Euro Pharmaceutics 2018: Impairment of KaRas signalling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miRa143- Yukihiro Akao- Gifu University

Despite considerable research on Kâ€ÂRas inhibitors, none had been established until now. We synthesized nucleaseâ€Âresistant synthetic miRâ€Â143 (miRâ€Â143#12), which strongly silenced Kâ€ÂRas, its effector signal molecules AKT and ERK, and the Kâ€ÂRas activator Sos1. We examined the antiâ€Âproliferative effect of miRâ€Â143#12 and the mechanism in human colon cancer DLDâ€Â1 cell (G13D) and other cell types harboring Kâ€ÂRas mutations. Cell growth was markedly suppressed in a concentrationâ€Âdependent manner by miRâ€Â143#12 (IC50: 1.32 nmol L−1) with a decrease in the Kâ€ÂRas mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of Kâ€ÂRas mRNA expression. MiRâ€Â143#12 silenced cytoplasmic Kâ€ÂRas mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miRâ€Â143#12 and a lowâ€Âdose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing Kâ€ÂRas by siRâ€ÂKRas instead of miRâ€Â143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miRâ€Â143#12 perturbed the Kâ€ÂRas expression system and Kâ€Â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 miRâ€Â143#12 enabled us to understand Kâ€ÂRas signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in Kâ€ÂRasâ€Âdriven colon cancer cell lines.

INTRODUCTION
The 3 classical mammalian ras genes, Kâ€Âras, Nâ€Âras and Hâ€Âras, encode 21â€ÂKd proteins that are members of the guanine nucleotideâ€Âbinding protein superfamily.1, 2 The canonical properties of Ras are those of a small GTPase that normally cycles between a GTPâ€Âbound active and a GDPâ€Âbound inactive state.This cycle is negatively regulated by GTPaseâ€Âactivating proteins that stimulate the intrinsic GTPase activity and are positively regulated by guanine nucleotide exchange factors (GEF). Ras is normally present in the GDPâ€Âbound 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€Âprotein 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€ÂRas account for approximately 85% of all Ras mutations, those in Nâ€ÂRas for approximately 15% and those in Hâ€ÂRas 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€ÂRas is also the predominantly mutated isoform, whereas Nâ€ÂRas mutations are infrequent, and Hâ€ÂRas mutations have not been detected.12 Cancerâ€Âcausing mutations impair the GTPase activity of Ras, causing Ras to accumulate in the GTPâ€Âbound 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€ÂPCR
•    Kâ€ÂRasâ€ÂGTP assay
•    Assay for luciferase activity
•    In vivo tumor model and administration of the synâ€ÂmiRâ€Â143
•    Statistics

RESULTS
Growth inhibition by synâ€ÂmiRâ€Â143 of Kâ€ÂRas mutant human colon cancer DLDâ€Â1 (Kâ€ÂRasG13D) cell line
To explore the use of miRâ€Â143 as a possible Kâ€ÂRas inhibitor for Kâ€ÂRas mutant colon cancer cells, we designed and synthesized some miRâ€Â143 having different structures of the double strand for acquiring nuclease resistance. These results indicated that synâ€ÂmiRâ€Â143 are potent growth suppressors at extremely low concentrations and possibly suppressed the expression and activation systems of Kâ€ÂRas in Kâ€ÂRasâ€Âdriven DLDâ€Â1 colon cancer cells, which was not observed with Am.
Kâ€ÂRas effector signaling pathways enhanced the transcription of Kâ€ÂRas itself
It has been reported that silencing Ras by miRâ€Â143 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€ÂmiRâ€Â143 and siRâ€ÂKRas, the latter being considered to be equal to Ras inhibitors on the cell growth of Kâ€ÂRas mutant cells that the expression level of Kâ€ÂRas was decreased by either of 2 different siRNA for Kâ€ÂRas, which bind to the ORF and 3′UTR regions of Kâ€ÂRas, 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€Âactin was used as an internal control
Synâ€ÂmiRâ€Â143 silenced Sos1 by RNAi we show that the ectopic expression of synâ€ÂmiRâ€Â143 decreased the level of Kâ€ÂRasâ€ÂGTP.  According to in silico prediction tools in TargetScan, Sos1 has an miRâ€Â143 binding site in its 3′UTR. To validate Sos1 as a target gene of miRâ€Â143, we performed a luciferase reporter assay. Growth inhibition by combined treatment with synâ€ÂmiRâ€Â143 and cetuximab
The effectiveness of cetuximab is now limited to patients with Kâ€ÂRas wildâ€Âtype tumors. Above we showed that the ectopic expression of synâ€ÂmiRâ€Â143 significantly decreased the levels of Kâ€ÂRas mRNA and Kâ€ÂRasâ€ÂGTP through perturbation of the positive circuit and activation of Kâ€ÂRasâ€ÂGDP
Tumor suppressive effect of synâ€ÂmiRâ€Â143 on in vivo experiment
To further validate the growth inhibitory effect of synâ€ÂmiRâ€Â143, we performed an in vivo study in which controlâ€ÂmiR or synâ€ÂmiRâ€Â143 were administrated systemically every 72 hours (750 μg/kg/administration) 4 times to nude mice that had been subcutaneously inoculated with DLDâ€Â1 (Kâ€ÂRasG13D) cells
Effects of synâ€ÂmiRâ€Â143 in other Kâ€ÂRas mutation harboring colon cancer cell lines

To further validate the effects of synâ€ÂmiRâ€Â143 on other Kâ€ÂRas wild and mutant human colon cancer cell lines, we performed the same experiments by using SW48 (Kâ€ÂRasWild/Bâ€ÂRafWild), HT29 (Kâ€ÂRasWild/Bâ€ÂRaf V600E) and SW480 (Kâ€ÂRasG12V/Bâ€ÂRafWild) cells.

DISCUSSION:
We demonstrated that chemicallyâ€Âmodified miRâ€Â143#12 exhibited a potent suppressive effect on Kâ€ÂRas networks and that Kâ€ÂRasâ€Âmutant colon cancer DLDâ€Â1 cells established a positive circuit through the constitutive Kâ€ÂRas activationâ€Âstimulation of effector signaling pathways, resulting in enhanced nuclear Kâ€ÂRas transcription, which was clearly disclosed by using the potent chemicallyâ€Âmodified miRâ€Â143#12 This positive circuit was also true in the case of another Kâ€ÂRas mutant, one in SW480 cells The miRâ€Â143#12 impaired Kâ€ÂRas networks including the positive circuit by silencing the key molecules of the networks Furthermore, a wellâ€Âmodified drug delivery system will be required to distribute miRâ€Â143#12 into tumors as an RNA medicine against Rasâ€Âdriven 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.
 

Author(s): Yukihiro Akao

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