Cancer is one of the leading causes of death worldwide. Among central nervous system tumors, glioblastoma is the most common and most aggressive tumor entity. Despite new therapeutic strategies and a better understanding of the molecular mechanisms leading to tumorigenesis, the median glioblastoma patient survival is only 15 months.A hypoxic tumor microenvironment is one of the characteristics of glioblastoma and tumor hypoxia has been linked to multiple hallmarks of cancer, including tumor promoting inflammation, metabolic reprogramming, angiogenesis, invasion and metastasis. The main effectors of the cellular response to hypoxia are the hypoxia-inducible transcription factors (HIFs), whose stability is tightly regulated through hydroxylation by the prolyl hydroxylase domain proteins (PHDs). The latter require oxygen and 2-oxoglutarate (2-OG) as co-substrates to hydroxylate specific residues within HIF-a, which eventually leads to HIF-a degradation under normoxia. Under hypoxia, PHDs are inactive and HIF-a is stabilized. Activation of the hypoxic response is not only mediated by hypoxia, but also by the accumulation of the oncometabolite 2-hydroxyglutarate, which can modulate PHD activity. Identification and characterization of oncogenic mutations leading to the accumulation of oncometabolites acting as 2-oxoglutarate (2-OG) analogs, showed the central role of 2-OG in the regulation of cellular responses. We therefore hypothesized that isocitrate dehydrogenases (IDHs), which convert isocitrate to 2-OG, might play a central role in the maintenance of 2-OG levels and hence the regulation of 2-OG dependent enzyme activity. In line with this hypothesis, we show that IDH1 knock-down results in reduction of 2-OG levels, accompanied by the activation of the hypoxic response as a direct result of PHD inhibition, evidenced by reduced HIF-a hydroxylation. In order to directly demonstrate the involvement of 2-OG in this process, we treated IDH1 knock-down cells with cell permeable 2-OG. Importantly, addition of 2-OG suppressed the IDH1 knock-down-mediated increase of HIF-a levels and HIF-a target gene expression. Since these findings demonstrated that IDH1-dependent changes in 2-OG control PHD activity, we examined if IDH1 silencing also influences other PHD-regulated signaling pathways, such as NF-?B signaling. In line with our previous findings, IDH1 silencing-mediated PHD inactivation led to increased NF-?B nuclear translocation and activation of NF-?B downstream targets. Additionally, we demonstrated that the cancer stem cell (CSC) phenotype, which has been closely linked to HIF-a activation, is also enhanced by IDH1 silencing and reverted by 2-OG treatment. Importantly, we could show that aberrant 2-OG levels also modulate invasion and the expression of Snail, a key regulator of mesenchymal transition, which plays a crucial role in the control of tumor invasion. In line with the cell culture-based data, our in vivo experiments verified the effects of IDH1 silencing on the induction of invasive characteristics, as well as activation of HIF-a and NF-?B signaling and the stem cell phenotype in tumors. Additionally, we identified TGFß, an inducer of mesenchymal transition, as a regulator of IDH1. Strikingly, in breast and lung cancer cells, tumor types in which metastasis is the primary cause of death, IDH1 silencing induced similar molecular changes, including activation of HIF-a signaling and increased expression of Snail. Most importantly, IDH1 silencing in this context resulted in increased metastasis. In addition to accumulating cancer cell-intrinsic changes, tumors reshape their own microenvironment, contributing to the acquisition of cancer hallmarks and tumor progression. One typical consequence of metabolic reprogramming under hypoxia is acidification of the tumor microenvironment. Therefore, in a second part of this work, we aimed to understand the molecular mechanisms controlled by the combination of acidosis and hypoxia in the tumor microenvironment, and its effect on tumorigenesis. We show that an acidic tumor microenvironment promotes the CSC phenotype and even further potentiates the hypoxic response, suggesting a role for acidosis in tumorigenesis and tumor therapy resistance. We revealed that acidosis controls HIF-a function through heat shock protein 90 (HSP90). Strikingly, absence or inactivation of HSP90 under acidic conditions reduced tumor initiation, tumor hypoxia and tumor growth. Importantly, we found that HSP90 levels correlate with hypoxic and stem cell marker genes in glioblastoma patients. Among the cancer hallmarks that are regulated by hypoxia, are the activation of invasion and the induction of angiogenesis. In the third part of this work, we focused on hypoxia-dependent mechanisms that regulate these capabilities. We show that hypoxia, which can be promoted by inhibition of angiogenesis, induces local glioma invasion and downregulates the cell adhesion molecule ephrinB2. Importantly, we reveal that ephrinB2 downregulation is directly controlled by ZEB2, a target of HIF-1a, establishing a critical role of a HIF-1a-ZEB2-EphrinB2 axis in glioma invasion, including invasion induced by anti-angiogenic agents. Notably, absence of ephrinB2 increased tumor invasiveness, whereas ephrinB2 overexpression decreased it, revealing a crucial function of ephrinB2 in the control of the invasive capacity of gliomas. Taken together, our results demonstrate the pivotal role of 2-OG for the control of PHD activity, the hypoxic response and downstream biological processes, such as CSC maintenance, invasion and metastasis. Additionally, we also revealed distinct molecular mechanisms through which hypoxia regulates cancer hallmarks, namely enhancing tumor invasion via a HIF-1a-ZEB2-EphrinB2 axis, or synergizing with acidosis to control the CSC phenotype and tumor growth through HSP90. These findings identify several mechanisms, centered around the control of hypoxic signaling in tumors, which regulate cancer hallmarks and may provide targets for future therapeutic strategies.
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