Long noncoding RNA FEZF1-AS1 predicts poor prognosis and modulates pancreatic cancer cell proliferation and invasion through miR-142/HIF-1α and miR-133a/EGFR upon hypoxia/normoxia

Nowadays, pancreatic cancer (PC) remains the most lethal tumor, partially due to the invasive and treatment-resistant phenotype induced by the extent of hypoxic stress within the tumor tissue. According to previous studies, miR-142/HIF-1α and miR-133a/EGFR could modulate PC cell proliferation under hypoxic and normoxic conditions, respectively. In the present study, FEZF1-AS1, a recently described oncogenic long noncoding RNA, was predicted to target both miR-142 and miR-133a; thus, we hypothesized that FEZF1-AS1 might affect PC cell proliferation through these two axes under hypoxic or normoxic conditions. In PC cell lines, FEZF1-AS1 acted as an oncogene via promoting PC cell proliferation and invasion through miR-142/HIF-1α axis under hypoxic condition; however, FEZF1-AS1 failed to affect the protein levels of HIF-1α and VEGF under the normoxic condition, suggesting the existence of another signaling pathway under normoxic condition. As predicted by an online tool, FEZF1-AS1 could target miR-133a to inhibit its expression; under the normoxic condition, FEZF1-AS1 exerted its effect on PC cell lines through miR-133a/EGFR axis. Taken together, FEZF1-AS1 might be a promising target in controlling the aberrant proliferation and invasion of PC cell lines.     

Astrocyte responses to injury: VEGF simultaneously modulates cell death and proliferation

Hypoxia is linked to changes in blood-brain barrier (BBB) permeability, and loss of BBB integrity is characteristic of many pathological brain diseases including stroke. In particular, astrocytes play a central role in brain homeostasis and BBB function. We investigated how hypoxia affects astrocyte survival and assessed whether VEGF release through hypoxia-inducible factor-1alpha (HIF-1alpha) induction plays a role in tolerance of these cells to insult. Thus primary astrocytes were subjected to normoxic (21% O(2)), hypoxic (1% O(2)), or near-anoxic (<0.1% O(2)) conditions in the presence or absence of glucose. Cell death was significantly initiated after combined oxygen glucose deprivation, and, surprisingly, astrocyte proliferation increased concomitantly. Near anoxic, but not hypoxic, conditions stabilized HIF-1alpha protein and provoked DNA binding activity, whereas oxygen and glucose deprivation accelerated HIF-1alpha accumulation. Unexpectedly, Hif-1alpha knockdown studies showed that elevated VEGF levels following increased insult was only partially due to HIF-1alpha induction, suggesting alternative mechanisms of VEGF regulation. Notably, endogenous VEGF signaling during insult was essential for cell fate since VEGF inhibition appreciably augmented cell death and reduced proliferation. These data suggest Hif-1 only partially contributes to VEGF-mediated astrocyte responses during chronic injury (as occurs in clinical hypoxic/ischemic insults) that may ultimately be responsible for disrupting BBB integrity.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value.

In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.     

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.     

Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value. In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.