ShRNA kinome screen identifies TBK1 as therapeutic target for HER2+ breast cancer
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Liu Jeff
PROVIDER: S-ECPF-GEOD-53658 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Microarray analysis was performed at the UHN Microarray Centre (UHNMAC, Ontario, Canada) using Illumina HumanHT-12 v4 BeadChip with 500 ng of total RNA prepared by RNeasy mini kit (QIAGEN, Cat. No. 74104). Samples from HCC1954 cells with 3-day treatment of TBK1-II at 4 uM were used to compare with vehicle-treated controls. Microarray data was processed and normalized by lumi package from BioConductor in R with Quantile Method. Difference between the samples were calculated by Bayesian statistic using limma package from BioConductor in R to obtain Moderated T value for subsequent Pathway analysis. Total RNA extracted from HER2+ HCC1954 cells after 3 days treatment of TBK1-II inhibitor compared with vehicle control
2013-12-26 | E-GEOD-53658 | biostudies-arrayexpress
Project description:Microarray analysis was performed at the UHN Microarray Centre (UHNMAC, Ontario, Canada) using Illumina HumanHT-12 v4 BeadChip with 500 ng of total RNA prepared by RNeasy mini kit (QIAGEN, Cat. No. 74104). Samples from HCC1954 cells with 3-day treatment of TBK1-II at 4 uM were used to compare with vehicle-treated controls. Microarray data was processed and normalized by lumi package from BioConductor in R with Quantile Method. Difference between the samples were calculated by Bayesian statistic using limma package from BioConductor in R to obtain Moderated T value for subsequent Pathway analysis.
2013-12-26 | GSE53658 | GEO
Project description:MicroRNAs (miRNAs) are implicated in numerous physiologic and pathologic processes, such as the development of resistance to chemotherapy. Determining the role of miRNAs in these processes is often accomplished through measuring miRNA abundance by polymerase chain reaction, sequencing, or microarrays. We have developed a system for the large-scale monitoring of dynamic miRNA activity and have applied this system to identify the contribution miRNA activity to the development of trastuzumab resistance in a cell model of HER2+ breast cancer. MiRNA activity measurements identified significantly different activity levels between BT474 cells (HER2 + breast cancer) and BT474R cells (HER2 + breast cancer cells selected for resistance to trastuzumab). We created a library of 32 miRNA reporter constructs, which were delivered by lentiviral transduction into cells, and miRNA activity was quantified by bioluminescence imaging. Upon treatment with the bioimmune therapy, trastuzumab, the activity of 11 miRNAs were significantly altered in parental BT474 cells, and 20 miRNAs had significantly altered activity in the therapy-resistant BT474R cell line. A combination of statistical, network and classification analysis was applied to the dynamic data, which identified miR-21 as a controlling factor in trastuzumab response. Our data suggested downregulation of miR-21 activity was associated with resistance, which was confirmed in an additional HER2 + breast cancer cell line, SKBR3. Collectively, the dynamic miRNA activity measurements and analysis provided a system to identify new potential therapeutic targets in treatment-resistant cancers.
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Project description:Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.
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Project description:IntroductionThe human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in breast cancer. Heterodimerization of HER2 with other HER family members results in enhanced tyrosine phosphorylation and activation of signal transduction pathways. HER2 overexpression increases the translation of fatty acid synthase (FASN), and FASN overexpression markedly increases HER2 signaling, which results in enhanced cell growth. However, the molecular mechanism and regulation of HER2 and FASN interaction are not well defined. Lapatinib is a small-molecule tyrosine kinase inhibitor that blocks phosphorylation of the epidermal growth factor receptor and HER2 in breast cancer cells, resulting in apoptosis. We hypothesized that FASN is directly phosphorylated by HER2, resulting in enhanced signaling and tumor progression in breast cancer cells.MethodsUsing mass spectrometry, we identified FASN as one of the proteins that is dephosphorylated by lapatinib in SKBR3 breast cancer cells. Immunofluorescence, immunoprecipitation, Western blotting, a kinase assay, a FASN enzymatic activity assay, an invasion assay, a cell viability assay and zymography were used to determine the role of FASN phosphorylation in invasion of SKBR3 and BT474 cells. The FASN inhibitor C75 and small interfering RNA were used to downregulate FASN expression and/or activity.ResultsOur data demonstrated that FASN is phosphorylated when it is in complex with HER2. FASN phosphorylation was induced by heregulin in HER2-overexpressing SKBR3 and BT474 breast cancer cells. Heregulin-induced FASN phosphorylation resulted in increased FASN enzymatic activity, which was inhibited by lapatinib. The FASN inhibitor C75 suppressed FASN activity by directly inhibiting HER2 and FASN phosphorylation. Blocking FASN phosphorylation and activity by lapatinib or C75 suppressed the activity of matrix metallopeptidase 9 and inhibited invasion of SKBR3 and BT474 cells.ConclusionsFASN phosphorylation by HER2 plays an important role in breast cancer progression and may be a novel therapeutic target in HER2-overexpressing breast cancer cells.
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