BMS-754807 is cytotoxic to non-small cell lung cancer cells and enhances the effects of platinum chemotherapeutics in the human lung cancer cell line A549

Background Despite advances in targeted therapy for lung cancer, survival for patients remains poor and lung cancer remains the leading cause of cancer-related deaths worldwide. The type I insulin-like growth factor receptor (IGF-IR) has emerged as a potential target for lung cancer treatment, however, clinical trials to date have provided disappointing results. Further research is needed to identify if certain patients would benefit from IGF-IR targeted therapies and the ideal approach to incorporate IGF-IR targeted agents with current therapies. Methods The dual IGF-IR/insulin receptor inhibitor, BMS-754807, was evaluated alone and in combination with platinum-based chemotherapeutics in two human non-small cell lung cancer (NSCLC) cell lines. Cell survival was determined using WST-1 assays and drug interaction was evaluated using Calcusyn software. Proliferation and apoptosis were determined using immunofluorescence for phospho-histone H3 and cleaved caspase 3, respectively. Results Treatment with BMS-754807 alone reduced cell survival and wound closure while enhancing apoptosis in both human lung cancer cell lines. These effects appear to be mediated through IGF-IR/IR signaling and, at least in part, through the PI3K/AKT pathway as administration of BMS-754807 to A549 or NCI-H358 cells significantly suppressed IGF-IR/IR and AKT phosphorylation. In addition of BMS-754807 enhanced the cytotoxic effects of carboplatin or cisplatin in a synergistic manner when given simultaneously to A549 cells. Conclusions BMS-754807 may be an effective therapeutic agent for the treatment of NSCLC, particularly in lung cancer cells expressing high levels of IGF-IR.


Background
Lung cancer remains the leading cause of cancer-related deaths worldwide [1], and non-small cell lung cancer (NSCLC) represents ~80 % of the cases of lung cancer [2]. Lung cancer is often not diagnosed until later stages [3,4] when it is no longer responsive to the standard treatment which is based on platinum chemotherapeutics and radiation [5][6][7]. Despite the development of a number of new targeted therapies against epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and anaplastic lymphoma kinase (ALK), overall survival rates remain poor [7][8][9][10]. There is a need to develop new treatment strategies including new drugs and understand the most effective way to incorporate them into current treatments. Several type I insulin-like growth factor receptor (IGF-IR) targeting agents have been in clinical development, including monoclonal antibodies and tyrosine kinase inhibitors, which are reviewed by Scagliotti and Novello [11] and Chen and Sharon [12]. Figitumumab, a monoclonal antibody against IGF-IR, reached a phase III clinical trial in non-adenocarcinoma NSCLC that was ended due to poor outcomes [13]. This

BMC Research Notes
*Correspondence: rmoorehe@uoguelph.ca Department of Biomedical Science, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, ON N1G2W1, Canada disappointing outcome leaves researchers asking if a better understanding of the role of IGF-IR in lung cancer would identify certain biomarkers, histological subtypes, and drug combinations or sequences that could improve the effectiveness of targeting the IGF-IR as part of lung cancer treatment.
The IGF-IR is a tyrosine kinase receptor which exists as a dimer (α 2 β 2 ): two monomers consisting of an alpha subunit and a beta subunit that are joined by disulfide bonds [49]. The IGF-IR signals through two main pathways, the PI3K/AKT pathway and the MAPK/ERK pathway [50,51]. The IGF-IR will form heterodimers with the closely related insulin receptor (IR), which shares 60 % structural similarity [52][53][54]. The IGF-IR responds to ligand stimulation by IGF-I, IGF-II, and insulin. While the IR is primarily activated by insulin, it will also bind to IGF-II and can bind both IGF-I and IGF-II when part of a heterodimer with IGF-IR [55][56][57][58][59][60]. Since both IGF-I and IGF-II are reported to be important activators of IGF-IR signaling in lung cancer [61][62][63][64], there is a good rationale for targeting the IR as well as the IGF-IR in order to achieve complete inhibition of the IGF axis. Additionally, in breast cancer cells and Ewing sarcoma the IR was identified as a mechanism of resistance to IGF-IR specific monoclonal antibodies [65,66]. There may be a therapeutic benefit to dual inhibition of both receptors over IGF-IR specific monoclonal antibodies. BMS-754807 is an ATP competitive inhibitor that targets both the IGF-IR and the IR [67,68].
This study evaluated the efficacy of BMS-754807 alone, and in combination with cisplatin or carboplatin, on two NSCLC cell lines in vitro. Our findings showed that BMS-754807 significantly inhibited IGF-IR/IR, AKT and ERK1/2 phosphorylation and reduced the survival of both NSCLC cell lines evaluated. BMS-754807 significantly increase apoptosis in both A549 and NCI-H358 cells and significantly reduced proliferation in A549 cells. In A549 but not NCI-H358 cells, BMS-754807 induced synergistic cytotoxicity when combined with cisplatin or carboplatin. These findings demonstrate the benefit of inhibiting IGF-IR/IR signaling in a pre-clinical setting and suggest inhibiting IGF-IR/IR signaling should be further evaluated as a potential therapeutic strategy for the treatment of NSCLC.

Cell lines and reagents
Human lung cancer cell lines, A549 and NCI-H358, were purchased from American Type Culture Collection (ATCC, Manassas, VA). A549 and NCI-H358 cells were cultured in RPMI 1640 medium supplemented with 10 % FBS and 1 % antibiotic/antimycotic (v/v) (Life Technologies, Burlington, ON). Cell were maintained in incubators at 37 °C and 5 % carbon dioxide. Cell identity was confirmed through STR profiling (The Centre for Applied Genomics at The Hospital for Sick Children, Toronto, ON).
BMS-754807 was purchased from Sellek Chemicals (Houston, TX). Cisplatin and carboplatin were both purchased from Sigma-Aldrich (Oakville, ON). BMS-754807 was dissolved in DMSO (Sigma-Aldrich, Oakville, ON) and stored at −20 °C while cisplatin was dissolved in saline and carboplatin was dissolved in water. Cisplatin and carboplatin solutions were made fresh for each experiment, and stored at 4 °C for the duration of the experiment.

WST-1 Assay
Cells were plated in a 96 well plate at a density of 2500 cells/well, and allowed to adhere for 4 h. BMS-754807, cisplatin, or carboplatin were added either alone or in combination or a solvent only control was added (0.1 % of total volume). Drugs and media were refreshed every 24 h. At 72 h, cell viability reagent WST-1 (Roche, Mississauga, ON) was added and absorbance was read using Universal Microplate Reader EL800 (Bio-Tek Insturments, Inc., Winooski, VT) after a 3 h incubation.

Analysis of single and combination drug response
Cell viability determined with WST-1 (Roche, Mississauga, ON) assay after single agent and combination treatments with BMS-754807, cisplatin, and carboplatin were used to determine the IC 50 and Combination Indices (CI) as calculated using Calcusyn software (Biosoft, Cambridge, UK). For combination effects: a synergistic effect is considered for a CI < 1; an additive effect is considered for a CI = 1; and an inhibitory effect is considered for a CI > 1.

Immunofluorescence
Cells were plated on coverslips in 6-well dishes at a density of 0.5-1 × 10 5 cells/well, allowed to adhere overnight, then treated with 0.5 μM of BMS-754807), or DMSO only (to 0.1 % v/v of total volume). Cells were fixed at 24 h post-treatment in 10 % buffered formalin (Fisher Scientific, Ottawa, ON) for 1 h. Cells were washed in PBS, permeabilized in 0.5 % Triton-X in PBS, and blocked in 5 % BSA in PBS. Then the cells were incuabated in primary antibodies against the proliferation marker Ki67 (1:400; cat# Ab15580) (Abcam, Toronto, ON) or apoptosis marker cleaved caspase 3 (1:200; cat# Ab3623) (Millipore, Etobicoke, ON) overnight at 4 °C. Cells were incubated with a fluorescent anti-rabbit secondary antibody (1:200; cat# A-21207) (Life Technologies, Burlington, ON) for 1 h at room temperature. Dapi nuclear stain (Life Technologies, Burlington, ON) was used as a counterstain, and coverslips were mounted using Prolong-gold (Life Technologies, Burlington, ON). Images were captured using Olympus BX61 microscope and Metamorph software (Molecular Devices, Sunnyvale, CA). Proliferation and apoptosis rates within cell populations were quantified by manual counts of the number of cells staining positive for Ki67 or cleaved caspase 3, respectively, and expressed as percentages relative to the total number of cells present as revealed by nuclear DAPI staining.

Wound healing assay
Cells were plated on 12 well plates at a density of 2.0 × 10 5 cells/well and cells typically reached 90-95 % confluency the following day. A wound was made through the centre of the well and cells were treated with 0.5 μM of BMS-754807, or solvent only (to 0.1 % v/v of total volume). Drugs and media were refreshed and wound closure was measured every 24 h. Images were captured using Olympus IX71 microscope and Q-Capture software (Q-Imaging, Surrey, BC).

Statistics
All experiments were repeated in triplicate or quadruplicate. Statistical analysis was performed using Graphpad Prism 5 (Graphpad Software, Inc., La Jolla, CA). Means were compared using a one-way ANOVA and post hoc Dunnett's test when multiple groups were compared back to a control value or a paired Student's T test when two means were compared. Error is represented by Standard Error of Measurement (SEM). Statistical significance is noted as p < 0.05.

IGF-IR Activity is Inhibited by BMS-754807
A549 and NCI-H358 cells were treated with the dual IGF-IR/IR inhibitor BMS-754807 under regular growth conditions. When BMS-754807 was used at concentrations of 0.25 and 0.5 μM, the levels of activated IGF-IR/ IR (pIGF-IR/IR) were reduced within 4 h, and a significant reduction in pIGF-IR/IR was observed at 24 h in both cell lines suggesting BMS-754807 inhibited IGF-IR/ IR activation (Fig. 1a-h). Furthermore, treatment with BMS-754807 caused a concurrent decrease in phosphorylated AKT in both lung cancer cell lines (Fig. 1a-h). ERK phosphorylation was significantly reduced following administration of 0.5 μM but not 0.25 μM BMS-754807 ( Fig. 1a-h).
To compare protein levels in A549 and NCI-H358 cells protein bands from the 4 h No Treatment groups were quantified. The IGF-IR protein was detected in both cell lines with A549 cells expressing more IGF-IR protein than NCI-H358 cells (Fig. 1a, b, i). A549 cells also possessed higher levels of phosphorylated IGF-IR/IR (pIGF-IR/IR) and phosphorylated AKT (pAKT) than NCI-H358 cells (Fig. 1a, b, i).

Treatment with BMS-754807 inhibits proliferation and induces apoptosis
Concentration-response curves were generated to determine the sensitivity of each lung cancer cell line to BMS-754807. BMS-754807 administration was sufficient to reduce cell viability in both A549 and NCI-H358 cells (Fig. 2a). The IC 50 values were determined using Calcusyn software (Table 1) to be 0.76 μM for NCI-H358 cells and 1.08 μM for A549 cells. In order to determine the specific cause of this reduction of cell viability, proliferation and apoptosis rates were determined in A549 and NCI-H358 cells following treatment with 0.5 μM BMS-754807 by immunoflourescence for Ki67 (Fig. 2b) or cleaved caspase 3 (Fig. 2c), respectively. BMS-754807 significantly inhibitied proliferation in A549 cells (Fig. 2d) but not NCI-H358 cells (Fig. 2e). Treatment with 0.5 μM BMS-754807 significantly increased apoptosis in both A549 (Fig. 2f ) and NCI-H358 cells (Fig. 2g).

Treatment with BMS-754807 or platinum chemotherapeutics reduces cell survival
Concentration-response curves were generated to determine the sensitivity of each lung cancer cell line to cisplatin and carboplatin when they were used as single agents. Both cell lines were sensitve to growth inhibition by cisplatin (Fig. 4a) and carboplatin (Fig. 4b) in a concentration dependant manner. The IC 50 values were determined using Calcusyn software (Table 1). A549 cells were more sensitive to platinum chemotherapeutics than NCI-H358 cells, with IC 50 values of 3.3 and 14.9 μM for cisplatin and 65.3 and 202.7 μM for carboplatin, respectively. Both cell lines were more sensitive to cisplatin than to carboplatin as reflected by the lower IC 50 values for cisplatin compared to carboplatin.

BMS-754807 synergizes with platinum chemotherapeutics to reduce A549 Cell viability
To determine if the addition of BMS-754807 could enhance the anticancer effects of platinum based chemotherapeutics at low concentrations, BMS-754807 was combined simultaneously with either cisplatin or carboplatin and the effects on cell viability were compared to those of each single agent. Three concentrations of BMS-754807 (0.01, 0.1, 0.25 μM in A549 cells and 2.5, 5.0, 10 µM in NCI H358 cells) were combined with three concentrations of cisplatin (0.5, 1, 2.5 μM) or carboplatin (10, 25, 50 μM in A549 cells and 50, 75 and 100 µM in NCI H358 cells), which based on the single agent concentrations were responsible for no more than approximately 25-30 % decrease in cell viability. At all concentrations of cisplatin and carboplatin used, the addition of BMS-754807 enhanced the effects of cisplatin and carboplatin (Fig. 5a, b). Combination indices were calculated using Calcusyn software to determine the nature of the interaction between cisplatin and BMS-754807 or carboplatin and BMS-754807. Representative plots of these interactions are presented in Fig. 5c, d. Combination indices <1 suggest synergistic interaction between the two drugs while combination indices of approximately 1 suggest additive drug interactions and combination indices >1 suggest antagonistic drug interactions. Based on the data presented in Fig. 5c, d, the combination of 0.25 μM BMS-754807 with cisplatin or carboplatin resulted in a synergistic reduction of cell survival in A549 cells (black symbols) but only additive or antagonistic interactions in NCI-H358 cells (white symbols). A complete list of the drug interactions for all the drug combinations tested are presented in Tables 2 and 3.

Discussion
The IGF-IR is frequently expressed at high levels in NSCLC and thus represents a potential therapeutic target. One compound that targets IGF-IR (as well as IR) is BMS-754807 and this drug was evaluated in two human non-small cell lung cancer cell lines, A549 and NCI-H358. Both A549 and NCI-H358 cells express IGF-IR protein and the IGF-IR is phosphorylated in both cell lines under normal growth conditions with A549 cells containing higher levels of IGF-IR and phosphorylated IGF-IR than the NCI-H358 cells. BMS-754807 signficantly reduced, IGF-IR/IR, AKT and ERK phosphorylation in both cell lines with the most dramatic effect being the reduction in AKT phosphorylation. When Wittman et al. [67] first reported the development of BMS-754807 they reported it to be more effective at ERK inhibition than AKT with IC 50 of 13 and 22 nM, respectively. However, subsequent reports in cell lines suggest the BMS-754807 is a more potent inhibitor of AKT than ERK phosporylation [68] which is consistent with our results. Furthermore, BMS-754807 was also reported to preferentialy inhibit the AKT pathway in breast cancer [70] and prostate cancer cells [71].
As    Tables 2 and 3 values (0.5-5 µM range) however proliferation and apoptosis were not specifically assessed [68]. Another novel finding of this study was that BMS-754807, as a single agent, could suppress wound closure. Since several studies have demonstrated an important role for IGF-IR in the motility and invasion of lung cancer cells [72][73][74][75][76] it is possible that BMS-874807 can negatively impact lung cancer cell migration and potentially metastais. However, this finding of reduced wound closure requires confirmation as alterations in proliferation and apoptosis can influence the rate of wound closure independent of migration.
Platinum-based chemotherapeutic agents are commonly employed in the treatment of unresectable NSCLC [77]. Therefore, we evaluated the efficacy of cisplatin and carboplatin in A549 and NCI-H358 cells and found that both cell lines were more sensitive to cisplatin than carboplatin. This is consistent with the study by Mahalingham et al. [8] that reported cisplatin was more effective than carboplatin in the treatment of NSCLC. To evaluate whether BMS-754807 could enhance the effects of platinum chemotherpeutics, A549 and NCI-H358 cell survival was assessed following simultaneous administration of either BMS-754807 and cisplatin or BMS-754807 and carboplatin. In almost all the combinations tested, administration of BMS-754807 with cisplatin or carboplatin resulted in a syngersitic reduction in cell survival in A549 cells. In NCI-H358 cells, combinations of BMS-754807 with cisplatin or carboplatin resulted in additive or antagonistic drug interactions with respect to cell survival. It remains unclear why the combinations of BMS-754807 with the platinum agents was more effective in the A549 cells. A549 cells express higher levels of IGF-IR than NCI-H358 cells and thus it is possible that A549 cells are more reliant on IGF-IR signaling for proliferation and survival. It is also possible that the intrinsic insensitivity of NCI-H358 cells to platinum chemotherapeutics (IC 50 for both cisplatin and carboplatin were considerably higher for NCI-H358 cells compared to A549 cells) impacted the interactions at the concentrations evaluated. A final potential explanation is that the different mutational profile of the two cells could influence drug interaction. While both cell lines harbour a mutation in K-ras, A549 cells have a mutation in Cdkn2a but contain wild type p53 while NCI-H358 express mutant p53 but wild type Cdkn2a (atcc.org). The only other study evaluating BMS-754807 in combination with chemotherapy in NSCLC found that BMS-754807 in combination with gefitinib resulted in synergistic reduction in cell survival in the human NSCLC cell line, NCI-H292 [78]. In small

Conclusions
In summary, this research demonstrates for the first time, the efficacy of BMS-754807 as a single agent in A549 and NCI-H358 cells and in combination with platinumbased chemotherapeutic agents in A549 cells. Therefore, BMS-754807 may be an effective therapeutic agent for the treatment of lung cancer, particularly in patients with lung tumors expressing high levels of IGF-IR.
Authors' contributions SEF performed the majority of the experiments and wrote the manuscript. RJ assisted with the drug combination assays while RB performed the wound closure assays on NCI-H358 cells. PM assisted with the immunofluorescence and RAM ran the project and edited the manuscript. All authors read and approved the final manuscript.