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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

Comparison of viability assay using Alamar Blue and proliferation assay using thymidine incorporation, and the cell cycle changes in drug treatment.The inhibitory effects of drug combinations on MDA-MB-231, MDA-MB-436, BT20 and HCC1143 breast cancer cell lines were compared using (A) H3-thymidine incorporation assay and (B) Alamar Blue viability assay. BEZ, BEZ235 (10nM); GSK, GSK2126458 (2.5nM); AZD, AZD8055 (10nM); EVL, everolimus (100nM). Averages of three independent experiments are shown. (C) Scatter plot showing the relative thymidine incorporation (x-axis) and Alamar Blue fluorescence (y-axis). Significant correlation (Pearson Correlation, R = 0.96; Linear Regression, P = 1.54 x 10−18) was found between the thymidine uptake assay and viability assay in MDA-MB-231 (blue), MDA-MB-436 (red), BT20 (yellow); HCC1143 (green). Averages of two independent experiments are shown. (D) Cell cycle analysis of treated MDA-MB-231 cells. Cells were treated with DMSO control, 100 nM everolimus (EVL), 100nM BEZ235 (BEZ), everolimus and BEZ235 (EVL BEZ) combination. Cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population.
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pone.0131400.g007: Comparison of viability assay using Alamar Blue and proliferation assay using thymidine incorporation, and the cell cycle changes in drug treatment.The inhibitory effects of drug combinations on MDA-MB-231, MDA-MB-436, BT20 and HCC1143 breast cancer cell lines were compared using (A) H3-thymidine incorporation assay and (B) Alamar Blue viability assay. BEZ, BEZ235 (10nM); GSK, GSK2126458 (2.5nM); AZD, AZD8055 (10nM); EVL, everolimus (100nM). Averages of three independent experiments are shown. (C) Scatter plot showing the relative thymidine incorporation (x-axis) and Alamar Blue fluorescence (y-axis). Significant correlation (Pearson Correlation, R = 0.96; Linear Regression, P = 1.54 x 10−18) was found between the thymidine uptake assay and viability assay in MDA-MB-231 (blue), MDA-MB-436 (red), BT20 (yellow); HCC1143 (green). Averages of two independent experiments are shown. (D) Cell cycle analysis of treated MDA-MB-231 cells. Cells were treated with DMSO control, 100 nM everolimus (EVL), 100nM BEZ235 (BEZ), everolimus and BEZ235 (EVL BEZ) combination. Cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population.

Mentions: We next assessed drug effects using Alamar Blue (to assess cell viability) and compared them with the results of the thymidine incorporation assay (which measures DNA synthesis) (Fig 7A and 7B). Viability assays in the four cell lines treated by single drug alone or combination using everolimus with BEZ235, GSK2126458 and AZD8055, respectively showed an excellent correlation (r = 0.96; P = 1.54 x 10−18) (Fig 7C).


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)

Comparison of viability assay using Alamar Blue and proliferation assay using thymidine incorporation, and the cell cycle changes in drug treatment.The inhibitory effects of drug combinations on MDA-MB-231, MDA-MB-436, BT20 and HCC1143 breast cancer cell lines were compared using (A) H3-thymidine incorporation assay and (B) Alamar Blue viability assay. BEZ, BEZ235 (10nM); GSK, GSK2126458 (2.5nM); AZD, AZD8055 (10nM); EVL, everolimus (100nM). Averages of three independent experiments are shown. (C) Scatter plot showing the relative thymidine incorporation (x-axis) and Alamar Blue fluorescence (y-axis). Significant correlation (Pearson Correlation, R = 0.96; Linear Regression, P = 1.54 x 10−18) was found between the thymidine uptake assay and viability assay in MDA-MB-231 (blue), MDA-MB-436 (red), BT20 (yellow); HCC1143 (green). Averages of two independent experiments are shown. (D) Cell cycle analysis of treated MDA-MB-231 cells. Cells were treated with DMSO control, 100 nM everolimus (EVL), 100nM BEZ235 (BEZ), everolimus and BEZ235 (EVL BEZ) combination. Cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131400.g007: Comparison of viability assay using Alamar Blue and proliferation assay using thymidine incorporation, and the cell cycle changes in drug treatment.The inhibitory effects of drug combinations on MDA-MB-231, MDA-MB-436, BT20 and HCC1143 breast cancer cell lines were compared using (A) H3-thymidine incorporation assay and (B) Alamar Blue viability assay. BEZ, BEZ235 (10nM); GSK, GSK2126458 (2.5nM); AZD, AZD8055 (10nM); EVL, everolimus (100nM). Averages of three independent experiments are shown. (C) Scatter plot showing the relative thymidine incorporation (x-axis) and Alamar Blue fluorescence (y-axis). Significant correlation (Pearson Correlation, R = 0.96; Linear Regression, P = 1.54 x 10−18) was found between the thymidine uptake assay and viability assay in MDA-MB-231 (blue), MDA-MB-436 (red), BT20 (yellow); HCC1143 (green). Averages of two independent experiments are shown. (D) Cell cycle analysis of treated MDA-MB-231 cells. Cells were treated with DMSO control, 100 nM everolimus (EVL), 100nM BEZ235 (BEZ), everolimus and BEZ235 (EVL BEZ) combination. Cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population.
Mentions: We next assessed drug effects using Alamar Blue (to assess cell viability) and compared them with the results of the thymidine incorporation assay (which measures DNA synthesis) (Fig 7A and 7B). Viability assays in the four cell lines treated by single drug alone or combination using everolimus with BEZ235, GSK2126458 and AZD8055, respectively showed an excellent correlation (r = 0.96; P = 1.54 x 10−18) (Fig 7C).

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