Profilin-1 overexpression upregulates PTEN and suppresses AKT activation in breast cancer cells

Abstract

Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has been regarded as a tumor-suppressor molecule for breast cancer. Since AKT signaling impacts cell survival and proliferation, in this study we investigated whether AKT activation in breast cancer cells is sensitive to perturbation of Pfn1 expression. We found that even a moderate overexpression of Pfn1 leads to a significant reduction in phosphorylation of AKT in MDA-MB-231 breast cancer cells. We further demonstrated that Pfn1 overexpression in MDA-MB-231 cells is associated with a significant reduction in the level of the phosphoinositide regulator of AKT, PIP(3), and impaired membrane translocation of AKT that is required for AKT activation, in response to EGF stimulation. Interestingly, Pfn1-overexpressing cells showed post-transcriptional upregulation of PTEN. Furthermore, when PTEN expression was silenced, AKT phosphorylation was rescued, suggesting PTEN upregulation is responsible for Pfn1-dependent attenuation of AKT activation in MDA-MB-231 cells. Pfn1 overexpression induced PTEN upregulation and reduced AKT activation were also reproducible features of BT474 breast cancer cells. These findings may provide mechanistic insights underlying at least some of the tumor-suppressive properties of Pfn1.

(c) 2008 Wiley-Liss, Inc.

Figures

Fig. 1. Effect of Pfn1 overexpression on AKT activation in MDA-231 cells

A) GFP and Pfn1 immunoblots of total cell lysates extracted from GFP and GFP-Pfn1 expressers of MDA-231. B) GFP and Pfn1 immunoblots of GFP-Pfn1 expresser lysate with known amounts of purified recombinant GFP (rGFP) or Pfn1 (rPfn1) proteins, respectively, were analyzed for quantitative estimation of the expression level of GFP-Pfn1 relative to that of endogenous Pfn1. C) Comparative profiles of phospho-AKT (T308 and S473), phospho-GSK3β (S9) between GFP and GFP-Pfn1 expressing MDA-231 cells in regular culture condition. D) Phospho-AKT profiles (T308 and S473) of GFP and GFP-Pfn1 expressers under basal and EGF-stimulated conditions confirm Pfn1-overexpression induced inhibition of AKT phosphorylation. AKT immunoblot shows comparable expression level of AKT between our two cell lines. E) Quantitative representation of the ratio of phospho-AKT (S473) to total AKT for GFP and GFP-Pfn1 expressers at different time points after EGF stimulation (data summarized from 3 independent experiments and * denotes p<0.05). The inset shows a representative phospho-EGFR immunoblot from one such experiment revealing higher tyrosine phosphorylation of EGFR in Pfn1 overexpressing cells. F and G) Immunoblots reveal that S241 phosphorylation (panel F) as well as the total expression level (panel G) of PDK1 for GFP and GFP-Pfn1 expressers are similar. PDK phosphorylation is insensitive to EGF treatment (panel D). For all experiments, GAPDH immunoblots serve as the loading controls.

Fig. 2. Membrane targeting of AKT is inhibited by Pfn1 overexpression

A) AKT immunostaining shows much more pronounced EGF-dependent membrane targeting of AKT (marked by arrow) in GFP-expressing cells compared to Pfn1-overexpressers (scale bar − 20 μm). B) Fluorescence intensity analyses of AKT-immunostaining images are summarized in a bar graph to reveal the relative ATI values of GFP and GFP-Pfn1 expressers in basal and EGF-stimulated conditions (n - number of analyzed cells pooled from 2 independent experiments). C) A bar graph summarizes the % of cells displaying membrane staining of AKT. These data are summarized from 2 independent experiments and * indicates p

Fig. 3. Pfn1 overexpression suppresses PIP3 generation

A) PIP3 immunostaining images of GFP and GFP-Pfn1 expressers at basal and 5 minutes after EGF stimulation (scale bar − 20 μm). B) Quantification of the average intensity of PIP3 staining of the two cell lines with or without EGF treatment (these data were normalized to the intensity value calculated for GFP cells in basal state; ‘n’ denotes number of cells for each experimental group pooled from 2 independent experiments). C) Quantification of the average intensity of PIP3 staining of GFP and GFP-Pfn1 expressers which were first pretreated with either 25 μM LY294002 or DMSO (vehicle control) for 30 minutes and then stimulated with EGF for 5 minutes shows a dramatic decrease in the intensity of PIP3 staining as a result of LY treatment (these data were normalized to the intensity value calculated for GFP cells under DMSO treatment; ‘n’ denotes number of cells for each experimental group). The inset shows a phospho-AKT (S473) immunoblot of GFP-expressers under both basal and EGF stimulation (30 min) in the presence of either 25 μM LY compound or DMSO (AKT immunoblot serves as the loading control). LY compound treatment complete suppresses EGF-dependent AKT phosphorylation thereby confirming inhibition of PI3K activity. D) i) Time-course of AKT phosphorylation of GFP and GFP-Pfn1 expressing MDA-231 cells in response to PDGF treatment show downregulation of AKT activation in Pfn1 overexpressers (GAPDH blot serves as the loading control). ii) Lipids extracted from GFP and GFP-Pfn1 expressers following 5 min of PDGF stimulation, when spotted on a membrane and probed with PIP3-specific detection system, show significantly reduced PIP3 level in GFP-Pfn1 overexpresser compared to GFP cell line. Known amounts of PIP3 (positive control) and other PIs (PI, PI3P – negative control) were pre-spotted on the membrane to demonstrate the specificity of the detection procedure. The dotted circles represent the spotted area. For all analyses * represents p<0.05.

Fig. 4. Pfn1 overexpression inhibits AKT activation in a PTEN-dependent manner

A) Phosphotyrosine immunoprecipitates from GFP and GFP-Pfn1 expressers, either in basal or EGF stimulated (10 minutes) conditions, were probed with antibodies specific for p85 and EGFR to reveal their tyrosine phosphorylation status. EGF-induced tyrosine phosphorylation of both p85 and EGFR were found to be higher in Pfn1 over-expressing cells. Immunoblot of total cell lysates shows comparable expression level of p85 between the two cell lines. B) Pfn1-overexpressing cells show marked increase in PTEN level. C) RT-PCR data show similar levels of PTEN transcript between GFP and GFP-Pfn1 expressers (GAPDH RT-PCR products serve as the loading control). D) PTEN immunoblot of total extracts from siRNA (either control or PTEN-specific)-transfected cells shows strong silencing of PTEN expression. Phospho-AKT (S473) immunoblot demonstrates that PTEN silencing rescues the inhibition of AKT phosphorylation in GFP-Pfn1 expressers. E) Comparative profiles of PTEN and pAKT (T308) between GFP and GFP-Pfn1 overexpressers of BT474 breast cancer cells. For all experiments, GAPDH blots serve as the loading controls.