ATP consumption assays

Parental and Abl-manipulated NMuMG or 4T1 cells were cultured on 10-cm plates, and on reaching 80% confluency, were rinsed once with PBS and immediately lysed in buffer H/0.1% Triton-X100 (37). The resulting whole-cell extracts were clarified by microcentrifugation, and 700 μg of total protein was immunoprecipitated with rotation overnight at 4°C with anti-c-Abl antibodies (BD Biosciences). Captured immunocomplexes were washed twice with PBS and once with kinase assay buffer (40 mM Tris, 20 mM MgCl₂, and 0.1 mg/ml BSA, pH 7.5) and subsequently were resuspended in 250 μl kinase assay buffer, which then was divided into 100-μl aliquots that received either diluent or Imatinib myselate (10 μM; LC Laboratories, Woburn, MA, USA), a pharmacological Abl kinase inhibitor. Abl protein kinase reactions were initiated by addition of 100 μl of 2× kinase reaction cocktail, which contained the Abl peptide substrate (10 μM; New England Biolabs, Ipswich, MA, USA) and ATP (1 μM). Positive controls contained activated Abl1 kinase (50 ng/reaction; Cell Signaling, Boston, MA, USA), which represented maximal (i.e., 100%) Abl kinase activity. Abl PTK reactions were allowed to proceed at room temperature for 1 h under continuous rotation, at which point 50 μl was removed, mixed with an equal volume of Kinase Glo-Max reagent (Promega), and incubated at room temperature for 10 min. Luminescence was quantified over a 0.5-s integration window on a luminescent microplate reader (Turner Biosystems, Sunnyvale, CA, USA). Specific Abl kinase activity was determined by subtracting the luminescence, measured in relative light units (RLU), observed in Imatinib-containing samples from that measured for their corresponding diluent containing counterparts, which subsequently was normalized to the RLU observed in Abl-positive control samples.

Three-dimensional (3-D) culture system

NMuMG or 4T1 cells were diluted to 3 × 10⁴ cells/ml in complete medium supplemented with 5% Cultrex reconstituted basement membrane (R&D Systems, Minneapolis, MN, USA), and subsequently they were plated in 48-well plates on top of a Cultrex cushion. Substrate stiffness was manipulated by adding type I collagen (0–3 mg/ml; BD Biosciences) to the Cultrex cushion. Cells were stimulated with TGF-β1 (5 ng/ml; R&D Systems) in the absence or presence of the following inhibitors: Imatinib myselate (20 μg/ml); MMP 2/3 (30 μM) or MMP 2/9 inhibitor (500 nM) (Calbiochem, San Diego, CA, USA); or DECMA-1/E-cadherin blocking antibody (0.5 mg/ml, Sigma, St. Louis, MO, USA). The medium/Cultrex mixture was replaced every 3 d, and organoids were allowed to grow for 4–7 d, at which point they were monitored by bright-field photography. The resulting organoids also were monitored by 3-D proliferation assays using the MTS reagent kit (Promega) according to the manufacturer’s instructions. Afterward, 100 μl of the medium/MTS mixture was transferred to a 96-well plate, and the absorbance at 450 nm was determined on a microplate reader (Turner Biosystems). Specific proliferation signals were calculated by subtracting reagent blanks from those containing cells.

In some experiments, the resulting parental and CST-Abl-expressing 4T1 acini were stained with FITC-phalloidin to visualize the actin cytoskeleton, and with DAPI to visualize nuclei. Briefly, 4T1 cell derivatives were cultured for 7 d on compliant Cultrex cushions in 8-well chamber slides (5000 cells/well; Thermo Fisher, Rochester, NY, USA), at which point the organoids were rinsed with PBS supplemented with CaCl₂ (0.5 mM) and MgCl₂ (0.9 mM) (PBS²⁺) and fixed in 4% paraformaldehyde/PBS²⁺. Afterward, the organoids were rinsed in PBS²⁺ and permeabilized with 0.1% Triton X-100/PBS²⁺ for 5 min, followed by an additional PBS²⁺ rinse prior to blocking in 1% BSA/PBS²⁺ for 45 min. Organoids were incubated with FITC-phalloidin (0.5 μM, Sigma) in BSA/PBS²⁺ for 30 min, followed by sequential PBS²⁺ and water washes. Afterward, the stained cultures were dried for 2 h at 37°C; subsequently, they were mounted with ProLong Gold Antifade that contained DAPI (Invitrogen, Carlsbad, CA, USA). Stained organoids were visualized on a Leica DM5000 microscope (×40; Leica Microsystems, Bannockburn, IL, USA) and photographed using Image-Pro Plus 5.1 software (Media Cybernetics, Inc., Bethesda, MD, USA).

MMP fluorimetric and immunoblot assays

NMuMG and 4T1 cells were plated onto 6-well tissue culture plates (3×10⁵/well) and allowed to adhere overnight. The following morning, the cells were rinsed once with PBS and incubated in serum-free medium (SFM; 2 ml/well) in the absence or presence of TGF-β1 (5 ng/ml) for 24 h. The resulting conditioned medium and paired whole-cell extract was harvested and analyzed for MMP expression or activity using two complementary methods: fluorimetric and immunoblotting assays. For fluorimetric assays, equal volumes of conditioned medium (1.5 ml) were immunoprecipitated with anti-MMP-9 antibody (2 μg; Sigma) overnight at 4°C under continuous rotation. The resulting immunocomplexes were rinsed 3 times with PBS and resuspended in 140 μl PBS, followed by their activation with APMA (1 mM; AnaSpec, San Jose, CA, USA) for 2 h at 37°C. The activated samples were assayed for MMP-9 activity using the SensoLyte 520 MMP Sampler Kit (AnaSpec) and the MMP substrate SB14 (10 μM; AnaSpec), according to the manufacturer’s instructions. Duplicate samples were incubated at room temperature for 60 min, followed by reading of fluorescence intensity at Ex/Em = 490/520 nm. In addition, equal volumes (30 μl) of paired whole-cell extracts prepared in buffer H/0.1% Triton X-100 (37) and clarified by microcentrifugation were immunoblotted for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a loading control. For MMP immunoblotting assays, 50 μl of conditioned medium was loaded onto SDS-PAGE and subsequently was immunoblotted for MMP-9 or MMP-3 (1:500 dilution; Sigma). β-Actin controls were performed in tandem as described above.

Invasion assays

Invasion assays were conducted by culturing 3.5 × 10⁵ cells/well on Matrigel-coated (diluted 1:25 in SFM; BD Biosciences) modified Boyden chambers, and the cells were induced to invade by addition of 4% serum in the absence or presence of TGF-β1 (5 ng/ml) for 48 h. The chambers were harvested as described elsewhere (37, 38), and specific cell invasion was quantified by measuring the optical density of stained chamber membranes in ImageJ 1.40g software (National Institutes of Health, Bethesda, MD, USA).

Semiquantitative real-time PCR

NMuMG cells were plated in 6-well plates (3.5×10⁵/well) and allowed to adhere overnight before adding fresh complete medium the following morning. Afterward, the cells were treated with TGF-β1 (5 ng/ml) in the absence or presence of Imatinib myselate (10 μg/ml) for 48 h before harvesting total RNA using the RNeasy kit (Qiagen, Valencia, CA, USA). Total RNA (1 μg) was reverse-transcribed to cDNA using the cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), and the resulting cDNAs (10 μl) were diluted 40-fold before analysis. Semiquantitative RT-PCR was performed in 25-μl reaction volumes that contained 12.5 μl 2× SYBR Green (Bio-Rad), 200 nM PCR primer set, 2 μl nuclease-free water, and 10 μl cDNA sample. PCR cycling was performed on a Bio-Rad MJ Mini thermal cycler with Opticon 3.0 software using the following parameters: 1) 95°C for 10 min (1 cycle), and 2) 95°C for 15 s, followed 55°C for 1 min (40 cycles). The threshold for quantity calculations was adjusted to the linear range of amplification for each experiment, which subsequently was normalized to corresponding GAPDH mRNA expression levels. Individual primer sequences are presented in Supplemental Table 1.

Actin cytoskeletal immunofluorescence

NMuMG cells were plated at 3 × 10⁵/well onto gelatin-coated (0.1%) glass coverslips in 24-well plates and allowed to adhere overnight. Afterward, the cells were incubated in the absence or presence of Imatinib (10 μg/ml) or TGF-β1 (5 ng/ml) as indicated. On completion of drug or agonist stimulation, the cells were fixed and stained with FITC-phalloidin (0.25 μM, Sigma), as described previously (39). Stained cells were visualized using an ×60 oil objective on a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) and photographed using SlideBook software (Intelligent Imaging Innovations, Inc., Denver, CO, USA).

Abl activation restores normal MEC morphology and TGF-β-mediated cytostasis in metastatic MECs grown in compliant microenvironments

Our findings in NMuMG cells, in which Abl inactivation induced an EMT-like phenotype and transcriptional signature, prompted us to ask whether expression of a CST-Abl mutant to mimic Abl activation in metastatic MECs could normalize and revert their morphologies. Thus, we stably expressed CST-Abl in metastatic murine 4T1 breast cancer cells, which resulted in low-level CST-Abl protein expression (Fig. 1A) that likely reflects the significantly shortened half-life previously ascribed to constitutively activated Abl kinases (40). However, despite the relatively low level of expression, CST-Abl cells did indeed possess significantly higher levels of Abl PTK activity as compared to their parental counterparts (Fig. 1B). In contrast, expression of the KD-Abl construct in 4T1 cells resulted in elevated Abl protein levels (Fig. 1A) but no measurable increase in the levels of Abl PTK activity (Fig. 1B). Similar to NMuMG cells, we found that expression of CST-Abl promoted stronger cell-cell junctions and reduced the spreading of 4T1 cells in 2-D cultures (Fig. 4A). However, culturing these same cells on compliant 3-D organotypic reconstituted basement membranes greatly magnified the subtle morphological differences observed in 2-D culture (Fig. 4B). Indeed, although both parental (i.e., empty vector) and KD-Abl-expressing 4T1 cells formed large, irregularly shaped organoids characteristic of malignant MECs, 4T1 cells that expressed CST-Abl formed smaller acinar structures that were perfectly spherical (Fig. 4B). To confirm that the overall reduction in Abl protein expression observed in CST-Abl cells was not responsible for the phenotypic reversion of 4T1 cell morphology, we also cultured Abl-deficient 4T1 cells in 3-D organotypic cultures and observed a phenotype that resembled that of their parental counterparts (Fig. 4B). Most strikingly, we found that parental 4T1 cells failed to form hollow acinar structures when grown in compliant 3-D-organotypic cultures, an expected outcome that was wholly consistent with the aggressive tumorigenic phenotype of 4T1 cells (Fig. 4C). In stark contrast, culturing CST-Abl-expressing 4T1 cells under identical conditions resulted in their formation of small, spherical acini that appeared to be partially hollow (Fig. 4C). This dramatic finding suggests that Abl activation was capable of inducing the morphological and phenotypic reversion of 4T1 cells. Moreover, although expression of CST-Abl tended to inhibit the proliferation of 4T1 cells, both parental and CST-Abl-expressing 4T1 cells failed to undergo growth arrest in response to TGF-β when cultured on tissue culture plastic (i.e., a stiff microenvironment; Fig. 4D). Quite surprisingly, significant proliferative differences between these cell lines did manifest on their growth in compliant 3-D organotypic cultures, which was wholly sufficient in restoring the cytostatic function of TGF-β (Fig. 4D). Note that while we failed to observe any effect of manipulating Abl expression or activity on the ability of TGF-β to activate Smad2/3 and p38 MAPK (Supplemental Fig. 2), we did find that expressing CST-Abl greatly potentiated the coupling of TGF-β to p21^Cip1 mRNA expression in 4T1 cells (Fig. 4E). Thus, the heightened induction of p21^Cip1 expression by TGF-β in CST-Abl-expressing cells may account for the significant suppression in their proliferation relative to that of their parental counterparts (Fig. 4D). In addition, we found that rendering normal MECs deficient in Abl had no effect on their cytostatic response to TGF-β, as parental and Abl-deficient NMuMG cells, both exhibited similar extents of growth arrest when stimulated with TGF-β (data not shown). Taken together, these findings show for the first time that exposing aggressive, metastatic mouse mammary carcinoma cells to compliant microenvironmental signals reinstates their cytostatic response to TGF-β, and in effect, partially reestablishes the tumor-suppressing functions of TGF-β in malignant MECs. Equally important, our findings show that Abl activation is sufficient to phenotypically and morphologically normalize the architecture of metastatic mouse mammary carcinoma cells.

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Figure 4.

Abl activation promotes normal MEC morphology and TGF-β-mediated cytostasis in metastatic MECs grown in compliant microenvironments. A, B) Bright-field images (×70) of parental, CST-Abl-, KD-Abl, or shAbl-expressing 4T1 cells grown in 2-D (A) or compliant 3-D organotypic (B) culture systems as indicated. Insets: magnified views of boxed regions. Images are representative of 3 independent experiments. C) Parental or CST-Abl-expressing 4T1 cells were grown in compliant 3-D organotypic cultures for 7 d as indicated. Afterward, resulting acinar structures were stained with FITC-phalloidin and DAPI. Insets: magnified views of boxed regions. Images are representative of 2 independent experiments. D) Parental or CST-Abl-expressing 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 48 h in 2-D (left panel) or compliant 3-D organotypic (right panel) cultures as indicated. Differences in proliferation were determined by employment of MTS proliferation assays. Data are means ± se; n = 2–5. *P < 0.01, ^‡P < 0.05 vs. untreated parental 4T1 cells; Student’s t test. E) 4T1 cells expressing empty vector, CST-Abl, or KD-Abl were stimulated with TGF-β1 (5 ng/ml) for 24 h. Afterward, relative mRNA expression of p21^Cip1 transcripts was determined by semiquantitative real-time PCR. Data are combined from 2 independent experiments.

Our findings above clearly show the ability of compliant organotypic cultures to restore the cytostatic activities of TGF-β in metastatic breast cancer cells, a physiological reaction that is independent of altered genotypes. Along these lines, several studies have found that increasing ECM stiffness and rigidity can promote the development of malignant phenotypes in 3-D organotypic cultures and that the nearly infinite stiffness of 2-D culture systems can mask important morphological and functional differences that exist between normal and metastatic MECs (41,42,43). We, therefore, investigated the effect of ECM stiffness on the morphological and proliferative responses of parental (i.e., empty vector) and CST-Abl-expressing 4T1 cells to TGF-β. To do so, we added increasing amounts of type I collagen to the Cultrex basement membrane cushions, which typically are compliant and possess an elastic modulus approximating that of normal breast tissue, while supplementation with type I collagen (3 mg/ml) approximates the stiffness of tumors and their accompanying stroma (44). Figure 5A shows that parental 4T1 cells became increasingly branched and dysmorphic in response to increasing ECM stiffness, while their CST-Abl-expressing counterparts were highly resistant to rigidity-induced morphological alterations. Indeed, CST-Abl-expressing 4T1 cells formed considerably smaller organoids as compared to parental 4T1 cells and failed to exhibit any overt morphological alterations until the highest concentrations of type I collagen were reached (3 mg/ml) (Fig. 5A). Most important, ECM rigidity was observed to dose dependently override the cytostatic activities of TGF-β. Indeed, parental 4T1 cells, which do not arrest growth in response to TGF-β in 2-D cultures (Fig. 4D), recovered a cytostatic response to TGF-β when grown in compliant 3-D organotypic cultures (Fig. 4D). However, increasing 3-D organotypic culture rigidity dose dependently reduced and ultimately ablated the cytostatic response of parental 4T1 cells to TGF-β (Fig. 5B), a finding consistent with a more malignant phenotype elicited by increasing microenvironmental stiffness (41,42,43). In stark contrast, 4T1 cells expressing CST-Abl retained their ability to undergo cytostasis in response to TGF-β even in rigid microenvironments (Fig. 5B). Collectively, these findings show for the first time that Abl activation can override microenviromental signals that promote tumorigenesis, and in doing so, can dictate normal MEC morphologies that restore the tumor-suppressing activities of TGF-β in metastatic breast cancer cells.

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Figure 5.

Abl activation abrogates rigidity-induced proliferation and maintains TGF-β cytostatic response in metastatic MECs. Parental and CST-Abl-expressing 4T1 cells were grown in the absence or presence of TGF-β1 (5 ng/ml) for 7 d in compliant or rigid (i.e., 1–3 mg/ml type I collagen) 3-D organotypic cultures as indicated. A) Left panels: differences in cell morphology and proliferation were monitored by bright-field microscopy (×70). Right panels: differences in organoid branching at 3 mg/ml collagen. B) Differences in organoid proliferation were monitored by MTS assays. Data are expressed as means ± se (n=3). *P < 0.01, ^‡P < 0.05 vs. untreated parental 4T1 cells; Student’s t test.

Abl activation alleviates basal and TGF-β-induced MMP expression and secretion

MMPs are enzymes capable of remodeling the ECM through the cleavage of fibronectins, laminins, collagens, and other components of the microenvironment, and through the release of a variety of second messengers that modify the ECM and cell behavior (45). A number of MMPs have been shown to be crucial for cancer cell motility, most notably MMPs 1-3 and -9 (46,47,48,49). In addition, in some contexts, TGF-β promotes cancer cell invasiveness by stimulating MMP expression or activity (9,10,11,12). The striking role of Abl in determining the morphological output of MECs in response to altered microenvironmental tension (Figs. 4 and ​and5)5) led us to investigate a function for Abl in regulating MMP expression and secretion. First, zymography of parental and Abl-expressing 4T1 cells showed that TGF-β readily induced MMP secretion in parental and KD-Abl-expressing cells; however, medium conditioned by CST-Abl-expressing cells contained markedly less gelatinase activity (Supplemental Fig. 3A). We also sought to identify which specific MMPs were being secreted by Abl-manipulated 4T1 cells through the employment of immunoblots and fluorimetric assays to detect MMP expression and activity. We found that TGF-β stimulated significant up-regulation of MMP-9 (Fig. 6) and MMP-13 (Supplemental Fig. 3B), but it downregulated that of MMP-3 (Fig. 6) in parental and KD-Abl-expressing 4T1 cells. Significantly, both basal and TGF-β-regulated secretion of MMPs 3, 9, and 13 was abrogated by CST-Abl expression in 4T1 cells (Fig. 6 and Supplemental Fig. 3B). Again, to rule out the possibility that Abl-deficiency (Fig. 1A) was responsible for these effects on MMP secretion, we also performed immunoblot analyses to monitor MMP-3 and MMP-9 secretion by Abl-deficient 4T1 cells, which expressed both MMPs in a manner identical to that of their parental counterparts (Fig. 6C). In addition, we found that Abl deficiency was not sufficient to induce MMP secretion in normal cells, as parental and Abl-deficient NMuMG cells secreted similar levels of MMP-3 and MMP-9 both basally and in response to TGF-β (data not shown). Taken together, these results show that CST-Abl is a potent and broad spectrum inhibitor of both basal and TGF-β-stimulated production of MMPs in metastatic breast cancer cells.

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Figure 6.

Abl activation alleviates basal and TGF-β-induced MMP expression and secretion. A) Quiescent parental, CST-Abl-, and KD-Abl-expressing 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 24 h, at which point the resulting conditioned medium (cdm) was collected and immunoblotted for MMPs 9 and 3 as indicated. Differences in cell numbers produced were monitored by immunoblotting for β-actin in corresponding whole-cell extracts (WCE). Graph depicts mean ± se ratios of MMP-9/β-actin signals; n = 5. *P < 0.01; Student’s t test. B) Conditioned medium was prepared as in A and was immunoprecipitated with anti-MMP-9 antibodies for use in fluorescent MMP-9 enzymatic assays. Symbols represent data obtained in 4 independent experiments; horizontal bars depict mean MMP-9 activity. C) Abl-deficient 4T1 cells (shAbl) were stimulated with TGF-β1 (5 ng/ml) for 24 h, at which point conditioned medium was collected and immunoblotted for MMPs 9 and 3 as indicated. Differences in cell numbers were monitored by immunoblotting corresponding whole-cell extracts with anti-β-actin antibodies. Images are from a representative experiment that was performed 2 times with identical results.

Considering the ability of CST-Abl expression to inhibit secretion of several MMPs (Fig. 6) and to dictate normal MEC morphology in 3-D organotypic cultures (Fig. 4), we endeavored to block MMP function pharmacologically in parental 4T1 cells to attempt to recapitulate the effects of CST-Abl expression. To do so, we cultured parental and CST-Abl-expressing 4T1 cells in compliant 3-D organotypic cultures and found that treatment with inhibitors against MMP2/3 and MMP2/9 both markedly reduced abnormal organoid branching and proliferation in parental 4T1 cells (Fig. 7). Predictably, these same experimental conditions had no measurable impact on organoid formation in CST-Abl-expressing cells. In view of the results involving NMuMG cells in which maintenance of E-cadherin expression correlated with normal MEC morphology (Fig. 3), we also treated these same 4T1 cell lines with a neutralizing E-cadherin antibody (DECMA-1) and observed a dramatic increase in organoid size and dysmorphic features only in parental 4T1 cells on E-cadherin inactivation (Fig. 7). Treatment with a nonspecific rat IgG (control) had no impact on organoid size (data not shown). Collectively, these findings coalesce to emphasize the importance of matrix remodeling and cell-cell contacts in determining MEC morphology and behavior, as well as their response to TGF-β.

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Figure 7.

MMP and E-cadherin inhibitors alter the morphology and proliferation of metastatic MECs in 3-D organotypic cultures. Parental and CST-Abl-expressing 4T1 cells were cultured in the absence or presence of an MMP 2/3 inhibitor (30 μM), an MMP 2/9 inhibitor (500 nM), or a neutralizing E-cadherin antibody (DECMA-1; 0.5 mg/ml) for 5 d in compliant 3-D organotypic cultures as indicated. Differences in cell morphology and proliferation were monitored by bright field microscopy (×70; A), or by MTS proliferation assays (B). Insets: magnified views of boxed regions. Data are means ± se; n = 3. ^‡P < 0.05 vs. untreated parental 4T1 cells; Student’s t test).

We thank Dr. Tony Hunter (Salk Institute, La Jolla, CA, USA) for providing wild-type, kinase-dead, and constitutively-active murine c-Abl (type IV) constructs. We also thank members of the W.P.S. laboratory for critical comments and reading of the manuscript. Research support was provided in part by the National Institutes of Health (CA129359) and the Komen Foundation (BCTR0706967) to W.P.S., and by the Department of Defense Predoctoral Training Fellowship (BC083323) to T.M.A.