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Gene expression profile in response to doxorubicin-rapamycin combined treatment of HER-2 overexpressing human mammary epithelial cell lines

ABSTRACT: HER-2 positive breast cancers frequently sustain elevated AKT/mammalian target rapamycin (mTOR) signaling which has been associated with resistance to doxorubicin treatment in the clinic. In our study we investigated if the mTOR inhibitor rapamycin increased the sensitivity to doxorubicin therapy in HB4a, a luminal normal mammary cell line; C5.2, a transformed cell derived from HB4a transfected with HER-2 and SKBR3 that exhibits HER-2 amplification. Flow cytometry analysis showed that the combination treatment for 24 hours with rapamycin 20nM and doxorubicin caused accumulation of HB4a and C5.2 cells in S-G2/M. Otherwise in SKBR3 cells, we observed a relative depletion of cells in S-G2/M and concomitant accumulation in G0/G1 of 10% of the cells. The analysis of IC50 of doxorubicin alone and in combination with rapamycin indicated that the sensitivity was increased 2.37 fold in HB4a, 2.46 in C5.2 and 1.87 in SKBR3, suggesting that rapamycin might have enhanced the effects of doxorubicin. Changes in gene expression resulting from co-treatment demonstrated that functional groups of genes with roles in cell cycle, proliferation, apoptosis regulation were represented in the 3 cells analysed. Other biological functions were exclusively associated with each cell suggesting that the inhibition of mTOR activation induced by HER-2 is complex and depends on the cellular context.

ORGANISM(S): Homo sapiens

PROVIDER: GSE26599 | GEO | 2011/01/13

SECONDARY ACCESSION(S): PRJNA136201

REPOSITORIES: GEO

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Project description:The mTOR (mammalian Target of Rapamycin) pathway is constitutively activated in Diffuse Large B-Cell Lymphoma (DLBCL). mTOR inhibition has been shown to have clinical activity in patients with DLBCL, although overall response rates remain low. We therefore evaluated differences in the transcriptome between DLBCL cell lines with differential sensitivity to the mTOR inhibitor Rapamycin, to (A) identify gene-expression patterns(GEP) capable of identifying sensitivity to Rapamycin, (B) understand the underlying mechanisms of resistance to Rapamycin in DLBCL and (C) identify bioactive molecules likely to synergize with mTOR inhibitors. Using Affymetrix HuGene ST 1.0 microarrays, we were able to identify a gene expression signature capable of accurately predicting sensitivity and resistance to Rapamycin in DLBCL cell lines. Pathway analysis identified the serine/threonine kinase Akt as central to the differentially-expressed gene network. Connectivity mapping of our datasets identified compounds targeting the AKT pathway with a high likelihood of reversing the GEP associated with resistance to Rapamycin. Specifically, we evaluated the HIV protease inhibitor (PI) Nelfinavir, which is known to have anti-cancer and Akt-inhibitory properties, as well as the small molecule Akt inhibitor MK-2206, for their potential to synergize with to Rapamycin in DLBCL. Nelfinavir and MK-2206 caused profound inhibition of cell viability in combination with Rapamycin in DLBCL cell lines. Low nanomolar concentrations of Rapamycin inhibited phosphorylation of Akt and also downstream targets of activated mTOR when used in combination with these Akt inhibitors. These findings have the potential to significantly improve patient selection for mTOR inhibitor therapy, and to improve rates and depths of response. More broadly, they support the use of global RNA expression and connectivity mapping to improve patient selection and identify synergistic drug combinations for cancer therapy. DLBCL cell lines were tested for Rapamycin sensitivity and classified as "sensitive" or "resistant." Genome-wide analysis of all cell lines were performed using the Affymetrix HuGene ST 1.0 Array Platform. Genes with differential expression between sensitive and resistant cell lines were analyzed using Statistical Analysis of Microarrays (SAM) software, and a signature of genes determnined. This signature was found to accurately predict sensitivity or resistance of other DLBCL cell lines, and to identify the protein kinase Akt as central to resistance.

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Gene expression profile in response to doxorubicin-rapamycin combined treatment of HER-2 overexpressing human mammary epithelial cell lines

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