Hyperactivation of Notch signaling and the cellular hypoxic response are frequently observed in cancers, with increasing reports of connections to tumor initiation and progression. The two signaling mechanisms are known to intersect, but while it is well established that hypoxia regulates Notch signaling, less is known about whether Notch can regulate the cellular hypoxic response. We now report that Notch signaling specifically controls expression of HIF2a, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2a by Notch under normoxic conditions leads to elevated HIF2a protein levels in primary breast cancer cells as well as in human breast cancer, medulloblastoma and renal cell carcinoma cell lines. The elevated level of HIF2a protein was in certain tumor cell types accompanied by down-regulation of HIF1a protein levels, indicating that high Notch signaling may drive a HIF1a-to-HIF2a switch. At the transcriptome level, the presence of HIF2a was required for approximately 21% of all Notch-induced genes: among the 1062 genes that were upregulated by Notch in medulloblastoma cells during normoxia, upregulation was abrogated in 227 genes when HIF2a expression was knocked down by HIF2a siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2a, which may be important for future cancer therapies. Overall design: DAOY-NERT2 cells, +/- Notch induction by Tamoxifen (TMX) for 48 hours, +/- hypoxia (1% O2) treatment for 48 hours, where HIF1a or HIF2a had been knocked down by siRNA, were subjected to RNA sequencing. The quality of the cDNA libraries was tested on an Agilent 2100 bioanalyzer. The libraries were sequenced on an Illumina HiSeq 2000 system, and the reads were aligned to the human genome (assembly hg19) and a transcriptome database (RefSeq and Ensembl) using bowtie. RPKM values were generated using rpkmforgenes.
Notch signaling promotes a HIF2α-driven hypoxic response in multiple tumor cell types.
Specimen part, SubjectView Samples
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.
Loss of CSL Unlocks a Hypoxic Response and Enhanced Tumor Growth Potential in Breast Cancer Cells.
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Brain tumors are among the most malignant cancers and can arise from neural stem cells or oligodendrocyte progenitor cells (OPCs). Glioma-propagating cells (GPCs) that have stem-like properties have been derived from tumor variants such as glioblastoma multiforme (GBM) and oligodendroglial tumors, the latter being more chemosensitive with better prognosis. It has been suggested that such differences in chemosensitivity arise from the different profiles of OPCs versus neural stem cells. We thus explored if GPCs derived from these glioma variants can serve as reliable in vitro culture systems for studies. We utilized gene expression analyses, since GBM and oligodendrogliomas can be molecularly classified. Accordingly, we derived a gene signature distinguishing oligodendroglial GPCs from GBM GPCs collated from different studies, which was enriched for the Wnt, Notch and TGF-beta pathways. Using a novel method in glioma biology, the Connectivity Map, we mapped the strength of gene signature association with patient gene expression profiles in 2 independent glioma databases [GSE16011, http://caintegrator-info.nci.nih.gov/rembrandt]. Our gene signature consistently stratified survival in glioma patients. This data would suggest that in vitro low passage GPCs are similarly driven by transcriptomic changes that characterize the favorable outcome of oligodendrogliomas over GBM. Additionally, the gene signature was associated with the 1p/19q co-deletion status, the current clinical indicator of chemosensitivity. Our gene signature detects molecular heterogeneity in oligodendroglioma patients that cannot be accounted for by histology or the 1p/19q status alone, and highlights the limitation of morphology-based histological analyses in tumor classification, consequently impacting on treatment decisions.
Progenitor-like traits contribute to patient survival and prognosis in oligodendroglial tumors.
Sex, Age, Disease stage, SubjectView Samples
The childhood brain tumour medulloblastoma includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant Beta-Catenin in WNT-medulloblastoma an essentially curable form of the disease induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoural chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma a less curable disease subtype contains an intact blood brain barrier, rendering this tumour impermeable and resistant to chemotherapy. Remarkably, the medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumour vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumours.
Medulloblastoma Genotype Dictates Blood Brain Barrier Phenotype.
Specimen partView Samples
Resistance to proteasome inhibitors (PIs) is a ubiquitous clinical concern in multiple myeloma (MM). We proposed that signaling-level responses after PI would reveal new means to enhance efficacy. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on spliceosome components. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis demonstrated broad-scale intron retention suggestive of PI-specific splicing interference. Direct spliceosome inhibition synergized with carfilzomib and showed potent anti-myeloma activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerabilityin myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma. Overall design: We examine 1) gene expression of MM cells in response to PI and 2)alternative splicing in response to PI and comparator chemotherapeutic compound. We further investigate splice factor mechanism in MM cells, by examining alternative splicing in MM with overexpression of wild type and mutant splice factor, SRSF1
Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma.
Cell line, Subject, Compound, TimeView Samples
Alterations in gene expression following fatty acid synthase inhibtion were evaluated in androgen sensitive LNCaP cells and castration resistant 22Rv1 and LNCaP-95 cells. Cell were exposed to 2 concentrations (0.1 and 0.5 uM) of FASN inhibitor IPI-9119 or DMSO for 6 days. Overall design: Differential gene expression anlaysis in 3 prostate cancer cell lines treated with FASN inhibitor IPI-9119
Inhibition of de novo lipogenesis targets androgen receptor signaling in castration-resistant prostate cancer.
Specimen part, Cell line, SubjectView Samples
We used Affymetrix HTA2.0 microarray profiling to analyze gene expression patterns in tumor and paired non-tumor tissue of HCC and CCA patients.
Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.
Specimen part, Disease, Disease stage, SubjectView Samples
Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. To identify mutations that drive medulloblastoma we sequenced the entire genomes of 37 tumours and matched normal blood. One hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma: several target distinct components of the epigenetic machinery in different disease subgroups, e.g., regulators of H3K27 and H3K4 trimethylation in subgroup-3 and 4 (e.g., KDM6A and ZMYM3), and CTNNB1-associated chromatin remodellers in WNT-subgroup tumours (e.g., SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours, identified genes that maintain this cell lineage (DDX3X) as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumourigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.
Novel mutations target distinct subgroups of medulloblastoma.