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IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-β1 resulting in epithelial to mesenchymal transition.

Walsh LA, Damjanovski S - Cell Commun. Signal (2011)

Bottom Line: Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways.In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7, Canada. sdamjano@uwo.ca.

ABSTRACT

Introduction: TGF-β signaling has been extensively studied in many developmental contexts, amongst which is its ability to induce epithelial to mesenchymal transitions (EMT). EMTs play crucial roles during embryonic development and have also come under intense scrutiny as a mechanism through which breast cancers progress to become metastatic. Interestingly, while the molecular hallmarks of EMT progression (loss of cell adhesion, nuclear localization of β-catenin) are straightforward, the cellular signaling cascades that result in an EMT are numerous and diverse. Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.

Methods: MCF-7 breast cancer cells treated with recombinant IGF-1 were assayed for metalloproteinase activity and invasiveness through a matrigel coated transwell invasion chamber. IGF-1 treatments were then followed by the addition of latent-TGF-β1 to determine if elevated levels of IGF-1 together with latent-TGF-β1 could cause EMT.

Results: Results showed that IGF-1 - a molecule known to be elevated in breast cancer is a regulator of matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 breast cancer cells. The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways. In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.

Conclusions: Taken together our data suggest a novel a link between IGF-1 levels, MMP activity, TGF-β signaling, and EMT in breast cancer cells.

No MeSH data available.


Related in: MedlinePlus

IGF-1 increased metalloproteinase activity and invasiveness in MCF-7 breast cancer cells via the PI3K and MAPK pathways. a) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 caused a 2.9 fold increase in protease activity as determined using a fluoregenic metalloproteinase substrate. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the increased protease activation and resulted in a 34% and 29% decrease in activity respectively compared to IGF-1 treatment alone. b) MCF-7 cell viability, assessed by a fluorogenic viability assay, was not significantly affected by IGF-1 and/or inhibitor treatments. c) Treatment of MCF-7 conditioned media with aforementioned reagents resulted in no significant changes in MMP activity. d-e) Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors reveals decreased expression of phospho-ERK and phospho-AKT expression respectively. f) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 increased invasiveness through a matrigel coated transwell chamber by ~400% compared to vehicle treatment (control). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to vehicle control. Each assay was repeated three times (three experimental repeats). All data are mean ± s.e.m, (n = 3) *P < 0.01.
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Figure 1: IGF-1 increased metalloproteinase activity and invasiveness in MCF-7 breast cancer cells via the PI3K and MAPK pathways. a) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 caused a 2.9 fold increase in protease activity as determined using a fluoregenic metalloproteinase substrate. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the increased protease activation and resulted in a 34% and 29% decrease in activity respectively compared to IGF-1 treatment alone. b) MCF-7 cell viability, assessed by a fluorogenic viability assay, was not significantly affected by IGF-1 and/or inhibitor treatments. c) Treatment of MCF-7 conditioned media with aforementioned reagents resulted in no significant changes in MMP activity. d-e) Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors reveals decreased expression of phospho-ERK and phospho-AKT expression respectively. f) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 increased invasiveness through a matrigel coated transwell chamber by ~400% compared to vehicle treatment (control). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to vehicle control. Each assay was repeated three times (three experimental repeats). All data are mean ± s.e.m, (n = 3) *P < 0.01.

Mentions: IGF-1 has been shown previously to stimulate protease activity in many cell types [13]. To determine if IGF-1 could stimulate metalloproteinase activity in MCF-7 cells they were treated with 100 nM recombinant IGF-1, and subsequently their conditioned media was used to assay for metalloproteinase activity using a broad-spectrum metalloproteinase (Mca-KPLGL-Dpa-AR-NH2) Fluorogenic Peptide Substrate. IGF-1 treatment resulted in a 2.9 fold increase in metalloproteinase activity compared to vehicle control (Figure 1a). To identify the pathway(s) regulating protease induction by IGF-I, pharmacological inhibitors were used that inhibit Phosphoinositide 3-kinases (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling - two known downstream IGF-I effectors [13]. Treatment with the PI3K inhibitor wortmannin (5 nm) significantly reduced IGF-I-mediated protease activation by 34% (Figure 1a). Treatment with the MAPK inhibitor SB 202190 (10 M) had a 29% decrease in IGF-1 induced protease activation (Figure 1a). Cell viability was unaltered by treatments (Figure 1b) confirming that the effects of the inhibitors were not due to alterations in cell viability. The specificity of the reagents on mediating cellular events and not simply altering activity in the culture media was assayed via the addition of the above reagents to cell free conditioned media where in resulted in no significant changes in MMP activity (Figure 1c). Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors revealed decreased levels of phospho-ERK and phospho-AKT moieties respectively, confirming the efficacy of the inhibitors (Figure 1d-e). Notably, the treatment of MCF-7 cells with IGF-1 increased invasiveness through a matrigel coated transwell invasion chamber by ~400% compared to control (Figure 1f). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to control (Figure 1f).


IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-β1 resulting in epithelial to mesenchymal transition.

Walsh LA, Damjanovski S - Cell Commun. Signal (2011)

IGF-1 increased metalloproteinase activity and invasiveness in MCF-7 breast cancer cells via the PI3K and MAPK pathways. a) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 caused a 2.9 fold increase in protease activity as determined using a fluoregenic metalloproteinase substrate. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the increased protease activation and resulted in a 34% and 29% decrease in activity respectively compared to IGF-1 treatment alone. b) MCF-7 cell viability, assessed by a fluorogenic viability assay, was not significantly affected by IGF-1 and/or inhibitor treatments. c) Treatment of MCF-7 conditioned media with aforementioned reagents resulted in no significant changes in MMP activity. d-e) Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors reveals decreased expression of phospho-ERK and phospho-AKT expression respectively. f) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 increased invasiveness through a matrigel coated transwell chamber by ~400% compared to vehicle treatment (control). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to vehicle control. Each assay was repeated three times (three experimental repeats). All data are mean ± s.e.m, (n = 3) *P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104381&req=5

Figure 1: IGF-1 increased metalloproteinase activity and invasiveness in MCF-7 breast cancer cells via the PI3K and MAPK pathways. a) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 caused a 2.9 fold increase in protease activity as determined using a fluoregenic metalloproteinase substrate. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the increased protease activation and resulted in a 34% and 29% decrease in activity respectively compared to IGF-1 treatment alone. b) MCF-7 cell viability, assessed by a fluorogenic viability assay, was not significantly affected by IGF-1 and/or inhibitor treatments. c) Treatment of MCF-7 conditioned media with aforementioned reagents resulted in no significant changes in MMP activity. d-e) Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors reveals decreased expression of phospho-ERK and phospho-AKT expression respectively. f) Treatment of MCF-7 cells with 100 nM recombinant IGF-1 increased invasiveness through a matrigel coated transwell chamber by ~400% compared to vehicle treatment (control). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to vehicle control. Each assay was repeated three times (three experimental repeats). All data are mean ± s.e.m, (n = 3) *P < 0.01.
Mentions: IGF-1 has been shown previously to stimulate protease activity in many cell types [13]. To determine if IGF-1 could stimulate metalloproteinase activity in MCF-7 cells they were treated with 100 nM recombinant IGF-1, and subsequently their conditioned media was used to assay for metalloproteinase activity using a broad-spectrum metalloproteinase (Mca-KPLGL-Dpa-AR-NH2) Fluorogenic Peptide Substrate. IGF-1 treatment resulted in a 2.9 fold increase in metalloproteinase activity compared to vehicle control (Figure 1a). To identify the pathway(s) regulating protease induction by IGF-I, pharmacological inhibitors were used that inhibit Phosphoinositide 3-kinases (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling - two known downstream IGF-I effectors [13]. Treatment with the PI3K inhibitor wortmannin (5 nm) significantly reduced IGF-I-mediated protease activation by 34% (Figure 1a). Treatment with the MAPK inhibitor SB 202190 (10 M) had a 29% decrease in IGF-1 induced protease activation (Figure 1a). Cell viability was unaltered by treatments (Figure 1b) confirming that the effects of the inhibitors were not due to alterations in cell viability. The specificity of the reagents on mediating cellular events and not simply altering activity in the culture media was assayed via the addition of the above reagents to cell free conditioned media where in resulted in no significant changes in MMP activity (Figure 1c). Western Blot analysis of MCF-7 cells treated with MAPK or PI3K inhibitors revealed decreased levels of phospho-ERK and phospho-AKT moieties respectively, confirming the efficacy of the inhibitors (Figure 1d-e). Notably, the treatment of MCF-7 cells with IGF-1 increased invasiveness through a matrigel coated transwell invasion chamber by ~400% compared to control (Figure 1f). Pre-treatment of cells with PI3K or MAPK inhibitors before IGF-1 treatment resulted in no significant change in invasiveness compared to control (Figure 1f).

Bottom Line: Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways.In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7, Canada. sdamjano@uwo.ca.

ABSTRACT

Introduction: TGF-β signaling has been extensively studied in many developmental contexts, amongst which is its ability to induce epithelial to mesenchymal transitions (EMT). EMTs play crucial roles during embryonic development and have also come under intense scrutiny as a mechanism through which breast cancers progress to become metastatic. Interestingly, while the molecular hallmarks of EMT progression (loss of cell adhesion, nuclear localization of β-catenin) are straightforward, the cellular signaling cascades that result in an EMT are numerous and diverse. Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.

Methods: MCF-7 breast cancer cells treated with recombinant IGF-1 were assayed for metalloproteinase activity and invasiveness through a matrigel coated transwell invasion chamber. IGF-1 treatments were then followed by the addition of latent-TGF-β1 to determine if elevated levels of IGF-1 together with latent-TGF-β1 could cause EMT.

Results: Results showed that IGF-1 - a molecule known to be elevated in breast cancer is a regulator of matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 breast cancer cells. The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways. In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.

Conclusions: Taken together our data suggest a novel a link between IGF-1 levels, MMP activity, TGF-β signaling, and EMT in breast cancer cells.

No MeSH data available.


Related in: MedlinePlus