Walsh LA and Damjanovski S (2011), IGF-1 increases invasive potential of MCF 7 bre...

XB-ART-44053

Cell Commun Signal 2011 May 02;91:10. doi: 10.1186/1478-811X-9-10.

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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 .

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

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Species referenced: Xenopus
Genes referenced: akt1 cdh1 cdh2 ctnnb1 igf1 itk mapk1 ocln pik3ca vim

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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 Â± s.e.m, (n = 3) *P < 0.01.
	Figure 2. IGF-1 and latent TGF-Î²1 caused morphological changes in MCF-7 cells consistent with EMT. Vehicle treated cells showed typical rounded epithelial morphology (a). Treatment of MCF-7 cells with 100 nM recombinant IGF-1 + 10 nM latent TGF-Î²1 caused a mesenchymal morphological phenotype characterized by a fibroblast-like appearance (g). Treatment with IGF-1 or latent TGF-Î²1 alone did not result in a morphological change (c, e). Pre-treatment of cells with PI3K or MAPK inhibitors abolished IGF-1 + latent TGF-Î²1 induced mesenchymal morphological phenotype (d, f). BB94 or TGF-Î²1 inhibitor prevented the morphological changes associated with IGF-1 + TGF-Î²1 treatment (b, h). Cytoskeleton was stained with phalloidin (red) and the nucleus with DAPI (blue). Images are representative of 3 independent experiments with consistent results.
	Figure 3. Semi-quantitative real time PCR showed IGF-1 and latent TGF-Î²1 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-Î²1, (iii) IGF-1+latent TGF-Î²1+PI3K inhibitor, (iv) IGF-1+latent TGF-Î²1+MAPK inhibitor, (v) IGF-1atent TGF-Î²1+TGF-Î²1 inhibitor or (vi) IGF-1+latent TGF-Î²1+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-Î²1 as compared to latent TGF-Î²1 treatment alone. As there was no significant difference between vehicle and latent TGF-Î²1 treatment, latent TGF-Î²1 treatment alone was used for comparison with other treatments to further highlight changes associates with the activation of TGF-Î²1 and EMT. The addition of PI3K, MAPK, MMP or TGF-Î²1 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-Î²1+inhibitors. All data are mean Â± s.e.m. (n = 3, three experimental repeats).
	Figure 4. Semi-quantitative real time PCR showed IGF-1 and latent TGF-Î²1 resulted in no changes in EMT marker gene expression consistent with EMT in Hs578t cells. Hs578t cells treated with (i) IGF-1, (ii) IGF-1+latent TGF-Î²1, (iii) IGF-1+latent TGF-Î²1+PI3K inhibitor, (iv) IGF-1+latent TGF-Î²1+MAPK inhibitor or (v) IGF-1+latent TGF-Î²1+TGF-Î²1 inhibitor were analyzed for levels of marker genes associated with EMT. Changes in N-cadherin, occludin, and vimentin expression were not consistent with EMT after treatment with IGF-1+TGF-Î²1 compared to TGF-Î²1 treatment alone. The addition of PI3K. MAPK or TGF-Î²1 inhibitors did not affect these changes in marker gene expression. Hs578t cells treated with TGF-Î²1 alone resulted in changes in marker gene expression that were also not consistent with EMT compared to vehicle control. All data are mean Â± s.e.m. (n = 3, three experimental repeats).
	Figure 5. IGF-1 and latent TGF-Î²1 resulted in nuclear localization of Î²-catenin in MCF-7 cells. Nuclei are stained with DAPI (blue) while Î²-catenin is localized with a specific antibody (green). Treatment of MCF-7 cells with 100 nM recombinant IGF-1 + 10 nM TGF-Î²1 resulted in nuclear (blue) localization of Î²-catenin (green) (green blue overlap g-h). Treatment with vehicle control (a-b), IGF-1 (c-d) or TGF-Î²1 alone (e-f) showed Î²-catenin primarily localized at the plasma membrane. Images are representative of 3 independent experiments with consistent results.
	Figure 6. Schematic Model of IGF-1 and TGF-Î²1 signaling in MCF-7 breast cancer cells. 1) IGF-1 binds IGF-1 receptor. 2) IGF-1 receptor signals through the PI3K and MAPK pathways. 3) IGF-1 signaling causes transcriptional activation of unknown target genes (?). 4) Unknown target protein(s) (?) are secreted and activate MMPs. 5) Active MMPs are available to activate latent molecules in the ECM, such as TGF-Î². 6) Active TGF-Î²1 binds TGF-Î²1 receptor. 7) TGF-Î² signaling causes nuclear localization of Î²-catenin. 8) TGF-Î² signaling causing transcriptional activation of genes that result in an EMT.

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Adams, Regulation of development and differentiation by the extracellular matrix. 1993, Pubmed