ToxSci Advance Access originally published online on May 21, 2007
Toxicological Sciences 2007 98(2):436-444; doi:10.1093/toxsci/kfm125
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A Proposed Mechanism for the Protective Effect of Dioxin against Breast Cancer
,2,3,1
* Molecular Toxicology Interdepartmental Doctoral Program, School of Public Health
Department of Pathology and Laboratory Medicine and Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California
1 To whom correspondence should be addressed at Department of Pathology and Laboratory Medicine, Box 951732 Center for Health Sciences, University of California Los Angeles, Los Angeles, CA 90095-1732. Fax: (310) 794-9272. E-mail: ohank{at}mednet.ucla.edu.
Received March 21, 2007; accepted May 14, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Although it is causative for many types of cancers, experimental and epidemiological evidence suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) may in fact protect against breast cancer. The mechanism(s) for this protection remain unclear. In an attempt to further elucidate this mechanism, we performed a microarray experiment to identify genes that were modulated upon dioxin treatment. We found that dioxin downregulated the messenger RNAs for the G-protein–coupled receptor, CXCR4, as well as its unique chemokine ligand, CXCL12, in MCF-7 breast cancer cells. We demonstrated that the corresponding proteins are also downregulated by dioxin. The interaction between CXCR4 and CXCL12 plays a central role in the metastasis of breast cancer, as disruption of the CXCL12/CXCR4 axis has been shown to limit the metastasis of breast cancer cells to the lung in mice. Utilizing an in vitro chemotaxis assay, we demonstrate that dioxin specifically inhibits the migration of MCF-7 cells toward CXCL12. We also show that dioxin reduces CXCR4 under hypoxia and CXCL12 under estradiol-induced conditions in MCF-7 cells. Finally, as the CXCR4/CXCL12 axis is implicated in the progression of numerous types of cancer, we identified several other cancer cell lines in which dioxin modulates CXCR4 and CXCL12 levels. We therefore propose that one mechanism whereby dioxin may protect against breast cancer is via downregulation of CXCR4 and CXCL12, thereby inhibiting progression of the disease. Further, other nontoxic ligands for the aryl hydrocarbon receptor (selective AHR modulators) may exert their protective effects by a similar mechanism.
Key Words: dioxin; CXCR4; CXCL12; breast cancer; AHR; chemokine receptor.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin) is one of the most potent carcinogens ever tested. Ubiquitous in the environment, dioxin can be detected in air, soil, and food products. In mice and rats, dioxin not only acts as a complete hepatocarcinogen, but has also been shown to cause thyroid follicular cell adenomas, squamous cell carcinomas of the nasal cavity, as well as numerous types of fibrosarcomas (IARC, 1997). Investigation of the Seveso, Italy population inadvertently exposed to dioxin after a chemical reactor explosion in 1976 revealed that high exposure levels resulted in an increased risk of all cancers combined. Upon analysis of four highly exposed industrial cohorts and based on sufficient animal and mechanistic data, the International Agency for Research on Cancer (IARC) officially classified dioxin as a group 1 human carcinogen in 1997 (IARC, 1997).
Although dioxin is clearly a potent tumor promoter, it may have a protective effect for breast cancer; exposure has been associated with a decreased incidence of breast carcinoma in both animal and human studies. Kociba et al. (1978)
found that lifetime dietary administration of low doses of dioxin to Sprague–Dawley rats resulted in a decrease in age-dependent spontaneous mammary and uterine tumors. Holcomb and Safe (1994) found that a nontoxic dose of dioxin coadministered with 7,12-dimethylbenzanthracene (DMBA) delayed mammary tumor formation in Sprague–Dawley rats. In addition, administration of dioxin to rats with established DMBA-induced mammary tumors inhibited the growth of those tumors. Finally, although there was a positive association between dioxin exposure and blood malignancies, sarcomas, gastrointestinal, prostate, bladder, and lung cancers (as well as all cancers combined) in the Seveso cohort, there was a decreased incidence of breast and endometrial cancers in exposed individuals (Bertazzi et al., 1997). This last association is not without controversy, however; in a follow-up study, it was determined that high levels of dioxin in the serum of women of the Seveso cohort (a 10-fold increase in serum levels) correlated with an increased breast cancer risk (Warner et al., 2002). The apparent contradictions in the Seveso studies could be attributed to a number of factors, including potential bias associated with disease survival, small case number, a relatively young cohort, and perhaps most importantly, developmental stage at exposure (Brown et al., 1998; Warner et al., 2002). All of the effects of dioxin are believed to be mediated by the aryl hydrocarbon receptor (AHR, Hankinson, 1995). When dioxin binds the AHR, the complex enters the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT), and modulates the transcription of an array of genes.
The identification of genes in breast cancer cells which show differential expression upon dioxin treatment could potentially identify novel pathways responsible for the inhibitory effects of dioxin on breast cancer. Through our microarray analysis, we have for the first time identified the G-protein–coupled chemokine receptor, CXCR4, and its unique ligand, CXCL12, as genes downregulated by dioxin in MCF-7 breast cancer cells. Furthermore, in contrast to some studies that maintain that the cancer-protective effect of dioxin depends upon its antiestrogenic activity, our studies suggest a novel, estrogen-independent mechanism to explain these protective effects.
CXCR4 and CXCL12 were originally studied in relation to their roles in regulating leukocyte trafficking. However, in 2001, strong evidence was obtained suggesting that the CXCR4/CXCL12 axis plays a central role in the development of breast cancer (Muller et al., 2001). Breast cancer cells were found to overexpress CXCR4, and that the principal organs to which breast cancer cells metastasize, the lung, liver, and bone, were found to express high levels of CXCL12. Furthermore, the administration of neutralizing antibodies to CXCR4 impaired the metastasis of breast cancer cells to the lung in mice. It was therefore proposed that the CXCR4/CXCL12 interaction plays a critical role in determining the metastatic destination of breast cancer cells (Muller et al., 2001). Subsequent studies have strongly supported and amplified this original hypothesis. For example, breast cancer cells selected for high metastatic potential for bone were found to overexpress CXCR4 (Kang et al., 2003), and CXCL12 was shown to enhance chemotaxis and invasion of breast cancer cells in culture (Fernandis et al., 2004). Recently, the CXCR4/CXCL12 axis has been shown to regulate metastasis of many other tumor cell types including pancreatic, bladder, prostate, and lung cancers (Marchesi et al., 2004; Phillips et al., 2003; Retz et al., 2004; Singh et al., 2004).
In addition to the homing of metastatic cells, the CXCR4/CXCL12 interaction has been shown to stimulate other stages of tumor progression, including migration and invasion of tumor cells (Kang et al., 2003) and the angiogenic response (Balkwill, 2004; Kryczek et al., 2005; Orimo et al., 2005). Interestingly, CXCL12 is also expressed in ovarian cancer cell lines and appears to stimulate cell proliferation in an autocrine manner via binding to CXCR4 on these same cells (Hall and Korach, 2003). Expression of CXCL12 in primary tumor cells can also stimulate malignancy by other means; for example, overexpression of CXCL12 in ovarian cancers appears to increase the malignancy of the cells due to the attraction of plasmocytoid dendritic cells (DC2s, which express CXCR4) to the tumor, thereby diminishing the host's immunological response to the tumor (Hall and Korach, 2003; Zou et al., 2001).
We have confirmed the downregulation of CXCR4 and CXCL12 messenger RNAs (mRNAs) and proteins in breast and ovarian cancer cells. We further substantiate the biological significance of this downregulation by demonstrating a specific inhibition by dioxin of MCF-7 cell chemotaxis towards a CXCL12 gradient. We also show that dioxin reduces CXCR4 expression under hypoxia as well as CXCL12 expression in the presence of estradiol. Finally, we have identified other cancer cell lines in which dioxin exerts a similar effect. A clearer understanding of this mechanism may validate the use of nontoxic AHR ligands such as diindolylmethanes (DIMs) for the future prevention or treatment of breast and other cancers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Cell culture, RNA extraction, and complementary DNA synthesis.
BG-1 cells were a kind gift from Dr Kenneth Korach (National Institute of Environmental Health Sciences, NC), A549 cells were provided by Dr Michael Roth (University of California, Los Angeles, CA), LNCaP cells were a gift from Dr Curtis Eckhert (University of California, Los Angeles, CA), and MB-MDA-175, MB-MDA-436, MB-MDA-468, BT-474, and T47D cells were generously provided by Dr Cindy Wilson (University of California, Los Angeles, CA). MCF-7, HepG2, Hep3B, RL95-2, A2058, Hs766T, and CaOV-3 cells were obtained from the American Type Culture Collection (Manassas, VA). MCF-7, BG-1, A549, Hep3B, and CaCo-2 cells were maintained in Minimal Essential Medium and RL95-2 cells were maintained in Dulbecco's Modified Eagle’s Medium (DMEM)/Ham's F12. T47D, HT-29, A2058, Hs766T, MB-MDA-231, CaOV-3 cells were maintained in DMEM containing 4mM L-glutamine. MB-MDA-175, MB-MDA-436, MB-MDA-468, LNCaP, 786-0, RL95-2, BT-474, and LS-180 cells were maintained in RPMI 1640 Medium. All media was purchased from Invitrogen (Carlsbad, CA) and supplemented with 10% fetal bovine serum (FBS; Omega, Tarzana, CA), 100 U/ml penicillin/100 µg/ml streptomycin solution (Gemini Bio-Products, West Sacramento, CA), and 0.25 U/ml Amphotericin B (Omega). Cells were maintained at 37%C under 5% CO2. For estradiol experiments, MCF-7 cells were grown in phenol red-free medium containing dextran charcoal-stripped serum for three days prior to initiation of the experiment, and cells were dissociated with phenol red-free TrypLE Express (Invitrogen). One-hundred nanomolars dioxin (Wellington Laboratories, Guelph, Ontario, Canada) dissolved in dimethyl sulfoxide (DMSO) was administered to cells, with a final concentration of DMSO at 0.1% in the medium. Standard precautions were taken to extract all dioxin-containing medium for proper disposal. For estradiol and hypoxia experiments, cells were treated with 10nM 17ß-estradiol (Sigma, St Louis, MO) or 1% O2 (Series II water-jacketed CO2 incubator with a HEPA filter (ThermoForma, Waltham, MA).
RNA was isolated using RNEasy Mini columns (Qiagen, Valencia, CA) according to the manufacturer's instructions and quantified on a SmartSpec 3000 spectrophotometer (BioRad, Hercules, CA). Five micrograms of total RNA was used for complementary DNA (cDNA) synthesis in a total reaction volume of 20 µl. Reverse transcription reactions were performed using Superscript III reverse transcriptase (Invitrogen) and primed with random hexamers (Invitrogen) according to the manufacturer's instructions. Following cDNA synthesis, each reaction was diluted into a total of 200 µl of RNAse-free water, which was then used as template for real-time PCR.
Gene chip microarrays.
Total RNA was isolated independently from three different cell cultures and used to generate cRNA, which was labeled with biotin as recommended by Affymetrix (Santa Clara, CA). cRNA was hybridized to Affymetrix HU133A and HU133B Gene arrays by the UCLA Microarray Core Facility. After washing, the arrays were scanned and analyzed using MicroArray Suite 5.0 software (Affymetrix). Average intensities for each array were globally scaled to a target intensity of 150. Three independent experiments were undertaken in order to confirm reproducibility.
The array data were imported into DNA-Chip Analyzer software (http:www.dchip.org) for normalization and model-based analysis. Four criteria were used to determine differentially expressed gene transcripts. First, probe sets on the array that were assigned as "absent" call in all samples were excluded. Secondly, a two-tailed Student's t-test was used for comparison of average gene expression signal intensity between the dioxin (n = 3) and control (n = 3) samples. A critical 
level of 0.05 was defined for statistical significance. Thirdly, an absolute difference (the absolute value mean difference between the dioxin and control groups) threshold equal to the median probe set expression over all arrays divided by five was used to screen out probe sets with expression values in the noise range. Finally, fold ratios were calculated for those gene transcripts that satisfied the first three criteria. Only those gene transcripts that exhibited a 1.8-fold change or greater were included for further analysis.
Real-time PCR.
SYBR Green and Taqman real-time PCR were performed according to standard protocols. The CXCR4 and CXCL12 cDNAs were amplified using Assays On Demand (Affinity Bioreagents, product numbers Hs00607978_s1 and Hs00171022_m1, respectively), which were provided as a x20 solution of primers and probe (sequences proprietary). Quantities were normalized to those for the 36B4 ribosomal housekeeping gene. The forward and reverse primers used for 36B4 quantification were 5'-CCACGGTGCTGAACATGCT-3' and 5'-TCGAACACCTGCTGGATGAC-3', respectively. For Taqman assays, the 36B4 probe sequence was 5'-FAM-ACCATCTCCCCCTTCTCCTTTGGGCT-TAMRA-3'. The forward and reverse primers used to quantify ALDH3 (by SYBR green) were 5'-AGAGTTCTACGGGGAAGATGCTAAG-3' and 5'-GCAAGGTGATGTGGAGGATGAC-3', respectively. All primers and probes were purchased from Integrated DNA Technologies. Real-time PCR was carried out using the ICycler IQ (BioRad) or 7500 Fast (Applied Biosystems, Foster City, CA) under standard protocols. Data were analyzed using the ICycler or ABI software and Microsoft Excel, and significance was evaluated by application of the Student's t-test.
Flow cytometry.
For intracellular staining of CXCL12 and surface staining of CXCR4, cells were grown to 70% confluence and treated with dioxin for 1, 2, or 3 days. For the quantification of CXCL12, cells were also treated with 1 µl/ml Brefeldin A (GlogiPlug, BD-PharMingen, Franklin Lakes, NJ) for the final 6 h of incubation to inhibit protein secretion. Cells were harvested by trypsinization, passed through an 18.5G needle, and pelleted. After washing in a phosphate buffered saline–bovine serum albumin (PBS–BSA) buffer, cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature. Cells were then pelleted and cells to be stained for CXCL12 were permeabilized in 0.2% Tween at 37%C for 15 min. Cells were then blocked with human AB serum for 30 min at room temperature and stained with either primary CXCL12 antibody (R&D Cat. # MAB350), primary CXCR4 antibody (Affinity Bioreagents Cat. # OPA1-01101, Golden, CO) or the appropriate IgG isotype control for 30 min at 4%C in the dark. After washing, cells were incubated with goat anti-mouse or anti-rabbit IgG-fluorescent isothiocyanate (FITC) secondary antibodies (BD-PharMingen and Caltag, Carlsbad, CA) for 40 min at 4%C in the dark. Cells were washed again, resuspended in PBS–BSA buffer, and analyzed immediately on a FACScan Analytic Flow Cytometer (Becton Dickinson). Data were analyzed using FCS Express3 Lite Software (DeNovo, Inc., Thornhill, Ontario, Canada).
Chemotaxis assays.
Cells were treated at 70% confluence with either 100nM dioxin or DMSO in complete media for 16 h and then incubated in serum-free medium supplemented with dioxin or DMSO for 8 h. Cells were dissociated with TrypLE Express (Invitrogen), pelleted, and resuspended in chemotaxis buffer (serum-free medium containing 0.1% BSA + either 100nM dioxin or DMSO vehicle at a final concentration of 0.1%). They were gently passed three times through an 18.5G needle, counted using a hemocytometer, and brought to a concentration of 1.5 x 106 cells/ml. One hundred microliters of cells was plated in replicates of four onto transwell inserts (6.5 mm diameter, 8 µm pores; Fisher Scientific) precoated with 50 µl of 8 µg/ml fibronectin (Sigma). Inserts were placed into wells containing either chemotaxis buffer with DMSO or chemotaxis buffer with dioxin. Lower chambers contained chemotaxis buffer supplemented with varying concentrations of CXCL12, 2.5% FBS, or an equivalent volume of water. Cells were allowed to migrate for 5 h, after which time the medium was removed from the upper and lower chambers and replaced with phenol red-free complete medium + 10% MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] tetrazolium reagent (Owen's reagent; Promega, Madison, WI). The MTS reagent is bioreduced by cells to a soluble formazan product which can be detected spectrophotometrically for the purpose of quantifying the number of viable cells in each insert. After incubation at 37%C for 1 h, medium was removed from the upper and lower chambers, combined, and scored for absorbance at 490 nm using an EL-800 Universal Microplate Reader (Bio-Tek Instruments). Meanwhile, cells on the inserts were washed with PBS and those on the upper layer were gently wiped off with a prewet q-tip. Cells on the lower layer were fixed in 100% methanol and stained with crystal violet. Membranes were excised from the inserts, mounted on glass slides, and with a marker, dived into eight equal sections. Cells in one random viewing field (at x 40 magnification) from each section were counted, and the average was calculated. Data from each insert were normalized to the equivalent MTS data to control for small differences in viability. Data are expressed as chemotaxis index, or normalized number of cells in experimental group/control group. Statistical analysis was performed using a two-tailed t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Genes Modulated upon Dioxin Treatment in MCF-7 Cells
In order to investigate dioxin-modulated gene expression in MCF-7 cells, we analyzed the expression profiles from three separate cell culture experiments. We used the Affymetrix Human Expression Arrays U133A and U133B, containing almost 45,000 probe sets, representing more than 39,000 transcripts, which were derived from
33,000 well-substantiated human genes. We set the limits at 1.8-fold to identify genes that were both upregulated and downregulated by dioxin. Several of the fifteen upregulated genes we identified were previously known to be dioxin-inducible. However, none of the 17 downregulated genes we identified have previously been reported to be downregulated by dioxin. Several of the genes we identified are known to be inducible by estradiol (E) or hypoxia (H) in certain cell lines (Tables 1 and 2). Results from the microarray experiments were confirmed for numerous transcripts by real-time PCR after treatment of MCF-7 cells for 16 h with 100nM dioxin.
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CXCR4 and CXCL12 are Downregulated upon Dioxin Treatment
As we were particularly interested in the fact that both CXCR4 and its unique ligand, CXCL12, were downregulated by dioxin, we confirmed by real-time PCR that both mRNAs were downregulated in a time-dependent manner at 8-, 16-, 24-, and 48-h postdioxin treatment (Figs. 1A and 1B). ALDH3 relative expression levels were upregulated at these same time points, serving as a positive control (Fig. 1C). We used flow cytometric analysis to determine whether dioxin affected CXCR4 cell surface expression and CXCL12 intracellular expression. Following treatment with dioxin, cells were harvested and analyzed for surface expression levels of CXCR4. One, 2, and 3 days after dioxin treatment, CXCR4 surface levels were significantly decreased relative to DMSO-treated cells (Fig. 2A). CXCL12 levels were also quantified using flow cytometry, and were found to be significantly decreased upon dioxin treatment for 1, 2, and 3 days relative to DMSO-treated cells (Fig. 2B).
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Dioxin Inhibits Chemotaxis of MCF-7 cells Toward a CXCL12 Gradient
In order to establish whether the downregulation of surface CXCR4 expression by dioxin has a consequential biological effect, we performed chemotaxis assays to determine whether dioxin affects the migration of MCF-7 cells towards a CXCL12 gradient. MCF-7 cells pretreated with DMSO or dioxin for 24 h were allowed to migrate through a fibronectin-coated membrane towards either CXCL12 at final concentrations of 50, 100, or 500 ng/ml, 2.5% FBS, or toward no chemoattractant. Cells treated with dioxin were significantly impaired in their ability to migrate through the membrane towards all three concentrations of CXCL12, with a resulting migration rate similar to the background rate (DMSO-treated cells exposed to no CXCL12 gradient, Fig. 3). This result indicates that the degree to which dioxin downregulates surface CXCR4 levels may be sufficient to significantly impact the homing of breast cancer cells to areas of high CXCL12 expression. Dioxin did not inhibit chemotaxis towards a gradient of 2.5% FBS, indicating that the inhibition of chemotaxis specifically affects that towards CXCL12 and is not merely a consequence of inhibition of motility. This last conclusion is supported by the observation that dioxin did not reduce the background migration rate (migration towards no chemoattractant).
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Dioxin Inhibits CXCR4 under Hypoxic Conditions and CXCL12 under Estradiol-Inducing Conditions
CXCR4 is induced in numerous cell types upon exposure to low oxygen tension (1% O2, Schioppa et al.). In light of this, we cotreated MCF-7 cells with hypoxia and dioxin to determine whether dioxin could inhibit the hypoxic induction of CXCR4 mRNA. We found that dioxin did indeed reduce CXCR4 under hypoxic conditions (Fig. 4A). The degree of downregulation elicited by dioxin, however, was similar under hypoxic (1.86-fold) and normoxic (1.82-fold) conditions, indicating that dioxin does not specifically affect the hypoxic response. We did not observe induction of CXCL12 by hypoxia in our cells.
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Since CXCL12 is inducible by estradiol in MCF-7 cells (Hall and Korach, 2003
), we also sought to determine whether dioxin could inhibit the expression of CXCL12 under estradiol-induced conditions in these cells. We found that dioxin did significantly reduced levels of CXCL12 mRNA (Fig. 4B). However, the degree of downregulation was similar in estradiol-treated and -untreated cells, again indicating that the effect of dioxin was not on the estradiol response per se. This conclusion is supported by our observation that both CXCR4 and CXCL12 were downregulated by dioxin in the presence of phenol red-free medium supplemented with dextran charcoal-stripped FBS (Figs. 5A and 5B). Since this medium lacks ligands for the Estrogen Receptor, these results therefore indicate that the effect is not antiestrogenic in nature. Dioxin has well-established antiestrogenic activity, so this is an important distinction between our observed effects and other consequences of dioxin action.
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Dioxin Modulates CXCR4 and CXCL12 mRNA Levels in Other Cell Lines
In order to determine whether dioxin affects CXCR4 and/or CXCL12 expression in other cell types, we used real-time PCR to screen cells of varying origins for changes in dioxin-induced gene expression. We found that dioxin downregulates CXCR4 mRNA in five of 19 cell lines tested. These lines originated from cancers of the breast, colon, skin, and ovary. Interestingly, dioxin upregulated CXCR4 mRNA in seven of the 19 cell lines tested. These cells originated from cancers of the colon, liver, lung, ovary, pancreas, and uterus. In five of the eight cell lines that expressed CXCL12, dioxin elicited a downregulation of this transcript. It had no effect on expression levels in the remaining three lines (Table 3). Thus, in all breast cancer cell lines tested, dioxin either downregulated or caused no change in CXCR4 or CXCL12 levels, whereas in colon and ovarian cancer cells, the direction of the response varied among cell lines.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Despite significant experimental and epidemiological evidence, the effects of dioxin on the development and progression of breast cancer remain unclear. In vitro and in vivo data suggest that dioxin may have a protective effect, as did early epidemiological evidence following the Seveso exposure. Follow-up studies on this last cohort, however, suggest that at least after prolonged exposure, this preliminary conclusion may be incorrect (Warner et al., 2002
). In order to further investigate this issue, we used microarray technology to identify genes differentially regulated by dioxin in breast cancer cells and found that dioxin downregulates CXCR4 and CXCL12, two gene products recently shown to mediate the progression of breast cancer.
The interaction between the chemokine CXCL12 (SDF-1) and its cognate receptor, CXCR4, is known to regulate a variety of immune processes, such as infection and inflammation, and many key signaling pathways involved in cell survival, proliferation, chemotaxis, migration, and adhesion (Luker and Luker, 2006). CXCL12 is the only known ligand for CXCR4, a relationship unique among chemokines and their receptors, and one which implicates a role for CXCL12 in all CXCR4-mediated events. The organ sites to which primary breast cancers typically metastasize, the lung and bone, constitutively express some of the highest levels of CXCL12 in the body. It is thought that preferential metastasis is mediated by chemoattraction between CXCR4 on the primary tumor cells and CXCL12 at the sites of metastasis, as neutralizing antibodies to CXCR4 significantly inhibit the metastasis of tumorigenic breast cancer cells to the lung (Muller et al., 2001). Patients with tumors expressing elevated levels of CXCR4 showed an increased frequency of lymph node metastasis (Kato et al., 2003), and high CXCR4 expression in tumors was associated with an overall decrease in patient survival (Li et al., 2004). Furthermore, CXCR4 may represent a predictor of poor prognosis for breast cancer (Luker and Luker, 2006). The inhibition of CXCR4/CXCL12 signaling by dioxin would therefore presumably have a protective effect for breast cancer.
CXCR4 is regulated at both the RNA and protein levels. Nuclear factor kappa B directly induces CXCR4 mRNA in breast cancer cells (Helbig et al., 2003) for example, while CXCL12 stimulates the internalization and subsequent recycling or degradation of CXCR4 on ovarian cancer cells (Scotton et al., 2001). Tumor necrosis factor-alpha (TNF-) can either downregulate or upregulate mRNA and surface CXCR4 expression, depending on the cell type, while CXCL12 has been shown to upregulate TNF- mRNA and protein secretion in neuroepithelial cells (Han et al., 2001). Previously, however, no connection has been made between CXCR4 or CXCL12 expression and gene regulation mediated by the AHR. Putative xenobiotic response elements exist in the CXCR4 (– 123, – 948) and CXCL12 (– 446, – 581) upstream promoter regions. It will be interesting to investigate whether the AHR/ARNT complex binds to these or other upstream regions of CXCR4 and CXCL12, and to identify the coactivators or corepressors associated with this complex.
The inhibition of CXCR4 by dioxin could potentially explain the experimental and epidemiological data suggesting a protective effect of dioxin for breast cancer. However, the fact that dioxin downregulates CXCL12 in addition to CXCR4 is also important, since CXCL12 has the potential to stimulate CXCR4 through autocrine and paracrine mechanisms. Earlier studies have focused on the role of CXCR4 and CXCL12 in the metastasis of breast cancer, but it has become apparent that this interaction also plays an important role in the primary tumor. The expression of CXCR4 has been shown to be necessary for the growth of breast tumor xenografts implanted into the mammary fat pad (Luker and Luker), and CXCL12 can support the survival and growth of a variety of normal and tumor cell types, including breast cancer cells (Orimo et al., 2005). CXCL12 has also been shown to promote angiogenesis by attracting endothelial cells to the tumor microenvironment (Burger and Kipps, 2006), and CXCL12 expression in primary tumors encourages the recruitment and accumulation of immune cells to the site of the primary tumor, which can subsequently induce neoangiogenesis (Kryczek et al., 2005). Therefore, in addition to metastasis, the downregulation of CXCL12 by dioxin may have an inhibitory effect on survival, proliferation, or angiogenesis of the primary tumor.
We found dioxin to inhibit chemotaxis of MCF-7 cells specifically toward CXCL12 without the addition of a golgi secretion inhibitor. Therefore, although these cells could secrete endogenous CXCL12 and presumably autostimulate, they were still able to migrate towards exogenous ligand, and dioxin was able to inhibit this chemotaxis. Although dioxin downregulated CXCR4 and CXCL12 only modestly, we observed an inhibition of chemotaxis to background levels or below after dioxin treatment. Due to the fact that the binding of CXCL12 to CXCR4 activates such an array of intracellular signal transduction pathways, thereby mediating a variety of biological effects, the number of effector molecules regulated by CXCR4 is likely to be great. It remains unclear, however, to what extent these effector molecules relate to particular functions in the cell. Therefore, modest modulation of CXCR4 levels may still significantly affect levels of effector molecules, thereby eliciting a proportionally greater biological effect.
Since hypoxia plays a key role in tumorigenesis and HIF-1 regulates CXCR4 expression (Schioppa et al., 2003), we sought to determine whether dioxin could affect the expression of CXCR4 under hypoxic conditions in breast cancer cells. We found that the combination of hypoxia and dioxin did reduce CXCR4 to control levels, but since the degree of reduction was similar under normoxia and hypoxia, the effect of dioxin is probably not on the hypoxic response per se. Therefore, the protective effects of dioxin against breast cancer are likely not through inhibition of hypoxia-mediated angiogenesis or hypoxia-induced invasive potential.
Likewise, CXCL12 has been shown to be a target of estrogen action in breast and ovarian cancer cells, and can mediate the proliferative effects of estradiol in those cells (Hall and Korach, 2003). We found that dioxin inhibited CXCL12 under estradiol-inducing conditions in MCF-7 cells, but the degree of reduction was not significantly greater in estradiol-treated than in estradiol-untreated cells. Therefore, although dioxin may inhibit estradiol-induced proliferation of primary tumor cells through downregulation of CXCL12, this outcome is unlikely to be a result of a direct effect on the estradiol response.
Inhibitors of CXCR4 function in breast and other cancers are being pursued as rationally based therapeutics. Thus far, CXCR4 antagonists have shown promising results in their antitumor activities (Liang et al., 2004). Since CXCR4 is a major coreceptor for some types of human immunodeficiency virus (HIV) viral entry, many CXCR4 inhibitors are also being investigated for their potential inhibition of HIV infection and/or AIDS progression (Hatse et al., 2005). Although it remains unclear whether dioxin has a protective effect for breast cancer in humans, our data provide a novel mechanism of action for this compound and a plausible explanation for existing evidence implicating its protective role for breast cancer. As dioxin exhibits tumor promoting activity, we propose that nontoxic ligands for the AHR, termed selective AHR modulators, such as 3,3'-DIM, may also downregulate CXCR4 and/or CXCL12 in breast cancer cells and represent potentially useful chemoprotective agents for breast cancer.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institutes of Health/National Cancer Institute (NIH/NCI CA28868); University of California Toxic Substances Research and Teaching Program fellowships to E.L.H. and R.T.T.
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NOTES |
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2 Current address: 6 Paddock Lane, Great Neck, New York 11020.
3 Current address: UT M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 0432, Houston, Texas 77030.
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ACKNOWLEDGMENTS |
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We thank Arnaud Colontonio and Beverly Redsar for sharing their flow cytometry expertise.
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