Combination of rapamycin and 17-allylamino-17-demethoxygeldanamycin (17-AAG) abrogates Akt activation and potentiates mTOR blockade in breast cancer cells


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Matthew Roforth, Chalet Tan

Mercer University, Atlanta, USA

Abstract

Introduction

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is therefore necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signalling.

Method

Human breast MCF-7 and MDA-MB-231 cells were treated with rapamycin and 17-AAG individually or in combination. Cell proliferation assay, cell cycle analysis and Western blotting were performed.

Results

Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. Akt activation in response to rapamycin is abrogated by 17-AAG. In combination 17-AAG confers potent suppression of Raf-MEK-ERK signalling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block mTOR phosphorylation at Ser2448, as well as mTORC1 downstream effectors S6K1 and 4E-BP1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signalling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy.

Conclusion

Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

Introduction

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is therefore necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signalling.

Method

Human breast MCF-7 and MDA-MB-231 cells were treated with rapamycin and 17-AAG individually or in combination. Cell proliferation assay, cell cycle analysis and Western blotting were performed.

Results

Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. Akt activation in response to rapamycin is abrogated by 17-AAG. In combination 17-AAG confers potent suppression of Raf-MEK-ERK signalling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block mTOR phosphorylation at Ser2448, as well as mTORC1 downstream effectors S6K1 and 4E-BP1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signalling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy.

Conclusion

Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

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