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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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Expression of constitutively active MEK in IGF-IR cells results in invasive structures and repression of E-cadherin. (A) IGF-IR or Neo cells overexpressing vector control or constitutively active MEK2 (MEKDD) were grown in monolayer cultures containing EGF and IGF-I (100 ng/ml). Bar, 50 μM. (B) Transwell migration assay and western analyses of IGF-IR or Neo cells overexpressing MEKDD were performed. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media containing 2% horse serum and 100 ng/ml IGF-I. Lysates were immunoblotted as indicated. Error bars represent means ± SD. (C) IGF-IR or Neo cells overexpressing MEKDD were grown in 3D Matrigel/collagen (50:50) cultures for 12 d. All cells were maintained in 2% horse serum, EGF, and 100 ng/ml IGF-I. Phase-contrast images are shown. Bar, 50 μM. (D) IGF-IR or Neo cells overexpressing vector control (V) or MEKDD were grown in monolayer cultures with 2% horse serum and 100 ng/ml IGF-I. Cells were lysed in NP-40 lysis buffer and lysates were immunoblotted with the indicated antibodies.
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fig6: Expression of constitutively active MEK in IGF-IR cells results in invasive structures and repression of E-cadherin. (A) IGF-IR or Neo cells overexpressing vector control or constitutively active MEK2 (MEKDD) were grown in monolayer cultures containing EGF and IGF-I (100 ng/ml). Bar, 50 μM. (B) Transwell migration assay and western analyses of IGF-IR or Neo cells overexpressing MEKDD were performed. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media containing 2% horse serum and 100 ng/ml IGF-I. Lysates were immunoblotted as indicated. Error bars represent means ± SD. (C) IGF-IR or Neo cells overexpressing MEKDD were grown in 3D Matrigel/collagen (50:50) cultures for 12 d. All cells were maintained in 2% horse serum, EGF, and 100 ng/ml IGF-I. Phase-contrast images are shown. Bar, 50 μM. (D) IGF-IR or Neo cells overexpressing vector control (V) or MEKDD were grown in monolayer cultures with 2% horse serum and 100 ng/ml IGF-I. Cells were lysed in NP-40 lysis buffer and lysates were immunoblotted with the indicated antibodies.

Mentions: To assess whether enhanced ERK activation was sufficient to induce phenotypic alterations in IGF-IR cells similar to those caused by Akt1 down-regulation, we overexpressed an activated variant of MEK2 (MEKDD) in IGF-IR or Neo cells (Fig. 6). Expression of MEKDD in IGF-I–stimulated IGF-IR cells enhanced the spindle-shaped morphology of these cells in monolayer cultures (Fig. 6 A). MEKDD overexpression also enhanced migration of both IGF-IR and Neo control cells in the presence of IGF-I (Fig. 6 B). However, in 3D cultures, only cells expressing both ectopic IGF-IR and MEKDD were capable of forming structures with invasive protrusions that resembled those observed with Akt1 down-regulation (Fig. 6 C). Despite their migratory capacity in transwell assays, MEKDD-overexpressing parental MCF-10A cells formed normal acinar structures. We have previously shown that MEKDD cells produce EGF receptor ligands, which could be responsible for the migration observed with MEKDD cells (Debnath et al., 2003b).


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)

Expression of constitutively active MEK in IGF-IR cells results in invasive structures and repression of E-cadherin. (A) IGF-IR or Neo cells overexpressing vector control or constitutively active MEK2 (MEKDD) were grown in monolayer cultures containing EGF and IGF-I (100 ng/ml). Bar, 50 μM. (B) Transwell migration assay and western analyses of IGF-IR or Neo cells overexpressing MEKDD were performed. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media containing 2% horse serum and 100 ng/ml IGF-I. Lysates were immunoblotted as indicated. Error bars represent means ± SD. (C) IGF-IR or Neo cells overexpressing MEKDD were grown in 3D Matrigel/collagen (50:50) cultures for 12 d. All cells were maintained in 2% horse serum, EGF, and 100 ng/ml IGF-I. Phase-contrast images are shown. Bar, 50 μM. (D) IGF-IR or Neo cells overexpressing vector control (V) or MEKDD were grown in monolayer cultures with 2% horse serum and 100 ng/ml IGF-I. Cells were lysed in NP-40 lysis buffer and lysates were immunoblotted with the indicated antibodies.
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Related In: Results  -  Collection

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fig6: Expression of constitutively active MEK in IGF-IR cells results in invasive structures and repression of E-cadherin. (A) IGF-IR or Neo cells overexpressing vector control or constitutively active MEK2 (MEKDD) were grown in monolayer cultures containing EGF and IGF-I (100 ng/ml). Bar, 50 μM. (B) Transwell migration assay and western analyses of IGF-IR or Neo cells overexpressing MEKDD were performed. Cells were starved in the absence of EGF for 24 h. Migration was assessed after 16–20 h in media containing 2% horse serum and 100 ng/ml IGF-I. Lysates were immunoblotted as indicated. Error bars represent means ± SD. (C) IGF-IR or Neo cells overexpressing MEKDD were grown in 3D Matrigel/collagen (50:50) cultures for 12 d. All cells were maintained in 2% horse serum, EGF, and 100 ng/ml IGF-I. Phase-contrast images are shown. Bar, 50 μM. (D) IGF-IR or Neo cells overexpressing vector control (V) or MEKDD were grown in monolayer cultures with 2% horse serum and 100 ng/ml IGF-I. Cells were lysed in NP-40 lysis buffer and lysates were immunoblotted with the indicated antibodies.
Mentions: To assess whether enhanced ERK activation was sufficient to induce phenotypic alterations in IGF-IR cells similar to those caused by Akt1 down-regulation, we overexpressed an activated variant of MEK2 (MEKDD) in IGF-IR or Neo cells (Fig. 6). Expression of MEKDD in IGF-I–stimulated IGF-IR cells enhanced the spindle-shaped morphology of these cells in monolayer cultures (Fig. 6 A). MEKDD overexpression also enhanced migration of both IGF-IR and Neo control cells in the presence of IGF-I (Fig. 6 B). However, in 3D cultures, only cells expressing both ectopic IGF-IR and MEKDD were capable of forming structures with invasive protrusions that resembled those observed with Akt1 down-regulation (Fig. 6 C). Despite their migratory capacity in transwell assays, MEKDD-overexpressing parental MCF-10A cells formed normal acinar structures. We have previously shown that MEKDD cells produce EGF receptor ligands, which could be responsible for the migration observed with MEKDD cells (Debnath et al., 2003b).

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