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Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition.

Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, Natesan S, Brugge JS - J. Cell Biol. (2005)

Bottom Line: In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation.The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT.These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

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Overexpression of Akt1 suppresses EGF-stimulated migration and ERK activation. (A) MCF-10A cells overexpressing HA-tagged wild-type Akt1 or -2 were generated. (top) Levels of overexpression were confirmed using antibodies against the HA tag or a panAkt antibody that recognizes all isoforms. (bottom) Levels of activated, phosphorylated Akt and phosphorylated GSK3β were assessed after EGF stimulation. (B) Migration of MCF-10A cells overexpressing vector control or wild-type HA-tagged Akt1 or -2 was assessed by transwell assay. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media with 2% horse serum ± 5 ng/ml EGF. A representative experiment is shown with mean values obtained by counting 10 separate fields. Error bars represent means ± SD. (C) MCF-10A cells overexpressing vector control or wild-type Akt1 or -2 were starved for 24 h and stimulated with media containing 2% horse serum ± 5 ng/ml EGF. Cells were lysed and immunoblotted with the indicated antibodies.
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fig9: Overexpression of Akt1 suppresses EGF-stimulated migration and ERK activation. (A) MCF-10A cells overexpressing HA-tagged wild-type Akt1 or -2 were generated. (top) Levels of overexpression were confirmed using antibodies against the HA tag or a panAkt antibody that recognizes all isoforms. (bottom) Levels of activated, phosphorylated Akt and phosphorylated GSK3β were assessed after EGF stimulation. (B) Migration of MCF-10A cells overexpressing vector control or wild-type HA-tagged Akt1 or -2 was assessed by transwell assay. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media with 2% horse serum ± 5 ng/ml EGF. A representative experiment is shown with mean values obtained by counting 10 separate fields. Error bars represent means ± SD. (C) MCF-10A cells overexpressing vector control or wild-type Akt1 or -2 were starved for 24 h and stimulated with media containing 2% horse serum ± 5 ng/ml EGF. Cells were lysed and immunoblotted with the indicated antibodies.

Mentions: To more directly assess the potential inhibitory effect of Akt1 on growth factor–induced migration and ERK activation, MCF-10A cells overexpressing wild-type or HA-tagged Akt1 or -2 were generated by retroviral infection. Comparable levels of overexpression were confirmed using antibodies that recognize the HA tag or all Akt isoforms (panAkt; Fig. 9 A). Because ligand-stimulated IGF-IR cells are not motile, we examined the effects of Akt1 or -2 overexpression on parental MCF-10A cells treated with EGF, which induces robust migration and strong activation of ERK. Comparable total levels of activated, phosphorylated Akt were induced after EGF stimulation (Fig. 9 A). Phosphorylation of glycogen synthase kinase-3 (GSK3)β, a known Akt substrate (Brazil et al., 2004), was also comparable in cells overexpressing Akt1 or -2, further confirming equivalent levels of Akt activity. In comparison to control MCF-10A cells, wild-type Akt1-overexpressing cells exhibited decreased migration in response to EGF stimulation (Fig. 9 B). These results are consistent with previous studies in which overexpression of wild-type or activated Akt1 in breast cancer cell lines slightly decreased migration and invasion (Shaw et al., 1997; Arboleda et al., 2003). In contrast, overexpression of wild-type Akt2 did not affect EGF-induced migration.


Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition.

Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, Natesan S, Brugge JS - J. Cell Biol. (2005)

Overexpression of Akt1 suppresses EGF-stimulated migration and ERK activation. (A) MCF-10A cells overexpressing HA-tagged wild-type Akt1 or -2 were generated. (top) Levels of overexpression were confirmed using antibodies against the HA tag or a panAkt antibody that recognizes all isoforms. (bottom) Levels of activated, phosphorylated Akt and phosphorylated GSK3β were assessed after EGF stimulation. (B) Migration of MCF-10A cells overexpressing vector control or wild-type HA-tagged Akt1 or -2 was assessed by transwell assay. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media with 2% horse serum ± 5 ng/ml EGF. A representative experiment is shown with mean values obtained by counting 10 separate fields. Error bars represent means ± SD. (C) MCF-10A cells overexpressing vector control or wild-type Akt1 or -2 were starved for 24 h and stimulated with media containing 2% horse serum ± 5 ng/ml EGF. Cells were lysed and immunoblotted with the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171329&req=5

fig9: Overexpression of Akt1 suppresses EGF-stimulated migration and ERK activation. (A) MCF-10A cells overexpressing HA-tagged wild-type Akt1 or -2 were generated. (top) Levels of overexpression were confirmed using antibodies against the HA tag or a panAkt antibody that recognizes all isoforms. (bottom) Levels of activated, phosphorylated Akt and phosphorylated GSK3β were assessed after EGF stimulation. (B) Migration of MCF-10A cells overexpressing vector control or wild-type HA-tagged Akt1 or -2 was assessed by transwell assay. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media with 2% horse serum ± 5 ng/ml EGF. A representative experiment is shown with mean values obtained by counting 10 separate fields. Error bars represent means ± SD. (C) MCF-10A cells overexpressing vector control or wild-type Akt1 or -2 were starved for 24 h and stimulated with media containing 2% horse serum ± 5 ng/ml EGF. Cells were lysed and immunoblotted with the indicated antibodies.
Mentions: To more directly assess the potential inhibitory effect of Akt1 on growth factor–induced migration and ERK activation, MCF-10A cells overexpressing wild-type or HA-tagged Akt1 or -2 were generated by retroviral infection. Comparable levels of overexpression were confirmed using antibodies that recognize the HA tag or all Akt isoforms (panAkt; Fig. 9 A). Because ligand-stimulated IGF-IR cells are not motile, we examined the effects of Akt1 or -2 overexpression on parental MCF-10A cells treated with EGF, which induces robust migration and strong activation of ERK. Comparable total levels of activated, phosphorylated Akt were induced after EGF stimulation (Fig. 9 A). Phosphorylation of glycogen synthase kinase-3 (GSK3)β, a known Akt substrate (Brazil et al., 2004), was also comparable in cells overexpressing Akt1 or -2, further confirming equivalent levels of Akt activity. In comparison to control MCF-10A cells, wild-type Akt1-overexpressing cells exhibited decreased migration in response to EGF stimulation (Fig. 9 B). These results are consistent with previous studies in which overexpression of wild-type or activated Akt1 in breast cancer cell lines slightly decreased migration and invasion (Shaw et al., 1997; Arboleda et al., 2003). In contrast, overexpression of wild-type Akt2 did not affect EGF-induced migration.

Bottom Line: In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation.The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT.These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

Show MeSH
Related in: MedlinePlus