Project description:Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process of effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results: Colony formation and sulforhodamine B (IC50 < 1nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI was significantly associated with tumor size or lymph node status. High (>75) percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.01) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In Wang dataset, RMI predicted time to disease relapse (P = 0.09). Conclusions: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. Mol Cancer. 2009 Sep 24;8(1):75. Experiment Overall Design: Rapamycin treatment of MDA-MB-468 breast cancer cell line: Experiment Overall Design: MDA-MB-468 cell line was treated by DMSO (vehicle) and 100 nM rapamycin for 24 hours. We sought to identify differentially expressed genes. Experiment Overall Design: Rapamycin treatment of breast tumor xenografts: Experiment Overall Design: MDA-MB-468 cells were inoculated in the mammary fat pad of female nude mice. When resulting tumor volumes had reached 75-150 mm3, the mice were divided in four groups. Groups 1 and 2 received a single injection of DMSO (vehicle) or rapamycin (15 mg/kg) intraperitoneally and sacrificied 24 h later (1-day groups). Groups 3 and 4 received weekly injections of DMSO or rapamycin for 3 weeks and sacrificied 24 h after the last injection (22-day groups).

Project description:BackgroundMutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action.MethodsMDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin.ResultsWe show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells.ConclusionThe current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.

Project description:Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process of effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results: Colony formation and sulforhodamine B (IC50 < 1nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI was significantly associated with tumor size or lymph node status. High (>75) percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.01) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In Wang dataset, RMI predicted time to disease relapse (P = 0.09). Conclusions: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. Mol Cancer. 2009 Sep 24;8(1):75. Experiment Overall Design: Rapamycin treatment of MDA-MB-468 breast cancer cell line: Experiment Overall Design: MDA-MB-468 cell line was treated by DMSO (vehicle) and 100 nM rapamycin for 24 hours. We sought to identify differentially expressed genes. Experiment Overall Design: Rapamycin treatment of breast tumor xenografts: Experiment Overall Design: MDA-MB-468 cells were inoculated in the mammary fat pad of female nude mice. When resulting tumor volumes had reached 75-150 mm3, the mice were divided in four groups. Groups 1 and 2 received a single injection of DMSO (vehicle) or rapamycin (15 mg/kg) intraperitoneally and sacrificied 24 h later (1-day groups). Groups 3 and 4 received weekly injections of DMSO or rapamycin for 3 weeks and sacrificied 24 h after the last injection (22-day groups).

Dataset Information

?-catenin knockdown enhances the effects of fluorouracil in the breast cancer cell line MDA-MB-468.

Publications

<i>β-catenin</i> knockdown enhances the effects of fluorouracil in the breast cancer cell line MDA-MB-468.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets