Abstract
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 HIF2α, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2α by Notch under normoxic conditions leads to elevated HIF2α 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 HIF2α protein was in certain tumor cell types accompanied by downregulation of HIF1α protein levels, indicating that high Notch signaling may drive a HIF1α-to-HIF2α switch. At the transcriptome level, the presence of HIF2α 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 HIF2α expression was knocked down by HIF2α siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2α, which may be important for future cancer therapies.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Interaction between signaling pathways is vital during normal development and tissue homeostasis
We noted that HIF2α mRNA expression levels were increased during conditions where Notch signaling was activated and, decreased under conditions of Notch blockage (Fig. 1a)
To test whether Notch regulates HIF2α mRNA expression, we expressed an activated form of Notch (Notch1 ICD) (Supplementary Figure 1A) in nine different human tumor cell lines derived from a range of tumors, and monitored HIF1α and HIF2α mRNA levels in normoxia (21% oxygen)
Summary
Interaction between signaling pathways is vital during normal development and tissue homeostasis. An improved understanding of how signaling pathways interact is warranted, as it may facilitate tailored therapy approaches based on identified pathway abnormalities. We addressed whether the Notch singling pathway modulates the cellular response to hypoxia, i.e., low oxygen conditions. The Notch signaling pathway is a highly evolutionarily conserved cell-cell contact-dependent signaling mechanism, which is activated when a ligand binds to a Notch receptor, leading to receptor cleavage and the release of the Notch intracellular domain (Notch ICD). Notch ICD subsequently translocates to the nucleus and forms a ternary transcriptional activation complex with CSL ( known as RBP-Jk) and Mastermind-like (MAML) to induce expression of downstream target genes, including Notch-regulated ankyrin repeat-containing protein (NRARP), Hes, or Hey genes [1, 2]. Notch mutations are found in several tumor types, having either oncogenic or tumor suppressor roles, depending on the type of tumor [3]
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