Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4/CXCL12 axis

MCF-7 human breast adenocarcinoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Sigma-Aldrich); H460 human non-small lung cancer cell (ATCC) were cultured in RPMI 1640 (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Sigma-Aldrich). The two cell lines were maintained at 37 °C in a humidified 5% CO2 atmosphere. HEK-293 T cells (Sigma-Aldrich) were cultured in adherent conditions in DMEM (Sigma-Aldrich) medium with 10% FBS and 1% Penicillin-Streptomycin at 37 °C in a humidified 5% CO2 atmosphere.

Lentiviral preparation and transduction were performed as described in Di Sanzo et al. [29]. Cells were stably transduced with a lentiviral DNA containing either an shRNA that targets the 196–210 region of the FHC mRNA (sh29432) (MCF-7^shFHC) (H460^shFHC) or a control shRNA without significant homology to known human mRNAs (MCF-7^shRNA) (H460^shRNA). A pool of clones were analysed after puromycin selection.

MCF-7^shFHC cells were seeded in six-well plates at 6 × 10⁵cells/well and grown overnight prior to transfection. All plasmids were transfected with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions.

FHC reconstitution was performed using 2 μg/μl of the expression vector containing the full length of human FHC cDNA (pcDNA₃/FHC) (MCF-7^shFHC/pcDNA ₃ ^FHC) while 2 μg/μl of pcDNA₃ plasmid was used as negative control (MCF-7^shFHC/pcDNA ₃). Transfection efficiency was tested 48 h. All transfection experiments were repeated in triplicate.

Total RNA was extracted from FHC-silenced MCF-7 and H460 (MCF-7^shFHC, H460^shFHC) and scrambled-shRNA MCF-7 and H460 (MCF-7^shRNA, H460^shRNA) by using Trizol method according to the manufacturer’s instructions (Thermo Fisher Scientific). All the RNA samples were DNase-1 treated (Ambion, Austin, TX, USA), and purity and integrity of the RNA was checked spectroscopically and by gel electrophoresis before use. Then, 1 μg of purified RNA was reverse-transcribed by using High capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) [30].

MCF-7 and H460 silenced and un-silenced cells were lysed in the following buffer (20 mM Hepes pH 7.9, 420 mM NaCl, 1% Triton ×-100, 1 mM EDTA, 25% glycerol, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM DTT, 1 μg/ml aprotinin, 1 μg/ml leupeptin) for 30 min on ice. After removing cell debris by centrifugation (12,000 g × 30 min), the concentration of protein extracts was measured by the Bio-Rad protein assay according to the manufacturer’s instructions (BioRad, California, USA). Total protein extract was boiled for 10 min in SDS sample buffer, separated by 10–12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Non-specific reactivity was blocked in nonfat dry milk in TPBS [5% (w/v) milk in PBS (pH 7.4) and 0.005% Tween 20] for 2 h at room temperature. The membrane was incubated with rabbit anti-H ferritin (1:200; Santa Cruz Biotechnology, Texas, USA), rabbit anti-CXCR4 antibody (1:500; Abcam, Cambridge, UK), rabbit anti-VIMENTIN, anti-E-CADHERIN antibodies, anti-phospho-AKT (Ser473), anti-AKT, anti-P70S6 K, anti-phospho-P70S6 K (T389) (1:1000; Cell Signaling Technology, Danvers, MA, USA) over-night at 4 °C, followed by incubation with goat anti-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology). The membrane was developed by ECL-Western blot detection reagents according to the manufacturer’s instructions (Santa Cruz Biotechnology). γ-Tubulin was used as a loading control.

Gene expression analysis was assessed by real-time PCR using the cDNA obtained from MCF-7^shRNA, MCF-7^shFHC, MCF-7^shFHC/pcDNA ₃ ^FHC and from H460^shRNA and H460^shFHC cells.

Primer sequences used for amplifications were as follows:

GAPDH-F 5′-aac acc acc atg gag aag gc −3′; GAPDH-R 5′-aca gcc ttg gca gca cca ct-3′;

FHC FW: 5′-cat caa ccg cca gat caa c-3′; FHC REV: 5′-gat ggc ttt cac ctg ctc at-3′.

Twist-F 5′-tga atc ttg ctc agc ttg tc-3′; Twist-R 5′-cgg gcg tcc gga gtc tta-3′;

Slug-F 5′-ggt gtc aga tgg agg agg g-3′; Slug-R 5′-cat gcc tgt cat acc aca ac-3′;

Vim-F 5′-agg aaa tgg ctc gtc acc ttc gtg-3′ Vim-R 5′-gga gtg tcg gtt gtt aag aac tag-3′;

E-cadherin-F 5′-tac gcc ggg act cca cct a-3′; E-cadherin-R 5′-cca gaa agc gag gcc tga t-3′.

20 ng of cDNA was amplified in 20 μl of reaction mix containing Power SYBR Green PCR Master mix (Thermo Fisher Scientific), 20 pmol of each primer pair and nuclease-free water. The thermal profile consisted of 1 cycle at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min. The human GAPDH cDNA fragment was amplified as the internal control. Data analysis was performed using the 2^-ΔΔCt [31].

MCF-7 and H460 cells were cultured on cover slip. After two wash in PBS, cells were fixed with 4% paraformaldehyde (PFA) (Sigma Aldrich) in Sodium Posphate solution 0.120 M pH = 7.4, for 20 min at 37 °C and then washed in PBS. Cells were washed twice with high salt solution (HS: NaCl 500 mM, Na₃PO₄ pH = 7.4 20 mM) and then permeabilized in HS buffer containing Triton-X-100 0.3% and jelly at 0.2% for 30 min at room temperature in a humidified room. To stain actin filaments, cells were incubated for 30 min in this buffer containing Oregon Green® 488 phalloidin at 1:400 dilution (Molecular Probes, Thermo Fisher Scientific), while primary antibodies (ferritin heavy chain, 1:100 Santa Cruz Biotechnology; Vimentin and E-Cadherin, 1:100, Cell Signaling) were diluted in the same buffer for 2 h at room temperature. Appropriate secondary antibodies (antimouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 555, Thermo Fisher Scientific) diluted in HS buffer were applied for 1 h at room temperature. After 3 washes with HS buffer and 1 wash with PBS, Nuclear Dapi (1:1000, Invitrogen, Carlsbad, CA) was added for 20 min (ProLong Gold antifade Reagent, Molecular Probes, Eugene, OR). The slides were mounted on microscope slides using a mounting solution ProLong Gold antifade reagent (Thermo Fisher Scientific). Images were collected using a Leica DM-IRB/TC-SP2 confocal microscopy system (40×, 63× or 100× objective) at 1024 × 1024 resolution pixel.

ROS were determined by incubating MCF-7 and H460 silenced and unsilenced cells with the redox-sensitive probe 2′-7′-DCF (CM-H2CFDA; Molecular Probes, Eugene, OR) [32]. Briefly, 1 × 10⁶ cells were plated in 96-well plates and incubated with Hanks balanced saline solution (HBSS), 10 mM glucose and 20 μm DCF for 15 min at 37 °C. After two cycle washes, cells were maintained in HBSS supplemented with 10 mM glucose. Fluorescence was measured using the FACS FORTESSA.

Cell viability was tested using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) assay. For the assay, 3 × 10³ FHC-silenced and unsilenced MCF-7 and H460 cells were plated in 96-well flat bottom tissue culture plate. At 24, 48 and 72 h of culture, 10 μl of MTT solution (2 mg/mL) were added per well. After 6 h of incubation crystals of formazan were solubilized with 50 μl of 2-Propanol (Sigma-Aldrich). Optical density (O.D.) was read on a multiwell scanning spectrophotometer (ELISA reader) at 450 and 595 nm.

To assess cell proliferation MCF-7 and H460 cells were plated in DMEM and RPMI complete media. Cell counts using trypan blue were performed at 0, 24, 48 and 72 h.

MCF-7 and H460 cells (1 × 10⁵) were seeded in 10 ml of DMEM (MCF-7) or RPMI 1640 (H460) (Sigma-Aldrich), supplemented with FBS and Penicillin-Streptomycin (Sigma-Aldrich) in 100 mm plates. After 24, 48, 72 and 96 h, 500 μl of supernatant were taken and glucose and lactate concentration was measured using COBAS 6000 instrument (Roche, Indianapolis, IN, USA). The assay was performed three times.

MCF-7 and H460 cells (1 × 10⁶) were harvested and rinsed twice, and 1% bovine serum albumin (BSA) in PBS solution was used to block the cells for 30 min in an ice bath. Then cells were stained with anti-CXCR4 PE-antibody (FAB170P, clone 12G5, R&D Systems, Minneapolis, MN, USA) for 1 h at 4 °C. After antibody staining, cells were rinsed with 1% BSA in PBS three times, resuspended in PBS, and evaluated by a FACS Canto II cytofluorometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA).

Migration was assayed in 24 transwell chambers (Corning Inc., Corning, NY, USA) using inserts with 8-μm pore membrane. MCF-7 and H460 cells were placed in the upper chamber (2 × 10⁵cells/well) in DMEM containing 0.5% BSA (migration media) plus/minus AMD3100. CXCL12 (100 ng/mL) was added to the lower chamber. After 18 h of incubation, cells on the upper surface of the filter were removed using a cotton wool swab; the cells that had migrated onto the lower surface of the membrane were stained with DAPI, photographed and visually counted in 10 random fields. Migration index is the ratio between number of migrated cells / number of migrating cells toward CXCL12 free media [33].

MCF-7^shRNA and MCF-7^shFHC cells were pre-incubated for 30 min at 37 °C with AMD3100 (10 μM). Subsequently forskolin (1 μM) for 20 min was added and stimulation with CXCL12 (100 ng/ml) for 10 min was done. Controls include cells stimulated with CXCL12 and forskolin, or forskolin alone in absence of anti-CXCR4 inhibitors. Then the cells were harvested and lysed with 0.1 M HCl and cAMP levels was assayed using a direct competitive enzyme immunoassay (BioVision, Milpitas, CA, USA).

Data are expressed as means ± SD of at least three independent experiments conducted in triplicates as indicated in the text and in the figure legends. Statistical significance was evaluated by t-test or Two-way ANOVA as indicated in the figure legends. Statistical significance was indicated as follows: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****).

We previously demonstrated that FHC intracellular amounts may regulate the expression of a number of miRNAs and EMT-related genes (miR-125b, Vimentin, and SPARC) in different cell types [17, 19, 34]. In this work, we evaluated the effects of FHC silencing on EMT in MCF-7 human breast cancer cells and in H460 human lung cancer cells. MCF-7 cells were stably transduced with a lentiviral DNA containing a Hferritin specific shRNA (MCF-7^shFHC) or with an shRNA without significant homology to known human mRNAs (MCF-7^shRNA). FHC mRNA and protein levels were measured in pools of silenced clones, as illustrated in Panel a of Fig. 1. Panel b shows a representative immunofluorescence for H ferritin subunit in MCF-7^shFHC and MCF-7^shRNA cells.

FHC silencing produced dramatic morphological changes in MCF-7 cells. Indeed, phase-contrast microscope analysis revealed that the MCF-7^shFHC cells underwent a consistent modification, passing from a cobblestone-like morphology to a spindle-shaped and fusiform features (Panel C of Fig. 1), as well as cytoskeleton disarray, shown by staining of actin filaments with phalloidin (Panel D of Fig. 1). To corroborate the morphological data, we analysed the expression of several EMT markers in the MCF-7^shFHC and MCF-7^shRNA cells. Western blot, immunofluorescence and qPCR, reported in Fig. 2, show that FHC-silencing is accompanied by a decreased expression of E-cadherin, by an increase of Vimentin and by an up-regulation of EMT-related transcription factors Twist, Slug and ZEB1 [35‐37].

To strengthen the relationship between FHC down-regulation and EMT induction, we rescued FHC expression in MCF-7^shFHC cells (Fig. 3, Panel A), and re-evaluated the EMT markers. As shown in Panel B of Fig. 3, the expression of Vimentin, Twist and Slug was consistently reduced upon FHC reconstitution, while E-Cadherin levels were unaffected. We believe that this last phenomenon may be determined by the fact that the partial rescue of Twist and Slug in transiently FHC-reconstituted MCF-7 cells is still able to efficiently repress the expression of E-Cadherin. This hypothesis is confirmed by several published data reporting that the transcriptional regulation is indeed the major mechanism controlling E-Cadherin expression [35, 36].

Epithelial cells undergoing EMT lose cell polarity and cell-to-cell adhesion capability, acquiring, in turn, enhanced migratory and invasive properties [37]. To further investigate the effects of FHC-silencing, we performed a wound healing assay on MCF-7^shFHC and control cells. The results of a triplicate set of independent assays (Panel A of Fig. 4) demonstrate that the MCF-7^shFHC cells possess, at 48 h, an enhanced migratory capability consistent with their EMT phenotype.

Next, we evaluated by methyl-thiazolyl-tetrazolium (MTT) assay the viability of FHC-silenced cells at 24, 48 and 72 h. The results, shown in Panel B of Fig. 4, indicate that FHC silencing increased cell proliferation with a maximum of about 1.8-fold at 72 h. Moreover, as further feature of cellular aggressiveness, the glucose/lactate levels were quantified over time in the MCF7^shFHC versus MCF7^shRNA cells. A time-dependent decrease in glucose concentration, paralleled by an increase in lactate levels were detected in the culture medium of MCF-7^shFHC, indicating that the MCF-7^shFHC cells are indeed consuming higher amounts of glucose for energy production (Panel C of Fig. 4).

Down-regulation of ferritin, and particularly of its heavy subunit, determines an increase of the intracellular Labile Iron Pool (LIP) [38‐41]. The free iron, in turn, induces the formation of Reactive Oxygen Species (ROS), which are implicated in EMT induction in different cell types [10, 42, 43]. Panel A of Fig. 5 shows that, in MCF-7^shFHC cells, ROS amount is about five-fold greater than that in MCF-7^shRNA cells. To investigate to what extent increased ROS are related to EMT in our experimental model, we treated MCF-7^shFHC cells with the ROS scavenger N-acetylcysteine (NAC), and analyzed the levels of the EMT markers. NAC treatment partially reversed the expression of Twist (about 25%), Slug and Vimentin (about 40%), while it left unaffected the amounts of E-cadherin (Panels B and C of Fig. 5). The addition of NAC to the culture medium of MCF-7^shFHC cells did not substantially modify their migration capability (Panel D of Fig. 5), while, on the contrary, it significantly reduced their proliferation rate (Panel E of Fig. 5). Moreover, the spindle-shaped morphology of the silenced cells remained substantially unaltered upon NAC addition (Panel F of Fig. 5).

It has been shown that the binding of the chemokine CXCL12 to its CXCR4 receptor induces EMT in several cell types including MCF-7 cells [26‐28, 44]. It has been also established that FHC binds the internalized CXCR4 receptor and impairs its transductional pathway [15] consisting, among others, in the inhibition of cAMP production and activation of ERK and AKT [45, 46]. In the subsequent experiments we investigated whether CXCR4 is activated in MCF-7^shFHC cells and to what extent this activation might play a role in the induction of the EMT phenotype. Upon FHC silencing, CXCR4 protein level increased of about two-fold, and the receptor was shifted towards the cell surface, even though not to a significant extent (Panels A and B of Fig. 6). In line with an activation of CXCR4 pathway in the silenced cells, the treatment with CXCL12 inhibited cAMP production more strongly in MCF-7^shFHC cells than in the control ones. Moreover, this effect was only partially rescued by the canonical CXCR4 inhibitor AMD3100 (Panel C of Fig. 6). We also evaluated the CXCR4-mediated activation of ERK1/2 and PI3K/Akt; the levels of phospho-ERK (pERK) were mildly increased in MCF-7^shFHC cells (Panel D of Fig. 6) and appeared substantially unaffected by treatment with CXCL12 and with AMD3100 (data not shown). Conversely, phospho-AKT (pAKT) was dramatically induced in MCF-7^shFHC (lanes 1 and 5 of upper panel E of Fig. 6) and the addition of CXCL12 did not further increase pAKT activity (lanes 2 and 6 of the same panel). The lower western blot in panel E of Fig. 6 shows that the addition of AMD3100 is unable to rescue the pAKT induction detected in MCF-7^shFHC. Since CXCR4 has been shown to activate mTOR pathway via AKT [22] we also evaluated the levels of p-S6 ribosomal kinase (S6 K), the downstream effector of mTOR [47‐49]. pAKT activation in MCF-7^shFHC cells corresponded to increased phosphorylation of S6 K, which is not inhibited by AMD3100 (Fig. 6 Panel F). Thus, the significant increase of pAKT and its downstream target S6 K, suggests a strong activation of survival pathways through CXCR4 following ferritin silencing.

To further explore the impact of CXCL12/CXCR4 axis activation on phenotypic changes produced by ferritin heavy chain silencing, we measured migration and proliferation in the presence of CXCL12 in MCF-7^shFHC versus MCF-7^shRNA cells. CXCL12 induces an approximately 3-fold increase of migration capability both in MCF-7^shRNA and MCF-7^shFHC cells. Interestingly, when MCF-7^shRNA and MCF-7^shFHC cells were exposed to AMD3100 a decrease in migration was detected (Panel G of Fig. 6). With regard to cell growth, the presence of CXCL12 in the culture medium did not produce any proliferative advantage in MCF-7^shFHC vs. MCF-7^shRNA and AMD3100 inhibited growth rate to the same extent both in FHC-silenced and unsilenced cells (data not shown).

To verify if the role of FHC silencing in EMT is restricted to MCF-7 cells or if it represents a more general phenomenon, we reproduced most of the analysis in the lung tumor NCI-H460 (H460) cells either FHC-silenced (H460^shFHC) or unsilenced (H460^shRNA) cells (Panel A of Fig.7). The reason for this choice lies in the fact that we recently published that FHC silencing of H460 cells determines increased ROS production, enhanced cell viability and activation of AKT [13]. Moreover, the analysis of glucose/lactate metabolism, shown in Panel B of Fig. 7, suggests a more aggressive phenotype of H460^shFHC compared to H460^shRNA cells. Consequently we evaluated the EMT markers by western blot and qPCR analysis. The results are shown in Panel C of Fig. 7.

As in MCF-7 cells, also in the H460 cells FHC knock-down is accompanied by a reduced expression of E-cadherin and by an up-regulation of Vimentin, Twist, Slug and ZEB1. Moreover, consistently with the acquisition of EMT properties, H460^shFHC cells gained greater migratory capacity than control cells at 48 h (Panel D of Fig. 7). The expression levels of these molecules were partially restored upon NAC treatment. Panel A of Fig. 8 shows that Twist levels are reduced of about 50% and that Slug and Vimentin are reduced of about 45% in the NAC-treated H460^shFHC compared to the untreated H460^shFHC. Unlike MCF-7 cells, ROS chelation was able to partially restore E-cadherin expression. ROS chelation also reduced the enhanced proliferative rate of H460^shFHC cells, as shown in Panel B of Fig. 8.

As demonstrated in MCF-7 cells, FHC-silencing deregulated CXCR4 expression also in H460 cells. The steady-state amount of the receptor, evaluated by western blot, was about doubled in H460^shFHC cells with an increased localization, even though not statistically significant on the cell surface (Fig. 9, Panel a and Panel b). Along with the activation of AKT, which has been already demonstrated [13], FHC knock-down increased pERK expression, as shown in Panel C of Fig. 9. H460^shFHC cells were also treated with CXCL12 and with AMD3100 and pERK and pAKT levels were evaluated. The results, shown in Panel C of Fig. 9, are similar to those obtained with MCF-7^shFHC cells, since the levels of the two kinases were unaffected by treatment with CXCL12 and AMD3100. Finally, we analyzed the migration of H460 cells in the presence of AMD3100 demonstrating that the CXCR4 inhibitor consistently reduced the migratory capability of the H460^shFHC cells treated with CXCL12 (Panel D of Fig. 9).

Taken all together these results indicate that, albeit less dramatically than in MCF-7 cells, FHC silencing induces EMT and enhanced aggressiveness also in the lung tumor H460 cells and that these phenomena are attributable to both ROS increase and CXCR4 activation.