mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt

Abstract

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.

Figure 1

mTOR inhibition activates Akt in tumor cells. A, 1 nmol/L rapamycin treatment induced S473 Akt and S21/9 GSK3α/β phosphorylation in vitro in a DU-145 prostate cancer cell line. Akt1, Akt2, and Akt3, total Akt, and total GSK3α/β did not change. Phosphorylation of p70/S6K decreased with rapamycin treatment whereas total p70/S6K levels did not change. B, 1 nmol/L rapamycin treatment induced S473 Akt and S21/9 GSK3α/β phosphorylation in vitro in a MCF-7 cancer cell line. Akt1, Akt2, and Akt3, total Akt, and total GSK3α/β did not change. Phosphorylation of p70/S6K decreased with rapamycin treatment whereas total p70/S6K levels did not change. C, 1 nmol/L rapamycin treatment for 24 hours induced S473 Akt in the breast cancer cell lines BT474, SkBr3, and the rhabdomyosarcoma cell line Rh30. Total Akt levels did not change. D, mTOR inhibition with 1 nmol/L rapamycin induced Akt Kinase activity in an IGF-IR-dependent manner. An Akt kinase assay in DU-145 cells confirmed that the increased pAkt (S473) resulted in increased Akt activity, which was abrogated by the inhibition of PI3K with the small-molecule LY294002 (10 μmol/L) and by inhibition of IGF-IR with either the small-molecule NVP-AEW541 (800 nmol/L) or the monoclonal antibody A12 (10 nmol/L). A Western blot of pAkt (S473) at the same time points in DU-145 showed the increased Akt phosphorylation which was abrogated by IGF-IR or PI3K inhibition. Although there is no detectable increase in pan-phospho-Akt as measured by Western blot, an Akt kinase assay in MDA-MB-468 cells reveals an induction of Akt activity and p-Akt1 (S473) that is abrogated by LY294002 as well as the IGF-IR inhibitors. E, treatment of MCF-7 cells with rapamycin (1 nmol/L) induced phosphorylation of the FoxO transcription factors, FKHR (S256 and T24), AFX (S193), and FKHRL1 (T32). The rapamycin-induced FoxO phosphorylation was abrogated by IGF-IR inhibition with 800 nmol/L NVP-AEW541. Total FKHR, AFX, and FKHRL1 levels did not change with rapamycin or NVP-AEW-541 treatment.

Figure 1

mTOR inhibition activates Akt in tumor cells. A, 1 nmol/L rapamycin treatment induced S473 Akt and S21/9 GSK3α/β phosphorylation in vitro in a DU-145 prostate cancer cell line. Akt1, Akt2, and Akt3, total Akt, and total GSK3α/β did not change. Phosphorylation of p70/S6K decreased with rapamycin treatment whereas total p70/S6K levels did not change. B, 1 nmol/L rapamycin treatment induced S473 Akt and S21/9 GSK3α/β phosphorylation in vitro in a MCF-7 cancer cell line. Akt1, Akt2, and Akt3, total Akt, and total GSK3α/β did not change. Phosphorylation of p70/S6K decreased with rapamycin treatment whereas total p70/S6K levels did not change. C, 1 nmol/L rapamycin treatment for 24 hours induced S473 Akt in the breast cancer cell lines BT474, SkBr3, and the rhabdomyosarcoma cell line Rh30. Total Akt levels did not change. D, mTOR inhibition with 1 nmol/L rapamycin induced Akt Kinase activity in an IGF-IR-dependent manner. An Akt kinase assay in DU-145 cells confirmed that the increased pAkt (S473) resulted in increased Akt activity, which was abrogated by the inhibition of PI3K with the small-molecule LY294002 (10 μmol/L) and by inhibition of IGF-IR with either the small-molecule NVP-AEW541 (800 nmol/L) or the monoclonal antibody A12 (10 nmol/L). A Western blot of pAkt (S473) at the same time points in DU-145 showed the increased Akt phosphorylation which was abrogated by IGF-IR or PI3K inhibition. Although there is no detectable increase in pan-phospho-Akt as measured by Western blot, an Akt kinase assay in MDA-MB-468 cells reveals an induction of Akt activity and p-Akt1 (S473) that is abrogated by LY294002 as well as the IGF-IR inhibitors. E, treatment of MCF-7 cells with rapamycin (1 nmol/L) induced phosphorylation of the FoxO transcription factors, FKHR (S256 and T24), AFX (S193), and FKHRL1 (T32). The rapamycin-induced FoxO phosphorylation was abrogated by IGF-IR inhibition with 800 nmol/L NVP-AEW541. Total FKHR, AFX, and FKHRL1 levels did not change with rapamycin or NVP-AEW-541 treatment.

Figure 2

mTOR inhibition activates Akt in humans. A, liver metastasis of a colorectal carcinoma from a patient treated with a daily administration of RAD001 for 4 weeks. pAkt was expressed in nuclei and cytoplasm of tumor cells (1) and levels of expression increased after therapy (2). Skin infiltration by ductal carcinoma in a patient treated with a weekly administration of RAD001 for 4 weeks. A similar increment of pAkt was observed before (3) and after therapy (4); (3,3′-diaminobenzidine, pAkt ×400). B, evaluation of levels of pAkt in paired tumor samples from patients treated with daily and weekly administration of RAD001 by immunohistochemistry. A significant increment (P = 0.018) of this protein was observed in eight patients. C, tumor and biopsy characteristics. The characteristics of each tumor biopsy and the dose schedule of the eight patients in the RAD001 clinical trial are described.

Figure 3

IGF-I signaling mediates AKT activation induced by mTOR inhibition. A and B, the induction of pAkt (S473) by rapamycin was abrogated by the small-molecule inhibitor of IGF-IR, NVP-AEW541, and by the monoclonal antibody against IGF-IR, A12, in both the DU-145 prostate cancer cell line (A) and the MCF-7 breast cancer cell line (B). Total Akt levels did not change. C, 1 nmol/L rapamycin treatment up-regulates IRS-1 levels in MCF-7, DU-145, and MDA-MB-468 cell lines by 1 hour, and this induction persists for 24 hours. IRS-2 levels remain unchanged.

Figure 4

IGF-I prevents and IGF-IR inhibitors enhance the antiproliferative effects of rapamycin. A, IGF-I (60 ng/mL) rescued MCF-7 cells from rapamycin’s antiproliferative effects in serum-free medium and the induction of pAKT was greater in cells cotreated with rapamycin and IGF-I than in cells treated with either single agent. B, the combination of mTOR inhibition with IGF-IR inhibition resulted in enhanced antitumor effects. In vitro, combined mTOR and IGF-IR inhibition with rapamycin (1 nmol/L) and NVP-AEW541 (1 μmol/L) resulted in additive inhibition of proliferation in DU-145 cells, MCF-7 cells, and MDA-MB-468 cells as compared with either single agent. C, in the breast cancer cell line, MCF-7, and the prostate cancer cell line, DU-145, 2 days of combined mTOR and IGF-IR inhibition with rapamycin (1 nmol/L) and NVP-AEW541 (1 μmol/L) resulted in enhanced G₁ arrest. In the breast cancer cell line, MDA-MB-468, 2 days of combination treatment resulted in enhanced apoptosis compared with single agent treatment groups.

Figure 4

IGF-I prevents and IGF-IR inhibitors enhance the antiproliferative effects of rapamycin. A, IGF-I (60 ng/mL) rescued MCF-7 cells from rapamycin’s antiproliferative effects in serum-free medium and the induction of pAKT was greater in cells cotreated with rapamycin and IGF-I than in cells treated with either single agent. B, the combination of mTOR inhibition with IGF-IR inhibition resulted in enhanced antitumor effects. In vitro, combined mTOR and IGF-IR inhibition with rapamycin (1 nmol/L) and NVP-AEW541 (1 μmol/L) resulted in additive inhibition of proliferation in DU-145 cells, MCF-7 cells, and MDA-MB-468 cells as compared with either single agent. C, in the breast cancer cell line, MCF-7, and the prostate cancer cell line, DU-145, 2 days of combined mTOR and IGF-IR inhibition with rapamycin (1 nmol/L) and NVP-AEW541 (1 μmol/L) resulted in enhanced G₁ arrest. In the breast cancer cell line, MDA-MB-468, 2 days of combination treatment resulted in enhanced apoptosis compared with single agent treatment groups.