S 4C and 4D).DiscussionThe ETS family of transcription factors is one of the largest involved in the regulation of a variety of different genes that play key roles in proliferation, apoptosis, differentiation, hematopoiesis, metastasis, tissue remodeling, angiogenesis and transformation [3?4]. Identification of the target genes for normal and oncogenic ETS genes may lead to a better understanding of the mechanisms underlying malignant transformation. All ETS family members bind to 59-GGAA/T-39 DNA sequences and ETS target genes may be identified upon the presence of a functional binding site in their regulatory regions [5?]. The crucial role of ETS chimeric proteins in the development of ESFT is well documented [45], and although it has been shown that EWSR1-FLI1 and EWSR1-ERG bind ETS sequences in vitro with similar specificities and affinities as the wild-type transcription factors [46?7], it has recently beenshown that EWSR1-ETS chimeric proteins induce chromatin structure alterations that lead to transcription dysregulation [48]. Contrary to ESFT, in PCa the most common ETS fusion member is ERG (about 90 of the fusion positive cases), and only 1?0 of the cases have fusion genes involving other ETS members, namely, ETV1, ETV4 and ETV5 (which cluster in the PEA3 subfamily), and FLI1 (which clusters with ERG in the ERG subfamily) [8,12?4,23]. The ETS domain of the PEA3 subfamily displays 60 homology with the ERG subfamily, but there is no significant homology outside the ETS domain as indicated by the presence of a PNT domain in the ERG subfamily but not in the PEA3 subfamily [6,23]. Recently, we showed that while some genes are specifically and differentially expressed between PCa harboring ERG or ETV1 rearrangements, others are commonly dysregulated between these tumor molecular subtypes and PCa without ETS rearrangements, with ETV4 and ETV5 positive tumors clustering together with those with ETV1 rearrangement [33]. As FLI1 and ERG belong to the same subfamily and share 98 of sequence homology in the DNA binding domain [23?4], we questioned whether in a different cell background they would show dysregulation of the expression of the same genes.ETS Fusion Targets in CancerWe started by analyzing the expression of four well-validated targets of the EWSR1-FLI1 24272870 CI 1011 web oncoprotein in ESFT and we used ARMS for control purposes. We validated the dysregulation in the expression of four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein in ESFT, showing overexpression of CAV1 and NR0B1 and underexpression of 3687-18-1 cost IGFBP3 and TGFBR2 [16,26?8]. We then evaluated the expression of these genes in PCa with and without ETS rearrangements. None of these genes showed significant expression differences between PCa 15857111 ERG+ and PCa ETS2, suggesting that ERG proteins do not regulate their expression in this tumor type. However, the expression of CAV1, IGFBP3 and TGFBR2 is decreased in PCa in general, suggesting a role in prostate carcinogenesis. Our data suggest that regulation of CAV1 expression may be, at least in part, controlled by promoter methylation, which has also been reported by others [49]. Although we found decreased CAV1 expression especially in PCa oETS+ and the ETV1-positive LNCaP cell line showed increased expression of CAV1 after DAC treatment, the methylation status of the CAV1 promoter in PCa oETS+ samples was heterogeneous, while in other tumor samples CAV1 was more consistently methylated (Supplementary Table S2). This s.S 4C and 4D).DiscussionThe ETS family of transcription factors is one of the largest involved in the regulation of a variety of different genes that play key roles in proliferation, apoptosis, differentiation, hematopoiesis, metastasis, tissue remodeling, angiogenesis and transformation [3?4]. Identification of the target genes for normal and oncogenic ETS genes may lead to a better understanding of the mechanisms underlying malignant transformation. All ETS family members bind to 59-GGAA/T-39 DNA sequences and ETS target genes may be identified upon the presence of a functional binding site in their regulatory regions [5?]. The crucial role of ETS chimeric proteins in the development of ESFT is well documented [45], and although it has been shown that EWSR1-FLI1 and EWSR1-ERG bind ETS sequences in vitro with similar specificities and affinities as the wild-type transcription factors [46?7], it has recently beenshown that EWSR1-ETS chimeric proteins induce chromatin structure alterations that lead to transcription dysregulation [48]. Contrary to ESFT, in PCa the most common ETS fusion member is ERG (about 90 of the fusion positive cases), and only 1?0 of the cases have fusion genes involving other ETS members, namely, ETV1, ETV4 and ETV5 (which cluster in the PEA3 subfamily), and FLI1 (which clusters with ERG in the ERG subfamily) [8,12?4,23]. The ETS domain of the PEA3 subfamily displays 60 homology with the ERG subfamily, but there is no significant homology outside the ETS domain as indicated by the presence of a PNT domain in the ERG subfamily but not in the PEA3 subfamily [6,23]. Recently, we showed that while some genes are specifically and differentially expressed between PCa harboring ERG or ETV1 rearrangements, others are commonly dysregulated between these tumor molecular subtypes and PCa without ETS rearrangements, with ETV4 and ETV5 positive tumors clustering together with those with ETV1 rearrangement [33]. As FLI1 and ERG belong to the same subfamily and share 98 of sequence homology in the DNA binding domain [23?4], we questioned whether in a different cell background they would show dysregulation of the expression of the same genes.ETS Fusion Targets in CancerWe started by analyzing the expression of four well-validated targets of the EWSR1-FLI1 24272870 oncoprotein in ESFT and we used ARMS for control purposes. We validated the dysregulation in the expression of four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein in ESFT, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 [16,26?8]. We then evaluated the expression of these genes in PCa with and without ETS rearrangements. None of these genes showed significant expression differences between PCa 15857111 ERG+ and PCa ETS2, suggesting that ERG proteins do not regulate their expression in this tumor type. However, the expression of CAV1, IGFBP3 and TGFBR2 is decreased in PCa in general, suggesting a role in prostate carcinogenesis. Our data suggest that regulation of CAV1 expression may be, at least in part, controlled by promoter methylation, which has also been reported by others [49]. Although we found decreased CAV1 expression especially in PCa oETS+ and the ETV1-positive LNCaP cell line showed increased expression of CAV1 after DAC treatment, the methylation status of the CAV1 promoter in PCa oETS+ samples was heterogeneous, while in other tumor samples CAV1 was more consistently methylated (Supplementary Table S2). This s.