Steroid hormone modulation of RET through two estrogen responsive enhancers in breast cancer

Abstract

RET, a gene causatively mutated in Hirschsprung disease and cancer, has recently been implicated in breast cancer estrogen (E2) independence and tamoxifen resistance. RET displays both E2 and retinoic acid (RA)-dependent transcriptional modulation in E2-responsive breast cancers. However, the regulatory elements through which the steroid hormone transcriptional regulation of RET is mediated are poorly defined. Recent genome-wide chromatin immunoprecipitation-based studies have identified 10 putative E2 receptor-alpha (ESR1) and RA receptor alpha-binding sites at the RET locus, of which we demonstrate only two (RET -49.8 and RET +32.8) display significant E2 regulatory response when assayed independently in MCF-7 breast cancer cells. We demonstrate that endogenous RET expression and RET -49.8 regulatory activity are cooperatively regulated by E2 and RA in breast cancer cells. We identify key sequences that are required for RET -49.8 and RET +32.8 E2 responsiveness, including motifs known to be bound by ESR1, FOXA1 and TFAP2C. We also report that both RET -49.8 regulatory activity and endogenous RET expression are completely dependent on ESR1 for their (E2)-induction and that ESR1 is sufficient to mediate the E2-induced enhancer activity of RET -49.8 and RET +32.8. Finally, using zebrafish transgenesis, we also demonstrate that RET -49.8 directs reporter expression in the central nervous system and peripheral nervous system consistent with the endogenous ret expression. Taken collectively, these data suggest that RET transcription in breast cancer cells is modulated by E2 via ESR1 acting on multiple elements collectively.

Figure 1.

E2- and RA-induced regulatory response of RET locus ESR1- and RARA-binding sites. (A) The human RET interval (chr10:42,770,285–42,950,127; hg18) contains 10 ESR1-binding sites and 2 RARA-binding sites identified from MCF-7 whole genome ChIP assays (20,–33). Amplicons encompassing them are displayed on a UCSC Genome Browser (genome.ucsc.edu) custom track are named according to their distance from the RET transcriptional start site (ESR1 containing amplicons, black rectangles; RARA containing amplicons, asterisks). (B) E2-induced luciferase activity directed by 10 amplicons containing putative ESR1-binding sites at RET, assayed in MCF-7 cells. E2-depleted (light gray bars) and E2-exposed (10 nm; black bars) pDSMA_RET construct expression values were normalized against a promoter-only construct (Prom. only). (C) RA-induced luciferase activity directed by two amplicons at RET that contain putative RA receptor binding sites was similarly assayed in MCF-7 cells. RA-depleted (light gray bars) and all trans RA-exposed (1 µm; black bars) pDSMA_RET expression values were normalized against a promoter-only construct (Prom. only), pDSMA construct. All assays were conducted in triplicate (error bars, standard deviation). ***P-value < 0.005.

Figure 2.

RET −49.8 reflects endogenous RET transcription. (A) E2- and RA-induced luciferase activity of RET −49.8 was assayed in MCF-7 cells. E2-depleted (blue bars), RA exposed (green bars), E2 exposed (red bars), E2 and RA combined (purple bars) pDSMA RET −49.8 construct expression values were normalized against a promoter-only construct. All assays were conducted in triplicate (error bars, standard deviation). (B) Quantitative real-time polymerase chain reaction (PCR) measures effects of E2 deprivation (blue bars), E2 (red bars), RA exposed (green bars) or E2 and RA combined (purple bars) on endogenous MCF-7 RET expression, normalized to 18S rRNA. RET expression levels in E2-deprived cells were set as a level of 1-fold expression level. **P-value < 0.005; ***P-value < 0.0005.

Figure 3.

Identification of sites critical for RET −49.8 and RET +32.8 E2 responsiveness in vitro. (A) Putative sites for TFAP2C, ESR1 and FOXA1 binding identified by in silico motif searching, manual curation and by imputation from ChIP-PET and ChIA-PET analyses [(30,37); filled black rectangles]. Sites are identified as: F1, FOXA1-like motif 1; F2, FOXA1-like motif 2; 9 CR, 9 bp ESR1 CR; 12 CR, 12 bp ESR1 CR; TFAP2C-like motif, AP2 S1; TFAP2C-like motif, AP2 S2. (B) Two FOXA1-type-binding sites, two TFAP2C-like motifs, a 9 bp CR and 12 bp ESR1 CRs from ESR1 ChIP-PET and ChIA-PET (30,37) were mutated independently and in combination. Gray bars represent RET −49.8 regions, with black boxes representing the six motifs. Light gray boxes indicate motifs mutagenized in the respective RET −49.8 constructs. E2-induced luciferase activity of RET −49.8 wild-type and mutagenized constructs were assayed in MCF-7 cells. E2-depleted (light gray bars) and E2-exposed (black bars) pDSMA RET −49.8 mutant construct expression values were normalized against a promoter-only construct. All assays were conducted in triplicate (error bars, standard deviation).

Figure 4.

Endogenous RET and RET −49.8 and RET +32.8 are dependent on ESR1. (A) Quantitative real-time PCR assay of the effects of ESR1 knockdown using siRNA on endogenous MCF-7 RET expression, normalized to 18S rRNA. RET expression levels for E2-deprived conditions were normalized as 1-fold expression. NT siRNA, non-targeting control siRNA; E2-deprived, gray bars; E2-treated, black bars. (B) Effects of ESR1 knockdown via siRNA on pDSMA RET −49.8 and RET +32.8 E2 responsiveness in MCF-7 cells (NT siRNA, non-targeting control siRNA). (C) Quantitative real-time PCR assay of the effects of FOXA1 knockdown using siRNA on endogenous MCF-7 RET expression, normalized to 18S rRNA. RET expression levels for E2-deprived conditions were normalized as 1-fold expression. NT siRNA, non-targeting control siRNA. E2 deprived, gray bars; E2-treated, Black bars. (D) Effects of FOXA1 knockdown via siRNA on pDSMA RET −49.8 E2 responsiveness in MCF-7 cells (NT siRNA, non-targeting control siRNA). E2 deprived, gray bars; E2-treated, black bars. (E) Ectopic ESR1 expression facilitates E2-induced luciferase expression directed by RET −49.8 and RET +32.8 in ESR1-negative MDA-MB-231 breast cancer cells. pC3.1 empty, pCDNA3.1 with no coding sequence; pC3.1 ESR1, pCDNA3.1 ESR1 expression vector; E2-deprived, gray bars; E2-treated, black bars. (F) Western blot confirms ESR1 knockdown in MCF-7 cells co-transfected with targeting or non-targeting (NT) siRNA co-transfected with pDSMA RET −49.8. (G) Western blot confirms FOXA1 knockdown in MCF-7 cells co-transfected with targeting or non-targeting (NT) siRNA co-transfected with pDSMA RET −49.8. (G) Western blot images demonstrating expression of ESR1 only in MDA-MB-231 cells transfected with pCDNA3.1 empty and pCDNA3.1 ESR1.

Figure 5.

RET locus interacting ESR1-binding sites do not exceed the activity of RET −49.8 and RET +32.8. The ESR1-binding sites present in each vector are represented graphically on the left of each numbered construct. Light gray box represents SV40 promoter, white box represent luciferase coding region, while black boxes represent RET locus ESR1-binding sites from left to right RET −49.8, RET −38.1, RET −31.7, RET +4.8 and RET +32.8. E2 responsive luciferase activity of the different combinations RET locus ESR1-binding sites predicted to interact in MCF-7 cells (37). E2-depleted (gray bars) and E2-exposed (10 nm; black bars) pDSMA RET* expression values were normalized against a promoter-only construct (Prom. only), pDSMA construct. Constructs are labeled 1 through 10. (1) Promoter-only construct; (2) RET −49.8; (3) RET −38.1; (4) RET −31.7; (5) RET +4.8; (6) RET +32.8; (7) RET −49−38−31+4+32; (8) RET −49−38−31; (9) RET −49+4+32; (10) RET −49+32; all assays were conducted in triplicate (error bars, SD).

Figure 6.

RET −49.8 directs ret appropriate reporter expression. (A) RET −49.8 directs eGFP reporter expression in the central nervous system in stable transgenic zebrafish at 24hpf (lateral image, with dorsal image inset). SC, spinal cord; HB, hindbrain. (B) RET −49.8 directs eGFP reporter expression in the central nervous and peripheral nervous system in stable transgenic zebrafish at 72hpf (lateral image, with close up on expression inset). HB, hindbrain, DSC, dorsal spinal cord; VSC, ventral spinal cord; LLG, lateral line ganglia.