A role for K-ras in conferring resistance to the MEK inhibitor, CI-1040

Abstract

PD184352/CI-1040 is a potent and selective MEK1/2 inhibitor that represents the first MEK-targeted agent to enter clinical trials. Here, we report the development and molecular characterization of CI-1040 resistance in the murine colon 26 (C26) carcinoma cell line. The growth rate of the resistant line (C26/CI-1040r) in the presence of 2 microM CI-1040 is comparable to that of parental C26 cells in the absence of CI-1040. C26/CI-1040r cells are approximately 100-fold more resistant than the parental line to CI-1040 inhibition in soft agar and are less sensitive to the induction of apoptosis that normally occurs in response to CI-1040 treatment. K-ras expression is significantly elevated in C26/CI-1040r cells. We confirmed a causative role for K-ras in conferring resistance to CI-1040 by transfecting K-ras into parental C26 cells, whereupon an elevation in the levels of phosphorylated ERK1/2 was observed in addition to resistance to CI-1040. Furthermore, an in vivo-derived MEK inhibitor-resistant line also shows increased K-ras expression. Our data suggest that increasing activated K-ras expression represents one potential mechanism by which tumor cells that initially are responsive to blockade of the MAP kinase pathway can overcome their sensitivity to MEK inhibition.

Figure 1

C26/CI-1040^r cells are resistant to the growth-inhibitory effects of CI-1040. (A) Dose response of CI-1040 in C26/CI-1040^r and its parental cell line C26 in a soft agar growth assay. (B) Cell morphology and cell density of C26 and C26/CI-1040^r in cell culture. Cells were seeded in six-well plates at 50,000 cells/well. One day after cell plating, the cells were treated with DMSO or 2 µM CI-1040 for 2 days. (C) Effect of CI-1040 on cell monolayer growth. After pictures were taken as shown in (B), both floating and attached cells were combined and then counted with a Coulter Counter. (D–F) Effect of CI-1040 on C26 parental cell growth was measured by [¹⁴C]thymidine incorporation.

Figure 1

C26/CI-1040^r cells are resistant to the growth-inhibitory effects of CI-1040. (A) Dose response of CI-1040 in C26/CI-1040^r and its parental cell line C26 in a soft agar growth assay. (B) Cell morphology and cell density of C26 and C26/CI-1040^r in cell culture. Cells were seeded in six-well plates at 50,000 cells/well. One day after cell plating, the cells were treated with DMSO or 2 µM CI-1040 for 2 days. (C) Effect of CI-1040 on cell monolayer growth. After pictures were taken as shown in (B), both floating and attached cells were combined and then counted with a Coulter Counter. (D–F) Effect of CI-1040 on C26 parental cell growth was measured by [¹⁴C]thymidine incorporation.

Figure 1

C26/CI-1040^r cells are resistant to the growth-inhibitory effects of CI-1040. (A) Dose response of CI-1040 in C26/CI-1040^r and its parental cell line C26 in a soft agar growth assay. (B) Cell morphology and cell density of C26 and C26/CI-1040^r in cell culture. Cells were seeded in six-well plates at 50,000 cells/well. One day after cell plating, the cells were treated with DMSO or 2 µM CI-1040 for 2 days. (C) Effect of CI-1040 on cell monolayer growth. After pictures were taken as shown in (B), both floating and attached cells were combined and then counted with a Coulter Counter. (D–F) Effect of CI-1040 on C26 parental cell growth was measured by [¹⁴C]thymidine incorporation.

Figure 2

The C26/CI-1040^r cell line is less sensitive to CI-1040-induced apoptosis. Cells were plated on regular or polyHEMA-coated 96-well tissue culture plates. One day after cell plating, the cells were treated with the indicated concentration of CI-1040. The cells were then harvested for the apoptosis assay 24 hours after compound treatment. (A) Apoptosis assay for C26 and C26/CI-1040^r cells grown on regular tissue culture plates. (B) Apoptosis assay for C26 and C26/CI-1040^r cells grown on polyHEMA-coated 96-well plates.

Figure 3

C26/CI-1040^r is less sensitive to CI-1040-induced cell cycle arrest than the parental C26 cell line. Cells were treated for 24 hours with CI-1040 and then cell cycle analysis was run on the BD LSR flow cytometer. A total of 25,000 events was collected per analysis.

Figure 4

The basal level of ERK1/2 phosphorylation increases in C26/CI-1040^r cells, but the sensitivity of MEK1/2 to CI-1040 inhibition is not significantly altered. (A) Effect of CI-1040 on ERK phosphorylation in C26 and C26/CI-1040^r cells. Cells were treated with the indicated concentration of CI-1040 for 1 hour and then harvested for Western analysis. Two different exposures of the same immunoblots are shown. (B) Effect of CI-1040 on MEK1 and MEK2 in C26 and C26/CI-1040^r cells. MEK1 or MEK2 from C26 or C26/CI-1040^r cells was immunoprecipitated with the corresponding antibody. The kinase activities were assayed with GST-ERK1K71R as substrate as described in the Materials and Methods section. The exposure time was adjusted to give a similar basal level of intensity (without compound treatment). (C) Effect of CI-1040 treatment on ERK and MEK phosphorylation. C26 (P) and C26/CI-1040^r (R) cells were treated with DMSO (-) or 2 µM CI-1040 (+) as described in the Materials and Methods section.

Figure 5

K-ras is activated and overexpressed in CI-1040-resistant cells. (A) K-ras mRNA is upregulated in C26/CI-1040^r cells. K-ras signal data are derived from genechip experiments (Table 1). (B and D) K-ras protein is overexpressed in both in vitro- and in vivo-derived resistant cells. The K-ras from C26 parental or resistant cells was immunoprecipitated with a pan-ras antibody (SC-35AC) and blotted with K-ras antibody (SC-30). (C) K-ras is activated. The Ras activation assay was performed with parental and C26/CI-1040^r cell lysate using Upstate's ras activation assay kit.

Figure 6

Overexpression of K-ras in C26 cells confers CI-1040 resistance. CI29-2, CI29-13, and CI29-16 are single clones from C26 parental cells transfected with K-rasV12 selected in the presence of 2 µM CI-1040. 33-10, 33-11, and 33-4 are single clones from C26 parental cells transfected with K-rasV12 selected in the absence of CI-1040. (A) Soft agar assay. (B) Overexpression of K-ras increased ERK1/2 phosphorylation.