From Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells.
The Journal of cell biology.
The Journal of cell biology.
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Figure 3. Attenuation of PI3K activity results in cross-modulation of other signaling pathways and intermediates. Cell lysates from S-1, T4-2, and T4-2+LY grown in 3D lrBM or on 2D plastic substrata for 10 d were analyzed for expression of (A) EGFR, ß1 integrin, phosphorylated Akt (serine 473)/total, phosphorylated GSK-3ß (serine 9)/total, and (B) PTEN (n = 3); E-cadherin was used as the loading control. It was shown previously that the total level of E-cadherin does not change under these conditions (Weaver et al., 1997).
- T4-2 cells treated with LY294002 show reduced levels of EGFR and ß1 integrin (Fig. 3 A).
- This effect depended upon 3D lrBM as it is not observed in cells cultured on two-dimensional (2D) plastic substrata (it should be noted that inhibition of PI3K activity, as measured by activation of downstream mediators Akt and GSK-3ß, was equally effective in cells on 2D or in 3D; Fig. 3 A).
- In addition, our results revealed that PTEN, the antagonist of PI3K that acts to dephosphorylate PIP3 and which becomes down-regulated in many carcinomas (Simpson and Parsons, 2001; Yamada and Araki, 2001), is also a component of the cross-modulated signaling network, as treatment of T4-2 cells with LY294002 resulted in an increase of PTEN to the level of the nonmalignant cells; this modulation, too, was seen only in cells cultured on 3D lrBM (Fig. 3 B).
- The evidence that inhibition of PI3K can affect crossmodulation of a number of distinct signaling pathways is a demonstration that pathways downstream of PI3K are integrated into transduction networks when cells are grown in the physiological 3D lrBM; consistent with this model, we found that reversion of the tumor cells to a normal phenotype was associated with increased expression of PTEN, the PI3K antagonist (Fig. 3 B).
- Normalization of signaling pathways in T4-2 cells in response to inhibition of PI3K is dependent upon culture in 3D lrBM, as T4-2 cells grown on 2D tissue culture plastic do not show the dramatic downmodulation of ß1 integrin and EGFR (Fig. 3 A), up-regulation of PTEN (Fig. 3 B), or the alterations in cellular morphology in response to treatment with inhibitors of PI3K (Figs.
- 2 and 3).
- We now show that components of the PI3K signaling pathway are involved in this cross-modulation process, as phenotypic reversion by inhibition of PI3K is associated with, and presumably, supported by, up-regulation of the PI3K antagonist, PTEN (Fig. 3).
- This also requires the establishment of organized structures in 3D lrBM, as treatment of T4-2 cells with PI3K inhibitors does not result in up-regulation of PTEN when cells are grown on 2D plastic substrata (Fig. 3 B).