Cell culture, cell transfection and generation of stable cell clones
MCF7 (# HTB-22, RRID:CVCL_0031), HCC38 (# CRL-2314, RRID:CVCL_1267), BT-549 (# HTB-122, RRID:CVCL_1092) and MDA-MB-231 (# HTB-26, RRID:CVCL_0062) cells were cultured in Roswell Park Memorial Institute medium, SKBR3 (# HTB-30, RRID:CVCL_0033) cells in McCoy’s 5A medium, BT-20 (# CRL-7912, RRID:CVCL_0178) cells in Eagle’s minimal essential medium and HEK 293T (# CRL-3216, RRID:CVCL_0063) cells in Dulbecco’s modified Eagle’s medium (Lonza Group, Basel, Switzerland). All media were supplemented with 10% fetal bovine serum and 2 mM L-glutamine (complete medium) (Lonza Group). Cells were grown at 37 °C in a H2O-saturated, 5% CO2 atmosphere. Cells were either bought from or authenticated by American Type Culture Collection (Manassas, VA, USA).
Co-transfection of HEK 293T cells with plasmids encoding FLAG-tagged SGK3 constructs and pEGFP-N1 L-plastinWT was performed using Lipofectamine 2000 following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h after transfection and used for immunoblot analysis.
For immunoprecipitation experiments, HEK 293T cells were transfected with the plasmids pEGFP-N1 L-plastinWT, pEGFP-N1 L-plastinS5E, pEGFP-N1 L-plastinS5A or pEGFP-N1 L-plastinEF-ABD1 using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 h after transfection and used for immunoprecipitation.
HEK 293T cells were used for the production of lentiviral particles. Briefly, HEK 293T cells were transiently transfected with third generation lentiviral vectors using Lipofectamine 2000. The virus-containing supernatant was harvested 24 h and 48 h after medium change, cleared by centrifugation at 2000 rpm and 4 °C for 10 min, and ﬁltered through a 0.45 mm ﬁlter. Concentration of lentiviral particles was performed by precipitation with PEG10000 (1:5 volume of 40% PEG10000 solution; Merck KGaA, Darmstadt, Germany) at 4 °C overnight, followed by centrifugation at 2800 rpm and 4 °C for 30 min. The virus pellet was resuspended in serum-free medium, divided in aliquots, and stored at -80 °C. Target cells were transduced in the presence of 8 mg/ml Polybrene (hexadimethrine bromide, Merck) for 16 h. The transduced cells, positive for green ﬂuorescent protein (GFP) expression, were selected with 1 mg/ml puromycin in complete medium for 48 h.
The plasmid pEGFP-N1 L-plastinWT used for transiently transfecting HEK 293T cells was generated from the previously described plasmid pDsRed-Monomer-N1 L-plastinWT (12). Briefly, the L-plastinWT 1.9 kb cDNA fragment obtained by EcoRI/AgeI restriction of pDsRed-Monomer-N1 L-plastinWT was inserted into the EcoRI/AgeI restricted pEGFP-N1 vector. The plasmid pEGFP-N1 L-plastinEF-ABD1 was generated by PCR amplification using the plasmid pEGFP-N1 L-plastinWT as a template and using primers that were designed to generate the restriction sites EcoRI and BamHI necessary for cloning the PCR-amplified cDNA into the pEGFP-N1 vector. The following primers were used: 5’-TATAGAATTCatggccagaggatc-3’ as forward primer and 5’- GCGGATCCGCTTTGTGCAGGGC-3’ as reverse complement primer. Lentiviral transduction was performed using third generation lentiviral vectors. The packaging vector psPAX2 and the envelope vector pMD2.G were obtained from Addgene (LGC Standards, Middlesex, United Kingdom). The transfer vector CD527A-1 carried the cDNAs corresponding to GFP, L-plastinWT-GFP, non-phosphorylatable L-plastinS5A-GFP or phosphomimetic L-plastinS5E-GFP. Briefly, the cDNA fragments were obtained by PCR amplification using the respective pEGFP-N1 plasmids as templates and using primers that were designed to generate the requested L-plastin mutation as well as the restriction sites necessary for cloning the PCR-amplified cDNAs into the CD527A-1 vector. For all cDNAs, XbaI and BamHI restriction sites were generated at the 5’- and 3’-ends, respectively. The following primers were used: 5’-TACTTCTAGAATGGCCAGAGGATCAGTGTC-3’ as forward primer for L-plastinWT-GFP, 5’-TACTTCTAGAATGGCCAGAGGAGCAGT-3’ as forward primer for L-plastinS5A-GFP, 5’-TACTTCTAGAATGGCCAGAGGAGAAGTGTC-3’ as forward primer for L-plastinS5E-GFP, 5’-TACTTCTAGAATGGTGAGCAAGGGCGA-3’ as forward primer for GFP and finally 5’-AGTAGGATCCCTTGTACAGCTCGTCCATGC-3’ as reverse complement primer for all constructs. All constructs were verified by sequencing. The GIPZ short hairpin RNA (shRNA) non-silencing lentiviral vector as well as the target shRNAs for L-plastin (GIPZ Lentiviral shRNA Library, pool of clones V2LHS_133928, V2LHS_133929, V2LHS_238253, V2LHS_311716 and V2LHS_311717) were purchased from GE Dharmacon (Diegem, Belgium). The FLAG-tagged SGK3 plasmids were a kind gift of Professor Dan Liu (Baylor College of Medicine, Houston, TX, US) (characterized in (22)).
Antibodies and reagents
Antibodies mouse anti-Src (L4A1, #2110, RRID:AB_10691385), rabbit anti-EGFR (#2232, RRID:AB_331707), rabbit anti-IGF-IRβ (D23H3, #9750, RRID:AB_10950969), rabbit anti-phosphoSrc (pTyr416, #2101, RRID:AB_331697), rabbit anti-phosphoAKT (pSer473, D9E, #4060, RRID:AB_2315049) and rabbit anti-phosphoSGK3 (pThr320, D30E6, #5642, RRID:AB_10694357) were from Cell Signaling Technology (Danvers, MA, USA), goat anti-AKT (N-19, #sc-1619, RRID:AB_671713), mouse anti-phosphoERK (pTyr204, E-4, #sc-7383, RRID:AB_627545), mouse anti-SGK3 (C-6, #sc-166847, RRID:AB_2188556) and rabbit anti-cortactin (H-191, #sc-11408, RRID:AB_2088281) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), rabbit anti-ERK (#M5670, RRID:AB_477216), mouse anti-cortactin (4F11, #05-180, RRID:AB_309647) and mouse anti-β-actin (#A5441, RRID:AB_476744) from Merck, rabbit anti-HGFR (22H22L13, #700261, RRID:AB_2532310) and mouse anti-L-plastin (LPL4A.1, #MA5-11921, RRID:AB_10979969) from Thermo Fisher Scientiﬁc (Waltham, MA, USA). The GFP-Trap_A antibody used for the nanotrap assays (#gta-20, RRID:AB_2631357) was from Chromotek (Planegg, Germany). The rabbit antibody specifically recognizing L-plastin phosphorylated on Ser5 (anti-Ser5-P) was raised against a peptide encoding L-plastin residues 2–17 where Ser5 was phosphorylated [ARGS(P)VSDEEMMELREA] (characterized in (11)).
The stimulators phorbol 12-myristate 13-acetate (PMA), human epidermal growth factor (EGF), human hepatocyte growth factor (HGF) and human insulin-like growth factor 1 (IGF) were purchased at Merck. The inhibitors FAK inhibitor II and RSK inhibitor II (Bi-D1870) were from Merck and AKT inhibitor VIII was from VWR (Oud-Heverlee, Belgium). The MEK inhibitor Trametinib and the dual PI3K/mTOR inhibitor Apitolisib were purchased at CliniSciences (Nanterre, France). Human tissue microarrays (TMAs) were purchased at AMS Biotechnology (Cambridge, MA, USA).
Treatment of cells with pharmacologic agents
Cells were cultured in the absence of serum for 16 h and then treatment was performed at 37 °C as follows: 0.1 mM PMA for 1 h, 1 ng/ml EGF for 15 min, 40 ng/ml HGF for 20 min, 100 ng/ml IGF for 20 min, 20 mM AKT inhibitor VIII for 1 h, 5 mM FAK inhibitor II for 1 h, 5 mM RSK inhibitor Bi-D1870 for 30 min, 5 nM Trametinib (Mekinist) for 1 h or 500 nM Apitolisib for 1 h. When activators and inhibitors were combined, the incubation with the inhibitors was performed first and their presence was maintained during the incubation with the activators.
In situ cell lysis was performed with a cell scraper in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 5 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 1% sodium-deoxycholate, 1 mM Na3VO4, 10 mM NaF, 100 mM leupeptin, and 100 mM E64D) containing a cocktail of protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Lysate clarification was done by centrifugation at 13200 rpm for 15 min at 4 °C and total protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Proteins (50 µg per lane) were resolved by SDS-PAGE in a 10% NuPAGE Tris-Base gel (Invitrogen) under reducing conditions, and transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) by semidry transfer. Membranes were saturated in Tris-buffered saline containing 1% bovine serum albumin and 0.1% Tween for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C and with secondary antibodies IRDye 680 RD donkey anti-mouse (#926-68072, RRID:AB_10953628, Thermo Fisher Scientiﬁc) and IRDye 800 CW goat anti-rabbit (#926-32211, RRID:AB_621843, Thermo Fisher Scientiﬁc) for 1 h at room temperature. Each antibody incubation was followed by at least three wash steps in Tris-buffered saline supplemented with 0.1% Tween. Signal intensities were quantified using the Odyssey Infrared Image System (LI-COR Biosciences, Lincoln, NE, USA). The ratio between the intensities obtained for phosphorylated protein versus total protein was calculated to make individual samples comparable and then normalized to the mean of all the ratios calculated for one blot to make blots comparable by accounting for technical day-to-day variability. For representative purposes, data were scaled to the controls present on each blot and are represented as means +/- SEM of three independent experiments. Raw images of the immunoblots are shown in the Supplementary Figure S1.
The candidate signalling network upstream of L-plastin was derived from the literature. The experimental data were obtained by immunoblot analysis as described above and the ratios of P-LPL/LPL, P-ERK/ERK, P-AKT/AKT and P-Src/Src were used for model contextualization as follows. Within the FALCON toolbox, Dynamic Bayesian Networks are used to quantitatively simulate the logic of signalling pathways (23). Briefly, networks are initialized in a random state and the activity of ‘input nodes’ is fixed according to the experimental conditions (presence or absence of growth factors and inhibitors). The signals are then propagated according to the laws of probability until convergence, when the activities of the ‘output nodes’ are compared with the measurements. A gradient descent algorithm is used to optimize the weights of the edges controlling the relative contributions of upstream nodes to downstream nodes in order to minimize the mean squared error (MSE) between the simulations and the measurements.
Regularized optimization was then used to put in evidence the specific differences in signalling between the cell lines. Two types of regularization were applied to the parameter space during joint optimization of the individual cell line-specific models. Firstly, we sought to decrease the influence of experimental noise on the results by including a group partial-norm term penalizing the concurrent activation of a node by more than one activator. The effect of such regularization is to prune the network of edges that are not well supported by experimental evidence. Secondly, uniformity regularization (24) was applied across the four cell lines for each parameter. This density-based regularization term stems from the biological assumption that differences between the cell lines are more likely due to a small number of differences than to large-scale rewiring, and its effect is to remove small differences between cell line-specific models unless they are well supported by the data. The combined effect of these two regularization terms is to reduce the size of the model and point to the signalling pathways that are differentially activated among the cell lines.
Regularized optimization with the FALCON toolbox was performed on the full dataset, after which the optimal model size was determined using the Bayesian Information Criterion (25) and the topology of the final multi-cell line model was fixed by removing edges with low (<0.01) flux and merging similar (<0.01 standard deviation) edges. This final model was re-optimized on the full dataset, using unregularized optimization, to retrieve unbiased estimations for the activity of the different signalling proteins and the strength of the interactions between them. To estimate the error on the parameters, we optimized 20 models with synthetic datasets by applying random Gaussian noise on the measurements proportionally to the measurement error.
Files containing the data used for the modelling can be found in the Supplementary Figures S3 - S14.
In vitro kinase assays of full-length recombinant L-plastin
The in vitro kinase assay was carried out as described before (Lommel, 2016). Briefly, full-length recombinant L-plastin (10 mg) was incubated with 50 mM ATP and 100 ng recombinant kinase SGK1, SGK2, SGK3 or RSK1 purchased at SignalChem (Richmond, BC, Canada) in a reaction volume of 25 ml, according to the manufacturer’s protocol. For the negative control, the respective kinase was omitted. The reaction with RSK1 was performed as a positive control. Following an incubation of 15 min at 30 °C, Laemmli buffer was added, and the samples were boiled at 100 °C for 5 min and then subjected to immunoblot analysis.
For immunoprecipitation, 6 x 106 HEK 293T cells were transiently transfected with expression vectors encoding GFP, L-plastinWT-GFP, L-plastinS5A-GFP, L-plastinS5E-GFP or L-plastinEF-ABD1-GFP. 24 h after transfection, cells were homogenized in 500 µl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% Triton, 1% glycerin, 1 mM PMSF, 1 mM sodium orthovanadate) containing a cocktail of protease inhibitors (Roche Diagnostics) and incubated on ice for 30 min. After a centrifugation step at 13200 rpm and 4 °C for 10 min, total protein concentration was determined by Bradford assay (Bio-Rad) and sample concentrations were adjusted with dilution buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, cocktail of protease inhibitors). 50 µl were added to SDS-containing sample buffer and used for SDS-PAGE (referred to as input). 25 µl of GFP-nanotrap beads (#gta-20, RRID:AB_2631357, Chromotek, Planegg, Germany) were added and incubated for 2 h on an end-over-end rotor at 4 °C. After a centrifugation step of 5 min at 3000 rpm at 4 °C, the supernatant was removed, and 50 µl of the supernatant were used for SDS-PAGE (referred to as non-bound). The bead pellet was washed four times with 300 µl dilution buffer. After the last washing step, the beads were resuspended in 2x SDS-containing sample buffer and boiled for 10 min at 95 °C (referred to as bound). The obtained samples were submitted to immunoblot analysis.
Transwell migration and invasion assays
For the transwell assays, cells were washed in phosphate-buffered saline (PBS) and resuspended in serum-free medium. A cell suspension containing 50000 cells was added to the upper well of transwell migration inserts (pore size: 8 μm, BD Biosciences, San Jose, CA, USA) or 100000 cells to BD BioCoatTM MatrigelTM invasion chambers (pore size: 8 μm, BD Biosciences). In the lower well, complete medium (700 μl) was used as chemoattractant. Cells were incubated for 24 h at 37 °C and 5% CO2, fixed in 4% PFA for 10 min and stained with DAPI for 10 min. Cells that did not migrate to the lower compartment were removed with a cotton swab. Inserts were mounted on glass slides and five random fields at a magnification of 20x were counted per sample.
Cells were plated on 0.1% gelatin-coated glass coverslips. Following incubation, cells were washed with PHEM buffer (2 mM HEPES, 10 mM EGTA, 2 mM MgCl2, 60 mM PIPES, pH 6.9) and fixed for 20 min with cold PFA 4%. Next, cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin in PHEM buffer for 30 min, and then incubated with mouse anti-cortactin (1:200, #05-180, RRID:AB_309647, Merck) and rabbit anti-Ser5-P-L-plastin (1:50) at 4 ºC overnight, followed by incubation with Alexa Fluor 405-conjugated goat anti-mouse IgG (1:250, #A31553, RRID:AB_221604, Thermo Fisher Scientific), Alexa Fluor 488-conjugated GFP booster (1:200, #gb2AF488-10, RRID:AB_2827573, Chromotek, Planegg, Germany), Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:250, #A11037, RRID:AB_2534095, Thermo Fisher Scientific) and Alexa Fluor 633-conjugated phalloidin (1:50, #A22284, Thermo Fisher Scientific) or Alexa Fluor 568-conjugated phalloidin (1:50, #12380, Thermo Fisher Scientific) at room temperature for 1 h. Coverslips were mounted using Vectashield Anti-fade Mounting Medium (#H-1000, RRID:AB_2336789, Vector Laboratories, San Francisco, CA, USA) and image acquisition was performed using the Andor Spinning Disk Revolution system (CSU-W1; Andor Technology, Belfast, United Kingdom) based on a Nikon Ti microscope (Nikon, Tokyo, Japan) with an Andor iXon Ultra EMCCD camera.
To quantify invadopodia formation, MDA-MB-231 cells were plated at low density on top of 0.1% gelatin-coated coverslips and cultured for 24 h. All samples from the same replicate were stained simultaneously as described above. Four random fields at a magnification of 40x were counted per sample using single confocal slices of the ventral surface of the cells. Image analysis was performed using ImageJ software (RRID:SCR_003070, National Institutes of Health, Bethesda, MD, USA). Firstly, the threshold “moments” was applied to the images of cells stained for F-actin and cortactin. To identify invadopodia, the tool “image calculator” was used to show dot-like structures that were present in both images. The GFP-positive invadopodia were determined in the same way using the result image obtained from the calculation of F-actin and cortactin, which was then compared with the GFP signal. Particle frequency was determined using the “analyze particle” command. A cut-off of 0.5-20 µm2 was set as the size range and a value of 0.2 as the minimal circularity shape.
Gelatin degradation assay
The gelatin degradation assay was adapted from a previously described protocol (26). Firstly, 0.2% gelatin solution (#9000-70-8, Merck) was labeled using the Alexa Fluor 568-gelatin labeling kit (#A10238, Thermo Fisher Scientific) and aliquots were stored at -20 ºC. To coat glass coverslips, the fluorescent gelatin stock was mixed in a proportion 4:1 with non-labeled 0.2% gelatin solution and kept at 50 ºC. A volume of 100 µl of this mixture was given on top of each coverslip and incubated for 5 min. The coverslips were lifted and submerged in PBS in separate wells in a 12-well cell culture plate. When all coverslips were coated, PBS was aspirated and coverslips were incubated for 15 min on ice with pre-chilled 0.5% glutaraldehyde solution. After washing, the coverslips were incubated for 3 min at room temperature with freshly prepared sodium borohydride solution (5 mg/ml). Finally, the coverslips were extensively washed and stored at 4 ºC in PBS for up to two weeks, protected from light.
To quantify the gelatin degradation ability, 80000 MDA-MB-231 cells were plated on top of Alexa Fluor 568-labeled gelatin-coated coverslips in 12-well cell culture plates and allowed to attach for 6 h. Coverslips were then submitted to immunofluorescence and six random fields at a magnification of 60x were examined per sample using single confocal slices of the ventral surface of the cells. The cell area was determined by the F-actin staining, using the “ROI manager” tool of ImageJ software. To determine degraded area, a threshold was applied to make visible the dark areas of degraded fluorescent gelatin and quantification was performed using the “analyze particle” command. Relative degradation area was determined as total degradation area divided by total cell area, normalized to the value obtained for MDA-MB-231 GFP control cells.
To analyze the activity of matrix metalloproteinases (MMPs), cells were cultured in complete medium until 70-80% confluence. Cells were then washed with PBS and cultured in serum-free medium for 24 h. The conditioned medium was collected, cleared by centrifugation and stored at -80 ºC. Zymography acrylamide gels (10%) were prepared according to standard procedures with gelatin added to a final gelatin concentration of 1 mg/ml. The cell-free supernatant was mixed with 5x non-reducing sample buffer, incubated at room temperature for 10 min, and a volume of 25 µl of the mixture was loaded on the gels. After electrophoresis, the gels were incubated in washing buffer (50 mM Tris-HCl pH 7.5, 5 mM CaCl2, 1 µM ZnCl2, 2.5% Triton X-100) for 30 min. Finally, the gels were kept at 37 ºC with gentle agitation in incubation buffer (50 mM Tris-HCl pH 7.5, 5 mM CaCl2, 1 µM ZnCl2, 1% Triton X-100) for at least 24 h. Gelatinase activity was visualized by staining the gels with Coomassie Brilliant Blue G250 (Merck) with subsequent destaining in acetic acid-methanol-H2O (1:3:6). To visualize the amount of protein loaded, a parallel 10% polyacrylamide gel was loaded with the same volume of each sample and stained with Roti-blue (Carl Roth, Karlsruhe, Germany) for 1 h. Areas of protease activity and Roti-blue stained gels were analyzed using the Odyssey Infrared Image System (LI-COR Biosciences).
All statistical analyses were carried out using Prism 5 (GraphPad Software, RRID:SCR_002798, San Diego, CA, USA). Results are expressed as means +/- SEM of three independent experiments. Statistical significance was assessed by performing unpaired Student’s t-test or ANOVA for multiple comparison tests.