The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells

The cell lines used are described in Table 1. The SUM149 and SUM190 cell lines were purchased from Asterand Inc. (Detroit, MI, USA). The MDA-IBC03 cells were obtained from W.A. Woodward, and KPL4 cells were obtained from N.T. Ueno, The University of Texas MD Anderson Cancer Center, TX, USA. All other cell lines, MDA-MB-231, MDA-MB-468, MCF-10A, and MCF-12A, were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The new model of IBC, designated as FC-IBC02, was previously developed in our laboratory, through the isolation of tumor cells from the pleural effusion of an IBC patient, as described elsewhere [10].

CEP-37440 was synthesized and provided by Teva Branded Pharmaceutical Products R&D, West Chester, PA, USA. CEP-37440 has modest plasma protein binding, high intrinsic solubility, reduced lipophilicity, favorable microsomal metabolic stability across species, reduced capacity for drug–drug interaction, and possesses favorable oral bioavailability and a lower clearance rate in vivo across multiple species [21]. For in vitro assays, CEP-37440 free base was dissolved in dimethyl sulfoxide (DMSO) at concentration of 4 mM. For in vivo studies, CEP-37440 tri-hydrochloride di-hydrate salt was used and the drug was dissolved in distilled water. The media for the different cell lines are described in (Additional file 12).

Cells were grown to 80 % confluence in 100 mm in diameter Petri dishes. Cells were collected in cold phosphate-buffered saline (PBS) by scraping from culture plates, centrifuged, and washed twice with cold PBS; proteins were extracted with 500 μl of cell extraction buffer (Invitrogen, Life Technologies, Carlsbad, CA, USA) containing a mixture of protease inhibitors (Protease Inhibitors Cocktail, Sigma-Aldrich, St. Louis, MO, USA) and 1 mM phenylmethanesulfonyl fluoride (PMSF). After extraction for 30 min on ice, the extracts were centrifuged at 14,000 × g for 10 min at 4 °C, and the supernatant was transferred to a fresh microcentrifuge tube and kept at −80 °C until use. The protein concentration in the cell extracts was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Total FAK1 was studied in the cell protein extracts using FAK (Total) enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, Frederick, MD, USA); cell extracts were diluted 1:40 in dilution buffer as was recommended by the manufacturer; total FAK1 was expressed as ng total FAK1/μg protein. To study phospho-FAK1, FAK (pY397) ELISA kits (Invitrogen, Life Technologies, Carlsbad, CA, USA) were used and cell extracts were diluted 1:5 in dilution buffer; phospho-FAK1 (Y397) was expressed as units of phospho-FAK1/μg protein.

Total ALK and phospho-ALK (Tyr 1604) were studied using PathScan Total ALK Sandwich ELISA and PathScan Phospho-ALK (Tyr 1604) Sandwich ELISA kits, respectively (Cell Signaling Technology, Inc., Danvers, MA, USA); protein extracts from Karpas 299 cells, a human T cell lymphoma cell line, were used as positive control.

For FC-IBC02, SUM190, MDA-IBC03, SUM149, MDA-MB-231, MDA-MB-468, MCF-10A, and MCF-12A, a total of 2000 cells were plated per well in a 96-well plate, except for KPL4 in which 1000 cells were plated. After the cells were plated, they were allowed to attach overnight and then treated with CEP-37440 during several time points (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h). Several CEP-37440 concentrations were evaluated (0, 3, 10, 30,100, 300, 1000, 2000, and 3000 nM), and each concentration was tested in quadruplicate; as controls, cells growing in regular media (without CEP-37440) and media with 0.075 % DMSO (vehicle) were included. After CEP-37440 treatments, the living cells were quantified using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Briefly, the media with the CEP-37440 was removed, and 100 μl of fresh media was added to each well; 20 μl 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) compound was added and the cells were incubated at 37 °C in a cell incubator for 4 h. The absorbance at 490 nm and 630 nm (reference wavelength) was measured with a 96-well microplate reader (iMark™, Bio-Rad Laboratories Inc., Hercules, CA, USA).

FC-IBC02 cells were treated with 1000 nM CEP-37440 for 48 h and RNA was isolated. Affymetrix Genechip® Human Gene 1.0 ST array (Affymetrix Inc., Santa Clara, CA, USA) were used for gene expression studies (Additional file 12). Data analyses were performed using GeneSpring software 13.1 (Agilent Technologies, Inc., Santa Clara, CA, USA). The criteria for differentially expressed genes were set at ≥ 2.0-fold changes. Statistical analysis was performed to compare two groups using t test unpaired with a p value less than or equal to 0.05. A heat map was generated from the differentially expressed gene list. The list of differentially expressed genes was loaded into Ingenuity Pathway Analysis (IPA) 8.0 software (http://​www.​ingenuity.​com) to perform biological network and functional analyses.

Studies were approved by the Institutional Animal Care Committee at Thomas Jefferson University. A total of 10⁶ cells were suspended in 100 μl PBS, mixed with 100 μl Matrigel (BD Biosciences, Bedford, MA, USA), and injected into the fourth left inguinal mammary fat pad of severe combined immune-deficient (SCID) mice. The animals were palpated daily for detection of tumor development and, once the breast tumor xenografts reached approximately 50–100 mm³ (approximately 20–30 days postinjection), the mice were randomly allocated into groups. Two doses of CEP-37440 were tested for mice harboring FC-IBC02 or SUM149 breast tumor xenografts; the mice allocated to treatment received either 30 mg/kg twice a day (bid) or 55 mg/kg bid by oral gavage in a volume of 100 μl, 5 days/week for 35–40 days. For mice harboring SUM190 breast tumor xenografts, only the higher CEP-37440 dose (55 mg/kg bid) was tested. The CEP-37440 doses were chosen based on preliminary experiments in order to achieve an optimal plasma concentration–response relationship [21]. Breast tumors were measured using a vernier caliper, and tumor volumes were calculated using the following equation: V = [(L1 + L2)/2] × L1 × L2 × 0.526 where L1 and L2 are the length and width of the tumor. After 40 days of treatment or when the primary tumor reached a volume of approximately 1 cm³, the animals were euthanized by carbon dioxide (CO₂) inhalation. Breast tumors and other organs (lungs, heart, liver, spleen, brain, ovaries, kidneys, and lymph nodes) were removed, fixed in 10 % neutral-buffered formalin and paraffin-embedded for histological examination (Additional file 12).

For the analyses of the cell proliferation data, the log-transformed response measures (Abs 490 nm and Abs 630 nm) were modeled using the linear mixed effects (LME) model adjusting for correlations between repeated measures over time. The fixed effects included the ten concentrations and linear time trends. For the analyses of in vivo tumor growth data, the log-transformed tumor volumes were modeled using LME models adjusting for correlations between repeated measures from the same animal. The fixed effects included the control group and treatment groups (30 mg/kg CEP-37440, and 55 mg/kg CEP-37440), and linear and quadratic time trends. The LME models included either only linear terms or both linear and quadratic terms as appropriate for specific time-dependent trends. Percent tumor growth inhibition (% TGI) was calculated as follows: 100 × [(tumor volume of the control group at the end of treatment − tumor volume of the treated group at the end of the treatment)/tumor volume of the control group at the end of the treatment] = 100 × (1-exp (mean difference in log volumes at the end of the treatment), where the mean difference in log volumes at the end of the treatment was estimated from the fitted LME models. The data were analyzed using R package ‘nlme’ (The R Foundation for Statistical Computing http://​www.​R-project.​org).

Using ELISA, total FAK1 and phospho-FAK1 (Tyr 397) were studied in the IBC cell lines KPL4, MDA-IBC03, FC-IBC02, SUM190, and SUM149, and in the non-IBC triple-negative cell lines MDA-MB-231 and MDA-MB-468. All of the cell lines expressed high levels of FAK1 (between 0.28 ng to 0.7 ng FAK1/μg protein), and no differences were observed between IBC and non-IBC cell lines (Fig. 1a). However, the levels of phospho-FAK1 (Tyr 397) differed between the cell lines (Fig. 1b). The percentage of phosphorylated FAK1 (Tyr 397) in relation of total FAK1 was higher in KPL4, MDA-MB-231 and MDA-MB-468 (Fig. 1c); 4.4 % of total FAK1 was phosphorylated in KPL4, and approximately 3.6 % was phosphorylated MDA-MB-231 and MDA-MB-468. A lower percentage of FAK1 was phosphorylated in FC-IBC02 (1.4 %), SUM149 (1.4 %), SUM190 (0.66 %), and MDA-IBC03 (0.54 %) (Fig. 1c). We also studied the expression of FAK1 and phospho-FAK1 in the human normal-like breast epithelial cells MCF-10A and MCF-12A; these cells express FAK1 (approximately 0.5 ng total FAK1/μg protein and 0.26 ng total FAK1/μg protein, respectively) but they did not express phospho-FAK1 (Tyr 397).

The IBC cell lines FC-IBC02, SUM190, KPL4, SUM149 and MDA-IBC03 were tested for expression of ALK by ELISA. The human T cell lymphoma cell line Karpas 299 that carries the NPM (nucleophosmin)-ALK fusion gene was used as positive controls and these cells were strongly positive for total ALK and phospho-ALK (Tyr 1604). All of the IBC cell lines tested were negative for total ALK and phospho-ALK (Tyr 1604), and the non-IBC cell lines MDA-MB-231 and MDA-MB-468 were also negative for total ALK and phospho-ALK expression (data not shown).

Cell proliferation rate was studied in the IBC cell lines in the presence of several concentrations of CEP-37440. The MTS assay that measures cellular respiration was used as a surrogate of cell proliferation. In the presence of 300 nM CEP-37440, FC-IBC02 significantly decreased in proliferation compared to the control without the drug, and 1000 nM CEP-37440 completely inhibited FC-IBC02 proliferation, killing these cells after 72 h of treatment (Fig. 2a). FC-IBC02 showed a positive linear growth rate when the cells were in the presence of lower concentrations of CEP-37440 (3 nM, 10 nM, 100 nM and 300 nM) (Additional file 1: Figure S1 and Additional file 2: Table S1); in contrast, the growth rates in the cells treated with high concentrations of CEP-37440 (1000 nM, 2000 nM and 3000 nM) were not significantly different from time 0 (Additional file 1: Figure S1 and Additional file 2: Table S1).

In KPL4 cells, treatment with 1000 nM CEP-37440 for 144 h reduced significantly the proliferation of cells compared to the control, and the growth rate was even lower in the presence of 2000 nM or 3000 nM CEP-37440 (Fig. 2b). The growth rate in the KPL4 cells treated with the 3000 nM CEP-37440 was the lowest (Additional file 3: Figure S2 and Additional file 4: Table S2).

In SUM190 cells, treatment with 1000 nM CEP-37440 for 144 h reduced proliferation to approximately 60 % (Fig. 2c). The response measures corresponding to all concentrations except for 3000 nM grew over time and were significantly different from zero rates (Additional file 5: Figure S3 and Additional file 6: Table S3). The IBC cell line MDA-IBC03 only responded to a very high concentration of CEP-37440 (Fig. 2d), and SUM149 showed a slight response to high CEP-37440 concentrations (Fig. 2e). We also tested the effect of CEP-37440 on the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468 (Fig. 3a and b). Treatment with 2000 nM CEP-37440 for 144 h reduced MDA-MB-231 and MDA-MB-468 proliferation to approximately 46–54 % (Fig. 3a, 3b). We also studied the potential cytotoxic effects of CEP-37440 on normal breast epithelial cells, MCF-10A and MCF-12A (Fig. 3c and d). We found that 300 nM or 1000 nM CEP-37440 did not affect the proliferation of these cells; 2000 nM CEP-37440 decreased their proliferation to approximately 50 % and 3000 nM CEP-37440 inhibited their proliferation almost completely (Fig. 3c, 3d). The drug concentration required to reduce growth rates to 50 % (GI₅₀) for each cell line are shown in Table 1.

In conclusion, CEP-37440 at low concentration specifically reduced the proliferation of three out of five IBC cell lines; FC-IBC02, KPL4 and SUM190 cells showed decreased proliferation in the presence of 1000 nM CEP-37440, and this same concentration did not significantly reduce the proliferation of normal breast epithelial cells. The CEP-37440 concentrations required to reduced the growth rate to 50 % (GI₅₀) for each cell line are indicated in Table 1.  The sensitivity of these IBC cell lines to CEP-37440 was not related to the cell subtype since FC-IBC02 is a triple-negative cell line, and KPL4 and SUM190 are human epidermal growth factor receptor 2; (ErbB2)-positive (Table 1). MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB231 and MDA-MB-468 were less sensitive to CEP-37440, and the IBC cell line SUM149 was slightly affected by the drug.

To test the effect of CEP-37440 on the autophosphorylation of FAK1 and its Tyr 397 site, IBC cell lines were treated with 1000 nM CEP-37440 during different time intervals (0 h, 48 h, 72 h, 96 h, and 120 h) and total FAK1 and phospho-FAK1 (Tyr 397) were studied (Fig. 4). In FC-IBC02 cells treated with CEP-37440, the levels of total FAK1 were similar compared to the control without treatment (0.5 ng to 0.6 ng total FAK1/μg protein) (Fig. 4a), but the levels of phospho-FAK1 decreased over time in the cells treated with CEP-37440, reaching a value lower than 0.002 units phospho-FAK1/μg protein at 72 to 120 h of treatment (Fig. 4b). In SUM190 cells, the levels of total FAK1 decreased to approximately 50 % in the cells treated with 1000 nM of CEP-37440 compared to the control without treatment (Fig. 4a), and the phospho-FAK1 (Tyr 397) decreased from 0.07 units/μg protein in the control to 0.0015 units/μg protein in the CEP-37440-treated cells after 48 to 120 h of treatment (Fig. 4b). In both FC-IBC02 and SUM190 cells, the levels of phospho-FAK1 (Tyr 397) reached similar levels (0.0015 to 0.002 units/μg protein) after the treatment with 1000 nM CEP-37440 for 48 h (Fig. 4b).

In KPL4 cells, the levels of total FAK1 were similar between cells treated with CEP-37440 and the control without treatment (Fig. 4a), however, there was a significant decrease in phospho-FAK1 (Tyr 397) after CEP-37440 treatment from 2.5 % in the control without treatment to 1.4 % in cells treated with CEP-37440 (Fig. 4b and 4c). In SUM149, the levels of total FAK1 in treated cells were similar to the control during the first 72 h of treatment, decreasing from 0.4–0.2 ng total FAK1/μg protein after 96–120 h of CEP-37440 treatment (Fig. 4a); however there was a slightly decrease of phospho-FAK1, decreasing from 0.019 Units phospho-FAK1/ μg protein in the control to 0.008-0.009 Units phospho-FAK1/ μg protein at 96-120 h of CEP-37440 treatment (Fig. 4b). In SUM149, there was a decrease from 4.9 % phospho-FAK1 in the control without treatment to 3.8 % phospho-FAK1 after 120 h of CEP-37440 treatment (Fig. 4c).

In conclusion, low concentration of CEP-37440 (1000 nM) was able to decreased phospho-FAK1 by half in FC-IBC02, SUM190, and KPL4 cells after 48 h of treatment compared to the controls without treatment, while in SUM149, it only decreased slightly.

FC-IBC02 IBC cells were selected for expression array analyses because our previous results showed that CEP-37440 at low concentration (1000 nM) was able to completely inhibit the proliferation of these cells in vitro. Cells were treated with 1000 nM CEP-37440 for 48 h, RNA was isolated from the cells, and expression studies were performed. The microarray data have been deposited into the NCBI’s gene expression omnibus (GEO) data sets (GSE73285). A total of 43 genes showed changes in their expression when cells were treated with CEP-37440 compared to the control (2.0-fold, p = 0.05), with ten downregulated and 33 upregulated genes (Fig. 5 and Table 2). Using IPA, the major functions affected by CEP-37440 were cell death and survival, cellular growth and proliferation, and motility. Among key upregulated genes, those related to interferon signaling and cytokines were upregulated as follows: IFI27 (4.3-fold), IFI6 (3.5-fold), IFI35 (2.4-fold), IRF7 (2.1-fold), CCL5 (4.6-fold), IL32 (3.6-fold), and IL23A (2.1-fold). Similarly, the expression of interferon-stimulated genes was upregulated, as well: OAS2 (5.8-fold), OAS3 (3.5-fold), OAS1 (3.3-fold), MX1 (2.6-fold), and ISG15 (2.4-fold). CEP-37440 upregulated two other genes related to cell death and survival: BIK (2.3-fold) and KDR (2.3-fold).

CEP-37440 was tested in vivo using the triple-negative FC-IBC02 and SUM149 and the ErbB2-positive SUM190 IBC breast xenograft models. The IBC cells were injected in the mammary fat pad of female SCID mice, and treatment with CEP-37440 began once the breast tumor xenografts reached approximately 50–100 mm³. All of the mice injected with FC-IBC02 or SUM149 developed breast tumor xenografts, and two doses of CEP-37440 (30 mg/kg and 55 mg/kg) were tested in these mice. In the animals harboring FC-IBC02 breast tumor xenografts, mice treated with 55 mg/kg bid CEP-37440 showed smaller tumor breast xenografts compared to the control group that did not receive the drug (Fig. 6). The breast tumor growth was significantly lower in the group treated with 55 mg/kg bid CEP-37440 than the control group without treatment over the 7-week study (p ≤ 0.001) (Additional file 7: Figure S4 and Additional file 8: Table S4). Also, mice treated with 55 mg/kg bid showed smaller tumors than the mice treated with 30 mg/kg bid CEP-37440 over the entire duration of the study (Additional file 7: Figure S4). At the end of the study, there was approximately 33 % reduction in the FC-IBC02 breast tumor xenograft size after the mice were treated with 55 mg/kg bid CEP-37440 (week 8; 40 days of treatment) (Fig. 6a) (p = 0.128). Furthermore, there was approximately 21 % reduction in the FC-IBC02 breast tumor xenografts size after the mice were treated with 30 mg/kg bid CEP-37440 at week 8 (40 days of treatment) (p = 0.380) (Fig. 6a). FC-IBC02 mice treated with CEP-37440 showed no signs of toxicity, such as hair loss or significant weight loss compared to the control group without treatment (Fig. 6b). Importantly, although our previous experiments showed that 20 % of the mice with FC-IBC02 breast xenografts developed spontaneous metastatic brain tumors (Fig. 7c), none of the mice treated with CEP-37440 (30 mg/kg or 55 mg/kg) developed brain tumors. No differences in the number of metastatic sites in the lungs were found when comparing the 55 mg/kg CEP-37440-treated mice (Fig. 7e) and the control group (Fig. 7b) .

In animals harboring SUM149 breast tumor xenografts, mice treated with CEP-37440 showed smaller tumor breast xenografts compared to the control group that did not receive the drug (Fig. 8a). The breast tumor growth was significantly lower in the mice treated with CEP-37440 than in the control group without treatment over the 7-week study, although the growth of the tumors was lower in the mice treated with 30 mg/kg bid than with 55 mg/kg bid CEP-37440 (Additional file 9: Figure S5 and Additional file 10: Table S5). At the end of the study (week 7, 35 days of treatment), there was approximately 43 % reduction in the SUM149 breast tumor xenograft size after the mice were treated with 30 mg/kg bid CEP-37440 (p = 0.021) and approximately 23 % reduction in the SUM149 breast tumor xenografts size after the mice were treated with 55 mg/kg bid CEP-37440 at week 7 (p = 0.302). SUM149 breast tumor xenograft mice treated with CEP-37440 showed no significant weight loss compared to the control group that was not treated with CEP-37440 (Fig. 8b).

CEP-37440 was also tested in mice harboring SUM190 ErbB2-positive breast tumor xenografts. A significant difference in the size of the SUM190 breast tumor xenografts was observed in the treated group when compared to mice in the control group that did not receive the drug (Fig. 9a). There was 79.7 % reduction in the SUM190 breast tumor xenograft size after the mice were treated with 55 mg/kg bid CEP-37440 for 35 days (week 7) (p = 0.001) (Additional file 11: Table S6). There were no significant differences between the weight of the control and CEP-37440-treated mice (Fig. 9b).