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Carcinogenesis Advance Access originally published online on July 13, 2006
Carcinogenesis 2007 28(2):267-279; doi:10.1093/carcin/bgl129
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

HGF induces CXCR4 and CXCL12-mediated tumor invasion through Ets1 and NF-{kappa}B

Paola Maroni{dagger}, Paola Bendinelli{dagger}, Emanuela Matteucci and Maria Alfonsina Desiderio*

Institute of General Pathology, School of Medicine University of Milan, via Luigi Mangiagalli, 31-20133 Milan, Italy

*To whom correspondence should be addressed. Tel: +39 025 031 5334; Fax: +39 025 031 5338; Email: a.desiderio{at}unimi.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
CXCR4 is a chemokine receptor probably involved in the homing of metastatic breast cancer, and its expression is modulated by tumor environmental stimuli such as hepatocyte growth factor (HGF) and hypoxia. Here, we demonstrate that, depending on the stimulus, different transcription factors can cooperate in enhancing CXCR4 transcription in MCF-7 breast cancer cell line. In HGF-treated MCF-7 cells, the DNA binding of Ets1 activated by MAPK1/ERK1/2 transduction pathway as well as the DNA binding of NF-{kappa}B played a critical role in CXCR4 transcription and protein induction. Under HGF stimulation, the blockade of these transcription factors by dominant negatives and inhibitors prevented the expression of CXCR4 receptor, the activity of a gene reporter driven by CXCR4 promoter sequence and the chemoinvasion toward the CXCL12 ligand. NF-{kappa}B was activated also by hypoxia and contributed, with HIF-1, to the increase in CXCR4 expression. The results suggest that Ets1, specifically activated by HGF, might cooperate with NF-{kappa}B activity to enhance the invasive/metastatic phenotype of breast carcinoma cells.

Abbreviations: EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; HGF, hepatocyte growth factor; MMP1, metalloprotease-1


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tumor development and progression involve the genotype of neoplastic cells, and are strongly influenced by the microenvironment (1). The accumulated evidence indicates that breast carcinoma cells actively recruit stromal cells, such as inflammatory cells, vascular cells and fibroblasts into the tumor, and that this recruitment is essential for the generation of a microenvironment that actively fosters tumor growth (1–4). Changes in cytokines (proinflammatory cytokines, chemokines and growth factors), produced by the stromal cells, and possible hypoxic niches in the tumor mass seem to influence gene expression profile in malignancy and to determine the cell fate. So, depending on the molecular context within the neoplastic cell and the external influences, different partnership of molecular events takes place with different outcomes in terms of their success at seeding cells to particular secondary sites (1,5).

CXCR4 is a chemokine receptor implicated in the homing of hemopoietic cells, but also expressed in some carcinomas such as breast cancer (6). Müller et al. (7) showed that the ligand, CXCL12, is present in lymph nodes, lungs, bone and liver, suggesting that this distribution could contribute to the tropism of breast cancer metastases for these sites. The hypothesis is that the CXCR4 ligand attracts circulating cells into the secondary sites where they then survive to form metastases. Silencing of CXCR4 blocks breast cancer metastasis (8).

We have demonstrated that two tumor environmental stimuli such as the multifunctional cytokine hepatocyte growth factor (HGF) and hypoxia transcriptionally induce the functional CXCR4 receptor in the low invasive breast carcinoma cells MCF-7. In the highly invasive MDA-MB 231 cells, CXCR4 is inducible only by hypoxia while being reduced by HGF treatment (9). This might be related to the constitutively elevated NF-{kappa}B activity and the low DNA methylase activity in MDA-MB 231 cells (10–12). HGF plays an important role in tumor cell invasion and metastasis that is mediated through the Met receptor tyrosine kinase (13). Paradoxically, tumor progression is associated with both increased microvascular density and intratumoral hypoxia, which represents a positive stimulus for invasion (14). Thus, our results are consistent with the role of chemotaxis in increasing tumor invasiveness, and HGF might be implicated in the homing phase.

In the present paper, we are interested in how the changes to the tumor cell microenvironment are integrated at transcriptional level to influence molecular events involved in tumor invasiveness. To this purpose, we chose the MCF-7 cells to study the transcriptional targets of HGF and hypoxia responsible for CXCR4 induction. Ets1, a member of a transcription factor family that shows a unique DNA-binding domain, is involved in tumor invasive growth (15–19). The activity of Ets1 may undergo a complex regulation by kinases and transcription factors (15). To extend the knowledge about the role and regulatory mechanisms of Ets1 DNA binding under our experimental conditions, we first assayed the activity of two CXCR4 gene reporters containing the consensus sequences for Ets1 and NF-{kappa}B but differing for the presence of HIF-1 binding sites. NF-{kappa}B and HIF-1 are heterodimeric transcription factors implicated in the response to hypoxic stress, and also in the expression of HGF target genes (20–23). Then, we evaluated the involvement of the mitogen-activated protein kinase ERK1/2 in Ets1 DNA binding to CXCR4 promoter sequences in response to HGF and hypoxia, and the possible cooperation between Ets1 and NF-{kappa}B or HIF-1. The biological significance of Ets1 and NF-{kappa}B activation in HGF-treated tumor cells was evaluated after blockade with the respective dominant negative and super-repressor by examination of CXCR4 RNA and protein expression and breast cancer cell migration.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
RPMI-1640 and fetal bovine serum (FBS) were from Sigma Chemical Co. (St Louis, MO). Recombinant human HGF, CXCL12 and anti-CXCR4 monoclonal antibody (MAB-172) were from R & D Systems (Abingdon, UK). [{alpha}-32P]dCTP (3000 Ci/mmol), [{gamma}32-P]ATP (3000 Ci/mmol), Hybond C-extra nylon filters, Hybond ECL nitrocellulose membranes and nick translation kit were from Amersham (Amersham Biosciences Europe GmbH, Italy). The monoclonal anti-human HIF-1{alpha} antibody Ab-2 (Clone OZ15) used for supershift was from NeoMarkers (Fremont, CA), and the anti-human HIF-1{alpha} antibody used for immunoblot was from Transduction Laboratories (Lexington, KY). Anti-Met(C12), anti-Ets1(C20), anti-phospho-ERK1/2(E4), anti-ERK1/2(K23), anti-Akt1/2 (H136), anti-B23 and anti-vinculin antibodies were purchased from Santa-Cruz Biotechnology (Santa-Cruz, CA). Anti-phosphotyrosine (4G10) antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-p50 and anti-p65 antibodies were a gift of Dr M. Ernst (NCI, Frederick, MD). Anti-phospho-Akt (Ser 473) was from Cell Signaling (Beverly, MA). Anti-p52 and anti-RelB antibodies were a gift of Dr G. Bonizzi (European Institute of Oncology, Milan, Italy). Alexa Fluor 488 secondary antibody was from Molecular Probes (Eugene, OR). PD 98059 was from Calbiochem (Darmstadt, Germany). pGL2 enhancer, pGEM-T Easy, pRL-TK, T4 ligase, calf intestinal alkaline phosphatase, Pfu DNA polymerase, Klenow polymerase and dual luciferase reporter assay system were from Promega (Madison, WI). All other chemicals were of the highest grade available.

Cell cultures
Human breast carcinoma cells MCF-7 were from the European Cell Cultures Collection. All cells, routinely maintained in RPMI-1640 medium containing 10% FBS, were starved (0.1% FBS) for 18–24 h before HGF (100 ng/ml) treatment. Some cells were treated with 10 µM PD 98059 for 30 min before HGF or hypoxia exposure (24). The inhibitor was dissolved in DMSO at the non-toxic final concentration of 0.02%. Hypoxia was performed in an incubator equilibrated with a gas mixture containing 5% CO2 and 1% O2 and nitrogen balanced, as reported previously (21). These cells were used for total RNA, nuclear and total protein extractions (21,22,25).

Northern blot and western blot assays
Northern blots of total RNA (30 µg) were performed. The filters were hybridized with the labeled 1.2 kb fragment (XbaI–EcoRI) from CXCR4 cDNA (furnished by TNC Wells, Serono Pharmaceutical Research Institute, Geneva, Switzerland), with the labeled 1.6 kb fragment (SmaI–KpnI) from Ets1 cDNA (kindly provided by Dr G. Gambarotta, University of Torino, Italy), or with the plasmid containing human HIF-1{alpha} cDNA (kindly provided by Dr R.D. Thornton, PCOM, Philadelphia, PA). As a loading standard we used the labeled probe for 36B4, which encodes acidic ribosomal phosphoprotein. This cDNA probe (651 bp) was prepared by polymerase chain reaction (PCR) using the following sense and antisense primers: 5'-CTATCCGCGGTTTCTGATTG-3'; 3'-TGCCCCTGGAGATTTTAGTG-5' (GeneBankTM accession no. M17885 [GenBank] ). To confirm that each lane contained equal amounts of total mRNA, the ribosomal RNA concentration in each lane was estimated visually in the ethidium bromide-stained gels. Western blots of total proteins (100 µg) were immunoblotted with anti-CXCR4 (5 µg/ml), anti-Ets1 (1 : 1000), anti-phospho-ERK 1/2 (1 µg/ml), anti-ERK 1/2 (1 : 200), anti-phospho-Akt (1 : 1000), anti-Akt1/2 (1 µg/ml) or anti-vinculin antibody, to confirm equal loading. Western blot of nuclear proteins (50 µg) were immunoblotted with anti-HIF-1{alpha} (1 : 350) antibody, and to evaluate the equal loading, immunoblot with B23/nucleophosmin was performed (26). Immunoprecipitation experiments for Met were described previously (27). Enhanced chemiluminescence kit (ECL or ECL-plus; Amersham Biosciences) was used for detection. The relative amounts of specific mRNAs and proteins were calculated after densitometric evaluation of the northern and western blots, respectively, and controls were assigned an arbitrary value of 1.

Plasmids
We used KpnI/XhoI to subclone the original gene reporter pCXCR4(–2632/+86)Luc (kindly provided as pGL2 basic construct by Dr A.J. Caruz, Universidad de Jaen, Madrid, Spain) in the pGL2-enhancer vector, previously cut with SmaI/KpnI. The pCXCR4(–600/+19)Luc fragment was amplified by PCR with Pfu DNA polymerase using pCXCR4(–2632/+86)Luc as template, and the following sense and antisense primers: 5'-TCCCGGGCTTCTGAAAGTATCTCCTAATTATCTG-3' and 3'-CAAACAACCGACGCCGTCGTCCATGGT-5' (Roche, Germany). PCR was performed in 50 µl of 1x PCR buffer containing 2 mM MgCl2, 1 mM dNTPs, 2 U of Pfu DNA polymerase, 10 µM DNA primers and 200 ng pCXCR4(–2632/+86)Luc. The samples were heated at 95°C for 120 s, then at 95°C for 45 s, 68°C for 45 s, 72°C for 120 s for 35 cycles and then at 74°C for 5 min. The PCR fragment was cloned in pGEM-T Easy and cut with SmaI and NotI, blunted with Klenow polymerase and cloned in pGL2-enhancer, previously cut with SmaI. The expression vector RSVIkB{alpha}MSS, coding for NF-{kappa}B super-repressor (ssNF{kappa}B), was kindly provided by Dr N.D. Perkins (University of Dundee, Dundee, UK); the dominant negative forms of ARNT (pcDNA3ARNTdelta_b, {Delta}ARNT) and of Ets1 ({Delta}Ets1) were, respectively, from Dr M. Schwarz (Institute for Toxicology, University of Tübingen, Germany) and from Dr J. Ghysdael (CNRS UMR, Orsay, France).

Transient transfection assay
For transient transfection experiments MCF-7 cells were seeded in 24-multiwell plates, in T25 or in T75 flasks, and were transfected at 70–80% of confluence (0.2 x 106, 6 x 106 and 18 x 106, respectively) using Fugene-6 (Roche). The cells in the multiwell plates were transfected with 200 ng reporter plasmid pCXCR4(–2632/+86)Luc or pCXCR4(–600/+19)Luc with or without 500 ng of ssNF{kappa}B and/or 1 µg of {Delta}Ets1 per well. For normalization the cells were transfected with pRL-TK (Renilla luciferase) vector. The cells in the flasks were transfected with 1 µg of ssNF{kappa}B, 2 µg of {Delta}ARNT or 2 µg of {Delta}Ets1 per 106 cells (22). After 8 h, all the cells underwent overnight starvation followed by HGF treatment (100–200 ng/ml), or complete medium was added and hypoxic treatment was performed. Then, the cells in the multiwell plates were collected 24 h after the treatments, and firefly and Renilla luciferase activities were measured with a dual luciferase assay system (22). The cells in the flasks were used 4 h after the treatments for total RNA extraction and northern blot analysis, or for nuclear protein extraction and electrophoretic mobility shift assay (EMSA) analysis.

Electrophoretic mobility shift assay
The EMSA method was used to analyze the DNA-binding activities of Ets1, NF-{kappa}B, HIF-1 and Octamer-1 transcription factors in nuclear extracts (21,22,25), prepared from 30 x 106 MCF-7 cells. Single-strand oligonucleotides with consensus binding sites for the transcription factors were labeled with T4 polynucleotide kinase (Amersham) by using [{gamma}-32P]ATP, annealed to the complementary strand, and purified by PAGE. After spectrophotometric measurement, nuclear proteins (10–15 µg) were incubated for 20 min at 25°C in the binding reaction mixtures, containing 0.5 ng 32P-labeled double-stranded sequences. For competition experiments, 50-fold molar excess of each specific unlabeled double-stranded sequence was added to the binding mixtures. DNA–protein complexes were resolved by electrophoresis on 5% native polyacrylamide gel at 4°C in 0.5, 1 and 0.3% TBE (1x TBE: 90 mM Tris–borate containing 2 mM EDTA, pH 8) for the study of Ets1, NF-{kappa}B and HIF-1 DNA binding, respectively. For super-gel shift assay, nuclear extracts were incubated without the oligonucleotide but with 1 µg anti-p50, anti-p65, anti-p52, anti-RelB or anti-HIF-1{alpha} antibody for 60 min on ice (21,22), or with 1 µg anti-Ets1 antibody for 15 min at room temperature (19), followed by incubation with the labeled oligonucleotide and electrophoresis as described above. The oligonucleotides containing the Ets1 consensus sequence were 5'-GATCGAGAGGATGTTATAAAGCA-3' present in the promoter of metalloprotease-1 (MMP1); 5'-CGCGGGGGAATGGGCGGTTGGAAGCCTGGC-3' (EtsA, containing two sites) and 5'-CCTCCGAAGGAAAGGATCTT-3' (EtsB, containing one site) present in the CXCR4 promoter, spanning at –397 to –412 and –478 to –481 from the transcription start site. The oligonucleotide containing the kB consensus sequence for NF-{kappa}B was 5'-TCCCCTGGGCTTCCCAAGCC-3' (‘p65’) present in the promoter of CXCR4 at –96 to –111. The oligonucleotides containing the HRE consensus sequence for HIF-1 were 5'-TTCTGATTCGTGCCAAAGCT-3' (HRE1, containing one site) and 5'-GGGTCCGTGTCGCGACGCACGCGCCT-3' (HRE2, containing two sites) present in the promoter of CXCR4, and spanning at –1017 to –1021 and at –1258 to –1273. The Octamer-1 binding site sequence was 5'-TGCGAATGCAAATCACTAGAA-3'. The oligonucleotides were synthesized by Roche.

Fluorescence microscopy
Cells were seeded at 4 x 104 per well on sterile coverslips, previously placed in 24-multiwell plates, and allowed to attach. Thus, the cells in the wells were transiently transfected with 1 µg {Delta}Ets1 and 500 ng ssNF-kB expression vectors alone or in combination. After starvation and treatment with HGF (200 ng/ml) the cells were fixed with 4% paraformaldehyde solution. These cells were washed and incubated with anti-CXCR4 antibody (10 µg/ml) in 0.2% BSA for 90 min, rinsed and then incubated with the secondary fluorescent antibody (1 : 500) in PBS for 30 min. After washing, the coverslips were applied on the slides with Entellan (Merck, Darmstadt, Germany). The cells were examined with a fluorescence microscope and images were collected through the specimens at x400 magnification and displayed on a computer screen (9).

Tumor cell chemoinvasion assay
Cultured cells were transiently transfected or not with 1 µg of ssNF{kappa}B and 2 µg of {Delta}Ets1 per 106 cells, and were treated for 16 h with HGF (200 ng/ml). Then, chemoinvasion assay was performed using a Matrigel invasion chamber from BD Biocoat Cellware (Becton Dickinson Labware, Bedford, MA). To this purpose, 8 x 104 transfected or un-transfected cells that have been exposed or not to HGF were seeded in the top chamber. CXCL12 (400 ng/ml) was added to the bottom chamber to induce cell migration through Matrigel. The multiwell chamber was incubated for 22 h in a humidified tissue culture incubator. After removal of non-invading cells from the top, invading cells were stained with Diff-Quik (Dade Behring, Switzerland) and counted (9). Ten fields under a x200 magnification were randomly selected and counted.

Statistical analysis
Densitometric values were analyzed by analysis of variance, with P < 0.05 considered significant. Differences from controls were evaluated on original experimental data, and then we assigned to controls the arbitrary value of 1.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Role of Ets1, NF-{kappa}B and HIF-1 in the transcriptional response of CXCR4 to HGF or hypoxia
The present investigation was performed in MCF-7 breast carcinoma cells because both HGF and hypoxia induced CXCR4 receptor in this cell line. As shown in Figure 1A and B, HGF and hypoxia increased mRNA and protein levels evaluated by northern and western blots, in agreement with our previous data (9). CXCR4 mRNA level increased after HGF between 4 and 12 h (of ~3-fold). After hypoxia, CXCR4 mRNA peaked at 4 h (2.8-fold) and slightly declined thereafter, remaining 1.8-fold above the control value at 16 h. Consistently, CXCR4 protein levels progressively increased until 24 h in HGF- and hypoxia-treated cells, reaching values 5- and 3-fold higher than those of controls (starved or 10% FBS-treated cells). Tyrosine phosphorylation of Met receptor was observed 30 min after HGF treatment (Figure 1C).


Figure 1
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  		Fig. 1 CXCR4 induction and gene reporter activity after HGF or hypoxia treatment. (A) Northern blot analysis of total RNA extracted from HGF- or hypoxia-treated MCF-7 cells and hybridization with labeled CXCR4 probe. The values at the bottom indicate the HGF- or hypoxia-induced variations relative to controls, that is, starved or 10% FBS-cultured cells, calculated from densitometric analysis. Hybridization with labeled 36B4 probe was used for normalization. Ethidium bromide-stained gel is shown. The experiment has been repeated three times with similar results. (B) Western blot analysis of total proteins extracted from HGF- or hypoxia-treated MCF-7 cells. The immunoblot was performed with anti-CXCR4 antibody. The values at the bottom indicate the HGF- or hypoxia-induced variations relative to controls, calculated from densitometric analysis. The immunoblot with anti-vinculin antibody was used for normalization. The experiment has been repeated three times with similar results. (C) Immunoprecipitation of Met using protein extracts from HGF-treated cells and immunoblotting with anti-Met or anti-p-Tyr antibody. (D) Structure of human CXCR4 promoter. The position of the consensus site for NF-{kappa}B ‘p65’, six Ets1-binding sites and five HRE binding sites for HIF-1 relative to the transcription start site (GenBankTM accession no. AJ224869), may be noted. (E) MCF-7 cells were transiently transfected with construct pCXCR4(–2632/+86)Luc or pCXCR4(–600/+19)Luc. Some cells were co-transfected with {Delta}Ets1 and ssNF{kappa}B alone or in combination. The histograms indicate the fold changes of luciferase activity in cells treated with HGF or hypoxia versus controls (i.e. starved or 10% FBS-treated cells). The data are the mean ± standard error (SE) of three independent experiments performed in triplicate. Where the SE are not shown, they fall within the columns. *P < 0.05 and **P < 0.005 versus controls; {Delta}P < 0.05 and {Delta}{Delta}P < 0.005 versus HGF- or hypoxia-treated cells. °P < 0.05 and °°°P < 0.001 versus pCXCR4(–2632/+86)Luc transfected cells exposed to HGF or hypoxia.

 
To better define the regulation of the human CXCR4 induction by the two tumor environmental stimuli, we analyzed the CXCR4 promoter using MatInspector analysis software (accession no. AJ224869 [GenBank] ). The promoter region of 700 bp upstream the TATA box contained six consensus sequences for Ets1 (5'-GGAA/T-3') and one NF-{kappa}B consensus site called ‘p65’ (5'-CCTGGGCTTCCCAAG-3') (28). Three forward HRE sites (F) (5'-CGTGC/T-3') and two reverse HRE sites (R) (5'-GCACG-3') were upstream between –825 and –1933 bp (Figure 1D).

We next determined the activity of the CXCR4 promoter (–2632/+86)Luc and of a construct (–600/+19)Luc, which was prepared by PCR to contain the multiple Ets1 consensus sites and the NF-{kappa}B consensus site. The MCF-7 cells were transfected with these gene reporters and were exposed to HGF or hypoxia, normalizing the transfection efficiency by co-transfecting the cells with pRL-TK vector. The luciferase/Renilla activity ratios were calculated by the software and used to evaluate the changes in luciferase activity after HGF or hypoxia compared with controls (starved or 10% FBS-treated cells). To evaluate the role played by the transcription factors in CXCR4 transactivation, we transiently co-transfected these cells with pCXCR4(–600/+19)Luc and {Delta}Ets1 and/or ssNF{kappa}B expression vectors (Figure 1E). ssNF{kappa}B is a highly specific and effective NF-{kappa}B inhibitor with mutations in the IkB{alpha}-conserved serine residues typically targeted for phosphorylation after cellular stimulation (29). {Delta}Ets1 is a dominant negative for Ets1, bearing only the DNA-binding site. HGF increased the luciferase activity of the two pCXCR4 gene reporters with a 20% difference between pCXCR4(–2632/+86)Luc and pCXCR4(–600/+19)Luc, indicating the principal role of Ets1 and NF-{kappa}B activities in the control of CXCR4 transactivation in response to the cytokine. Hypoxia stimulated 4-fold the activity of pCXCR4(–2632/+86)Luc and doubled the activity of the short PCR fragment, suggesting that the HIF-1 binding to HRE sequences might play an important role in CXCR4 transcription. In HGF-treated cells, {Delta}Ets1 and ssNF{kappa}B alone prevented, up to 38 and 42%, the increase of pCXCR4(–600/+19)Luc activity, while their combination impaired the activation. In MCF-7 cells transfected with pCXCR4(–600/+19)Luc and exposed to hypoxia, {Delta}Ets1 was ineffective and ssNF{kappa}B completely prevented the increase in the gene reporter activity. As shown in the Figure 1E, the co-transfection of the expression vectors did not modify pCXCR4(–600/+19)Luc activity.

Effect of transcription factor blockade on CXCR4 mRNA and protein expression in cells stimulated by HGF or hypoxia
On the basis of the results of transcription factor transactivation, we blocked Ets1, NF-{kappa}B and HIF-1 activities and analyzed CXCR4 mRNA expression under our experimental conditions (Figure 2A). To this purpose, HGF- or hypoxia-treated MCF-7 cells were transfected with the expression vectors for inhibitors and dominant negatives of the studied transcription factors, that is, ssNF{kappa}B, {Delta}Ets1 and {Delta}ARNT. {Delta}ARNT codes for a mutant HIF-1ß subunit form that lacks the basic domain and is therefore still capable of heterodimerizing with the {alpha}-subunit but cannot bind DNA. In 4 h HGF-treated MCF-7 cells, CXCR4 mRNA induction was prevented up to 60% by ssNF{kappa}B and {Delta}Ets1, and 30% by {Delta}ARNT. This result might also be explained by considering that HGF-activated NF-{kappa}B controls HIF-1{alpha} expression and HIF-1 activity (23). In the cells exposed to 4 h hypoxia, the expression vectors for ssNF{kappa}B and {Delta}ARNT transfected were effective in preventing up to 79 and 43% the induction of CXCR4. {Delta}Ets1 did not affect the augment of CXCR4 transcript level after hypoxia treatment, and seemed to slightly enhance the basal level of the messenger.


Figure 2
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  		Fig. 2 CXCR4 mRNA and protein expression in HGF- or hypoxia-treated cells transfected with ssNF{kappa}B, {Delta}ARNT and {Delta}Ets1. (A) Northern blot analysis of total RNA extracted from MCF-7 cells transiently transfected with ssNF{kappa}B, {Delta}ARNT or {Delta}Ets1 expression vector and exposed to HGF or hypoxia. Hybridization was performed with labeled CXCR4 or with 36B4 for normalization. Ethidium bromide-stained gels are shown. Each value is the mean ± SE of experiments performed in triplicate. *P < 0.05 and **P < 0.005 versus controls, that is, starved or 10% FBS-cultured cells; {Delta}P < 0.05, {Delta}{Delta}P < 0.005 and {Delta}{Delta}{Delta}P < 0.001 versus HGF- or hypoxia-treated cells. (B) The cells on coverslips were transfected with {Delta}Ets1 and ssNF{kappa}B alone or in combination, and then were starved and treated with HGF. After 16 or 24 h of HGF treatment, the cells were fixed with 4% paraformaldehyde, incubated with the primary anti-CXCR4 antibody and then with the secondary fluorescent antibody. Images were taken under fluorescence microscope at x400 magnification. (C) Western blot analysis of HGF-treated cells and immunoblot with anti-CXCR4 antibody. The values at the bottom indicate the HGF-induced variations relative to starvation, calculated from densitometric analysis. The immunoblot with anti-vinculin antibody was used for normalization.

 
To study the possible correspondence between CXCR4 mRNA and protein expression pattern, we evaluated the receptor protein under HGF treatment by cell immunofluorescence and western blot analysis (Figure 2B and C). The times examined were 16 and 24 h after HGF exposure, when the CXCR4 protein levels showed the highest increases by western blot. We observed an enhanced fluorescent signal at 16 and 24 h in HGF-treated MCF-7 cells, which was prevented by {Delta}Ets1 and ssNF{kappa}B. The transfection of {Delta}Ets1 alone or in combination with ssNF{kappa}B caused the earliest (16 h) blockade of CXCR4 protein induction after HGF, resulting in a strong decrease of the fluorescent signal. It is worth noting that the anti-CXCR4 antibody used detects multiple conformations of the receptor, which is present not only on cell surface but also in the cytosol (30,31). Thus, the increased production of the CXCR4 receptor protein after HGF was probably mediated by the cooperation of Ets1 and NF-{kappa}B transcription factor activities. These results were confirmed and quantified by the western blot analysis of CXCR4 protein (Figure 2C). In 24 h hypoxia-treated cells, CXCR4 expression examined by immunofluorescence and western blot was reduced by the combination of ssNF{kappa}B and {Delta}ARNT (data not shown).

Ets1 and NF-{kappa}B activity blockade impaired MCF-7 cell migration toward CXCL12 in HGF-treated cells
The Matrigel chemoinvasion chamber may be considered an in vitro model for metastasis. The low invasive MCF-7 cells did not migrate spontaneously through the Matrigel-coated filter. HGF pretreatment or CXCL12 exposure (bottom chamber) did not result in any significant cell invasion (Figure 3A) (9). The MCF-7 cells pretreated with HGF for 16 h acquired responsiveness to CXCL12, present in the bottom chamber (CXCL12 + HGF) and displayed significant chemoinvasion properties, which were prevented by co-transfection with {Delta}Ets1 and ssNF{kappa}B (Figure 3B). The cell count-mean per field was 30 ± 4 for the cells pretreated with HGF and exposed to CXCL12 (CXCL12 + HGF), and 1.1 ± 0.2 for the cells transfected with {Delta}Ets1 and ssNF{kappa}B before HGF treatment and CXCL12 exposure (Figure 3C).


Figure 3
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  		Fig. 3 MCF-7 cell migration toward CXCL12 in response to HGF. (A) MCF-7 cells treated with HGF for 16 h were used for Matrigel invasion assay with CXCL12 added to the bottom chamber. (B) MCF-7 cells were transiently transfected or not with {Delta}Ets1 and ssNF{kappa}B before 16 h HGF treatment. These cells were used for Matrigel invasion assay, with CXCL12 added to the bottom chamber (CXCL12 + HGF pretreatment). We estimated invasion by counting the number of invading cells on the lower side of the membrane after staining (x200 magnification of selected fields). (C) The total number of migrated cells in 10 fields was counted, and the mean value for one field has been reported. The experiment was repeated three times, and the means ± SE are shown. Where the SE are not shown, they fall within the columns. ***P < 0.001 versus CXCL12 exposed cells; {Delta}{Delta}{Delta}P < 0.001 versus HGF-treated cells exposed to CXCL12.

 
Characterization and regulation of Ets1 expression and binding to CXCR4 promoter after HGF or hypoxia treatment
To evaluate the involvement of the Ets1 transcription factor in CXCR4 transcription, in a first series of experiments we measured endogenous Ets1 mRNA and protein expression at various times after HGF or hypoxia treatment of MCF-7 cells. Using the same protein extracts we analyzed also the phosphorylation state of mitogen-activated protein kinase ERK 1/2, which is one of the kinases that positively regulates Ets1 activity by phosphorylation of threonine 38 (15) (Figure 4).


Figure 4
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  		Fig. 4 Ets1 expression and ERK1/2 phosphorylation after HGF or hypoxia treatment. (A) Northern blot analysis of total RNA extracted from HGF- or hypoxia-treated MCF-7 cells and hybridization with labeled Ets1 cDNA. The values at the bottom indicate the variations of treated cells relative to controls, that is, starved or 10% FBS-cultured cells, calculated from densitometric analysis. Hybridization with labeled 36B4 probe was used for normalization. Ethidium bromide-stained gel is shown. The experiment has been repeated three times with similar results. (B) Western blot analysis of total and nuclear proteins prepared from HGF- or hypoxia-treated MCF-7 cells. Immunoblots were performed with the antibodies against the indicated proteins. Anti-vinculin and anti-B23 antibodies were used for normalization of total and nuclear proteins, respectively. Relative amounts of Ets1, CXCR4 and HIF-1{alpha} proteins, and the ratio between phosphorylated/unphosphorylated forms of ERK 1/2 in treated cells relative to control cells were calculated from densitometric analysis. Each value is the mean ± SE of experiments performed in triplicate. *P < 0.05 and ***P < 0.001 versus controls, that is, starved or 10% FBS-cultured cells. Starved (open squares) and HGF-treated (filled squares) cells; FBS (10%) (open triangles) and hypoxia-treated (filled triangles) cells.

 
Total Ets1 mRNA and protein levels were analyzed by northern and western blots (Figure 4A and B). After both the stimuli, Ets1 transcript levels increased at 4 h, reaching at 8 h values 1.7-fold and 1.4-fold higher than those of controls (Figure 4A). It is worth noting that starvation (0.1% FBS) for 24 h did not influence the basal-control value of Ets1 messenger. Four-hour HGF treatment increased (2-fold) Ets1 protein level, which almost tripled at 12 h, slightly decreasing thereafter (Figure 4B). After hypoxia, a progressive increase in protein level was observed between 4 and 24 h (2- to 3-fold).

As shown in Figure 4B, ERK 1/2 proteins (p42 and p44) were markedly phosphorylated 4 h after HGF treatment, while no changes in the phosphorylation state were observed after hypoxia. The treatments did not modify the level of unphosphorylated ERK 1/2 proteins during the entire observation period. The graphic reports the changes in the ratio between the levels of phosphorylated and unphosphorylated ERK 1/2 proteins, showing a 6-fold enhancement in 4 h HGF-treated cells. Also at 2 h, the ERK 1/2 phosphorylation state was elevated (data not shown), and the enzyme might phosphorylate Ets1 transcription factor probably contributing to its binding activity. The levels of Akt protein and of the phosphorylated form were unchanged by both the treatments. CXCR4 protein levels progressively increased both after HGF and hypoxia treatments, reaching at 24 h values 5- and 3-fold higher than controls. Nuclear HIF-1{alpha} protein levels were enhanced between 4 and 18 h after both treatments. The densitometric evaluations have been reported in the graphics (Figure 4B).

Then, we investigated the functional significance of Ets1 transcription factor by studying the Ets1 binding to the consensus sequences present in the CXCR4 promoter under the two studied stimuli (Figure 5A and B). This was achieved by EMSA, using nuclear extracts from HGF- or hypoxia-treated cells and the following oligonucleotide probes: MMP1, containing the Ets1 binding site of the MMP1 promoter, EtsA and EtsB that correspond to a couple and single consensus sites present in the CXCR4 promoter. The Ets1 DNA binding was stimulated (3- to 4-fold) between 2 and 8 h after HGF treatment using the three oligonucleotides, and 2- to 5.5-fold starting from 2 h in hypoxia-exposed cells, remaining practically constant until 4 h. It is worth noting that in HGF-treated cells, the earliest (2 h) and highest activation (3.2-fold) of the binding was observed using the oligonucleotide EtsA, containing two adjacent Ets1 consensus sequences of the CXCR4 promoter. At 8 h also the binding to EtsB was remarkable (4-fold). However, in hypoxia-treated cells the strongest Ets1 activation was observed using the MMP1 oligonucleotide. The specificity of the complexes was examined by competition experiments using 50-fold molar excess of unlabeled oligonucleotides (comp), which almost completely suppressed the complex formation.


Figure 5
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  		Fig. 5 Analysis of Ets1 DNA binding in HGF- or hypoxia-treated cells. EMSA of Ets1 binding to oligonucleotides containing consensus sequences present in the promoters of MMP1 and of CXCR4 (EtsA with two sites and EtsB with one site). Nuclear extracts from (A) HGF or (B) hypoxia (Hyp) treated MCF-7 cells were used. comp, specific competition with 50-fold excess cold oligonucleotide and nuclear protein samples prepared at 4 h of the treatments. unsp, non-specific binding. Representative autoradiograms from three independent experiments. The values at the bottom indicate the HGF- or hypoxia-induced variations relative to controls, that is, starved or 10% FBS-cultured cells, calculated from densitometric analysis. (C) MCF-7 cells treated with PD 98059 before 4 h HGF or hypoxia exposure. (D) MCF-7 cells transfected with ssNF{kappa}B before 4 h HGF or hypoxia treatment. Ets1-DNA complex formation at 4 h of treatment was supershifted (SS) by the specific anti-Ets1 antibody (Ab). Representative autoradiograms from three independent experiments.

 
DNA-binding activity of the constitutively expressed ubiquitous Octamer-1 transcription factor was unaffected by the treatments (data not shown).

To deepen the knowledge about the regulation of Ets1 activity in MCF-7 cells exposed to HGF or hypoxia, we considered the role of ERK 1/2 activity and the possible cooperation with NF-{kappa}B activity (Figure 5C and D). The first regulatory mechanism was evaluated by MCF-7 cell pretreatment with PD 98059 that inhibits MEK 1/2 activity, blocking downstream the phosphorylative activation of its substrate ERK 1/2. The inhibitor markedly prevented the DNA binding of Ets1 to the MMP1 consensus sequence only in the cells exposed to 4 h HGF treatment (Figure 5C). The Ets1 binding complex was formed by two subunits p54/p42, as reported in literature (19), and seemed to be regulated through phosphorylation. In fact, after PD 98059 treatment we observed a reduction of the DNA binding that ranged from 60% (lower band) to 100% (upper band). In the supershift experiments, the addition of anti-Ets1 (p54) antibody markedly reduced the intensity of the DNA binding and caused a band shift (SS), thus indicating a specific interference with the Ets1 protein–DNA complex formation under both the experimental conditions. The specificity of the binding was also confirmed by the specific competition with 50-fold excess of unlabeled oligonucleotide. Thus, only in HGF-treated cells the enhanced DNA binding activity involved Ets1 subunit phosphorylation via ERK 1/2.

We studied the possible cooperation of Ets1 with NF-{kappa}B activity by transfecting the cells with ssNF{kappa}B before the 4 h HGF or hypoxia exposure (Figure 5D). NF-{kappa}B activity blockade largely prevented the formation of the complex between Ets1 and the MMP1 oligonucleotide in the cells exposed to HGF (lane 3), but was ineffective in hypoxia-treated cells (lane 7). The specificity of Ets1 binding was evaluated by preincubation with the anti-Ets1 antibody, which disrupted the labeled DNA probe–Ets1 protein complex causing a shift (SS) both using HGF- or hypoxia-treated cells (lanes 4 and 8).

DNA-binding activity of the constitutively expressed ubiquitous Octamer-1 transcription factor was unaffected by all the treatments (data not shown).

NF-{kappa}B and HIF-1 binding to specific consensus sequences in CXCR4 promoter in MCF-7 cells exposed to HGF or hypoxia
It is well known that the response element ‘p65’ in the CXCR4 promoter (~–70 bp upstream the TATA box) is the NF-{kappa}B consensus sequence most probably important for CXCR4 transcription regulation in response to cytokines (28). We synthesized this oligonucleotide called ‘p65’, which was used for EMSA analysis of nuclear extracts from HGF- or hypoxia-treated cells (Figure 6A). It is worth noting that the strongest increase in NF-{kappa}B activity (~6-fold) was observed 4 h after HGF. The NF-{kappa}B DNA binding was stimulated <2-fold at 2 and 4 h after hypoxia. These DNA probe–protein complexes were disrupted by the antibody against p65 or against p50, indicating that the transcription factor was a heterodimer p50/p65. This finding is in agreement with Helbig et al. (28) who demonstrate the direct binding of p50 and p65 subunits to the CXCR4 promoter consensus sequence. The antibodies anti-p52 and anti-RelB did not cause any supershift (data not shown). The addition of 50-fold excess of unlabeled oligonucleotide resulted in the displacement of the heterodimer binding to the labeled probe at 4 h of each treatment.


Figure 6
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  		Fig. 6 Analysis of NF-{kappa}B and HIF-1 DNA bindings in HGF- or hypoxia-treated cells. (A) EMSA of NF-{kappa}B binding to the oligonucleotide corresponding to the consensus site in CXCR4 promoter. NF-{kappa}B–DNA complex formation was competed by 50-fold excess cold oligonucleotide (comp) and supershifted by the specific anti-p50 or anti-p65 antibody. unsp, non-specific binding. The values at the bottom indicate the HGF- or hypoxia-induced variations relative to the controls, that is, starved or 10% FBS-cultured cells, calculated from densitometric analysis. (B) Northern blot and western blot analyses of total RNA and nuclear proteins extracted from HGF- or hypoxia-treated MCF-7 cells. Northern blot was hybridized with the labeled HIF-1{alpha} cDNA probe. The values at the bottom indicate the HGF- or hypoxia-induced variations relative to controls, that is, starved or 10% FBS-cultured cells, calculated from densitometric analysis. Hybridization with labeled 36B4 probe was used for normalization. Ethidium bromide-stained gel is shown. Immunoblot was performed with anti-HIF-1{alpha} antibody, and anti-B23 antibody for normalization. The experiments have been repeated three times with similar results. (C) EMSA of HIF-1 using HRE1 (one consensus site) or HRE2 (two consensus sites) labeled oligonucleotides, corresponding to the HIF-1 consensus sites in CXCR4 promoter, and nuclear proteins extracted from HGF- or hypoxia-treated cells. HIF-1–DNA complex formation was competed by 50-fold excess cold oligonucleotide (comp) and supershifted by the specific anti-HIF-1{alpha} antibody (Ab). Arrows indicate the specific HIF-1 binding. const, constitutive. Representative autoradiograms from three independent experiments.

 
Figure 6B shows the time courses of HIF-1{alpha} mRNA and protein levels in response to HGF and hypoxia. The HIF-1{alpha} steady-state transcript level peaked 4–8 h after HGF treatment (12- to 7-fold increases), and progressively declined thereafter, remaining 3-fold above starvation at 16 h. HIF-1{alpha} mRNA level was unaffected by hypoxia during the entire observation period. Western blot of nuclear proteins showed HIF-1{alpha} migrating as a series of bands from 106 to 116 kDa (21). HGF raised nuclear HIF-1{alpha} protein levels 2.9- and 2.5-fold between 4 and 6 h. After 4–6 h of hypoxia treatment, we observed a strong HIF-1{alpha} induction (15-fold).

Finally, we performed EMSA analysis of HIF-1 using two oligonucleotides corresponding to one (HRE1) or two (HRE2) HRE consensus sequences in the CXCR4 promoter (Figure 6C). Only HRE1 seemed to be functional, and DNA bindings were observed with nuclear extracts from 2 h and 4 h HGF- or 2 h hypoxia-treated cells. The HRE1 DNA probe–protein complexes were reduced by the specific antibody against HIF-1{alpha} (Ab), indicating the specific presence of this subunit in the complex. The specificity of the binding complex was verified by competition with the unlabeled oligonucleotide (50-fold molar excess).

DNA-binding activity of the constitutively expressed ubiquitous Octamer-1 transcription factor was unaffected by the treatments (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Aberrations in the reciprocal interaction of neoplastic and stromal cell types with each other through cytokines and with other components of the tumor microenvironment, such as hypoxic niches, may influence breast cancer progression. These stimuli control, for example, the expression of the chemokine receptor CXCR4, which confers invasive capacity to carcinoma cells (5,6,9,32). The experiments of the present paper were performed in MCF-7 cells, a hormone-sensitive and low-invasive cell line, which is responsive to HGF and hypoxia by inducing CXCR4. Consistent with the activation of different signaling pathways by the two studied stimuli, we demonstrate that different combinations of transcription factors were responsible for the expression of CXCR4 and for invasiveness.

In a database-assisted bioinformatics analysis of the human CXCR4 promoter, we have found multiple Ets1 binding sites within the 700 bp sequence spanning upstream the TATA box and containing also the response element for NF-{kappa}B, called ‘p65’. The latter maps to nucleotides –96 to –111 relative to the transcription start site (28,33). Here, we demonstrate that both the Ets1 dominant negative and the super-repressor of NF-{kappa}B prevented the HGF-dependent transactivation and induction of CXCR4, thus showing the principal role of these transcription factors in CXCR4 transcriptional response to HGF.

Although Ets proteins bind to DNA recognition sites bearing 5'-GGA(A/T)-3' central core, flanking sequences influence their binding specificity (15). The functionality of the consensus sequences for Ets1 and NF-{kappa}B in the CXCR4 promoter was, therefore, evaluated by performing EMSA analysis with the corresponding oligonucleotides. Remarkable DNA-binding activity of nuclear extracts from HGF-treated MCF-7 cells was observed both with EtsA and EtsB as well as with ‘p65’ oligonucleotides containing the consensus sites for Ets1 and NF-{kappa}B of the CXCR4 promoter. Activated NF-{kappa}B was a heterodimer p50/p65, which is known to be that endowed with transactivating function (29,34). As reported in literature, in MCF-7 cells p50 subunit is abundantly present in the nucleus of unstimulated cells (35), while p65 translocates from the cytosol to the nucleus after cytokine stimulation (36). DNA binding of Ets1 and NF-{kappa}B transcription factors to the respective CXCR4 promoter sequences was activated also by hypoxia, even if to a lower extent relative to HGF. So, in hypoxic cells the discrepancy occurring with the dominant negative for Ets1, which did not prevent CXCR4 induction, seemed to be only apparent.

Ets1 is involved in cell differentiation, proliferation, transformation, apoptosis, angiogenesis, motility and invasion (15,37,38). Unlike other types of transcription factors, the members of Ets family do not appear to associate as homodimers and heterodimers and by themselves may display only weak transactivation properties. Cancer signaling pathways and protein interactions control multiple Ets1 functions and may contribute to divergent Ets1 roles in various biological settings (15). However, the molecular mechanisms involved in Ets1 activation by HGF and hypoxia are not well defined.

First, several protein kinases have been demonstrated to phosphorylate Ets1 and modulate its activity. We demonstrate that HGF enhanced ERK 1/2 phosphorylation, while hypoxia was ineffective. In agreement, the blockade of MEK1/ERK1/2 transduction pathway prevented Ets1 DNA-binding activity in HGF-treated MCF-7 cells. This finding is consistent with the early critical involvement of MEK1/ERK1/2 transduction pathway in HGF-dependent CXCR4 induction (9). We did not study the role of PKC{alpha} because it is practically absent in MCF-7 cells (39). Moreover, Akt phosphorylation was very low and probably was not involved in Ets1 expression and activity. Secondly, the Ets family may form complexes with unrelated factors to initiate or enhance transcription. The blockade of the functional NF-{kappa}B transcription factor in HGF-treated MCF-7 cells by using ssNF{kappa}B strongly reduced Ets1 DNA-binding activity. Thirdly, HIF-1 activity might be involved in Ets1 expression directly in agreement with the presence of HREs in Ets1 promoter (40), and consistent with the blockade of HGF- and hypoxia-dependent Ets1 induction observed with ARNT dominant negative (data not shown). Also, Ets1 synthesis seems to depend on HGF via activation of Ras/Raf/MEK1/ERK 1/2 pathway (15). Ets1 protein level is low in MCF-7 cells (19), but was inducible by the two studied stimuli. Consistently, we report here that both HGF and hypoxia treatments of MCF-7 cells increased the expression of the {alpha}-subunit of HIF-1 and HIF-1 binding activity to HRE1 sequence present in CXCR4 promoter. HRE2 was not functional, probably being constituted by two palindromic sequences. The HIF-1{alpha} protein accumulation seemed to be dependent on different mechanisms, that is, increased transcription after HGF and possibly stabilization after hypoxia (21,41). HIF-1/Ets1 activity interaction in the transactivation of specific genes such as CXCR4 cannot be excluded.

The protein interaction at the promoter level may be influenced by the convergence of multiple intracellular and extracellular signals in conjunction with cell-type protein expression and DNA methylation state, which may vary with tumor cell progression and also environmental stimuli (12). HGF might affect DNA methylation differently from hypoxia, thus influencing the expression of specific genes in breast carcinoma cells (42). Because Ets1 binding sites close to NF-{kappa}B ‘p65’ consensus sequence might be methylated, on the basis of computer analysis of CG-dinucleotide-rich sequences, the DNA binding of the Ets1 dominant negative in the MCF-7 cells probably depends on this epigenetic regulation. Thus, we hypothesize that Ets1 blockade actually prevented CXCR4 mRNA expression in HGF-treated MCF-7 cells because DNA methylation was probably reduced. In EMSA experiments, the Ets1 binding to synthesized unmethylated oligonucleotides took place, however, after both stimuli.

Our data suggest that tumor environmental stimuli may activate different sets of transcription factors. In hypoxia-treated cells, we give direct proof that HIF-1 and NF-{kappa}B activities were involved in CXCR4 induction, consistent with the proposed mechanisms for redox-dependent and reciprocal regulation of these transcription factors (22,23). The gene reporter pCXCR4(–2632/+86) containing HRE and NF-{kappa}B consensus sites had the highest activity. In agreement, {Delta}ARNT and ssNF{kappa}B reduced CXCR4 expression in hypoxia-treated cells.

In conclusion, it is likely that the Ets1 cluster of binding sites near the NF-{kappa}B consensus sequence contributed to CXCR4 protein expression and MCF-7 cell invasiveness toward CXCL12 after HGF exposure. This region of the promoter showed the maximal luciferase activity. The Ets1/NF-{kappa}B synergy on CXCR4 promoter might be related to the positional relationship between the respective consensus sites (16). By computer analysis, we found another NF-{kappa}B consensus site in the CXCR4 promoter localized, however, >600 bp upstream the region rich in Ets1 sites.

Studies are in progress to evaluate CXCR4 expression in human breast tumors in vivo also in relationship with HGF production and Ets1/NF-{kappa}B activities. No data regarding a direct relationship are reported in literature. In VHL defective clear-renal carcinoma and hemangioblastomas, CXCR4 was studied comparatively with the expression of HIF-1{alpha} and of HIF-1 target genes (31). All these findings might be important for devising targeted therapies delivered to tumor or even better to cells of the microenvironment producing HGF. HGF-Met autocrine loops have been detected in breast carcinomas, and are often associated with malignant progression of the tumors and with poor prognosis (43). However, Met-expressing breast tumors may respond to HGF highly produced by stromal cells. It is tempting to speculate that HGF of stroma origin may play a crucial role in facilitating invasiveness and metastasis (44). One mechanism might be the activation of specific molecular targets within the adjacent carcinoma cells such as transcription factors, widely investigated for individualized cancer therapy (15,31,45–47). Modulation of tumor cytokine network could have a therapeutic potential in malignant diseases probably because it is easier to modulate the phenotype than the genotype.


   Footnotes
 
{dagger}These authors have contributed equally to this work. Back


   Acknowledgments
 
We thank Dr S. Pece of the European Institute of Oncology and the University (Milan, Italy) for fluorescence microscope analysis. The paper was supported by grants from MIUR and Ministero della Salute, Italy.

Conflict of Interest Statement: None declared.


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 Introduction
 Materials and methods
 Results
 Discussion
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Received February 23, 2006; revised May 29, 2006; accepted June 16, 2006.


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P. Bendinelli, E. Matteucci, P. Maroni, and M. A. Desiderio
NF-{kappa}B Activation, Dependent on Acetylation/Deacetylation, Contributes to HIF-1 Activity and Migration of Bone Metastatic Breast Carcinoma Cells
Mol. Cancer Res., August 1, 2009; 7(8): 1328 - 1341.
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 CarcinogenesisHome page
E. Matteucci, P. Bendinelli, and M. A. Desiderio
Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells
Carcinogenesis, June 1, 2009; 30(6): 937 - 945.
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 Cancer Res.Home page
B. Sung, S. Jhurani, K. S. Ahn, Y. Mastuo, T. Yi, S. Guha, M. Liu, and B. B. Aggarwal
Zerumbone Down-regulates Chemokine Receptor CXCR4 Expression Leading to Inhibition of CXCL12-Induced Invasion of Breast and Pancreatic Tumor Cells
Cancer Res., November 1, 2008; 68(21): 8938 - 8944.
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 Mol Cancer ResHome page
E. Matteucci, E. Ridolfi, P. Maroni, P. Bendinelli, and M. A. Desiderio
c-Src/Histone Deacetylase 3 Interaction Is Crucial for Hepatocyte Growth Factor Dependent Decrease of CXCR4 Expression in Highly Invasive Breast Tumor Cells
Mol. Cancer Res., August 1, 2007; 5(8): 833 - 845.
[Abstract] [Full Text] [PDF]

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