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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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PP242 transiently inhibits mTORC2 activity in colorectal carcinoma cells.(A) Western blot analysis for the presence of phosphorylated (p-) and unphosphorylated AKT and S6 in matched colorectal carcinoma tumor (T) and adjacent normal colorectal tissues (N). β-actin was used as protein loading control. The numbers label the cases. (B) The colorectal carcinoma cell lines as indicated were treated with PP242 in the indicated concentrations and then subjected to cell viability assay. The experiment was repeated three times and the data presented as mean + SD (standard deviation). (C) Each of the cell lines as indicated to the top of the panel were untreated or treated with PP242 (1 μM) for the indicated hours and then examined by western blotting using antibodies against the phosphorylated (p-) and unphosphorylated proteins as indicated to the left of the panel.
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pone-0073175-g001: PP242 transiently inhibits mTORC2 activity in colorectal carcinoma cells.(A) Western blot analysis for the presence of phosphorylated (p-) and unphosphorylated AKT and S6 in matched colorectal carcinoma tumor (T) and adjacent normal colorectal tissues (N). β-actin was used as protein loading control. The numbers label the cases. (B) The colorectal carcinoma cell lines as indicated were treated with PP242 in the indicated concentrations and then subjected to cell viability assay. The experiment was repeated three times and the data presented as mean + SD (standard deviation). (C) Each of the cell lines as indicated to the top of the panel were untreated or treated with PP242 (1 μM) for the indicated hours and then examined by western blotting using antibodies against the phosphorylated (p-) and unphosphorylated proteins as indicated to the left of the panel.

Mentions: The expression of mTOR, RACTOR, RICTOR, p70S6K and 4E-BP1 is elevated in colorectal carcinoma [44,45]. Because mTORC2 phosphorylates AKT at S473 (AKTS473) [9–11], we thought to identify the AKTS473 and determine whether the mTORC2 are activated in the carcinoma. Western blotting revealed that AKTS473 were elevated in the carcinoma tissues as compared with the matched normal tissues (Figure 1A), thus suggesting the activation of mTORC2 in the carcinomas; however, non-phosphorylated AKT was also elevated, which could not rule out the possibility that the overexpression might cause the phosphorylation in the cancer tissues. During the activation of mTORC1, its substrate p70S6K phosphorylates ribosomal protein S6 [7]. The expression of S6 protein was consistent in matched carcinoma and normal tissues, but detection of the phosphorylated S6 at S235/236 (S6S235/236) in the carcinoma but not matched normal tissues suggested the elevated mTORC1 activity in the carcinomas (Figure 1A).


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)

PP242 transiently inhibits mTORC2 activity in colorectal carcinoma cells.(A) Western blot analysis for the presence of phosphorylated (p-) and unphosphorylated AKT and S6 in matched colorectal carcinoma tumor (T) and adjacent normal colorectal tissues (N). β-actin was used as protein loading control. The numbers label the cases. (B) The colorectal carcinoma cell lines as indicated were treated with PP242 in the indicated concentrations and then subjected to cell viability assay. The experiment was repeated three times and the data presented as mean + SD (standard deviation). (C) Each of the cell lines as indicated to the top of the panel were untreated or treated with PP242 (1 μM) for the indicated hours and then examined by western blotting using antibodies against the phosphorylated (p-) and unphosphorylated proteins as indicated to the left of the panel.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073175-g001: PP242 transiently inhibits mTORC2 activity in colorectal carcinoma cells.(A) Western blot analysis for the presence of phosphorylated (p-) and unphosphorylated AKT and S6 in matched colorectal carcinoma tumor (T) and adjacent normal colorectal tissues (N). β-actin was used as protein loading control. The numbers label the cases. (B) The colorectal carcinoma cell lines as indicated were treated with PP242 in the indicated concentrations and then subjected to cell viability assay. The experiment was repeated three times and the data presented as mean + SD (standard deviation). (C) Each of the cell lines as indicated to the top of the panel were untreated or treated with PP242 (1 μM) for the indicated hours and then examined by western blotting using antibodies against the phosphorylated (p-) and unphosphorylated proteins as indicated to the left of the panel.
Mentions: The expression of mTOR, RACTOR, RICTOR, p70S6K and 4E-BP1 is elevated in colorectal carcinoma [44,45]. Because mTORC2 phosphorylates AKT at S473 (AKTS473) [9–11], we thought to identify the AKTS473 and determine whether the mTORC2 are activated in the carcinoma. Western blotting revealed that AKTS473 were elevated in the carcinoma tissues as compared with the matched normal tissues (Figure 1A), thus suggesting the activation of mTORC2 in the carcinomas; however, non-phosphorylated AKT was also elevated, which could not rule out the possibility that the overexpression might cause the phosphorylation in the cancer tissues. During the activation of mTORC1, its substrate p70S6K phosphorylates ribosomal protein S6 [7]. The expression of S6 protein was consistent in matched carcinoma and normal tissues, but detection of the phosphorylated S6 at S235/236 (S6S235/236) in the carcinoma but not matched normal tissues suggested the elevated mTORC1 activity in the carcinomas (Figure 1A).

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