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AKT inhibition overcomes rapamycin resistance by enhancing the repressive function of PRAS40 on mTORC1/4E-BP1 axis.

Mi W, Ye Q, Liu S, She QB - Oncotarget (2015)

Bottom Line: Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1.However, treatment with both drugs resulted in profound effects in vitro and in vivo.Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation.

View Article: PubMed Central - PubMed

Affiliation: Markey Cancer Center and Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY, USA.

ABSTRACT
The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

No MeSH data available.


Related in: MedlinePlus

The effects of AKT and mTORC1 activation on cell migration and invasion are mediated by 4E-BP1A, B. Transwell migration A. and invasion B. analyses of MCF7 and HCT116 cells were performed in the presence of 50 nM rapamycin (Rap) and 1 μM MK2206, alone or in combination, or DMSO as control for 6 h (A) and 30 h (B), respectively. The results represent the mean number of migrated (A) or invaded (B) cells per field ± S.E.M. (n = 3). *P < 0.03 for combination of Rap and MK2206 versus DMSO Ctrl, Rap or MK2206. C. Migration analysis of HCT116 cells with stable expression of control shRNA or 4E-BP1 shRNA was performed in the presence of the drugs as indicated in (A) for 6 h. The results are expressed as the inhibition of migration relative to the DMSO-treated controls. **P < 0.02 for combination of Rap and MK2206 in Sh 4E-BP1 cells versus that in Sh Ctrl cells. Data shown in graphs represent the mean ± S.E.M. (n = 3).
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Figure 3: The effects of AKT and mTORC1 activation on cell migration and invasion are mediated by 4E-BP1A, B. Transwell migration A. and invasion B. analyses of MCF7 and HCT116 cells were performed in the presence of 50 nM rapamycin (Rap) and 1 μM MK2206, alone or in combination, or DMSO as control for 6 h (A) and 30 h (B), respectively. The results represent the mean number of migrated (A) or invaded (B) cells per field ± S.E.M. (n = 3). *P < 0.03 for combination of Rap and MK2206 versus DMSO Ctrl, Rap or MK2206. C. Migration analysis of HCT116 cells with stable expression of control shRNA or 4E-BP1 shRNA was performed in the presence of the drugs as indicated in (A) for 6 h. The results are expressed as the inhibition of migration relative to the DMSO-treated controls. **P < 0.02 for combination of Rap and MK2206 in Sh 4E-BP1 cells versus that in Sh Ctrl cells. Data shown in graphs represent the mean ± S.E.M. (n = 3).

Mentions: Our recent studies show that 4E-BP1-regulated cap-dependent translation also plays an important role in controlling cancer cell motility and metastasis [9, 10]. Using Boyden chamber assays described previously [9], treatment with rapamycin or MK2206 alone for 6 h had only a modest effect on MCF7 and HCT116 cell migration. However, a combination of both drugs was effective in inhibiting their migration (Figure 3A). Similar results were observed in the ability of HCT116 cells that invade through Matrigel 30 h after drug exposure (Figure 3B). Notably, knockdown of 4E-BP1 expression in HCT116 cells profoundly reduced the inhibitory effect of combined treatment on cell migration compared with that in the control cells (Figure 3C).


AKT inhibition overcomes rapamycin resistance by enhancing the repressive function of PRAS40 on mTORC1/4E-BP1 axis.

Mi W, Ye Q, Liu S, She QB - Oncotarget (2015)

The effects of AKT and mTORC1 activation on cell migration and invasion are mediated by 4E-BP1A, B. Transwell migration A. and invasion B. analyses of MCF7 and HCT116 cells were performed in the presence of 50 nM rapamycin (Rap) and 1 μM MK2206, alone or in combination, or DMSO as control for 6 h (A) and 30 h (B), respectively. The results represent the mean number of migrated (A) or invaded (B) cells per field ± S.E.M. (n = 3). *P < 0.03 for combination of Rap and MK2206 versus DMSO Ctrl, Rap or MK2206. C. Migration analysis of HCT116 cells with stable expression of control shRNA or 4E-BP1 shRNA was performed in the presence of the drugs as indicated in (A) for 6 h. The results are expressed as the inhibition of migration relative to the DMSO-treated controls. **P < 0.02 for combination of Rap and MK2206 in Sh 4E-BP1 cells versus that in Sh Ctrl cells. Data shown in graphs represent the mean ± S.E.M. (n = 3).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4546444&req=5

Figure 3: The effects of AKT and mTORC1 activation on cell migration and invasion are mediated by 4E-BP1A, B. Transwell migration A. and invasion B. analyses of MCF7 and HCT116 cells were performed in the presence of 50 nM rapamycin (Rap) and 1 μM MK2206, alone or in combination, or DMSO as control for 6 h (A) and 30 h (B), respectively. The results represent the mean number of migrated (A) or invaded (B) cells per field ± S.E.M. (n = 3). *P < 0.03 for combination of Rap and MK2206 versus DMSO Ctrl, Rap or MK2206. C. Migration analysis of HCT116 cells with stable expression of control shRNA or 4E-BP1 shRNA was performed in the presence of the drugs as indicated in (A) for 6 h. The results are expressed as the inhibition of migration relative to the DMSO-treated controls. **P < 0.02 for combination of Rap and MK2206 in Sh 4E-BP1 cells versus that in Sh Ctrl cells. Data shown in graphs represent the mean ± S.E.M. (n = 3).
Mentions: Our recent studies show that 4E-BP1-regulated cap-dependent translation also plays an important role in controlling cancer cell motility and metastasis [9, 10]. Using Boyden chamber assays described previously [9], treatment with rapamycin or MK2206 alone for 6 h had only a modest effect on MCF7 and HCT116 cell migration. However, a combination of both drugs was effective in inhibiting their migration (Figure 3A). Similar results were observed in the ability of HCT116 cells that invade through Matrigel 30 h after drug exposure (Figure 3B). Notably, knockdown of 4E-BP1 expression in HCT116 cells profoundly reduced the inhibitory effect of combined treatment on cell migration compared with that in the control cells (Figure 3C).

Bottom Line: Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1.However, treatment with both drugs resulted in profound effects in vitro and in vivo.Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation.

View Article: PubMed Central - PubMed

Affiliation: Markey Cancer Center and Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY, USA.

ABSTRACT
The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

No MeSH data available.


Related in: MedlinePlus