Hypoxia-induced release, nuclear translocation, and signaling activity of a DLK1 intracellular fragment in glioma

Abstract

Glioblastoma multiforme is characterized in part by severe hypoxia associated with tumor necrosis. The cellular response to hypoxia can influence several properties of tumor cells associated with aggressive tumor growth, including metabolic adaptations and tumor cell migration and invasion. Here, we found that Delta Like Non-Canonical Notch Ligand 1 (DLK1) expression was elevated as compared with normal brain in a genetically engineered mouse model of glioma, and that DLK1 expression increased with tumor grade in human glioma samples. DLK1 expression was highest in hypoxic and perivascular tumor areas, and we found that hypoxia induced the release and nuclear translocation of an intracellular fragment of DLK1 in murine glioma as well as in human glioma cultures. Release of the intracellular fragment was dependent on ADAM17 and Hypoxia-inducible Factor 1alpha and 2alpha (HIF-1alpha/HIF-2alpha), as ADAM17 inhibitors and HIF1A/HIF2A siRNA blocked DLK1 cleavage. Expression of a cleavable form of DLK1 amplified several hypoxia-induced traits of glioma cells such as colony formation, stem cell marker gene expression, a PI3K-pathway-mediated metabolic shift, and enhanced invasiveness. Effects of DLK1 were dependent on DLK1-cleavage by ADAM17, as expression of non-cleavable DLK1 could not replicate the DLK1-induced hypoxic phenotype. Finally, forced expression of DLK1 resulted in more invasive tumor growth in a PDGFB-induced glioma mouse model without affecting overall survival. Together, our findings suggest a previously undescribed role for DLK1 as an intracellular signaling molecule.

Niche-derived soluble DLK1 promotes glioma stemness and growth

“…overexpression specifically in the context of glioblastoma, we show that the previously reported association between DLK1 expression and tumor grade in glioma (Grassi et al, 2020, Yin et al, 2006 As with astrocyte-derived DLK1 in regulation of normal neural stem cells, the exact mechanism(s) by which soluble DLK1 signals to glioma cells remains to be investigated. We show here that soluble DLK1 can contribute to a stronger and more prolonged response to hypoxia, as mediated by increased HIF-2alpha stabilization in DLK1 treated cells.…”

“…To test whether tumor-associated astrocytes could be a source of DLK1 in the glioma tumor microenvironment, we generated PDGFB/shp53-induced murine gliomas using the RCAS/tv-a system as previously described (Grassi et al, 2020), then costained tumors for the astrocyte marker GFAP and DLK1. While the bulk of the tumor cells appeared negative for DLK1 expression, DLK1 signal was detected in areas of GFAP staining both in perinecrotic and perivascular tumor areas, and both in GFAP positive and negative cells, suggesting DLK1 expression in astrocytes and tumor cells in these areas ( Fig.…”

“…Delta Like Non-Canonical Notch Ligand 1 (DLK1) is a transmembrane protein in the Notch family of ligands, that is capable of signaling in a Notch-dependent andindependent manner depending on cellular context (Baladrón et al, 2005;Ceder, Jansson, Helczynski, & Abrahamsson, 2008;Falix, Aronson, Lamers, & Gaemers, 5 2012; Ferrón et al, 2011;Huang et al, 2019;Li et al, 2014). Expression of DLK1 is increased with tumor grade in glioma, and its signaling has been associated with various tumor cell properties such as proliferation, invasion, and stemness (Grassi, Pantazopoulou, & Pietras, 2020;Yin et al, 2006). The mechanisms underlying these effects on tumor cell behavior remain poorly understood, but likely include signaling from the extracellular, soluble domain of DLK1 (Wang & Sul, 2006).…”

Niche-derived soluble DLK1 promotes glioma growth

“…To test whether tumor-associated astrocytes could be a source of DLK1 in the glioma tumor microenvironment, we generated PDGFB/shp53-induced murine gliomas using the RCAS/tv-a system as previously described [14] , then co-stained tumors for the astrocyte marker glial fibrillary acidic protein (GFAP) and DLK1. While the bulk of the tumor cells appeared negative for DLK1 expression, DLK1 signal was detected in areas of GFAP staining both in perinecrotic and perivascular tumor areas, and both in GFAP positive and negative cells, suggesting DLK1 expression in astrocytes, tumor cells, and potentially other cell types present in these areas ( Figure 1 A−C, F−G and Supplementary Figure 1 A−B).…”

“…Delta Like noncanonical Notch ligand 1 (DLK1) is a transmembrane protein in the Notch family of ligands, that is capable of signaling in a Notch-dependent and -independent manner depending on cellular context [8] , [9] , [10] , [11] , [12] , [13] . Expression of DLK1 is increased with tumor grade in glioma, and its signaling has been associated with various properties of aggressive tumor cells [14 , 15] . The mechanisms underlying these effects on tumor cell behavior remain poorly understood, but likely include signaling from the extracellular, soluble domain of DLK1 [16] .…”