Limits...
A mechanism for synergy with combined mTOR and PI3 kinase inhibitors.

Yang S, Xiao X, Meng X, Leslie KK - PLoS ONE (2011)

Bottom Line: Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone.Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27.While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.

Show MeSH

Related in: MedlinePlus

Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244.A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198385&req=5

pone-0026343-g003: Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244.A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation.

Mentions: To overcome the compensatory Akt phosphorylation induced by temsirolimus treatment as a mechanism of acquired cell resistance, six candidate inhibitors were chosen based on previously reported efficacy (Supporting Table S2). Ishikawa H (type I endometrial cancer cells) and Hec50co cells (type II endometrial cancer cells) [22] were first used to test the efficacy of these agents alone and in combination with temsirolimus. As shown in Figure 3A, Ishikawa H and Hec50co cells displayed a basal level of Akt phosphorylation that was further increased by temsirolimus treatment, with Ishikawa H cells showing higher levels compared with Hec50co cells, as discussed above (also, Fig. 1C, D). BEZ235 (dual mTOR/PI3K inhibitor) and ZSTK474 (PI3K inhibitor) decreased the basal level of Akt phosphorylation in both cell lines tested when used alone (Fig. 3A). Strikingly, when combined with temsirolimus, both BEZ235 and ZSTK474 blocked the temsirolimus-induced hyper-phosphorylation of Akt. We tested four other endometrial cancer cell lines and observed a similar pattern of inhibition of Akt phosphorylation (Fig. 3B, left panel). In order to demonstrate the effect of drug treatment even on resistant KLE and Hec1A cells, which have very low levels of basal phospho-Akt, we exposed the immunoblot for extended periods of time (Fig. 3B). The effect of the treatment regimens was preserved in these cells as well even though the baseline levels of phospho-Akt were substantially reduced. A marker for temsirolimus activity is the loss of phosphorylation of p70S6K, which is directly downstream of mTORC1. Accordingly, we detected a loss of phospho-S6K whereas total S6K levels were generally unchanged (Fig. 3B, right panel). This result confirms that whether temsirolimus is used alone or in combination, this cellular response is maintained.


A mechanism for synergy with combined mTOR and PI3 kinase inhibitors.

Yang S, Xiao X, Meng X, Leslie KK - PLoS ONE (2011)

Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244.A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198385&req=5

pone-0026343-g003: Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244.A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation.
Mentions: To overcome the compensatory Akt phosphorylation induced by temsirolimus treatment as a mechanism of acquired cell resistance, six candidate inhibitors were chosen based on previously reported efficacy (Supporting Table S2). Ishikawa H (type I endometrial cancer cells) and Hec50co cells (type II endometrial cancer cells) [22] were first used to test the efficacy of these agents alone and in combination with temsirolimus. As shown in Figure 3A, Ishikawa H and Hec50co cells displayed a basal level of Akt phosphorylation that was further increased by temsirolimus treatment, with Ishikawa H cells showing higher levels compared with Hec50co cells, as discussed above (also, Fig. 1C, D). BEZ235 (dual mTOR/PI3K inhibitor) and ZSTK474 (PI3K inhibitor) decreased the basal level of Akt phosphorylation in both cell lines tested when used alone (Fig. 3A). Strikingly, when combined with temsirolimus, both BEZ235 and ZSTK474 blocked the temsirolimus-induced hyper-phosphorylation of Akt. We tested four other endometrial cancer cell lines and observed a similar pattern of inhibition of Akt phosphorylation (Fig. 3B, left panel). In order to demonstrate the effect of drug treatment even on resistant KLE and Hec1A cells, which have very low levels of basal phospho-Akt, we exposed the immunoblot for extended periods of time (Fig. 3B). The effect of the treatment regimens was preserved in these cells as well even though the baseline levels of phospho-Akt were substantially reduced. A marker for temsirolimus activity is the loss of phosphorylation of p70S6K, which is directly downstream of mTORC1. Accordingly, we detected a loss of phospho-S6K whereas total S6K levels were generally unchanged (Fig. 3B, right panel). This result confirms that whether temsirolimus is used alone or in combination, this cellular response is maintained.

Bottom Line: Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone.Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27.While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.

Show MeSH
Related in: MedlinePlus