| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 INSERM,, U844, Site Saint Eloi-Bat INM, 80, rue Augustin Fliche, Montpellier F-34091, France 2 University of Montpellier I,, Montpellier F-34090, France 3 INSERM,, U735, Laboratoire d'Oncogénétique and 4 Laboratoire d'Anatomo-Cytopathologie,, Centre René Huguenin, 35 rue Dailly, St-Cloud F-92211, France
(Correspondence should be addressed to G Lazennec, INSERM, U844, Site Saint Eloi-Bat INM, 80, rue Augustin Fliche, BP 74103 Montpellier Cedex 5, France; Email: lazennec{at}montp.inserm.fr)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
(ER)-negative breast cell lines. Analysis of CXCL8 chromosomal location showed that several CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8) were localized in the same narrow region (360 kb in size) of chromosome 4. We thus hypothesized that they could belong to the same cluster. Quantification of these chemokines in breast tumors showed that samples expressing high CXCL8 also produced elevated levels of CXCL1, CXCL3, and CXCL5, and displayed low content of ER. CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 were co-regulated both in tumors and in breast cancer cell lines. CXCL5 and CXCL8 were mainly produced by epithelial cells, whereas CXCL1, CXCL2, and CXCL3 had a high expression in blood cells. The overexpression of these chemokines in tumor cells was not the result of gene amplification, but rather of an enhanced gene transcription. Our data suggest that high CXCL8 expression in tumors is mainly correlated to activating protein-1 (AP-1) pathway and to a minor extent to NF-
B pathway. Interestingly, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 chemokines were present at higher levels in metastases when compared with grade I and III biopsies. High levels of CXCL8, CXCL1, and CXCL3 accounted for a shorter relapse-free survival of ER-positive patients treated with tamoxifen. In summary, we present evidences that multiple CXC chemokines are co-expressed in CXCL8-positive breast tumors. In addition, these chemokines could account for the higher aggressiveness of these types of tumors.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
(ER). The lack of ER expression in breast tumors is associated with poorer prognosis (Skoog et al. 1987, Lazennec et al. 2001). However, little is known about the possible factors involved in this difference.
Recently, chemokines have emerged as potential factors involved in breast carcinogenesis (Freund et al. 2003, Balkwill 2004, Walser & Fulton 2004). It has been reported that chemokines are implicated in many aspects of carcinogenesis like tumor growth, angiogenesis, and metastasis development (Balkwill 2004). Some tumor cells not only regulate their chemokine expression to help recruit inflammatory cells, but also use these factors to stimulate tumor growth and progression (Zhu & Woll 2005). Chemokines are small heparin-binding proteins that activate and stimulate the infiltration of circulating lymphocytes into tissue from peripheral blood and serve as mediators of inflammation (Luster 1998, Rossi & Zlotnik 2000, Proudfoot 2002). Chemokines can be divided into four groups, namely C, CC, CXC, and CX3C, according to cysteine residues in the NH2-terminal part of the protein. We and others have recently shown that CXCL8, a CXC member was expressed in breast cancer cell lines (Freund et al. 2003, Lin et al. 2004). CXC members exhibit two cysteines separated by a non-conserved amino acid residue. CXC chemokine subfamily comprises 16 members, among which CXCL1 (GRO-), CXCL2 (GRO-β), CXCL3 (GRO-
), CXCL4 (PF4), CXCL4V1 (PF4V1), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL7 (NAP-2), CXCL8 (IL-8), CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC), and CXCL12 (SDF-1) are the most studied members. The NH2-terminus of several CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) contains three amino acid residues (Glu-Leu-Arg; ‘ELR motif’), which confers them a chemotactic activity for neutrophils and proangiogenic properties (Hebert et al. 1991, Clark-Lewis et al. 1993, Strieter et al. 1995, 2005). CXC chemokines mediate their effects via binding and activating seven transmembrane receptors, namely CXCR1–CXCR6 (Mantovani 1999). CXCR1 binds only CXCL6 and CXCL8, while CXCR2 binds all ELR+ CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8; Strieter et al. 2005). Recent studies have shown that CXCR1 and CXCR2 were poorly expressed in breast tumors, while CXCR4 was overexpressed in metastatic breast tumors (Muller et al. 2001).
We demonstrated earlier that CXCL8 expression is negatively linked to ER status and is associated with higher invasiveness potential of breast cancer cells (Freund et al. 2003). CXCL1 has been also shown to promote growth of tumor cells and to be involved in angiogenesis of colon cancer and melanoma (Loukinova et al. 2000, Li et al. 2004). Since several CXC chemokines, including CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8, are located to a short region (360 kb) of the long arm of the chromosome 4, we hypothesized that this gene cluster could exhibit similar regulation in breast cancer. By analyzing the expression of these chemokines in breast tumors, we demonstrate that CXCL1, CXCL3, CXCL5, CXCL6, and CXCL8 are co-regulated and display a higher expression in ER-negative tumors. These chemokines are produced by tumor cells and also by fibroblasts, endothelial, or blood cells. At least for CXCL1 and CXCL8, the high expression of these chemokines involved gene regulation events and not gene amplification. We also observed that CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL8 chemokines were present at higher levels in metastases when compared with grade I and grade III biopsies. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 were then investigated in an independent panel of ER-positive tumors from 48 postmenopausal breast cancer patients treated with adjuvant tamoxifen alone, of whom 24 relapsed. Overexpression of CXCL1, CXCL3, and CXCL8 was associated with significantly shorter relapse-free survival (RFS) in univariate analysis. Our data suggest that multiple chemokines, including in particular CXCL8 could be involved in the higher aggressiveness of breast tumors.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We analyzed samples of 48 primary breast tumors excised from women at our institution from 1980 to 1994. Samples containing more than 70% of tumor cells were considered suitable for this study. Immediately following surgery, the tumor samples were placed in liquid nitrogen until RNA extraction. The patients met the following criteria: primary unilateral non-metastatic postmenopausal breast carcinoma; ER positivity (as determined at the protein level by biochemical methods (Dextran-coated charcoal method until 1988 and enzymatic immunoassay thereafter) and confirmed by ER real-time quantitative RT-PCR assay); complete clinical, histological, and biological information available; no radiotherapy or chemotherapy before surgery; and full follow-up at our institution. The malignancy of infiltrating carcinomas was scored according to Scarff–Bloom and Richardson's histoprognostic system (Bloom & Richardson 1957). The characteristics of the patients are shown in Table 1. The patients had physical examinations and routine chest radiography every 3 months for 2 years, then annually. Mammograms were done annually. The median follow-up was 7.2 years (range 1.5–10 years). All the patients received postoperative adjuvant endocrine therapy (tamoxifen, 20 mg daily for 3–5 years), and no other treatment. Twenty-four patients relapsed. The distribution of first relapse events was as follows: 22 metastases and 2 local and/or regional recurrences with metastases.
|
expression status, we also analyzed 48 additional primary breast tumors: 24 ER-negative and 24 ER-positive tumors. The characteristics of these 48 patients are shown in Table 2.
|
We analyzed five ER-positive cell lines (MCF7, T47-D, ZR75-1, MDA-MB361, and HCC1500) and seven ER-negative cell lines (BT-20, MDA-MB468, SK-BR-3, MDA-MB157, MDA-MB231, MDA-MB435, and MDA-MB436), obtained from the American Tissue Type Culture Collection.
Specimens of adjacent normal breast tissue from six breast cancer patients, and normal breast tissue from three women undergoing cosmetic breast surgery, were used as sources of normal RNA.
Differential isolation of epithelial cells and fibroblasts from breast tumors
Breast tumors were minced with scalpels and incubated overnight with Liberase Blendzyme 2 (Roche Applied Science) for enzymatic dispersion. Organoids and aggregated cells (epithelial fraction) and isolated cells (fibroblast fraction) were isolated from the digest by differential centrifugation followed by culture in selective media as described earlier (Speirs et al. 1998). Fibroblast fraction was cultured in Ham's F10 medium containing L-glutamine (3 mM), insulin (5 µg/ml), thyroid hormone tri-iodothyronine (1 nM), hydrocortisone (1 µg/ml), and 10% serum calf fetal. Epithelial fraction was cultured under the same conditions plus epidermal growth factor (5 ng/ml), transferrin (5 µg/ml), and with 5% human serum (without serum calf fetal). Cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C, and the medium was changed three times a week.
Endothelial and blood cells isolation
HUVEC (human umbilical vein endothelial cells) were isolated from human umbilical veins and maintained in endothelial cell growth medium (EGM-2) medium (Smadja et al. 2005). Total RNA of blood-circulating cells was extracted from whole blood of normal healthy donors.
Real-time RT-PCR
Reactions are characterized by the point during cycling when amplification of the PCR product is first detected, rather than the amount of PCR product accumulated after a fixed number of cycles. The larger the starting quantity of the target molecule, the earlier a significant increase in fluorescence is observed. The parameter Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence generated by SYBR green dye-amplicon complex formation passes a fixed threshold above baseline. The increase in fluorescent signal associated with exponential growth of PCR products is detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin–Elmer Applied Biosystems, Foster City, CA, USA), using PE Biosystems analysis software according to the manufacturer’s manuals.
The precise amount of total RNA added to each reaction mixture (based on optical density) and its quality (i.e., lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of two endogenous RNA control genes involved in two cellular metabolic pathways, namely TBP (Genbank accession NM_003194 [GenBank] ), which encodes the TATA box-binding protein (a component of the DNA-binding protein complex TFIID), and RPLP0 (NM_001002 [GenBank] ), which encodes the human acidic ribosomal phosphoprotein P0. Each sample was normalized on the basis of its TBP (or RPLPO) content.
Results expressed as N-fold differences in target gene expression relative to the TBP (or RPLPO) gene, and termed ‘RNA Ntarget’, were determined as 
, where the 
Ct value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the TBP (or RPLP0) gene (Bieche et al. 1999, 2001). Target genes were considered to be detectable but not quantifiable when the Ct value was above 35.
Primers for the target genes and the endogenous control genes used in the different RT-PCR assays were chosen with the assistance of the computer program Oligo 5.0 (National Biosciences, Plymouth, MN, USA). We conducted BLASTN searches against dbEST and nr (the non redundant set of GenBank, EMBL, and DDBJ database sequences) to confirm the total gene specificity of the nucleotide sequences chosen for the primers, and the absence of DNA polymorphisms. In particular, the primer pair was selected to be unique relative to the sequences of closely related family member genes or of the corresponding retropseudogenes. To avoid amplification of contaminating genomic DNA for the quantitative RT-PCR, one of the two primers was placed at the junction between two exons, or in a different exon, if possible. The nucleotide sequences of the oligonucleotide primers for real-time RT-PCR are shown in Table 3. Gel electrophoresis was used to verify the specificity of PCR amplicons. For the selected primer pair, we performed no template control, which produced negligible signals (usually >40 in Ct value), suggesting that primer–dimer formation effects were negligible.
|
Elisa
CXCL8 and CXCL1 concentrations in culture supernatants were determined by ELISA using Duoset kits (R&D Systems, Minneapolis, MN, USA) as recommended by the manufacturer.
Statistical analysis
As the mRNA levels did not fit a Gaussian distribution, a) the mRNA levels in each subgroup of samples were characterized by their median values and ranges, rather than their mean values and coefficients of variation and b) relationships between the molecular markers and clinical and biological parameters were tested using the nonparametric Mann–Whitney U test (Mann & Whitney 1947). Differences between two populations were judged significant at confidence levels >95% (P<0.05).
To visualize the capacity of a given molecular marker to discriminate between two populations (in the absence of an arbitrary cut-off value), we summarized the data in a receiver operating characteristics (ROC) curve (Hanley & McNeil 1982). When a molecular marker has no discriminative value, the ROC curve lies close to the diagonal and the area under curve (AUC) is close to 0.5. When a marker has strong discriminative value, the ROC curve moves to the upper left-hand corner (or to the lower right-hand corner) and the AUC is close to 1.0 (or 0).
Hierarchical clustering was performed using the GenANOVA software (Didier et al. 2002).
RFS was determined as the interval between diagnosis and detection of the first relapse (local and/or regional recurrence, and/or metastasis). Survival distributions were estimated by the Kaplan–Meier method (Kaplan & Meier 1958), and the significance of differences between survival rates was ascertained using the log-rank test (Peto et al. 1977).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-positive breast tumors. (Sheikh et al. 1994, Osborne 1998). We have demonstrated recently an inverse correlation between expression of the CXC chemokine CXCL8 and ER levels in human breast cancer cell lines (Freund et al. 2003), which could account for their higher aggressiveness. Interestingly, CXCL8 gene is located with a number of other CXC chemokines in a short region (360 kb) of chromosome 4 (4q21; Fig. 1 and Table 4). The goal of this study was to determine whether the chemokines of this cluster were co-regulated and what could be the factors controlling their expression.
|
|
We first analyzed by real-time PCR the expression in breast cancer biopsies of a number of genes located on 4q21 (Table 4). This included in particular CXC chemokines CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8. The 48 samples (Table 2) were divided into two groups according to their low (–/+) or high (++) CXCL8 content (see Statistical analysis section in Materials and methods). In the two groups, the RNA levels of the genes located in 4q21 location, as well as the expression of ER, KI67, and CXCR1 and CXCR2 genes were determined (Table 5). We observed that CXCL8++ tumors expressed significantly lower levels of ER (Table 5), which is in agreement with our and other group data on breast cancer cell lines and breast cancer biopsies (Freund et al. 2003, Lin et al. 2004). CXCL4, CXCL4V1, CXCL7, and albumin genes were not significantly expressed. In addition, CXCL1, CXCL3, CXCL5, and CXCL6 were overexpressed in CXCL8++ tumors. CXCL2 also displayed a trend of overexpression in CXCL8 tumors. All the other genes of the 4q21 region exhibited a similar expression in both types of tumors (Table 5). CXCR1 and CXCR2, the receptors mediating the effects of the chemokines present on chromosome 4, as well as KI67, displayed a similar expression in the two CXCL8 groups.
|
To determine which cells of the tumor could produce chemokines, we measured the RNA levels of the 4q chemokines cluster in breast cancer cell lines. By comparing the levels of chemokines in cell lines producing low or high levels of CXCL8 (Table 6), we observed that CXCL6, CXCR1, and CXCR2 were poorly expressed. In contrast, CXCL1, CXCL2, CXCL3, and CXCL5 were more expressed in CXCL8-positive cell lines when compared with CXCL8 low or negative cell lines. Interestingly, with the exception of ZR-75-1 cells, all cell lines which expressed high levels of CXCL8 were ER-negative. Low producers of CXCL8 were mostly composed of ER-positive cell lines and a few ER-negative cells (MDA-MB468 and SKBR-3), which is in agreement with our previous data (Freund et al. 2003).
|
|
To address the question of the co-regulation of these chemokines, we first hypothesized that some of the overexpressed chemokines could be the result of genomic DNA amplification. To test this hypothesis, we analyzed five genomic DNA samples from normal patients, four breast tumors with low CXCL8 mRNA expression, and four breast tumors with high CXCL8 mRNA overexpression. Among the eight tumors tested, no tumor showed 4q21 DNA amplification when looking at CXCL1 and CXCL8 genes (data not shown).
We then designed an assay to evaluate the functionality of 4q21 chemokine genes in cell lines. Cells were treated with tumour necrosis factor (TNF)-, a general activator of cytokine expression and we first monitored the RNA levels the expression of CXCL1, CXCL2, CXCL3, and CXCL8 in MCF-7 (low CXCL8) and MDA-MB231 (high CXCL8) cells. TNF- strongly induced the expression of the chemokines tested, suggesting that all four genes were fully active in the two cell lines (Table 8). Genes surrounding the chemokine cluster were only modestly (AREG, EREG, and ADAMTS3) or not (RASSF6) regulated by TNF- in the two cell lines. Finally, we also observed that CXCL8 and CXCL1 protein secretions were up-regulated by TNF- (Fig. 2).
|
|
B site at the promoter level (Freund et al. 2004), we undertook to analyze whether CXCL8 RNA levels found in breast tumors could be correlated with the amounts of AP-1 and NF-B members or known target genes (Table 9 and Supplemental Data Table 1, which is now online at http://erc.endocrinology-journals.org/content/vol14/issue4/). We observed that CXCL8 RNA levels were strongly correlated to those of AP-1 members such as ATF3, c-Jun, or JunB, and to their target genes such as MMP1, MMP9, or TNC (Table 9). CXCL8 expression was also correlated GADD45B, a typical NF-B target gene, and also to TNC and MMP9, but not with most of NF-B members or target genes tested.
|
Many CXC chemokines are strong inducers of chemotaxy. We thus hypothesized that they could also be involved in tumor progression and that they could increase metastases formation. To evaluate this, we analyzed CXCL8 expression in 11 grade I tumors, 37 grade III tumors, and 24 metastases in primary tumors and distant metastases (Table 10). We observed that CXCL8 expression raised from grade I to grade III primary tumors and exhibited a strong increase (eightfold increase) when looking at distant metastases, suggesting that this chemokine could be a key player in metastasis. CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6 also showed increased levels from grade III to metastases but not between grade I and grade III patients (Table 10).
|
ER-negative tumors are generally more aggressive than ER-positive tumors. However, some ER-positive tumors display aggressive features too. It is of great interest to understand why this subpopulation of ER-positive tumors is more metastatic. To address this issue, we measured the expression levels of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, as well as HER2 by real-time RT-PCR in a cohort of 24 ER-positive breast tumor patients who relapsed, and 24 ER-positive breast tumor patients who did not relapse (Table 1). All these 48 ER-positive tumors were from postmenopausal patients treated with primary surgery followed by adjuvant tamoxifen alone. We used univariate analysis (log-rank test) to further study the prognostic value of these seven genes. For each gene, the 48 ER-positive breast tumors were divided into two equal groups of 24 tumors with ‘low’ and ‘high’ CXC chemokine mRNA levels. Univariate analysis showed that a high expression level of CXCL1, CXCL3, and CXCL8 correlated with significantly shorter relapse-free survival (RFS; Figs 3 and 4). The outcome of the 24 patients with high mRNA levels of these three genes was significantly worse than those of the 24 patients with low CXCL1, CXCL3, and CXCL8 mRNA levels. No significant prognostic value was associated with the other four genes, CXCL2 (P=0.59), CXCL5 (P=0.47), CXCL6 (P=0.16), and HER2 (P=0.83). Moreover, using a Cox proportional hazards model, we also assessed the prognostic value, for RFS, of parameters that were significant or near significant (P<0.1) in univariate analysis, i.e., SBR grade, lymph node status (Table 1), and the genes CXCL1, CXCL3, and CXCL8 (Figs 3 and 4). Only the prognostic significance of CXCL8 mRNA status shows a trend toward significance (P=0.068).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
, which is in agreement with previous work (Freund et al. 2003, Lin et al. 2004). It is interesting to note that CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 were all ELR (glutamate–leucine–arginine)-positive chemokines (Moser et al. 2004). This appears quite important as the NH2-terminal motif has been shown to be responsible for the ability of chemokines to attract neutrophils as well as a necessary motif involved in angiogenic properties of these chemokines (Strieter et al. 2005). The co-regulation of CXCL1, CXCL3, CXCL5, CXCL6, and CXCL8 in tumors was puzzling. Our data suggest that this high expression does not arise from genomic amplification, but rather from an increased transcriptional activity of these genes. In addition, we showed that CXCL1, CXCL2, CXCL3, and CXCL8 were induced by TNF in breast cancer cells, demonstrating the genes were functional. Our previous work on CXCL8 gene regulation in breast cancer cells had shown that CXCL8 overexpression in ER-negative breast cancer cell lines was mainly mediated through an overexpression of NF-B and AP-1 transcription factors, which in turn could induce CXCL8 promoter activity (Freund et al. 2004). To date, only a few data are available concerning the regulation of CXCL1, 3, 5, and 6 gene promoters. Of particular note is the fact that CXCL1 promoter displays many features common to CXCL8, with in particular also a crucial NF-B site involved in constitutive expression of CXCL1 gene in melanoma (Wood & Richmond 1995, Yang & Richmond 2001). So, we hypothesized that tumors producing high amounts of CXCL8 could also have deregulated NF-B and AP-1 pathways. To test this hypothesis, we measured the expression levels of a series of NF-B and AP-1 transcription factors or target genes. Our data showed that CXCL8 expression was correlated primarily to AP-1 transcription factors such as ATF3, c-Jun, or JunB and typical AP-1 target genes, but to a lesser extent to NF-B pathway.
To determine whether tumor cells themselves were producing these chemokines, we first analyzed breast cancer cell lines. Our data confirmed that cell lines overexpressing CXCL8 were producing CXCL1, CXCL2, CXCL3, and CXCL5, which is in good agreement with the overall expression of breast tumors. Of particular note, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 were also produced mostly by ER-negative cell lines (Table 6), which constitutes the first report of such a co-regulation. To go further, we next measured the chemokine content of intratumoral fibroblast and epithelial cells, as well as the one of blood and endothelial cells. Interestingly, we found that CXCL5, CXCL6, and CXCL8 were mostly produced by intratumoral epithelial cells, whereas CXCL1, CXCL2, and CXCL3 were synthetized in priority by blood cells. But other types of cells could also produce CXCL8. Indeed, CXCL8 was also produced at relatively high levels by endothelial cells and intratumoral fibroblasts. In addition, CXCR1 and CXCR2 were mainly detected in endothelial cells, even though CXCR2 is considered to be the primary receptor responsible for endothelial cell chemotaxis (Addison et al. 2000, Salcedo et al. 2000).
Next, we focused our attention to CXCL8 to evaluate its potential prognosis value. Our data showed that CXCL8 content in the primary tumor was increased in grade III patients when compared with grade I patients. This is in agreement with other reports showing that CXCL8 is overexpressed in breast tumors when compared with normal tissues and that breast cancer patients display high seric levels of CXCL8 (Green et al. 1997, Kozlowski et al. 2003). Moreover, distant metastases were extensively producing CXCL8, suggesting that CXCL8 could be one of the factors leading to metastasis. Indeed, our previous work showed that CXCL8 could increase the invasion potential of breast cancer cells, one of the first steps before metastasis (Freund et al. 2003). Benoy et al. (2004) have also shown that patients with metastatic breast tumors displayed high seric amounts of CXCL8. In vivo studies in athymic mouse models also corroborate these data, suggesting that breast tumors secreting high levels of CXCL8 metastasize more easily to bone (De Larco et al. 2001, Bendre et al. 2002). We then wondered whether the other chemokines of the cluster were following the same trend. We observed that CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6 were also more expressed in metastasis when compared with grade I and grade III patients, suggesting that they could also be involved in metastasis. If CXCL1 has been shown to increase breast cancer cell invasion in vitro (Youngs et al. 1997), so far, this is the first report on the involvement of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6 in breast cancer metastasis.
From this point, we knew that CXCL8, 1, 2, 3, 5, and 6 chemokines were mainly produced by ER-negative tumors, and were globally overexpressed in breast cancer metastases. However, one crucial question was to look at the case of patients with ER-positive tumors who were treated with tamoxifen and had an impaired survival. Indeed, it is quite difficult to understand why a subpopulation of ER-positive breast cancer patients will still have a bad outcome. So, our goal was to determine whether CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, or CXCL8 could discriminate among ER-positive breast cancer patients, those who will develop metastasis rapidly. Univariate analysis showed that, among these six genes (as well as HER2), only CXCL1, CXCL3, and CXCL8 correlated with RFS. In multivariate analysis, only CXCL8 shows a trend toward significance to be an independant predictor of response to tamoxifen therapy. These data, which need to be confirmed on a larger set of patients, suggest that CXCL8 could help to improve the prognosis of ER-positive patients who are treated with tamoxifen.
In conclusion, our work suggest that breast tumors expressing high levels of CXCL8 have a bad prognosis evolution which could be the reflect of their higher content of ELR-positive CXC chemokines such as CXCL1, CXCL3, CXCL5, and CXCL6. These chemokines belong to a hyperactive gene cluster, which is likely to be regulated by AP-1 and to a lesser extent by NF-B pathways. Moreover, ER-positive breast cancer patients with high levels of CXCL1, CXCL3, or CXCL8 display a shorter relapse-free survival. It will be interesting to dissect further the mechanisms of regulation, as a potential therapeutic approach could be to identify the common factors up-regulating these chemokines in the same subset of tumors.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Balkwill F 2004 Cancer and the chemokine network. Nature Reviews. Cancer 4 540–550.[CrossRef][Web of Science][Medline]
Bendre MS, Gaddy-Kurten D, Mon-Foote T, Akel NS, Skinner RA, Nicholas RW & Suva LJ 2002 Expression of interleukin 8 and not parathyroid hormone-related protein by human breast cancer cells correlates with bone metastasis in vivo. Cancer Research 62 5571–5579.
Benoy I, Salgado R, Colpaert C, Weytjens R, Vermeulen PB & Dirix LY 2002 Serum interleukin 6, plasma VEGF, serum VEGF, and VEGF platelet load in breast cancer patients. Clinical Breast Cancer 2 311–315.[Medline]
Benoy IH, Salgado R, Van Dam P, Geboers K, Van Marck E, Scharpe S, Vermeulen PB & Dirix LY 2004 Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clinical Cancer Research 10 7157–7162.
Bieche I, Onody P, Laurendeau I, Olivi M, Vidaud D, Lidereau R & Vidaud M 1999 Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. Clinical Chemistry 45 1148–1156.
Bieche I, Parfait B, Le Doussal V, Olivi M, Rio M-C, Lidereau R & Vidaud M 2001 Identification of CGA as a novel estrogen receptor-responsive gene in breast cancer: an outstanding candidate marker to predict the response to endocrine therapy. Cancer Research 61 1652–1658.
Bloom HJ & Richardson WW 1957 Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. British Journal of Cancer 11 359–377.[Web of Science][Medline]
Clark-Lewis I, Dewald B, Geiser T, Moser B & Baggiolini M 1993 Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. PNAS 90 3574–3577.
Didier G, Brezellec P, Remy E & Henaut A 2002 GeneANOVA – gene expression analysis of variance. Bioinformatics 18 490–491.
Freund A, Chauveau C, Brouillet JP, Lucas A, Lacroix M, Licznar A, Vignon F & Lazennec G 2003 IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells. Oncogene 22 256–265.[CrossRef][Web of Science][Medline]
Freund A, Jolivel V, Durand S, Kersual N, Chalbos D, Chavey C, Vignon F & Lazennec G 2004 Mechanisms underlying differential expression of interleukin-8 in breast cancer cells. Oncogene 23 6105–6114.[CrossRef][Web of Science][Medline]
Green AR, Green VL, White MC & Speirs V 1997 Expression of cytokine messenger RNA in normal and neoplastic human breast tissue: identification of interleukin-8 as a potential regulatory factor in breast tumours. International Journal of Cancer 72 937–941.[CrossRef][Web of Science][Medline]
Hanley JA & McNeil BJ 1982 The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 143 29–36.
Hebert CA, Vitangcol RV & Baker JB 1991 Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. Journal of Biological Chemistry 266 18989–18994.
Jiang XP, Yang DC, Elliott RL & Head JF 2000 Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status. Cytokine 12 458–465.[CrossRef][Web of Science][Medline]
Kaplan E & Meier P 1958 Nonparametric estimation from incomplete observations. Journal of the American Statistical Association 53 457–481.[CrossRef][Web of Science]
Kozlowski L, Zakrzewska I, Tokajuk P & Wojtukiewicz MZ 2003 Concentration of interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-10 (IL-10) in blood serum of breast cancer patients. Roczniki Akademii Medycznej w Bialymstoku 48 82–84.[Medline]
De Larco JE, Wuertz BR, Manivel JC & Furcht LT 2001 Progression and enhancement of metastatic potential after exposure of tumor cells to chemotherapeutic agents. Cancer Research 61 2857–2861.
Lazennec G, Bresson D, Lucas A, Chauveau C & Vignon F 2001 ER beta inhibits proliferation and invasion of breast cancer cells. Endocrinology 142 4120–4130.
Li A, Varney ML & Singh RK 2004 Constitutive expression of growth regulated oncogene (gro) in human colon carcinoma cells with different metastatic potential and its role in regulating their metastatic phenotype. Clinical and Experimental Metastasis 21 571–579.
Lin Y, Huang R, Chen L, Li S, Shi Q, Jordan C & Huang RP 2004 Identification of interleukin-8 as estrogen receptor-regulated factor involved in breast cancer invasion and angiogenesis by protein arrays. International Journal of Cancer 109 507–515.[CrossRef][Web of Science][Medline]
Loukinova E, Dong G, Enamorado-Ayalya I, Thomas GR, Chen Z, Schreiber H & Van Waes C 2000 Growth regulated oncogene-alpha expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC receptor-2 dependent mechanism. Oncogene 19 3477–3486.[CrossRef][Web of Science][Medline]
Luster AD 1998 Chemokines–chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338 436–445.
Mann H & Whitney D 1947 On a test of whether one of two random variables is stochastically larger than the other. Annals of Mathematical Statistics 18 50–60.[CrossRef][Web of Science]
Mantovani A 1999 The chemokine system: redundancy for robust outputs. Immunology Today 20 254–257.[CrossRef][Web of Science][Medline]
Moser B, Wolf M, Walz A & Loetscher P 2004 Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25 75–84.[CrossRef][Web of Science][Medline]
Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN et al. 2001 Involvement of chemokine receptors in breast cancer metastasis. Nature 410 50–56.[CrossRef][Medline]
Osborne CK 1998 Steroid hormone receptors in breast cancer management. Breast Cancer Research and Treatment 51 227–238.[CrossRef][Web of Science][Medline]
Penson RT, Kronish K, Duan Z, Feller AJ, Stark P, Cook SE, Duska LR, Fuller AF, Goodman AK, Nikrui N et al. 2000 Cytokines IL-1beta, IL-2, IL-6, IL-8, MCP-1, GM-CSF and TNFalpha in patients with epithelial ovarian cancer and their relationship to treatment with paclitaxel. International Journal of Gynecological Cancer 10 33–41.[Web of Science][Medline]
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson K, Peto J & Smith PG 1977 Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. British Journal of Cancer 35 1–39.[Web of Science][Medline]
Proudfoot AE 2002 Chemokine receptors: multifaceted therapeutic targets. Nature Reviews. Immunology 2 106–115.[CrossRef][Web of Science][Medline]
Rossi D & Zlotnik A 2000 The biology of chemokines and their receptors. Annual Review of Immunology 18 217–242.[CrossRef][Web of Science][Medline]
Salcedo R, Resau JH, Halverson D, Hudson EA, Dambach M, Powell D, Wasserman K & Oppenheim JJ 2000 Differential expression and responsiveness of chemokine receptors (CXCR1-3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB Journal 14 2055–2064.
Sheikh MS, Garcia M, Pujol P, Fontana JA & Rochefort H 1994 Why are estrogen-receptor-negative breast cancers more aggressive than the estrogen-receptor-positive breast cancers? Invasion and Metastasis 14 329–336.
Skoog L, Humla S, Axelsson M, Frost M, Norman A, Nordenskjold B & Wallgren A 1987 Estrogen receptor levels and survival of breast cancer patients. A study on patients participating in randomized trials of adjuvant therapy. Acta Oncologica 26 95–100.
Smadja DM, Bieche I, Uzan G, Bompais H, Muller L, Boisson-Vidal C, Vidaud M, Aiach M & Gaussem P 2005 PAR-1 activation on human late endothelial progenitor cells enhances angiogenesis in vitro with upregulation of the SDF-1/CXCR4 system. Arteriosclerosis, Thrombosis, and Vascular Biology 25 2321–2327.
Speirs V, Green AR, Walton DS, Kerin MJ, Fox JN, Carleton PJ, Desai SB & Atkin SL 1998 Short-term primary culture of epithelial cells derived from human breast tumours. British Journal of Cancer 78 1421–1429.[Web of Science][Medline]
Strieter RM, Polverini PJ, Arenberg DA & Kunkel SL 1995 The role of CXC chemokines as regulators of angiogenesis. Shock 4 155–160.[Web of Science][Medline]
Strieter RM, Burdick MD, Gomperts BN, Belperio JA & Keane MP 2005 CXC chemokines in angiogenesis. Cytokine and Growth Factor Reviews 16 593–609.[CrossRef][Web of Science][Medline]
Walser TC & Fulton AM 2004 The role of chemokines in the biology and therapy of breast cancer. Breast Disease 20 137–143.[Medline]
Wood LD & Richmond A 1995 Constitutive and cytokine-induced expression of the melanoma growth stimulatory activity/GRO alpha gene requires both NF-B and novel constitutive factors. Journal of Biological Chemistry 270 30619–30626.
Yang J & Richmond A 2001 Constitutive IkappaB kinase activity correlates with nuclear factor-B activation in human melanoma cells. Cancer Research 61 4901–4909.
Youngs SJ, Ali SA, Taub DD & Rees RC 1997 Chemokines induce migrational responses in human breast carcinoma cell lines. International Journal of Cancer 71 257–266.[CrossRef][Web of Science][Medline]
Zhu YM & Woll PJ 2005 Mitogenic effects of interleukin-8/CXCL8 on cancer cells. Future Oncology 1 699–704.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  Z. Saidak, R. Mentaverri, and E. M. Brown The Role of the Calcium-Sensing Receptor in the Development and Progression of Cancer Endocr. Rev., April 1, 2009; 30(2): 178 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chavey, M. Muhlbauer, C. Bossard, A. Freund, S. Durand, C. Jorgensen, C. Jobin, and G. Lazennec Interleukin-8 Expression Is Regulated by Histone Deacetylases through the Nuclear Factor-{kappa}B Pathway in Breast Cancer Mol. Pharmacol., November 1, 2008; 74(5): 1359 - 1366. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
