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Transl Oncol
2019 Jan 01;121:134-142. doi: 10.1016/j.tranon.2018.09.001.
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The Evolutionarily Conserved Cassette Exon 7b Drives ERG's Oncogenic Properties.
Jumbe SL
,
Porazinski SR
,
Oltean S
,
Mansell JP
,
Vahabi B
,
Wilson ID
,
Ladomery MR
.
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The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.
Figure 1. Evolutionary conservation of ERG's cassette exon 7b. (A) Alignment of the 24 amino acids encoded in frame by exon 7b in a range of species: Homo sapiens (human) NP_001129626.1, Mus musculus (mouse) NP_598420.1, Gallus gallus (chicken) XP_015155736.1, Danio rerio (zebrafish) NP_001008616.1, and Strongylocentrotus purpuratus (sea urchin) XP_011672837.1. A putative ERK phosphorylation site is shown. (B) Alignment of a sample of corresponding genomic sequences surrounding the splice sites of the cassette exon. Sequences were obtained using BLAST (NCBI) and aligned with Clustal Omega.
Figure 2. SSO-induced ERG exon 7b skipping in MG63 and VCaP cells. (A) The 5′ and 3′ splice sites of exon 7b were targeted using morpholino SSOs. The positions and sequences of the SSOs and the positions of ERG PCR primers are indicated. (B) Treatment with 3 μM of the E7b3 SSO resulted in near-complete exon 7b skipping in MG63 and VCaP cells 24 hours after transfection. The percent spliced in (PSI, ψ) ratio is shown. (C) Ninety-six hours after transfection with E7b5 and E7b3 SSOs, levels of ERG protein isoforms lacking the 24 amino acids (52 kDa) increased in both cell lines. A β-actin loading control was included in the western blot (cropped to highlight relevant bands).
Figure 3. Effect of SSO-induced exon 7b skipping on cell proliferation (A,B) and apoptosis (C,D) in MG63 and VCaP cells. After 72 hours of treatment with 3 μM SSO, Ki-67 expression was determined using immunofluorescent microscopy. Ki-67 foci were overlaid with DAPI. The graphs show the percentage of Ki-67–positive cells **P = ≤ .01, N = 3 repeats. Caspase 3/7–positive foci were quantified using immunofluorescent microscopy to determine apoptosis in MG63 (C) and VCaP (D) cells following treatment with 3 μM SSO for 72 hours. The percentage of caspase 3/7–positive cells is shown (*P < 0.05, **P < 0.01, ***P < 0.001). Error bars show 95% confidence interval (95% CI); N = 3 repeats.
Figure 4. Effect of SSO-induced exon 7b skipping on invasion and xenograft growth. Transwell invasion assay in MG63 cells (A) and VCaP cells (B) 48 hours following SSO transfection. Fold change is relative to PBS control; N = 3 repeats in each case. (C) A total of 7 × 106 MG63 cells were injected subcutaneously into 2-month-old immunocompromised nude mice which were dosed intraperitoneally with saline, control SSO, or E7b3 SSO at 12.5 mg/kg twice weekly once tumors reached 3 mm by 3 mm in size. Tumors were measured with a caliper twice weekly over a period of 56 days. (*P < 0.05, ***P < 0.001). Error bars in (A) and (B) show 95% confidence interval (95% CI); N = 3 repeats. For the xenograft experiments (C) groups of six mice were used.
Figure 5. TNSALP induction is attenuated by SSO-induced skipping of exon 7b. (A) Treatment with 1,25D and FHBP induced TNSALP activity measured indirectly via the production of p-NP. (B) Cell viability measurements confirm that changes were not caused by reduced cell number or viability. (C) TNSALP expression levels were measured by qPCR, expressed as % of total RNA input. (D) The addition of 1,25D or FHBP alone did not induce exon 7b skipping. N = 5 repeats in each case. (E) Analysis of the TNSALP promoter reveals the presence of at least one putative ERG binding site 960 bp upstream of the transcriptional start site. Primer positions and sequence are shown. (F) Chromatin immunoprecipitation analysis; TNSALP promoter DNA is present in the ERG immunoprecipitate. IgG, negative control; RNA polymerase II, positive IP control.
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