Figure 1.
Effects of mTORC1/2 inhibition on cytotoxicity and Ser-473-Akt phosphorylation in PTEN-negative cancer cell lines.
(A) Differential susceptibility of LNCaP, PC-3, and MDA-MB-468 (468) cells to Ku-0063794-mediated suppression of cell viability in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent inhibitory effects of Ku-0063794 on mTOR signaling, as indicated by p70SK phosphorylation, and Akt phosphorylation at Ser-473 versus Thr-308. Representative blots from three independent experiments are shown.
Figure 2.
Evidence that ILK acts as a Ser-473-Akt kinase in PC-3 and MDA-MB-468 cells.
(A) Dose-dependent effect of T315 on the viability of LNCaP, PC-3, and MDA-MB-468 (468) cells in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent effects of T315 on the phosphorylation of Akt at Ser473 versus Thr308 and GSK3β. Suppression of GSK3β phosphorylation served as a marker for T315-mediated ILK inhibition. (C) Left panel, co-treatment with 3 µM T315 did not enhance the ability of Ku-0063793 to inhibit Ser473-Akt phosphorylation in LNCaP cells. Right panel, co-treatment with 0.8 µM Ku-0063793 did not enhance the ability of T315 to inhibit Ser473-Akt phosphorylation in PC-3 cells. Cells were exposed to test agents at the indicated concentrations for 24 h in 5% FBS-supplemented medium. (D) Left, representative Western blot of the phosphorylation and/or expression levels of Akt, ILK, and the components of mTORC complexes mTOR, raptor, and rictor in PC-3, LNCaP, and MDA-MB-468 (468) cancer cell lines. Right, relative expression levels of p-Ser-473- and p-Thr-308-Akt, Akt, ILK, mTOR, raptor, and rictor in PC-3 and MDA-MB-468 cells relative to those in LNCaP cells. Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry and normalized to β-actin. Signals from phosphorylated Akt were first normalized to that of total Akt and then to that of β-actin. The abundance of each protein in PC-3 and MDA-MB-231 cells was expressed as a percentage of that in LNCaP cells. Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. *P<0.05, compared to LNCaP cells.
Figure 3.
Evidence that mTORC2 is the Ser-473-Akt kinase in LNCaP cells.
(A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of siRNA-mediated ILK knockdown, alone or in combination with Ku-0063794 (0.4 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Dose-dependent effect of ectopic expression of ILK, alone (left panel) or in combination with Ku-0063794 (0.4 µM, 24 h), on the phosphorylation of Ser473-Akt and GSK3β. All immunoblots are representative of three independent experiments.
Figure 4.
Evidence that ILK is the Ser473-Akt kinase in PC-3 and MDA-MB-468 cells.
(A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of shRNA-mediated rictor knockdown, alone or in combination with T315 (2 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Western blot analysis of the effect of shRNA-mediated repression of ILK (left) or rictor (right) on the phosphorylation of Akt at Ser473 versus Thr308, GSK3β (ILK target) and p70S6K (mTOR target) in MDA-MB-468 cells. For induction of ILK shRNA, cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. All immunoblots are representative of three independent experiments.
Figure 5.
Evidence that ILK binds and phosphorylates Akt in PC-3 cells.
(A) Co-immunoprecipitation analysis reveals the association of ILK with Akt in PC-3 cells, which can be attenuated by T315. PC-3 cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted (WB) with antibodies against ILK, p-Ser473-Akt, or Akt (Input; lower panel), or were immunoprecipitated (IP) with anti-Akt antibody and protein A/G agarose followed by immunoblotting with antibodies against ILK, p-Ser473-Akt, or Akt (upper panel). (B) Effect of T315 on the kinase activity of ILK. Equal amounts of immunocomplexes obtained by immunoprecipitation of ILK from PC-3 cell lysates were incubated with bacterially expressed GST-Akt and 200 mM of ATP in the presence of DMSO or T315 at indicated concentrations for 20 min, and were then subjected to Western blotting with antibodies against p-Ser-473-Akt, Akt, or ILK. The immunoprecipitation procedure using IgG was performed as control. Left panel, Western blot analysis of the dose-dependent effect of T315 on the kinase activity of ILK. Right panel, relative expression levels of phosphorylated Ser-473-Akt after normalization to total Akt and subtraction from control (time zero). Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry. The abundance of phosphorylated Ser-473-Akt in T315-treated cells was expressed as a percentage of that in the DMSO control cells (0 µM). Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. *P<0.05, compared to DMSO control (0 µM).
Figure 6.
ILK interacts with rictor in PC-3 and MDA-MB-468, but not in LNCaP cells.
Co-immunoprecipitation analysis reveals that ILK associates with rictor in MDA-MB-468 and PC-3 cells, which can be attenuated by T315. No such association was observed in LNCaP cells. Cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted with antibodies against ILK, rictor or β-actin (Input; lower panels), or were immunoprecipitated (IP) with anti-ILK antibody and protein A/G agarose followed by immunoblotting (WB) with antibodies against ILK or rictor (upper panels).
Figure 7.
Pharmacologic inhibition or genetic silencing of ILK suppresses EMT and invasive phenotype.
(A) Inhibition of ILK by si/shRNA-mediated silencing or treatment with T315 reduces the expression of Snail in association with an increase in that of E-cadherin and concomitant reduction in that of vimentin in PC-3 (left panel) and MDA-MB-468 (right panel) cells. Cells were treated with T315 for 24 h. For induction of ILK shRNA, MDA-MB-468 cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. Immunoblots are representative of three independent experiments. (B) Images of invasive colonies after growth of PC-3 cells on basement membrane matrix for 6 days in the presence of DMSO control versus T315 at the indicated concentrations. Bars, 100 μm. (C) Dose-dependent effects of T315 and Ku-0063794 on the EGF-induced phosphorylation of Akt at Ser-473 versus Thr-308 in the PTEN-functional MDA-MB-231 cell line. Cells were co-treated with inhibitor and EGF for 24 h. Representative blots from three independent experiments are shown.