Targeted Cancer Cell Killing by Highly Selective miRNA-Triggered Activation of a Prokaryotic Toxin–Antitoxin System

Abstract

Although not evolved to function in eukaryotes, prokaryotic toxin Kid induces apoptosis in human cells, and this is avoided by co-expression of its neutralizing antitoxin, Kis. Inspired by the way Kid becomes active in bacterial cells we had previously engineered a synthetic toxin-antitoxin system bearing a Kis protein variant that is selectively degraded in cells expressing viral oncoprotein E6, thus achieving highly selective killing of cancer cells transformed by human papillomavirus. Here we aimed to broaden the type of oncogenic insults, and therefore of cancer cells, that can be targeted using this approach. We show that appropriate linkage of the kis gene to a single, fully complementary, target site for an oncogenic human microRNA enables the construction of a synthetic toxin-antitoxin pair that selectively kills cancer cells overexpressing that particular microRNA. Importantly, the resulting system spares non-targeted cells from collateral damage, even when they overexpress highly homologous, though non-targeted, microRNAs.

Bacterial Toxin-Antitoxin Systems’ Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology

“…Similarly, alterations in the miR-21 miRNA expression within the MCF-7 breast cancer cells were used for the development of a yefM-yoeB- based (type II TA system) strategy for the selective killing of malignant cells in vitro. The 3′-untranslated region of yefM antitoxin gene was enriched in the miR-21 target sites, which ensured the antitoxin mRNA degradation within the cancer cells, and led to selective in vitro ablation of MCF-7 cancer cells [ 181 , 182 ].…”

Functional and Structural Diversity of Bacterial Contact-Dependent Growth Inhibition Effectors

“…When the toxin and immunity genes are transcribed, the oncomiRNA binds the complementary sequence and initiates degradation of the immunity mRNA. Thus, in cancer cells, transcription of the immunity gene is silenced while toxin transcription continues, leading to toxin-induced cell death ( Turnbull et al, 2019 ; Houri et al, 2020 ).…”

“…Anti-cancer applications to date involve the design of TA pairs where the antitoxin is specifically degraded in cancer cells freeing the toxin to eradicate cancer cells ( Preston et al, 2016 ; Turnbull et al, 2019 ; Houri et al, 2020 ). For example, in human papilloma virus (HR-HPV) induced cervical cancer cells, oncoprotein E6 binds and induces the polyubiquitination of specific target proteins, resulting in target protein degradation.…”

“…Another approach takes advantage of microRNA-driven oncogenic stress, where miRNA (noncoding RNA) base pairs with complementary target sites (miRts) to inhibit translation or induce degradation of target mRNAs. Here, a cancer cell specific miRt is encoded downstream of the antitoxin gene, resulting in cancer cell specific silencing of the antitoxin resulting in toxin activation ( Figure 5D ) ( Turnbull et al, 2019 ; Houri et al, 2020 ). This strategy has been utilized by two different TA pairs along with two different miRNA/miRt sequences indicating that this approach could be applied to target specific cancer cells utilizing a variety of different TA systems.…”