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Potentiation of Growth Inhibitory Responses of the mTOR Inhibitor Everolimus by Dual mTORC1/2 Inhibitors in Cultured Breast Cancer Cell Lines.

Leung EY, Askarian-Amiri M, Finlay GJ, Rewcastle GW, Baguley BC - PLoS ONE (2015)

Bottom Line: We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested.The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested.The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland, New Zealand; Department of Molecular Medicine and Pathology, University of Auckland, Grafton, Auckland, New Zealand.

ABSTRACT
The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

No MeSH data available.


Related in: MedlinePlus

Signaling pathway usage and growth inhibitory response of MDA-MB-231, MDA-MB-436, BT20 and HCC1143 exposed to different drugs.(A) IC50 values (50% inhibition of thymidine incorporation) are shown for BEZ235 (BEZ), GSK2126458 (GSK) and AZD8055 (AZD). Cells were treated with drugs for 3 days and cell proliferation was measured by the thymidine incorporation assay. Results are shown as the mean ± standard error from triplicate experiments. (B) Signaling pathway usage as measured by basal protein phosphorylation of AKT, p70S6K, rpS6 and 4E-BP1 in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Actin is the loading control.
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pone.0131400.g004: Signaling pathway usage and growth inhibitory response of MDA-MB-231, MDA-MB-436, BT20 and HCC1143 exposed to different drugs.(A) IC50 values (50% inhibition of thymidine incorporation) are shown for BEZ235 (BEZ), GSK2126458 (GSK) and AZD8055 (AZD). Cells were treated with drugs for 3 days and cell proliferation was measured by the thymidine incorporation assay. Results are shown as the mean ± standard error from triplicate experiments. (B) Signaling pathway usage as measured by basal protein phosphorylation of AKT, p70S6K, rpS6 and 4E-BP1 in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Actin is the loading control.

Mentions: We chose four everolimus resistant triple-negative breast cancer cell lines, MDA-MB-231, MDA-MB-436, BT20 and HCC1143, characterized by a diversity of activated signaling pathways and proliferation inhibition by ATP competitive inhibitors to mTOR. The cell lines were assayed for sensitivity to BEZ235, GSK2126458 and AZD8055, using a 3-day 3H-thymidine incorporation assay (Fig 4A). PI3K/AKT/mTOR pathway usage was analyzed by western blots (Fig 4B). MDA-MB-436 showed the highest AKT phosphorylation, relatively low phosphorylated rpS6, and had the lowest IC50 of the three inhibitors tested as compared to MDA-MB-231, BT20 and HCC1143.


Potentiation of Growth Inhibitory Responses of the mTOR Inhibitor Everolimus by Dual mTORC1/2 Inhibitors in Cultured Breast Cancer Cell Lines.

Leung EY, Askarian-Amiri M, Finlay GJ, Rewcastle GW, Baguley BC - PLoS ONE (2015)

Signaling pathway usage and growth inhibitory response of MDA-MB-231, MDA-MB-436, BT20 and HCC1143 exposed to different drugs.(A) IC50 values (50% inhibition of thymidine incorporation) are shown for BEZ235 (BEZ), GSK2126458 (GSK) and AZD8055 (AZD). Cells were treated with drugs for 3 days and cell proliferation was measured by the thymidine incorporation assay. Results are shown as the mean ± standard error from triplicate experiments. (B) Signaling pathway usage as measured by basal protein phosphorylation of AKT, p70S6K, rpS6 and 4E-BP1 in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Actin is the loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492962&req=5

pone.0131400.g004: Signaling pathway usage and growth inhibitory response of MDA-MB-231, MDA-MB-436, BT20 and HCC1143 exposed to different drugs.(A) IC50 values (50% inhibition of thymidine incorporation) are shown for BEZ235 (BEZ), GSK2126458 (GSK) and AZD8055 (AZD). Cells were treated with drugs for 3 days and cell proliferation was measured by the thymidine incorporation assay. Results are shown as the mean ± standard error from triplicate experiments. (B) Signaling pathway usage as measured by basal protein phosphorylation of AKT, p70S6K, rpS6 and 4E-BP1 in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Actin is the loading control.
Mentions: We chose four everolimus resistant triple-negative breast cancer cell lines, MDA-MB-231, MDA-MB-436, BT20 and HCC1143, characterized by a diversity of activated signaling pathways and proliferation inhibition by ATP competitive inhibitors to mTOR. The cell lines were assayed for sensitivity to BEZ235, GSK2126458 and AZD8055, using a 3-day 3H-thymidine incorporation assay (Fig 4A). PI3K/AKT/mTOR pathway usage was analyzed by western blots (Fig 4B). MDA-MB-436 showed the highest AKT phosphorylation, relatively low phosphorylated rpS6, and had the lowest IC50 of the three inhibitors tested as compared to MDA-MB-231, BT20 and HCC1143.

Bottom Line: We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested.The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested.The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland, New Zealand; Department of Molecular Medicine and Pathology, University of Auckland, Grafton, Auckland, New Zealand.

ABSTRACT
The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

No MeSH data available.


Related in: MedlinePlus