Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen.

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.

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma. Significance: These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

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