Impairment of K-Ras signaling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miR-143

Abstract

Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC₅₀ : 1.32 nmol L^-1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines.

Keywords: K-Ras; Ras inhibitor; Sos1; epidermal growth factor receptor; miR-143.

Figure 1

Synthetic miR‐143 showed a marked growth inhibition in colon cancer DLD‐1 cells. A, Sequences of synthetic miR‐143 used in this study. F RNA, Fluoro‐RNA; Ome RNA, O‐Methyl RNA; PS, phosphorothioate. B, Remaining percentage of each miR‐143, Am, #1 and #12, remaining in the presence of FBS evaluated by performing RT‐qPCR. The 0‐min value of each miR‐143 is indicated as 100%. The mean value was taken for each time. C, DLD‐1 cells were transfected with control‐miR, Am, #1 or #12 for 72 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control is indicated as 100%. The IC₅₀ values are indicated in the figure. D, Western blot analysis was performed to determine the levels of the ERK5, total Ras, ERK1/2 and AKT after transfection with each miR‐143 at 10 nmol L⁻¹. β‐actin was used as an internal control. E, Expression levels of K‐Ras mRNA as relative ratios with respect to the GAPDH expression level. The expression level of mRNA was calculated using the ΔΔCt method. F, DLD‐1 cells were transfected with control‐miR, Am, #1 or #12 at 10 nmol L⁻¹ for 72 h. The level of K‐Ras‐GTP was estimated by performing western blot analysis at 72 h after the transfection. The levels of GTP‐bound K‐Ras were quantified by densitometry scanning and normalized to the total levels of K‐Ras. *P < .05 vs control (Student's t test) in the figures

Figure 2

Contribution of K‐Ras to growth and its signaling networks. A, DLD‐1 cells were transfected with control‐miR or siR‐KRas (ORF or 3′UTR) for 48 h. Western blot analysis was performed to determine the level of total Ras. β‐actin was used as an internal control. B, DLD‐1 cells were transfected with control‐miR or siR‐KRas for 48 h. The cell viability was estimated at 48 h after the transfection. The cell viability of the control is indicated as 100%. C, DLD‐1 cells were transfected with control‐miR, siR‐KRas, #1 or #12 for 24, 48, 72 or 96 h. Western blot analysis was performed to determine the levels of Ras, p‐AKT, AKT, p‐ERK1/2 and ERK1/2. β‐actin was used as an internal control. The relative ratios of band density of Ras with respect to those of β‐actin were quantified by densitometry. Then, the comparative ratios of the controls as “1” are indicated in the Figure. D and F, DLD‐1 cells were treated with an AKT inhibitor (AKT inhibitor IV) or MEK inhibitor (PD98059) for 24 h. The K‐Ras mRNA level was estimated at 24 h after the treatment. The mRNA level of the control (0; DMSO alone) is indicated as 100%. E and G, Western blot analysis was performed to determine the levels of Ras at 24 h after the treatment. β‐actin was used as an internal control. *P < .05 vs control (Student's t test) in the figures

Figure 3

Sos‐1 is a target of the miR‐143. A, Vector with the binding site for miR‐143 is indicated as Wild; and that without it as Mutant. DLD‐1 cells were transfected with Wild/control‐miR, Wild/#12, Mutant/control‐miR or Mutant/#12 for 48 h. The luciferase activities were estimated at 48 h after the transfection. The luciferase activity of the control (Cont; control‐miR) is indicated as 100%. B, DLD‐1 cells were transfected with control‐miR, A, #1 or #12 at 10 nmol L⁻¹ for 48 h or 72 h. The expression level of Sos1 mRNA, given as relative ratios with respect to the GAPDH expression level, was evaluated by RT‐qPCR. The expression level of control (Cont; control‐miR alone) is indicated as “1.” C, Western blot analysis was performed to determine the level of Sos1. β‐actin was used as an internal control. D, DLD‐1 cells were co‐transfected with anti‐miR‐143 inhibitor and/or each miR for 72 h. The cell viability was estimated at 72 h after the transfection. The cell viability control (Control: control‐miR alone) is indicated as 100%. E, Western blot analysis was performed to determine the levels of ERK5 and Sos1. β‐actin was used as an internal control. The relative ratios of the band density of ERK5 and Sos1 with respect to those of β‐actin were quantified by densitometry. Then, the comparative ratios of the controls as “1” are indicated in the Figure. F, DLD‐1 cells were transfected with siR‐Sos1 (2, 5 nmol L⁻¹) for 48 h. The cell viability was estimated at 48 h after the transfection. The cell viability control (0; control‐miR alone) is indicated as 100%. G, Western blot analysis was performed to determine the levels of Sos1, Ras, p‐AKT, AKT, p‐ERK1/2 and ERK1/2 proteins. β‐actin was used as an internal control. H, DLD‐1 cells were transfected with siR‐Sos1 (2, 5 nmol L⁻¹) for 48 h. The expression level of K‐Ras mRNA as relative ratios with respect to the GAPDH expression level was evaluated by RT‐qPCR. The expression level of control (0; control‐miR alone) is indicated as “1.” I, DLD‐1 cells were transfected with control or siR‐Sos1 for 48 h. The level of K‐Ras‐GTP was estimated by performing western blot analysis at 48 h after the transfection. The levels of GTP‐bound K‐Ras were quantified by densitometry scanning and normalized to the total levels of K‐Ras. *P < .05 vs control (Student's t test) in the figures

Figure 4

Schematic diagram indicating the “positive circuit” of K‐Ras mRNA expression and the target genes of miR‐143#12

Figure 5

Ectopic expression of Syn‐miR‐143 recovered the efficacy of EGFR inhibitors. A, DLD‐1 cells were treated with cetuximab for 24 h. The cell viability was estimated at 24 h after the treatment. The cell viability of the control (0; PBS alone) is indicated as 100%. B, DLD‐1 cells were transfected with control‐miR, #1 or #12 for 48 h and then treated with cetuximab for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (Control; control‐miR and PBS) is indicated as 100%. C, Western blot analysis was performed to determine the levels of ERK5, Ras, p‐ERK1/2, ERK1/2, p‐AKT, AKT and PARP‐1 proteins. β‐actin was used as an internal control. D, DLD‐1 cells were transfected with siR‐KRas (3′UTR) for 48 h and then treated with cetuximab for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (Control; control‐miR and PBS) is indicated as 100%. E, Western blot analysis was performed to determine the levels of ERK5, Ras, p‐ERK1/2, ERK1/2, p‐AKT, AKT and PARP‐1 proteins. β‐actin was used as an internal control. F, DLD‐1 cells were treated with lapatinib for 24 h. The cell viability was estimated at 24 h after the treatment. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (0; PBS alone) is indicated as 100%. G, DLD‐1 cells were transfected with control‐miR, #1 or #12 for 48 h and then treated with lapatinib for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (Control; control‐miR and PBS) is indicated as 100%. *P < .05 vs control (Student's t test) in the figures

Figure 6

Tumor suppressive effect of the miR‐143 on DLD‐1 cell xenografted mice. A, DLD‐1 cells were injected subcutaneously into BALB/c nude mice. When the tumor volume had reached 100 mm³, 8 mice were sorted into each group. Then, control‐miR, #1 or #12 was intravenously injected every 72 h. At the second administration of #12, cetuximab was intraperitoneally injected every 72 h. The tumor volume was calculated as 0.5236 L₁ (L₂)², where L₁ is the long axis and L₂ is the short axis of the tumor. C vs #1; P = .03 (day 16), C vs #12; P = .02 (day 13), C vs #12 + E; P = .05 (day 10), #12 vs #12 + E; P = .04 (day 19). The dose of 750 μg/kg/injection was given intravenously to each treated mouse every 3 d. The protocol is shown in the figure. B, The body weight was measured at every administration time. C, Western blot analysis was performed to determine the levels of K‐Ras, p‐AKT, AKT, p‐ERK1/2, ERK1/2 and ERK5 proteins in the xenografted tumor samples. β‐actin was used as an internal control. *P < .05 vs control (Student's t test)

Figure 7

Effects of the miR‐143 on other types of K‐Ras mutant cells. A, B and C, SW48, HT29 and SW480 cells, respectively, were transfected with control‐miR, #1 or #12 for 72 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control is indicated as 100%. D and H, SW48, HT29 and SW480 cells were treated with cetuximab or lapatinib for 24 h. The cell viability was estimated at 24 h after the treatment. The cell viability of the control (0; PBS alone) is indicated as 100%. E, F and G, SW48, HT29 and SW480 cells, respectively, were transfected with #1 or #12 for 48 h and then treated with cetuximab for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (Control; control‐miR and PBS) is indicated as 100%. I, J and K, SW48, HT29 and SW480 cells, respectively, were transfected with #1 or #12 for 48 h and then treated with lapatinib for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (Control; control‐miR and PBS) is indicated as 100%. *P < .05 vs control (Student's t test)