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Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

Lee SL, Chou CC, Chuang HC, Hsu EC, Chiu PC, Kulp SK, Byrd JC, Chen CS - PLoS ONE (2013)

Bottom Line: Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells.In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells.Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.

No MeSH data available.


Related in: MedlinePlus

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.
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pone-0067149-g002: 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.

Mentions: In contrast, T315 was effective in suppressing the viability of all three cell lines with IC50 values in the range of 1.5–2 µM (MDA-MB-468, 1.5 µM; PC-3 and LNCaP, 2 µM) (Fig. 2A). The ability of T315 to inhibit ILK in these cell lines was evident in the dose-dependent suppression of relevant biomarkers, including the phosphorylation of GSK3β [5], [6], [7], [22] and the expression of the oncogenic factor Y-box binding protein (YB)-1 [32], [51], [56] (Fig. 2B). However, the selective downregulation of Akt phosphorylation at Ser-473 by T315 was observed in PC-3 and MDA-MB-468 cells, but not in LNCaP cells (Fig. 2B). These data suggest that mTORC2 and ILK might play mutually exclusive roles in mediating the phosphorylation of Ser473-Akt in these cell lines. This premise was supported by the inability of T315 to enhance Ku-0063794-mediated suppression of Ser473-Akt phosphorylation in LNCaP cells relative to Ku-0063794 alone (Fig. 1B), and vice versa in PC-3 cells (Fig. 2C versus 2B).


Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

Lee SL, Chou CC, Chuang HC, Hsu EC, Chiu PC, Kulp SK, Byrd JC, Chen CS - PLoS ONE (2013)

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.
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pone-0067149-g002: 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.
Mentions: In contrast, T315 was effective in suppressing the viability of all three cell lines with IC50 values in the range of 1.5–2 µM (MDA-MB-468, 1.5 µM; PC-3 and LNCaP, 2 µM) (Fig. 2A). The ability of T315 to inhibit ILK in these cell lines was evident in the dose-dependent suppression of relevant biomarkers, including the phosphorylation of GSK3β [5], [6], [7], [22] and the expression of the oncogenic factor Y-box binding protein (YB)-1 [32], [51], [56] (Fig. 2B). However, the selective downregulation of Akt phosphorylation at Ser-473 by T315 was observed in PC-3 and MDA-MB-468 cells, but not in LNCaP cells (Fig. 2B). These data suggest that mTORC2 and ILK might play mutually exclusive roles in mediating the phosphorylation of Ser473-Akt in these cell lines. This premise was supported by the inability of T315 to enhance Ku-0063794-mediated suppression of Ser473-Akt phosphorylation in LNCaP cells relative to Ku-0063794 alone (Fig. 1B), and vice versa in PC-3 cells (Fig. 2C versus 2B).

Bottom Line: Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells.In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells.Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.

No MeSH data available.


Related in: MedlinePlus