TGF-β drives epithelial-mesenchymal transition through δEF1-mediated downregulation of ESRP

Abstract

Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair and cancer progression in adult tissues. We have recently shown that transforming growth factor (TGF)-β-induced EMT involves isoform switching of fibroblast growth factor receptors by alternative splicing. We performed a microarray-based analysis at single exon level to elucidate changes in splicing variants generated during TGF-β-induced EMT, and found that TGF-β induces broad alteration of splicing patterns by downregulating epithelial splicing regulatory proteins (ESRPs). This was achieved by TGF-β-mediated upregulation of δEF1 family proteins, δEF1 and SIP1. δEF1 and SIP1 each remarkably repressed ESRP2 transcription through binding to the ESRP2 promoter in NMuMG cells. Silencing of both δEF1 and SIP1, but not either alone, abolished the TGF-β-induced ESRP repression. The expression profiles of ESRPs were inversely related to those of δEF1 and SIP in human breast cancer cell lines and primary tumor specimens. Further, overexpression of ESRPs in TGF-β-treated cells resulted in restoration of the epithelial splicing profiles as well as attenuation of certain phenotypes of EMT. Therefore, δEF1 family proteins repress the expression of ESRPs to regulate alternative splicing during TGF-β-induced EMT and the progression of breast cancers.

Figure 1

Changes in alternative splicing during TGF-β-induced EMT. (a) Changes in alternative splicing of CD44. Specific primers to detect v1-v10 variants of CD44 are shown as arrows (top panel). GAPDH was used as internal control. (b) The total level of CD44 mRNA was evaluated by quantitative RT–PCR analysis. (c) Specific primers to detect splicing variants of Mena are shown as arrows (top panel). GAPDH was used as internal control. (d) NMuMG cells treated with TGF-β for 24 h were prepared for Mouse Exon 1.0 ST Array. The ARH method was adapted to identify candidate genes at the exon level whose expressions changed during EMT. (e) The ratio of expression changes of each exon calculated by probe signal value in CD44 is shown. Red circles indicate the exons whose probe signals were altered by TGF-β treatment and reported to be spliced by ESRPs (Warzecha et al., 2009a).

Figure 2

Requirement of de novo protein synthesis for downregulation of ESRP2 by TGF-β. (a) Effect of TGF-β on the expression of ESRPs in NMuMG cells (left) and EpRas cells (right) was examined by quantitative RT–PCR analysis. n.d., not detected. (b) After treatment of EpRas cells with TGF-β or transfection with ESRP1 siRNA, the levels of ESRP1 were evaluated by immunoblot analysis. α-tubulin was used as a loading control. (c) JygMC(A) cells were treated with 10 μM of TβR-I inhibitor (SB431542) for 48 h. The levels of ESRP1 and ESRP2 were evaluated by quantitative RT–PCR analysis. SB, SB431542. (d) NMuMG cells transfected with both Smad2 and Smad3 siRNAs were stimulated with 1 ng/ml TGF-β for 24 h, and then examined by quantitative RT–PCR analysis for the expression levels of ESRP2. NC, control siRNA. (e) NMuMG cells pretreated with 3 μM cycloheximide (CHX) for 1 h were stimulated with 1 ng/ml TGF-β for 24 h, and examined by quantitative RT–PCR analysis for PAI1 (left), SIP1 (center) and ESRP2 levels (right).

Figure 3

Regulation of ESRP2 expression by δEF1 and SIP1. (a) After treatment with 1 ng/ml of TGF-β, the kinetics of ESRP2, δEF1, SIP1 and E-cadherin expressions were examined in NMuMG cells by quantitative RT–PCR analysis. The ratio of the mRNA levels in TGF-β-treated cells as compared with that in non-treated cells is shown. (b) NMuMG cells were transfected with mouse ESRP2 promoter-reporter construct (ESRP2-Luc) in combination with various amounts of caTβR-I, δEF1 and SIP1 plasmids. At 48 h after transfection, cells were harvested and assayed for luciferase activities. (c) mRNA levels of ESRP2 in NMuMG cells infected with null, δEF1 or SIP1 adenoviruses were determined by quantitative RT–PCR. (d) ChIP analysis was performed using NMuMG and EpRas cells in the presence or absence of 1 ng/ml TGF-β. FLAG-SIP1 adenovirus was infected into NMuMG and EpRas cells 24 h before ChIP analysis. Endogenous δEF1 and FLAG-SIP1 were immunoprecipitated with anti-δEF1 antibody and with anti-FLAG antibody, respectively. Eluted DNAs from NMuMG cells and from EpRas cells were subjected to conventional PCR for ESRP2 promoter (left) and for ESRP1 promoter (right), respectively. HBB promoter was used as negative control. Primers used are shown as arrows. (e) NMuMG cells transfected with siRNA against δEF1, SIP1, or both (siδEF1+siSIP1) were stimulated with 1 ng/ml TGF-β for 48 h and examined by quantitative RT–PCR analysis for δEF1 (left), SIP1 (center) and ESRP2 levels (right). NC, control siRNA.

Figure 4

Isoform switching of FGFRs induced by TGF-β. (a) NMuMG cells were treated with 1 ng/ml TGF-β for 24 h and the expression of FGFR1 (left) and FGFR2 (center) was determined by quantitative RT–PCR. Expression of FGFR isoforms was analyzed by conventional RT–PCR using specific primers for IIIb or IIIc (right). (b) After EpRas cells were treated with 1 ng/ml TGF-β, the total levels of FGFR1 (left) and FGFR2 (center) and expression of FGFR isoforms (right) were examined. (c) NMuMG cells transfected with siRNA against mouse ESRP2 (siESRP2) were incubated for 48 h, and then analyzed by quantitative RT–PCR to determine the levels of endogenous ESRP2 (left). Expression of FGFR2 isoform was analyzed by conventional RT–PCR (right). TF(−), no transfection; NC, control siRNA. (d) EpRas cells transfected with siRNAs against both ESRP1 (siESRP1) and ESRP2 (siESRP2) were incubated for 48 h, and analyzed by quantitative RT–PCR to determine the levels of endogenous ESRP1 (left) and ESRP2 (center). Expression of FGFR isoforms was analyzed by conventional RT–PCR (right). TF(−), no transfection; NC, control siRNA. (e) NMuMG cells infected with GFP or FLAG-ESRP2 lentiviruses were treated with TGF-β for 24 h and analyzed by immunoblot analysis (left) and conventional RT–PCR to determine the levels of IIIb and IIIc isoforms of FGFR1 (right). IF(−), no infection. (f) After NMuMG cells were treated with 1 ng/ml TGF-β for 48 h or transfected with both δEF1 and SIP1 siRNAs, conventional RT–PCR were performed to detect expression of FGFR1 isoforms. NC, control siRNA

Figure 5

Expression profiles of ESRP1/2 and δEF1/SIP1 in breast cancer cells. (a) mRNA levels of the expression of ESRP1, ESRP2, δEF1 and SIP1 were determined by quantitative RT–PCR and compared among 23 human breast cancer cell lines. Gene cluster shown is reported by Neve et al. (2006) and Charafe-Jauffret et al. (2006). Basal A subtype reveals basal-like signature with basal cytokeratin (K5/K14) positive, and basal B subtype exhibits a stem-cell like expression profile with vimentin positive and may reflect the clinical triple-negative tumor type (Neve et al., 2006). (b) The expression of FGFRs isoforms in human breast cancer cell lines was determined by conventional RT–PCR. (c) MDA-MB-231 and BT549 cells were transfected with siRNAs against δEF1 and SIP1, and mRNA levels of ESRP1 and ESRP2 were examined by quantitative RT–PCR. NC, control siRNA. (d) Representative images of hematoxylin and eosin (HE) staining and immunohistochemical staining of cytokeratin 19 (K19), ESRP1, and δEF1 in primary tumor samples from breast cancer patients are shown (# 1 and 2).

Figure 6

ESRPs attenuate malignant phenotypes of cancer cells. (a--e) MDA-MB-231 cells were infected with lentiviruses encoding ESRP1 and ESRP2. The cells were examined for anchorage-independent growth in soft agar (left in a) and quantified (right in a). Expression of E-cadherin in the cells was evaluated by quantitative RT–PCR (b), immunohistochemical (d) and immunoblot (e) analyses. (c) Morphology of the cells were analyzed by phase-contrast microscopy (d). α-tubulin levels were monitored as a loading control (e). (f–h) NMuMG cells infected with lentivirus encoding GFP or FLAG-ESRP2 were treated with TGF-β for 36 h. The cells were analyzed by phase-contrast microscopy (f), immunoblot analyses with the indicated antibodies (g), and immunohistochemical analyses with anti-E-cadherin (green in h) and anti-FLAG (red in h) antibodies, and by TOTO3 to detect nuclei (blue in h). α-tubulin levels were monitored as a loading control. Ratio of E-cadherin to α-tubulin is shown at bottom (g). IF(−), no infection.