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How moderate changes in Akt T-loop phosphorylation impact on tumorigenesis and insulin resistance.

Wullschleger S, Sakamoto K, Johnstone L, Duce S, Fleming S, Alessi DR - Dis Model Mech (2010)

Bottom Line: Using insulin-resistant PDK1(K465E/K465E) PH domain knock-in mice, we found that introducing the PTEN(+/-) mutation to slightly stimulate Akt restored normal insulin sensitivity.This was also accompanied by slower growth of B cell follicular lymphomas, as monitored by magnetic resonance imaging.Our findings imply that signal transduction inhibitors that lead to a modest reduction in Akt activity would not only delay onset of tumours possessing elevated phosphoinositide 3-kinase pathway activity but would also reduce the growth rate of developed tumours.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

ABSTRACT
The Akt signalling pathway plays vital roles in controlling cellular responses to insulin as well as in proliferation and survival. Inhibition of Akt signalling leads to insulin resistance and type 2 diabetes, whereas hyperactivation of Akt promotes tumorigenesis. In this study, we investigate how modest changes in the activity of the Akt signalling pathway, to an extent that might be achieved by drug treatment, would impact on insulin resistance and tumorigenesis. Using insulin-resistant PDK1(K465E/K465E) PH domain knock-in mice, we found that introducing the PTEN(+/-) mutation to slightly stimulate Akt restored normal insulin sensitivity. Introducing the PDK1(K465E/K465E) PH domain knock-in mutation into cancer-prone PTEN(+/-) mice, lowered Akt activity only by about 50%, but led to a delay in tumour onset of ∼4 months in a broad range of tumours. This was also accompanied by slower growth of B cell follicular lymphomas, as monitored by magnetic resonance imaging. Our findings imply that signal transduction inhibitors that lead to a modest reduction in Akt activity would not only delay onset of tumours possessing elevated phosphoinositide 3-kinase pathway activity but would also reduce the growth rate of developed tumours.

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Reduced tumour growth in PDK1K465E/K465EPTEN+/− mice. (A) The size of B cell follicular lymphomas of 11-month-old littermate PDK1+/+PTEN+/− and PDK1K465E/K465E PTEN+/− mice were determined by MRI imaging. Individual tumours from the same animal were measured at 0-, 3- and 6-week intervals. The data are depicted as relative tumour volume. PDK1+/+ PTEN+/− n=3, PDK1K465E/K465E PTEN+/− n=2. (B) Lysates of B cell follicular lymphomas from PDK1+/+ PTEN+/− and PDK1K465E/K465E PTEN+/− mice were subjected to immunoblot analysis using the indicated antibodies. (C) Immunohistochemical analysis of B cell follicular lymphomas using the indicated antibodies.
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f4-0040095: Reduced tumour growth in PDK1K465E/K465EPTEN+/− mice. (A) The size of B cell follicular lymphomas of 11-month-old littermate PDK1+/+PTEN+/− and PDK1K465E/K465E PTEN+/− mice were determined by MRI imaging. Individual tumours from the same animal were measured at 0-, 3- and 6-week intervals. The data are depicted as relative tumour volume. PDK1+/+ PTEN+/− n=3, PDK1K465E/K465E PTEN+/− n=2. (B) Lysates of B cell follicular lymphomas from PDK1+/+ PTEN+/− and PDK1K465E/K465E PTEN+/− mice were subjected to immunoblot analysis using the indicated antibodies. (C) Immunohistochemical analysis of B cell follicular lymphomas using the indicated antibodies.

Mentions: To assess the growth rate of tumours formed in PDK1+/+PTEN+/−and PDK1K465E/K465EPTEN+/− mice, the volumes of B cell follicular lymphomas present in the animals were measured at 3-week intervals over a 6-week period by magnetic resonance imaging (MRI). For these experiments, littermate mice of the same age and possessing tumours were selected. We observed that the volume of B cell follicular lymphomas in PDK1+/+PTEN+/− mice increased 1.7-fold over this period of analysis (Fig. 4A). By contrast, the size of tumours observed in PDK1K465E/K465EPTEN+/− mice did not appreciably increase over 6 weeks. After this period, total cell extracts were generated from the B cell follicular lymphomas studied by MRI. Phospho-immunoblotting revealed that Akt phosphorylation at T308, but not S473, was significantly reduced in the B cell follicular lymphomas derived from PDK1K465E/K465EPTEN+/− mice compared with PDK1+/+PTEN+/−animals (Fig. 4B). We did not detect significant differences in the phosphorylation of Akt substrates, such as PRAS40, FOXO1 and GSK3α/β, comparing lymphomas from PDK1+/+PTEN+/− with those from PDK1K465E/K465EPTEN+/− mice. Similarly, there was no significant difference in the phosphorylation of other PDK1 substrates, S6K and SGK [assessed by phosphorylation of its substrate NDRG1 (Garcia-Martinez and Alessi, 2008)], in tumours derived from PDK1+/+PTEN+/− as compared with PDK1K465E/K465EPTEN+/− mice. To further assess the properties of the tumours formed in PDK1+/+PTEN+/− and PDK1K465E/K465EPTEN+/− mice, sections of B cell follicular lymphomas were analysed by immunohistochemistry. PTEN staining of the lymphoma was detected, which indicates that these lymphomas have not lost all expression of PTEN. We also investigated the subcellular localisation of the FOXO1 transcription factor, which is localised in the cytoplasm after phosphorylation by Akt. In both centroblasts and smaller centrocytes, FOXO1 staining was predominantly cytoplasmic, irrespective of genotype, indicating that Akt was activated. Strong cytoplasmic staining for phosphorylation of S6 in centroblasts was observed, suggesting that S6K was active in tumours of either genotype. There was no significant difference between the genotypes in the proportion of the proliferative marker Ki67. These data indicate that although Akt phosphorylation at T308 was reduced in tumours from PDK1K465E/K465EPTEN+/− mice, once tumours had formed there was no significant difference in the signalling pathway downstream of Akt, which is consistent with previous studies (Bayascas et al., 2005).


How moderate changes in Akt T-loop phosphorylation impact on tumorigenesis and insulin resistance.

Wullschleger S, Sakamoto K, Johnstone L, Duce S, Fleming S, Alessi DR - Dis Model Mech (2010)

Reduced tumour growth in PDK1K465E/K465EPTEN+/− mice. (A) The size of B cell follicular lymphomas of 11-month-old littermate PDK1+/+PTEN+/− and PDK1K465E/K465E PTEN+/− mice were determined by MRI imaging. Individual tumours from the same animal were measured at 0-, 3- and 6-week intervals. The data are depicted as relative tumour volume. PDK1+/+ PTEN+/− n=3, PDK1K465E/K465E PTEN+/− n=2. (B) Lysates of B cell follicular lymphomas from PDK1+/+ PTEN+/− and PDK1K465E/K465E PTEN+/− mice were subjected to immunoblot analysis using the indicated antibodies. (C) Immunohistochemical analysis of B cell follicular lymphomas using the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008965&req=5

f4-0040095: Reduced tumour growth in PDK1K465E/K465EPTEN+/− mice. (A) The size of B cell follicular lymphomas of 11-month-old littermate PDK1+/+PTEN+/− and PDK1K465E/K465E PTEN+/− mice were determined by MRI imaging. Individual tumours from the same animal were measured at 0-, 3- and 6-week intervals. The data are depicted as relative tumour volume. PDK1+/+ PTEN+/− n=3, PDK1K465E/K465E PTEN+/− n=2. (B) Lysates of B cell follicular lymphomas from PDK1+/+ PTEN+/− and PDK1K465E/K465E PTEN+/− mice were subjected to immunoblot analysis using the indicated antibodies. (C) Immunohistochemical analysis of B cell follicular lymphomas using the indicated antibodies.
Mentions: To assess the growth rate of tumours formed in PDK1+/+PTEN+/−and PDK1K465E/K465EPTEN+/− mice, the volumes of B cell follicular lymphomas present in the animals were measured at 3-week intervals over a 6-week period by magnetic resonance imaging (MRI). For these experiments, littermate mice of the same age and possessing tumours were selected. We observed that the volume of B cell follicular lymphomas in PDK1+/+PTEN+/− mice increased 1.7-fold over this period of analysis (Fig. 4A). By contrast, the size of tumours observed in PDK1K465E/K465EPTEN+/− mice did not appreciably increase over 6 weeks. After this period, total cell extracts were generated from the B cell follicular lymphomas studied by MRI. Phospho-immunoblotting revealed that Akt phosphorylation at T308, but not S473, was significantly reduced in the B cell follicular lymphomas derived from PDK1K465E/K465EPTEN+/− mice compared with PDK1+/+PTEN+/−animals (Fig. 4B). We did not detect significant differences in the phosphorylation of Akt substrates, such as PRAS40, FOXO1 and GSK3α/β, comparing lymphomas from PDK1+/+PTEN+/− with those from PDK1K465E/K465EPTEN+/− mice. Similarly, there was no significant difference in the phosphorylation of other PDK1 substrates, S6K and SGK [assessed by phosphorylation of its substrate NDRG1 (Garcia-Martinez and Alessi, 2008)], in tumours derived from PDK1+/+PTEN+/− as compared with PDK1K465E/K465EPTEN+/− mice. To further assess the properties of the tumours formed in PDK1+/+PTEN+/− and PDK1K465E/K465EPTEN+/− mice, sections of B cell follicular lymphomas were analysed by immunohistochemistry. PTEN staining of the lymphoma was detected, which indicates that these lymphomas have not lost all expression of PTEN. We also investigated the subcellular localisation of the FOXO1 transcription factor, which is localised in the cytoplasm after phosphorylation by Akt. In both centroblasts and smaller centrocytes, FOXO1 staining was predominantly cytoplasmic, irrespective of genotype, indicating that Akt was activated. Strong cytoplasmic staining for phosphorylation of S6 in centroblasts was observed, suggesting that S6K was active in tumours of either genotype. There was no significant difference between the genotypes in the proportion of the proliferative marker Ki67. These data indicate that although Akt phosphorylation at T308 was reduced in tumours from PDK1K465E/K465EPTEN+/− mice, once tumours had formed there was no significant difference in the signalling pathway downstream of Akt, which is consistent with previous studies (Bayascas et al., 2005).

Bottom Line: Using insulin-resistant PDK1(K465E/K465E) PH domain knock-in mice, we found that introducing the PTEN(+/-) mutation to slightly stimulate Akt restored normal insulin sensitivity.This was also accompanied by slower growth of B cell follicular lymphomas, as monitored by magnetic resonance imaging.Our findings imply that signal transduction inhibitors that lead to a modest reduction in Akt activity would not only delay onset of tumours possessing elevated phosphoinositide 3-kinase pathway activity but would also reduce the growth rate of developed tumours.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

ABSTRACT
The Akt signalling pathway plays vital roles in controlling cellular responses to insulin as well as in proliferation and survival. Inhibition of Akt signalling leads to insulin resistance and type 2 diabetes, whereas hyperactivation of Akt promotes tumorigenesis. In this study, we investigate how modest changes in the activity of the Akt signalling pathway, to an extent that might be achieved by drug treatment, would impact on insulin resistance and tumorigenesis. Using insulin-resistant PDK1(K465E/K465E) PH domain knock-in mice, we found that introducing the PTEN(+/-) mutation to slightly stimulate Akt restored normal insulin sensitivity. Introducing the PDK1(K465E/K465E) PH domain knock-in mutation into cancer-prone PTEN(+/-) mice, lowered Akt activity only by about 50%, but led to a delay in tumour onset of ∼4 months in a broad range of tumours. This was also accompanied by slower growth of B cell follicular lymphomas, as monitored by magnetic resonance imaging. Our findings imply that signal transduction inhibitors that lead to a modest reduction in Akt activity would not only delay onset of tumours possessing elevated phosphoinositide 3-kinase pathway activity but would also reduce the growth rate of developed tumours.

Show MeSH
Related in: MedlinePlus