Trametinib potentiates TRAIL‐induced apoptosis via FBW7‐dependent Mcl‐1 degradation in colorectal cancer cells

Abstract Trametinib is a MEK1/2 inhibitor and exerts anticancer activity against a variety of cancers. However, the effect of Trametinib on colorectal cancer (CRC) is not well understood. In the current study, our results demonstrate the ability of sub‐toxic doses of Trametinib to enhance TRAIL‐mediated apoptosis in CRC cells. Our findings also indicate that Trametinib and TRAIL activate caspase‐dependent apoptosis in CRC cells. Moreover, Mcl‐1 overexpression can reduce apoptosis in CRC cells treated with Trametinib with or without TRAIL. We further demonstrate that Trametinib degrades Mcl‐1 through the proteasome pathway. In addition, GSK‐3β phosphorylates Mcl‐1 at S159 and promotes Mcl‐1 degradation. The E3 ligase FBW7, known to polyubiquitinate Mcl‐1, is involved in Trametinib‐induced Mcl‐1 degradation. Taken together, these results provide the first evidence that Trametinib enhances TRAIL‐mediated apoptosis through FBW7‐dependent Mcl‐1 ubiquitination and degradation.


| INTRODUC TI ON
Colorectal cancer (CRC) is the second most commonly reported cancer and a major cause of cancer-related death worldwide. 1,2 The median survival time of patients with metastatic colorectal cancer has been reported to be approximately 8 months with palliative treatment, and the median survival time extends to 25.8-31.4 months when standard chemotherapy is administered. 3 As the major driving events of CRC progression, RAF and RAS mutations, along with TNM staging, may help in the clinical management of CRC. 4 In addition, pathological staging and MSI status can help clinicians choose adjuvant therapy. 5 Furthermore, the mutation states of PIK3CA, BRAF (V600E) and KRAS suggest the possibility of anti-EGFR treatment in CRC. 6 Current CRC treatment involves adjuvant therapy with irinotecan/oxaliplatin and 5-fluorouracil, which increases patient survival by 1 year. 7,8 Monoclonal antibody therapy, including treatment with cetuximab and bevacizumab, has advanced CRC treatment, [9][10][11] but the availability of other potent CRC drugs is lacking. New and effective anti-CRC therapies are therefore urgently required.
Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent. 12,13 TRAIL (part of the TNF-α superfamily) binds to specific death receptors termed TRAIL-R1 (DR4) and TRAIL-R2 (DR5) to induce tumour lethality through the extrinsic and intrinsic apoptotic pathways. 12,14,15 Through extrinsic signalling, TRAIL forms a multiprotein cell death signalling axis involving DR4 and DR5, FADD, and effector caspase 8. 16 Signalling through this complex leads to the cleavage and activation of caspase-3 and the subsequent apoptotic cell death. [16][17][18] The intrinsic pathway involves cell death mediated through mitochondrial events. 18,19 TRAIL-mediated tumour cell death can occur in response to a range of anticancer drugs. 20 However, various cancers exhibit resistance to TRAIL, which raises questions about its efficacy as a monotherapy. 12,21 This resistance can be circumvented through dual therapies that sensitize cancer cells to TRAIL, but the discovery of such agents is challenging. 22 It has been reported that Bcl-2 and Bcl-xL inhibitors can enhance cancer cell sensitivity to TRAIL. 23,24 Trametinib (Mekinist) is a selective MEK1/2 inhibitor with activity against BRAF V600 melanomas. 25,26 Trametinib is currently approved for cases of metastatic, unresectable melanomas harbouring the BRAF-V600E/K mutation and can be combined with dabrafenib to improve its therapeutic efficacy. 27,28 However, the effect and mechanism of Trametinib on CRC cells have not been well studied.
Mcl-1 is a very unstable protein, and the degradation of Mcl-1 can be triggered by a variety of stresses, including anticancer drugs. 29 Mcl-1 protein stability and activity are regulated by posttranslational modifications, such as phosphorylation. 30 The Mcl-1 protein contains a proline/glutamic acid/serine/threonine (PEST) region, which is phosphorylated. 31  phorylation also affects its anti-apoptotic activity and interactions with other Bcl-2 family proteins. 7,8 Here, we assessed the ability of Trametinib to sensitize CRC tumours to TRAIL-mediated cell death in CRC. We provide the first evidence of the ability of Trametinib to enhance CRC apoptosis in combination with TRAIL, and this effect is mediated by Mcl-1 degradation.

| Gene silencing (siRNA)
siRNAs against Mcl-1 (sc-35877) and FBW7 (sc-37547) and a control siRNA (scrambled; sc-37007) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The indicated cells were seeded in 12-well plates for 24 hours. The Lipofectamine RNAi Max reagent (Invitrogen) was used for siRNA transfections for 24 hours. The cells were treated with Trametinib/TRAIL for 24 hours for further analysis.

| MTT assay
To assess the viability, 1 × 10 4 cells in 96-well flat-bottom plates were treated with increasing concentrations of Trametinib or TRAIL as indicated for 72 hours. To each well, 20 μL of 5 mg/mL MTT reagent (Roche, Basel, Switzerland) was added, and the plates were incubated for 1 hour in the tissue culture incubator at 37°C. The crystals of formazan were solubilized with 150 μL DMSO after the media were removed. The absorbance at 450 nm was determined by microplate reader.

| Colony formation assay
For the colony-forming assays, the HCT116 cells were treated with Trametinib, TRAIL or their combination for 24 hours, plated in 12well plates at equal numbers (500 cells) and cultured for 2 weeks.
The cells were washed with PBS, and the colonies were fixed (methanol ~95%) and stained using crystal violet solution.

| Assessment of apoptosis
Apoptotic cells were identified using the FITC Annexin V/PI Apoptosis Detection Kit, and fragmented nuclei were assessed through Hoechst 33258 staining. Briefly, the cells were exposed to Trametinib/TRAIL for 24 hours in binding buffer and labelled Annexin V-FITC was added for 15 minutes. The apoptotic cells were assessed by flow cytometry (BD FACSCanto II).

| Co-immunoprecipitation
For Co-IP assays, the cells were lysed by scrape-harvesting and suspended in 1 mL of lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.5% Nonidet P-40) supplemented with a protease inhibitor cocktail (Sigma). The cell lysates were collected and centrifuged for 5 minutes at 12 600 g (4°C). The clarified lysates were labelled with 2 μg of primary antibodies (ON, 4°C) followed by the addition of protein G beads for 1 hour at 4°C. The beads were then washed with cold lysis buffer and centrifuged. The bound proteins were extracted from the beads using 2× Lamelli buffer and assessed by Western blot assay.

| Statistical analysis
Statistical analysis was carried out using GraphPad InStat V software (GraphPad Software Inc., San Diego, CA, USA). The results are expressed as the mean of arbitrary values ± SD. All the results were evaluated using unpaired Student's t test. P < 0.05 was considered significant.

| Trametinib/TRAIL synergistically promote CRC apoptosis
Trametinib is known to induce cell death in melanoma, leukaemia and lung cancer cells. 35 10 μmol/L ( Figure 1A). Since HCT116 cells are more sensitive than other cell lines, we selected this cell line for the subsequent experiments. Moreover, we analysed the effect of TRAIL on HCT116 cells.
We found that the IC50 was higher than 50 ng/mL ( Figure 1B). For the combination, we found strong synergistic effects of Trametinib and TRAIL in HCT116 cells ( Figure 1C). The combination index (CI) values of Trametinib and TRAIL are shown in Figure 1D. Thus, for the following experiments, we used 10 ng/mL for the next combination treatments. When Trametinib and TRAIL were combined, higher levels of cytotoxicity were observed in HCT116 and DLD1, RKO and HT29 cells ( Figure 1E-H), while NCM356 cells showed minimal losses in viability ( Figure 1I); those outcomes indicates that the F I G U R E 2 Trametinib sensitizes TRAIL-induced apoptosis in CRC cells. A, HCT116 cells were treated with 0.1 μmol/L Trametinib, 10 ng/ mL TRAIL or their combination 24 h. Cell morphology was examined under a light microscope. Attached cells were counted. B, HCT116 cells plated in six-well cell culture plates were treated with 0.1 μmol/L Trametinib, 10 ng/mL TRAIL, or their combination for 24 h. After 14 days, the plates were stained for cell colonies with crystal violet dye, and photographs of colonies taken using a digital camera. C, HCT116 cells were treated with 0.1 μmol/L Trametinib, 10 ng/mL TRAIL or their combination for 24 h. Apoptosis was analysed by a nuclear fragmentation assay. D, HCT116 cells were treated with 0.1 μmol/L Trametinib, 10 ng/mL TRAIL, or their combination for 24 h. Apoptosis was analysed by Annexin V/PI staining followed by flow cytometry. E, HCT116 cells were treated with 0.1 μmol/L Trametinib, 10 ng/mL TRAIL, or their combination for 24 h. Indicated proteins were analysed by Western blotting. F, HCT116 cells pre-treated with 10 μmol/L z-VAD-fmk for 1 h were treated with 0.1 μmol/L Trametinib, 10 ng/mL TRAIL, or their combination for 24 h.

| Trametinib enhances TRAILmediated apoptosis
We next assessed the synergism of Trametinib/TRAIL by investigating their effects on HCT116 morphology (Figure 2A). We Thus, Trametinib enhances TRAIL-induced apoptosis through the induction of extrinsic and intrinsic apoptosis.

| Trametinib down-regulates Mcl-1 to sensitize CRC cells to TRAIL
We next investigated whether Trametinib sensitizes HCT116 cells to TRAIL through the stimulation of death receptor pathways. As shown in Figure 3A Our findings demonstrated that Trametinib exerts its effects by decreasing Mcl-1 expression.

| Trametinib induces Mcl-1 degradation in a ubiquitin-proteasome manner
Given these findings, we further examined the relationship between Therefore, we next assessed the influence of MG132, a proteasome inhibitor, on Trametinib-induced Mcl-1 degradation. Figure 4D shows that MG132 significantly inhibited Mcl-1 degradation in response to Trametinib. Our findings also showed that Trametinib promoted Mcl-1 ubiquitination in HCT116 cells ( Figure 4E). The data described above indicate that Trametinib down-regulates Mcl-1 levels in a ubiquitin-proteasome-dependent manner.

| Trametinib enhances the Mcl-1 and FBW7 interaction in CRC cells
FBW7 is an E3 ligase known to ubiquitinate Mcl-1 and target it for proteasomal degradation. 38 We therefore investigated the effect of
In agreement with this finding, GSK3β silencing also inhibited the effects of Trametinib on Mcl-1 ( Figure 6C). We also observed a reduced ability of Trametinib to degrade Mcl-1 when S159 of Mcl-1 was mutated to S159A ( Figure 6D). Taken together, these data revealed that pS159 of Mcl-1 is required for its Trametinib-stimulated degradation. The phosphorylation of Mcl-1 at S159 has been previously identified as a key signal that depends on GSK-3β to degrade S159 phosphorylated Mcl-1. 50 Our findings show that FBW7-mediated Mcl-1 degradation requires S159 phosphorylation and GSK-3β activation.

| D ISCUSS I ON
In addition, S159 phosphorylation regulates the binding of FBW7 to Mcl-1. Overall, these results indicate that FBW7 mediates the S159 phosphorylation-dependent Mcl-1 protein turnover.
In summary, our findings demonstrate a new mechanism of Trametinib in sensitization to TRAIL. These findings add new knowledge to our understanding of the role of Trametinib in the pathophysiology and treatment of CRC. To the best of our knowledge, these are the first data to reveal that Trametinib enhances TRAIL sensitization by targeting Mcl-1 via ubiquitin-proteasome degradation.

ACK N OWLED G EM ENTS
This work was supported by Natural Science Foundation of Liaoning Province (grant no. 2019-ZD-0922).

CO N FLI C T O F I NTE R E S T
The authors have declared that no conflicts of interest exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.