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CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT

View Article: PubMed Central - PubMed

ABSTRACT

The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3′-untranslated region (3′-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3′-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.

No MeSH data available.


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CELF1 expression and function are conserved in multiple cellular models of EMT.(a) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. HSP90 serves as a loading control. (b) Quantification of cellular migration and invasion in transwell assays in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. (c,d) As in a and b, but in response to stable Renilla Luciferase or CELF1 overexpression in the indicated cell lines, respectively. In c, HSP90 serves as a loading control. (e,f) As in a and b, but in response to stable knockdown of β-galactosidase (shGLB1) or two distinct CELF1 shRNAs (shCELF1A, shCELF1B) in the indicated cell lines, respectively. In e, HSP90 serves as a loading control. All panels are representative of a minimum of three individual experimental replicates. For immunoblots depicted, samples were derived from the same experiment and gels were processed in parallel. Error bars depict s.d. of the mean. *P≤0.05 (Student's t-test). See also Supplementary Fig. 4. Full scans of blots are shown in Supplementary Fig. 11.
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f5: CELF1 expression and function are conserved in multiple cellular models of EMT.(a) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. HSP90 serves as a loading control. (b) Quantification of cellular migration and invasion in transwell assays in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. (c,d) As in a and b, but in response to stable Renilla Luciferase or CELF1 overexpression in the indicated cell lines, respectively. In c, HSP90 serves as a loading control. (e,f) As in a and b, but in response to stable knockdown of β-galactosidase (shGLB1) or two distinct CELF1 shRNAs (shCELF1A, shCELF1B) in the indicated cell lines, respectively. In e, HSP90 serves as a loading control. All panels are representative of a minimum of three individual experimental replicates. For immunoblots depicted, samples were derived from the same experiment and gels were processed in parallel. Error bars depict s.d. of the mean. *P≤0.05 (Student's t-test). See also Supplementary Fig. 4. Full scans of blots are shown in Supplementary Fig. 11.

Mentions: Treatment of the MCF10AT1 line with TGF-β for 72 h resulted in increased expression of CELF1 protein and EMT (Fig. 5a,b, Supplementary Fig. 4a). As in the parental MCF10A line, stable overexpression of CELF1 promoted EMT in MCF10AT1 cells independent of TGF-β stimulus (Fig. 5c,d, Supplementary Fig. 4b). In contrast to the MCF10AT1 line, the MCF10CA1a cells expressed CELF1 protein a priori even in the absence of TGF-β treatment (Fig. 5a). Strikingly, shRNA-mediated knockdown of CELF1 in MCF10CA1a cells significantly decreased the migratory and invasive potential of these cells, increased the relative expression of E-cadherin, and decreased the relative expression of mesenchymal markers (Fig. 5e,f, Supplementary Fig. 4b).


CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT
CELF1 expression and function are conserved in multiple cellular models of EMT.(a) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. HSP90 serves as a loading control. (b) Quantification of cellular migration and invasion in transwell assays in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. (c,d) As in a and b, but in response to stable Renilla Luciferase or CELF1 overexpression in the indicated cell lines, respectively. In c, HSP90 serves as a loading control. (e,f) As in a and b, but in response to stable knockdown of β-galactosidase (shGLB1) or two distinct CELF1 shRNAs (shCELF1A, shCELF1B) in the indicated cell lines, respectively. In e, HSP90 serves as a loading control. All panels are representative of a minimum of three individual experimental replicates. For immunoblots depicted, samples were derived from the same experiment and gels were processed in parallel. Error bars depict s.d. of the mean. *P≤0.05 (Student's t-test). See also Supplementary Fig. 4. Full scans of blots are shown in Supplementary Fig. 11.
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f5: CELF1 expression and function are conserved in multiple cellular models of EMT.(a) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. HSP90 serves as a loading control. (b) Quantification of cellular migration and invasion in transwell assays in untreated or TGF-β-treated MCF10A, MCF10AT1, MCF10CA1a and HMLE cells, and in untreated or EGF-treated MDA-MB-468 and MDA-MB-231 cells. (c,d) As in a and b, but in response to stable Renilla Luciferase or CELF1 overexpression in the indicated cell lines, respectively. In c, HSP90 serves as a loading control. (e,f) As in a and b, but in response to stable knockdown of β-galactosidase (shGLB1) or two distinct CELF1 shRNAs (shCELF1A, shCELF1B) in the indicated cell lines, respectively. In e, HSP90 serves as a loading control. All panels are representative of a minimum of three individual experimental replicates. For immunoblots depicted, samples were derived from the same experiment and gels were processed in parallel. Error bars depict s.d. of the mean. *P≤0.05 (Student's t-test). See also Supplementary Fig. 4. Full scans of blots are shown in Supplementary Fig. 11.
Mentions: Treatment of the MCF10AT1 line with TGF-β for 72 h resulted in increased expression of CELF1 protein and EMT (Fig. 5a,b, Supplementary Fig. 4a). As in the parental MCF10A line, stable overexpression of CELF1 promoted EMT in MCF10AT1 cells independent of TGF-β stimulus (Fig. 5c,d, Supplementary Fig. 4b). In contrast to the MCF10AT1 line, the MCF10CA1a cells expressed CELF1 protein a priori even in the absence of TGF-β treatment (Fig. 5a). Strikingly, shRNA-mediated knockdown of CELF1 in MCF10CA1a cells significantly decreased the migratory and invasive potential of these cells, increased the relative expression of E-cadherin, and decreased the relative expression of mesenchymal markers (Fig. 5e,f, Supplementary Fig. 4b).

View Article: PubMed Central - PubMed

ABSTRACT

The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3′-untranslated region (3′-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3′-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.

No MeSH data available.


Related in: MedlinePlus