Membrane cholesterol depletion reduces breast tumor cell migration by a mechanism that involves non-canonical Wnt signaling and IL-10 secretion
© The Author(s) 2016
Received: 12 January 2016
Accepted: 5 August 2016
Published: 30 August 2016
Migration and invasion are hallmarks of cancer cells that allow their dissemination to other tissues. Studying the cellular and molecular basis of cancer cell migration can help us to understand and control cancer metastasis. Many membrane molecules have been described as being involved in tumor cell migration, and among them is cholesterol.
In the present study we investigated the role of membrane cholesterol in the migration of breast tumor cells. The human mammary gland/breast epithelial cell line MDA-MB 231 was used and membrane cholesterol was depleted with methyl-β-cyclodextrin (MbCD). Cell migration was measured in a cell-based scratch assay and the involvement of the Wnt signaling was tested using Lef-1/TCF reporter activation in permanently transfected MDA-MB 231 cells. Cell morphology was analyzed using fluorescent phalloidin to label F-actin structures.
Our results show that membrane cholesterol depletion reduces cell migration, induces changes in cell morphology and in membrane protrusions. Cholesterol depletion also induces an increase in the secretion of IL-10, a cytokine involved in the control of cell migration. In addition, we show that these effects do not involve the activation of the canonical Wnt/beta-catenin pathway. Interestingly, Wnt5a inhibited cell migration in similar levels as MbCD, pointing to a role for the non-canonical Wnt pathway and IL-10 in this process.
Taken together, our results suggest a correlation between membrane cholesterol, non-canonical Wnt signaling, IL-10 secretion and breast tumor cell migration. These results could contribute to the understanding of breast tumor cell migration and may be used in the development of new therapeutic strategies targeted to breast cancer metastasis.
A breast tumor is composed of many different cells that are present in the inner and the outer region of the tumor mass. These cells are embedded in a complex extracellular matrix, which contains many soluble factors. A minority of these cells can undergo changes that enable then to migrate to a blood vessel and then to other tissues during the metastasis process. The molecules and the processes that can initiate and control tumor cell migration are poorly known. Understanding the molecular and cellular basis of breast cancer metastasis is crucial for the development of therapeutic strategies devoted to breast cancer treatment. A variety of membrane proteins and lipids have been implicated in the regulation of cancer cell adhesion and migration, and cholesterol in particular, has been related to several aspects of cancer pathology. Several studies support that cholesterol content in tumor cells is higher than in normal cells [1–5]. Transcriptional profiling by microarray has demonstrated that refractory cancers exhibit significant overexpression of a number of genes involved in the cholesterol biosynthetic pathway . Cholesterol is an integral component of cellular membranes, determines membrane rigidity and fluidity, and plays a crucial role in membrane organization, dynamics, and function . In the present study we investigated the possible role of membrane cholesterol in the regulation of tumor cell migration. We used the breast tumor cell line MDA-MB 231 to test the effects of cholesterol depletion on cell migration. To deplete cholesterol from the plasma membrane we used the substance methyl-β-cyclodextrin (MbCD), a compound that has a hydrophobic cavity with a high affinity for cholesterol [8, 9]. In the last years our group has shown that cholesterol depletion induces the release of Wnt molecules, which in turn induces the proliferation and subsequent fusion of cultured skeletal muscle cells [10, 11]. Here we show that cholesterol depletion from MDA-MB 231 cells result in a decrease in cell migration, which is accompanied by changes in cell morphology and by the secretion of IL-10. The knowledge of the role of membrane cholesterol in the cellular response to migration could contribute to our understanding of tumor cell migration and may be used in the development of new therapeutic strategies targeted to breast cancer metastasis.
This study using human cell lines was approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro. All cell culture reagents were purchased from Invitrogen (São Paulo, Brazil). The human mammary gland/breast epithelial cell line MDA-MB 231 was obtained from the American Type Culture Collection (ATCC® HTB-26™). Cells were routinely grown in RPMI medium containing 10 % fetal bovine serum (FBS), 1 % L-glutamine and 1 % penicillin-streptomycin, in a humidified 5 % CO2 atmosphere at 37 °C. Cells were cultured up to 70 %-100 % confluence and then some cultures were treated with the cholesterol depleting agent methyl-β-cyclodextrin (MbCD, Sigma, USA) at different concentrations for 2 h. After MbCD treatment, cultures were washed with fresh cultured medium (RPMI with 2 % FBS) and grown in culture medium with 2 % FBS for the next 2 to 48 h. Some cultures were grown in the presence of Wnt3a- or Wnt5a-enriched medium, which was obtained respectively from mouse Wnt3a or Wnt5a transfected L-cells. Other cells were grown in the presence of the Wnt activator 6-bromoindirubin-3'-oxime (Bio; Sigma) at a final concentration of 5 M or with the Wnt inhibitor Dkk1 (recombinant mouse Dkk1 protein; R&D Systems, USA) at a final concentration of 0.1 mg/ml.
Mouse L-cells culture and Wnt3a- and Wnt5a-conditioned medium preparation
L-Wnt3a (ATCC® CRL-2647™) and L-Wnt5a cells (ATCC® CRL-2814™) were cultured in DMEM supplemented with 10 % FBS and 0.4 mg/ml neomycin (Invitrogen) to maintain transgene expression during cell culture expansion. Conditioned medium from L-Wnt3a and from L-Wnt5a were collected as described below. Briefly, 1.3 x 106 cells were plated in 75 cm2 culture flasks with 14 ml of medium without antibiotics, and left to grow for four days. The first batch of medium was collected and replaced with 14 ml of fresh medium for another three days. The second batch of medium was then collected and the cells discarded. Both batches were mixed, sterile-filtered (0.22 μm) and stored at −20 °C for further assays.
Cell viability assay
Cell viability was determined using 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma). Briefly, cells were plated at an initial density of 2.5 x 104 cells per well in 96-well plates and incubated for 24 h at 37 °C under 5 % CO2. Twenty-four-h cultures were treated with MbCD at a final concentration of 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 2, 5 or 10 mM for 2 h. Cells treated with medium only (RPMI medium containing 10 % FBS, 1 % L-glutamine and 1 % penicillin-streptomycin) served as a negative control group. After 24 or 48 h of treatment, the supernatant of each well was removed and cells were washed twice with PBS. Then, 10 μl of MTT solution (5 mg/ml in PBS) and 100 μl of medium were then added to each well and incubated for 4 h at 37 °C, 5 % CO2. The resultant formazan crystals were dissolved in dimethyl sulfoxide (100 μl) and absorbance intensities were measured in a microplate reader (FlexStation® 3 Reader, Molecular Devices, USA) at 570 nm. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated control cells.
Trypan blue exclusion assay
MbCD treated and non-treated cells were stained with 0.4 % trypan blue, and then examined microscopically to determine the total number of cells and trypan blue positive cells. The ratio of trypan blue positive cells was calculated as following: [(the number of trypan blue positive cells)/(the number of total cells)] × 100.
Plasma membrane integrity assay
To measure plasma membrane integrity, we assayed serum lactate dehydrogenase (LDH) levels and calculated percentages of LDH release to the medium. LDH activity was measured spectrophotometrically using a commercial kit (Doles, Goiás, Brazil). After MbCD treatment, 50 μl of the supernatant were transferred to an enzymatic assay plate and 50 μL of LDH substrate plus 5 μL ferric alumem were added and incubated for 3 min at 37 °C, protected from light. Then, 10 μl of NAD were added and absorbance intensities were measured in a microplate reader (FlexStation® 3 Reader, Molecular Devices, USA) at 490 nm. The percentage of LDH release was calculated by ([LDH] sample × 100)/total [LDH]. [LDH] sample was the LDH level of the sample (released in medium) and total [LDH] was the LDH content in the wells after addition of lysis solution (0.9 % Triton X-100).
Lipid extraction and cholesterol determination
The liquid culture media from untreated and MbCD treated cell cultures were collected after 2, 5, 8, 12, and 24 h after treatment for cholesterol determination. The extraction started by adding 4 ml of chloroform:methanol:HCl (2:1:0.075, v/v) and 1 ml of HCl 0.6 M to 1 ml the samples in glass tubes, followed by agitation and centrifugation (10 min, 300 x g). The organic phase (containing the lipids) was collected and 1 ml of chloroform:methanol:HCl 0,6 M (3:48:47; v/v) was added, followed by agitation and centrifugation (10 min, 300 x g). The final organic phase from each sample was dried under N2 gas and quantified gravimetrically. Cholesterol was determined according to the method described by Courchaine et al. . Briefly, the dried lipids from each sample in duplicate were solubilized in 750 μl of acetic acid, followed by the addition of 500 μl of a color reagent (0.05 g FeCl3, 2 ml H3PO4, 23 ml H2SO4), vigorously homogenized in a vortex mixer, and kept for 10 min at room temperature. The absorbance of each sample was determined in a spectrophotometer (FlexStation® 3 Reader, Molecular Devices, USA) at 550 nm. Cholesterol (Sigma) was used as the standard. The results are expressed as mg cholesterol per ml of sample. Aliquots (10 μl) from each sample were used for protein determination by BCA reagent (Thermo Scientific, USA), using bovine serum albumin as the standard.
Cell-based scratch assay
Cells were cultured in 24-well culture plates for 24 h up to 90 %-100 % confluence. Scratched wound lines on the upside of cultured cells were created by 200 μl yellow micropipette tip. The scratched cells were washed with PBS after removal of culture media. The cells were cultured for 2 h with 2 mM MbCD and after the removal of culture media cells were cultured for the next 2, 8, 12 or 24 h. All cell-based scratch assays were performed in the presence of the anti-mitotic reagent cytosine arabinoside (ara-c; Sigma) at a final concentration of 10−5 M in order to inhibit cell proliferation and analyze only cell migration. The wound area was measured from the image taken with an Axiovert 100 microscope (Carl Zeiss, Germany) by Image J program (NIH, USA) at 3 different sites from each wound area of gaps. Three independent experiments were performed.
Cells were rinsed with PBS and fixed with 4 % paraformaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.5 % Triton-X 100 in PBS for 30 min. Cells were then washed in PBS for 30 min and incubated with Texas Red Phalloidin (Molecular Probes, USA) for 1 h at 37 °C. After incubation, cells were washed for 30 min with PBS. Nuclei were labeled with DAPI (4,6-Diamino-2-phenylindole dyhydrochloride; 0.1 μg/ml in 0.9 % NaCl) for 5 min. Cells were mounted in ProLong Gold antifade reagent (Molecular Probes) and examined with an Axiovert 100 microscope (Carl Zeiss, Germany). Images were acquired with an Olympus DP71 digital camera (Olympus, Japan).
Cell electroporation and luciferase assay
Electroporation was performed as previously described . Briefly, 2 x 106 MDA-MB 231 cells were ressuspended in 100 μl of electroporation buffer (5 mM KCl; 15 mM MgCl2; 120 mM Na2HPO4/NaH2PO4 pH 7.2; 25 mM Sodium Succinate; 25 mM Manitol) containing 4 μg of the reporter system plasmid 7xTcf-FFluc//SV40-PuroR (7TFP, Addgene plasmid 24308) to evaluate the activation of Wnt signaling  and 0.4 μg of TK-Renilla plasmid (Promega, USA). The cells were immediately transferred to a sterile 0.2 cm cuvette (Mirus Biotech®, USA) and electroporated using the program X-013 of the Lonza® Nucleofactor® II electroporation system. After electroporation, cells were plated in 12-well plates at an initial density of 105 cells/well and left to adhere overnight at 37 °C and 5 % CO2. On the following day, cultures were rinsed with PBS and incubated in triplicates with 2 mM MbCD, 10 % Wnt3a-medium or 5 μM Bio for 24 h. Cells were lysed with 80 μl of Reporter Lysis buffer/well (Promega, USA) and processed to a single freeze/thaw cycle at −80 °C to achieve complete lysis. After a brief centrifugation at 4 °C, supernatants were used to quantify luciferase activity through light detection by luminometer (Glomax, Promega, USA) after adding the Luciferase Assay Reagent according to the manufacture’s protocol.
The levels of IL-10 in cultured media were determined by enzyme-linked immunosorbent assay (ELISA) protocol as standardized by BD Biosciences (USA). Conditioned media were collected from untreated cultures and after 2, 8 and 24 h of treatment with 2 mM MbCD or 10 % Wnt3a or 10 % Wnt5a. Samples were centrifuged and the supernatants used for measurement of the concentration of IL-10. Briefly, the anti-mouse IL-10 capture monoclonal antibody (2 μg/ml, R&D Systems, USA) was absorbed on a polystyrene 96-well plate (Maxisorb, NUNC, Denmark), and the IL-10 present in the sample was bound to the antibody-coated wells. The recombinant mouse IL-10 (16 pg/ml to 0.125 pg/ml, R&D Systems, USA) was added to standard wells. The biotinylated anti-mouse IL-10 detecting mAb (0.1 μg/ml, R&D Systems, USA) was added to bind the IL-10 captured by the first antibody. After washing, streptavidin-horseradish peroxidase (streptavidin-HRP, Zymed, USA) was added to the wells to detect the biotinylated detecting antibody, and finally a Tetramethyl Benzidine chromogenic solution (TMB, Zymed) was added as a substrate, and a colored product formed in proportion to the amount of IL-10 present in the samples was stopped with HCl 1 N. Absorbance was read using 450 nm as wavelength. All samples were analyzed in duplicate. Results of cytokine concentration were expressed in pg/ml and were mean of three different experiments.
All the values were represented as the mean ± standard error. Statistical analysis was performed with one-way ANOVA on Ranks with Newman-Keuls Post Hoc test and statistical significance was defined as *p < 0.05 and ***p < 0.001.
Results and Discussion
It has been demonstrated that MbCD extracts not only cholesterol from plasma membranes, but also membrane proteins . So, we decided to analyze the amount of proteins present in the MbCD-conditioned media. We found a significant increase in the amount of proteins in the conditioned media collected 12 h after MbCD treatment (Fig. 2b), when compared to conditioned media collected from untreated cultures (control). These data suggest that MbCD induces the release and/or secretion of proteins from the cells to the culture media. These results are in agreement with our previous work showing that MbCD induces the release of proteins in cultured cells .
Since we found that treatment of MDA-MB 231 cells with 2 mM of MbCD for 2 h was able to remove high levels of cellular cholesterol without interfering with cell viability (Figs. 1 and 2), we decided to use this concentration of MbCD and duration of treatment for all the subsequent experiments in order to deplete membrane cholesterol and analyze its effects in cell migration.
In the last years our group has shown that membrane cholesterol depletion induces the release of Wnt molecules to the culture medium, which in turn induces the proliferation and differentiation of cultured skeletal muscle cells [10, 11, 17, 23]. Thus, we decided to study the possible involvement of the Wnt/beta-catenin pathway in the reduction in cell migration observed after cholesterol depletion. We analyzed the effects of Wnt3a and Bio, two activators of the Wnt pathway, and Dkk-1, an inhibitor of the Wnt pathway, in the migration of MDA-MB 231 cells. Quantification of the shaved area showed that 70 % of the scratched area was still empty after 24 h of Bio treatment, whereas cells treated with Dkk-1 covered the scratched area of the dish after 8 h of culture (Figs. 5 and 6). In addition, we found that only 30 % of the shaved area was clear of cells after 24 h of Wnt3a treatment (Fig. 6). Interestingly, when added together, Dkk-1 could rescue part of the inhibitory effects of MbCD in cell migration (Fig. 6). These results suggest that the inhibition of MDA-MB 231 cell migration caused by MbCD-induced cholesterol depletion is, at least partially, mediated by the Wnt pathway.
Our results show a minor inhibition of Wnt3a in the migration of MDA-MB 231 cells. In contrast to our data, Matsuda and colleagues  described that Wnt3a increased the migratory ability of MDA-MB 231 cells in a wound closure assay. The apparent discrepancy between our results and theirs could be attributed to their lack of use of an anti-mitotic reagent (such as cytosine arabinoside or mitomycin) to inhibit cell proliferation in the wound closure assay. Wnt3a molecules have been shown to have a proliferative role in many different cell types and tissues . Thus, the increased migratory ability of MDA-MB 231 cells after exposure to Wnt3a observed by Matsuda et al.  could be explained by an increase in cell proliferation and not cell motility.
Interestingly, quantification of the shaved area after 24 h of Wnt5a treatment showed the same extent of cell migration as obtained for MbCD (Fig. 6), suggesting that MbCD could be acting by a non-canonical Wnt pathway. The results with Wnt5a (Fig. 6) together with data showing that the canonical Wnt/beta-catenin pathway is not involved (Fig. 7), lead us to hypothesize that the non-canonical Wnt pathway could be involved in the inhibition of cell migration observed after MbCD treatment.
Wnt5a activates the beta-catenin-independent Wnt pathway, in which small G proteins, such as Rac and Rho, and protein kinases, such as protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase (CaMK), are involved . The beta-catenin-independent pathway is thought to regulate the cytoskeleton, thereby coordinating cell migration and polarity. Our data showing changes in the distribution of the actin cytoskeleton (Figs. 3 and 4), in cell polarity (Figs. 3 and 4) and in cell migration (Figs. 5 and 6) after cholesterol depletion is in agreement with the expected role of Wnt5a.
The collection of our results suggests the involvement of membrane cholesterol in the control of protein secretion that in turns regulates breast tumor cell migration. How membrane cholesterol controls protein secretion is not known. It has been shown that cholesterol depletion by MbCD facilitates both redistribution of SNAP25 (synaptosome-associated protein of 25 kDa) and sequential exocytosis . SNAP25 is a component of the SNARE complex, which is involved in membrane fusion during exocytosis. The authors hypothesized that cholesterol depletion may facilitate the diffusion of SNAP25 protein along the plasma membrane and thereby enhance the redistribution of SNAP25 into fused granules or that alternatively cholesterol depletion may alter properties of the fusion pore. Interestingly, SNAP-25 is anchored to the cytosolic face of membranes via palmitoyl side chains and many effects of palmitoylation on modified proteins are thought to reflect changes in association with cholesterol-rich membrane domains [32, 33].
We have previously shown that cholesterol depletion induces the release of Wnt molecules to the culture medium in primary cultures of myoblast cells [10, 11]. Several Wnt proteins (such as Wnt-5a) are palmitoylated and palmitoylated proteins are frequently targeted to cholesterol-enriched membrane domains. Thus, in face of our results we postulate that depletion of membrane cholesterol could alter Wnt availability and release.
Here we show for the first time a correlation between membrane cholesterol, non-canonical Wnt signaling, IL-10 secretion and breast tumor cell migration. Our results show that membrane cholesterol depletion reduces cell migration, induces changes in cell morphology and in membrane protrusions. Cholesterol depletion also induces an increase in the secretion of IL-10. In addition, we show that Wnt5a inhibited cell migration in similar levels as MbCD, pointing to a role for the non-canonical Wnt pathway and IL-10 in this process. These results could contribute to the understanding of breast tumor cell migration and may be used in the development of new therapeutic strategies targeted to breast cancer metastasis.
The authors thank Juliana Lourenço and Alan Minho for their expert technical assistance. This work was supported by Brazilian grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação do Câncer/Programa de Oncobiologia.
FSG carried out the cell cultures, immunofluorescence, ELISA and cell viability experiments. LSS carried out the cholesterol determination assays. MB carried out the electroporations. MIDR and SK participated in the luciferase reporter experiments. MLC participated in image analysis and in the interpretation of data. CM and PF conceived the study, participated in its design, in the interpretation of data, coordination of the study and in the writing of the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no conflicts of interest.
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