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Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

Chen L, Xu B, Liu L, Liu C, Luo Y, Chen X, Barzegar M, Chung J, Huang S - Oncotarget (2015)

Bottom Line: mTOR is a central controller for cell growth/proliferation and survival.Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells.In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China.

ABSTRACT
mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

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Inhibition of mTORC1/2 by PP242 potently suppresses the basal or IGF-1-stimulated cell adhesionSerum-starved Rh30 and HT29 cells were treated with or without PP242 (1 μM) or rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 1 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies, showing that PP242 potently inhibited the basal or IGF-1-stimulated phosphorylation of Akt (Ser473) and S6K1 (Thr389) in Rh30 and HT29 cells. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) Cell adhesion was determined using CN IV-coated cell adhesion assay, showing that inhibition of mTORC1/2 by PP242 dramatically suppressed the basal and IGF-1-stimulated adhesion in Rh30 and HT29 cells, and the inhibitory effect of PP242 was more potent that of Rapa. Results are means ± SE (n = 6). **P < 0.01, difference versus control group; ##P < 0.01, difference versus IGF-1 group; ++P < 0.01, PP242 group or PP242+IGF-1 group versus Rapa group or Rapa+IGF-1 group.
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Figure 4: Inhibition of mTORC1/2 by PP242 potently suppresses the basal or IGF-1-stimulated cell adhesionSerum-starved Rh30 and HT29 cells were treated with or without PP242 (1 μM) or rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 1 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies, showing that PP242 potently inhibited the basal or IGF-1-stimulated phosphorylation of Akt (Ser473) and S6K1 (Thr389) in Rh30 and HT29 cells. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) Cell adhesion was determined using CN IV-coated cell adhesion assay, showing that inhibition of mTORC1/2 by PP242 dramatically suppressed the basal and IGF-1-stimulated adhesion in Rh30 and HT29 cells, and the inhibitory effect of PP242 was more potent that of Rapa. Results are means ± SE (n = 6). **P < 0.01, difference versus control group; ##P < 0.01, difference versus IGF-1 group; ++P < 0.01, PP242 group or PP242+IGF-1 group versus Rapa group or Rapa+IGF-1 group.

Mentions: Since rictor also has mTORC2-independent functions related to regulation of cytoskeleton and cell migration [39–42], to confirm the role of mTORC2 in the regulation of cell adhesion, PP242, an mTOR kinase inhibitor that blocks both mTORC1 and mTORC2, was used. As predicted, PP242 inhibited both mTORC1-mediated phosphorylation of S6K1 and mTORC2-mediated phosphorylation of Akt in Rh30 and HT29 cells (Figure 4A). Interestingly, PP242 (inhibition of mTORC1/2) suppressed the basal or IGF-1-stimulated cell adhesion of Rh30 and HT29 cells more potently than rapamycin (inhibition of mTORC1) (Figure 4B). Collectively, the above results demonstrate that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.


Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

Chen L, Xu B, Liu L, Liu C, Luo Y, Chen X, Barzegar M, Chung J, Huang S - Oncotarget (2015)

Inhibition of mTORC1/2 by PP242 potently suppresses the basal or IGF-1-stimulated cell adhesionSerum-starved Rh30 and HT29 cells were treated with or without PP242 (1 μM) or rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 1 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies, showing that PP242 potently inhibited the basal or IGF-1-stimulated phosphorylation of Akt (Ser473) and S6K1 (Thr389) in Rh30 and HT29 cells. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) Cell adhesion was determined using CN IV-coated cell adhesion assay, showing that inhibition of mTORC1/2 by PP242 dramatically suppressed the basal and IGF-1-stimulated adhesion in Rh30 and HT29 cells, and the inhibitory effect of PP242 was more potent that of Rapa. Results are means ± SE (n = 6). **P < 0.01, difference versus control group; ##P < 0.01, difference versus IGF-1 group; ++P < 0.01, PP242 group or PP242+IGF-1 group versus Rapa group or Rapa+IGF-1 group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466674&req=5

Figure 4: Inhibition of mTORC1/2 by PP242 potently suppresses the basal or IGF-1-stimulated cell adhesionSerum-starved Rh30 and HT29 cells were treated with or without PP242 (1 μM) or rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 1 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies, showing that PP242 potently inhibited the basal or IGF-1-stimulated phosphorylation of Akt (Ser473) and S6K1 (Thr389) in Rh30 and HT29 cells. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) Cell adhesion was determined using CN IV-coated cell adhesion assay, showing that inhibition of mTORC1/2 by PP242 dramatically suppressed the basal and IGF-1-stimulated adhesion in Rh30 and HT29 cells, and the inhibitory effect of PP242 was more potent that of Rapa. Results are means ± SE (n = 6). **P < 0.01, difference versus control group; ##P < 0.01, difference versus IGF-1 group; ++P < 0.01, PP242 group or PP242+IGF-1 group versus Rapa group or Rapa+IGF-1 group.
Mentions: Since rictor also has mTORC2-independent functions related to regulation of cytoskeleton and cell migration [39–42], to confirm the role of mTORC2 in the regulation of cell adhesion, PP242, an mTOR kinase inhibitor that blocks both mTORC1 and mTORC2, was used. As predicted, PP242 inhibited both mTORC1-mediated phosphorylation of S6K1 and mTORC2-mediated phosphorylation of Akt in Rh30 and HT29 cells (Figure 4A). Interestingly, PP242 (inhibition of mTORC1/2) suppressed the basal or IGF-1-stimulated cell adhesion of Rh30 and HT29 cells more potently than rapamycin (inhibition of mTORC1) (Figure 4B). Collectively, the above results demonstrate that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

Bottom Line: mTOR is a central controller for cell growth/proliferation and survival.Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells.In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China.

ABSTRACT
mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

Show MeSH
Related in: MedlinePlus