- Research article
- Open Access
- Open Peer Review
SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction
© Pasquier et al. 2015
- Received: 7 January 2015
- Accepted: 14 July 2015
- Published: 1 August 2015
The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites.
Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases.
We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion.
We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.
- Breast cancer
- Tumor microenvironment
- Stromal cells
Development of distant metastasis in breast cancer is responsible for the majority of cancer related deaths . Metastasis happens through highly organized and organ specific sequential steps . Among chemokines implicated in this cascade; SDF-1α/CXCR4 regulates organ specific colonization of metastatic tumor cells [3–6]. The stromal cell derived factor 1-α (SDF-1α) or CXCL-12 is physiologically expressed by mesenchymal stromal cells of metastasized breast cancer host organs such as liver, lungs, lymphatic tissues or bone marrow . CXCR4 is over-expressed in many breast cancer cells (BCC), promoting cancer cell migration and invasion . BCC differential chemokine receptor expression is correlated with their metastatic behavior . CXCR4 expression predicts bone metastasis in breast cancer patients . Two new ligands, the ubiquitin and the macrophage migration inhibitory factor were recently discovered to bind CXCR4, however their role in cancer biology has not been documented as much as SDF-1α [11–14].
Among many effects, SDF-1α/CXCR4 interaction regulates cancer cell motility and adhesion. . Muller et al. showed that CXCR4 expression on breast cancers related to their migratory/metastatic behavior. They also illustrated that the inhibition of SDF-1α/CXCR4 interaction resulted in reduced metastasis in breast cancer xenograft models . Concordantly, multiple studies showed that in different tumor types SDF-1α/CXCR4 interaction resulted in increased metastasis. SDF-1α signaling is involved in cell migratory properties, cell survival, homing and resistance to treatment [5, 17–19]. The mechanism through which SDF-1α can regulate such different proprieties as migration and adhesion (implicated in homing) is not clearly established.
It has been shown that CXCR4/SDF-1α interactions induced increased migration, proliferation and adhesion of breast cancer cells through different signaling pathways such as calcium mobilization , phosphorylation of src and fak , and phosphatidylinositol 3-kinase . In multiple melanomas, SDF-1α increases homing, adhesion and invasiveness of cancer through the activation of GTPases of the Ras superfamily, RhoA and Rac1 . Small GTPases play important roles in basic cellular processes such as cell proliferation, invasion, chemotaxis and adhesion . Rho-protein-dependent cell signaling is important for malignant transformations . RhoA activation triggers many pathways including Rho-associated protein kinase (ROCK) responsible for actin polymerization required for cell locomotion . We have previously illustrated the role of Rho GTPases modulation in different neoplasic context such as melanoma, breast and ovarian cancers [27–31].
Here, we investigated the effect of different concentrations of SDF-1α in the modulation of cancer cell migration and adhesion. We studied how the Rho GTPases mediated SDF-1α effect, by demonstrating that RhoA and Rac1 were sequentially activated at different concentration of SDF-1α, thus, promoting different metastatic properties through the modulation of cancer cells phenotype.
Breast cancer cell line MDA-MB231, MCF7, SK-BR-3, MDA-MB261, Hs578T, T47D was purchased from ATCC and cultured following ATCC recommendations (ATCC, Manassas, VA, USA). DMEM high glucose (Hyclone, Thermo Scientific), 10 % FBS (Hyclone, Thermo Scientific), 1 % Penicillin-Streptomycin-Amphotericyn B solution (Sigma), 1X Non-Essential Amino-Acid (Hyclone, Thermo Scientific) and 1 % L-glutamine. MDA-MB231 cell lines were stably transduced by lentiviral vectors encoding eGFP (Genethon, Evry). Bone Marrow host cells (BMHCs) are mesothelial cells extracted from bone marrow aspirates of donors within a bone marrow transplantation program in the Hematology Department of Hôtel-Dieu in Paris . The samples were obtained with the approval of an appropriate ethics committee and are in compliance with the Helsinki Declaration. BMHCs were maintained and expanded in culture using DMEM low glucose (Hyclone, Thermo Scientific), 30 % FBS (Hyclone, Thermo Scientific), 1 % Penicillin-Streptomycin-Amphotericyn B solution (Sigma). All cultured cells were incubated as monolayers at 37 °C under a water-saturated 95 % air-5 % CO2 atmosphere and media are renewed every 2–3 days.
Bone marrow samples were obtained from the Hematology Department of Hôtel-Dieu in Paris. All necessary ethical approval for the collection and use of the tissue samples and cell lines were obtained. The Hotel Dieu IRB is the ethics committee who approved the bone marrow samples and reviewed the project. All donors were healthy donors in a bone marrow graft program and informed consent was given. Written informed consent for participation in the study was obtained from participants or, where participants are children, a parent or guardian. All samples obtained were de-identified.
Tissue micro-array construction and immunohistochemistry
Immunohistochemistry was performed on 5-μm thick routinely processed paraffin sections. Using a tissue microarray instrument (Beecher Instruments, Alphelys™), we removed representative areas of the tumor from paraffin embedded tissue blocks. The antibodies were incubated for 30 or 60 min and then revealed by a system of polymers coupled to the peroxidase (EnVision™ kit, Dako Cytomation, Glostrup, Denmark).
Cell proliferation assay
Cells were plated at 50,000 cells per well in a 6 well plate in medium without FBS. Cells were then counted with a hemocytometer for the following six days every two days. Two wells were counted per conditions. For co-cultures, only the green cells (MDA-GFP) were counted. The experiment was performed in triplicates.
Live-cell microscopy was used to analyze co-culture of mesothelial and tumor cells. Cells were labeled with 1 mg/ml Alexa FluorW 594 conjugated wheat germ agglutinin (WGA, Invitrogen SARL, Cergy Pontoise, France) at 5 μg/ml for 10 min at 37 °C in the dark. WGA is a probe for detecting glycoconjugates, which selectively binds to N-acetylglucosamine and Nacetylneuraminic acid residues of cell membranes. Confocal microscopy was performed on fixed cells in 3.7 % formaldehyde. Cells were stained with a 50 μg/ml AF647-conjugated phalloidin (Sigma) to label actin filaments. Slides were mounted in a mounting media SlowFade® Gold Antifade Reagent with DAPI (Invitrogen). Imaging was performed using a Zeiss confocal Laser Scanning Microscope 710 (Carl Zeiss). Post-acquisition image analysis was performed with Zeiss LSM Image Browser Version 126.96.36.199 (Carl Zeiss).
Co-culture of MDA-MB231 and BMHC were established for 48 h. Cells were subsequently washed with PBS and fixed for 45 min in 30 % formaldehyde +5 % glutaraldehyde. Fixed cells were then centrifuged, treated with 50 mM ammonium chlorate, dehydrated and enveloped in Epoxy resin at low temperature at polymerization conditions. The micro sections (600–800 Au) were performed and colored with uranyl acetate and lead and visualized on a Philips CM 10 electron microscope as previously described .
Motility assay in agarose gel
Our agarose gel assay was conceived based on the publication of Mousseau et al. . First, we designed two molds using 15 ml tube lids, one with 3 lids allowing us to quantify the motility of the cells between a control wells, and a treated one and one with 5 lids for the competition experiments.
Agarose gel well formation
A 1 % solution of agarose was prepared in medium composed of 50 % phosphate-buffered saline (PBS) and 50 % DMEM (Gibco®; Invitrogen, Carlsbad, CA, USA) supplemented with 10 % heat-inactivated FBS and 2 mM L-glutamine (Invitrogen). For a 100-mm diameter Petri dish, 20 mL final agarose solution was needed. Type II agarose (Sigma-Aldrich) was added to PBS. After agarose was dissolved in PBS in a microwave oven, the solution was autoclaved and sterile DMEM was added. The agarose solution was poured into the Petri dish around the specific molds to give the well shape (Additional file 1: Figure S1). After 20–30 min of cooling, the gel was humidified with 5 mL DMEM, and the template was removed. Before performing the cell assay, 5 mL FBS-free DMEM were added to the gel for 1–6 h in order to stabilize the pH, for saturation of the gel and to prevent culture medium from diffusing in the gel during the experiment.
Chemotaxis assay and measurements during cell migration
Cells were seeded at a density of 80 000 cells per well in a complete medium with FBS. After 24 h, the medium was replaced with a starving medium with FBS. For the 3 wells experiments, the MDA-MB231 were seeded in the middle well, starving medium was poured as negative control on one side, on the other side BMHC or SDF-1α concentration tested was used. For the 5 wells experiments, MDA-MB231 were seeded on the middle well, one well was poured with starving medium as negative control and different concentrations of SDF-1α were added in the three other wells. Due to the short SDF1-α half-life, the medium was replaced every day . Image capture and measurements were performed using an AMG Evos microscope (Fisher Scientific). The number of migrating cells was evaluated by measuring the distance traveled by the cells. The starting reference point used was the beginning of the agarose wall.
Wound closure assay
Migration was assessed by wound closure assay as previously described . Cells cultured at confluence in 24-well plates were scratched with a small tip along the ruler. Cells were then cultured for 24 h in starvation media with or without SDF-1α.
For the calcein-AM assay, cells were prepared as previously described . Briefly, cells were stained with 0,25 μM of calcein-AM. After 15 min incubation at 37 °C, cells were washed twice with PBS.
Tube formation assay
A Matrigel-based capillary-genesis assay was performed on cells to assess their ability to form an organized tubular network as previously described . Cells were starved for 6 h then 100,000 cells were cultured on 250 μl of Matrigel (BD bioscience). The degree of tube formation was quantified at different time-points by measuring the intersection of tubes in five randomly chosen fields from each well using ImageJ.
Western blot analysis
Western blot were carried out as previously described . Immunostaining was carried out using a goat monoclonal antibodies against RhoA (2117), Rock2 (9029), Rac1 (2465), Cdc42 (2466), SDF-1α (3740), integrin (α4-4600; α5-4705; αV-4711; β3-4702; β4-4707; β5-4708), actin (3200) (1/1000, Cell signaling) and a secondary polyclonal mouse anti-goat antibody HRP conjugated (1/2000, cell signaling). Blots were developed using HRP and chemiluminescent peroxidase substrate (#CPS1120, Sigma). Data were collected using Geliance CCD camera (Perkin Elmer), and analyzed using ImageJ software (NIH).
Cells were treated as indicated with SDF-1α. Pulldown assays were performed according to the manufacturer's protocol (Rho activation assay kit 17–294 and Rac1 activation assay kit 17–441, both from Millipore, Billerica, MA).
Primers Sequence used for RT-PCR
siRNA against human RhoA (Santa Cruz biotechnology) were introduced into cells by lipid mediated transfection using siRNA transfection medium, reagent and duplex (Santa Cruz biotechnology) following manufacturer recommendations. Briefly the day before transfection cells were platted at 2,5 .105 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. Cells were incubated until they reach 60–80 % confluence. The duplex solution containing the siRNA is then added to the cells. After 5 to 7 h, antibiotic are added in each well and the cells are incubated for 24 h more. The media is then replaced by normal growth media and cells are used for experiments and assay by RT-PCR to analyze the expression of RhoA gene.
RNA silencing and generation of lentiviral particles
Stable lentiviral particles expressing small hairpin interfering RNAs (shRNA) targeting human Rac1 mRNA in MDA-MB231 cells were generated using cDNA lentiviral shRNA vector (MISSION® shRNA Plasmid DNA, Sigma Aldrich). The sequence was: 5′-CCGGCCTTCTTAACATCACTGTCTTCTCGAGAAGACAGTGATGTTAAGAAGGTTTTTG-3′. We used a scramble non-sense RNAi sequence with no homology in the mouse genome (shScramble) to control the unspecific effects of shRNA (Sigma Aldrich). In brief, 293 T cells were co-transfected with shRNA lentiviral plasmid or shScramble lentiviral plasmid plus the lentiviral packaging and envelope plasmids (Sigma Aldrich) using lipofectamin2000 and following manufacturer’s instructions. Medium containing generated viral particles was collected three days post transfection. Generated shRac1 lentiviral particles were used to infect MDA-MB231 cells using 4 μg/ml polybrene in order to generate stable shRac1 expressing cells. Puromycin selection (2 μg/ml) was used to select the infected cells.
adhesion assay Tissue culture plates (96-well) were pre-coated with bone marrow host cells to reach 70 % confluency or with nonspecific attachment factors (Chemicon) following manufacturers’ instructions, or with human endothelial cells. MDA-MB-231 previously transfected with eGFP were seeded at 5 *104/well in 200 ml serum-free medium, and allowed to attach for 1 h at 37 °C with BMHC. Non-adherent cells were removed by gentle washing with PBS. The adherent cells were quantified by quantifying the fluorescence at 560 nm in each well using a Wallac Flite fluorescence reader. In order to determine the role of the different GTPases in adhesion to stromal cells we used specific siRNA transfected MDA-MB-231.
All quantitative data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with SigmaPlot 11 (Systat Software Inc., Chicago, IL). A Shapiro-Wilk normality test, with a p = 0.05 rejection value, was used to test normal distribution of data prior further analysis. All pairwise multiple comparisons were performed by one way ANOVA followed by Holm-Sidak posthoc tests for data with normal distribution or by Kruskal-Wallis analysis of variance on ranks followed by Tukey posthoc tests, in case of failed normality test. Paired comparisons were performed by Student’s t-tests or by Mann–Whitney rank sum tests in case of unequal variance or failed normality test. Statistical significance was accepted for p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). All experiments were performed in triplicates.
Breast cancer cells interact with bone marrow host cells (BMHC)
SDF-1α/CXCR4 regulates adhesion to BMHCs
The specific adhesion between MDA-MB231 and BMHC was significantly reduced with the monoclonal antibody against SDF-1α or CXCR4 but not significantly increased by SDF-1α treatment, suggesting that BMHC secreted SDF-1α already induced optimal adhesion (Fig. 2g). MDA-MB361, MCF7 and T47D cell lines showed also an increased adhesion to BMHC, and the monoclonal antibody against SDF-1α was able to reduce it (Fig. 2h).
SDF-1α has a concentration dependent effect on MDA-MB231
We hypothesized that during the migratory process BCC are exposed to different concentrations of SDF-1α. Muller et al. established that the optimal migration/invasion of MDA-MB231 during SDF-1α treatment was obtained at 100 ng/ml with lower migration and invasion at low and high doses . We selected 3 different concentrations of SDF-1α, 50, 100 and 200 ng/ml and investigated the dose dependent response for adhesion, migration, invasion or proliferation of MDA-MB231 cells.
To confirm this result, we developed an agarose gel assay to test the chemotactic properties of different concentration of SDF-1α (Fig. 3e left graph, Additional file 1: Figure S2). All concentrations of SDF-1α significantly attracted MDA-MB231 cells as compared to well with only media in it. In a 4 well setting, MDA-MB231 cells were more attracted toward 100 ng/ml of SDF-1α as compared to control and 50 or 200 ng/ml (Fig. 3e right graph, Additional file 1: Figure S3).
Finally SDF-1α treatment increased the number of cells in S and G2/M at 50 and 100 ng/ml (Fig. 3f). Altogether we confirmed the previously described role of SDF-1α on breast cancer migration and invasion. However, we also illustrated that high concentration of SDF-1α does not induce similar phenotypic modulation. As we verified that CXCR4 expression was not modified by high SDF-1α concentration (receptor endocytosis or down regulation leading to loss of effect) (Additional file 1: Figure S4A), we hypothesized that different downstream effectors could play a role in mediating the concentration dependent phenotypic modulation.
SDF-1α mediated Rho GTPase and integrin regulation is concentration dependent
As the changes in expression do not necessarily correlate with activation of Rho GTPases, we confirmed increased activation of RhoA and Rac1 using a GTP pull-down assay (Additional file 1: Figure S4E). Among the mediators of migration, invasion and adhesion integrin play a major role. The shift of integrin profile has been associated to the acquisition of a metastatic phenotype . Thus we investigated their expression on MDA-MB231 after SDF-1α treatment (Fig. 4c and Additional file 1: Figure S5A). Western blot data show an up-regulation of αV, β1 and β3 protein after 4 h of stimulation with 200 ng/ml of SDF-1α. Moreover, using an inhibition strategy with monoclonal antibody, we were able to confirm the role of the αV, β1 and β3 integrin in the adhesion of MDA-MB231 (Additional file 1: Figure S5B).
A balance between RhoA and Rac1 activation mediates differential effect of SDF-1α
We demonstrated that the migration and adhesion sequences of breast cancer cells, induced by SDF-1α gradients, involves successively the activation and inactivation of RhoA and an increased expression of Rac1 through the gradient.
Krook et al. recently underlined the role of Rac1 and Cdc42 for the CXCR4 dependent metastasis of Ewing sarcoma cells to SDF-1α-rich microenvironments such as lungs and bone marrow . Cytokine mediated tumor cell migration or chemo invasion, is an important early step in cancer metastasis. Muller et al. have shown that SDF-1α was mainly produced by organs that are frequent sites of breast cancer metastasis . Experimental metastatic mouse models have shown that targeting or silencing CXCR4 inhibited development of metastasis in breast cancer [16, 46–49].
While the role of SDF-1α in the metastatic spread in solid tumors has been clearly established, its role in activation of RhoGTPases has only been described in the context of multiple myeloma where SDF-1α binding to its receptor CXCR4 induces chemotaxis and motility through RhoA activation .
However, it is essential to understand how a single cytokine can modulate apparently contradictory effects. The importance of cytokine gradients has been illustrated in the developmental context, where SDF-1α gradient is primordial during migration of the zebrafish posterior lateral line primordium . Kim et al. have investigated the role of SDF-1α gradient and their data is concordant with our findings as they demonstrated reduction of MDA-MB231 velocity at high concentration of SDF-1α (above 150nM) . Similarly, the migration of leukemic cell lines (KG-1v, KG-1a, HL-60, and leucapheresis-derived CD34+) was reduced at high concentration of SDF-1α (180 vs 60nM) .
Our main hypothesis is that breast cancer cells are not exposed to similar concentration of SDF-1α during the metastatic process. The differential tissue concentration of cytokines has been shown in different physiological and pathological contexts such as ischemia and tumor grade in glioblastoma [53, 54].
We have shown for example that endothelial cells from the bone marrow secrete a high concentration of SDF-1α as compared to endothelial cells from other organs . Such differential organ concentration can influence cancer cell plasticity. Indeed extensive work from Massague clearly demonstrates that the microenvironment of the host organ plays a role in selecting specific cancer cell clones or phenotype. Interestingly in their data and among the genes involved in Bone Marrow metastasis, CXCR4 expression was significantly increased .
SDF-1α induced-RhoGTPases activation (expression) in cancer has been previously linked to cell migration. In our settings, CXCR4 expression was not modified with low and high concentration of SDF-1α. Hence, suggesting different mechanisms for the differential regulation of RhoA and Rac1 expression. The differential regulation of RhoA and Rac1 has been previously suggested, where by the expression of dominant negative Rho family GTPases mimics activation of other member of the Rho GTPases family . Inactivation of Rac1 can result in an inversion of polarity associated to an activation of RhoA . Metastatic cells interacting with bone marrow cells display higher levels of Rac1 in vitro and in vivo [59–62].
We found that SDF-1α concentration level radically modifies the integrin expression profile, where high SDF-1α concentration increased in αV, β1 and β3. αVβ3 integrin regulates Rac1 in endothelial migration and angiogenesis . αVβ1 activates Rac1 in CHO cells and stop cell migration and increase adhesion through cell polarization . Rac1 up regulation has been associated to RhoA inhibition and linked to the modulation of the cytoskeleton .
If the clinical relevance of our findings is confirmed, then one might think that targeting RhoA could induce increased adhesion and potential homing; down-regulating the Rac1 signaling would induce increase migratory proprieties. SDF-1α blockade is currently used in hematopoietic stem cell mobilization, and is under evaluation in the treatment of leukemia and solid tumors .
We would like to thank warmly Jenine Davidson for her help with the design of the conclusion figure. We would like to appreciate greatly the help of Mariam El Bakry for the order and all her administrative work.
We thank the Flow Cytometry Facility within the Microscopy Core at Weill Cornell Medical College in Qatar for contributing to these studies. The Core is supported by the “Biomedical Research Program at Weill Cornell Medical College in Qatar”, a program funded by Qatar Foundation.
Financial support: This publication was made possible by grants from the Qatar National Research Fund under its National Priorities Research Program award number NPRP 09-1174-3-291 and NPRP 4-640-1-096. Its contents are solely the responsibility of the authors and do not necessarily represent the views of the Qatar National Research Fund.
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