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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 and erlotinib treatment suppresses xenograft progression in mice.(A) DLD-1 cells were injected subcutaneously in mice for xenograft formation and then treated with oral gavage of saline in control group of mice, PP242 in monotherapy, and PP242 plus erlotinib in combination treatment for the indicated days. The tumor volumes from the same group mice were grouped and presented as mean + SD. (B) At necropsy, the representative mice bearing subcutaneous xenografts were shown from the left to the right with PP242 and erlotinib combination, PP242 alone and saline. (C) The xenografts were dissected from mice and their weights were presented mean + SD. *, p < 0.05; **, p < 0.01. (D) The xenografts were lysed and subjected to western blotting for the presence of phosphorylated and unphosphorylated proteins in the xenograft tissues.
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pone-0073175-g005: PP242 and erlotinib treatment suppresses xenograft progression in mice.(A) DLD-1 cells were injected subcutaneously in mice for xenograft formation and then treated with oral gavage of saline in control group of mice, PP242 in monotherapy, and PP242 plus erlotinib in combination treatment for the indicated days. The tumor volumes from the same group mice were grouped and presented as mean + SD. (B) At necropsy, the representative mice bearing subcutaneous xenografts were shown from the left to the right with PP242 and erlotinib combination, PP242 alone and saline. (C) The xenografts were dissected from mice and their weights were presented mean + SD. *, p < 0.05; **, p < 0.01. (D) The xenografts were lysed and subjected to western blotting for the presence of phosphorylated and unphosphorylated proteins in the xenograft tissues.

Mentions: To evaluate therapeutic potential of the combination of PP242 and erlotinib in treating colorectal carcinoma, we generated subcutaneous xenografts by injecting subcutaneously DLD-1 cells in athymic (nu/nu) mice. Once the xenografts were formed at approximately 150-200 mm3 sizes, the mice were treated for 21 days through oral gavage of saline as control or PP242 (50mg/kg), alone or in combination with erlotinib (75 mg/kg). Our earlier work [55] and the data presented in Figure 2 have shown that erlotinib treatment alone has no effects on DLD-1 cell growth and, thus, we omitted erlotinib treatment alone and saved a group of mice. Tumor volumes were measured once every 3 days and mice were followed closely for 21 days. The results showed that the combination of PP242 and erlotinib significantly enhanced the therapeutic effects of PP242 treatment (Figure 5A). At necropsy, the significant difference in the tumor sizes was observed among the three groups of mice (Figure 5B). The tumors were removed and the tumor weights further indicated the significant synergy of the combination treatment (Figure 5C). Western blotting of fresh tumor tissues showed that the combination treatment abolished the mTORC1 substrate, S6S235/236 and the mTORC2 substrate AKTS473. Collectively, these results indicate that the combination treatment of PP242 and erlotinib completely blocks the mTORC1 and mTORC2 kinase activity, induce apoptosis in colorectal carcinoma cells and suppresses the carcinoma xenograft progression.


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 and erlotinib treatment suppresses xenograft progression in mice.(A) DLD-1 cells were injected subcutaneously in mice for xenograft formation and then treated with oral gavage of saline in control group of mice, PP242 in monotherapy, and PP242 plus erlotinib in combination treatment for the indicated days. The tumor volumes from the same group mice were grouped and presented as mean + SD. (B) At necropsy, the representative mice bearing subcutaneous xenografts were shown from the left to the right with PP242 and erlotinib combination, PP242 alone and saline. (C) The xenografts were dissected from mice and their weights were presented mean + SD. *, p < 0.05; **, p < 0.01. (D) The xenografts were lysed and subjected to western blotting for the presence of phosphorylated and unphosphorylated proteins in the xenograft tissues.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750018&req=5

pone-0073175-g005: PP242 and erlotinib treatment suppresses xenograft progression in mice.(A) DLD-1 cells were injected subcutaneously in mice for xenograft formation and then treated with oral gavage of saline in control group of mice, PP242 in monotherapy, and PP242 plus erlotinib in combination treatment for the indicated days. The tumor volumes from the same group mice were grouped and presented as mean + SD. (B) At necropsy, the representative mice bearing subcutaneous xenografts were shown from the left to the right with PP242 and erlotinib combination, PP242 alone and saline. (C) The xenografts were dissected from mice and their weights were presented mean + SD. *, p < 0.05; **, p < 0.01. (D) The xenografts were lysed and subjected to western blotting for the presence of phosphorylated and unphosphorylated proteins in the xenograft tissues.
Mentions: To evaluate therapeutic potential of the combination of PP242 and erlotinib in treating colorectal carcinoma, we generated subcutaneous xenografts by injecting subcutaneously DLD-1 cells in athymic (nu/nu) mice. Once the xenografts were formed at approximately 150-200 mm3 sizes, the mice were treated for 21 days through oral gavage of saline as control or PP242 (50mg/kg), alone or in combination with erlotinib (75 mg/kg). Our earlier work [55] and the data presented in Figure 2 have shown that erlotinib treatment alone has no effects on DLD-1 cell growth and, thus, we omitted erlotinib treatment alone and saved a group of mice. Tumor volumes were measured once every 3 days and mice were followed closely for 21 days. The results showed that the combination of PP242 and erlotinib significantly enhanced the therapeutic effects of PP242 treatment (Figure 5A). At necropsy, the significant difference in the tumor sizes was observed among the three groups of mice (Figure 5B). The tumors were removed and the tumor weights further indicated the significant synergy of the combination treatment (Figure 5C). Western blotting of fresh tumor tissues showed that the combination treatment abolished the mTORC1 substrate, S6S235/236 and the mTORC2 substrate AKTS473. Collectively, these results indicate that the combination treatment of PP242 and erlotinib completely blocks the mTORC1 and mTORC2 kinase activity, induce apoptosis in colorectal carcinoma cells and suppresses the carcinoma xenograft progression.

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