Combination of AURKA inhibitor and MEK inhibitor strongly enhances G1 arrest and induces synergistic antitumor effect on KRAS or BRAF mutant colon cancer cells

doi:10.21203/rs.3.rs-4340988/v1

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Combination of AURKA inhibitor and MEK inhibitor strongly enhances G1 arrest and induces synergistic antitumor effect on KRAS or BRAF mutant colon cancer cells

https://doi.org/10.21203/rs.3.rs-4340988/v1

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Background

In colorectal cancer, RAS and BRAF are major mutation points in the RAS-MAPK signaling pathway. These gene mutations are known to be important causes of resistance to anti-EGFR antibody therapies. MEK inhibitors have been hoped to be an effective therapy for RAS or BRAF mutation tumors; however, their suppression effect for the RAS-MAPK signaling pathway is not sufficient when used as a single agent. Aurora kinase A (AURKA), one of the mitotic kinases, is expected to be a novel therapeutic target in cancers. Recently, it has been reported that AURKA interacts with the EGFR-RAS-MAPK signaling pathway. In this study, we examined whether the combination of MK-5108 (AURKA inhibitor) and trametinib (MEK inhibitor) enhanced the antitumor effect for colon cancer cell lines.

Methods

We used four cell lines, HCT116, LoVo (TP53 wild, KRAS mutant), DLD1 (TP53 mutant, KRAS mutant), and HT29 (TP53 mutant, BRAF mutant). To determine the antitumor effects, a WST-8 assay was performed. Combination index was used to evaluate the efficacy of the combination of MK-5108 and trametinib. EdU assay and PI staining were performed to estimate cell cycle arrest and cell apoptosis. To identify the molecular mechanisms of the antitumor effects of the combination therapy, protein expressions were evaluated by immunoblot analysis.

Results

The combination of MK-5108 and trametinib showed synergistic enhancements of antitumor effect in all cell lines. MK-5108 and trametinib induced G2/M arrest and G1 arrest, respectively, and the two-drug combination further enhanced G1 arrest. The addition of MK-5108 to trametinib enhanced the suppression of p-ERK and other G1/S progression-related proteins expression. In HCT116 cells, harboring wild-type TP53, the combination therapy induced more potent cell proliferation suppression and apoptosis induction than in TP53 knockout cells. These were related to enhancement of p53 expression and caspase activation.

Conclusion

The combination of MK-5108 and trametinib showed synergistic enhancement of antitumor effect with either KRAS or BRAF mutation. Furthermore, the combination therapy could be more effective in wild-type TP53 cells.

AURKA

MEK

colon cancer

KRAS

BRAF

ERK

cell cycle

apoptosis

Recent developments in chemotherapeutic strategies for colorectal cancer have dramatically improved the prognosis, especially since the use of molecular targeted therapeutics. However, the presence of several genetic variants causes resistance to some molecular targeted therapeutics.

The epidermal growth factor receptor (EGFR) has multiple downstream signaling pathways, which are known to play an important role in the oncogenesis, growth and progression of various cancers [1]. In colorectal cancer, anti-EGFR antibodies are frequently used as one of the standard therapies. However, several activating mutations have been reported in the RAS-MAPK signaling pathway, one of the major downstream pathways of EGFR [2]. Activating mutations in RAS or BRAF genes have been found in about 50% of colorectal cancers, and these mutations are important causes of resistance to anti-EGFR antibody therapies [3, 4]. Although targeted therapies for a few types of RAS/RAF mutant colorectal cancers (such as those with KRAS^G12C and BRAF^V600E mutations) have been clinically applied in recent years [5, 6], these mutations are relatively rare and no effective treatment has been developed for the majority of patients with other RAS/BRAF mutations.

MEK is a downstream molecule of the RAS-MAPK signaling pathway and was hoped to be a new target of treatment for RAS/RAF mutant tumors. Many MEK inhibitors have been developed, and some have progressed to clinical trials for several tumors, including colon cancer. Regrettably, the antitumor effect of MEK inhibitors is not sufficient as monotherapy because reactivation of RAS-MAPK signaling is caused by a feedback mechanism and reduces the MEK inhibition effect [7, 8]. It is thought that the RAS-MAPK signaling pathway should be suppressed at multiple points to enhance the antitumor effect of MEK inhibitors. Nowadays, MEK inhibitors are used clinically in combination with other drugs for tumors with specific genetic mutations.

Aurora kinase A (AURKA) is one of the mitotic kinases, a family of proteins regulating mitosis. AURKA is known to play an important role in the late G2 phase to the early M phase of cell division, including centrosome maturation, spindle assembly, entry to mitosis, and cytokinesis [9, 10]. Gene amplification and overexpression of AURKA have been observed in various cancers, and AURKA gene amplification has been reported in about 50% of colorectal cancers [11]. A relationship between AURKA overexpression and poor prognosis has also been reported, suggesting that AURKA may function as an oncogenic driver [12]. Inhibition of AURKA activity induces suppression of cell proliferation, and many AURKA inhibitors have been developed. MK-5108 is a highly selective small molecular inhibitor of AURKA. MK-5108 induces G2/M arrest and apoptosis and has shown antitumor effects both in vitro and in vivo [13, 14]. AURKA is also known to interact with various cancer-related signals, such as activation of the Wnt signaling pathway, and suppression of p53 and BRCA function [15, 16]. Recently, several studies have pointed out that AURKA interacts with the EGFR-RAS-MAPK signaling pathway and that they upregulate each other [17–20]. Thus, we hypothesized that the addition of an AURKA inhibitor to a MEK inhibitor may serve to enhance suppression of the RAS-MAPK signaling pathway. In the present study, we investigated whether a combination of the AURKA inhibitor MK-5108 and the MEK inhibitor trametinib shows a synergistic antitumor effect in colon cancer cell lines harboring several different phenotypes (TP53 wild/mutant and KRAS or BRAF mutant). Furthermore, we explored the molecular mechanisms of the antitumor effect of the combination therapy both in terms of cell cycle arrest and apoptosis.

Reagents

The selective aurora kinase A inhibitor MK-5108 was purchased from Chemscene (Monmouth Junction, NJ, US), and the MEK inhibitor trametinib was purchased from Cayman Chemical (Ann Arbor, MI, US). MK-5108 and trametinib were dissolved in DMSO to concentrations of 10 mmol/L and 324 µmol/L, respectively.

Cell Lines and Culture

Four human colon cancer cell lines (HCT116, LoVo, DLD1, HT29) and one isogenic variant (HCT116 p53-/-) were used. HCT116 and HCT116 p53-/- were obtained from Horizon Discovery (Cambridge, UK). LoVo, DLD1, and HT29 were obtained from ATCC (Rockville, MD, US). HCT116, HCT116 p53-/-, and DLD1 cells were cultured in RPMI 1640 medium (MilliporeSigma, Burlington, MA, US) supplemented with 10% fetal bovine serum (FBS) (MilliporeSigma). LoVo cells were cultured in Ham’s F-12 medium (Nacalai Tesque, Kyoto, JP) with 10% FBS. HT29 cells were cultured in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, US) with 10% FBS.

Cell Viability Analysis and Combination Index

Cell viabilities were measured by WST-8 assay with Cell Count Reagent SF (Nacalai Tesque) according to the manufacturer’s protocol. Cells plated in 96-well plates were cultured overnight. MK-5108 and trametinib were added alone or in combination and cultured for 3 days, then analyzed using the iMark microplate reader (Bio-Rad, Hercules, CA, US). The absorbance of the plates was read at wavelengths of 540 nm and 620 nm. The efficacy of the combination therapy was evaluated using the Chou-Talalay method for drug combination using CalcuSyn software (Biosoft, Cambridge, UK) [21]. A combination index (CI) < 0.9 indicates synergism, CI = 0.9–1.1 indicates additivity, and CI > 1.1 indicates antagonism.

Cell Cycle Analysis

Cells were plated in 60 mm dishes. After overnight culture, the medium was changed, agents were added, and then cultured for another 22 hours. EdU was then added to the medium and cultured for another 2 hours. EdU assay was performed for harvested cells using Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher) according to the manufacturer’s protocol. Flow cytometry was performed using BD FACSVerse, and the percentage of each cell cycle phase was calculated using FlowJo software (both produced by BD Biosciences, Franklin Lakes, NJ, US).

Cell Proliferation Analysis

Cells were plated in 90 mm dishes and cultured overnight. Subsequently, cells were stained with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher). After 3 days of culture, cells were harvested, and flow cytometry was performed using Attune NxT (Thermo Fisher) and analyzed with FlowJo. Cells cultured in serum-free medium were used as non-proliferating cells. The amount of cell proliferation was calculated using the division index.

Cell Death Analysis

To evaluate the apoptotic cell population, propidium iodide staining was performed. After adding the agents, cells were cultured for 24 hours and then harvested. Cells were stained with propidium iodide using Cycletest Plus DNA reagent kit (BD Biosciences). Flow cytometry was performed using BD FACSVerse, and the sub-G1 fraction was analyzed using FlowJo. Cytotoxicity was determined by LDH assay using the Cytotoxicity LDH Assay Kit-WST (Dojindo, Kumamoto, JP). To reduce measuring LDH contained in FBS, cells were cultured in Opti-MEM I (Thermo Fisher) with 2.5% FBS. After 3 days of culture, cytotoxicity and cell viability were determined from LDH amounts in medium and in live cells, respectively, according to the manufacturer's protocol.

Immunoblot Analysis

Cell lysates were prepared using a RIPA buffer supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Both SDS-PAGE and immunoblot analysis were performed as previously described [22]. The following are the primary antibodies used in this study: mouse monoclonal antibody against p21, rabbit monoclonal antibody against FAS, cleaved-Caspase 8, rabbit polyclonal antibody against PUMA, cleaved-PARP, cleaved-Caspase 9 (Cell Signaling Technology, Danvers, MA, US), mouse polyclonal antibody against phospho-ERK (p-ERK) (Thr202/Tyr204) (Abcepta, San Diego, CA, US), mouse monoclonal antibody against p53 (MONOSAN, Uden, NL), mouse monoclonal antibody against E2F1 (KH20/KH95) (MilliporeSigma), mouse monoclonal antibody against Rb (BD Biosciences), and rabbit polyclonal antibody against β-actin (MBL, Tokyo, JP). Both HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG antibodies (GE Healthcare, Chicago, IL, US) were used as secondary antibodies. Chemiluminescent reactions were visualized by ECL Select Western Blotting Detection Reagent (GE Healthcare), and Ez-Capture Imaging System (Atto, Tokyo, JP).

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Analysis of statistical significance between three or more groups was performed using one-way ANOVA with Tukey's test for post hoc analysis. If equality of variance was not shown by Levine's test, the significance was evaluated using the Games-Howell test. P values < 0.05 were considered statistically significant. Statistical analysis was carried out using SPSS version 26.0 (IBM, Armonk, NY, US).

Combination of MK-5108 and trametinib shows a synergistic antitumor effect

To investigate the efficacy of the combination of MK-5108 and trametinib, we examined the antitumor effect in HCT116 cells, colon cancer cells harboring wild-type (wt) TP53 and mutant-type (mt) KRAS, by WST-8 assay (Fig. 1A). Monotherapy of MK-5108 or trametinib reduced cell viabilities in a dose-dependent manner. Furthermore, CIs indicated that the combination therapy of these two drugs synergistically enhanced the antitumor effect. We also examined the efficacy of the combination therapy in other colon cancer cell lines: LoVo harboring wt TP53 and mt KRAS, DLD1 harboring mt TP53 and mt KRAS, and HT29 harboring mt TP53 and mt BRAF (Figs. 1B-D). The cell viabilities of these three cell lines were almost dose-dependently suppressed in both the monotherapies and in the combination therapy, similarly to HCT116 cells. CIs of these cell lines were below 0.9 except for the lowest dose level of HT29, indicating that the combination therapy also synergistically enhances the antitumor effect. In all cell lines, CIs were relatively lower during the combination therapy with nearly 50% of the suppression dose levels of MK-5108 and trametinib. Therefore, we set the concentrations of MK-5108 and trametinib as follows: HCT116 (0.4µM, 8nM), LoVo (0.8µM, 4nM), DLD1 (16µM, 320nM), and HT29 (3µM, 1nM).

The combination therapy induces G1 phase arrest and inhibits cell cycle progression

Since both MK-5108 and trametinib induce cell cycle arrest at different phases, we examined the influence of the combination therapy on the cell cycle. In HCT116 cells, MK-5108-alone increased the G2/M phase and polyploid cell fractions, trametinib-alone increased the G1 phase cell fraction, and both monotherapies decreased the S phase cell fraction (Figs. 2A and S1A). Furthermore, the combination therapy reduced the S phase cell fraction more pronouncedly. These results indicate that MK-5108 and trametinib, both of which inhibit cell cycle progression at different points, induced an enhanced cell cycle arrest in the combination therapy. Next, we evaluated the number of cell divisions of HCT116 in MK-5108, trametinib, and the combination therapy. As shown in Fig. 2F, the division index of the combination therapy was less than that of MK-5108-alone or trametinib-alone therapies. Compared with the control, the combination therapy reduced the division index by 74%. This result shows that MK-5108 and trametinib do indeed inhibit cell division, and that the combination therapy inhibits it more potently. Additionally, in the other cell lines, the combination therapy pronouncedly reduced the S phase cell fraction (Figs. 2B-D, S1B-D). Interestingly, although the G2/M phase and polyploid cell fractions appeared to increase most with the combination therapy, the percentage of positive cells for phospho-Histone H3 (p-HH3), known as a mitosis marker, was lower with the combination therapy than with MK-5108-alone therapy (Figure S2). This result shows that the increase of the G2/M phase and polyploid cell fractions with the combination therapy did not indicate an increase of mitotically arrested cells. Mitotic arrest results in three types of cell fates: complete cell division, cell death, and mitotic slippage which induces polyploidy [23]. Our findings suggest that the combination therapy mainly increased diploid or more polyploid cells induced by mitotic slippage, and that those cells were in the G1 phase. Thus, we expect that cell cycle arrest, and especially the G1 phase arrest, plays an important role in enhancing the antitumor effects of the combination of MK-5108 and trametinib.

The combination therapy inhibits the expression of G1/S progression-related proteins

To explore the molecular mechanism of G1 arrest promoted by the combination therapy, we examined G1/S progression-related proteins using immunoblot analysis (Fig. 3A). In all cell lines, the combination therapy reduced expressions of p-ERK, a downstream factor of MEK and an important factor for cell cycle progression, especially G1/S progression. pp-Rb and E2F1, two other important factors of G1/S progression, also tended to decrease more with the combination therapy. In addition to the G1 arrest effect of trametinib and the G2/M arrest effect of MK-5108, synergistic suppression of ERK activity and other downstream factors may induce a strong cell cycle arrest, especially G1 arrest. For HCT116 and LoVo cells, both harboring wt TP53, we examined the expression of the CDK inhibitor p21, which suppresses G1/S progression, and p53, a transcription factor of p21. As shown in Fig. 3B, p53 and p21 expressions were enhanced by MK-5108 in both cell lines. Especially in HCT116 cells, the expressions of p53 and p21 were further increased by the addition of trametinib. AURKA and p53 mutually inhibit each other, and it is known that inhibition of AURKA increases p53 expression in wt TP53 tumor cells. Activation of the p53 function may contribute to the acceleration of cell cycle arrest. The cell cycle of HCT116p53-/- cells (p53 knockout HCT116 cells) was analyzed to assess the contribution of p53 function to cell cycle arrest. As shown in Figs. 2E and S1E, the combination therapy also reduced the S phase fraction in HCT116p53-/- cells. However, the reduction degree of the S phase fraction of HCT116p53-/- cells from the control to the combination therapy was lower than that of HCT116 cells. In HCT116p53-/-, the number of cell divisions was also lowest with the combination therapy, but it only reduced the division index by 64% compared with the control (Figure S3). Expressions of cell cycle-related proteins were also examined in HCT116p53-/- cells (Fig. 3C). While p-ERK expression was strongly suppressed by the combination therapy, there was no significant increase in p21 expression. The suppression of pp-Rb expression with the combination therapy was also weaker than that achieved in HCT116 cells. These results suggest that the combination therapy may induce cell cycle arrest more strongly in wt TP53 cells.

Apoptosis is promoted with the combination therapy in wt TP53 cells

Next, we analyzed the effect of the combination of MK-5108 and trametinib on the induction of cell death (Fig. 4A). In HCT116 cells, MK-5108-alone therapy increased the sub-G1 fraction, known to reflect apoptosis, while trametinib-alone therapy did not show a clear increase. However, the addition of trametinib to MK-5108 increased the sub-G1 fraction more than the MK-5108-alone therapy. To confirm whether the combination therapy induced cell death, the LDH assay was performed in HCT116 cells (Fig. 4F). Compared with MK-5108-alone or trametinib-alone therapies, the combination therapy increased the number of dead cells relative to viable cells. These results suggest that the combination therapy also promoted cell death more effectively than monotherapy in HCT116 cells. However, the same results were not obtained in all other cell lines. As shown in Fig. 4B-E, the MK-5108-alone therapy increased the sub-G1 fraction in all cell lines, but trametinib could not enhance the sub-G1 increasing effect of MK-5108 except in LoVo cells. In HCT116p53-/- cells, sub-G1 fractions were markedly decreased with the MK-5108-alone therapy or the combination therapy when compared with sub-G1 fractions in HCT116 cells. Next, we examined the expression of cleaved-PARP, known as a marker of apoptosis, in each cell line by immunoblot analysis (Fig. 4G). In HCT116 and LoVo cells, looking at the wt TP53 cell lines, the cleaved-PARP expression level was increased in the order of the combination, MK-5108-alone, and trametinib-alone, and it was correlated with the change of the sub-G1 fraction. On the other hand, DLD1 cells did not show an increase of cleaved-PARP expression with the combination therapy, and HT29 cells showed only a small amount of cleaved-PARP expression. In HCT116p53-/- cells, as with the sub-G1 fraction, cleaved-PARP expression was also markedly decreased compared with its expression in HCT116 cells. The LDH assay also showed that cell death was actually reduced in HCT116p53-/- (Figure S4). Similarly, in LoVo cells, cleaved-PARP expression was reduced by knockdown of p53 (Figure S5). Apoptosis-related proteins were examined in HCT116 and LoVo cells, in which cell death was promoted by the combination therapy (Fig. 4H). Both cleaved-Caspase-8 and cleaved-Caspase-9 (apoptosis activation markers of extrinsic and intrinsic pathways) were upregulated by the combination therapy. These results suggest that activated p53 promotes apoptosis with the combination therapy in the wt TP53 cell lines, which may contribute to the synergistic enhancement of the antitumor effect.

The combination therapy may be more effective for wt TP53 cells

Finally, we investigated the antitumor effect of the combination of MK-5108 and trametinib in HCT116p53-/- cells (Fig. 4I). The CIs were below 0.9 at most concentrations, indicating that the combination therapy synergistically enhanced the antitumor effect even in HCT116 cells with p53 knocked out.

In this study, we showed that the combination of MK-5108 and trametinib has a synergistic antitumor effect on colon cancer cell lines. Previously, two basic studies about the effectiveness of the combination of AURKA inhibitor and MEK inhibitor have been reported, one on melanoma cell lines [24] and the other on colorectal cancer cell lines [25]. However, these studies have not described the involved mechanisms in detail. The main contributions of our study are showing that the combination of AURKA inhibitor and MEK inhibitor is effective regardless of TP53 mutation and RAS or BRAF mutation, and revealing in detail the mechanisms of the efficacy of the combination therapy.

We showed that efficient induction of cell cycle arrest is the most important mechanism in the synergistic enhancement of antitumor effects of the combination therapy. The results of the EdU assay revealed that the G2/M phase and polyploid cell fractions were increased in the combination therapy. Whereas it appeared that the combination therapy had promoted mitotic arrest, in fact, mitotic arrest was not promoted. Anti-mitotic drugs (such as microtubule targeting agents and mitotic kinase inhibitors) induce mitotic arrest. It is known that some tumor cells exit from mitotic arrest without normal cell division and become polyploid cells [23]. This phenomenon is referred to as ‘mitotic slippage’ and is considered to be one of the major causes of resistance to chemotherapy [26]. Many studies have reported that AURKA inhibitors also promote polyploid cells [12, 27, 28], so the increased G2/M phase and polyploid cell fractions with the combination therapy may have included a significant number of polyploid cells in the G1 phase. Therefore, in addition to the G1 arrest effect of trametinib and the G2/M arrest effect of MK-5108, the enhancement of G1 arrest by the combination therapy may be an important factor in promoting cell cycle arrest.

We focused on ERK activity, an important factor of cell cycle progression, as a mechanism of G1 arrest promoted by the combination of MK-5108 and trametinib. ERK is a downstream factor of the RAS-MAPK signaling pathway; however, the AURKA inhibitor also decreased ERK activity in this study. The relationships between AURKA and the RAS-MAPK signaling pathway have been reported. Although the detailed mechanisms have not yet been revealed, it is thought that AURKA upregulates the RAS-MAPK signaling pathway [17, 29, 30]. Some previous studies have reported that AURKA inhibition by a small molecular inhibitor [19] or small interference RNA [29] decreased ERK activity in RAS mutant cell lines. In this study, MK-5108 enhanced the effect of trametinib on ERK activity reduction, which could be an important factor in the enhancement of G1 arrest.

Furthermore, we showed that wild-type p53 may contribute to the induction of cell cycle arrest. In wt TP53 cell lines, MK-5108 increased the expression of p53 and its transcriptional target p21. The relationship between AURKA and p53 has been studied extensively, and it is known that these two molecules form a negative feedback loop. AURKA phosphorylates p53, therefore inhibiting the transcriptional activity of p53 and promoting an MDM2-mediated p53 degradation by ubiquitination [16, 31]. As shown in our study, it is also known that suppressing AURKA activity increases the expression of p53 [32, 33]. In this study, especially in HCT116 cells, adding trametinib to MK-5108 enhanced even more the expression of p53 and p21. Several studies showed that ERK also suppresses p53 via an upregulation of MDM2 [34, 35]. The combination of MK-5108 and trametinib strongly induced p53 expression, and it may coordinately promote G1 arrest with decreased ERK activity.

In all cell lines, MK-5108 increased the sub-G1 fraction and the expression of cleaved-PARP, indicating apoptosis induction. When mitotic arrest is prolonged in tumor cells, while some cells complete normal division or transition to polyploid cells through mitotic slippage, other cells fail to escape mitotic arrest leading to cell death [23, 36]. Though the mechanism is unclear, caspase-mediated pathways have been reported as being involved in this process [37]. AURKA also inhibits apoptosis via suppression of FADD [38], induction of Bcl-2 [38, 39], and activation of NF-κB [40]. This indicates that AURKA inhibition promotes acceleration and derepression of apoptosis. In this study, while trametinib did not enhance the apoptosis promotion effect of MK-5108 in mt TP53 cells, the combination therapy enhanced it in wt TP53 cells. In HCT116 cells, the expressions of p53, Fas, cleaved-Caspase 8, and cleaved-PARP were correlated. This suggests that the combination of MK-5108 and trametinib activated the extrinsic apoptosis pathway via p53 activation, inducing apoptosis synergistically. In fact, compared to what occurred in HCT116 cells, cell death and apoptosis-related protein expression were reduced in HCT116p53-/- cells. It also suggested that p53 may contribute significantly to apoptosis. However, in LoVo cells, while both cleaved-Caspase 8 and cleaved-Caspase 9 correlated with cleaved-PARP, there was no correlation with the expressions of Fas and PUMA, their upstream factors and transcription targets of p53. There seems to be another molecular pathway involved in apoptosis in LoVo cells. Although we could not elucidate it, p53 knockdown decreased the expression of cleaved-PARP, indicating that p53 also plays an important role in apoptosis in LoVo cells.

In this study, we demonstrated that the combination of MK-5108 and trametinib shows a synergistic antitumor effect in RAS/RAF mutant colon cancer and that the combination therapy may be more effective in wt TP53 cells (Fig. 5). The combination of AURKA inhibitor and MEK inhibitor could be a novel strategy for RAS/RAF mutant colon cancer. Additionally, in RAS/RAF wild colon cancer, it has been reported that some tumors acquiring resistance to anti-EGFR antibody therapy harbor new mutations in the RAS-MAPK signaling pathway [41]. The combination therapy may constitute salvage therapy for such patients. However, this study was carried out using only in vitro experiments. Moreover, there were no statistically significant differences in the combination index between HCT116 and HCT116p53-/-. Hence, further in vivo evaluation is needed, including a comparison of the presence of the TP53 mutation.

In conclusion, the combination therapy of an AURKA inhibitor (MK-5108) and a MEK inhibitor (trametinib) exerts a synergistic antitumor effect on colon cancer cells regardless of TP53 and RAS/RAF mutation status via suppression of cell division by promotion of cell cycle arrest. The presence of wt TP53 may more effectively enhance the antitumor effect through strong cell cycle arrest induction and through synergistic promotion of apoptosis.

AURKA

Aurora kinase A

EGFR

epidermal growth factor receptor

combination index

p-ERK

phospho-ERK

p-HH3

phospho-Histone H3

ppRB

hyperphosphorylated Rb

Acknowledgements

We would like to thank Cosmin Florescu from the Medical English Communications Center, University of Tsukuba for English language editing.

Authors’ contributions

MS, YY, TM and KF conceived and designed the study. MS performed the experiments. MS, YY, and KT confirm the authenticity of all the raw data. MS performed the statistical analyses. MS, YY, and KT wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the scholarship donation from the Pharmaceutical Business Division of Yakult Honsha Co.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Gastroenterology, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575

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No competing interests reported.

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Combination of AURKA inhibitor and MEK inhibitor strongly enhances G1 arrest and induces synergistic antitumor effect on KRAS or BRAF mutant colon cancer cells

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials and Methods

Reagents

Cell Lines and Culture

Cell Viability Analysis and Combination Index

Cell Cycle Analysis

Cell Proliferation Analysis

Cell Death Analysis

Immunoblot Analysis

Statistical Analysis

Results

Combination of MK-5108 and trametinib shows a synergistic antitumor effect

The combination therapy induces G1 phase arrest and inhibits cell cycle progression

The combination therapy inhibits the expression of G1/S progression-related proteins

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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