ELF3 is a repressor of androgen receptor action in prostate cancer cells - PubMed (original) (raw)

ELF3 is a repressor of androgen receptor action in prostate cancer cells

A Shatnawi et al. Oncogene. 2014.

Abstract

The androgen receptor (AR) has a critical role in the development and progression of prostate cancer (PC) and is a major therapeutic target in this disease. The transcriptional activity of AR is modulated by the coregulators with which it interacts, and consequently deregulation of cofactor expression and/or activity impacts the expression of genes whose products can have a role in PC pathogenesis. Here we report that E74-like factor 3 (ELF3), a member of the ETS family of transcription factors, is a repressor of AR transcriptional activity. Exogenous expression of ELF3 represses AR transcriptional activity when assessed using reporter-based transfection assays or when evaluated on endogenous AR target genes. Conversely, ELF3 knock down increases the AR transcriptional activity. Biochemical dissection of this activity indicates that it results from the physical interaction between ELF3 and AR and that this interaction inhibits the recruitment of AR to specific androgen response elements within target gene promoters. Significantly, we observed that depletion of ELF3 expression in LNCaP cells promotes cell migration, whereas increased ELF3 expression severely inhibits tumor growth in vitro and in a mouse xenograft model. Taken together, these results suggest that modulation of ELF3 expression and/or AR/ELF3 interaction may have utility in the treatment of PC.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

ELF3 expression is down regulated in PC tissues and interacts with AR. A, Waterfall plot of ELF3 expression in paired-sample data. Whole-transcript data was downloaded from the Gene Expression Omnibus under accession GSE21034 and only the paired-sample data was used for analysis. ELF3 expression was downregulated in 20/29 patients in primary tumors compared to normal adjacent sites_. B,_ Interaction between ELF3 and AR in vivo. Lysates from LNCaP cells treated with vehicle or R1881 immunoprecipitation (IP) and immunoblot (WB) with the indicated antibodies. C, Direct interaction between ELF3 and AR. In vitro translated full length AR or deletion mutants as indicated by the schematic representation (left) were subjected to pull-down analysis with full length GST-ELF3 (right). D, ELF3 colocalized with AR in LNCaP cells. Fluorescence microscopy images of LNCaP cells expressing endogenous AR (green) and Flag-ELF3 (red) and treated with either vehicle (top panel) or R1881 (bottom panel) for 16 hrs and probed with anti-rabbit AR or Flag anti-mouse followed by specific Alexa-conjugated secondary antibody.

Figure 2

ELF3 represses AR-mediated transcription. A, Repression of 2XARE, PSA and MMTV luciferase reporter activity by ELF3 co-transfected with AR in HepG2 cells treated with vehicle or 10 nM R1881 with an immunoblot (inset) showing the expression levels of AR, Flag Elf3 and RPLP0 serving as a control. Error bars, SD; *, P<0.05. B, Repression of PSA, KLF5 and MMTV luciferase reporter activity by ELF3 transfected in LNCaP cells treated with vehicle or 10 nM R1881 with an immunoblot (inset) showing the expression levels of AR, Flag Elf3 and RPLP0 as a loading control. Error bars, SD; *, P<0.05. C, PSA promoter activity in HepG2 cells co-transfected with AR and ELF3 or control (C) expression vectors and treated with increasing doses of R1881. Error bars, SD; *, P<0.05. D, Effect of CSX treatment on the PSA promoter activity in R1881-treated HepG2 cells co-transfected with AR and ELF3 or control (C) expression vectors. Error bars, SD; *, P < 0.05.

Figure 3

Identification of ELF3 domains involved in repression of AR transcriptional activity. Left, schematic diagram displaying ELF3 functional PNT, TAD, SAR, AT-hook and ETS domains, the T7-ELF3 fragment obtained in the original screen and the ELF3 deletion and point mutation constructs used in this study. Right, bar graph showing the % repression of the PSA promoter activity in HepG2 cells co-transfected with control ELF3 or ELF3 derivatives described previously in material and methods and AR and treated with 10 nM R1881. Error bars, SD; *, P<0.05. B, WB of showing the expression levels of AR, Flag-ELF3 full length (FL), Flag-ELF3 mutations and deletions, V5 tag ELF3 (FL) and its V5 tag mutation relative to RPLP0 loading control in HepG2 cells transfected and treated as described in A.

Figure 4

ELF3 modulates the expression of AR target genes and prevent its recruitment to AR response elements. A, Western blot analysis showing the protein levels of AR and ELF3 in LNCaP cells transfected with either a control siRNA (siC), a siRNA targeting AR (siAR) or two different siRNAs targeting ELF3 (siELF3#1 and #2) and treated with vehicle (−) or 10 nM R1881. RPLP0 served as a control. B, Heatmap indicating the differential induction of endogenous AR target genes in LNCaP cells in response to 10 nM R1881 as monitored by qRT-PCR upon AR or ELF3 knockdown. C, Western blot analysis of the inducible TetOn-Flag-ELF3-LNCaP stable cell line showing the expression level of ELF3 after 24h induction by Dox. GAPDH served as a control. D, Differential induction of AR target genes in R1881-treated TetOn-Flag-ELF3-LNCaP cells upon ELF3 induction by Dox as monitored by qRT-PCR. Error bars, SD; *, P<0.05. E, Enrichment of AR and Flag-Elf3 binding to PSA, NKX3.1 and TMPRSS2 regulatory regions as assayed by standard ChIP upon induction of ELF3 expression by Dox in TetOn-Flag-ELF3-LNCaP cells. Error bars, SD; *, P<0.05.

Figure 5

ELF3 knockdown promotes androgen-induced LNCaP cell migration and ELF3 ecptopic expression inhibits cell proliferation. A, Boyden chamber migration assays was used to assess the migration of LNCaP cells following ELF3 knockdown and treatment with either vehicle (top) or 10 nM R1881 (bottom). Migrated cells were stained with crystal violet and imaged at X100 magnification. B, Bar graphs representing average migrated cell numbers of four different microscopic fields of vehicle or R1881-treated cells following ELF3 knockdown. Error bars, SD; *, P<0.05. B, TetOn-Flag-ELF3-LNCaP Real Time proliferations assay. Cells were grown in CS-RPMI1640 for 48h and treated with ±Dox and with vehicle or R1881 as indicated for additional 24h. Then cells were plated in E16-plates and kept with the same treatment for the entire period. The proliferation signals were recorded by xCELLigence machine 5h after cell plating, the time required for cell adhesion and before proliferation.

Figure 6

ELF3 reduces tumor growth in a non-castrated mice xenograft assay. A, Inducible TetOn-Flag-ELF3-LNCaP stable cells were transplanted subcutaneously into the right flanks of none castrated SCID male mice. The animals were divided into two groups, one fed with Dox (n=7) and the other group fed with control diet C (n=6) for a total period of 7 weeks. The growth curve of the xenografts with a representative image (inset) of immunoblotting analysis showing the expression levels of ELF3 and AR in tumors of Dox fed mice versus controls. RPLP0 served as a control. B, Tumor weight at 7 weeks after of implantation (bottom) together with a representative photograph of tumors excised from Dox and C fed mice (top). C, Expression levels of AR target genes in tumors excised from Dox and C fed mice as monitored by qRT-PCR. Error bars, SD; *, P<0.05.

References

1. Ryan CJ, Tindall DJ. Androgen receptor rediscovered: the new biology and targeting the androgen receptor therapeutically. J Clin Oncol. 2011;29:3651–3658. - PubMed
1. Knudsen KE, Kelly WK. Outsmarting androgen receptor: creative approaches for targeting aberrant androgen signaling in advanced prostate cancer. Expert Rev Endocrinol Meabt. 2011;6:483–493. - PMC - PubMed
1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29. - PubMed
1. Yap TA, Zivi A, Omlin A, de Bono JS. The changing therapeutic landscape of castration-resistant prostate cancer. Nat Rev Clin Oncol. 2011;8:597–610. - PubMed
1. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer. 2001;1:34–45. - PubMed

ELF3 is a repressor of androgen receptor action in prostate cancer cells - PubMed (original) (raw)