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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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Related in: MedlinePlus

Baseline expression of PTEN, phospho-Akt and phospho-PDK1.Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. β-actin expression served as a loading control.
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pone-0026343-g002: Baseline expression of PTEN, phospho-Akt and phospho-PDK1.Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. β-actin expression served as a loading control.

Mentions: To understand the basis for cell sensitivity to temsirolimus as a single agent, we turned to an analysis of Akt activation, both at baseline and in response to treatment. We found that the baseline constitutive activation of Akt was predictive of cell sensitivity with the most resistant cells having very low basal S473 and T308 phosphorylation (Fig. 1C, D). After treatment with temsirolimus, a compensatory increase in Akt phosphorylation at both sites was detected in the most sensitive endometrial cancer cell lines tested (Fig. 1C), but the primarily resistant cells (KLE) demonstrated no Akt phosphorylation at either site, and another resistant line, Hec50co, showed reduced phosphorylation (Fig. 1D). Thus, in contrast to sensitive cells, primarily resistant cells have low basal Akt phosphorylation and do not respond with compensatory hyper-phosphorylation after temsirolimus treatment. Of note, phospho-PDK1, the kinase responsible for Akt phosphorylation at T308, is low in several resistant cell lines such as Hec1A and KLE (Fig. 2). This indicates primary cell resistance and a general lack of dependence on the Akt signaling pathway for proliferation. On the other hand, the finding of compensatory activation of Akt in responsive cells is consistent with previous reports in the literature of rapalog-induced Akt phosphorylation in many cancer cell lines, human xenograft models, and patient tumors [16]. Compensatory hyper-Akt phosphorylation suggests one potential mechanism whereby cells that are initially sensitive escape the growth inhibitory effects of the drug and become secondarily resistant. From these data, we distinguish between primary resistance with a lack of baseline Akt dependence versus acquired resistance demonstrated by hyper-Akt activation in response to temsirolimus.


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

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

Baseline expression of PTEN, phospho-Akt and phospho-PDK1.Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. β-actin expression served as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026343-g002: Baseline expression of PTEN, phospho-Akt and phospho-PDK1.Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. β-actin expression served as a loading control.
Mentions: To understand the basis for cell sensitivity to temsirolimus as a single agent, we turned to an analysis of Akt activation, both at baseline and in response to treatment. We found that the baseline constitutive activation of Akt was predictive of cell sensitivity with the most resistant cells having very low basal S473 and T308 phosphorylation (Fig. 1C, D). After treatment with temsirolimus, a compensatory increase in Akt phosphorylation at both sites was detected in the most sensitive endometrial cancer cell lines tested (Fig. 1C), but the primarily resistant cells (KLE) demonstrated no Akt phosphorylation at either site, and another resistant line, Hec50co, showed reduced phosphorylation (Fig. 1D). Thus, in contrast to sensitive cells, primarily resistant cells have low basal Akt phosphorylation and do not respond with compensatory hyper-phosphorylation after temsirolimus treatment. Of note, phospho-PDK1, the kinase responsible for Akt phosphorylation at T308, is low in several resistant cell lines such as Hec1A and KLE (Fig. 2). This indicates primary cell resistance and a general lack of dependence on the Akt signaling pathway for proliferation. On the other hand, the finding of compensatory activation of Akt in responsive cells is consistent with previous reports in the literature of rapalog-induced Akt phosphorylation in many cancer cell lines, human xenograft models, and patient tumors [16]. Compensatory hyper-Akt phosphorylation suggests one potential mechanism whereby cells that are initially sensitive escape the growth inhibitory effects of the drug and become secondarily resistant. From these data, we distinguish between primary resistance with a lack of baseline Akt dependence versus acquired resistance demonstrated by hyper-Akt activation in response to temsirolimus.

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