Limits...
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.


Related in: MedlinePlus

GREs encode gene products required for EMT.(a) Immunoblot analysis of epithelial and mesenchymal cell markers in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. HSP90 serves as a loading control. (b) Quantification of relative cellular migration and invasion in transwell assays in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. (c,d) As in a and b except that untreated MCF10A cells were stably transduced with pL6.3-Renilla Luciferase or pL6.3-driving the expression of the indicated coding sequences, respectively. All panels are representative of a minimum of three 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. 2. Full scans of blots are shown in Supplementary Fig. 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121338&req=5

f3: GREs encode gene products required for EMT.(a) Immunoblot analysis of epithelial and mesenchymal cell markers in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. HSP90 serves as a loading control. (b) Quantification of relative cellular migration and invasion in transwell assays in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. (c,d) As in a and b except that untreated MCF10A cells were stably transduced with pL6.3-Renilla Luciferase or pL6.3-driving the expression of the indicated coding sequences, respectively. All panels are representative of a minimum of three 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. 2. Full scans of blots are shown in Supplementary Fig. 9.

Mentions: To determine whether the GRE-containing mRNAs functionally contribute to EMT, we transiently transfected MCF10A cells with siRNAs targeting Firefly luciferase (negative control) or each of the 13 GRE-containing transcripts, and treated the transfectants with TGF-β for 72 h (Supplementary Fig. 2a). After 72 h, EMT of the transfectants was monitored via immunoblot analysis of molecular markers, and migration and invasion in standard transwell assays. Via these criteria, 10 of the 13 GRE-containing mRNAs tested blocked TGF-β-induced EMT (Fig. 3a,b, Supplementary Fig. 2b), indicating that these mRNAs are necessary for TGF-β-induced EMT. Importantly, these effects were independent of changes in cellular proliferation or apoptosis (Supplementary Fig. 2c,d).


CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT
GREs encode gene products required for EMT.(a) Immunoblot analysis of epithelial and mesenchymal cell markers in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. HSP90 serves as a loading control. (b) Quantification of relative cellular migration and invasion in transwell assays in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. (c,d) As in a and b except that untreated MCF10A cells were stably transduced with pL6.3-Renilla Luciferase or pL6.3-driving the expression of the indicated coding sequences, respectively. All panels are representative of a minimum of three 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. 2. Full scans of blots are shown in Supplementary Fig. 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121338&req=5

f3: GREs encode gene products required for EMT.(a) Immunoblot analysis of epithelial and mesenchymal cell markers in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. HSP90 serves as a loading control. (b) Quantification of relative cellular migration and invasion in transwell assays in TGF-β-treated MCF10A cells transiently transfected with siRNAs targeting Firefly Luciferase or the indicated mRNAs. (c,d) As in a and b except that untreated MCF10A cells were stably transduced with pL6.3-Renilla Luciferase or pL6.3-driving the expression of the indicated coding sequences, respectively. All panels are representative of a minimum of three 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. 2. Full scans of blots are shown in Supplementary Fig. 9.
Mentions: To determine whether the GRE-containing mRNAs functionally contribute to EMT, we transiently transfected MCF10A cells with siRNAs targeting Firefly luciferase (negative control) or each of the 13 GRE-containing transcripts, and treated the transfectants with TGF-β for 72 h (Supplementary Fig. 2a). After 72 h, EMT of the transfectants was monitored via immunoblot analysis of molecular markers, and migration and invasion in standard transwell assays. Via these criteria, 10 of the 13 GRE-containing mRNAs tested blocked TGF-β-induced EMT (Fig. 3a,b, Supplementary Fig. 2b), indicating that these mRNAs are necessary for TGF-β-induced EMT. Importantly, these effects were independent of changes in cellular proliferation or apoptosis (Supplementary Fig. 2c,d).

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