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Negative regulation of fibroblast growth factor 10 (FGF-10) by polyoma enhancer activator 3 (PEA3).

Chioni AM, Grose R - Eur. J. Cell Biol. (2009)

Bottom Line: Knockdown of PEA3 message led to increased Fgf-10 expression, whereas overexpression of PEA3 resulted in decreased Fgf-10 expression.Furthermore, over-expression of PEA3 in these cells resulted in impaired cell migration, which was rescued by treatment with FGF-10.Thus, PEA3 can regulate the transcription of Fgf-10 and such modulation can control breast cancer cell behaviour.

View Article: PubMed Central - PubMed

Affiliation: Centre for Tumour Biology, Institute of Cancer, Barts & The London School of Medicine & Dentistry, London EC1M 6BQ, UK.

ABSTRACT
FGF-10 plays an important role in development and disease, acting as the key ligand for FGFR2B to regulate cell proliferation, migration and differentiation. Aberrant FGF signalling is implicated in tumourigenesis, with several cancer studies reporting FGF-10 or FGFR2B upregulation or identifying activating mutations in Fgfr2. We used 5' RACE to identify a novel transcription start site for murine Fgf-10. Conventional in silico analysis predicted multiple binding sites for the transcription factor PEA3 upstream of this site. Binding was confirmed by chromatin immunopreciptation, and functional significance was studied by both RNAi knockdown and transient over-expression of PEA3. Knockdown of PEA3 message led to increased Fgf-10 expression, whereas overexpression of PEA3 resulted in decreased Fgf-10 expression. Thus, we have identified PEA3 as a negative regulator of Fgf-10 expression in a murine cell line and confirmed that activity also is seen in human breast cancer cell lines (MCF-7 and MDA-MB-231). Furthermore, over-expression of PEA3 in these cells resulted in impaired cell migration, which was rescued by treatment with FGF-10. Thus, PEA3 can regulate the transcription of Fgf-10 and such modulation can control breast cancer cell behaviour.

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Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2−ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t-test. Results were considered significant at P<0.05 (*).
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fig4: Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2−ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t-test. Results were considered significant at P<0.05 (*).

Mentions: Firstly, we cloned the full-length human PEA3-encoding cDNA sequence into a mammalian expression vector, pcDNA4/TO, and confirmed orientation and fidelity by sequencing the whole insert (data not shown). Within 48 h of transient transfection with our PEA3 expression construct, real-time RT-PCR revealed that levels of PEA3 mRNA expression in MDA-MB-231 cells increased by over 3000-fold compared to the negligible levels seen in cells transfected with empty pcDNA4/TO vector (Fig. 4A). Similarly, PEA3 protein was detected readily in PEA3-transfected MDA-MB-231 cells, but not in empty vector control cells (Fig. 4B). Crucially, at the same 48 h timepoint, PEA3-transfected cells showed a 73% reduction in FGF-10 mRNA expression relative to controls (Fig. 4A). Similar results were obtained at the protein level, with FGF-10 protein reduced by 70% (Fig. 4B). Thus, over-expression of PEA3 in MDA-MB-231 cells decreased Fgf-10 expression. Similar results were obtained with MCF-7 cells (data not shown). These results are consistent with the RNAi-mediated knockdown of PEA3 in mouse lung endothelial cells, suggesting that PEA3 is a negative regulator of Fgf-10 expression.


Negative regulation of fibroblast growth factor 10 (FGF-10) by polyoma enhancer activator 3 (PEA3).

Chioni AM, Grose R - Eur. J. Cell Biol. (2009)

Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2−ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t-test. Results were considered significant at P<0.05 (*).
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fig4: Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2−ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t-test. Results were considered significant at P<0.05 (*).
Mentions: Firstly, we cloned the full-length human PEA3-encoding cDNA sequence into a mammalian expression vector, pcDNA4/TO, and confirmed orientation and fidelity by sequencing the whole insert (data not shown). Within 48 h of transient transfection with our PEA3 expression construct, real-time RT-PCR revealed that levels of PEA3 mRNA expression in MDA-MB-231 cells increased by over 3000-fold compared to the negligible levels seen in cells transfected with empty pcDNA4/TO vector (Fig. 4A). Similarly, PEA3 protein was detected readily in PEA3-transfected MDA-MB-231 cells, but not in empty vector control cells (Fig. 4B). Crucially, at the same 48 h timepoint, PEA3-transfected cells showed a 73% reduction in FGF-10 mRNA expression relative to controls (Fig. 4A). Similar results were obtained at the protein level, with FGF-10 protein reduced by 70% (Fig. 4B). Thus, over-expression of PEA3 in MDA-MB-231 cells decreased Fgf-10 expression. Similar results were obtained with MCF-7 cells (data not shown). These results are consistent with the RNAi-mediated knockdown of PEA3 in mouse lung endothelial cells, suggesting that PEA3 is a negative regulator of Fgf-10 expression.

Bottom Line: Knockdown of PEA3 message led to increased Fgf-10 expression, whereas overexpression of PEA3 resulted in decreased Fgf-10 expression.Furthermore, over-expression of PEA3 in these cells resulted in impaired cell migration, which was rescued by treatment with FGF-10.Thus, PEA3 can regulate the transcription of Fgf-10 and such modulation can control breast cancer cell behaviour.

View Article: PubMed Central - PubMed

Affiliation: Centre for Tumour Biology, Institute of Cancer, Barts & The London School of Medicine & Dentistry, London EC1M 6BQ, UK.

ABSTRACT
FGF-10 plays an important role in development and disease, acting as the key ligand for FGFR2B to regulate cell proliferation, migration and differentiation. Aberrant FGF signalling is implicated in tumourigenesis, with several cancer studies reporting FGF-10 or FGFR2B upregulation or identifying activating mutations in Fgfr2. We used 5' RACE to identify a novel transcription start site for murine Fgf-10. Conventional in silico analysis predicted multiple binding sites for the transcription factor PEA3 upstream of this site. Binding was confirmed by chromatin immunopreciptation, and functional significance was studied by both RNAi knockdown and transient over-expression of PEA3. Knockdown of PEA3 message led to increased Fgf-10 expression, whereas overexpression of PEA3 resulted in decreased Fgf-10 expression. Thus, we have identified PEA3 as a negative regulator of Fgf-10 expression in a murine cell line and confirmed that activity also is seen in human breast cancer cell lines (MCF-7 and MDA-MB-231). Furthermore, over-expression of PEA3 in these cells resulted in impaired cell migration, which was rescued by treatment with FGF-10. Thus, PEA3 can regulate the transcription of Fgf-10 and such modulation can control breast cancer cell behaviour.

Show MeSH
Related in: MedlinePlus