It has been previously suggested that overexpression of mitochondrial superoxide dismutase (SOD) attenuates cancer development; however, the exact mechanism remains unclear. In this work, we have studied the direct effect of the mitochondria-targeted superoxide scavenger, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), on B16-F0 mouse melanoma cells and tumor growth in a nude mouse model of human melanoma. We show that scavenging of mitochondrial superoxide inhibited cell growth, reduced viability, and induced apoptosis in melanoma cells, but did not affect nonmalignant skin fibroblasts. Diminished mitochondrial superoxide inhibited redox-dependent Akt, restored activity of mitochondrial pyruvate dehydrogenase, and reduced HIF1-α and lactate dehydrogenase expression in cancer cells. Suppression of glycolysis in mitoTEMPO-treated melanoma cells resulted in a significant drop of cellular adenosine-5'-triphosphate and induced cell death. In vivo mitoTEMPO treatment effectively suppressed growth of established tumor in the mouse model of human melanoma. Therefore, our data lead to the hypothesis that scavenging of mitochondrial superoxide selectively inhibits redox-sensitive survival and metabolic pathways, resulting in cancer cell death. In contrast to existing anticancer therapies, inhibition of mitochondrial superoxide may represent a novel specific anticancer treatment with reduced cytotoxic side effects.

Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF protein levels in conditioned media, with no effects on VEGF mRNA levels. This increase in VEGF protein levels promoted paracrine activation of EC, including VEGF receptor-2 phosphorylation, and activation of downstream signaling proteins such as p44/42 MAPK and p38 MAPK. In addition, the elevated VEGF levels induced EC migration and sprouting, two key steps of tumor angiogenesis in vivo. It was concluded that hypoxia regulates VEGF and SEMA3F activities through transcriptional repression of their common receptor NRP2, providing a novel mechanism by which hypoxia induces tumor angiogenesis, growth and metastasis.

One key to malignant progression of pancreatic cancer (PC) is the acquired ability of tumour cells to escape immune-mediated lysis. Hypoxic microenvironment plays a causal role in PC metastasis. According to previous studies, hypoxia could induce the upregulation of HIF1A, ADAM10 and sMICA, leading to decreased NKG2D in NK cells and tumour cells escape from immune surveillance and NK cell-mediated lysis. In the present study, in NK cells derived from high-HIF1A expression patients, the levels of internalization of MICA/B and NKG2D were obviously higher than those in low-HIF1A expression group; hypoxia dramatically upregulated the levels of sMICA culture supernatant of Panc-1 cells. Regarding the molecular mechanism, dysregulated circRNAs and miRNAs that might modulate HIF1A-mediated immune escape were selected and examined for detailed functions. The expression of circ_0000977 could be induced by hypoxia, and circ_0000977 knockdown enhanced the killing effect of NK cells on PC cells under hypoxia through HIF1A and ADAM10. HIF1 and ADAM10 were direct downstream targets of miR-153; circ_0000977 served as a sponge for miR-153 to counteract miR-153-mediated repression of HIF1 and ADAM10 mRNA through direct targeting in both 293T cells and Panc-1 cells. miR-153 inhibition exerted an opposing effect on HIF1A-mediated immune escape of PC cells to circ_0000977 knockdown; the effect of circ_0000977 knockdown were partially attenuated by miR-153 inhibition. In summary, circ_0000977/miR-153 axis modulates HIF1A-mediated immune escape of PC cells through miR-153 downstream targets HIF1A and ADAM10. We provided a novel mechanism of HIF1A-mediated immune escape of PC cells from the perspective of circRNAs-miRNA-mRNA axis. Abbreviations: Pancreatic cancer (PC); peripheral blood lymphocytes (PBLs); A Disintegrin and Metalloproteinase Domain 10 (ADAM10); MHC class I-related molecule A (MICA); soluble MICA (sMICA); membrane MICA (mMICA); Hypoxia-inducible factor 1-alpha (HI1FA); long non-coding RNAs (lncRNAs); non-coding RNAs (ncRNAs); natural killer (NK); Haematoxylin and eosin (H&E); Immunohistochemistry (IHC); natural killer group 2 member D (NKG2D).

C-abl oncogene 1, nonreceptor tyrosine kinase (ABL1) kinase inhibitors such as imatinib mesylate (imatinib) are effective in managing chronic myeloid leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase-independent pathways support LSC survival. Given that the bone marrow (BM) hypoxic microenvironment supports hematopoietic stem cells, we investigated whether hypoxia similarly contributes to LSC persistence. Importantly, we found that although breakpoint cluster region (BCR)-ABL1 kinase remained effectively inhibited by imatinib under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of imatinib. Hypoxia-inducible factor 1 α (HIF1-α), which is the master regulator of the hypoxia transcriptional response, is expressed in the BM specimens of CML individuals. In vitro, HIF1-α is stabilized during hypoxia, and its expression and transcriptional activity can be partially attenuated by concurrent imatinib treatment. Expression analysis demonstrates at the whole-transcriptome level that hypoxia and imatinib regulate distinct subsets of genes. Functionally, knockdown of HIF1-α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1-α signaling supports LSC persistence independent of BCR-ABL1 kinase activity. Thus, targeting HIF1-α and its pathway components may be therapeutically important for the complete eradication of LSCs.

BACKGROUND

Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and fibrosis. The hepatocyte alterations accompanying the initiation of steatosis are not yet clearly defined.

METHODS

Induction of hepatosteatosis by chronic ethanol consumption was investigated using the Lieber-DeCarli (LD) high fat diet model. Effects were assessed by immunohistochemistry and blood and tissue enzymatic assays. Cell culture models were employed for mechanistic studies.

RESULTS

Pair feeding mice ethanol (LD-Et) or isocaloric control (LD-Co) diets for 6 weeks progressively increased hepatocyte triglyceride accumulation in morphological, biochemical, and zonally distinct cytoplasmic lipid droplets (CLD). The LD-Et diet induced zone 2-specific triglyceride accumulation in large CLD coated with perilipin, adipophilin (ADPH), and TIP47. In LD-Co-fed mice, CLD were significantly smaller than those in LD-Et-fed mice and lacked perilipin. A direct role of perilipin in formation of large CLD was further suggested by cell culture studies showing that perilipin-coated CLD were significantly larger than those coated with ADPH or TIP47. LD-Co- and LD-Et-fed animals also differed in hepatic metabolic stress responses. In LD-Et but not LD-Co-fed mice, inductions were observed in the following: microsomal ethanol-oxidizing system [cytochrome P-4502E1 (CYP2E1)], hypoxia response pathway (hypoxia-inducible factor 1 alpha, HIF1α), endoplasmic reticulum stress pathway (calreticulin), and synthesis of lipid peroxidation products [4-hydroxynonenal (4-HNE)]. CYP2E1 and HIF1 α immunostaining localized to zone 3 and did not correlate with accumulation of large CLD. In contrast, calreticulin and 4-HNE immunostaining closely correlated with large CLD accumulation. Importantly, 4-HNE staining significantly colocalized with ADPH and perilipin on the CLD surface.

CONCLUSIONS

These data suggest that ethanol contributes to macrosteatosis by both altering CLD protein composition and inducing lipid peroxide adduction of CLD-associated proteins.

Hypoxia is a microenvironmental stress in many pathological conditions, including wound healing and tumor invasion. Under hypoxia, the cells are forced to adapt alternative and self-supporting mechanisms. Understanding these mechanisms may lead to new insights into human disorders. We report here a novel autocrine signaling mechanism by which hypoxia promotes human keratinocyte (HK) migration. First, hypoxia triggers HKs to secrete heat shock protein 90-alpha (HSP90alpha) via a HIF1-dependent pathway. The secreted HSP90alpha in turn promotes migration, but not proliferation, of the cells. Disruption of the secretion or extracellular function of HSP90alpha blocked hypoxia-stimulated HK migration. The ubiquitously expressed surface receptor, LRP1 (LDL-receptor-related protein 1), mediates the HSP90alpha signaling. Inhibition of LRP1 binding to extracellular HSP90alpha by neutralizing antibodies or genetic silencing of the LRP1 receptor by RNAi completely nullified hypoxia-driven HK migration. Finally, re-introducing a RNAi-resistant LRP1 cDNA into LRP1-downregulated HKs rescued the motogenic response of the cells to hypoxia. We propose that the hypoxia-HSP90alpha-LRP1 autocrine loop provides previously unrecognized therapeutic targets for human disorders such as chronic wounds and cancer invasion.

OBJECTIVE

In an attempt to elucidate the relationship between biomarkers of tumor hypoxia, glycolysis and angiogenesis, we tested the hypothesis that intratumoral gene expression of the hypoxia response (hypoxia inducible factor [HIF1 alpha and 2 alpha]), glycolysis (lactate dehydrogenase A [LDHA]), glucose metabolism (glucose transporter-1 [Glut-1]) and genes involved in angiogenesis (i.e., VEGFA, VEGFR1-3, and neuropilin [NRP]1) are upregulated in metastatic colorectal cancer (mCRC) patients with high serum lactate dehydrogenase (LDH).

METHODS

78 formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from 36 patients with mCRC. Tumor gene expression was correlated with serum LDH levels from the same group of patients. FFPE tissues were dissected using laser-captured microdissection and analyzed for gene expression using a quantitative real-time RT-PCR method.

RESULTS

Intratumoral gene expression of VEGFA and VEGFR1 showed a statistically significant correlation with serum LDH levels (p = 0.006, r = 0.45 and p = 0.004, r = 0.50, respectively). Intratumoral expression of LDHA gene showed a significant correlation with Glut-1, VEGF, HIF1 alpha, HIF2 alpha and VEGFR1 (p = 0.007, r = 0.44; p < 0.001, r = 0.57; p = 0.013, r = 0.41; p = 0.044, r = 0.34; p = 0.026, r = 0.40). Serum LDH levels also correlated with microvessel density analyzed by immunohistochemical analysis.

CONCLUSIONS

The results demonstrated a significant correlation between the intratumoral gene expression of LDHA, HIF1 alpha, HIF2 alpha, Glut-1, NRP1, VEGFA and VEGFR1. Patients with high serum LDH have increased intratumoral gene expression of VEGFA and VEGFR1. The results also support the hypothesis that serum LDH levels may serve as a surrogate marker for activation of the HIF-related genes in the tumor.

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

The hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1alpha of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1alpha in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1alpha accumulation and subcellular localization as well as the transcriptional activity of the HIF1alpha-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O2), normoxia (20% O2) or high oxygen concentration (42% O2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1alpha can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl2, a known inhibitor of HIF1alpha degradation, show even higher levels of HIF1alpha, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1alpha in controls as well as in CoCl2-treated cells is found in the nuclei. CdCl2 inhibits nuclear import of HIF1alpha at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1alpha antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO2 than at low pO2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1alpha, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei.

Based on a previous finding that endothelial cell-specific molecule-1 (ESM-1) is a potential serum marker for colorectal cancer (CRC), the aim of this study was to clarify the clinicopathological significance of ESM-1 expression in CRC, and to explore the correlation between ESM-1 and HIF-1α in the tumorigenesis of CRC related to hypoxic conditions. ESM-1 mRNA expression was examined in CRC and corresponding normal mucosal tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. This experiment confirmed that ESM-1 levels were high in CRC. We screened the tissue samples of 143 CRC patients. By immunohistochemistry, we determined that the ESM-1 immunoreactivity was significantly correlated with the tumor size, depth of invasion, nodal status, distant metastasis and Dukes' stage, and was an independent prognostic factor for disease recurrence and worse survival outcome (P=0.001). The modulation of ESM-1 under hypoxia was investigated, and it was confirmed that ESM-1 expression was induced by HIF1-α and significantly attenuated by small interfering RNA (siRNA) targeting HIF-1α in CRC cells. These results showed that ESM-1 is significantly overexpressed, which is regulated by HIF-1α in CRC patients, and can be used as a potential biomarker and a therapeutic target for CRC.

All of the over 1 million total joint replacements implanted in the US each year are expected to eventually fail after 15-25 years of use, due to slow progressive subtle inflammation at the bone implant interface. This inflammatory disease state is caused by implant debris acting, primarily, on innate immune cells, that is, macrophages. This slow progressive pathological bone loss or "aseptic loosening" is a potentially life-threatening condition due to the serious complications in older people (>75 yrs) of total joint replacement revision surgery. In some people implant debris (particles and ions from metals) can influence the adaptive immune system as well, giving rise to the concept of metal sensitivity. However, a consensus of studies agrees that the dominant form of this response is due to innate reactivity by macrophages to implant debris where both danger (DAMP) and pathogen (PAMP) signalling elicit cytokine-based inflammatory responses. This paper discusses implant debris induced release of the cytokines and chemokines due to activation of the innate (and the adaptive) immune system and the subsequent formation of osteolysis. Different mechanisms of implant-debris reactivity related to the innate immune system are detailed, for example, danger signalling (e.g., IL-1β, IL-18, IL-33, etc.), toll-like receptor activation (e.g., IL-6, TNF-α, etc.), apoptosis (e.g., caspases 3-9), bone catabolism (e.g., TRAP5b), and hypoxia responses (Hif1-α). Cytokine-based clinical and basic science studies are in progress to provide diagnosis and therapeutic intervention strategies.

Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by poor prognosis and a low survival rate. VEGF-A is the most established angiogenic factor involved in the angiogenic-regulated tumor progression. WISP-1/CCN4 is an extracellular matrix-related protein that belongs to the Cyr61, CTGF, Nov (CCN) family and regulates many biological functions, such as angiogenesis. Previous studies indicated the role of WISP-1 in tumor progression. However, the angiogenic property of WISP-1 in the cancer microenvironment has never been discussed. Here, we provide novel insights regarding the role of WISP-1 in the angiogenesis through promoting VEGF-A expression. In this study, the correlation of WISP-1 and VEGF-A was confirmed by IHC staining of specimens from patients with OSCC. In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC. This pathway in turn induces the recruitment of endothelial progenitor cells and triggers the neovascularization in the tumor microenvironment. Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth. Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

In addition to its role as a toxicological signal mediator, the Aryl Hydrocarbon Receptor (AHR) is also a transcription factor known to regulate cellular responses to oxidative stress and inflammation through transcriptional regulation of molecules involved in the signaling of nucear factor-erythroid 2-related factor-2 (Nrf2), p53 (TRP53), retinoblastoma (RB1), and NFκB. Recent research suggests that AHR activation of these signaling pathways may provide the molecular basis for understanding AHR's evolving role in endogenous developmental functions during hematopoietic stem-cell maintenance and differentiation. Recent developments into the hematopoietic roles for AHR are reviewed, aiming to reconcile divergent findings as to the endogenous function of AHR in hematopoiesis. Potential mechanistic explanations for AHR's involvement in hematopoietic differentiation are discussed, focusing on its known role as a cell cycle mediator and its interactions with Hypoxia-inducible transcription factor-1 alpha (HIF1-α). Understanding the physiological mechanisms of AHR activation and signaling have far reaching implications ranging from explaining the action of various toxicological agents to providing novel ways to expand stem cell populations ex vivo for use in transplant therapies.

Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase (PDH) activity and thus promotes energetic switch from mitochondrial glucose oxidation to cytoplasmic glycolysis in cancerous cells (a phenomenon known as the 'Warburg effect') for their energy need, which facilitates the cancer progression by resisting induction of apoptosis and promoting tumor metastasis. Thus, in the present investigation, we explored the molecular mechanisms of the tumoricidal action of dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, on cells of a murine T cell lymphoma, designated as Dalton's lymphoma (DL). In vitro treatment of tumor cells with DCA inhibited their survival accompanied by a modulation of the biophysical composition of tumor-conditioned medium with respect to pH, glucose and lactate. DCA treatment also altered expression of HIF1-α and pH regulators: VATPase and MCT1 and production of cytokines: IL-10, IL-6 and IFN-γ. Moreover, we also observed an alteration in the expression of other apoptosis and cell survival regulatory molecules: PUMA, GLUT1, Bcl2, p53, CAD, caspase-3 and HSP70. The study discusses the role of novel molecular mechanisms underlying DCA-dependent inhibition of tumor cell survival. This study shows for the first time that DCA-dependent alteration of tumor cell survival involves altered pH homeostasis and glucose metabolism. Thus, these findings will provide a new insight for therapeutic applications of DCA as a novel antineoplastic agent against T cell lymphoma.

Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia-reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin-adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), and the oxygen-sensor neuroglobin increased significantly. Up-regulation of the HO-1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti-oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti-oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti-oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro-protective drug.

von Hippel-Lindau (VHL) gene inactivation occurs in von Hippel-Lindau (VHL) disease. The protein pVHL functions in a multi-subunit E3 ubiquitin ligase that targets the hypoxia-inducible transcription factor Hif1 alpha for proteasomal degradation during normoxia. We establish that pVHL binds to Tat-binding protein-1 (TBP-1), a component of the 19S regulatory complex of the proteasome. TBP-1 associates with the beta-domain of pVHL and complexes with pVHL and Hif1 alpha in vivo. Overexpression of TBP-1 promotes degradation of Hif1 alpha in a pVHL-dependent manner that requires the ATPase domain of TBP-1. Blockade of TBP-1 expression by small interfering RNA (siRNA) causes prolonged degradation kinetics of Hif1 alpha. Several distinct mutations in exon 2 of VHL disrupt binding of pVHL to TBP-1. A pVHL mutant containing a P154L substitution coimmunoprecipitates with Hif1 alpha, but not TBP-1, and does not promote degradation of Hif1 alpha. Thus, the ability of pVHL to degrade Hif1 alpha depends in part on its interaction with TBP-1 and suggests a new mechanism for Hif1 alpha stabilization in some pVHL-deficient tumors.

Hypoxia occurs in cancer, prolonged exercise, and long-term ischemia with durations of several hours or more, and the hypoxia-inducible factor 1 (HIF1) pathway response to these conditions differs from responses to transient hypoxia. We used computational modeling, validated by experiments, to gain a quantitative, temporal understanding of the mechanisms driving HIF1 response. To test the hypothesis that HIF1 alpha protein levels during chronic hypoxia are tightly regulated by a series of molecular feedbacks, we took into account protein synthesis and product inhibition, and analyzed HIF1 system changes in response to hypoxic exposures beyond 3 to 4 h. We show how three autocrine feedback loops together regulate HIF 1 alpha hydroxylation in different microenvironments. Results demonstrate that prolyl hydroxylase, succinate and HIF1 alpha feedback determine intracellular HIF1 alpha levels over the course of hours to days. The model provides quantitative insight critical for characterizing molecular mechanisms underlying a cell's response to long-term hypoxia.

The von Hippel-Lindau tumor-suppressor protein (pVHL) regulates the stability of HIF1 alpha and HIF2 alpha and thus is pivotal in cellular responses to changes in oxygen tension. Paradoxically, human cytotrophoblasts proliferate under hypoxic conditions comparable to those measured in the early gestation placenta (2% O(2)), but differentiate into tumorlike invasive cells under well-oxygenated conditions such as those found in the uterus. We sought to explain this phenomenon in terms of pVHL expression. In situ, pVHL immunolocalized to villous cytotrophoblast stem cells, and expression was enhanced at sites of cell column initiation; in both of these relatively hypoxic locations, cytoplasmic staining for HIF2 alpha was also detected. As cytotrophoblasts attached to and invaded the uterus, which results in their increased exposure to oxygen, pVHL staining was abruptly downregulated concordant with localization of HIF2 alpha to the nucleus. In vitro, hypoxia (2% O(2)) upregulated cytotrophoblast pVHL expression together with HIF2 alpha, which localized to the cytoplasm; culture under well-oxygenated conditions greatly reduced levels of both molecules. These results, together with the placental defects previously observed in VHL(-/-) mice, suggest that pVHL is a component of the mechanism that transduces local differences in oxygen tension at the maternal-fetal interface to changes in the biological behavior of cytotrophoblasts. Furthermore, these data provide the first example of oxygen-dependent changes in pVHL abundance.

Telomerase plays an important role during immortalization and malignant transformation as crucial steps in the development of human cancer. In a cellular model of oral-esophageal carcinogenesis, recapitulating the human disease, immortalization occurred independent of the activation of telomerase but through the recombination-based alternative lengthening of telomeres (ALT). In this stepwise model, additional overexpression of EGFR led to in vitro transformation and activation of telomerase with homogeneous telomere elongation in already immortalized oral squamous epithelial cells (OKF6-D1_dnp53). More interestingly, EGFR overexpression activated the PI3K/AKT pathway. This strongly suggested a role for telomerase in tumor progression in addition to just elongating telomeres and inferring an immortalized state. Therefore, we sought to identify the regulatory mechanisms involved in this activation of telomerase and in vitro transformation induced by EGFR. In the present study we demonstrate that telomerase expression and activity are induced through both direct phosphorylation of hTERT by phospho-AKT as well as PI3K-dependent transcriptional regulation involving Hif1-alpha as a key transcription factor. Furthermore, EGFR overexpression enhanced cell cycle progression and proliferation via phosphorylation and translocation of p21. Whereas immortalization was induced by ALT, in vitro transformation was associated with telomerase activation, supporting an additional role for telomerase in tumor progression besides elongating telomeres.

Hypoxia inducible factors (HIF) are transcription factors regulating expression of several genes related to oxygen homeostasis in response to hypoxic stress. Although HIF1-alpha and platelet derived growth factor-B (PDGF-B) are expressed in glioma tissue and closely related to tumor angiogenesis mediating vascular endothelial growth factor (VEGF) activity, their direct relationship has not yet been clarified. The aim of this study is to investigate whether HIF1-alpha regulates PDGF-B expression. The human glioblastoma cell lines, U87MG, U251MG, and A172, were exposed to 1-21% oxygen for 24 h. PDGF-B mRNA expression were quantitatively analyzed by real time RT-PCR, their intracellular protein levels were determined by computerized image analysis supported by flow cytometry to detect intracellular PDGF-B, and the concentration of secreted PDGF-B protein was assayed by ELIA. We also assayed following transfection of the cells with short interference RNA (siRNA) targeting HIF1-alpha mRNA. Relative PDGF-B mRNA and secretion of PDGF-B protein were significantly elevated at 1% oxygen. Following transfection of HIF1-alpha siRNA at 1% oxygen, PDGF-B expression was significantly suppressed at mRNA level. Our findings indicated that HIF1-alpha up-regulated expression of PDGF-B in human glioblastoma cells and showed the feasibility of siRNA technology in glioblastoma cell lines.

Although the causes of prostate cancer are still unknown, numerous studies support the role of genetic factors in the development and progression of this disease. Single nucleotide polymorphisms (SNPs) in key angiogenesis genes have been studied in prostate cancer. In this review, we provide an overview of the current knowledge of the role of genetic variants in the angiogenesis pathway in prostate cancer risk and progression. Of the 17 prostate cancer genome-wide association studies (GWAS) conducted to date, only one identified disease-associated SNPs in a region of an angiogenesis pathway gene. An association was observed between aggressive disease and three intergenic SNPs (rs11199874, rs10749408 and rs10788165) in a region on chromosome 10q26 that encompasses FGFR2. The majority (27/32, 84.4%) of primary candidate gene studies reviewed had a small (n < 800, 20/32, 62.5%) to medium sample size (n = 800-2000, 7/32, 21.9%), whereas only five (15.6%) had a large sample size (n ≥ 2000). Results from the large studies revealed associations with risk and aggressive disease for SNPs in NOS2A, NOS3 and MMP-2 and risk for HIF1-α. Meta-analyses have so far been conducted on FGFR2, TGF-β, TNF-α, HIF1-α and IL10 and the results reveal an association with risk for SNPs in FGFR2 and TGF-β and aggressive disease for SNPs in IL-10. Thus, existing evidence from GWAS and large candidate gene studies indicates that SNPs from a limited number of angiogenesis pathway genes are associated with prostate cancer risk and progression.

Tumor hypoxia is often linked to decreased survival in patients with breast cancer and current therapeutic strategies aim to target the hypoxic response. One way in which this is done is by blocking hypoxia-induced angiogenesis. Antiangiogenic therapies show some therapeutic potential with increased disease-free survival, but these initial promising results are short lived and followed by tumor progression. We hypothesized that this may be due to altered cancer stem cell (CSC) activity resulting from increased tumor hypoxia. We studied the effects of hypoxia on CSC activity, using in vitro mammosphere and holoclone assays as well as in vivo limiting dilution experiments, in 13 patient-derived samples and four cell lines. There was a HIF-1α-dependent CSC increase in ER-α-positive cancers following hypoxic exposure, which was blocked by inhibition of estrogen and Notch signaling. A contrasting decrease in CSC was seen in ER-α-negative cancers. We next developed a xenograft model of cell lines and patient-derived samples to assess the hypoxic CSC response. Varying sizes of xenografts were collected and analyzed for HIF1-α expression and CSC. The same ER-α-dependent contrasting hypoxic-CSC response was seen validating the initial observation. These data suggest that ER-α-positive and negative breast cancer subtypes respond differently to hypoxia and, as a consequence, antiangiogenic therapies will not be suitable for both subgroups.

Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1 alpha oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a "branching motif" immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best "hits" stabilize HIF1 alpha, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.