Background
The RAS/RAF/MEK/ERK pathway consists of numerous signaling elements that, when affected by mutations, can promote tumorigenesis and tumor progression [
1]. One of the most frequent cause of RAS/RAF/MEK/ERK pathway aberrant activation is BRAF gain-of function mutation (7–15%) [
2,
3]. More than 80% of BRAF mutations are single amino acid substitutions of valin for glutamic acid in position 600 (V600E). Additionally, BRAF V600 K mutations also occur frequently in BRAF-mutated melanoma (7–19%) [
3]. However incidence of non-V600 BRAF mutation is not negligible in certain cancer types. Lung adenocarcinoma, melanoma and colorectal cancer with BRAF mutation showed 50–86, 34 and 23% non-V600 BRAF mutation frequency, respectively [
4‐
6]. Despite the notable number of the cases, there is no effective targeted therapy against non-V600 BRAF mutant tumors.
RAS/RAF/MEK/ERK pathway vertical inhibition in treatment of V600 BRAF mutant melanoma is already approved in clinical practice. Compared to V600 mutant BRAF single inhibition, combination inhibition of V600 mutant BRAF and MEK displayed elevated progression free and overall survival in clinical trials with metastatic BRAFV600 mutant melanoma patients [
7,
8]. Also recent studies have shown that in NRAS mutant melanoma, RAS/RAF/MEK/ERK pathway vertical inhibition leads to a synergistic inhibitory effect [
9,
10]. Pan-RAF and MEK double treatment proved to be an effective therapeutic strategy in vitro in NRAS mutant melanomas, when RAS/RAF/MEK/ERK pathway is activated and dependence of proliferation and survival on this pathway is demonstrable [
9]. Furthermore MEK and ERK1/2 combination inhibition induced a high level of apoptosis in NRAS mutant melanoma cell lines [
10]. Also in KRAS mutant colorectal and lung cancer cell lines RAF and MEK combination inhibition abrogates ERK1/2 signaling and triggers apoptosis [
11]. Enhanced therapeutic effects of MEK inhibitor and pan-RAF inhibitor (sorafenib) combination therapies have already been described in hepatocellular, thyroid, lymphoma and renal carcinoma [
12‐
15].
Non-V600 BRAF mutant tumors often carry concomitant mutations in RAS or PI3K [
6]. NRAS or KRAS mutations frequently occur concurrently with kinase dead BRAF mutations and play a role in MAPK pathway activity maintenance via CRAF [
6,
16,
17]. Also the MAPK pathway could be still activated through dimerization of BRAF with reduced kinase activity and wild-type CRAF [
2,
6,
18]. Since in non-V600 BRAF mutant tumor cells the MAPK pathway could still play a dominant role via CRAF in proliferation, survival and migration, we hypothesized that vertical inhibition of RAF and MEK may lead to enhanced therapeutic effects as found previously in V600 BRAF mutant cells.
The aim of our study was to investigate the effect of pan-RAF inhibitors (sorafenib and AZ628) and MEK inhibitor (selumetinib) combination treatment on non-V600 BRAF mutant tumor cell lines with various BRAF activities (Table
1). Sorafenib has broad preclinical activity across tumor types. While sorafenib had the lowest IC50 values against CRAF and wild-type BRAF, 6 and 22 nM, respectively, and thus can be considered a pan-RAF inhibitor, it also targets a variety of other kinases including VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, c-Kit, Flt-3 and RET. [
19,
20]. AZ628 has strong selectivity for RAF kinases with an IC50 value of 30 nM for BRAF V600E and wild-type CRAF and of 100 nM for wild-type BRAF [
21]. Selumetinib is a non-ATP competitive and highly selective MEK 1/2 inhibitor. In clinical trials with selumetinib, patients harboring RAS/RAF mutations had higher objective response rate than patients with wild-type RAS/RAF [
22].
Table 1
Cell lines by tissue type, BRAF and RAS mutational status
A375 | melanoma | V600E | high | wild type | |
CRL5885 | lung | G466 V | low | wild type | |
WM3629 | melanoma | D594G | low | G12D (N) | |
WM3670 | melanoma | G469E | low | G12D (N) | |
MDAMB231 | breast | G464 V | intermediate | G13D (K) | |
CRL5922 | lung | L597 V | intermediate | Q61K (N) | |
Here, we report that in preclinical models of non-V600 BRAF mutant tumors, combination of sorafenib/AZ628 and selumetinib shows enhanced therapeutic effects compared to single treatment.
Methods
Reagents and cell lines
Selumetinib, sorafenib and AZ628 were obtained from Selleck Chemicals (Houston, TX), LC Laboratories (Woburn, MA) and Sigma-Aldrich (St. Louis, MO), respectively. Six human tumor cell lines were used in the experiments. Cell lines A375 (ATCC, CRL-1619), CRL5885 (ATCC, CRL-5885), CRL5922 (ATCC, CRL-5922) and MDAMB231 (ATCC, HTB-26) are available from ATCC. WM3629 (Coriell Cat# WC00117, RRID:CVCL_C275), WM3670 (Coriell Cat# WC00119, RRID:CVCL_6799) cell lines were obtained from the Wistar Institute
(Table
1). Activation status of the BRAF mutations are from Zheng et al., 2015 [
6]. Cell lines have no known additional mutations in PI3K or PTEN [
23‐
27].
All cell lines were cultured in DMEM (Lonza, Switzerland; with 4500 mg/dm3 glucose, piruvate and L-glutamine) supplemented with 10% foetal calf serum (Gibco) and 1% penicillin-streptomycin-amphotericin (Lonza). Cells were maintained in tissue culture flasks at 37 Co with 5% CO2.
Sulforhodamine B (SRB) assay and drug combinatory index assessment
Long-term antiproliferative effects of sorafenib and selumetinib treatment were assessed by performing clonogenic SRB (Sigma-Aldrich, St. Louis, MO) assays. Briefly, 2000 cells (WM3629, WM3670, CRL5885, CRL5922) or 250 cells (A375, MDAMB231) were plated in 24-well plate format and cultured overnight. Next day, the cells were treated with different concentrations of sorafenib or AZ628 and selumetinib and with combined treatment for 10 days. After 10 days, cell monolayers were fixed with 10% trichloroacetic acid and stained for 15 min with SRB. Excess stain was discarded and cells were washed with 1% acetic acid solution. Stained cells were dissolved in 10 mM Tris-HCl pH 8 and OD was measured at 570 nm using a microplate reader (EL800, BioTec Instruments, Winooski, VT). Interactions between drugs were tested on the basis of calculating the combination index (CI) according to Chou and Talalay [
28] with CompuSyn software (ComboSyn Inc). CI values CI < 1, CI = 1 or CI > 1 represents synergism, additive effects, and antagonism, respectively.
Immunoblot analysis
Immunoblot analysis was performed to demonstrate the effect of selumetinib and sorafenib treatment on the activation of CRAF, Erk1/2, Akt (at Ser473) and S6 protein. Induction of apoptosis upon selumetinib and sorafenib/AZ628 treatment was detected by total PARP and cleaved PARP. Cells were seeded in six-well plates and maintained overnight. Next day selumetinib and sorafenib/AZ628 or combination treatment were applied for 4 h or 48 h to determine the activation changes of CRAF, Erk, Akt and S6 or cleavage of PARP, respectively. For p-CRAF/CRAF, p-Erk1/2/Erk1/2, p-Akt/Akt, p-S6/S6 detection ice-cold 6% trichloroacetic acid (TCA) were used to precipitate cells. Then cells were centrifuged for 15 min at 12000×g at 4 °C. Modified Läemmli-type sample buffer containing 90 mM Tris-HCl, pH 7.9, 2% SDS, 10% glycerol, 5 mM EDTA, 125 mg/ml urea, 100 mM dithiothreitol (DTT), 0.02% bromophenol blue was used to dissolve protein pellets. Protein concentrations were measured by the modified Lowry method using bovine serum albumin as standard. To detect total/cleaved PARP cells were lysed with RIPA Buffer (Thermo Scientific, Waltham, MA) supplemented with 1% Halt Protease Inhibitor Single-Use Cocktail (Thermo Scientific). Total protein concentrations were measured with Pierce BCA Protein Assay kit (Thermo Scientific). Protein samples were separated by SDS-PAGE (10%) and transferred to PVDF membranes (Thermo Scientific). Primary antibodies to antiPARP/cleaved-PARP (Merck Millipore AM30, Cell Signaling; #9541) and anti p-Erk1/2/Erk1/2, p-Akt/Akt, p-S6/S6, p-CRAF/CRAF (Cell Signaling; #9101, #9102, #4058, #9272 #2215, #2217, #9427, #9422, respectively) and as loading control anti β-tubulin or β-actin (Cell Signaling #2128 and #4970), overnight at 4 °C in a dilution of 1:1000 were applied. Secondary HRP-conjugated anti-rabbit or anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) was used (1:10000, 1 h) at room temperature. Pierce ECL Western Blotting Substrate (Thermo Scientific) was used to visualize the protein bands.
TUNEL assay
Cells were seeded in 24 well plates (50,000 cells/well) and next day selumetinib or sorafenib or a combined treatment were applied. After 48 h of treatment 4% buffered formalin was used to fix the cells. Labelling of terminal deoxynucleotidyl transferase—mediated dUTP nick end (TUNEL) was performed according to the supplier’s recommendation (Roche Diagnostics, Basel, Switzerland). DAPI stained and TUNEL positive nuclei on at least three 10× microscopic fields were counted to quantify the images.
Cell cycle analysis
To determine cell cycle change upon selumetinib and sorafenib treatment, cells were treated with the inhibitors for 48 h in 6-well plates. Cell cycle analysis was carried out as described earlier [
29]. Briefly, cells were trypsinized and lysed before staining with DAPI for 5 min at 37 °C. After adding the stabilization buffer, samples was loaded onto an 8-well NC slide. NucleoCounter NC-3000™ system (Chemometec, Allerod, Denmark) was used to quantify cellular fluorescence.
Time-lapse video microscopy
Video microscopy measurements were performed and analyzed as described previously [
30]. The parameter migrated distance is calculated by averaging for each cell the displacement for the 48–60 h interval after treatment, in at least three independent experiments and three microscopic fields.
Mammary xenografts of MDAMB231 breast cancer cells
Animal experiments were carried out at the Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary and the animal-model experiments were conducted following the standards and procedures approved by the Animal Care and Use Committee of the National Institute of Oncology, Budapest (license number: PEI/001/2574–6/2015). 14-weeks-old female NSG mice were used as animal model, since previous work described NSG mice as a suitable model for study human breast cancer [
31]. Mice were bred and maintained in specific pathogen-free facility. MDAMB231 cells (2 × 10
6 in 50 μl serum-free DMEM) were injected into the mammary fat pad of female NSG mice. Two weeks after injection, mice were randomly and evenly divided into four groups (10 mice/group) and treated with vehicle, 25 mg/kg sorafenib and 10 mg/kg selumetinib or both intraperitoneally (i.p) every day except weekends for 18 days. Controls received equivalent amounts of DMSO as treated animals. All animals were included in the analysis. The changes of the body weight were also determined throughout the study (Fig.
5b). No adverse events were observed during the experiment. Tumors were measured with caliper twice a week and tumor sizes were calculated with the formula for the volume of a prolate ellipsoid (length x width
2 x (4π/3)) and then transformed into relative values (V) using the formula: V = Vt/V0, where V0 is the initial tumor volume and Vt is the tumor volume at the indicated time point. Eighteen days after the first treatment, mice were euthanized and the tumor tissue was removed and weighed.
Statistics
All statistical analyses were performed in GraphPad Prism 5 (GraphPad Software Inc., USA, San Diego, CA). One-way ANOVA followed by Tukey’s post hoc test was used to establish whether significant differences existed between groups. Differences were considered significant at p < 0.05.
Discussion
Currently there are no approved targeted therapeutic options for non-V600 BRAF mutant tumors. In our study we have demonstrated that in non-V600 BRAF mutant cells the combination of sorafenib and selumetinib treatment synergistically enhances growth inhibition compared to single treatment. Combination treatment also improves apoptosis induction and Erk1/2 inhibition compared to monotherapy and, moreover, also decreases migratory capacity of the cells. We have also performed growth inhibition and PARP cleavage assay with highly selective pan-RAF inhibitor AZ628 to confirm that effects of sorafenib treatment on non-V600 BRAF mutant cells are due to its pan-RAF inhibitor activity. We found that also AZ628 and selumetinib combination treatment resulted in synergistically increased growth inhibition and PARP cleavage in non-V600E BRAF mutant cells.
While our study is the first report on sorafenib/AZ628 and selumetinib combination, there are already studies on non-V600 BRAF mutant lung cancer cell lines using the BRAF V600E mutant selective inhibitors dabrafenib or vemurafenib and the MEK inhibitor trametinib [
32,
33]. In the H1666 (CRL5885) and H1395 non-V600 BRAF mutant lung cancer cell lines and the H508 colorectal cancer cell line dabrafenib and trametinib combination treatment showed enhanced anti-proliferative effects and caspase3/7 activation, however these results were not significant in all cell lines [
33]. In the H1755 non-V600 BRAF mutant lung cancer cell line vemurafenib and trametinib combination treatment caused a small but significant increase in apoptosis when compared to either single agent [
32]. It was also shown that in low but not in high activity BRAF mutant melanoma cell lines (WM3670, WM3629) sorafenib treatment could reduce tumor growth and induce apoptosis [
34]. We observed similar effects upon sorafenib treatment in these cells; however, adding selumetinib further increased inhibition of tumor growth and migration as well as enhanced the apoptosis inducing effects of sorafenib.
Targeted therapy could induce adverse effects on parallel signaling pathways. It was previously described that sorafenib treatment tends to elevate Akt activation in cell lines with KRAS or BRAF mutations [
35]. Also MEK inhibition could increase p-Akt level in RAF mutant cells [
36] and KRAS mutant cells [
37]. We observed that after 4 h treatment of selumetinib p-Akt level increased in WM3670 and WM3629 cells that have low activity non-V600 BRAF and additional NRAS mutation. Furthermore, we found that in the WM3629 cell line selumetinib caused significantly elevated migration which could be diminished by adding sorafenib. Our findings suggest that the potentially adverse signaling effects of selumetinib could be reversed by adding sorafenib.
We found significant tumor growth inhibition in the in vivo xenograft model of the MDAMB231 cell line upon sorafenib and/or selumetinib treatment. Although the enhanced tumor growth inhibition did not reach statistical significance between the selumetinib and the combination treatment group, in the combination group the variance between relative tumor volumes was significantly lower than in the selumetinib group (
p = 0.017, F-test). This observation indicates that combination treatment reduced the number of weak-responders in comparison to selumetinib single treatment. Furthermore, a recent study showed that the selumetinib and sorafenib combination treatment almost eliminated tumor growth and reduced metastatic pulmonary tumor burden in in vivo experiments with MDAMB231 cells [
38]. Nevertheless, additional in vivo preclinical models need to be investigated to clarify whether there is a therapeutic benefit from the combination in non-V600 BRAF mutant tumors.
There is an urgent and unmet need to find effective therapeutic treatment for non-V600 BRAF mutant tumors. Recent studies show that advanced melanoma and colorectal cancer patients with non-V600 BRAF mutation have longer overall survival compared those with both V600E BRAF mutant and wild-type BRAF [
39,
40]. However, in non-V600 BRAF mutant melanoma patients the BRAF V600E selective inhibitors (vemurafenib or dabrafenib) had only disease progression as the best response to therapy [
39]. Importantly, advanced lung cancer patients with non-V600 BRAF mutation has worse overall survival than V600E mutant patients [
5,
41,
42]. Of note, it was reported in a case study, that a female patient with non-V600 BRAF (G469R) mutated lung adenocarcinoma showed dramatic response to sorafenib treatment [
43].
Based on our preclinical findings, sorafenib treatment combined with selumetinib might result in enhanced therapeutic effect also in patients with non-V600 BRAF mutant tumors. Since our study investigated only one in vivo preclinical model, further studies are warranted to confirm whether combinatorial treatment delivers additional benefit compared to monotherapy. Regarding the safety of the proposed combination, a recent phase I study with sorafenib and selumetinib combination showed promising effectiveness and tolerable adverse effects in advanced hepatocellular carcinoma patients [
44].
Conclusion
Currently, there are no approved targeted therapies for non-V600 BRAF mutant cancer patients. Our in vitro data suggests that combination inhibition of RAF and MEK with sorafenib/AZ628 and selumetinib, respectively, should be further explored as a potential approach for inhibiting tumor growth in non-V600 BRAF mutant malignancies.
Acknowledgements
The authors thank Luca Hegedűs for her help in cell-cycle assays and Irén Bodrogi-Mayer, Anita Hídvégi and Violetta Piurkó for their excellent technical assistance.