Cellular and Molecular Biology

Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer

Abstract

Background

The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4.

Methods

We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5′ and 3′ splice sites of ERG’s exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo.

Results

In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member β-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3′ splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3′ splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo.

Conclusions

We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.

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Fig. 1: Validation of splice-switching oligonucleotides targeting ERG exon 4 in VCaP cells.
Fig. 2: Splice-switching caused by ERG exon 4 SSOs reduces ERG protein levels in two cancer cell lines.
Fig. 3: ERG exon 4 SSOs affect VCaP cancer cell behaviour and signalling.
Fig. 4: ERG exon 4 SSOs have anti-tumour effects in vivo.

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Acknowledgements

We would like to thank Dr Lee Spraggon for valuable help with designing the SSOs.

Funding information

: S.P. was supported by a Research Innovation Award from Prostate Cancer UK (RIA-030-15). We are very grateful to GeneTools LLC, Alexandra Vincent and Jim Summerton for technical advice and for the provision of reagents.

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Authors

Contributions

S.P., L.L., L.H., L.P., B.C. and E.N. performed experiments and analysed data. S.H. performed patient identification, tumour targeting, sampling and provided ex vivo culture support. A.H. and A.F. performed tumour targeting and sampling and provided pathology support. A.S., G.S. and J.K. performed patient identification, consent, surgery and coordination. S.P., S.O. and M.L. designed the experiments. M.L. and S.O., with input from I.W. and H.W., supervised the study. S.P. and M.L. wrote the paper with input from all co-authors.

Corresponding authors

Correspondence to Sean Porazinski or Michael Ladomery.

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Ethics approval and consent to participate

All animal operations were approved by the Animal Ethics Committee, University of Exeter, U.K. Patients were recruited and consented under Genomics England’s 100,000 Genome Project ethics at University College Hospital, with a subset of patients also consented under UCL/UCLH Biobank ethics (REC 15/YH/0311). The study was performed in accordance with the declaration of Helsinki.

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All subjects gave written consent for publication.

Data availability

Materials, data and associated protocols are available upon request. Supplementary information is available for this paper online.

Competing interests

The authors declare no competing interests.

Funding information

S.P. was supported by a Research Innovation Award from Prostate Cancer UK (RIA-030-15). We are very grateful to GeneTools LLC, Alexandra Vincent and Jim Summerton for technical advice and for the provision of reagents.

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Li, L., Hobson, L., Perry, L. et al. Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer. Br J Cancer (2020). https://doi.org/10.1038/s41416-020-0951-2

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