Despite considerable research on Kâ€ÂÂÃâÃÂRas inhibitors, none had been established until now. We synthesized nucleaseâ€ÂÂÃâÃÂresistant synthetic miRâ€ÂÂÃâÃÂ143 (miRâ€ÂÂÃâÃÂ143#12), which strongly silenced Kâ€ÂÂÃâÃÂRas, its effector signal molecules AKT and ERK, and the Kâ€ÂÂÃâÃÂRas activator Sos1. We examined the antiâ€ÂÂÃâÃÂproliferative effect of miRâ€ÂÂÃâÃÂ143#12 and the mechanism in human colon cancer DLDâ€ÂÂÃâÃÂ1 cell (G13D) and other cell types harboring Kâ€ÂÂÃâÃÂRas mutations. Cell growth was markedly suppressed in a concentrationâ€ÂÂÃâÃÂdependent manner by miRâ€ÂÂÃâÃÂ143#12 (IC50: 1.32 nmol L−1) with a decrease in the Kâ€ÂÂÃâÃÂRas mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of Kâ€ÂÂÃâÃÂRas mRNA expression. MiRâ€ÂÂÃâÃÂ143#12 silenced cytoplasmic Kâ€ÂÂÃâÃÂRas mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miRâ€ÂÂÃâÃÂ143#12 and a lowâ€ÂÂÃâÃÂdose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing Kâ€ÂÂÃâÃÂRas by siRâ€ÂÂÃâÃÂKRas instead of miRâ€ÂÂÃâÃÂ143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miRâ€ÂÂÃâÃÂ143#12 perturbed the Kâ€ÂÂÃâÃÂRas expression system and Kâ€ÂÂÃâÃÂRas 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 miRâ€ÂÂÃâÃÂ143#12 enabled us to understand Kâ€ÂÂÃâÃÂRas signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂdriven colon cancer cell lines.
INTRODUCTION
The 3 classical mammalian ras genes, Kâ€ÂÂÃâÃÂras, Nâ€ÂÂÃâÃÂras and Hâ€ÂÂÃâÃÂras, encode 21â€ÂÂÃâÃÂKd proteins that are members of the guanine nucleotideâ€ÂÂÃâÃÂbinding protein superfamily.1, 2 The canonical properties of Ras are those of a small GTPase that normally cycles between a GTPâ€ÂÂÃâÃÂbound active and a GDPâ€ÂÂÃâÃÂbound inactive state.This cycle is negatively regulated by GTPaseâ€ÂÂÃâÃÂactivating proteins that stimulate the intrinsic GTPase activity and are positively regulated by guanine nucleotide exchange factors (GEF). Ras is normally present in the GDPâ€ÂÂÃâÃÂbound inactive state, which can be changed to the activated state by extracellular stimuli such as the presence of mitogens, cytokines and growth factors. On activation, Ras exerts its functions through proteinâ€ÂÂÃâÃÂprotein interactions with effectors, such as Raf kinase and PI3K, to promote cell growth and survival.
In 1982, mutant Ras genes were detected in human cancers, marking the first discovery of mutated genes in cancer patients. Indeed, Ras mutations are genetic events that have been detected in 30% of all human cancers, with the specific Ras isoform generally differing according to the cancer type.6 Mutations in Kâ€ÂÂÃâÃÂRas account for approximately 85% of all Ras mutations, those in Nâ€ÂÂÃâÃÂRas for approximately 15% and those in Hâ€ÂÂÃâÃÂRas for less than 1%; these are single base missense mutations, mainly in codons 12, 13 or 61 of exons 2 and 3.5, In colon and rectal carcinomas, Kâ€ÂÂÃâÃÂRas is also the predominantly mutated isoform, whereas Nâ€ÂÂÃâÃÂRas mutations are infrequent, and Hâ€ÂÂÃâÃÂRas mutations have not been detected.12 Cancerâ€ÂÂÃâÃÂcausing mutations impair the GTPase activity of Ras, causing Ras to accumulate in the GTPâ€ÂÂÃâÃÂbound active state, which transmits strong downstream signals
MATERIALS AND METHODS
• Cell culture and cell viability
• Assay for stability of miRNA in vitro
• Western blotting
• Cell transfection with miRNA or siRNA
• Quantitative RTâ€ÂÂÃâÃÂPCR
• Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂGTP assay
• Assay for luciferase activity
• In vivo tumor model and administration of the synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143
• Statistics
RESULTS
Growth inhibition by synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 of Kâ€ÂÂÃâÃÂRas mutant human colon cancer DLDâ€ÂÂÃâÃÂ1 (Kâ€ÂÂÃâÃÂRasG13D) cell line
To explore the use of miRâ€ÂÂÃâÃÂ143 as a possible Kâ€ÂÂÃâÃÂRas inhibitor for Kâ€ÂÂÃâÃÂRas mutant colon cancer cells, we designed and synthesized some miRâ€ÂÂÃâÃÂ143 having different structures of the double strand for acquiring nuclease resistance. These results indicated that synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 are potent growth suppressors at extremely low concentrations and possibly suppressed the expression and activation systems of Kâ€ÂÂÃâÃÂRas in Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂdriven DLDâ€ÂÂÃâÃÂ1 colon cancer cells, which was not observed with Am.
Kâ€ÂÂÃâÃÂRas effector signaling pathways enhanced the transcription of Kâ€ÂÂÃâÃÂRas itself
It has been reported that silencing Ras by miRâ€ÂÂÃâÃÂ143 inhibits the growth of Ras mutant human cancer cell lines both in vitro and in vivo.32, 33 When we compared the effects of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 and siRâ€ÂÂÃâÃÂKRas, the latter being considered to be equal to Ras inhibitors on the cell growth of Kâ€ÂÂÃâÃÂRas mutant cells that the expression level of Kâ€ÂÂÃâÃÂRas was decreased by either of 2 different siRNA for Kâ€ÂÂÃâÃÂRas, which bind to the ORF and 3′UTR regions of Kâ€ÂÂÃâÃÂRas, respectively, 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. βâ€ÂÂÃâÃÂactin was used as an internal control
Synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 silenced Sos1 by RNAi we show that the ectopic expression of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 decreased the level of Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂGTP. According to in silico prediction tools in TargetScan, Sos1 has an miRâ€ÂÂÃâÃÂ143 binding site in its 3′UTR. To validate Sos1 as a target gene of miRâ€ÂÂÃâÃÂ143, we performed a luciferase reporter assay. Growth inhibition by combined treatment with synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 and cetuximab
The effectiveness of cetuximab is now limited to patients with Kâ€ÂÂÃâÃÂRas wildâ€ÂÂÃâÃÂtype tumors. Above we showed that the ectopic expression of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 significantly decreased the levels of Kâ€ÂÂÃâÃÂRas mRNA and Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂGTP through perturbation of the positive circuit and activation of Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂGDP
Tumor suppressive effect of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 on in vivo experiment
To further validate the growth inhibitory effect of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143, we performed an in vivo study in which controlâ€ÂÂÃâÃÂmiR or synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 were administrated systemically every 72 hours (750 μg/kg/administration) 4 times to nude mice that had been subcutaneously inoculated with DLDâ€ÂÂÃâÃÂ1 (Kâ€ÂÂÃâÃÂRasG13D) cells
Effects of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 in other Kâ€ÂÂÃâÃÂRas mutation harboring colon cancer cell lines
To further validate the effects of synâ€ÂÂÃâÃÂmiRâ€ÂÂÃâÃÂ143 on other Kâ€ÂÂÃâÃÂRas wild and mutant human colon cancer cell lines, we performed the same experiments by using SW48 (Kâ€ÂÂÃâÃÂRasWild/Bâ€ÂÂÃâÃÂRafWild), HT29 (Kâ€ÂÂÃâÃÂRasWild/Bâ€ÂÂÃâÃÂRaf V600E) and SW480 (Kâ€ÂÂÃâÃÂRasG12V/Bâ€ÂÂÃâÃÂRafWild) cells.
DISCUSSION:
We demonstrated that chemicallyâ€ÂÂÃâÃÂmodified miRâ€ÂÂÃâÃÂ143#12 exhibited a potent suppressive effect on Kâ€ÂÂÃâÃÂRas networks and that Kâ€ÂÂÃâÃÂRasâ€ÂÂÃâÃÂmutant colon cancer DLDâ€ÂÂÃâÃÂ1 cells established a positive circuit through the constitutive Kâ€ÂÂÃâÃÂRas activationâ€ÂÂÃâÃÂstimulation of effector signaling pathways, resulting in enhanced nuclear Kâ€ÂÂÃâÃÂRas transcription, which was clearly disclosed by using the potent chemicallyâ€ÂÂÃâÃÂmodified miRâ€ÂÂÃâÃÂ143#12 This positive circuit was also true in the case of another Kâ€ÂÂÃâÃÂRas mutant, one in SW480 cells The miRâ€ÂÂÃâÃÂ143#12 impaired Kâ€ÂÂÃâÃÂRas networks including the positive circuit by silencing the key molecules of the networks Furthermore, a wellâ€ÂÂÃâÃÂmodified drug delivery system will be required to distribute miRâ€ÂÂÃâÃÂ143#12 into tumors as an RNA medicine against Rasâ€ÂÂÃâÃÂdriven cancers.
Note: This work is partly presented at 17th Annual Congress on Pharmaceutics & Drug Delivery Systems on September 20-22, 2018 held in Prague, Czech Republic.
Journal of Clinical Medicine and Therapeutics received 95 citations as per Google Scholar report