MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

Immune-compromised NODScidIL2gamma^–/– mice were housed in SPF conditions in a 12-hour:12-hour light:dark cycle and given food and water ad libitum. Doxycycline (DOX)-containing food (700 mg/kg) was purchased from Gordon’s Specialty Stock Feeds and replaced weekly. Intraductal injections were modified from a previously described protocol without a Y incision in the abdomen [18]. For longitudinal studies, mice were randomized into DOX-treated or control-treated groups and monitored twice weekly for tumor growth, and measurements were taken until an ethical end point of 10% tumor burden or prior if the animal succumbed to tumor/metastasis-induced morbidity. For cross-sectional studies, mice were again randomized into DOX-treated or control-treated groups and sacrificed at 9 weeks (MDA-MB-231-2A xenografts) or 12 weeks (MDA-MB-468-2A xenografts) post tumor cell inoculation. For tail vein injections, mice were injected (with 1,500,000 MDA-MB-231-2A cells or with 2,000,000 MDA-MB-468-2A and MDA-MB-157 cells) using a 100 μl injection into the dorsal tail vein before harvest at 9 weeks post injection. At the end of the experiment, as indicated in the figures, mice were euthanized with CO₂ asphyxiation and the mammary glands, tumor and lungs were harvested and fixed for 4 hours in 10% buffered formalin at room temperature. Where possible, mammary glands were whole mounted and tissues were processed for histology as described previously [19]. After fixation, the mammary glands, tumors or lungs were sectioned and either stained with hematoxylin and eosin for routine histochemistry or stained with BIM (CST 2933), high molecular weight cytokeratin (Leica 34BE12), Vimentin (Leica NCL-L-VIM-V9), MCL-1 (ThermoScience MA5-13932), Cleaved Caspase-3 (CST ASP175 9664), multi-cytokeratin (Leica C-11) and Ki67 (ThermoScientific SP6) using DAKO immunohistochemistry as per the manufacturer’s instructions. All sections from tumors and lungs in each model were cut, sectioned, retrieved and stained at the same time permitted with each antigen.

Pools of BIMs2A or empty vector (EV) cells were made by routine cloning into an all-in-one tetracycline inducible vector (SH570MK as detailed in Additional file 1) and selected using Puromycin. BIMs2A expression was induced with 2 μg/ml DOX or vehicle control daily in the media and cells were harvested at the time points indicated in the figures. Annexin V PI staining was performed using the Annexin V-FITC Apoptosis Kit (Biovision, CA, USA) as per the manufacturer’s instructions. ABT-263 (5 μg/ml) was added to the media at the indicated times. For siRNA experiments, 5 nM siRNAs targeting MCL-1 (DHA-L-004384-00-0005) or non-targeting control siRNA (D-001206-14-05) was premixed with RNAiMAX (ThermoFisher) and cells were transfected the day after plating at a density of 1 × 10⁵ cells per well with ON-TARGETplus SmartPools of MCL-1 or nontargeting controls siRNAs as per the manufacturer’s instructions. A1210477 and UMI-77 (Selleckchem, MA, USA) was added to the media at 5 or 10 μM respectively as indicated in the figures. Dasatinib (Bristol-Myers Squibb, Princeton, NJ, USA) was added to the media in 2D and 3D at a concentration of 1 μM and 200 nM respectively. 3D collagen I/fibroblast models were performed as described previously [20].

Immunohistochemistry was used to assay MCL-1 protein expression using a mouse monoclonal antibody to MCL-1 (ThermoFischer (Pierce) MA5-13932) on TMAs constructed from tumors from a cohort of 292 patients diagnosed with invasive ductal breast carcinoma described in [21]. MCL-1 protein could only be detected in a subset of 246 of these cases due to missing or folded cores. The cohort consists of cases of invasive ductal carcinoma of no special type, median age 54 (range 24–87), with a median follow-up of 64 months (range 0–152.1). Of these, 68.6% were ER+, 57.1% were PR+, 18.7% were HER-2 amplified (by FISH) and 43.3% were lymph node-positive. Endocrine therapy (TAM) was given to 49.3% of patients and chemotherapy (AC or CMF) to 38%.

Immunoprecipitates were made using antibodies to MCL-1 (ThermoScience MA5-13932), BCL-2 (Millipore 05-729) and BCL-XL (CST 2764) using the TrueBlot kit according to the manufacturer’s instructions (Rockland Immunochemicals). Then 20 μg reduced protein was loaded in each well of 12% NuPAGE SDS polyacrylamide gels (Life Technologies) and separated using electrophoresis. Proteins were transferred to Immun-Blot PVDF (Biorad) and analyzed by western blot for mouse MCL-1 (CST 5453), BCL-2 (Millipore 05-729), BIM (CST 2933), PUMA (CST 4976), NOXA (ENZO ALX-804-C10), p53 (Epitomics 1026-1), Cofilin (total) (CST 5175), S3-Cofilin (CST 3313) and beta-ACTIN (Santa Cruz AC-74, A5316).

The production of contracted matrices is described elsewhere [20] and detailed methods are provided in Additional file 1. Contracted matrices were seeded with 1 × 10⁵ MDA-MB-231-2A cells and allowed to grow for 4 days, mounted on a metal grid and raised to an air–liquid interface to initiate invasion, which resulted in the matrix being fed from below with the media supplemented with either vehicle, DOX, dasatinib or a combination of DOX and dasatinib commencing at seeding (day 1) or 5 days after seeding (day 5). All treatments were performed on three independent matrices. Cells were allowed to invade for a total of 10 days towards the chemo-attractive gradient created by the air–liquid interface and then harvested for immunohistochemistry for multi-cytokeratin (Leica-Novocastra C-11) (invasion), Ki67 (ThermoScientific SP6) (proliferation) or Cleaved Caspase 3 (Cell Signaling Asp175 5A) (apoptosis) and scored as detailed in Additional file 1.

Quantification of the number and size of metastases (using sections stained with an antibody against human-Vimentin) and BIM intensity (using an antibody raised against human BIM) was performed using macros designed using FIJI image analysis software (http://​fiji.​sc/​Fiji) that are available from the corresponding author and are described in Additional file 1. Chi-squared analysis, Kaplan–Meier survival analysis and univariate analysis (UVA) using the Cox proportional hazards model were performed to determine correlations between MCL-1, clinicopathological features and outcome performed in Statview SE. All other data and statistics were analyzed in Prism6 for MacOSX. Data were graphed and parametric or nonparametric tests, as indicated in the figure legends, were used for normally distributed and skewed data respectively, and statistically significant groups were determined using bars as shown in the figures.

We investigated the levels of MCL-1 and the other BCL-2 family members BCL-2, MCL-1, BIM, PUMA and NOXA in 32 human breast cancer and immortalized breast epithelial cell lines (Fig. 1a). Variable levels of MCL-1 and BCL-XL were detected in all cell lines. BCL-2 levels were more variable, with only a small proportion (5/32, 15%) displaying high expression. MCL-1 was present in 17 cell lines with low or zero levels of BCL-2 (Fig. 1a). The MCL-1-interacting BH3-only proteins BIM, PUMA and NOXA were expressed at varying levels in all cell lines tested (Fig. 1a).

We investigated the importance of MCL-1 copy number variations and expression in breast tumors using publically available datasets. Gains and amplifications in MCL-1 copy number were observed in 14% of all cases in the TCGA dataset (CBioPortal [22, 23]). Increased MCL-1 copy number was also more frequently observed in invasive ductal carcinoma compared with normal tissues (TCGA, Oncomine [24]; Additional file 2: Figure S1A). MCL-1 mRNA expression correlated with advancing disease stage in the METABRIC dataset (Additional file 2: Figure S1B). Using publically available data sets obtained from NCBI GEO (KM Plotter [25]), we then investigated whether MCL-1 expression correlates with overall outcome. Three independent MCL-1 mRNA probes corresponding to Variant 1 (200798_x_at) and full-length MCL-1 (214057_at and 214056_at) were investigated, and the breast cancer cohort was split into untreated and treated cases (Additional file 2: Figure S1C, D). High MCL-1 mRNA expression predicted poor outcome in the untreated cases and the effects were reversed in the treated patients (Additional file 2: Figure S1C, D), but the hazard ratios in all cases were modest. Basal and HER2 amplified cases showed poor outcome when MCL-1 expression was low, with much stronger hazard ratios of 0.3 (n = 94 cases) and 0.08 (n = 30 cases) respectively. A similar nonsignificant trend was observed in the luminal A breast cancers (n = 68) but not in the luminal B subtypes (n = 115).

We then explored the prognostic significance of MCL-1 using tissue microarrays from a cohort of 246 patients diagnosed with invasive breast carcinoma [21]. MCL-1 immunohistochemistry displayed a variable expression pattern across tumors, ranging from absent to strong expression (0–3+) (Fig. 1b). MCL-1 expression was detected in the cytoplasm and in the nucleus of invasive carcinoma cells, with some heterogeneity observed within tumors (Fig. 1c, d). Cytoplasmic MCL-1 levels were lower in HER2-positive tumors compared with luminal A tumors but no other significant differences were observed (Fig. 1c, d). High cytoplasmic MCL-1 levels (Histoscore > 100) were modestly associated with improved breast cancer specific survival (log-rank Mantle Cox p = 0.04, hazard ratio = 0.54, 95% confidence interval 0.295–0.975; Fig. 1e). Nuclear MCL-1 expression was not predictive of outcome and no other significant effects on survival were observed when the cases were split into subtypes. High cytoplasmic MCL-1 expression was also positively correlated with the expression of the pro-apoptotic protein PUMA (chi-squared p < 0.0001, relative risk 1.75, 95% confidence interval 1.302–2.338).

To investigate whether MCL-1 antagonism was sufficient to induce cell death, genetic models were generated using the MCL-1 positive breast cancer cell lines MDA-MB-468 and MDA-MB-231. These lines are representative of the triple-negative subtype, which is often the most difficult to treat due to rapid disease progression [26]. The MDA-MB-468 cell line is characteristic of Basal A tumors (Fig. 1a indicated in red) while the MDA-MB-231 cell line is typical of claudin-low tumors (Fig. 1a indicated in blue). MCL-1 protein is found in both cell lines with lower levels observed in the MDA-MB-231 cell line.

To antagonize MCL-1, we expressed the BIMs2A sequence, a BIMs mutant (L62A/F69A) that selectively binds MCL-1 over BCL-2, BCL-XL, BCL-W and BFL-1 [15], using a DOX-inducible expression system. BIMs2A mimics small molecule BH3 mimetics by binding to the hydrophobic pocket and stabilizing MCL-1 levels. DOX treatment strongly induced BIMs2A by 24 hours in both MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cell lines (Fig. 2a, b). Increased MCL-1 was observed in both cell lines with no alteration in the levels of BCL-2 or BCL-XL (Fig. 2a, b), consistent with the effects observed in mouse embryonic fibroblasts [15]. MCL-1, BCL-XL and BCL-2 immune precipitates showed that endogenous MCL-1:BIM, BCL-2:BIM and BCL-XL:BIM complexes were present in both cell lines in control conditions (Fig. 2c, d). Induction of BIMs2A resulted in BIMs2A binding preferentially to MCL-1 as expected (Fig. 2c, d).

MCL-1 antagonism induced apoptosis of MDA-MB-468-2A cells grown as monolayers on plastic, seen as increased caspase 3 cleavage (Fig. 2a) and Annexin V/PI positivity (Fig. 2e), 48 hours after induction of BIMs2A. There was no effect observed in EV control cells treated with DOX (Fig. 2e). No significant death was observed in MDA-MB-231-2A cells grown on plastic (Fig. 2f). MDA-MB-468-2A cells were slightly more sensitive to the BCL-2/BCL-XL inhibitor ABT-263 after 24 hours of DOX exposure (Fig. 2g), suggesting that BCL-2/BCL-XL conferred partial resistance to MCL-1 inhibition and/or that MCL-1 antagonism sensitized the cells to ABT-263. MDA-MB-231-2A cells were highly sensitive to ABT-263 as described previously [14], but no additional effects were observed when combined with MCL-1 inhibition (Fig. 2h).

MCL-1 knockdown was compared with induction of BIMs2A in all cell lines (Additional file 3: Figure S2A). Cultures of MDA-MB-468-2A and MDA-MB-468-EV cells were mostly destroyed by MCL-1 knockdown via siRNAs targeting MCL-1, with significant effects of MOCK or nontargeting transfection alone (Additional file 3: Figure S2A, upper panels). This observation illustrates an artifact produced by knocking down an apoptosis suppressor using reagents that induce apoptosis. MDA-MB-231-2A or MDA-MB-231-EV cells, which have low levels of MCL-1, were insensitive to knockdown of MCL-1, as they were to BIMs2A. These data support our findings using the BIMs2A antagonist, and show the benefit of the mimetic approach over knockdown.

We then examined the effects of small molecule inhibitors of MCL-1, A1210477 [4] and UMI-77 [27], on apoptosis in these 2D models (Additional file 3: Figure S2B). Treatment with 5 μM A1210477 or UMI-77 was as effective as BIMs2A in MDA-MB-468-2A cells, with greater effects observed at 10 μM. Once again MDA-MB-231-2A cells were largely insensitive. A1210477 and UMI-77 also produced a significant increase in death in MDA-MB-157 cells, which also have relatively high levels of MCL-1, but no significant effects of these agents were observed in the MCL-1 low HCC1937 cells. Hence, the current pharmaceutical inhibitors of MCL-1 mimic induction of BIMs2A.

The effects of MCL-1 antagonism were also examined in a 3D organotypic in vitro model of invasion that more accurately recapitulates key aspects of the in vivo microenvironment [20]. MDA-MB-231-2A cells were seeded onto contracted collagen I matrices embedded with fibroblasts (Fig. 3a), transferred to an air–liquid interface and allowed to invade within the chemo-attractive gradient over a period of 10 days (Fig. 3b). BIMs2A was induced with supplementation of DOX in the media on day 1 of invasion (Fig. 3c–h, day 1). DOX treatment resulted in a significant reduction in the ability of MDA-MB-231-2A cells to invade (Fig. 3c, d), independent of potential effects on proliferation (Fig. 3e, f). In a post-invasion scenario, where we allowed the cells to invade for 5 days before DOX treatment (Fig. 3c–h, day 5), invasion was also significantly reduced (Fig. 3c, d) and proliferation was unaffected (Fig. 3e, f). As expected, induction of BIMs2A resulted in a modest increase in apoptosis in both conditions; however, apoptotic cells accounted for only a small proportion of the total population of cells and therefore could not account for the observed large reduction in invasion (Fig. 3g, h). MCL-1 antagonism prior to or after the initiation of invasion thus has anti-invasive effects in vitro.

The effects of MCL-1 antagonism on invasion were examined in vivo using a mammary intraductal injection technique [18], which requires cells to breach the basement membrane of the mammary duct to become locally invasive or metastatic (Fig. 4a). Mammary intraductal injection places breast cancer cells in a mammary tissue niche during tumor establishment and this maintains the phenotypic characteristics of the xenograft [28]. We observed that intraductal injection promotes metastatic progression in MDA-MB-468-2A cells. In mice bearing xenografts of MDA-MB-468-2A cells, induction of BIMs2A significantly reduced the rate of tumor growth, doubling the survival times of control mice (median survival: DOX 165 days vs Control 74 days; Fig. 4b, c and Additional file 3: Figure S2A, B). Induction of BIMs2A reduced the rate of onset of MDA-MB-231-2A xenografts (Fig. 4d), but there was no significant effect of BIMs2A on subsequent time to ethical end point (median survival: DOX 38 days vs Control 40 days; Fig. 4e). Immunohistochemistry confirmed strong and heterogeneous expression of BIMs2A in MDA-MB-468-2A primary tumors (Fig. 4f, DOX) and MDA-MB-231-2A primary tumors (Fig. 4g, DOX), confirming that BIMs2A was induced in the tumors.

We next investigated the effects of MCL-1 antagonism on the ability of triple-negative breast cancer cells to disseminate to the lung, using cohorts of mice bearing MDA-MB-468-2A and MDA-MB-231-2A mammary intraductal xenografts. Tumors and lungs were collected from these mice at 9 (MDA-MB-231-2A) and 12 weeks (MDA-MB-468-2A) (Fig. 4a), and lung metastases including single cell foci were visualized using anti-human pan-Cytokeratin or Vimentin immunohistochemistry (Fig. 4h and i, respectively). BIMs2A levels in tumors and lungs was analyzed using anti-human BIM immunohistochemistry (Additional file 4: Figure S4A and B respectively). A range of BIMs2A expression was observed among the individual cells of the primary tumors produced from MDA-MB-468-2A and MDA-MB-213-2A cell lines in mice fed DOX food (Additional file 4: Figure S4A). This observation was expected because the xenografted material was derived from pools of SH570MK-BIMs2A-infected cells. There were fewer and smaller metastases in the lungs from DOX-fed mice (Additional file 4: Figure S4C, D), however, these metastases lacked BIMs2A expression despite a predominance of cells expressing BIMs2A in the primary tumors (Additional file 4: Figure S4B), showing that only cells which did not express the BIMs2A antagonist could metastasize. To statistically analyze this effect we corrected these data using levels of BIMs2A expression observed in primary tumors (Additional file 4: Figure S4A, quantified in Additional file 4: Figure S4E, F), and showed a significant reduction in both the size and number of metastases in mice bearing both MDA-MB-468-2A and MDA-MB-231-2A xenografts (Fig. 4j, k and Additional file 4: Figure S4G, H). These data show, through a cellular competition assay, that MCL-1 antagonism by BIMs2A potently suppresses the ability of a tumor cell to metastasize.

We investigated whether MCL-1 antagonism could suppress cell seeding to the lung via tail vein injection (Additional file 4: Figure S4I–L). In this model, MCL-1 antagonism produced a trend towards reduced number and area of lung metastases produced from tail vein injections of MDA-MB-468-2A (Additional file 4: Figure S4I), but significantly suppressed the ability of MDA-MB-231-2A cells to survive and colonize the lung (Additional file 4: Figure S4J), where we observed an almost complete reduction in the size and number of lung metastases. Immunohistochemistry once again revealed that all cells that had colonized the lungs did not express the BIMs2A antagonist (Additional file 4: Figure S4K, L), indicating (as observed in the earlier intraductal models of metastasis) that only those cells which did not express the BIMs2A antagonist were able to survive and colonize the lung. Hence, expression of the BIMs2A ligand inhibited lung colonization. In addition we treated a cohort of mice injected with MDA-MB-157 cells with A1210477 or vehicle and observed a trend toward reduced metastases size that did not reach statistical significance (Additional file 4: Figure S4K). Importantly these mice showed no overt signs of systemic toxicity, suggesting that a therapeutic window for systemic MCL-1 suppression can be achieved. Together our data show that MCL-1 antagonism suppresses metastatic progression.

We then explored how MCL-1 might be regulating metastatic progression by profiling the kinome of MDA-MB-468-2A cells 24 hours after BIMs2A induction, prior to the onset of apoptosis, using Kinexus Antibody Microarrays. MCL-1 antagonism altered the levels of a large number of proteins important for invasion (Additional file 5: Figure S5A) [29, 30]. We observed changes in many SRC family kinases and their targets important in invasion, including a small increase in total CSK (cSRC tyrosine kinase), a negative regulator of SRC family kinases [31] (gain +53%). We also observed decreased total levels of the SRC family kinase, FYN, and the SFK target, ABL, which was increased by MCL-1 antagonism (63% and –20%, respectively). Loss of phosphorylation was also detected for Paxillin (–63%) and Vimentin (–71%), targets of SRC family kinase activity [32, 33]. MCL-1 antagonism also decreased the auto-phosphorylation site Y1148 in EGFR (–67%), proposed to be involved in the regulation of invasion by Src [34]. These data suggest that MCL-1 antagonism modulated the output of the SRC family kinases.

We also detected an increase in S3 phosphorylation of Cofilins 1 and 2 (+31% and +138%, respectively), an additional protein important for invasion. Cofilin is localized to invadopodia in MDA-MB-231-2A cells where phosphorylation of serine 3 suppresses actin-severing and polymerization function [29, 30]. Western blot analysis revealed that the ratio of S3 Cofilin to total Cofilin was significantly greater in MDA-MB-231-2A intraductal xenografts treated with DOX compared with control, with a similar trend observed in MDA-MB-468-2A xenografts (Additional file 5: Figure S5C). Immunofluorescence using antibodies against total Cofilin and phosphorylated Cofilin in MDA-MB-231-2A cells grown as monolayers also showed the same effect, with DOX treatment resulting in lower total Cofilin levels and increased serine 3 phosphorylation (Additional file 5: Figure S5D). Finally, proximity ligation assays using specific antibodies to MCL-1 and Cofilin in MDA-MB-231-2A cells revealed a novel interaction of MCL-1 with Cofilin that occurs throughout the content of MDA-MB-231-2A cells (Additional file 5: Figure S5E). Hence our data suggest that MCL-1 may regulate two modes of invasion: firstly those regulated by the SRC family kinases, and secondly those that involve the cytoskeletal remodeling protein Cofilin.

Because there are pharmaceutical compounds that target SRC family kinases, such as dasatinib, which have been shown to be anti-invasive in multiple cancer types [35‐37], we assessed the efficacy of combining MCL-1 antagonism with dasatinib treatment. When grown as monolayers on plastic, MDA-MB-468-2A cells were sensitive to dasatinib as a single agent alone, but DOX induction of BIMs2A amplified these effects (Additional file 6: Figure S6, top left panel). MDA-MB-468-EV control cells were equally sensitive to dasatinib and DOX treatment had no further effect (Additional file 6: Figure S6, lower left panel). MDA-MB-231-2A and MDA-MB-231-EV cells were largely insensitive to dasatinib as a single agent when grown as monolayers in 2D (Additional file 6: Figure S6, right panels). In contrast, when seeded onto contracted collagen I matrices, dasatinib significantly reduced their invasive capacity (Fig. 5a, b), and induction of BIMs2A significantly enhanced this effect, particularly when treatment was administered at day 1 of matrix seeding (Fig. 5a, b). BIMs2A, dasatinib or their combination had no effect on cell proliferation over the 10-day course of the experiment, whether treated at seeding or 5 days of invasion (Fig. 5a, c). Dasatinib also had no effects on apoptosis in MDA-MB-231-2A cells as a single agent (Fig. 5a, d). BIMs2A produced a small but significant increase in apoptosis in these cells when treatment was initiated at seeding or after 5 days of invasion, and dual therapy increased these effects (Fig. 5a, d).

In MDA-MB-231-2A intraductal xenografts in vivo, single-agent dasatinib had limited effects. Induction of MCL-1 inhibition with DOX alone had a slightly greater effect than dasatinib alone. Treatment with DOX and dasatinib significantly delayed the onset of mammary tumorigenesis (Fig. 5f, g), and metastasis to the lung was inhibited in all conditions compared with control (Fig. 5h).