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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.

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BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression.A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 µM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments.
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pone-0026343-g005: BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression.A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 µM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments.

Mentions: Temsirolimus and BEZ235 have been reported to inhibit cancer cell growth by inducing G0/G1 cell cycle arrest [26], [27]. Since we have two cell populations whose proliferation is differentially affected by temsirolimus treatment (sensitive vs. resistant), we chose two representative cell lines (AN3CA-sensitive; Hec50co-resistant) as models to test the combination approach on cell cycle progression. Cell cycle content analysis of AN3CA cells demonstrated that temsirolimus treatment increased the percent of cells in G1 from 42% (DMSO control) to 71% after only 24 hours (Fig. 5A). Co-treatment with BEZ235 and temsirolimus led to a slight increase of cells in G1 compared to temsirolimus alone at 24 hours (Fig. 5A). In contrast, treatment of Hec50co cells with temsirolimus alone caused a 4% increase in the G1 population, which required 72 hours of prolonged exposure to demonstrate (Fig. 5B). However, when temsirolimus was combined with BEZ235, the G1 population increased to 60% by 72 hours (Fig. 5B). Further, ZSTK474 effects in combination with temsirolimus (Fig. 5C) replicated those of BEZ235 (Fig. 5A), suggesting that G1 arrest is a common method by which combined inhibition of PI3K and mTOR achieves the decrease in cell viability observed in Figure 4. Also of note, the G1 block was achieved rapidly in sensitive cells, but eventually was observed even in resistant cells.


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

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

BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression.A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 µM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments.
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Related In: Results  -  Collection

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

pone-0026343-g005: BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression.A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 µM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments.
Mentions: Temsirolimus and BEZ235 have been reported to inhibit cancer cell growth by inducing G0/G1 cell cycle arrest [26], [27]. Since we have two cell populations whose proliferation is differentially affected by temsirolimus treatment (sensitive vs. resistant), we chose two representative cell lines (AN3CA-sensitive; Hec50co-resistant) as models to test the combination approach on cell cycle progression. Cell cycle content analysis of AN3CA cells demonstrated that temsirolimus treatment increased the percent of cells in G1 from 42% (DMSO control) to 71% after only 24 hours (Fig. 5A). Co-treatment with BEZ235 and temsirolimus led to a slight increase of cells in G1 compared to temsirolimus alone at 24 hours (Fig. 5A). In contrast, treatment of Hec50co cells with temsirolimus alone caused a 4% increase in the G1 population, which required 72 hours of prolonged exposure to demonstrate (Fig. 5B). However, when temsirolimus was combined with BEZ235, the G1 population increased to 60% by 72 hours (Fig. 5B). Further, ZSTK474 effects in combination with temsirolimus (Fig. 5C) replicated those of BEZ235 (Fig. 5A), suggesting that G1 arrest is a common method by which combined inhibition of PI3K and mTOR achieves the decrease in cell viability observed in Figure 4. Also of note, the G1 block was achieved rapidly in sensitive cells, but eventually was observed even in resistant cells.

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