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Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

Pant A, Lee II, Lu Z, Rueda BR, Schink J, Kim JJ - PLoS ONE (2012)

Bottom Line: Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein.Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1.Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume.

View Article: PubMed Central - PubMed

Affiliation: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, United States of America.

ABSTRACT
Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.

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The AKT inhibitor, MK-2206 inhibits AKT.PRB-specific Ishikawa (PRB23) cells were treated with A) increasing concentrations (0–1 uM) of MK-2206 for 24 h and B) 100 nM MK-2206 for various times (0–24 h). Protein levels of p(Ser473)-AKT, AKT and actin were measured by Western blot. C) PRB23 cells were treated with 100 nM MK-2206 for 4 h or 24 h and levels of p(Thr308)-AKT, AKT, p(Thr246)PRAS40, PRAS40, p(Thr202/Tyr204) ERK, ERK, p(Thr183/Tyr185) SAPK/JNK, JNK and actin were measured by western blot.
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pone-0041593-g001: The AKT inhibitor, MK-2206 inhibits AKT.PRB-specific Ishikawa (PRB23) cells were treated with A) increasing concentrations (0–1 uM) of MK-2206 for 24 h and B) 100 nM MK-2206 for various times (0–24 h). Protein levels of p(Ser473)-AKT, AKT and actin were measured by Western blot. C) PRB23 cells were treated with 100 nM MK-2206 for 4 h or 24 h and levels of p(Thr308)-AKT, AKT, p(Thr246)PRAS40, PRAS40, p(Thr202/Tyr204) ERK, ERK, p(Thr183/Tyr185) SAPK/JNK, JNK and actin were measured by western blot.

Mentions: It has previously been reported that Ishikawa cells carry a PTEN mutation and this results in constitutive downstream activation of AKT [7], [8]. In order to determine whether MK-2206 inhibited AKT in the PRB23 cells, cells were treated with increasing concentrations of MK-2206 (0–1 µM) and incubated for various times (0–24 h) (Fig. 1A, 1B). Phosphorylated (Ser473)-AKT was detected in cells treated with vehicle and levels progressively declined with increasing concentrations of MK-2206 (Fig. 1A). Total AKT levels were not affected by treatment with MK-2206. Levels of p(Ser473)-AKT decreased, as early as 1 h, and remained lower than vehicle treated cells at 24 h (Fig. 1B). Levels of p(Thr308)-AKT also declined with 100 nM MK-2206 treatment at both 4 h and 24 h (Fig. 1C). PRAS40 is a direct substrate of AKT and levels of p(Thr246)-PRAS40 decreased upon MK-2206 treatment at 4 h and 24 h, indicating that the AKT activity is decreased (Fig. 1C). Next, in order to demonstrate that MK-2206 was specific to the AKT pathway in PRB23 cells, levels of other phosphorylated kinases, which are not direct targets of AKT were measured. Levels of p(Thr202/Tyr204)-ERK as well as p(Thr183/Tyr185)-JNK did not change upon MK-2206 treatment either at 4 h or 24 h (Fig. 1C).


Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

Pant A, Lee II, Lu Z, Rueda BR, Schink J, Kim JJ - PLoS ONE (2012)

The AKT inhibitor, MK-2206 inhibits AKT.PRB-specific Ishikawa (PRB23) cells were treated with A) increasing concentrations (0–1 uM) of MK-2206 for 24 h and B) 100 nM MK-2206 for various times (0–24 h). Protein levels of p(Ser473)-AKT, AKT and actin were measured by Western blot. C) PRB23 cells were treated with 100 nM MK-2206 for 4 h or 24 h and levels of p(Thr308)-AKT, AKT, p(Thr246)PRAS40, PRAS40, p(Thr202/Tyr204) ERK, ERK, p(Thr183/Tyr185) SAPK/JNK, JNK and actin were measured by western blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404036&req=5

pone-0041593-g001: The AKT inhibitor, MK-2206 inhibits AKT.PRB-specific Ishikawa (PRB23) cells were treated with A) increasing concentrations (0–1 uM) of MK-2206 for 24 h and B) 100 nM MK-2206 for various times (0–24 h). Protein levels of p(Ser473)-AKT, AKT and actin were measured by Western blot. C) PRB23 cells were treated with 100 nM MK-2206 for 4 h or 24 h and levels of p(Thr308)-AKT, AKT, p(Thr246)PRAS40, PRAS40, p(Thr202/Tyr204) ERK, ERK, p(Thr183/Tyr185) SAPK/JNK, JNK and actin were measured by western blot.
Mentions: It has previously been reported that Ishikawa cells carry a PTEN mutation and this results in constitutive downstream activation of AKT [7], [8]. In order to determine whether MK-2206 inhibited AKT in the PRB23 cells, cells were treated with increasing concentrations of MK-2206 (0–1 µM) and incubated for various times (0–24 h) (Fig. 1A, 1B). Phosphorylated (Ser473)-AKT was detected in cells treated with vehicle and levels progressively declined with increasing concentrations of MK-2206 (Fig. 1A). Total AKT levels were not affected by treatment with MK-2206. Levels of p(Ser473)-AKT decreased, as early as 1 h, and remained lower than vehicle treated cells at 24 h (Fig. 1B). Levels of p(Thr308)-AKT also declined with 100 nM MK-2206 treatment at both 4 h and 24 h (Fig. 1C). PRAS40 is a direct substrate of AKT and levels of p(Thr246)-PRAS40 decreased upon MK-2206 treatment at 4 h and 24 h, indicating that the AKT activity is decreased (Fig. 1C). Next, in order to demonstrate that MK-2206 was specific to the AKT pathway in PRB23 cells, levels of other phosphorylated kinases, which are not direct targets of AKT were measured. Levels of p(Thr202/Tyr204)-ERK as well as p(Thr183/Tyr185)-JNK did not change upon MK-2206 treatment either at 4 h or 24 h (Fig. 1C).

Bottom Line: Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein.Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1.Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume.

View Article: PubMed Central - PubMed

Affiliation: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, United States of America.

ABSTRACT
Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.

Show MeSH
Related in: MedlinePlus