Horizontal Combination of MEK and PI3K/mTOR Inhibition in BRAF Mutant Tumor Cells with or without Concomitant PI3K Pathway Mutations

Abstract

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

Keywords: BEZ235; BRAF; PI3K; PTEN; combination therapy; selumetinib.

Figure 1

Short term (72 h) effect of selumetinib and BEZ235 on the cell viability (SRB assay). (a,b) Selumetinib was more effective on BRAF mutant cell lines (blue) than on BRAF + PI3K/PTEN mutant cell lines (green). After treatment with BEZ235, sensitivity difference was not detected in the mutational groups. (c,d) Average cell viability upon treatment with selumetinib or BEZ235 in the two mutational groups. Data is shown as mean ± SEM from at least three independent experiments.

Figure 2

Colony formation inhibitory effect of selumetinib and BEZ235 for 10 days. (a,b) BRAF mutant (blue marked) cell lines were more sensitive to selumetinib than the BRAF + PI3K/PTEN mutant (green) cells; on the other hand, BEZ235 inhibited the cell lines with BRAF + PI3K/PTEN mutation more effectively than the BRAF mutant cell lines. (c,d) Average clonogenic potential in the mutational groups after treatment with selumetinib or BEZ235. Data is shown the mean ± SEM from at least three independent experiments.

Figure 3

Combinatory effect of selumetinib + BEZ235 on the cell lines for 10 days. (a) Combination index (CI) was calculated from long term (10 days) clonogenic assay results upon treatment with a combination of selumetinib and BEZ235 in different concentrations. CI < 1, CI ≈ 1, and CI > 1 mean synergistic, additive, and antagonistic effect, respectively. In most of the cell lines, the combination treatments were closely additive except for WM239 and SW1417, where synergy was detected. Data is from at least three independent experiments. (b) The average CI values of the cells. Data is shown as mean ± SEM from at least three independent experiments. Combination index in WM239 and SW1417 cell lines was significantly lower than 1. Asterisks mean a significant difference between CI ≈ 1 and the given CI value of the cell by * p < 0.05.

Figure 3

Combinatory effect of selumetinib + BEZ235 on the cell lines for 10 days. (a) Combination index (CI) was calculated from long term (10 days) clonogenic assay results upon treatment with a combination of selumetinib and BEZ235 in different concentrations. CI < 1, CI ≈ 1, and CI > 1 mean synergistic, additive, and antagonistic effect, respectively. In most of the cell lines, the combination treatments were closely additive except for WM239 and SW1417, where synergy was detected. Data is from at least three independent experiments. (b) The average CI values of the cells. Data is shown as mean ± SEM from at least three independent experiments. Combination index in WM239 and SW1417 cell lines was significantly lower than 1. Asterisks mean a significant difference between CI ≈ 1 and the given CI value of the cell by * p < 0.05.

Figure 4

3D spheroid growth inhibition results upon treatment with single compounds or combination for 6 days. A375, WM239, HT29, and SW1417 cell lines were involved into 3D investigations. (a) Representative images of SW1417 spheroids from treatment day 0 and day 6. Scale bar indicate 500 µm. (b) Spheroid volume was detected by taking pictures during the treatment and (c) spheroid proliferation by CCK8 at the end of the experiment. Colors indicate the therapy as blue, red means selumetinib, BEZ235 and purple indicates the combination of them, respectively. The combination treatment was significantly more effective than single treatments only in the case of SW1417 spheroids. Data is shown as mean ± SEM from three independent experiments. Asterisks mean a significant difference between the treatment groups by * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Protein expression investigation by Western blot upon treatment with selumetinib and/or BEZ235. (a) For c-/PARP and PCNA detection 100nM selumetinib (S) and/or 10nM BEZ235 (B) was applied for 48 hours. Apoptosis induction (c-PARP) was detected upon treatment with selumetinib and selumetinib + BEZ235 in case of A375 and WM239. Decreased expression of PCNA upon selumetinib or combination treatment was detected in A375 and WM239 cells. (b) For signaling pathway element detection (Akt, Erk, S6), 50nM of selumetinib, 5nM of BEZ235 or the combination of them was used for 4 hours. In all four cell lines, S6 and Erk activation decreased upon treatment with BEZ235/selumetinib + BEZ235 and selumetinib/selumetinib + BEZ235, respectively. However, Akt activation decrease was detected only in case of SW1417 cell line. Blots are representative pictures from three independent experiments.

Figure 6

Subcutaneous xenograft model was established in NOD-SCID mice from SW1417 cells to determinate the effect of the single treatment and combinational therapy. Daily oral treatment of selumetinib (25 mg/kg) and BEZ235 (15 mg/kg) and the combination of them was used for 17 days. (a) Graph indicates tumor volume growth. A stronger effect of the combination treatment was detected compare to the single agents from the first measurement of the tumor volumes (third day). (b) Changes in the body weight during the therapy. (c) Pictures of the tumors from the animal experiment upon treatment with the indicated drug for 17 days. (d) Tumor mass upon 17-day treatment with vehicle (black), selumetinib (blue), BEZ235 (red), or combination (purple). The combination treatment had the highest inhibitory effect on tumor mass compare to the single treatments. Data is shown as average ± SEM from n = 9−10 groups. Asterisks indicate a significant difference between the treatment groups and the control by * p < 0.05, ** p < 0.01 and *** p < 0.001.