Samples investigated in the study.

1<p>All 38 investigated fusion genes are ranked based on the likelihood of being the correct fusion gene in the particular sample. Lower numbers indicate higher likelihood.</p>2<p>Information about RT-PCR protocols for fusion gene detection. In house: protocol not previously published. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070649#s2" target="_blank">Materials and Methods</a> for more details. NK: Normal karyotype.</p

Top ranked fusion genes in two sarcoma samples.

<p>A. <i>EWSR1</i>-<i>NR4A3</i> in the 168/97 sample. B. <i>SS18</i>-<i>SSX1</i> in the 9972 sample. I) Intensity heat map of chimeric oligos. Each square represents one possible exon-exon boundary between the two gene partners. One square is highly expressed (A: 13-3, B: 10-6) and reflects the presence of chimeric RNA covering the corresponding exon-exon boundary. This fusion breakpoint corresponds with a shift in relative expression measured by intragenic oligos covering both the upstream (II) and downstream (III) fusion gene partners. Blue and red colours represent the two possible chimeric transcripts generated from the fusion gene.</p

Potential Downstream Target Genes of Aberrant ETS Transcription Factors Are Differentially Affected in Ewing’s Sarcoma and Prostate Carcinoma

<div><p><em>FLI1</em> and <em>ERG</em>, the major ETS transcription factors involved in rearrangements in the Ewing’s sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa <em>ERG+</em>) and without (PCa ETS-) ETS fusion genes (<em>HIST1H4L</em>, <em>KCNN2, ECRG4</em> and <em>LDOC1</em>), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (<em>NR0B1</em>, <em>CAV1</em>, <em>IGFBP3</em> and <em>TGFBR2</em>). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of <em>CAV1</em> and <em>NR0B1</em> and underexpression of <em>IGFBP3</em> and <em>TGFBR2</em> in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa <em>ERG</em>+ and PCa ETS-, <em>CAV1, IGFBP3</em> and <em>TGFBR2</em> were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for <em>ECRG4</em> and <em>LDOC1</em>, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both <em>HIST1H4L</em> and <em>KCNN2</em> are significantly overexpressed in PCa <em>ERG+</em> and that ERG binds to the promoter of these genes. Conversely, <em>KCNN2</em> was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.</p> </div

Box-plot distribution of <i>CAV1</i> and <i>IGFBP3</i> expression in PCa sample subgroups.

<p><b>A)</b><i>CAV1</i> expression; <b>B)</b><i>IGFBP3</i> expression. A <i>p</i> value is shown whenever the differences in each two group comparison reach significance (<i>p</i><0.05).</p

Box-plot representation of the qRT-PCR data for the four genes described as EWSR1-FLI1 targets and associated with PCa samples harboring <i>ERG</i> rearrangements (<i>HIST1H4L</i>, <i>KCNN2</i>, <i>ECRG4</i> and <i>LDOC1</i>).

<p><b>A)</b> ESFT <i>versus</i> ARMS samples; <b>B)</b> PCa samples <i>versus</i> NPT samples. A <i>p</i> value is shown whenever the differences in each two group comparison reach significance (<i>p</i><0.05).</p

Analyses of <i>HIST1H4L</i> and <i>KCNN2</i> expression and their regulation by ERG in PCa samples harboring <i>ERG</i> rearrangements.

<p><b>A)</b> and <b>B)</b> Box-plot distribution of <i>HIST1H4L</i> and <i>KCNN2</i> expression in PCa sample subgroups, respectively. A <i>p</i> value is shown whenever the differences in each two group comparison reach significance (<i>p</i><0.05). <b>C)</b> and <b>D)</b> qPCR of ERG-immunoprecipitated chromatin from VCaP cells showing ERG binding to three regions of the <i>HIST1H4L</i> promoter and to two regions of the <i>KCNN2</i> promoter, respectively.</p

Box-plot representation of the qRT-PCR data for the four genes described as EWSR1-FLI1 targets (<i>CAV1</i>, <i>NR0B1</i>, <i>IGFBP3</i> and <i>TGFBR2</i>).

<p><b>A)</b> ESFT <i>versus</i> ARMS samples; <b>B)</b> PCa <i>versus</i> NPT samples. A <i>p</i> value is shown whenever the differences in each two group comparison reach significance (<i>p</i><0.05).</p