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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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IGF-IR hyperstimulation induces morphological changes in mammary epithelial cells. (A) MCF-10A cells overexpressing human IGF-IR or vector control (Neo) were grown in monolayer cultures containing EGF ± IGF (100 ng/ml). Lysates were immunoblotted with antibody against phosphorylated IGF-IR or total receptor. (B) Phase-contrast images of IGF-IR or Neo cells grown in 3D Matrigel cultures for 16 d in media containing 2% horse serum and EGF ± IGF-I (100 ng/ml). B (C) IGF-IR, Neo, or MCF-10A cells overexpressing a variant IGF-IR (Y950F) were grown in 3D Matrigel cultures for 7 or 16 d in 2% horse serum, EGF, and 100 ng/ml IGF-I. (D) Lysates of IGF-IR, Neo, or Y950F cells grown in monolayer cultures in 2% horse serum, EGF, and 100 ng/ml IGF-I were immunoblotted with antibody against phosphorylated Akt or phosphorylated, activated ERK. Bars, 50 μM.
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fig1: IGF-IR hyperstimulation induces morphological changes in mammary epithelial cells. (A) MCF-10A cells overexpressing human IGF-IR or vector control (Neo) were grown in monolayer cultures containing EGF ± IGF (100 ng/ml). Lysates were immunoblotted with antibody against phosphorylated IGF-IR or total receptor. (B) Phase-contrast images of IGF-IR or Neo cells grown in 3D Matrigel cultures for 16 d in media containing 2% horse serum and EGF ± IGF-I (100 ng/ml). B (C) IGF-IR, Neo, or MCF-10A cells overexpressing a variant IGF-IR (Y950F) were grown in 3D Matrigel cultures for 7 or 16 d in 2% horse serum, EGF, and 100 ng/ml IGF-I. (D) Lysates of IGF-IR, Neo, or Y950F cells grown in monolayer cultures in 2% horse serum, EGF, and 100 ng/ml IGF-I were immunoblotted with antibody against phosphorylated Akt or phosphorylated, activated ERK. Bars, 50 μM.

Mentions: To examine the effects of enhanced IGF-I stimulation on mammary acinar architecture, MCF-10A cells overexpressing IGF-IR cells were generated using a retroviral vector encoding the human IGF-IR complementary DNA. MCF-10A is an immortalized breast epithelial cell line that has been reported to express IGF-IR (Tannheimer et al., 1998); however, levels of endogenous receptor were barely detectable by Western blotting (Fig. 1 A). Despite significant overexpression, activation of the receptor remained ligand dependent (Fig. 1 A). In monolayer cultures, IGF-I stimulation of IGF-IR cells induced a subtle, but recognizable, conversion from a cuboidal, epithelial morphology to a more spindle-shaped morphology (unpublished data).


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)

IGF-IR hyperstimulation induces morphological changes in mammary epithelial cells. (A) MCF-10A cells overexpressing human IGF-IR or vector control (Neo) were grown in monolayer cultures containing EGF ± IGF (100 ng/ml). Lysates were immunoblotted with antibody against phosphorylated IGF-IR or total receptor. (B) Phase-contrast images of IGF-IR or Neo cells grown in 3D Matrigel cultures for 16 d in media containing 2% horse serum and EGF ± IGF-I (100 ng/ml). B (C) IGF-IR, Neo, or MCF-10A cells overexpressing a variant IGF-IR (Y950F) were grown in 3D Matrigel cultures for 7 or 16 d in 2% horse serum, EGF, and 100 ng/ml IGF-I. (D) Lysates of IGF-IR, Neo, or Y950F cells grown in monolayer cultures in 2% horse serum, EGF, and 100 ng/ml IGF-I were immunoblotted with antibody against phosphorylated Akt or phosphorylated, activated ERK. Bars, 50 μM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171329&req=5

fig1: IGF-IR hyperstimulation induces morphological changes in mammary epithelial cells. (A) MCF-10A cells overexpressing human IGF-IR or vector control (Neo) were grown in monolayer cultures containing EGF ± IGF (100 ng/ml). Lysates were immunoblotted with antibody against phosphorylated IGF-IR or total receptor. (B) Phase-contrast images of IGF-IR or Neo cells grown in 3D Matrigel cultures for 16 d in media containing 2% horse serum and EGF ± IGF-I (100 ng/ml). B (C) IGF-IR, Neo, or MCF-10A cells overexpressing a variant IGF-IR (Y950F) were grown in 3D Matrigel cultures for 7 or 16 d in 2% horse serum, EGF, and 100 ng/ml IGF-I. (D) Lysates of IGF-IR, Neo, or Y950F cells grown in monolayer cultures in 2% horse serum, EGF, and 100 ng/ml IGF-I were immunoblotted with antibody against phosphorylated Akt or phosphorylated, activated ERK. Bars, 50 μM.
Mentions: To examine the effects of enhanced IGF-I stimulation on mammary acinar architecture, MCF-10A cells overexpressing IGF-IR cells were generated using a retroviral vector encoding the human IGF-IR complementary DNA. MCF-10A is an immortalized breast epithelial cell line that has been reported to express IGF-IR (Tannheimer et al., 1998); however, levels of endogenous receptor were barely detectable by Western blotting (Fig. 1 A). Despite significant overexpression, activation of the receptor remained ligand dependent (Fig. 1 A). In monolayer cultures, IGF-I stimulation of IGF-IR cells induced a subtle, but recognizable, conversion from a cuboidal, epithelial morphology to a more spindle-shaped morphology (unpublished data).

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