The Y-box binding protein-1 (YB-1) transcription factor is associated with unfavorable clinical outcomes. However, the mechanisms underlying this association remain to be fully elucidated. We demonstrate that YB-1 phosphorylation, indicative of YB-1 activation, is a powerful marker of outcomes for ovarian cancer patients. In ovarian cancer, YB-1 phosphorylation is induced by activation of the lysophosphatidic acid (LPA) receptor (LPAR) via SRC-dependent transactivation of the epidermal growth factor receptor (EGFR) that is coupled to MAPK/p90 ribosomal S6 kinase (p90RSK), but not phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Activation of the LPAR/SRC/EGFR/MAPK/p90RSK/YB-1 axis leads to production of the EGFR ligand amphiregulin (AREG). AREG induces ongoing YB-1 phosphorylation as well as YB-1-dependent AREG expression thus constituting an AREG/YB-1 self-reinforcing loop. Disruption of transactivation of the EGFR and the downstream self-reinforcing loop decreases invasiveness of ovarian cancer cells in vitro and limits ovarian cancer growth in xenograft models. These findings established the regulation and significance of YB-1 phosphorylation, therefore further exploration of this signaling axis as a therapeutic avenue in ovarian cancer is warranted.
Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with no known molecular targets for treatment. Although erythema is commonly associated with IBC, the molecular mechanism of inflammation in the pathogenesis of IBC remains unknown. We have previously shown that EGFR is an emerging target in IBC (Zhang D. et al Clin Can Res 2009). As crosstalk between EGFR and COX-2 plays an important role in the inflammatory response in several cancers, including breast cancer, we hypothesized that COX-2 promotes the tumorigenesis and metastasis of IBC cells. Methods: Using clinically derived IBC and non-IBC tumor samples, a Spearman's Rank correlation coefficient analysis was performed to analyze the expression levels of COX-2 and EGFR in IBC and non-IBC. The levels of COX-2 metabolites, prostaglandins (PGs) PGE2 and PGF2α, were measured in IBC and non-IBC cell lines by HPLC/MS method. Cell migration and invasion assays were performed using SUM149 and KPL-4 IBC cell lines treated with PGs or the COX-2 inhibitor, celecoxib. We evaluated the epithelial to mesenchymal transition (EMT)-like phenotype in 3D culture of SUM149 cells treated with celecoxib, and the stem-like population by mammosphere formation, and CD44+/CD24− and aldefluor+ population by FACS. We treated preclinical IBC xenograft mice with celecoxib and measured tumor growth, PGs levels, and the expression of EMT protein markers. Nodal, a stem cell regulator and potential biomarker for breast cancer progression, was evaluated in IBC cells following treatment with celecoxib and recombinant Nodal or transfection with Nodal cDNA. Results: EGFR and COX-2 expression levels positively correlated within IBC, but not non-IBC tumors. Elevated levels of PGE2 and PGF2α were identified in multiple IBC cell lines suggesting that COX activity is elevated within IBC compared to non-IBC cells. PGs altered EMT protein markers and promoted cell migration and invasion, while Celecoxib inhibited EMT and migration and invasion in SUM149 and KPL-4 cells. Celecoxib treatment inhibited tumor growth in mice, and downregulated the expression of EMT protein markers, including Nodal. Celecoxib decreased the stem-like CD44+/CD24−, and aldefluor+ population and the formation of mammospheres. Exogenous Nodal mitigated the effects of celecoxib on cell migration and invasion and the stem-like population in SUM149 cells. Conclusion: We conclude that activation of the COX-2 inflammatory signaling pathway is critical in the development and progression of IBC. This study provides a novel insight into how inflammation may regulate cancer stem cells via Nodal, and will guide future research into the development of stem cell targeted therapies for IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-11.
Background: Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) is a powerful method commonly used to study global protein-DNA interactions including both transcription factors and histone modifications. We have found that the choice of ChIP-Seq library preparation protocol plays an important role in overall ChIP-Seq data quality. However, very few studies have compared ChIP-Seq libraries prepared by different protocols using multiple targets and a broad range of input DNA levels. Results: In this study, we evaluated the performance of 4 ChIP-Seq library preparation protocols (New England Biolabs [NEB] NEBNext Ultra II, Roche KAPA HyperPrep, Diagenode MicroPlex, and Bioo [now PerkinElmer] NEXTflex) on 3 target proteins, chosen to represent the 3 typical signal enrichment patterns in ChIP-Seq experiments:sharp peaks (H3K4me3), broad domains (H3K27me3), and punctate peaks with a protein binding motif (CTCF). We also tested a broad range of different input DNA levels from 0.10 to 10 ng for H3K4me3 and H3K27me3 experiments. Conclusions: Our results suggest that the NEB protocol may be better for preparing H3K4me3 (and potentially other histone modifications with sharp peak enrichment) libraries; the Bioo protocol may be better for preparing H3K27me3 (and potentially other histone modifications with broad domain enrichment) libraries, and the Diagenode protocol may be better for preparing CTCF (and potentially other transcription factors with well-defined binding motifs) libraries. For ChIP-Seq experiments using novel targets without a known signal enrichment pattern, the NEB protocol might be the best choice, as it performed well for each of the 3 targets we tested across a wide array of input DNA levels.
BackgroundChIP-Seq is a powerful method commonly used to study global protein-DNA interactions including both transcription factors and histone modifications. We have found that the choice of ChIP-Seq library preparation protocol plays an important role in overall ChIP-Seq data quality. However, very few studies have compared ChIP-Seq libraries prepared by different protocols using multiple targets and a broad range of input DNA levels.ResultsIn this study, we evaluated the performance of four ChIP-Seq library preparation protocols [NEB NEBNext Ultra II, Roche KAPA HyperPrep, Diagenode MicroPlex, and Bioo (now PerkinElmer) NEXTflex] on three target proteins, chosen to represent the three typical signal enrichment patterns in ChIP-Seq experiments: sharp peaks (H3K4me3), broad domains (H3K27me3) and punctate peaks with a protein binding motif (CTCF). We also tested a broad range of different input DNA levels from 0.10 to 10 ng for H3K4me3 and H3K27me3 experiments.ConclusionsOur results suggest that the NEB protocol may be better for preparing H3K4me3 (and potentially other histone modifications with sharp peak enrichment) libraries; the Bioo protocol may be better for preparing H3K27me3 (and potentially other histone modifications with broad domain enrichment) libraries, and the Diagenode protocol may be better for preparing CTCF (and potentially other transcription factors with well-defined binding motifs) libraries. For ChIP-Seq experiments using novel targets without a known signal enrichment pattern, the NEB protocol might be the best choice as it performed well for each of the three targets we tested across a wide array of input DNA levels.
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