October 11, 2022
Development of a potential microRNA medicine against KRAS mutantcancers
The main points of this study are as follows.

・ KRAS mutations occur in approximately 40% of all human colorectal
cancers and approximately 90% of all pancreatic cancers. Although KRAS
inhibitors have been developed worldwide, this has been greatly hamperedby the lack of protein structural pockets for drug binding. In recent years,
compounds that can selectively inhibit KRAS with a specific mutation
have been used as drugs to treat lung cancer. However, even such targetedKRAS inhibitors are only temporarily effective due to the high rate of thedevelopment of drug resistance.
・ KRAS signaling feeds into a broad cell proliferation signaling network.
Targeting just KRAS, therefore, does not fully inhibit this signaling
network. Targeting multiple key genes involved in this network by
microRNAs is, therefore, a more effective strategy.
・ The chemically modified microRNA143 that we developed is notreadily degraded, remaining stable in mouse blood, and it suppresses
the expression of several key genes in the KRAS signaling network,
thereby disabling the KRAS signaling network and inducing cell deathat extremely low doses in cancer cells (IC50: ranging from 0.11 –11.17 nM against colon cancer cell lines tested). The anticancer effectof this microRNA was more potent than suppression of the expressionof the individual genes.
・ In a mouse model, the KRAS signaling network was inhibited andtumor growth was significantly reduced. New blood vessels wereformed in the tumors, indicating that the effect of the chemicallymodified microRNA143 may have been enhanced.
KRAS mutations are frequently observed in various types of cancers. However, thedevelopment of effective KRAS-targeting anticancer drugs has been greatly hamperedbecause the KRAS protein structure lacks adequate drug-binding pockets and because
alternative signaling pathways can lead to activation of the broader KRAS signaling
network. Aberrant activation of the PI3K/AKT and MAPK/ERK KRAS-effector
pathways occurs often in many types of cancers, and many therapeutic agents targeting
components of these pathways have been developed. Targeting these pathways is an
important strategy for treating KRAS-mutant cancers. Numerous inhibitors targetingthese pathways have been clinically evaluated. For instance, for the MAPK pathway,inhibitors of RAF, MEK, and ERK have been tested clinically, as have inhibitors of thePI3K/AKT pathway. However, suppressing KRAS-driven cancers remains a challenge.
Global suppression of these signaling pathways is crucial for achieving a durable effecton cancers because inhibition of only a single component or pathway in the signaling
network generally results in resistance to the agent as a result of activation of pararell
signaling pathways. Therefore, there is a pressing need for an effective strategy tobroadly target KRAS-driven cancers. In light of the cellular localization of KRAS, there
is little scope for the development of antibody-based medicines. However, an approachthat appears to be quite promising is downregulation of KRAS expression using smallinterfering RNAs (siRNAs) or microRNAs.
We have developed more than 100 different chemically modified miR143 derivatives
for two main reasons. The first reason is to render the miR143 RNase-resistant andavoid happen immune response (Figure 1). The other reason is to enhance the bindingof target genes of miR143 and thereby suppress the translation of these genes. Amongthese derivatives, a chemically modified miR143-3p#12 (miR-143#12), in which only
the passenger strand is chemically modified, exhibits very potent antiproliferative
activity with good RNase resistance.

Figure 1. Sequence of chemically modified miR-143#12 and nuclease resistance.
Only one RNA strand is chemically modified in miR-143#12 (upper panel), and it
was stable and resistant to degradation in bovine serum (lower panel, left) and mouseblood (lower panel, right).
MiR-143#12 systemically inhibited the KRAS signaling network, which includes theKRAS-MAPK and PI3K-AKT effector signaling pathways, by silencing KRAS, SOS1,AKT, and ERK both in vitro and in vivo in colon cancer cells (1). In the same study, wedemonstrated a synergistic effect of an EGFR inhibitor and miR-143#12 inKRAS-mutant cells. In addition to colorectal cancer, miR-143#12 exhibited tumor
suppressor activity in various types of cancers. We found that miR-143#12 efficiently
suppressed the KRAS signaling network in KRAS-mutant bladder cancer (2), gastriccancer (3), renal cancer (4), and rhabdomyosarcoma cells (5) by systematicallysuppressing not only KRAS but also the PI3K/AKT and MAPK/ERK pathways of thesignaling network. MiR-143#12 has revealed an elaborate miR-143/KRAS cascade,which has not been previously demonstrated in KRAS-mutant colorectal cancer cells(Figure 2).
Figure 2. The entire KRAS signaling
network.
A positive KRAS regulatory feedback loop
exists downstream of AKT and ERK, which
induces expression of KRAS mRNA toreplenish the upstream KRAS protein. The
presence of a KRAS positive regulatory
circuit means that even when KRAS isinhibited,KRAS

signalingisonlytemporarily inhibited as new KRAS protein
is generated by the AKT and ERK effector
signals.
Local administration of miR-143#12 to nude mice implanted subcutaneously withhuman colon cancer cells resulted in marked inhibition of tumor growth, even at low
doses. Analysis of protein expression in treated mouse tumor samples revealed reduced

expression of KRAS network components (KRAS, AKT, ERK, and SOS1). The
antitumor effects of miR-143#12 have also been demonstrated in animal studies. Inaddition, as a new finding, staining of tumor samples with the CD31 marker of bloodvessels showed significant staining at the sites where miR-143#12 had penetrated. Thissuggests that miR-143#12 may induce normal angiogenesis, thereby enhancing its
effects (Figure 3).

Figure 3. Antitumor effect of miR-143#12 in a mouse cancer model.
(Upper left) Change in tumor size over time after miR-143#12 administration, with lessincrease in size for the miR-143#12-treated group compared to the control group.
(Bottom left) Suppression of KRAS signaling network protein expression in tumor
samples of the miR-143#12-treated group. (Right) CD31 staining revealing enhanced
angiogenesis in the miR-143#12-treated group.
Future Prospects
Although KRAS is a key driver of oncogenesis, it has become clear that inhibition ofKRAS, AKT, and ERK alone is not sufficient to cure cancers with KRAS mutations.
However, inhibition of the broader KRAS signaling network by our chemicallymodified miR-143#12 is highly effective. Furthermore, angiogenesis was observed in amouse cancer model, and this was accompanied by antitumor effects, even at low
concentrations. Future development of a drug delivery system that allows exploitationof the effects of systemic administration of miR-143 #12 is desirable.

Journal Title: Molecular Therapy - Nucleic Acids
Title: Chemically modified MIR143-3p exhibited anti-cancer effects by impairing theKRAS network in colorectal cancer cells
Author： Nobuhiko Sugito, Kazuki Heishima, Yukihiro Akao
DOI number: 10.1016/j.omtn.2022.09.001
Publication URL:
https://www.sciencedirect.com/science/article/pii/S2162253122002396
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growth factor receptor inhibitors by a novel synthetic miR-143.
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Mol Ther Methods Clin

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Int J Mol Sci. 2019 Apr

5;20(7):1697. doi: 10.3390/ijms20071697.PMID: 30959742(4) Synthetic miR-143 Exhibited an Anti-Cancer Effect via the Downregulation ofK-RAS Networks of Renal Cell Cancer Cells In Vitro and In Vivo.

Takai T, Tsujino T, Yoshikawa Y, Inamoto T, Sugito N, Kuranaga Y, Heishima K,Soga T, Hayashi K, Miyata K, Kataoka K, Azuma H, Akao Y.

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May 8;27(5):1017-1027. doi: 10.1016/j.ymthe.2019.03.004. Epub 2019 Mar 13.
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＜Research Matters＞
United Graduate School of Drug Discovery and Medical Information Sciences,
Gifu University
Yukihiro Akao
Phone:058-230-7607
E-mail: yakao@gifu-u.ac.jp＜Media Relations＞
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Gifu University
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