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. 2008 Jul 24:9:348.
doi: 10.1186/1471-2164-9-348.

Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer

Henna Heinonen  1 , Anni Nieminen, Matti Saarela, Anne Kallioniemi, Juha Klefström, Sampsa Hautaniemi, Outi Monni
Affiliations

Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer

Henna Heinonen et al. BMC Genomics. .

Abstract

Background: The 70 kDa ribosomal protein S6 kinase (RPS6KB1), located at 17q23, is amplified and overexpressed in 10-30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of RPS6KB1 in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.

Results: Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to RPS6KB1. Furthermore, 17 genes including VTCN1 and CDKN2B showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway.

Conclusion: Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value.

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Figures

Figure 1
Figure 1
Interfering the PI3K/mTOR/p70S6K signaling pathway. Schematic representation of the PI3K/mTOR/p70S6K pathway illustrates that p70S6K is located downstream of PI3K and mTOR. In this study, PI3K was inhibited with Ly294002, mTOR with rapamycin and p70S6K with siRNAs targeting RPS6KB1.
Figure 2
Figure 2
Protein-level validation of p70S6K suppression after RPS6KB1 siRNA treatments in breast cancer cell lines. BT-474 and MCF-7 cells were treated with three siRNAs against RPS6KB1 and one scramble oligo for 72 hours and the protein expressions were detected by Western immunoblotting. The p70S6K expression was significantly downregulated in siRNA-transfected samples when compared to the scramble oligo transfected samples. Beta-actin was used as a loading control.
Figure 3
Figure 3
Effects of rapamycin and Ly294002 treatments on phosphorylation of p70S6KThr389 in breast cancer cell lines. The cells were treated with A) 100 nM rapamycin and B) 50 μM Ly294002 for 24 hours and the phosphorylation status of p70S6K Thr389 was detected by Western immunoblotting. The plus (+) sign indicates the inhibitor-treated samples and the minus (-) sign the non-treated samples. Both rapamycin and Ly294002 blocked Thr389 phosphorylation of p70S6K. The total p70S6K expression was used as a reference.
Figure 4
Figure 4
Protein-level downregulation of eIF4G1 in rapamycin-treated BT-474 cells and in Ly294002-treated SK-BR-3 cells. The breast cancer cells were treated with 100 nM rapamycin and 50 μM Ly294002 for 24 hours and the protein expression of eIF4G1 was detected by Western immunoblotting. The plus (+) sign indicates the inhibitor-treated sample and the minus (-) sign the non-treated sample. A) Rapamycin and B) Ly294002 downregulated eIF4G1 expression at protein level in BT-474 and SK-BR-3 cells, respectively. Beta-actin was used as a loading control.
Figure 5
Figure 5
Effects of rapamycin, Ly294002 and RPS6KB1 siRNA treatments on apoptosis of breast cancer cell lines. A) The cells were treated with 100 nM rapamycin and 50 μM Ly294002 for 48 hours and the cell morphology was evaluated under the light microscope. B) Caspase-3 activity assay of rapamycin- and Ly294002-treated breast cancer cell lines. C) Caspase-3 activity assay of RPS6KB1 siRNA-treated breast cancer cell lines. The values are averages from two or three independent experiments. Ly294002 induced apoptosis in BT-474 and MDA-361 breast cancer cells, whereas rapamycin had no effect. Similarly, knock-down of RPS6KB1 did not have any effect on apoptosis in BT-474 and MCF-7 breast cancer cells.
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