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Background and purpose-Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance.

Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.

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Adaptation to tumor hypoxia is mediated in large part by changes in protein expression. These are driven by multiple pathways, including activation of the hypoxia inducible factor-1 (HIF-1) transcription factor and the PKR-like endoplasmic reticulum kinase PERK, a component of the unfolded protein response. Through gene expression profiling we discovered that induction of the HIF-1 target gene CA9 was defective in mouse embryo fibroblasts derived from mice harboring an eIF2␣ S51A knock-in mutation. This finding was confirmed in two isogenic human cell lines with an engineered defect in eIF2␣ phosphorylation. We show that impaired CA9 expression was not due to changes in HIF activity or CA9 mRNA stability. Using chromatin immunoprecipitation we show that the eIF2␣-dependent translationally regulated gene ATF4 binds directly to the CA9 promoter and is associated with loss of the transcriptional repressive histone 3 lysine 27 tri-methylation mark. Loss or overexpression of ATF4 confirmed its role in CA9 induction during hypoxia. Our data indicate that expression of CA9 is regulated through both the HIF-1 and unfolded protein response hypoxia response pathways in vitro and in vivo.Mammalian cells have evolved a number of sophisticated mechanisms to allow adaptation to changes in the supply of essential metabolic factors. These include mechanisms for sensing the presence of nutrients, energy, and oxygen. Many of these mechanisms are exploited by tumor cells to facilitate survival in the unique conditions found within the microenvironment of solid human tumors (1). In particular, adaptation to hypoxia is considered to be an important factor that contributes to the poor prognosis of patients with high levels of tumor hypoxia (2). The hypoxia-inducible factor (HIF) 2 plays an essential role in this adaptation by transcriptionally regulating the expression of a large number of genes involved in angiogenesis, glycolysis, invasion, and metastasis (3).HIF-1 is a heterodimeric transcription factor consisting of ␣ and ␤ subunits that are both ubiquitously expressed. Under aerobic conditions HIF-1␣ is ubiquitinated by the von Hippel-Lindau tumor suppressor protein causing its rapid degradation within the 26 S proteasome (4). This process relies on hydroxylation of proline residues 402 and 564 by a family of oxygen-sensitive prolylhydroxylases 1-3. Under hypoxic conditions hydroxylation cannot occur, resulting in HIF-1␣ stabilization and formation of a functional transcription factor with HIF-1␤. This complex drives transcription of HIF-responsive genes by binding to hypoxia-responsive elements (HREs) in their promoters.One of the best characterized targets of HIF-1 is carbonic anhydrase 9 (CA9). The CA9 promoter contains a single HRE element that is essential for its transcriptional increase during hypoxia (5). CA9 is frequently overexpressed in human tumors and is associated with poor prognosis (6, 7). It belongs to a family of zinc metalloenzymes involved in regulating pH by catalyzing the reversible conversion of carbon dio...

Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII+) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII+ cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII+ tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII+ cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII+ tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII+ glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII+ glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII+ glioblastoma.

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