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

Maria J Camões,João D Barros-Silva,Ragnhild A Lothe,Vera L Costa,Mafalda Almeida,Manuel R Teixeira,Rui Henrique,Paula Paulo,Nuno Cerveira,Franclim R Ribeiro,Carmen Jerónimo,Rolf I Skotheim,Sean Bong Lee

doi:10.1371/journal.pone.0049819

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.

Highlights

The involvement of ETS genes in cancer was first demonstrated by the presence of the oncogene v-ets as part of the gag-myb-ets transforming fusion protein of an avian retrovirus, E26 [1]
CAV1 Relative Expression CAV1 was significantly overexpressed in Ewing’s sarcoma family of tumors (ESFT) when compared to alveolar rhabdomyosarcomas (ARMS), showing a median 4.9 fold increase (Figure 1A)
There was no significant difference in CAV1 expression between Prostate cancer (PCa) ERG+ and PCa ETS2, CAV1 expression in PCa oETS+ was significantly lower when compared to PCa ETS2, with a median 5.5 fold decrease (Figure 2A)

Summary

Introduction

The involvement of ETS genes in cancer was first demonstrated by the presence of the oncogene v-ets as part of the gag-myb-ets transforming fusion protein of an avian retrovirus, E26 [1]. Less common gene fusions (1–10%), involve additional ETS family members, such as ETV1, ETV4, ETV5, and FLI1 [12,13,14] In both ESFT and PCa these ETS chimeric genes function as aberrant transcription factors, having a pivotal role in promoting transformation and oncogenesis. This hypothesis is consistent with experiments showing that EWSR1-FLI1 knockdown is correlated with decreased cell invasion and increased apoptosis [15,16] and with reports showing that overexpression of ERG and ETV1 in benign prostate cells induces a transcriptional program associated with invasion [17,18]

Methods

Results

Conclusion