Rational development of synergistic combinations of chemotherapy and molecular targeted agents for colorectal cancer treatment

The HCT116 (CCL-247), SW48 (CCL-231), SW480 (CCL-228), LS174T (CCL-188) and HT29 (HTB-38) colon adenocarcinoma cell lines from the American Type Culture Collection (ATCC, Manassas, Virginia) were grown in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS) and 2 mmol/L L-glutamine at 37 °C in a humidified atmosphere with 5% CO₂. The SN38–resistant HCT116 clones were obtained as previously described [24]. Briefly, the reference SN38–sensitive HCT116 clone (HCT116-s) was incubated with 10 nmol/L or 15 nmol/L SN38 and cloned to obtain the HCT116-SN6 clone and the HCT116-SN50 clone, respectively. All cell lines were cultured in drug-free medium at least for five days before any experiment.

Ultra-low attachment, round-bottomed 96-well plates (Corning Costar) were used for spheroid formation. HCT116 cells were seeded at a density of 150 cells/well. Cells aggregated and merged to form 3D spheroids within 24-72 h. Spheroids were then grown in the presence of BKM120 (75, 150, 300, 600 and 1200 nM) and MEK162 (0.3, 1, 3, 10, 30, 90, 270 nM) (added at day 4 and day 10 of culture) and experiments ended 14 days after seeding. Images of wells were taken with a phase-contrast microscope using a 10X objective. Cell viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). After addition of 100 μl of CellTiter Glo reagent to each well for 10 min, luminescence was measured on a 1450 MicroBeta TriLux Luminescence microplate reader (Perkin Elmer).

All in vivo experiments were performed under the supervision of an accredited researcher (Dr. Céline Gongora, accreditation #34–142) in compliance with the French regulations and ethical guidelines for experimental animal studies. Female athymic nu/nu mice were purchased from Harlan Laboratories and used at 6 to 8 weeks of age and were maintained in a specific and opportunistic pathogen-free (SOPF) facility in an accredited establishment (Agreement No. C34–172-27). All animals were healthy and drug naïve before experiments, and their weight was 21 to 30 g. To evaluate antitumor effect of drugs, 1 × 10⁶ HT29 or HCT116 tumour cells were injected subcutaneously (s.c.) in the left flank of each mouse. Tumours were detected by palpation and measured periodically with callipers. When tumours reached 100 mm³, mice were randomized in groups of 6 to 9 animals, to be treated with the indicated drugs or with vehicle (0.9% sodium chloride, controls).

The irinotecan stock solution was diluted in 0.9% sodium chloride and administered by intraperitoneal (i.p.) injection (four injections of 20 mg/kg, one every 4 days). Mice in the control group received 0.2 mL of 0.9% sodium chloride solution (i.p. injection) according to the same schedule.

BKM120 and MEK162 (0.3, 1, 3, 30 and 0.3, 1, 3, 10, 30 mg/kg, respectively) were administered by i.p. injection to tumour-bearing mice 5 times per week. Mice in the control group received 0.2 mL of 0.9% sodium chloride solution according to the same schedule.

Mice were euthanized by cervical dislocation when the tumour volume reached 1500 mm³ and explanted tumour xenografts were frozen for phosphokinome analysis.

Xenografts from nude mice were isolated and proteins extracted as follows. Tumours were cut and lysed in 500 μL of M-PER lysing buffer complemented with protease and phosphatase inhibitors and then homogenized with beads using the MixerMill apparatus. Extracts were centrifuged and proteins in the supernatants were quantified with the Bradford assay. Kinase phosphorylation was then profiled using the PamGene technology. Specifically, phosphotyrosine kinase (PTK) and phosphoserine/threonine kinase (STK) activity were assessed using the PTK and STK PamChip arrays and 1 and 0.5 μg of protein extracts, respectively, on a PamStation12.

On each array, 144 peptides with tyrosine or serine/threonine phosphorylation sites were immobilized. FITC-labelled anti-phosphotyrosine or anti-phosphoserine/threonine antibodies were used to detect peptide phosphorylation. Peptide phosphorylation was monitored by taking images with a charge coupled device (CCD) camera in combination with the Evolve software v1.2 (PamGene) that allows the real-time recording of the reaction kinetics. After array washing, fluorescence was detected at different exposure times (20, 50, 100 and 200 ms). Luminescence signal acquisition was performed over 200 ms and the 100 ms values estimated by linear regression were normalized to the mean of the single array means and used for analysis. A Z score for the treated versus control sample values was calculated.

After counting, cells were lysed in SDS buffer (bromophenol blue, 5% β-mercaptoethanol, 2% SDS, 10% glycerol, 62.5 mM Tris-HCl pH 6.8). Extracts were treated with benzonase, boiled for 5 min and separated by SDS-PAGE. Proteins were electro-transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, AB, Sweden). Primary antibodies were against AKT, ERK and MEK1 and their phosphorylated forms, and against GAPDH and tubulin (loading controls). Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgGs (Sigma Aldrich). Proteins were detected by enhanced chemoluminescence (ECL) with the ECL detection system from GE Healthcare Life Sciences (Buckinghamshire, UK) and images recorded using the G/BOX iChemi imaging system (Syngene).

Cell growth inhibition and cell viability after incubation with SN38, BKM120 and MEK162 were assessed using the sulforhodamine B (SRB) assay. Exponentially growing cells (1000 cells/well) were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% FCS. After 24 h, serial dilutions of the tested drugs were added and each concentration was tested in duplicate or triplicate. After 96 h, cells were fixed with 10% trichloroacetic acid and stained with 0.4% SRB in 1% acetic acid (Sigma Aldrich). SRB fixed to the cells was dissolved in 10 mmol/L Tris–HCl and absorbance at 540 nm was read using an MRX plate reader (Dynex, Inc., Vienna, VA, USA).

The interaction between the drugs tested in vitro and in vivo was investigated with a dose matrix test, in which increasing doses of each single drug were tested with all possible dose combinations of the other drugs. For each dose combination, the percentage of surviving cells expected in the case of additivity was calculated according to the Loewe equation [25]:where d_A and d_B are the doses of the inhibitory drugs A and B, and IC_X,A and IC_x,B their isoeffective doses that result in an effect of x%. The percentage of expected surviving cells in the case of effect independence was calculated according to the Bliss equation [25]:where fu_c is the expected fraction of cells unaffected by the drug combination in the case of effect independence, and fu_A and fu_B are the fractions of cells unaffected by treatment A and B, respectively. The generalized form of the Bliss equation for a combination of n treatments:was used to analyse the interaction effect of the combination of three drugs. The difference between the fu_c value and the fraction of living cells in the cytotoxicity test was considered as an estimation of the interaction effect, with positive values indicating synergism and negative values antagonism. In addition to this point-by-point estimation, a synthetic index of interaction for the entire dose matrix (called combination index, CI) was calculated using the method proposed by Lehár [26‐28]:where f_A and f_B are the dilution factors used in the cytotoxicity assay for drugs A and B, respectively, and Z_O and Z_E are the matrices of the survival percentage for the experimental data and for the corresponding Loewe additivity or Bliss independence data, respectively. CI is a positive-gated, effect-weighted volume over Loewe additivity or Bliss independence, adjusted for the variable dilution factors f_A and f_B. R scripts used for interaction analysis are available in Additional file 1.

Trysined cells were fixed with 4% paraformaldehyde and permeabilised with methanol. Then the cells were incubated with anti-caspase-3 antibody (CST-9661) followed with a secondary anti-rabbit-Alexa Fluor488 from Invitrogen (A1108). The analysis were performed on FC500 Beckman coulter.

Cell proliferation was measured by incorporating 5-ethynyl-2′-deoxyuridine (EdU) into DNA during active DNA synthesis (i.e., the last 16 h of culture). After cell fixation and permeabilization in 75% ethanol/PBS, incorporated EdU was labelled and detected with the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Invitrogen, Carlsbad, CA, USA). Analyses were done on a FC500 Beckman Coulter Flow Cytometer. EdU-positive cells were quantified using the Flow Jo analysis software (Treestar Inc., Ashland, Oregon, USA).

Cells were seeded in 25 cm² flasks (2 × 10⁴ cells/flask). After 24 h, cells were incubated with 1 μmol/L BKM120, 0.1 μmol/L MEK162 or both for 96 h. One million cells were then pelleted, washed with PBS, fixed in 75% ethanol and stained with 40 μg/mL of propidium iodide solution containing 100 μg/mL of RNase). Cell cycle distribution was determined with a FC500 Beckman Coulter Flow Cytometer using the FL-3 channel. Cells were gated on a dot plot that displayed DNA pulse-peak versus DNA-pulse area to exclude doublets. Cell cycle distributions were illustrated using the Flow Jo analysis software (Treestar Inc).

For in vitro experiments, data were compared using the unpaired Student’s t test.

For the mouse models, a linear mixed-regression model, containing both fixed and random effects, was used to determine the relationship between tumour growth and number of days after grafting. Ata were first transformed using the natural log scale to better fit the assumptions of the linear mixed model. The fixed part of the model included variables corresponding to the number of post-graft days and the different treatments. Interaction terms were built into the model; random intercepts and random slopes were included to take time into account. The coefficients of the model were estimated by maximum likelihood and considered significant at the 0.05 level. The log-rank test was used to compare survival curves between groups. Statistical analysis was performed using the STATA 10.0 software (StataCorp).

To evaluate the effect of irinotecan treatment on CRC phosphokinome profile, tumours from nude mice xenografted with HT29 cells and treated with irinotecan (n = 6) or saline solution (n = 6) for 19 days (Fig. 1a) were analysed using PTK and STK PamChip arrays. Analysis of the phosphorylation changes of the tested kinase (described using Z-scores) (Fig. 1b) indicated that INSR, AKT and MEK1 (MP2K1) were among the kinases with a Z-score greater than 1 (Fig. 1c). The highest Z-score was attributed to insulin receptor (INSR), that could be partially responsible for MEK1 and AKT activation. The irinotecan-induced AKT and MEK1 phosphorylation increase was confirmed by western blotting of HT29 cell xenografts (Fig. 1d).

We decided to target AKT and MEK in order to inhibit the effect of INSR activation and, at the same time, to evaluate drug combinations suitable for clinical application. Then, the interaction of BKM120 (PI3K inhibitor), MEK162 (MEK inhibitor) and SN38 was evaluated using a full-range dose matrix approach and SRB cytotoxicity tests for two-drug combinations (BKM120-MEK162, MEK162-SN38, and BKM120-SN38) in seven molecularly characterized CRC cell lines (Table 1). First, the combinations, using seven different BKM120 doses, ten MEK162 doses and eleven SN38 doses, were analysed in HCT116 cells (Fig. 2a) and then in the other cell lines (summary in Fig. 2b). Results are presented as the percentage of cell survival (blue matrix) and values of the additive, synergistic and antagonistic effects (black, red and green matrices, respectively) (Fig. 2a for HCT116 cells). A synergistic interaction was observed mainly with the BKM120-MEK162 combinations (Fig. 2a, upper panels), with a mean combination index of 1.94 (range: 1.22 to 2.27) in all the tested cell lines (Fig. 2b). Drug combinations including chemotherapy showed less positive interactions. A slight synergistic interaction was detected between SN38 and MEK162 (Fig. 2a, center matrices), with a mean CI of 1.10 (range: 0.04 to 2.05; Fig. 2b). Combinations of SN38 and BKM120 were mainly additive (Fig. 2a, bottom panels), with a mean CI of - 0.31 (range: - 1.43 to 0.60; Fig. 2b). The distribution of the interaction effects was not homogeneous over the dose matrices. For instance, analysis of the matrices for the BKM120-MEK162 combinations (Fig. 2a, upper panels) showed focal areas with higher synergy that were located in dose regions corresponding to 90% to 30% of cell survival with BKM120 and 80% to 65% with MEK162. Analysis of the matrices for the BKM-SN38 combinations showed focal areas of antagonism located in dose regions that corresponded to 85% to 70% of cell survival with BKM120 and 95% to 70% with SN38 (Fig. 2a, bottom panels). No clear correlation was detected between the drug interactions and the KRAS and p53 mutational status or sensitivity to irinotecan of the used CRC cell lines (Table 1 and Fig. 2b). Further analysis of the BKM120-MEK162 combination with more doses (Fig. 2c) showed that the more synergistic doses were between 21 and 219 μM for MEK162 and 344 and 791 μM for BKM120.

Then, the same approach was used to assess the BKM120-MEK162 interaction in HCT116 spheroids (Fig. 3a). These experiments confirmed the presence of an area of significant synergism (i.e., more than 15% of cytotoxicity due to the synergistic interaction) in dose regions that corresponded to 98% to 59% of cell survival with BKM120 and 97% to 91% with MEK162, with a peak of synergism (i.e., ≥25% of cytotoxicity due to the synergistic interaction) in the dose regions of 97% to 59% of cell survival for BKM120 and 91% for MEK162 (Fig. 3b). The cytotoxic effect detected in the dose area of intense synergism corresponded to 60% to 29% of cell survival, and the synergistic interaction accounted for 28% to 25% of the drug effect, which corresponded to 70% and 35% of the total cytotoxic effect, respectively. The most synergistic doses were between 150 and 600 nM for BKM120 and 3 nM for MEK162.

As the BKM120-MEK162 combination was the most efficient in SRB assays, its functional effects were evaluated in HCT116 cells (Fig. 4). Apoptosis analysis by quantifying the percentage of propidium iodide-positive cells (i.e., cells in the subG1 phase of the cell cycle, representing late apoptosis) and of cleaved caspase 3-positive cells (early apoptosis) (Fig. 4a and b) showed that the BKM120 and MEK162 combination at synergistic doses (1 μM and 0.1 μM, respectively) increased apoptosis, compared with each drug on its own or absence of treatment (NT). Similarly, analysis of cell proliferation by EdU incorporation showed that proliferation was inhibited only in cultures incubated with the drug combination and not with each drug alone (Fig. 4a and b). In agreement, cell number was reduced only by the MEK162-BKM120 treatment (Fig. 4b). Overall these results indicate that the BKM120-MEK162 combination has synergistic cytotoxic and cytostatic effects.

To evaluate in vivo the effect of the BKM120-MEK162 combination, preliminary experiments were carried out to select doses associated with a limited effect of each single drug on tumour growth in xenografted mice (Fig. 5a and b). To this aim, groups of six mice were treated or not (saline, NT) with different concentrations of MEK162 (0.3, 1, 3 and 30 mg/kg) or BKM120 (0.3, 1, 3, 10 and 30 mg/kg). Treatments did not induce any side effect. For MEK162, the best antitumor effect (compared with control) was observed with the 30 mg/kg dose, and a mild tumour growth inhibition with 0.3, 1 and 3 mg/kg. On the other hand, 30 mg/kg was the only BKM120 dose that induced tumour growth inhibition. Then, mice xenografted with HCT116 cells (n = 6 per group) were treated with the BKM120-MEK162 combination (12 possible dose combinations, BKM120 alone (0, 0.3 or 3 mg/kg) or MEK162 alone (0, 0.3, 1 or 3 mg/kg i.p.) (Fig. 5c). A synergistic effect was detected with BKM120-MEK162 combinations at doses that did not show any significant effect as single drugs (i.e., a fold increase ratio between tested dose and control higher than 5%). Particularly, combining 0.3 mg/kg BKM120 with 0.3 or 1 mg/kg MEK162 led to a reduction in the median tumour volume fold increase of 24% and 21%, which is nearly entirely due to the synergistic interaction between these drugs (Fig. 5c).

Finally, as irinotecan induces AKT and MEK phosphorylation in CRC cells (Fig. 1c), the interaction of the SN38, BKM120-MEK162 combination was evaluated in HCT116 cells using the SRB cytotoxicity assay and a three-dimensional dose matrix with increasing doses of the three drugs (five doses for BKM120, five doses for MEK162 and one dose for SN38) (Fig. 6a). Again, an important synergistic effect (red squares on the synergy matrices) were observed at doses not associated with significant single-drug activity (detailed in the blue survival matrix). Importantly, this effect was due to the three-drug combination because addition of 4 nM SN38 (inactive dose on its own) to BKM120 and MEK162 increased the cytotoxicity over the dose areas where synergism was detected for the other two drugs, and expanded the synergism areas towards lower doses of BKM120 and MEK162. Western blot analysis showed that in HCT116 cells incubated with the three-drug combination (BKM120, MEK162 and SN38), AKT and MEK phosphorylation were reduced (Fig. 6b).