Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma

Abstract

FLI1 and ERG, 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 ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). 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 CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 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.

Figure 1. Box-plot representation of the qRT-PCR data for the four genes described as EWSR1-FLI1 targets (CAV1, NR0B1, IGFBP3 and TGFBR2).

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

Figure 2. Box-plot distribution of CAV1 and IGFBP3 expression in PCa sample subgroups.

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

Figure 3. Box-plot representation of the qRT-PCR data for the four genes described as EWSR1-FLI1 targets and associated with PCa samples harboring ERG rearrangements (HIST1H4L, KCNN2, ECRG4 and LDOC1).

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

Figure 4. Analyses of HIST1H4L and KCNN2 expression and their regulation by ERG in PCa samples harboring ERG rearrangements.

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