Illumina HiSeq 2000 sequencing; GSM2048425: WT_Tumor_11_5; Homo sapiens; RNA-Seq

WT_Tumor_11_5

Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1± protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a novel role for CSL in tumorigenesis and regulation of the cellular hypoxic response. Overall design: CSL +/+ and CSL -/- MDA-MB-231 were subjected to Notch activation/inhibition and xenograft experiment. Total RNA were extracted from the samples and sent to NGS. Single Cell RNA-sequencing was also performed from cells isolated from xenograft tumors.

Loss of CSL unlocks a hypoxic response and enhanced tumor growth potential in breast cancer cells

Submitted by Gene Expression Omnibus on 29-FEB-2016

Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1Â± protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a novel role for CSL in tumorigenesis and regulation of the cellular hypoxic response. Overall design: CSL +/+ and CSL -/- MDA-MB-231 were subjected to Notch activation/inhibition and xenograft experiment. Total RNA were extracted from the samples and sent to NGS. Single Cell RNA-sequencing was also performed from cells isolated from xenograft tumors.

Tumors with surrounding stromal tissues from xenografts were minced and nonenzymaticallydigested using cell dissociation enzyme-free PBS-based (Gibco) for 30 minutes at 37o C withoccasional pipetting. The dissociated cells were then filtered through a 40 mm nylon mesh strainer. Single cellsuspension was obtained by re-suspension in CO2 -independent medium (Gibco) for single cell picking. Singlecells of xenografts were manually picked under the microscope as previously described (Picelli et al., 2013).Briefly, a single cell was picked using mouth pipette under 20X magnification in 0.5 ml solution. The pickedsingle cell was released into RNase-free PCR strip tube containing hypotonic Smart-seq RNA lysis bufferconsisting of 2.3 ml of 19:1 ratio of 0.4% TritonX-100 and RNase inhibitor (Clontech), 1 ml of 10 mM dNTP(Fermentas), 1 ml of 10 mM oligo-dT primer, and ERCC spike-in (Ambion) at 72o C for 3 min. Total RNA from tumors and cells was purified using the RNeasy Mini  Kit(QIAGEN), according to the manufacturer’s instructions. RNA concentration and RNA integrity number (RIN)were calculated by using the Agilent RNA 6000 Nano  Kit (Agilent Technologies). Two biological replicates for each RNA sample were processed to cDNA library preparations using the Illumina TruSeq™ RNA sample preparation  kit (Low-Throughput protocol) according to the manufacturer’s protocol. Quantification and qualitycontrol of the cDNA libraries were done using the Agilent DNA 1000 Kit (Agilent Technologies). The libraries were sequenced on an Illumina HiSeq 2000 system.

Loss of CSL unlocks a hypoxic response and enhanced tumor growth potential in breast cancer cells

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