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GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

Koo J, Wang X, Owonikoko TK, Ramalingam SS, Khuri FR, Sun SY - Oncotarget (2015)

Bottom Line: GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding.Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition.Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA, USA.

ABSTRACT
The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

No MeSH data available.


Related in: MedlinePlus

Rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through facilitating their degradation (A and B), which is mediated by either FBX4 (C) or FBXW7 (D and E)(A), The indicated cell lines were pre-treated with 10 μM MG132 for 30 min and then co-treated with 10 nM rapamycin for 6 h. (B), H460 cells were treated with DMSO or 10 nM of rapamycin for 6 h. The cells were then washed with PBS 3 times and re-fed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J Software and were normalized to tubulin. (C) The indicated A549 transfectants were exposed to 10 nM rapamycin for 6 h. (D) The indicated cells were transfected with the given siRNAs and after 48 h were exposed to 10 nM rapamycin for an additional 6 h. (E) The indicated cell lines were exposed to 10 nM rapamycin for 6 h. After the aforementioned treatments (A, C–E), the cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blotting to detect the given proteins. Cellular total RNA was also extracted from the indicated cell lines in E for RT-PCR detection of FBXW7.
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Figure 5: Rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through facilitating their degradation (A and B), which is mediated by either FBX4 (C) or FBXW7 (D and E)(A), The indicated cell lines were pre-treated with 10 μM MG132 for 30 min and then co-treated with 10 nM rapamycin for 6 h. (B), H460 cells were treated with DMSO or 10 nM of rapamycin for 6 h. The cells were then washed with PBS 3 times and re-fed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J Software and were normalized to tubulin. (C) The indicated A549 transfectants were exposed to 10 nM rapamycin for 6 h. (D) The indicated cells were transfected with the given siRNAs and after 48 h were exposed to 10 nM rapamycin for an additional 6 h. (E) The indicated cell lines were exposed to 10 nM rapamycin for 6 h. After the aforementioned treatments (A, C–E), the cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blotting to detect the given proteins. Cellular total RNA was also extracted from the indicated cell lines in E for RT-PCR detection of FBXW7.

Mentions: We were interested in knowing how inhibition of GSK3 blocks rapamycin-induced reduction of cyclin D1, c-Myc and Mcl-1 without interfering with the suppression of mTORC1 signaling and cap-binding by rapamycin. Considering that GSK3 is involved in regulating degradation of these proteins [19–21], we asked whether rapamycin-induced reduction of these proteins is due to enhanced protein degradation. To this end, we first compared the effects of rapamycin on cyclin D1 reduction in the absence and presence of the proteasome inhibitor, MG132. We observed that rapamycin-induced cyclin D1 reduction was prevented by the presence of MG132 in all three tested cell lines (Fig. 5A). Similarly, the presence of MG132 rescued rapamycin-induced reduction of both c-Myc and Mcl-1 (Fig. 4D). Moreover, we determined whether rapamycin affects the stabilities of these proteins. Compared with DMSO control, rapamycin apparently shortened the half-lives of not only cyclin D1, but also c-Myc and Mcl-1 (Fig. 5B), indicating that rapamycin decreases the stabilities of these proteins. Collectively, these data clearly suggest that rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through promoting their degradation.


GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

Koo J, Wang X, Owonikoko TK, Ramalingam SS, Khuri FR, Sun SY - Oncotarget (2015)

Rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through facilitating their degradation (A and B), which is mediated by either FBX4 (C) or FBXW7 (D and E)(A), The indicated cell lines were pre-treated with 10 μM MG132 for 30 min and then co-treated with 10 nM rapamycin for 6 h. (B), H460 cells were treated with DMSO or 10 nM of rapamycin for 6 h. The cells were then washed with PBS 3 times and re-fed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J Software and were normalized to tubulin. (C) The indicated A549 transfectants were exposed to 10 nM rapamycin for 6 h. (D) The indicated cells were transfected with the given siRNAs and after 48 h were exposed to 10 nM rapamycin for an additional 6 h. (E) The indicated cell lines were exposed to 10 nM rapamycin for 6 h. After the aforementioned treatments (A, C–E), the cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blotting to detect the given proteins. Cellular total RNA was also extracted from the indicated cell lines in E for RT-PCR detection of FBXW7.
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Related In: Results  -  Collection

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Figure 5: Rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through facilitating their degradation (A and B), which is mediated by either FBX4 (C) or FBXW7 (D and E)(A), The indicated cell lines were pre-treated with 10 μM MG132 for 30 min and then co-treated with 10 nM rapamycin for 6 h. (B), H460 cells were treated with DMSO or 10 nM of rapamycin for 6 h. The cells were then washed with PBS 3 times and re-fed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J Software and were normalized to tubulin. (C) The indicated A549 transfectants were exposed to 10 nM rapamycin for 6 h. (D) The indicated cells were transfected with the given siRNAs and after 48 h were exposed to 10 nM rapamycin for an additional 6 h. (E) The indicated cell lines were exposed to 10 nM rapamycin for 6 h. After the aforementioned treatments (A, C–E), the cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blotting to detect the given proteins. Cellular total RNA was also extracted from the indicated cell lines in E for RT-PCR detection of FBXW7.
Mentions: We were interested in knowing how inhibition of GSK3 blocks rapamycin-induced reduction of cyclin D1, c-Myc and Mcl-1 without interfering with the suppression of mTORC1 signaling and cap-binding by rapamycin. Considering that GSK3 is involved in regulating degradation of these proteins [19–21], we asked whether rapamycin-induced reduction of these proteins is due to enhanced protein degradation. To this end, we first compared the effects of rapamycin on cyclin D1 reduction in the absence and presence of the proteasome inhibitor, MG132. We observed that rapamycin-induced cyclin D1 reduction was prevented by the presence of MG132 in all three tested cell lines (Fig. 5A). Similarly, the presence of MG132 rescued rapamycin-induced reduction of both c-Myc and Mcl-1 (Fig. 4D). Moreover, we determined whether rapamycin affects the stabilities of these proteins. Compared with DMSO control, rapamycin apparently shortened the half-lives of not only cyclin D1, but also c-Myc and Mcl-1 (Fig. 5B), indicating that rapamycin decreases the stabilities of these proteins. Collectively, these data clearly suggest that rapamycin decreases the levels of cyclin D1, c-Myc and Mcl-1 through promoting their degradation.

Bottom Line: GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding.Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition.Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA, USA.

ABSTRACT
The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

No MeSH data available.


Related in: MedlinePlus