Corbin et al. · Molecular Therapy Nucleic Acids · 2021

Seed-mediated RNA interference of androgen signaling and survival networks induces cell death in prostate cancer cells

Corbin Joshua M., Georgescu Constantin, Wren Jonathan D., Xu Chao, Asch Adam S., Ruiz-Echevarria Maria J.

The study

What was asked, and what was found

Corbin et al., Molecular Therapy Nucleic Acids, 2021, set out to study what happens to prostate cancer cells when the tumour suppressor TMEFF2 is lost, and found something else. Short hairpin RNAs designed against TMEFF2 killed the cells and significantly inhibited xenograft growth in mice, and nine independent hairpins against the gene collapsed androgen receptor signalling on one blot. They also killed cell lines that do not express TMEFF2 at all, which is the first sign that the target was not the cause.

Settling that needed a reagent that silences TMEFF2 and nothing else. The group used a pool of four antisense oligonucleotides against TMEFF2 from AUM BioTech, with a non-target control oligo, in LNCaP prostate cancer cells. TMEFF2 protein fell, and the androgen receptor and prostate specific antigen did not move. A CRISPR-Cas9 knockdown of the same gene gave the matching answer on viability. Together those two arms showed that the short hairpin RNA phenotype was independent of TMEFF2, and the paper goes on to trace it to short seed matches that let those hairpins behave like microRNAs against many survival genes at once.

The delivery matters for anyone reading this as product evidence. In this study the oligos were transfected into the cells at 250 nM with a lipid reagent, not added to the medium, and the paper does not say which AUM line was supplied. What the experiment does show well is specificity: an antisense pool against one transcript moved that transcript and left the surrounding signalling network alone, in a paper whose entire argument is that a widely used RNA interference tool did not.

Key findings

Nine independent short hairpin RNAs against TMEFF2 were read side by side in LNCaP prostate cancer cells, with the androgen receptor and two androgen responsive proteins on the same blot.(Fig. 2A legend)
A pool of four antisense oligonucleotides against TMEFF2 lowered TMEFF2 protein in LNCaP prostate cancer cells while leaving the androgen receptor and prostate specific antigen untouched.(Results, Fig. S7B)
That clean result is what exposed the artifact: short hairpin RNAs aimed at the same gene had collapsed androgen signalling and killed the cells, and the antisense arm showed those effects had nothing to do with silencing TMEFF2.(Results)
The paper traces the short hairpin RNA effects to short seed matches in the untranslated regions of many genes at once, a mechanism that kills the cell rather than silencing the intended target.(Introduction)
A CRISPR-Cas9 knockdown of the same gene also failed to reproduce the loss of viability, which agrees with the antisense result and rules out the target itself.(Results, TMEFF2-targeted shRNAs reduce cell viability and growth of PCa cells)

Product: AUMsilence
Targets: TMEFF2 · human · mRNA
Models: LNCaP androgen dependent human prostate cancer cells · cell line
Delivery: transfected, not self-delivered: 250 nM oligo with 0.19% Dharmafect 3 transfection reagent, then hormone starved and stimulated
Readouts: western blot, quantitative reverse transcription PCR, RNA sequencing, trypan blue viability, xenograft tumour growth
Measured: oligo concentration and delivery: 250 nM with 0.19% Dharmafect 3 (pool of four against TMEFF2, or a non-target control, in LNCaP cells)schedule after transfection: 48 h, then 24 h in charcoal stripped serum, then 24 h with or without 10 nM hormone (10 nM dihydrotestosterone or 0.0001% ethanol vehicle)what the antisense knockdown did and did not do: TMEFF2 protein reduced; prostate specific antigen and androgen receptor unchanged (against the non-target control oligo, with and without hormone)what that result established for the paper as a whole: the short hairpin RNA phenotype was independent of TMEFF2 silencing (read together with the CRISPR knockout arm)
Figures: Supplementary Fig. S7B Western blot of prostate specific antigen, TMEFF2 and androgen receptor in LNCaP cells given the pooled targeting oligo or the non-target control, with and without hormone.Supplementary Fig. S2A and S2B Viability of LNCaP cells after CRISPR-Cas9 knockdown of TMEFF2 against a control guide, which is the companion negative result.
Citation: Corbin et al. Seed-mediated RNA interference of androgen signaling and survival networks induces cell death in prostate cancer cells. Molecular Therapy Nucleic Acids 2021;24:337-351. doi:10.1016/j.omtn.2021.03.002

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