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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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Combination treatment induces autophagy and PARP cleavage.(A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa).
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pone-0026343-g006: Combination treatment induces autophagy and PARP cleavage.(A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa).

Mentions: We next characterized the mechanism of cell death in response to combination therapy. Examination of several apoptotic markers, including caspase 3, revealed that the cells did not undergo caspase-dependent apoptosis (data not shown). However, several studies have demonstrated that both down-regulation of Akt [28], [29] and treatment with rapalogs [26] promote autophagy, which can lead to caspase-independent apoptosis characterized by poly (ADP-ribose) polymerase (PARP) cleavage [30], [31]. Therefore, we first tested processing of microtubule-associated protein light chain 3-I (LC3-I), which is a common marker for autophagy [32]. As noted in Figure 6A, under our experimental conditions, no loss of LC3-I occurred upon temsirolimus treatment when compared with control. In contrast, BEZ235 alone dramatically reduced the level of LC3-I in all tested cells. Furthermore, combination of temsirolimus with ZSTK474 reduced levels of LC3-I compared with ZSTK474 alone. The reduction in LC3-I levels was accompanied by the expected increase or maintenance of LC3-II in some cell lines (Ishikawa H and Hec50co), further confirming that the cells are undergoing autophagy (Fig. 6A). Since others have reported that BEZ235 induces caspase-independent apoptosis through PARP cleavage [30], we treated cells for longer periods of time (48 to 72 hrs) and examined PARP cleavage as a marker of apoptotic cell death. In both sensitive and resistant cell lines, we observed PARP cleavage following treatment with BEZ235 or ZSTK474, and addition of temsirolimus did not substantially increase the effect (Fig. 6B). Collectively, these data suggest that the mechanism of cell death involves massive autophagy which proceeds into caspase-independent apoptosis.


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

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

Combination treatment induces autophagy and PARP cleavage.(A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026343-g006: Combination treatment induces autophagy and PARP cleavage.(A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa).
Mentions: We next characterized the mechanism of cell death in response to combination therapy. Examination of several apoptotic markers, including caspase 3, revealed that the cells did not undergo caspase-dependent apoptosis (data not shown). However, several studies have demonstrated that both down-regulation of Akt [28], [29] and treatment with rapalogs [26] promote autophagy, which can lead to caspase-independent apoptosis characterized by poly (ADP-ribose) polymerase (PARP) cleavage [30], [31]. Therefore, we first tested processing of microtubule-associated protein light chain 3-I (LC3-I), which is a common marker for autophagy [32]. As noted in Figure 6A, under our experimental conditions, no loss of LC3-I occurred upon temsirolimus treatment when compared with control. In contrast, BEZ235 alone dramatically reduced the level of LC3-I in all tested cells. Furthermore, combination of temsirolimus with ZSTK474 reduced levels of LC3-I compared with ZSTK474 alone. The reduction in LC3-I levels was accompanied by the expected increase or maintenance of LC3-II in some cell lines (Ishikawa H and Hec50co), further confirming that the cells are undergoing autophagy (Fig. 6A). Since others have reported that BEZ235 induces caspase-independent apoptosis through PARP cleavage [30], we treated cells for longer periods of time (48 to 72 hrs) and examined PARP cleavage as a marker of apoptotic cell death. In both sensitive and resistant cell lines, we observed PARP cleavage following treatment with BEZ235 or ZSTK474, and addition of temsirolimus did not substantially increase the effect (Fig. 6B). Collectively, these data suggest that the mechanism of cell death involves massive autophagy which proceeds into caspase-independent apoptosis.

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