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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

Semi-quantitative real time PCR showed IGF-1 and latent TGF-β1 resulted in changes in marker gene expression consistent with EMT in MCF-7 cells. MCF-7 cells treated with (i) IGF-1, (ii) IGF-1+latent TGF-β1, (iii) IGF-1+latent TGF-β1+PI3K inhibitor, (iv) IGF-1+latent TGF-β1+MAPK inhibitor, (v) IGF-1atent TGF-β1+TGF-β1 inhibitor or (vi) IGF-1+latent TGF-β1+MMP inhibitor were analyzed for levels of marker genes associated with EMT. E-cadherin, and occludin levels decreased by 21 and 6 fold respectively while N-cadherin and vimentin levels increased by 24 and 8 fold respectively when treated with IGF1-+ latent TGF-β1 as compared to latent TGF-β1 treatment alone. As there was no significant difference between vehicle and latent TGF-β1 treatment, latent TGF-β1 treatment alone was used for comparison with other treatments to further highlight changes associates with the activation of TGF-β1 and EMT. The addition of PI3K, MAPK, MMP or TGF-β1 inhibitors attenuated these changes in marker gene expression. With the exception of vimentin, IGF-1 treatment alone caused changes in marker gene expression similar to that seen with IGF-1+ TGF-β1+inhibitors. All data are mean ± s.e.m. (n = 3, three experimental repeats).
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Figure 3: Semi-quantitative real time PCR showed IGF-1 and latent TGF-β1 resulted in changes in marker gene expression consistent with EMT in MCF-7 cells. MCF-7 cells treated with (i) IGF-1, (ii) IGF-1+latent TGF-β1, (iii) IGF-1+latent TGF-β1+PI3K inhibitor, (iv) IGF-1+latent TGF-β1+MAPK inhibitor, (v) IGF-1atent TGF-β1+TGF-β1 inhibitor or (vi) IGF-1+latent TGF-β1+MMP inhibitor were analyzed for levels of marker genes associated with EMT. E-cadherin, and occludin levels decreased by 21 and 6 fold respectively while N-cadherin and vimentin levels increased by 24 and 8 fold respectively when treated with IGF1-+ latent TGF-β1 as compared to latent TGF-β1 treatment alone. As there was no significant difference between vehicle and latent TGF-β1 treatment, latent TGF-β1 treatment alone was used for comparison with other treatments to further highlight changes associates with the activation of TGF-β1 and EMT. The addition of PI3K, MAPK, MMP or TGF-β1 inhibitors attenuated these changes in marker gene expression. With the exception of vimentin, IGF-1 treatment alone caused changes in marker gene expression similar to that seen with IGF-1+ TGF-β1+inhibitors. All data are mean ± s.e.m. (n = 3, three experimental repeats).

Mentions: To investigate if the IGF-1 and latent TGF-β1 induced changes in morphology were consistent with changes in expression of EMT marker genes, real-time PCR was performed with primers for E-cadherin, N-cadherin, occludin and vimentin. Treatment with latent TGF-β1 alone did not promote significant changes in marker gene expression compared to vehicle treatment (control -data not shown). Expression levels after latent TGF-β1 treatments were therefore used for comparisons with other treatments. MCF-7 cells treated with IGF-1 + latent TGF-β1 showed ~24 and ~8 fold increases in mesenchymal markers N-cadherin and vimentin respectively, and ~21 and ~6 fold decreases in epithelial markers E-cadherin and occludin respectively compared to cells treated with latent TGF-β1 alone (Figure 3). This expression pattern is consistent with EMT seen in other cell types [16]. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the IGF-1 + latent TGF-β1 mediated changes in EMT marker gene expression (Figure 3). IGF-1 treatment alone increased vimentin, but this in itself is not indicative of EMT. MCF-7 cells treated with a TGF-β1 inhibitor or the MMP inhibitor BB94 also did not show changes in marker gene expression consistent with EMT.


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)

Semi-quantitative real time PCR showed IGF-1 and latent TGF-β1 resulted in changes in marker gene expression consistent with EMT in MCF-7 cells. MCF-7 cells treated with (i) IGF-1, (ii) IGF-1+latent TGF-β1, (iii) IGF-1+latent TGF-β1+PI3K inhibitor, (iv) IGF-1+latent TGF-β1+MAPK inhibitor, (v) IGF-1atent TGF-β1+TGF-β1 inhibitor or (vi) IGF-1+latent TGF-β1+MMP inhibitor were analyzed for levels of marker genes associated with EMT. E-cadherin, and occludin levels decreased by 21 and 6 fold respectively while N-cadherin and vimentin levels increased by 24 and 8 fold respectively when treated with IGF1-+ latent TGF-β1 as compared to latent TGF-β1 treatment alone. As there was no significant difference between vehicle and latent TGF-β1 treatment, latent TGF-β1 treatment alone was used for comparison with other treatments to further highlight changes associates with the activation of TGF-β1 and EMT. The addition of PI3K, MAPK, MMP or TGF-β1 inhibitors attenuated these changes in marker gene expression. With the exception of vimentin, IGF-1 treatment alone caused changes in marker gene expression similar to that seen with IGF-1+ TGF-β1+inhibitors. All data are mean ± s.e.m. (n = 3, three experimental repeats).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Semi-quantitative real time PCR showed IGF-1 and latent TGF-β1 resulted in changes in marker gene expression consistent with EMT in MCF-7 cells. MCF-7 cells treated with (i) IGF-1, (ii) IGF-1+latent TGF-β1, (iii) IGF-1+latent TGF-β1+PI3K inhibitor, (iv) IGF-1+latent TGF-β1+MAPK inhibitor, (v) IGF-1atent TGF-β1+TGF-β1 inhibitor or (vi) IGF-1+latent TGF-β1+MMP inhibitor were analyzed for levels of marker genes associated with EMT. E-cadherin, and occludin levels decreased by 21 and 6 fold respectively while N-cadherin and vimentin levels increased by 24 and 8 fold respectively when treated with IGF1-+ latent TGF-β1 as compared to latent TGF-β1 treatment alone. As there was no significant difference between vehicle and latent TGF-β1 treatment, latent TGF-β1 treatment alone was used for comparison with other treatments to further highlight changes associates with the activation of TGF-β1 and EMT. The addition of PI3K, MAPK, MMP or TGF-β1 inhibitors attenuated these changes in marker gene expression. With the exception of vimentin, IGF-1 treatment alone caused changes in marker gene expression similar to that seen with IGF-1+ TGF-β1+inhibitors. All data are mean ± s.e.m. (n = 3, three experimental repeats).
Mentions: To investigate if the IGF-1 and latent TGF-β1 induced changes in morphology were consistent with changes in expression of EMT marker genes, real-time PCR was performed with primers for E-cadherin, N-cadherin, occludin and vimentin. Treatment with latent TGF-β1 alone did not promote significant changes in marker gene expression compared to vehicle treatment (control -data not shown). Expression levels after latent TGF-β1 treatments were therefore used for comparisons with other treatments. MCF-7 cells treated with IGF-1 + latent TGF-β1 showed ~24 and ~8 fold increases in mesenchymal markers N-cadherin and vimentin respectively, and ~21 and ~6 fold decreases in epithelial markers E-cadherin and occludin respectively compared to cells treated with latent TGF-β1 alone (Figure 3). This expression pattern is consistent with EMT seen in other cell types [16]. Pretreatment of cells with PI3K or MAPK inhibitors attenuated the IGF-1 + latent TGF-β1 mediated changes in EMT marker gene expression (Figure 3). IGF-1 treatment alone increased vimentin, but this in itself is not indicative of EMT. MCF-7 cells treated with a TGF-β1 inhibitor or the MMP inhibitor BB94 also did not show changes in marker gene expression consistent with EMT.

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