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 differentially regulates cell viability and Akt phosphorylation in a dose-dependent manner.A, B, Endometrial cancer cells were treated with increasing doses of temsirolimus for 72 hrs. Results are separated by (A) sensitivity or (B) resistance as determined by cell viability. C, D, Phosphorylation of rS6 (P-rS6) and Akt (P-Akt T308 and P-Akt S473) after treatment with indicated doses of temsirolimus for 72 hrs in temsirolimus-sensitive (C) or temsirolimus-resistant (D) cells was determined by Western blotting. Expression of total rS6 and Akt protein serves as loading controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198385&req=5

pone-0026343-g001: Temsirolimus differentially regulates cell viability and Akt phosphorylation in a dose-dependent manner.A, B, Endometrial cancer cells were treated with increasing doses of temsirolimus for 72 hrs. Results are separated by (A) sensitivity or (B) resistance as determined by cell viability. C, D, Phosphorylation of rS6 (P-rS6) and Akt (P-Akt T308 and P-Akt S473) after treatment with indicated doses of temsirolimus for 72 hrs in temsirolimus-sensitive (C) or temsirolimus-resistant (D) cells was determined by Western blotting. Expression of total rS6 and Akt protein serves as loading controls.

Mentions: To establish a cell-based model system to understand the efficacy of mTOR inhibitors in endometrial cancer patients, we tested the growth-inhibitory properties of temsirolimus on eight endometrial cancer cell lines using in vitro proliferation assays. Proliferation of four endometrial cancer cell lines (SKUT1B, AN3CA, RL95-2, and ECC-1) was inhibited at low nanomolar concentrations of temsirolimus (Fig. 1A). The temsirolimus IC50 for these sensitive cells was approximately 1 nM (Supporting Table S1), indicating a strong growth inhibitory impact. In contrast, Ishikawa H, Hec50co, Hec1A and KLE cells were more resistant to treatment, and the average IC50, when reached, was at least 10-fold higher (Fig. 1B, Supporting Table S1).


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

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

Temsirolimus differentially regulates cell viability and Akt phosphorylation in a dose-dependent manner.A, B, Endometrial cancer cells were treated with increasing doses of temsirolimus for 72 hrs. Results are separated by (A) sensitivity or (B) resistance as determined by cell viability. C, D, Phosphorylation of rS6 (P-rS6) and Akt (P-Akt T308 and P-Akt S473) after treatment with indicated doses of temsirolimus for 72 hrs in temsirolimus-sensitive (C) or temsirolimus-resistant (D) cells was determined by Western blotting. Expression of total rS6 and Akt protein serves as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026343-g001: Temsirolimus differentially regulates cell viability and Akt phosphorylation in a dose-dependent manner.A, B, Endometrial cancer cells were treated with increasing doses of temsirolimus for 72 hrs. Results are separated by (A) sensitivity or (B) resistance as determined by cell viability. C, D, Phosphorylation of rS6 (P-rS6) and Akt (P-Akt T308 and P-Akt S473) after treatment with indicated doses of temsirolimus for 72 hrs in temsirolimus-sensitive (C) or temsirolimus-resistant (D) cells was determined by Western blotting. Expression of total rS6 and Akt protein serves as loading controls.
Mentions: To establish a cell-based model system to understand the efficacy of mTOR inhibitors in endometrial cancer patients, we tested the growth-inhibitory properties of temsirolimus on eight endometrial cancer cell lines using in vitro proliferation assays. Proliferation of four endometrial cancer cell lines (SKUT1B, AN3CA, RL95-2, and ECC-1) was inhibited at low nanomolar concentrations of temsirolimus (Fig. 1A). The temsirolimus IC50 for these sensitive cells was approximately 1 nM (Supporting Table S1), indicating a strong growth inhibitory impact. In contrast, Ishikawa H, Hec50co, Hec1A and KLE cells were more resistant to treatment, and the average IC50, when reached, was at least 10-fold higher (Fig. 1B, Supporting Table S1).

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