PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

Abstract

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent. MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation. Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.

Fig. 1

Scheme of interactions between cell surivival (PI3K/Akt) and mitogenic (Ras/MAPK) signaling pathways (A) and modulation of ERK activity by phosphatases and their upstream effectors (B). Arrows show activatory- and blunt-end lines - inhibitory interactions

Fig. 2. Time courses of EGF-induced ERK and Akt activation in T47D and MCF7 breast cancer cells in the presence of MEK/ERK or PI3K/Akt inhibitors

Serum-starved T47D (A) or MCF7 (B) cells were either left untreated (control, black circles) or treated with wortmannin (200 nM, 30 min; black triangles down), Akt-VIII inhibitor (5 μM, 1 h; grey circles), OSU-03012 (50 μM, 1 h; grey squares) or U0126 (10 μM, 30 min; white circles) before stimulation with EGF (1 nM) for the indicated time intervals (min). Equal amounts of total cell lysates were resolved by NuPAGE and subjected to Multistrip Western blotting. Immunoblots (IB) were probed with antibodies against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204 and Thr204/Tyr187) and p44/42 MAPK (ERK1/2) (loading control) or phospho-Akt (Ser473) and Akt (loading control). Each graph represents the intensity of protein activation in arbitrary units (AU) plotted as the ratio between signals of phosphorylated and non-phosphorylated protein forms.

Fig. 3. A. Effects of MEK inhibitor U0126 on ERK phosphorylation upon treatment with different doses of EGF (0.01, 0.1, 1 nM)

Serum-starved cells were incubated with U0126 (10 μM) for 30 min before stimulation with indicated EGF doses for the indicated time intervals (min). Equal amounts of total cell lysates were resolved by NuPAGE and subjected to Multistrip Western blotting. Immunoblots (IB) were probed with anti-phospho-p44/42 MAPK (ERK1/2) (labeled as p-ERK). B. Effects of structurally distinct MEK inhibitors on ERK phosphorylation in T47D cells. Cells were preincubated with PD 098059 (50 μM) or PD 198306 (200 nM) for 30 min and stimulated with EGF (1 nM) for the indicated time intervals (min). C. The effect of suppression of MEK protein levels on ERK phosphorylation in the presence or absence of MEK inhibitor. T47D cells were transfected with either 200 nM of Scrambled (Scr) or MEK1/2 siRNA as described in “Materials and Methods”. Serum-starved cells were pretreated with U0126 (10 μM, 30 min) and stimulated with 1 nM EGF for 30 min. Suppression levels of MEK1/2 were assessed by probing immunoblots with anti-MEK1/2 antibody. GAPDH levels were used as loading controls.

Fig. 4. Effects of various growth factors on p-ERK resistance to MEK inhibition by U0126

Serum-starved T47D cells were incubated with U0126 (10 μM) for 30 min before stimulation with EGF (1 nM), TGF-α (10 nM), HRG-β (1 nM), IGF-1 (10 nM) and insulin (50 nM) for the indicated time intervals (min) (left panel) or with EGF (1 nM), PDGF-BB (1 nM), FGF-basic (10 nM), prolactin (PRL, 10 nM), VEGF (1 nM), IGF-1 (50 nM) and 5% FBS for 30 min (right panel). Equal amounts of total cell lysates were resolved by NuPAGE and subjected to Western blotting. Immunoblots (IB) were probed with antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt1/2/3 (Ser473) or GAPDH (loading control).

Fig. 5. MEK-independent ERK activation depends on kinase(s) located downstream of PI3K

A. Serum-starved T47D cells were either left untreated (Control, blot strip (1); black circles) or treated with U0126 alone (10 μM, 30 min; blot strip (2); white circles) or in combination with SQ22536 (50 μM, 1 h; blot strip (3); grey circles), Gö6850 (5 μM, 1 h; blot strip (4); black squares), Gö6983 (5 μM, 1 h; blot strip (5); white squares), Su6656 (10 μM, 1 h; blot strip (6); grey squares), wortmannin (WT) (200 nM, 30 min; blot strip (7); black diamonds), OSU-03012 (50 μM, 1 h; blot strip (8); white diamonds) and Akt-VIII inhibitor (5 μM, 1 h; blot strip (9); grey diamonds) before stimulation with EGF (1 nM) for the indicated time intervals (min). Total cell lysates were subjected to Multistrip Western blotting for ERK activation as described previously. The graph show the intensity of ERK activation in arbitrary units (AU) plotted as the ratio between phospho-ERK1/2 and total ERK1/2 signal. B. T47D cells were transfected with either 50 nM of PIK3R1 (p85-α regulatory subunit of PI3K), PIK3CA (p110-α catalytic subunit of PI3K), Akt1, Akt2 siRNA or with AllStar negative control siRNA as described in “Materials and Methods”. Serum-starved cells were pretreated with U0126 (10 μM, 30 min) and stimulated with 1 nM EGF for 30 min. The ratios between phospho-ERK1/2 and total ERK1/2 signals are shown as a percentage of siRNA control. Values represent a mean ± SD of three signal measurements.

Fig. 6. MEK-independent ERK phosphorylation is not mediated by p38 MAPK (A), PP2A (B) or GSK-3 (C)

A. Left panel. Serum-starved T47D cells were either left untreated (Control) or treated with wortmannin (WT) (200 nM, 30 min) before stimulation with EGF (1 nM) for the indicated time intervals (min). Total cell lysates were subjected to Multistrip Western blotting with anti-phospho-p38 MAPK (Thr180/Tyr182) antibodies. Right panel. Serum-starved T47D cells were treated with either wortmannin alone (200 nM, 30 min; WT) or with wortmannin plus PD169316 (5 μM, 60 min), U0126 (10 μM, 30 min) and their combination (U0126 + PD 169316 + WT) before stimulation with EGF (1 nM) for the indicated time intervals (min). Total cell lysates were subjected to Multistrip Western blotting for ERK activation as described previously. B. Similarly, T47D cells were treated with U0126 in the presence or absence of endothall (2 μM, 60 min) before the stimulation with EGF (1 nM) for the indicated time intervals (min) and ERK activation was measured. C. T47D cells were treated with Akt-VIII (5 μM, 1 h), SB 216763 (7 μM, 1 h) or their combination in the presence or absence of U0126 (10 μM, 30 min) before the stimulation with EGF (1 nM) for 30 min. ERK and Akt activation was measured.

Fig. 7. Synergistic downregulation of ERK phosphorylation and its downstream effectors by combined inhibition of MEK and PI3K activities

A-B. Serum-starved T47D cells were either left untreated (Control) or treated with U0126 (10 μM, 30 min), wortmannin (WT) (200 nM, 30 min) or both before stimulation with EGF (1 nM) for the indicated time intervals (min) (A) or for 60 min (B) A. Total cell lysates were subjected to Multistrip Western blotting with anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and anti-p44/42 MAPK (Erk1/2) antibodies (left panel). The graph on the right panel shows the synergistic inhibitory effect of combined treatment with MEK and PI3K inhibitors on ERK activation at 30 min following EGF stimulation (grey bars) compared to estimated theoretical value of additive effect (white bar) ± SD (error bars) (n=3, p<0.05). B. Total cell lysates were subjected to immunoblotting with antibodies against phospho-c-Raf (Ser338), c-Raf, phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Akt1/2/3 (Ser473), Akt1/2/3, phospho-p90RSK (Ser380), phospho-Elk1 (Ser383), phospho-STAT3 (Ser727), phospho-Estrogen receptor-alpha (Ser118), c-Fos, c-Myc or GAPDH (loading control).

Fig. 8. The effect of single or dual inhibition of Akt and MEK kinases on EGF-induced cell proliferation of T47D and MCF7 cells

Equal amounts of serum-starved cells were plated into 96-well plates at a density of 25000 cells/ml and maintained in 2 nM EGF-containing medium without FBS in the presence or absence of U0126 (10 μM), different concentrations of Akt-VIII (1, 2 or 4 μM) or their mixtures for 72 hours. The numbers of viable cells were determined with AlamarBlue assay. Each data point is the mean ± SD of 12 replicates and is representative of three independent trials.

Fig. 9. A. Effects of siRNA-mediated suppression of TOPK, biliverdin reductase, Fer and RIP2 on EGF-induced ERK phosphorylation upon MEK inhibition

T47D cells were transfected with either 50 nM of siRNA against indicated genes or AllStar negative control siRNA as described in “Materials and Methods”. Serum-starved cells were pretreated with U0126 (10 μM, 30 min) and stimulated with 1 nM EGF for 30 min. The ratios between phospho-ERK1/2 and total ERK1/2 signals are shown as a percentage of siRNA control. Values represent a mean ± SD of three signal measurements. B. Proposed mechanism of MEK-independent ERK activation downstream of PI3K/Akt involving TOPK or other unidentified kinases. Blunt-end arrows show some inhibitors used in this study.