Role of the 5′ UTR region in ERG and Tmprss2:ERG variants expression.

<p>(<b>A</b>) The native 5′ UTR of ERG-1b, ERG-1c and T1:E4 were replaced with a common one from the expression vector (in orange), and an optimized Kozak sequence. The replaced ATGs are in exon 3 (M3), 1c (M1c) and 4 (M4a) (<b>B</b>) Context of multiple in-frame ATG used as start codons in various ERG and Tmprss2:ERG variants. The sequence around the ATG is aligned to a consensus Kozak sequence, with the important conserved positions at -3 (R) and +4 (G) highlighted in red (counting the A as +1). Context is evaluated as ‘strong’ (S) if both positions are conserved, and ‘weak’ (W) if they are not. An expanded analysis of ATGs in the 5′UTR region of ERG and Tmprss2:ERG is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>C</b>) WB analysis of the variants and mutants represented in (A). Improving the M3 context favors its use at the expenses of the M4a ATG. (<b>D</b>) uORFs that might influence translation efficiency of ERG variants in the 5′ UTRs of ERG-1b and several common Tmprss2:ERG variants. Red ticks indicate ATGs, boxes below indicate the putative uORF generated and their approximate length (drawing not to scale). Red boxes indicate strong ATG contexts and green weak ones (as defined in (B)). More details about the uORFs are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>E</b>) Effect of independently mutating uORFs’ ATG in ERG-1b: mutation of the first ATG in exon 2 releases suppression of translation and increases levels of ERG from the ATG in exon 4. (<b>F</b>) Expression of ERG and Tmprss2:ERG variants<b>.</b> Full-length cDNAs for the variants, including their entire 5′ UTRs, were expressed and lysates from transiently transfected HeLa cells were analyzed by western blot.</p

Mapping of translation initiation of ERG isoforms.

<p>(<b>A</b>) N-terminal heterogeneity in human native ERG variants. The 5′ regions for the indicated variants are reproduced. Boxes represent exons with the ORF in blue. Red ticks below the exons indicate in-frame ATGs in exons 1c and exons 3 to 5. The out-of-frame products caused by exon skipping, from the otherwise in frame ATGs are indicated in red. (<b>B</b>) Western Blot (WB) of transient expression of the variants in HeLa cells. Antibody C20 recognizes exogenous protein and an endogenous band at ∼44 KDa corresponding in size to Δ4 variants. Antibody C17 preferentially recognizes exogenous ERG bands. (<b>C</b>) Mutations that independently eliminate the 3 in-frame ATGs in exon 4 were introduced into the cDNA of T1:E4 and analyzed as above. (<b>D</b>), Similarly, mutations were introduced to eliminate the ATGs in exon 3 (ERG-1b) or in exon 1c (ERG-1c) by themselves or in combination with mutations in the first in-frame ATG (M4a) in exon 4 (ERG-1b and ERG-1c).</p

5′ UTR Control of Native ERG and of Tmprss2:ERG Variants Activity in Prostate Cancer

<div><p>ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5′ UTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF) in their 5′ UTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4) with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4) fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumor–associated and normal ERG.</p> </div

Human ERG gene structure and main isoforms.

<p>(<b>A</b>) Top: The ∼300 Kb human ERG locus, drawn roughly to scale. Approximate intron sizes are indicated, along with exons position (bars). Red indicates first exons, blue common alternative ones and gray uncommon ones. Middle: Exon structure, with exon sizes at the bottom. Blue boxes indicate the main predicted ORFs, white boxes the untranslated regions and gray the uncommon exons. Red circles indicate polyA sites. Bottom: alignment of the exons forming the main ORF (ERG-1b) with the protein’s domains. Numbers indicate size in amino acids. PNT = pointed domain, AD = alternative domain, Ets = Ets domain, TAD = transactivation domain. Asterisk and circle indicate position of the first and second ATG. (<b>B</b>) Human ERG main variants. Alignment of exons forming the 30 main RNA variants of human ERG. Blue indicates the ORF, light blue the additional region from the ATG in exon 3. For each variant, the proposed name is indicated next to previous nomenclature (if available). The proposed protein name is reported along the predicted size in aa and KDa. Variants derived from the alternative usage of promoter 1a and 1b are paired as they lead to related mRNAs and identical proteins.</p

Differential ERG variant expression in normal tissues and PCa cells.

<p>Quantification by qPCR of alternative ERG variants in normal tissue (<b>A, D</b>), primary PCa samples (<b>B, E</b>) and PCa cell lines. (<b>C, F</b>). (<b>A–C</b>) Quantification of promoter usage. Primer sets specific for the 3 different ERG promoters and for the Tmprss2:ERG fusion where used, along with a primer set spanning exons 5–7 to quantify total ERG. Normal tissues do not express the fusion product. Expression of variants 1a and 1c is virtually undetectable in the PCa cell lines analyzed. For panel C, open symbols indicate LnCap, DU145 and C4-2 (not expressing Tmprss2:ERG fusion), full symbols indicate VCap and NCI-H660 (expressing Tmprss2:ERG fusion). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> A–C. (<b>D–F</b>) Quantification by qPCR of alternative polyadenylation usage. Primer sets specific for the 3 different polyA sites where used, along with a primer set to quantify total ERG and one set to quantify total exon 11 levels in order to infer 11SpA usage. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> D–F. In all cases, each indicated value represents averages of ≥3 independent experiments and is presented as ΔC(t) normalized to the housekeeping gene GAPDH, therefore a “high” ΔC(t) value means low levels of expression and a “low” value means high level of expression. Horizontal bars indicate the mean. Asterisks indicate that the product was not detected in the sample and would therefore be equivalent to an experimental point at the top of the table, but it is not reflected by the mean.</p

Activity of ERG and Tmprss2:ERG variants.

<p>(<b>A</b>) Stable expression of ERG variants in NIH-3T3 cells. Expression was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone-0049721-g003" target="_blank">Fig 3</a>. (<b>B–C</b>) Cells from (A) were assayed for their invasion potential using a matrigel invasion assay (B) and for their motility using a wound healing assay (C). Representative images are shown. (<b>D</b>) Quantification of (C), using ImageJ software (≥4 independent experiments). Bars indicate standard deviations. (<b>E</b>) Transient transcriptional activation of ERG-dependent promoters. Firefly luciferase cDNA was cloned downstream of the VE-Cadherin or MMP-1 promoters as depicted. The VE-Cadherin (red) and the MMP-1 (blue) promoters contain respectively 4 and 11 Ets binding sites (blue boxes, not to scale). Luciferase reporters were transiently co-expressed in HeLa cells with Tmprss2:ERG variants. A renilla luciferase construct was used to normalize for variation in transfection efficiency. Dual-luciferase assay was performed and activity is represented as fold-activity over that of co-transfected empty vector. Data represent averages of at least 3 independent experiments, with bars indicating standard deviations.</p

HPLC profiles of thalassemic specimens analyzed after differentiation from CD34-derived cultures.

<p>The HPLC profiles of the same thalassemic β0/0 (A) and β+/+ (B) specimens were analyzed at steady state (left) and following treatment with AnkT9W (right) after differentiation starting from either CD34⁺ cells (top) or ErPCs (bottom). In the same β0/0 specimen, AnkT9W contributes to increasing Hb A synthesis from from 0% to 62% (VCN = 0.92), in the CD34⁺-derived cells or to 73% (VCN = 0.97), in the ErPCs-derived cells. In the cells from the β+/+ patient, the net Hb increase was 35% (VCN = 0.88), if CD34⁺-derived or 23% (VCN = 0.94), if ErPCs-derived.</p

Sequences of exon 1 and 2 in AnkT9W and AnkT9Wsil1/2 silent mutants.

<p>Original and corresponding mutated bases of exons 1 and 2 inside constructs are in bold letters.</p

AnkT9W improves Hb synthesis and hematological parameters of thalassemic mice.

<p>Red cell hemolysates were analyzed monthly by HPLC for up to 6 months post BM transplant. Chimeric (A) and total absolute Hb content (B) in T9W-<i>Hbb^th3/+</i> and AnkT9W-<i>Hbb^th3/+</i> chimeras (n = 4 and 6, respectively). Hβ:mα Hb detected by HPLC and relative VCN (C and D) in mice chimeras obtained by transplanting <i>Hbb^th3+</i> BM treated with T9W or AnkT9W vectors into lethally irradiated <i>Hbb^th3+</i> recipient mice. (E, F) Hematologic parameters in WT, <i>Hbb^th3/+</i>, T9W-<i>Hbb^th3/+</i> or AnkT9W-<i>Hbb^th3/+</i> chimeras. On average, the hematocrits in AnkT9W-treated chimeras were 26% and 10% higher, the reticulocyte counts 71% and 49% lower, and the RDWs 35% and 22% lower than in <i>Hbb^th3/+</i> and T9W-<i>Hbb^th3/+</i> mice, respectively. For reticulocyte counts, Hb, RDW, p<0.0001,and for HCT, p = 0.0002.</p

AnkT9W increases HbA synthesis to therapeutic levels in most of the thalassemic ErPCs.

<p>(A) Net increase of Hb A % (ΔHb A, calculated by subtracting Hb A after treatment from that following treatment) is plotted against VCN (on the X axis) in the three patients groups, β0/0, β0/+ and β+/+. The total Hb A% is also represented as percentage of Hb A before (light grey) and after transduction (dark grey) with AnkT9W in the same groups (B). Figures C and D show the concomitant percentages of Hb F and α-aggregates, both of which are reduced in thalassemic ErPCs after treatment with AnkT9W. The tie bars under the X axes group different specimens from the same individual. Several specimens were harvested and transduced at different times so as to expose the variability in such experiments. “*” Sign indicates specimens treated with AnkT9Wsil2.</p