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 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

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

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