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Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

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The combination of P242 and erlotinib inhibits the carcinoma cell growth.(A) DLD-1 cells grown in EGF (50 ng/ml) containing medium were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and analyzed by western blotting for the indicated proteins (left). (B) DLD-1 and HT29 cells were treated with PP242 (1 μM) and erlotinib (2 μM), alone or in combination, and then subjected to cell viability assay. The data were presented as mean + SD. **, p < 0.01. (C) Colony formation assay of DLD-1 cells, treated or untreated with PP242 (1 μM) and/or erlotinib (2 μM), shows the colony densities in culture wells. (D) The colony numbers from the colony formation assay as described above in C were accounted and presented as mean + SD. NS, no significance; **, p < 0.01.
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pone-0073175-g003: The combination of P242 and erlotinib inhibits the carcinoma cell growth.(A) DLD-1 cells grown in EGF (50 ng/ml) containing medium were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and analyzed by western blotting for the indicated proteins (left). (B) DLD-1 and HT29 cells were treated with PP242 (1 μM) and erlotinib (2 μM), alone or in combination, and then subjected to cell viability assay. The data were presented as mean + SD. **, p < 0.01. (C) Colony formation assay of DLD-1 cells, treated or untreated with PP242 (1 μM) and/or erlotinib (2 μM), shows the colony densities in culture wells. (D) The colony numbers from the colony formation assay as described above in C were accounted and presented as mean + SD. NS, no significance; **, p < 0.01.

Mentions: The parallel increase in EGFRT1068 and AKTS473 suggests the possibility that the EGFR activation may contribute to the resume of mTORC2 activity in colorectal carcinoma cells under PP242 treatment. To test this notion, we treated DLD-1 cells with PP242 (1 μΜ), alone or in the combination with the EGFR inhibitor, erlotinib (2 μM). The erlotinib treatment blocked the phosphorylation of EGFR, temporally inhibited S6 phosphorylation but had no effect on 4E-BP1 phosphorylation (Figure 3A). In contrast, the combination treatment blocked the phosphorylation of EGFR (EGFRT1068) and mTORC1 substrates (S6S235/236; 4E-BP1T36/45); thus, EGFR activation contributes to the incomplete inhibition of mTORC1 by PP242 and in combination with the EGFR inhibitor erlotinib, PP242 can completely block the mTORC1 kinase activity. The treatment of PP242 (Figure 2B) and erlotinib (Figure 3A) alone did not affect the p-AKT; but the combination treatment ablated the p-AKT (Figure 3A). The data suggest that the inhibition of p-AKT is due to the synergistic effects; however, the mechanism remains to be elucidated.


Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

The combination of P242 and erlotinib inhibits the carcinoma cell growth.(A) DLD-1 cells grown in EGF (50 ng/ml) containing medium were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and analyzed by western blotting for the indicated proteins (left). (B) DLD-1 and HT29 cells were treated with PP242 (1 μM) and erlotinib (2 μM), alone or in combination, and then subjected to cell viability assay. The data were presented as mean + SD. **, p < 0.01. (C) Colony formation assay of DLD-1 cells, treated or untreated with PP242 (1 μM) and/or erlotinib (2 μM), shows the colony densities in culture wells. (D) The colony numbers from the colony formation assay as described above in C were accounted and presented as mean + SD. NS, no significance; **, p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750018&req=5

pone-0073175-g003: The combination of P242 and erlotinib inhibits the carcinoma cell growth.(A) DLD-1 cells grown in EGF (50 ng/ml) containing medium were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and analyzed by western blotting for the indicated proteins (left). (B) DLD-1 and HT29 cells were treated with PP242 (1 μM) and erlotinib (2 μM), alone or in combination, and then subjected to cell viability assay. The data were presented as mean + SD. **, p < 0.01. (C) Colony formation assay of DLD-1 cells, treated or untreated with PP242 (1 μM) and/or erlotinib (2 μM), shows the colony densities in culture wells. (D) The colony numbers from the colony formation assay as described above in C were accounted and presented as mean + SD. NS, no significance; **, p < 0.01.
Mentions: The parallel increase in EGFRT1068 and AKTS473 suggests the possibility that the EGFR activation may contribute to the resume of mTORC2 activity in colorectal carcinoma cells under PP242 treatment. To test this notion, we treated DLD-1 cells with PP242 (1 μΜ), alone or in the combination with the EGFR inhibitor, erlotinib (2 μM). The erlotinib treatment blocked the phosphorylation of EGFR, temporally inhibited S6 phosphorylation but had no effect on 4E-BP1 phosphorylation (Figure 3A). In contrast, the combination treatment blocked the phosphorylation of EGFR (EGFRT1068) and mTORC1 substrates (S6S235/236; 4E-BP1T36/45); thus, EGFR activation contributes to the incomplete inhibition of mTORC1 by PP242 and in combination with the EGFR inhibitor erlotinib, PP242 can completely block the mTORC1 kinase activity. The treatment of PP242 (Figure 2B) and erlotinib (Figure 3A) alone did not affect the p-AKT; but the combination treatment ablated the p-AKT (Figure 3A). The data suggest that the inhibition of p-AKT is due to the synergistic effects; however, the mechanism remains to be elucidated.

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

Show MeSH
Related in: MedlinePlus