Low‑dose trametinib and Bcl‑xL antagonist have a specific antitumor effect in KRAS‑mutated colorectal cancer cells

Abstract

KRAS-mutant colorectal cancer (CRC) is a highly malignant cancer with a poor prognosis, however specific therapies targeting KRAS mutations do not yet exist. Anti-epidermal growth factor receptor (EGFR) agents, including cetuximab and panitumumab, are effective for the treatment of certain patients with CRC. However, these anti-EGFR treatments have no effect on KRAS-mutant CRC. Therefore, new therapeutic strategies targeting KRAS-mutant CRC are urgently needed. To clarify the direct effect of KRAS gene mutations, the present study transduced mutant forms of the KRAS gene (G12D, G12V and G13D) into CACO-2 cells. A drug-screening system (Mix Culture assay) was then applied, revealing that the cells were most sensitive to the MEK inhibitor trametinib among tested drugs, Cetuximab, Panitumumab, Regorafenib, Vemurafenib, BEZ-235 and Palbociclib. Trametinib suppressed phosphorylated ERK (p-ERK) expression and inhibited the proliferation of KRAS-mutant CACO-2 cells. However, low-dose treatment with trametinib also increased the expression of the anti-apoptotic protein Bcl-xL in a dose-dependent manner, leading to drug resistance. To overcome the resistance of KRAS-mutant CRC to apoptosis, the combination of trametinib and the Bcl-xL antagonist ABT263 was assessed by in vitro and in vivo experiments. Compared with the effects of low-dose trametinib monotherapy, combination treatment with ABT263 had a synergistic effect on apoptosis in mutant KRAS transductants in vitro. Furthermore, in vivo combination therapy using low-dose trametinib and ABT263 against a KRAS-mutant (G12V) xenograft synergistically suppressed growth, with an increase in apoptosis compared with the effects of trametinib monotherapy. These data suggest that a low dose of trametinib (10 nM), rather than the usual dose of 100 nM, in combination with ABT263 can overcome the resistance to apoptosis induced by Bcl-xL expression, which occurs concurrently with p-ERK suppression in KRAS-mutant cells. This strategy may represent a promising new approach for treating KRAS-mutant CRC.

Drug sensitivity profile of minor KRAS mutations in colorectal cancer using mix culture assay: The effect of AMG‑510, a novel KRAS G12C selective inhibitor, on colon cancer cells is markedly enhanced by the combined inhibition of MEK and BCL‑XL

“…The method of creating these vectors is shown in the paper by Koyama et al ( 9 ).…”

“…We have developed and reported a mixed culture assay for stable and reliable screening of effective therapeutic targets, transduced with the pMXs-IRES-GFP vector, and analyzed using a flow cytometer ( 8 , 9 ). For this assay, we retrovirally transduced wild-type KRAS, major mutant KRAS genes (G12D, G12V, G13D) and minor mutant KRAS genes (G12A, G12C, G12S, Q61H, and A146) into CACO-2 cells using the pMX-IRES-GFP vector.…”

“…It is impossible to keep the GFP positive rate constant at 50%. Approximately plus or minus 10% is considered to be an acceptable range ( 8 , 9 ). On the first day, the mixed cells were seeded at a 20% confluency on a 12-well plate and were cultured for 12 days with molecular targeting agents.…”

“…We calculated the relative proliferation ratio (RPR) using the following formula, the day 0 GFP-positive rate (%) (A), and the day 12 GFP-positive rate (%) (B). The outline of this experimental system, the calculation method for RPR, and the experimental example are shown in our previous report ( 9 ).…”

“…Otherwise, the next most frequent G12A, G12C, G12S, Q61H and A146T were described as minor KRAS mutations. To assess the sensitivity of molecularly targeted drugs for KRAS mutations, Mix Culture Assays ( 8 , 9 ) were performed. First, we evaluated the resistance of EGFR drugs to minor KRAS mutations in colorectal cancer cells, the sensitivity of MEK and BCL-XL inhibitors, and their combined effects.…”