br tem effector signaling pathways of
tem (effector-signaling pathways of MAPK/Erk and PI3K/Akt tran-scription factor K-RAS and/or H-RAS).
We speculate that RTK mutations, such as those of EGFR and FGFR, would induce the overexpression of RAS in the downstream. Since miR-143#12 also silenced Akt and Erk, which inactivated growth and survival-related transcription factors, miR-143#12 could sup-press the RAS-signaling networks, including the RAS-positive circuit,
Molecular Therapy: Methods & Clinical Development
to inhibit cell proliferation along with apoptosis in BC cells. On the other hand, the inhibitors for MEK and Akt suppressed the mRNA levels of both K-RAS and H-RAS, which indicated the positive circuit of their expression. K-RAS would contribute to this positive circuit rather than H-RAS, because the silencing of K-RAS, but not that of H-RAS, induced inactivation of both effector-signaling pathways. Therefore, miR-143#12, which extremely silenced K-RAS and its effector-signaling molecules Akt and Erk, as well as SOS1, could
Figure 5. In Vivo Xenografted Mouse
induce a potent anti-tumor effect with apoptosis in K-RAS-dominant 253J-BV BC cells, which reflected the induction of apoptosis in the case of K-RAS mutant T24 BC cells. Previously, we confirmed that ectopic expression of miR-143 induced the inhibition of cancer cell growth of BC through the regulation of cell cycle-associ-ated genes.33 Of course, miR-143#12 may have other anti-cancer mechanisms for BC cells.
We focused on the effect of miR-143#12 by the intravesical infusion against orthotopic BC. As a result, the significant efficacy was obtained. Before, we reported the efficacy of miR-145 by intravesical infusion.34 In this present study, the novel synthetic miR-143#12 as well as miR-145 also exhibited a potent anti-tumor ac-tivity against BC by intravesical infusion with a PIC carrier, which was more excellent in view of the miR-143 levels in the tumor and blood sam-ples, as well as in terms of the anti-tumor activ-ity estimated by western blot analysis of the tumor samples. Interestingly, our result sug-gested that the expression levels of miR-143 were highly expressed even in brain. Therefore, miR-143#12/PIC may acquire the ability to pass the blood-brain barrier after taken by aminonucleoside of xenografted tumor. Further investigation is required in order to qualify the ADMET (ab-sorption, distribution, metabolism, excretion, and toxicity) of miR-143#12. Also, the finding of a potent anti-tumor effect of miR-143#12/
PIC was shown in the H&E-stained tumor samples, as evidenced by severe tumor cell death with fibrosis (Figures S4B and S4D).
As to the PIC groups, we did not obtain pathologically abnormal find-ings in the organs such as liver and kidney in the in vivo experiments (Figures S4B and S4D). The cationic liposomes have been reported to be toxic to hepatocytes,35 which were also observed in our systemic treatment. In both in vivo studies, we were able to confirm the effect
of miR-143#12 through RNAi, as estimated by the result of the Ago2-binding assay using samples of xenografted tumors. It is considered that the PIC nanocarrier with a hydrodynamic diameter of more than 10 nm (Figure S4A) can accumulate in the tumor, probably due to the enhanced permeability and retention (EPR) effect.32,36 In the case of intravesical administration, we consider that PIC-conju-gated miR-143 was taken into blood through the transplanted intratu-moral blood vessel; however, it is not clear whether this effect worked in our mouse model based on the data of the miR-143 tissue distribu-tion in the blood and tumor tissues (Figure 5). Further validation of the anti-cancer effect of miR-143#12, delivered by the PIC nanocarrier, on K-RAS-driven gastrointestinal cancers is now underway.
A novel chemically modified miR-143 systemically suppressed the RAS-signaling networks, including SOS1/RAS-, MAPK/Erk-, and
Figure 6. In Vivo Orthotopic Model Mouse
(A) Changes in tumor weight after treatment with control-miR/PIC or miR-143#12/PIC by intravesical injection. Approximately half of the mice were sacrificed, and the tumor weight was measured at around 30 days after the inoculation. Seven mice were assigned to each group. (B) Survival curves of miR-143#12/PIC and control-miR/PIC groups. (C) Changes in body weight of control-miR/PIC and miR-143#12/PIC groups. (D) Results of Ago2 loading assay using tumor samples from the miR-143#12/PIC group. (E) Levels of RAS and RAS-related proteins in tu-mor samples from the miR-143#12/PIC group. A repre-sentative protein expression profile estimated by western blot is shown. (F) Tissue distribution of miR-143 in the miR-143#12/PIC group (blood, tumor, kidney, and liver).