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

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordBackground 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.Prostate Cancer U

TGF beta-induced switch from adipogenic to osteogenic differentiation of human mesenchymal stem cells: identification of drug targets for prevention of fat cell differentiation

Background: Patients suffering from osteoporosis show an increased number of adipocytes in their bone marrow, concomitant with a reduction in the pool of human mesenchymal stem cells (hMSCs) that are able to differentiate into osteoblasts, thus leading to suppressed osteogenesis.Methods: In order to be able to interfere with this process, we have investigated in-vitro culture conditions whereby adipogenic differentiation of hMSCs is impaired and osteogenic differentiation is promoted. By means of gene expression microarray analysis, we have investigated genes which are potential targets for prevention of fat cell differentiation.Results: Our data show that BMP2 promotes both adipogenic and osteogenic differentiation of hMSCs, while transforming growth factor beta (TGF beta) inhibits differentiation into both lineages. However, when cells are cultured under adipogenic differentiation conditions, which contain cAMP-enhancing agents such as IBMX of PGE2, TGF beta promotes osteogenic differentiation, while at the same time inhibiting adipogenic differentiation. Gene expression and immunoblot analysis indicated that IBMX-induced suppression of HDAC5 levels plays an important role in the inhibitory effect of TGF beta on osteogenic differentiation. By means of gene expression microarray analysis, we have investigated genes which are downregulated by TGF beta under adipogenic differentiation conditions and may therefore be potential targets for prevention of fat cell differentiation. We thus identified nine genes for which FDA-approved drugs are available. Our results show that drugs directed against the nuclear hormone receptor PPARG, the metalloproteinase ADAMTS5, and the aldo-keto reductase AKR1B10 inhibit adipogenic differentiation in a dose-dependent manner, although in contrast to TGF beta they do not appear to promote osteogenic differentiation.Conclusions: The approach chosen in this study has resulted in the identification of new targets for inhibition of fat cell differentiation, which may not only be relevant for prevention of osteoporosis, but also of obesity