Project description: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.
Project description:Tumor hypoxia is linked to increased metastatic potential, but the molecular mechanisms coupling hypoxia to metastasis are poorly understood. Here, we show that Notch signaling is required to convert the hypoxic stimulus into epithelial-mesenchymal transition (EMT), increased motility, and invasiveness. Inhibition of Notch signaling abrogated hypoxia-induced EMT and invasion, and, conversely, an activated form of Notch could substitute for hypoxia to induce these processes. Notch signaling deploys two distinct mechanisms that act in synergy to control the expression of Snail-1, a critical regulator of EMT. First, Notch directly up-regulated Snail-1 expression by recruitment of the Notch intracellular domain to the Snail-1 promoter, and second, Notch potentiated hypoxia-inducible factor 1alpha (HIF-1alpha) recruitment to the lysyl oxidase (LOX) promoter and elevated the hypoxia-induced up-regulation of LOX, which stabilizes the Snail-1 protein. In sum, these data demonstrate a complex integration of the hypoxia and Notch signaling pathways in regulation of EMT and open up perspectives for pharmacological intervention with hypoxiainduced EMT and cell invasiveness in tumors.
Project description:Background: Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Results: Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: i) a larger set of genes induced by one pathway and not significantly affected by the activity status of the other pathway; and ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling. In addition, a number of genes were induced by Notch and hypoxia independently, and a final category of genes required simultaneous activation of Notch and hypoxia to be significantly induced. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Conclusions: We identify novel Notch and hypoxia downstream genes and genes co-regulated by the two pathways, providing a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer. Overall design: Total RNA was extracted from 7 treatments in 4 biological replicates of mouse embryonic stem (ES) cells. The 7 treatments consist of 1) ES cell medium for 6 hrs (ESC), 2) N2B27 medium for 6 hrs which is the control condition (N2B27), 3) N2B27 medium supplemented with doxycyline (Dox), 4) Pretreatment with DAPT in ES cell medium for 4 hours followed by switch to N2B27 medium for 6 hours (DAPT), 5) N2B27 under 1% oxygen hypoxic conditions (1% O2), 6) N2B27 supplemented with doxycycline under 1% oxygen hypoxic conditions (1% O2+Dox), 7) Pretreatment with DAPT in ES cell medium for 4 hours followed by switch to N2B27 medium for 6 hours (DAPT) under 1% oxygen hypoxic conditions (1% O2+DAPT).
Project description:Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown.The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1alpha was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits.Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1alpha and HIF-2alpha in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1alpha was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1alpha with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion.Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.
Project description:Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1?/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1?/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.
Project description:The Notch-3 receptor is a recognized key regulator of vascular responses and is increasingly associated with tumorigenesis. Hypoxia-inducible factors activate specific signaling pathways such as Notch in a number of cellular models. This study aimed to evaluate the regulation of Notch-3 by hypoxia in prostate cancer cells. Notch-3 gene and protein expression was established in a panel of aerobic and hypoxic prostate cell lines in vitro, the CWR22 xenograft model and RNA extracted from low grade (Gleason score < = 6); high grade (Gleason score > = 7); non-hypoxic (low HIF, low VEGF); hypoxic (high HIF, high VEGF) patient FFPE specimens. NOTCH-3 was upregulated in PC3 (3-fold), 22Rv1 (4.1-fold) and DU145 (3.8-fold) but downregulated in LnCaP (12-fold) compared to the normal cell lines. NOTCH-3 expression was modified following hypoxic exposure in these cells. NOTCH-3 was upregulated (2.2-fold) in higher grade and hypoxic tumors, when compared to benign and aerobic pools. In the CWR22 xenograft model, Notch-3 expression was restored in castrate resistant tumors. Nuclear translocation of the Notch-3 intracellular domain was no longer detected following exposure of cells to hypoxia but not associated with a change in expression of HES-1. Our data further identifies Notch-3 as a potentially key hypoxic-responsive member of the Notch pathway in prostate tumorigenesis.
Project description:The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1? (HIF-1?) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1? protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1? protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1? mutant into Mel285 cells with low HIF-1? baseline levels. In contrast, HIF-1? knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1? baseline levels. Pharmacologic blockade of HIF-1? protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1? signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1? pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.
Project description:Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro.Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and ?3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur.Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.
Project description:Hypoxia induces the expansion of glioblastoma stem cells (GSCs), but the mechanism underlying it is still unclear. Here, we supply evidence that hypoxia-inducible factor-1? (HIF-1?) induced activation of Notch pathway is essential for hypoxia-mediated maintenance of GSC. Either depletion of HIF-1? or inactivation of Notch signaling partly inhibits the hypoxia-mediated maintenance of GSC. Further data suggest a role for HIF-1? in the interaction and stabilization of intracellular domain of Notch (NICD), and activation of Notch signaling. The mRNA level of HIF-1? and Notch target gene FABP7 was elevated in GSC. And the STAT3 pathway responsible for the HIF-1? gene transcription, the phosphatidylinositol 3-kinase-Akt and ERK1/2, both of which are contributed to HIF-1? protein translation, are also preferentially activated in GSC. Inhibition of these pathways partly reduces the hypoxia-induced activation of the Notch pathway and subsequent GSC maintenance. Taken together, our findings suggest that HIF-1? requires Notch pathway to drive the maintenance of GSC. The activated regulation of HIF-1? makes GSC more sensitive to hypoxia-mediated maintenance. These findings enhance our understanding of mechanism of hypoxia-mediated GSC expansion and provide HIF-1? as an attractive target for glioblastoma therapy.
Project description:Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.