Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo

The Function of Hypoxia-Inducible Factor (HIF) Is Independent of the Endoplasmic Reticulum Protein OS-9

The protein “amplified in osteosarcoma-9” (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization

Hypoxia: an alarm signal during intestinal inflammation

Intestinal epithelial cells that line the mucosal surface of the gastrointestinal tract are positioned between an anaerobic lumen and a highly metabolic lamina propria. As a result of this unique anatomy, intestinal epithelial cells function within a steep physiologic oxygen gradient relative to other cell types. Furthermore, during active inflammatory disease such as IBD, metabolic shifts towards hypoxia are severe. Studies in vitro and in vivo have shown that the activation of hypoxia-inducible factor (HIF) serves as an alarm signal to promote the resolution of inflammation in various mouse models of disease. Amelioration of disease occurs, at least in part, through transcriptional upregulation of nonclassic epithelial barrier genes. There is much interest in harnessing hypoxia-inducible pathways, including stabilizing HIF directly or via inhibition of prolyl hydroxylase enzymes, for therapy of IBD. In this Review, we discuss the signaling pathways involved in the regulation of hypoxia and discuss how hypoxia may serve as an endogenous alarm signal for the presence of mucosal inflammatory disease. We also discuss the pros and cons of targeting these pathways to treat patients with IBD.Deposited by bulk impor

Hypoxia: an alarm signal during intestinal inflammation

Intestinal epithelial cells that line the mucosal surface of the gastrointestinal tract are positioned between an anaerobic lumen and a highly metabolic lamina propria. As a result of this unique anatomy, intestinal epithelial cells function within a steep physiologic oxygen gradient relative to other cell types. Furthermore, during active inflammatory disease such as IBD, metabolic shifts towards hypoxia are severe. Studies in vitro and in vivo have shown that the activation of hypoxia-inducible factor (HIF) serves as an alarm signal to promote the resolution of inflammation in various mouse models of disease. Amelioration of disease occurs, at least in part, through transcriptional upregulation of nonclassic epithelial barrier genes. There is much interest in harnessing hypoxia-inducible pathways, including stabilizing HIF directly or via inhibition of prolyl hydroxylase enzymes, for therapy of IBD. In this Review, we discuss the signaling pathways involved in the regulation of hypoxia and discuss how hypoxia may serve as an endogenous alarm signal for the presence of mucosal inflammatory disease. We also discuss the pros and cons of targeting these pathways to treat patients with IBD.Deposited by bulk impor

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated withparaneo plastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growthby developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4- hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated withattenuate d HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1a. pRCC is known for its tendency to avascularity, usually associated witha lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker

Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability