV irradi- showed a comparable percentage of apoptotic cells in ated (30 J/m , C), and then incubated at 37C for the times indicated (for mock and KDEL32 derived from MCF-7 (Fig. 3 C) or CCL5) or for 30 min (for UV irradiation). Cell lysates were analyzed by Western blot with anti–phospho-p38 (pp38) or anti-p38–specific anti- MDA-MB-231 (Fig. 3 D) tumors, indicating that CCR5 bodies. One representative experiment of three is shown. (D and E) expression did not affect apoptosis. Mock-, dnMKK3-, and dnMKK6-expressing cells and cells coexpressing WAF1 p21 levels were increased in mock- compared with dnMKK3 and dnMKK6 were stimulated with CCL5 (D) or UV irradi- 2 KDEL32-expressing MCF-7 xenografts (Fig. 4 A). Again, ated (30 J/m , E), and then incubated at 37C as above. CCL5-induced WAF1 p21 mdm2 up-regulation was visualized by Western blot (D). In the KDEL32 overexpression in MDA-MB-231 cells produced WAF1 case of UV irradiation (E), cell lysates were analyzed by Western blot no differences in p21 levels (Fig. 4 B). In agreement, with anti–phospho-p38 (pp38) or anti-p38–specific antibodies. In all WAF1 nuclear localization of p21 was clearly increased in mock cases, dnMKK3 and dnMKK6 expression was detected using an anti-Flag compared with KDEL32-MCF-7 xenografts, but not in antibody. One representative experiment of two is shown. those derived from MDA-MB-231 cells (Fig. 4 C). Collec- tively, these data indicate that a reduction in functional cell vation. p38 phosphorylation was nonetheless similar in surface CCR5 may increase the proliferation rate of tumor mock and KDEL32 cells after UV irradiation, indicating cells bearing wild-type p53, probably by diminishing levels WAF1 that KDEL32 expression does not affect p38 activation of the CDK inhibitor p21 . To analyze whether p38 CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine Figure 3. Cell surface levels of CCR5 specifically affect the pro- liferation of breast tumor xe- nografts with functional p53. BrdU (A and B) and TUNEL (C and D) staining of mock- and KDEL32-expressing MCF-7 (A and C) or MDA-MB-231 (B and D) xenografts. Counterstaining was done with hematoxylin and eosin for BrdU and nuclei were DAPI stained for TUNEL. 40. Data points at the right represent the percentage of BrdU or TUNEL nuclei determined in four random fields for each tumor analyzed (two-tailed Mann- Whitney test). *, P 0.05. MAPK was involved in this pathway, we stained mock and erence 16). In addition, the genotype distribution observed KDEL32-MCF-7 xenograft sections with an antibody in breast cancer patients was in accordance with the Hardy- against the active form of p38 MAPK. Mock-derived xe- Weinberg equilibrium law ( 0.57, P 0.75 with two nografts showed greater cytoplasmic and nuclear anti–phos- degrees of freedom). These data suggest that the 32 allele pho-p38 staining than those derived from KDEL32-MCF-7 is not associated with susceptibility to breast cancer. (percent nuclei stained for phospho-p38: mock 13.67  We next explored the role of the 32 allele in breast 3.1%, KDEL32 5.44  1.9%; Fig. 4 D). Thus, the results cancer by evaluating allelic frequency and genotypes of this suggest that the CCR5-p53 pathway is operative and regu- marker in patients grouped according to clinical, patholog- lates in vivo breast cancer cell proliferation. ical, or molecular parameters (Table I). We found no bias The CCR5-p53 Pathway Influences Human Breast Cancer in 32 frequency when tumors were grouped according to Progression. Chemokine and/or chemokine receptor hormone receptor (estrogen or progesterone), ErbB-2, or polymorphisms in the human population represent a angiogenin expression. Significant differences in allele fre- unique model by which to study the contribution of spe- quencies between groups were observed, however, when cific chemokines to pathogenesis. 32 is a 32-bp deletion classified by tumor size. Specifically, patients with the larg- within the CCR5 coding region, which results in a frame est tumor size (T4) had a 32 allelic frequency of 14.3%, a shift that generates a nonfunctional receptor (21–23). Ho- significant increase compared with the expected frequency mozygotes for this mutation (32/32) do not express for this mutation in the cohort ( 4.07, P 0.043). CCR5 on the cell surface, and receptor levels are also We analyzed whether the CCR5-induced p53 activation greatly reduced in 32 heterozygotes (32/). Both 32/ observed in breast cancer cell lines influenced breast cancer 32 and 32/ individuals appear healthy and show no progression in humans. A larger proportion of tumors de- apparent phenotype. Nonetheless, mutant 32 allele ex- rived from 32 patients tended to be negative for p53 ex- pression is associated with (a) resistance to HIV-1 infection pression than those derived from CCR5 wild-type individu- in homozygotes and slow progression to AIDS in heterozy- als (Table I). Absence of aberrant p53 expression, which gotes (21–23), (b) decreased severity of rheumatoid arthritis usually corresponds to p53 mutation and is associated with (24–26) and (c) multiple sclerosis (27), (d) long-term sur- better prognosis in breast cancer (30), is considered an indica- vival of renal transplants (28), and (e) reduced risk of myo- tion that this tumor suppressor gene is not mutated in these cardial infarction (29). These observations suggest a func- cancers. We analyzed DFS in the cohort, sorted according to tional role for CCR5 in these pathogenic processes. p53 and CCR5 status. Because of the small number of ho- To analyze the relevance of CCR5 in human breast can- mozygous patients, 32/32 and 32/ individuals were cer, we determined the allelic frequency and genotypes of grouped together. DFS differed significantly between 32 32 polymorphism in 547 patients diagnosed of primary and wild-type CCR5 patients only in those individuals with (nonmetastatic) breast cancer. The median follow-up period tumors with wild-type p53 (i.e., p53 tumors; Fig. 5, bot- was 83 mo. Six patients were 32/32 and 74 were 32/. tom; P 0.021, log-rank test). DFS was comparable be- The allelic frequency of 32 in the cohort was 7.86% tween 32 and wild-type CCR5 patients whose tumors ex- (86 of 1,094 chromosomes). No differences were detected pressed a mutated p53 form (i.e., p53 tumors; Fig. 5, in allelic frequencies between breast cancer patients and the bottom; P 0.752, log-rank test). When samples were general population (P 0.97, with Yates correction; ref- grouped according to CCR5 status (Fig. S1, available at Mañes et al. The Journal of Experimental Medicine Table I. Clinical Characteristics of Breast Cancer Patients (n 547) Allele frequency Clinical parameter CCR5wt CCR532 P ( Age 50 91.35 8.65 0.59 50 92.46 7.53 Menopausal status Pre-Peri 92.72 7.28 0.73 Post 91.96 8.04 Histologic grade I-II 91.64 8.36 0.86 III 91.07 8.93 Tumor size T1 92.82 7.18 T2 92.44 7.56 T3 92.04 7.95 T4 85.71 14.29 0.04 Lymph node infiltration 0 92.41 7.58 1–3 93.08 6.91 4–9 92.96 7.04 10 89.58 10.4 0.36 Stage I 92.02 7.97 II 92.62 7.38 III 89.24 10.76 0.22 Estrogen receptor Neg 91.21 8.79 0.52 Pos 92.54 7.46 Progesterone receptor Neg 91.63 8.37 0.76 Pos 92.32 7.68 ErbB-2 Neg 92.87 7.13 0.62 Pos 91.48 8.52 Angiogenin Neg 94.11 5.88 0.63 Pos 92.21 7.79 p53 Neg 91.11 8.89 Pos 94.11 5.89 0.1 WAF1 Figure 4. p21 and phospho-p38-MAPK detection in xenografts from mock- and KDEL32-expressing tumor cells. (A and B) Lysates Estrogen receptor defined as 10 fmol/mg, progesterone receptor as 20 fmol/mg (Abbott), ErbB-2 as 15 HNU/g (Oncogene from mock- and KDEL32-expressing MCF-7 (A) or MDA-MB-231 WAF1 (B) xenografts were analyzed sequentially with anti-p21 , anti-Mdm2, Science), angiogenin as 5 ng/mg (R&D Systems), and p53 defined anti-p53, and anti-tubulin antibodies by Western blot. (C) Cryosections by Western blot with pAb 181 (Oncogene Science) according to of xenografts derived from mock- and KDEL32-expressing MCF-7 and established standards. WAF1 anti- MDA-MB-231 cells, as indicated, were stained with anti-p21 body followed by a peroxidase-labeled second antibody. Counterstaining was performed with hematoxylin and eosin. (D) Paraffin sections from mock- and KDEL32-expressing MCF-7 xenografts were stained with ence in DFS between the p53-mutated versus the wild-type an anti–phospho-p38 antibody followed by a peroxidase-labeled second groups in 32 patients (P 0.68, log-rank test). antibody, and were hematoxylin and eosin counterstained. For C and D, the background staining with the second antibody was also analyzed (not depicted). 40. Discussion Here we present genetic evidence for the role of the CCR5 chemokine receptor in human breast cancer pro- gression. First, we show that stimulation of a human breast http://www.jem.org/cgi/content/full/jem.20030580/DC1), it was found that those wild-type CCR5 patients whose tu- adenocarcinoma cell line with the chemokine CCL5 results in activation of the tumor suppressor p53 through a mech- mors had wild-type p53 showed a better prognosis than those with mutant p53 (P 0.0051, log-rank test), concur- anism that depends on specific cell surface CCR5 expres- sion and G , JAK2, and p38-MAPK activation. Second, us- ring with the previously reported prognostic value of p53 (30). In contrast, there was no statistically significant differ- ing a mouse xenograft model, we show that the link CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine tivation at the cell surface was CCR5 expression depen- dent. Moreover, the active p38 MAPK form stained more intensely in mock-derived xenografts than in those derived from cells expressing the KDEL32 mutant, indicating that the CCR5-p38 MAPK pathway probably operates in this in vivo model. Because p38 MAPK is a prominent p53 ac- tivator in response to stress signals and certain anti-cancer drugs (34, 35), CCR5-induced p38 phosphorylation can WAF1 activate p53 transcriptional activity, leading to p21 in- duction and slowing of tumor cell growth. Concurring with this hypothesis, the data from our co- hort suggest that CCR5 influences the p53 mutation rate in human tumors. The p53 tumor suppressor gene is inacti- vated by mutation in approximately half of all human tu- mors. Evidence suggests that tumor cells have developed mechanisms other than mutation to “silence” p53 function (36). We found that mutation of p53 affected 46% of tu- mors from CCR5 wild-type individuals, but only 35% of tumors from CCR532 patients. Moreover, no tumors from the 32/32 patients showed mutant p53. These data strongly suggest that there is less selective pressure for p53 mutation in tumors from CCR532 individuals than from those bearing CCR5 wild-type alleles. In CCR532 pa- tients, all pathways downstream of CCR5 are abolished Figure 5. The 32 polymorphism affects human breast cancer progression. (homozygous) or severely impaired (heterozygous) and Kaplan-Meier DFS curves for the 547 breast cancer patients evaluated CCR5-mediated p53 activation is consequently diminished. according to p53 expression and the 32 polymorphism, as indicated. CCR5 wild-type (/) patients, solid line; 32/ and 32/32 patients, It is thus possible that p53 would be silenced in CCR532 dashed line. patients under conditions in which this tumor suppressor gene is activated in CCR5 wild-type individuals. In apparent contradiction to our results, elevated CCL5 between CCR5 and p53 is operative in regulating in vivo levels are reported in patients with progressive breast cancer proliferation of tumor cells. Abrogation of cell surface (8, 9), although it is noteworthy that these studies provide CCR5 expression enhanced proliferation of tumor cells no information on CCR5 and p53 expression. In one of bearing wild-type p53, but not of tumor cell lines express- these reports, markedly elevated plasma levels of CCL5 ing a mutated p53 form. Finally, based on the CCR532 were found in 27% of patients with progressive cancer, polymorphism in humans, which renders a nonfunctional raising the possibility that the CCL5 mediates tumor pro- CCR5 receptor, we found that CCR5 status influenced gression in some patients but not in others (9). Indeed, in disease progression in a genotyped cohort of 547 patients our cohort, the 32 polymorphism affects only breast can- diagnosed with primary (nonmetastatic) breast cancer. We cer progression in patients whose tumors expressed wild- found that DFS was shorter in CCR532 than in CCR5 type p53. wild-type patients with wild-type p53 tumors. Conversely, CCR5 may also have an indirect effect on cancer pro- DFS was comparable between CCR532 and CCR5 gression by controlling the antitumor immune response. wild-type patients whose tumors had mutant p53. CCR5 participates in chemotaxis of memory and activated Our results suggest a negative correlation between naive T cells and is required for T cell activation (37). Ele- CCR5 expression and the growth of human breast tumors expressing wild-type p53. As seen in our tumor xenograft models, CCL5 production in the tumor environment does not increase apoptosis, but restricts growth of cancer cells Table II. Lymphocyte Infiltration in Breast Tumors expressing wild-type p53 in a CCR5-dependent manner. These results concur with previous data showing the ability Infiltration grade  32/32/32 of CCL5 to inhibit growth factor–induced cell prolifera- tion (13), as well as CCL5-induced stabilization of p53 in 52 4 neuronal and astrocytic nuclei (17). In some cases, CCL5 11 0 can also activate the apoptotic pathway in CCR5-express- 10 0 ing cells (31). How CCR5 transmits the signal to p53 re- quires future investigation, but the data presented here sug- gest a role for p38 MAPK in this pathway. In agreement Lymphocyte infiltration 50% (), equal to 50% (), or 50% with others (32, 33), we found that CCL5-induced p38 ac- () per field at low magnification (10). Mañes et al. The Journal of Experimental Medicine vated lymphocyte infiltration is a rare event in breast can- Goeddel, S. Rosenberg, and T. Schall. 1996. RANTES se- cretion by gene-modified tumor cells results in loss of tumor- cer, as confirmed by analysis of lymphocyte infiltration in igenicity in vivo: role of immune cell subpopulations. Hum. 14 random samples from the cohort (Table II). Although Gene Ther. 7:1545–1553. additional research is needed to clarify the relevance of 32 8. Luboshits, G., S. Shina, O. Kaplan, S. Engelberg, D. Nass, B. polymorphism in the antitumor immune response, it is Lifshitz-Mercer, S. Chaitchik, I. Keydar, and A. Ben-Baruch. noteworthy that DFS curves in our cohort were similar be- 1999. Elevated expression of the CC chemokine regulated on tween 32 and wild-type patients whose tumors expressed activation, normal T cell expressed and secreted (RANTES) mutant p53. This suggests that the CCR5-dependent anti- in advanced breast carcinoma. Cancer Res. 59:4681–4687. tumor immune response does not have a major impact on 9. Niwa, Y., H. Akamatsu, H. Niwa, H. Sumi, Y. Ozaki, and this cancer progression. Alternatively, immune system im- A. Abe. 2001. Correlation of tissue and plasma RANTES pairment in 32 patients may only be important in those levels with disease course in patients with breast or cervical cancer. Clin. Cancer Res. 7:285–289. tumors with a benign prognosis (wild-type p53 tumors). 10. Mañes, S., E. Mira, C. Gomez-Mouton, R. Lacalle, P. In summary, we have identified a G -, JAK2-, and p38- Keller, J. Labrador, and C. Martinez-A. 1999. Membrane raft dependent pathway by which CCR5 regulates p53 tran- microdomains mediate front-rear polarity in migrating cells. scriptional activity. This pathway appears to be silenced or EMBO J. 18:6211–6220. severely impaired in individuals bearing the mutated 11. Mira, E., R. Lacalle, M. Gonzalez, C. Gomez-Mouton, J. CCR5 32 allele. As a consequence, breast tumors with Abad, A. Bernad, C. Martinez-A., and S. Mañes. 2001. 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Noncompetitive, chemokine-medi- MKK3 and MKK6 mutants, R. Sánchez-Prieto for helpful discus- ated inhibition of basic fibroblast growth factor-induced en- sions, D. Balomenos for aid with in vivo proliferation studies, L. dothelial cell proliferation. J. Biol. Chem. 273:7911–7919. Gómez for animal handling, and C. Mark for editorial assistance. 14. Del Real, G., S. Jiménez-Baranda, R. Lacalle, E. Mira, P. Lu- S. Jiménez-Baranda receives a fellowship from the Spanish Pro- cas, C. Gomez-Mouton, A. Carrera, C. Martinez-A., and S. grama de Formación de Personal Universitario. This work was sup- Mañes. 2002. Blocking of HIV-1 infection by targeting CD4 ported by grants from the Spanish MCyT. The Department of Im- to nonraft membrane domains. J. Exp. Med. 196:293–301. munology and Oncology was founded and is supported by the 15. Serrano, M., A. Lin, M. McCurrach, D. Beach, and S. Lowe. Spanish Council for Scientific Research and by Pfizer. 1997. 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CCR5 Expression Influences the Progression of Human Breast Cancer in a p53-dependent Manner

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Copyright © 2003, The Rockefeller University Press
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10.1084/jem.20030580
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Abstract

Chemokines are implicated in tumor pathogenesis, although it is unclear whether they affect human cancer progression positively or negatively. We found that activation of the chemokine receptor CCR5 regulates p53 transcriptional activity in breast cancer cells through pertussis toxin–, JAK2-, and p38 mitogen–activated protein kinase–dependent mechanisms. CCR5 blockade significantly enhanced proliferation of xenografts from tumor cells bearing wild-type p53, but did not affect proliferation of tumor xenografts bearing a p53 mutation. In parallel, data obtained in a primary breast cancer clinical series showed that disease-free survival was shorter in individuals bearing the CCR532 allele than in CCR5 wild-type patients, but only for those whose tumors expressed wild-type p53. These findings suggest that CCR5 activity influences human breast cancer progression in a p53-dependent manner. Key words: chemokine receptor • breast cancer • p53 • CCR5 polymorphism • p38 Introduction Many cancers express an extensive network of chemo- Current evidence does not establish whether these che- kines and chemokine receptors (1, 2). Studies in animal mokine biological activities in the tumor microenviron- models suggest that chemokines may act on tumor cells ment contribute to cancer growth and spread, or to host and/or tumor-infiltrating leukocytes (3–6). Tumor-pro- antitumor response and cancer regression. Studies using the duced chemokines are thought to have distinct roles in CC chemokine CCL5 (RANTES) as a model reported that the biology of primary and metastatic disease, including elevated CCL5 levels in the tumor environment contribute (a) directing leukocyte infiltration into the tumor, (b) reg- to improving the immune response against breast carcino- ulating the antitumor immune response, (c) controlling mas (7), but also correlate with poor prognosis in breast can- tumor angiogenesis, (d) functioning as autocrine or para- cers (8, 9). CCL5 also has a role in the chemotaxis and me- crine growth and survival factors, and (e) controlling tumor tastasis of breast cancer cell lines (10–12). Selected cell lines cell movement. with an enhanced chemotactic response to CCL5 neverthe- less showed decreased growth potential in nude mice, sug- gesting an inverse correlation between CCL5-induced The online version of this article contains supplemental material. chemotactic and proliferation signals (12). Compatible with Address correspondence to Santos Mañes, Department of Immunology this, basic fibroblast growth factor–induced proliferation of and Oncology, Centro Nacional de Biotecnología, Campus de Canto- endothelial cells was inhibited by distinct chemokines, in- blanco, Universidad Autonoma de Madrid, E-28049 Madrid, Spain. Phone: 34-91-585-4660; Fax: 34-91-372-0493; e-mail: [email protected] cluding CCL5, via a noncompetitive mechanism (13). R. Colomer’s present address is Instituto Catalan de Oncologia, Medical These results indicate that the role of chemokines in tumor Oncology Division, Hospital Josep Truet, Fança s/n, E-17007 Gerona, progression is complex and poorly understood. Spain. S. Montero’s present address is Pharma Mar S.A., Avda de los Reyes 1, Colmenar Viejo, E-28770 Madrid, Spain. Abbreviations used in this paper: BrdU, bromodeoxyuridine; DFS, disease- K. Harshman’s present address is Center for Integrative Genomics, free survival; MAPK, mitogen-activated protein kinase; siRNA, small University of Lausanne, CH-1015 Lausanne, Switzerland. interfering RNA. 1381 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2003/11/1381/9 $8.00 Volume 198, Number 9, November 3, 2003 1381–1389 http://www.jem.org/cgi/doi/10.1084/jem.20030580 The Journal of Experimental Medicine Here we studied the role of CCR5 in breast tumor pro- two intraperitoneal injections of bromodeoxyuridine (BrdU; 10 mg/ml, 50 l/mouse) 6 h apart, and primary tumors were re- gression by analyzing proliferation of xenografts derived moved 4 h after the last injection. Some tumors were fixed in from tumor cells expressing wild-type or mutated p53 in 70% ethanol, paraffin embedded, and analyzed for BrdU incorpo- combination with wild-type or mutated CCR5. We also ration using anti-BrdU antibody (Becton Dickinson) or active took advantage of the fact that 1% of the Caucasian pop- p38 MAPK using anti–phospho-p38 antibody (no. 9216; New ulation is homozygous for the CCR532 polymorphism, England BioLabs, Inc.). Other tumors were snap frozen in Tissue which renders a nonfunctional CCR5 receptor, to study Freezing Medium (Sakura Finetek) and cryosections were used the effect of this mutation in a clinical series of breast can- for in situ apoptosis determination by the TUNEL method cer patients. We describe a new mechanism by which (MEBSTAIN Apoptosis kit II; Immunotech) or p21 detection. CCR5 influences human breast cancer progression, de- Finally, some tumors were mechanically disrupted. Cell lysates pending on the status of the p53 tumor suppressor. were prepared with RIPA buffer and used both to measure hu- man CCL5 (Cytoscreen; Biosource International) and for p53, p21, and Mdm2 analysis in Western blot. CCL5 levels, BrdU in- Materials and Methods corporation, and TUNEL data were compared using the two- tailed Mann-Whitney test. Cell Lines and Stimulation. MCF-7 and MDA-MB-231 cells 32 Genotyping of the Breast Cancer Cohort. The breast can- (American Type Culture Collection) were transduced with retro- cer cohort comprised primary (nonmetastatic) breast cancer pa- viral pLZ-KDEL32-IRES-gfp or pLZ-IRES-gfp (mock) super- tients diagnosed at the Hospital Universitario 12 de Octubre, natants, and green fluorescent protein–expressing cells were se- Madrid, Spain, from January 1992 to December 1995. Stage IV lected by fluorescence-activated cell sorting (11). The percentage patients at diagnosis were excluded from this series. DNA was of CCR5 cells observed after subtracting the control value was isolated from biopsies of 547 patients and the CCR532 allele multiplied by average fluorescence intensity to calculate surface determined with an automated method based on real-time PCR CCR5 expression in mock and KDEL32-expressing cells (14). 175H in a Lightcycler system (16). Patients included in this study were MCF-7-p53 cells were obtained by transfection with pWZL- 175H disease free after surgery and showed no initial metastases or me- Hygro-p53 (provided by M. Serrano, Centro Nacional de tastases within 3 mo after surgery. Biotechnologia, Madrid, Spain; reference 15), followed by hy- Chemotherapy was administered to premenopausal patients gromycin selection. p53 silencing by small interfering RNA with tumors larger than 1 cm, postmenopausal patients with neg- (siRNA) was performed by transfecting MCF-7 cells with a pool ative estrogen receptor expression, and patients showing affected of p53-specific siRNA (SMARTpool kit; Dharmacon) at 100 lymph nodes. Patients with conservative surgery and those show- nM using Oligofectamine (GIBCO BRL). Transfection effi- ing four or more axillary lymph nodes affected were treated with ciency was 75  5.1% as estimated using a fluorescent-labeled radiotherapy. Patients whose tumors expressed hormone recep- siRNA. As a control, MCF-7 cells were transfected with nonspe- tors were treated with tamoxifen for 5 yr. Clinical follow-up was cific pooled siRNA duplexes (Dharmacon). Finally, MCF-7 cells performed according to institutional protocols by physical exami- were transfected with Flag-tagged dominant negative mutants for nation every 3 mo in the first 2 yr after surgery, every 6 mo in the MAP kinase kinases 3 and 6 (dnMKK3 and dnMKK6; provided next 3 yr, and every year after the sixth year. Mammography was by R.J. Davis, Howard Hughes Medical Institute, University of performed yearly and other diagnostic tests were performed when Massachusetts, Worcester, MA). 175H relapse was suspected. Disease-free survival (DFS) was defined as Serum-depleted mock, KDEL32-, p53 -, siRNA-, the time (in months) from diagnosis of disease, usually coinciding dnMKK3-, dnMKK6-, or dnMKK3 plus dnMKK6–expressing with surgery, to first relapse or to the last clinical revision for pa- MCF-7 cells were stimulated with 100 nM CCL5 (PeproTech), tients with no recurrence. A total of 133 out of 541 patients re- irradiated (30 Gy) or UV irradiated with UVC (254 nm, 30 2 lapsed (24.5%). J/m ), and then incubated at 37C for the times indicated. Equal Allelic frequencies between patient groups where compared protein amounts from cell lysates prepared with RIPA buffer (50 using standard tests with Yates correction, the Mantel-Haens- mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxy- zel test, or Fisher’s exact test where necessary, using Statcalc soft- cholate, 1% NP-40) were analyzed by Western blot with anti- ware (EpiInfo 5.1). For statistical analysis of genotypes, 32/ p53 (Oncogene Research Products), anti-p21 and anti-Mdm2 and 32/32 individuals were grouped to avoid values of 7. (Santa Cruz Biotechnology, Inc.), anti-p38 and anti–phospho- DFS was estimated using the Kaplan-Meier method and com- p38 mitogen-activated protein kinase (MAPK; no. 9211; New pared by the log-rank test. England BioLabs, Inc.), and anti-Flag (Sigma-Aldrich) antibodies. Online Supplemental Material. Fig. S1 shows DFS curves in Protein loading was confirmed with anti-tubulin antibody breast cancer patients grouped according to CCR5 status. Fig. (Sigma-Aldrich). For some experiments, cells were pretreated for S1 is available at http://www.jem.org/cgi/content/full/jem. 14 h with 100 M tyrphostin AG 490, 10 M LY 294002, 10 20030580/DC1. M PD 98059, 10 M SB 203580 (all from Calbiochem), 0.75 g/ml pertussis toxin (Sigma-Aldrich), or DMSO before CCL5 stimulation. Densitometric analysis of Western blots was per- formed with NIH Image software. Results Breast Tumor Xenograft Models and Immunohistochemistry. Eight CCR5 Signaling Increases p53 Transcriptional Activity. female BALB/c-SCID mice were injected subcutaneously in CCR5-mediated signals may regulate transcription of sev- both flanks with 3  10 mock or KDEL32-expressing MDA- 6 eral p53 target genes in certain cell types (17). To study the MB-231 or 5  10 MCF-7 cells. In the case of MCF-7, mice association between CCR5 and p53 in cancer cells, we an- were pretreated with 1 g/ml 17 -ethynyl estradiol (Sigma- alyzed CCR5-dependent signaling in the MCF-7 breast Aldrich) in drinking water for 1 wk before cell injection and during the experimental period. 4 wk after cell injection, mice received carcinoma line, which expresses CCR5 and wild-type p53. CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine Figure 1. CCR5 regulates p53 transcrip- tional activity. (A) Time course induction of WAF1 p53 (dotted line, ), p21 (solid line, ), and Mdm2 (dashed line, ) in CCL5-stimulated MCF-7 cells. Western blots from four indepen- dent experiments were quantified by densitometry and the values were normalized using the tubulin loading control. Data points are plotted relative to mean values obtained before chemokine addi- tion (n 5). (B) CCL5-induced p53 targets in 175H MCF-7 cells expressing the p53 mutant. One representative experiment of three is shown. (C and D) MCF-7 cells transfected with control or p53-specific siRNA oligonucleotides were stimulated with CCL5 (C) and incubated at 37C for the times indicated or  irradiated (30 Gy; D) and incubated for 3 h at 37C. Cell lysates were analyzed by Western blot. One representative experiment of three is shown. (E) CCR5 was detected in live mock- or KDEL32-expressing MCF-7 cells by FACS . Surface CCR5 expression in mock- and KDEL32-expressing cells was estimated by multiplying average fluorescence intensity by the percentage of CCR5 cells (n 3). (F and G) Mock- and KDEL32-expressing cells were stimulated with CCL5 (F) or  irradiated (30 Gy; G) and incubated at 37C for the times indicated. Cell lysates were analyzed by Western blot. One representative experiment of three is shown. Stimulation of MCF-7 cells with CCL5, a ligand for surface (18). KDEL32 expression reduced CCR5 levels by WAF1 CCR5, increased levels of well-established p53-regulated 70% (Fig. 1 E). CCL5 did not induce p21 or Mdm2 WAF1/CIP1 genes, such as p21 and Mdm2 (Fig. 1 A). p53 pro- in KDEL32-expressing MCF-7 cells (Fig. 1 F), indicating tein levels were essentially unaltered after CCL5 stimula- that CCR5 is the specific receptor by which CCL5 induces WAF1 tion, although a slight increase was observed 60 min after these genes. p21 and Mdm2 up-regulation were similar, stimulation. To determine whether CCL5-mediated however, in mock- and KDEL32-expressing MCF-7 cells WAF1 p21 and Mdm2 up-regulation is dependent on p53 after  irradiation, suggesting that KDEL32 expression did transcriptional activity, MCF-7 cells stably expressing a not affect the p53 pathway in these cells (Fig. 1 G). 175H dominant negative p53 mutant (15) were stimulated p53 Transcriptional Activity Requires CCR5-mediated p38 WAF1 with CCL5. CCL5 did not increase p21 or Mdm2 Activation. To study the mechanism by which CCR5 ac- 175H protein levels in MCF-7-p53 cells (Fig. 1 B). Concur- tivates p53, we used chemical inhibitors to block CCR5- rently, transfection of MCF-7 cells with p53-specific mediated signaling. Binding of chemokines to their recep- WAF1 siRNA prevented CCL5-induced p21 and Mdm2 up- tors leads to activation of heterotrimeric G proteins and regulation (Fig. 1 C). As a control, p53-specific siRNA also JAK family tyrosine kinases. These in turn initiate multiple inhibited  irradiation–induced up-regulation of p53 activ- signaling cascades, including phosphatidylinositol-3 kinase ity (Fig. 1 D). These results suggest that CCL5-induced as well as p38 and p42/p44 MAPK pathways (19). Incuba- transcriptional activation of p53 target genes, such as tion of MCF-7 cells with the inhibitors LY 294002 and PD WAF1 p21 and Mdm2, requires functional p53 protein. 98059, which block phosphatidylinositol-3 kinase and To analyze whether CCL5-induced p53 activation re- p42/p44 MAPK pathways, has only a marginal effect on WAF1 quires CCR5-mediated signaling at the cell surface, we spe- CCR5 induction of p21 and Mdm2 (Fig. 2 A). In con- WAF1 cifically blocked CCR5 in MCF-7 cells with a CCR5 trast, CCR5-mediated induction of p21 and Mdm2 dominant negative mutant. We previously showed that was inhibited by treatment of MCF-7 cells with pertussis overexpression of a CCR5 deletion mutant (KDEL32), toxin, AG 490, and SB 203580 (Fig. 2 A), which impede which is phenotypically similar to the natural CCR532 G , JAK-2, and p38 MAPK activation, respectively. In ad- (32) mutant, abrogates CCR5 function in MCF-7 cells dition, CCL5 induced p38 phosphorylation in mock-trans- (11). This effect is probably due to a trans inhibitory effect fected MCF-7 cells, but not in KDEL32-expressing cells of the KDEL32 mutant on CCR5 transport to the cell (Fig. 2 B), indicating that CCR5 mediates p38 MAPK acti- Mañes et al. The Journal of Experimental Medicine per se (Fig. 2 C). Finally, we used dominant negative mu- tants for MKK3 and MKK6 to block p38 MAPK activation (20). Overexpression of dnMKK3 or dnMKK6 mutants produced only modest inhibition of the CCL5-induced in- WAF1 crease in p21 and Mdm2 levels. Maximum inhibition was observed when dnMKK3 and dnMKK6 were co- expressed (Fig. 2 D). Likewise, UV-induced p38 phos- phorylation was maximally inhibited in cells coexpressing dnMKK3 and dnMKK6 (Fig. 2 E). The results suggest that specific CCR5 activation at the cell surface enhances p53 transcriptional activity in MCF-7 breast cancer cells by a G -, JAK-, and p38-dependent mechanism(s). The CCR5-p53 Pathway Regulates In Vivo Proliferation of Human Breast Tumor Xenografts. To study the CCR5-p53 pathway in tumor growth, we compared in vivo prolifera- tion of mock- and KDEL32-expressing MCF-7 (wild- type p53) or MDA-MB-231 (mutated p53) cells implanted into SCID mice. As for MCF-7, KDEL32 mutant ex- pression in MDA-MB-231 cells reduced surface CCR5 levels by 62  4.2%. We measured the amount of human CCL5 in extracts from mock and KDEL32 xenografts from both cell lines. CCL5 levels were comparable in mock and KDEL32 tumors (MCF-7-mock: 35.1  7.5 pg/100 g, n 8; MCF-7-32: 41.7  10.6 pg/100 g, n 8, P 0.5; MDA-MB-231-mock: 33.06  5.34 pg/ 100 g, n 5; MDA-MB-231-32: 50.21  17 pg/100 g, n 7, P 0.15, Mann-Whitney test), suggesting that all tumor types were equally exposed to CCL5. BrdU incorporation experiments using the MCF-7 xe- nografts showed that the percentage of BrdU cells was significantly higher in KDEL32 than in mock tumors (Fig. 3 A; P 0.05, Mann-Whitney test). It should be noted, however, that subcutaneous MCF-7 xenograft im- plantation and growth is dependent on estradiol supple- mentation during the experimentation period, and that estradiol induces equal proliferation of mock and KDEL32- expressing cells in vitro (unpublished data). This may at- Figure 2. CCR5 regulates p53 activity through in a G -, JAK2-, and tenuate the differences observed in BrdU incorporation WAF1 and Mdm2 p38 MAPK–dependent manner. (A) Measurement of p21 between mock- and KDEL32-expressing MCF-7 xe- induction analyzed by Western blot after CCL5 stimulation of MCF-7 nografts. Similar BrdU chase experiments using MDA- cells pretreated with the indicated chemical inhibitors. Data points represent MB-231 tumors showed no BrdU incorporation differ- the mean  SD of densitometric values obtained in two independent experiments for each inhibitor. Data are plotted relative to those obtained ences when mock- and KDEL32-expressing xenografts in DMSO-treated cells before CCL5 addition. (B and C) Mock- and were compared (Fig. 3 B). TUNEL assays of the xenografts KDEL32-expressing cells were stimulated with CCL5 (B) or UV irradi- showed a comparable percentage of apoptotic cells in ated (30 J/m , C), and then incubated at 37C for the times indicated (for mock and KDEL32 derived from MCF-7 (Fig. 3 C) or CCL5) or for 30 min (for UV irradiation). Cell lysates were analyzed by Western blot with anti–phospho-p38 (pp38) or anti-p38–specific anti- MDA-MB-231 (Fig. 3 D) tumors, indicating that CCR5 bodies. One representative experiment of three is shown. (D and E) expression did not affect apoptosis. Mock-, dnMKK3-, and dnMKK6-expressing cells and cells coexpressing WAF1 p21 levels were increased in mock- compared with dnMKK3 and dnMKK6 were stimulated with CCL5 (D) or UV irradi- 2 KDEL32-expressing MCF-7 xenografts (Fig. 4 A). Again, ated (30 J/m , E), and then incubated at 37C as above. CCL5-induced WAF1 p21 mdm2 up-regulation was visualized by Western blot (D). In the KDEL32 overexpression in MDA-MB-231 cells produced WAF1 case of UV irradiation (E), cell lysates were analyzed by Western blot no differences in p21 levels (Fig. 4 B). In agreement, with anti–phospho-p38 (pp38) or anti-p38–specific antibodies. In all WAF1 nuclear localization of p21 was clearly increased in mock cases, dnMKK3 and dnMKK6 expression was detected using an anti-Flag compared with KDEL32-MCF-7 xenografts, but not in antibody. One representative experiment of two is shown. those derived from MDA-MB-231 cells (Fig. 4 C). Collec- tively, these data indicate that a reduction in functional cell vation. p38 phosphorylation was nonetheless similar in surface CCR5 may increase the proliferation rate of tumor mock and KDEL32 cells after UV irradiation, indicating cells bearing wild-type p53, probably by diminishing levels WAF1 that KDEL32 expression does not affect p38 activation of the CDK inhibitor p21 . To analyze whether p38 CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine Figure 3. Cell surface levels of CCR5 specifically affect the pro- liferation of breast tumor xe- nografts with functional p53. BrdU (A and B) and TUNEL (C and D) staining of mock- and KDEL32-expressing MCF-7 (A and C) or MDA-MB-231 (B and D) xenografts. Counterstaining was done with hematoxylin and eosin for BrdU and nuclei were DAPI stained for TUNEL. 40. Data points at the right represent the percentage of BrdU or TUNEL nuclei determined in four random fields for each tumor analyzed (two-tailed Mann- Whitney test). *, P 0.05. MAPK was involved in this pathway, we stained mock and erence 16). In addition, the genotype distribution observed KDEL32-MCF-7 xenograft sections with an antibody in breast cancer patients was in accordance with the Hardy- against the active form of p38 MAPK. Mock-derived xe- Weinberg equilibrium law ( 0.57, P 0.75 with two nografts showed greater cytoplasmic and nuclear anti–phos- degrees of freedom). These data suggest that the 32 allele pho-p38 staining than those derived from KDEL32-MCF-7 is not associated with susceptibility to breast cancer. (percent nuclei stained for phospho-p38: mock 13.67  We next explored the role of the 32 allele in breast 3.1%, KDEL32 5.44  1.9%; Fig. 4 D). Thus, the results cancer by evaluating allelic frequency and genotypes of this suggest that the CCR5-p53 pathway is operative and regu- marker in patients grouped according to clinical, patholog- lates in vivo breast cancer cell proliferation. ical, or molecular parameters (Table I). We found no bias The CCR5-p53 Pathway Influences Human Breast Cancer in 32 frequency when tumors were grouped according to Progression. Chemokine and/or chemokine receptor hormone receptor (estrogen or progesterone), ErbB-2, or polymorphisms in the human population represent a angiogenin expression. Significant differences in allele fre- unique model by which to study the contribution of spe- quencies between groups were observed, however, when cific chemokines to pathogenesis. 32 is a 32-bp deletion classified by tumor size. Specifically, patients with the larg- within the CCR5 coding region, which results in a frame est tumor size (T4) had a 32 allelic frequency of 14.3%, a shift that generates a nonfunctional receptor (21–23). Ho- significant increase compared with the expected frequency mozygotes for this mutation (32/32) do not express for this mutation in the cohort ( 4.07, P 0.043). CCR5 on the cell surface, and receptor levels are also We analyzed whether the CCR5-induced p53 activation greatly reduced in 32 heterozygotes (32/). Both 32/ observed in breast cancer cell lines influenced breast cancer 32 and 32/ individuals appear healthy and show no progression in humans. A larger proportion of tumors de- apparent phenotype. Nonetheless, mutant 32 allele ex- rived from 32 patients tended to be negative for p53 ex- pression is associated with (a) resistance to HIV-1 infection pression than those derived from CCR5 wild-type individu- in homozygotes and slow progression to AIDS in heterozy- als (Table I). Absence of aberrant p53 expression, which gotes (21–23), (b) decreased severity of rheumatoid arthritis usually corresponds to p53 mutation and is associated with (24–26) and (c) multiple sclerosis (27), (d) long-term sur- better prognosis in breast cancer (30), is considered an indica- vival of renal transplants (28), and (e) reduced risk of myo- tion that this tumor suppressor gene is not mutated in these cardial infarction (29). These observations suggest a func- cancers. We analyzed DFS in the cohort, sorted according to tional role for CCR5 in these pathogenic processes. p53 and CCR5 status. Because of the small number of ho- To analyze the relevance of CCR5 in human breast can- mozygous patients, 32/32 and 32/ individuals were cer, we determined the allelic frequency and genotypes of grouped together. DFS differed significantly between 32 32 polymorphism in 547 patients diagnosed of primary and wild-type CCR5 patients only in those individuals with (nonmetastatic) breast cancer. The median follow-up period tumors with wild-type p53 (i.e., p53 tumors; Fig. 5, bot- was 83 mo. Six patients were 32/32 and 74 were 32/. tom; P 0.021, log-rank test). DFS was comparable be- The allelic frequency of 32 in the cohort was 7.86% tween 32 and wild-type CCR5 patients whose tumors ex- (86 of 1,094 chromosomes). No differences were detected pressed a mutated p53 form (i.e., p53 tumors; Fig. 5, in allelic frequencies between breast cancer patients and the bottom; P 0.752, log-rank test). When samples were general population (P 0.97, with Yates correction; ref- grouped according to CCR5 status (Fig. S1, available at Mañes et al. The Journal of Experimental Medicine Table I. Clinical Characteristics of Breast Cancer Patients (n 547) Allele frequency Clinical parameter CCR5wt CCR532 P ( Age 50 91.35 8.65 0.59 50 92.46 7.53 Menopausal status Pre-Peri 92.72 7.28 0.73 Post 91.96 8.04 Histologic grade I-II 91.64 8.36 0.86 III 91.07 8.93 Tumor size T1 92.82 7.18 T2 92.44 7.56 T3 92.04 7.95 T4 85.71 14.29 0.04 Lymph node infiltration 0 92.41 7.58 1–3 93.08 6.91 4–9 92.96 7.04 10 89.58 10.4 0.36 Stage I 92.02 7.97 II 92.62 7.38 III 89.24 10.76 0.22 Estrogen receptor Neg 91.21 8.79 0.52 Pos 92.54 7.46 Progesterone receptor Neg 91.63 8.37 0.76 Pos 92.32 7.68 ErbB-2 Neg 92.87 7.13 0.62 Pos 91.48 8.52 Angiogenin Neg 94.11 5.88 0.63 Pos 92.21 7.79 p53 Neg 91.11 8.89 Pos 94.11 5.89 0.1 WAF1 Figure 4. p21 and phospho-p38-MAPK detection in xenografts from mock- and KDEL32-expressing tumor cells. (A and B) Lysates Estrogen receptor defined as 10 fmol/mg, progesterone receptor as 20 fmol/mg (Abbott), ErbB-2 as 15 HNU/g (Oncogene from mock- and KDEL32-expressing MCF-7 (A) or MDA-MB-231 WAF1 (B) xenografts were analyzed sequentially with anti-p21 , anti-Mdm2, Science), angiogenin as 5 ng/mg (R&D Systems), and p53 defined anti-p53, and anti-tubulin antibodies by Western blot. (C) Cryosections by Western blot with pAb 181 (Oncogene Science) according to of xenografts derived from mock- and KDEL32-expressing MCF-7 and established standards. WAF1 anti- MDA-MB-231 cells, as indicated, were stained with anti-p21 body followed by a peroxidase-labeled second antibody. Counterstaining was performed with hematoxylin and eosin. (D) Paraffin sections from mock- and KDEL32-expressing MCF-7 xenografts were stained with ence in DFS between the p53-mutated versus the wild-type an anti–phospho-p38 antibody followed by a peroxidase-labeled second groups in 32 patients (P 0.68, log-rank test). antibody, and were hematoxylin and eosin counterstained. For C and D, the background staining with the second antibody was also analyzed (not depicted). 40. Discussion Here we present genetic evidence for the role of the CCR5 chemokine receptor in human breast cancer pro- gression. First, we show that stimulation of a human breast http://www.jem.org/cgi/content/full/jem.20030580/DC1), it was found that those wild-type CCR5 patients whose tu- adenocarcinoma cell line with the chemokine CCL5 results in activation of the tumor suppressor p53 through a mech- mors had wild-type p53 showed a better prognosis than those with mutant p53 (P 0.0051, log-rank test), concur- anism that depends on specific cell surface CCR5 expres- sion and G , JAK2, and p38-MAPK activation. Second, us- ring with the previously reported prognostic value of p53 (30). In contrast, there was no statistically significant differ- ing a mouse xenograft model, we show that the link CCR5 Influences Breast Cancer Progression The Journal of Experimental Medicine tivation at the cell surface was CCR5 expression depen- dent. Moreover, the active p38 MAPK form stained more intensely in mock-derived xenografts than in those derived from cells expressing the KDEL32 mutant, indicating that the CCR5-p38 MAPK pathway probably operates in this in vivo model. Because p38 MAPK is a prominent p53 ac- tivator in response to stress signals and certain anti-cancer drugs (34, 35), CCR5-induced p38 phosphorylation can WAF1 activate p53 transcriptional activity, leading to p21 in- duction and slowing of tumor cell growth. Concurring with this hypothesis, the data from our co- hort suggest that CCR5 influences the p53 mutation rate in human tumors. The p53 tumor suppressor gene is inacti- vated by mutation in approximately half of all human tu- mors. Evidence suggests that tumor cells have developed mechanisms other than mutation to “silence” p53 function (36). We found that mutation of p53 affected 46% of tu- mors from CCR5 wild-type individuals, but only 35% of tumors from CCR532 patients. Moreover, no tumors from the 32/32 patients showed mutant p53. These data strongly suggest that there is less selective pressure for p53 mutation in tumors from CCR532 individuals than from those bearing CCR5 wild-type alleles. In CCR532 pa- tients, all pathways downstream of CCR5 are abolished Figure 5. The 32 polymorphism affects human breast cancer progression. (homozygous) or severely impaired (heterozygous) and Kaplan-Meier DFS curves for the 547 breast cancer patients evaluated CCR5-mediated p53 activation is consequently diminished. according to p53 expression and the 32 polymorphism, as indicated. CCR5 wild-type (/) patients, solid line; 32/ and 32/32 patients, It is thus possible that p53 would be silenced in CCR532 dashed line. patients under conditions in which this tumor suppressor gene is activated in CCR5 wild-type individuals. In apparent contradiction to our results, elevated CCL5 between CCR5 and p53 is operative in regulating in vivo levels are reported in patients with progressive breast cancer proliferation of tumor cells. Abrogation of cell surface (8, 9), although it is noteworthy that these studies provide CCR5 expression enhanced proliferation of tumor cells no information on CCR5 and p53 expression. In one of bearing wild-type p53, but not of tumor cell lines express- these reports, markedly elevated plasma levels of CCL5 ing a mutated p53 form. Finally, based on the CCR532 were found in 27% of patients with progressive cancer, polymorphism in humans, which renders a nonfunctional raising the possibility that the CCL5 mediates tumor pro- CCR5 receptor, we found that CCR5 status influenced gression in some patients but not in others (9). Indeed, in disease progression in a genotyped cohort of 547 patients our cohort, the 32 polymorphism affects only breast can- diagnosed with primary (nonmetastatic) breast cancer. We cer progression in patients whose tumors expressed wild- found that DFS was shorter in CCR532 than in CCR5 type p53. wild-type patients with wild-type p53 tumors. Conversely, CCR5 may also have an indirect effect on cancer pro- DFS was comparable between CCR532 and CCR5 gression by controlling the antitumor immune response. wild-type patients whose tumors had mutant p53. CCR5 participates in chemotaxis of memory and activated Our results suggest a negative correlation between naive T cells and is required for T cell activation (37). Ele- CCR5 expression and the growth of human breast tumors expressing wild-type p53. As seen in our tumor xenograft models, CCL5 production in the tumor environment does not increase apoptosis, but restricts growth of cancer cells Table II. Lymphocyte Infiltration in Breast Tumors expressing wild-type p53 in a CCR5-dependent manner. These results concur with previous data showing the ability Infiltration grade  32/32/32 of CCL5 to inhibit growth factor–induced cell prolifera- tion (13), as well as CCL5-induced stabilization of p53 in 52 4 neuronal and astrocytic nuclei (17). In some cases, CCL5 11 0 can also activate the apoptotic pathway in CCR5-express- 10 0 ing cells (31). How CCR5 transmits the signal to p53 re- quires future investigation, but the data presented here sug- gest a role for p38 MAPK in this pathway. In agreement Lymphocyte infiltration 50% (), equal to 50% (), or 50% with others (32, 33), we found that CCL5-induced p38 ac- () per field at low magnification (10). Mañes et al. The Journal of Experimental Medicine vated lymphocyte infiltration is a rare event in breast can- Goeddel, S. Rosenberg, and T. Schall. 1996. 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