Overexpression Of Hypoxia-inducible Factor Research Articles

Hypoxia inducible factor: a potential prognostic biomarker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common oral cancer. Hypoxia inducible factor (HIF) is involved in many malignant tumors' growth and metastasis and upregulated by hypoxia, including oral cancer. Many studies have studied about the prognostic value of HIF expression in OSCC; however, they do not get the consistent results. Therefore, this study explored the correlation between the HIF expression and the prognosis of OSCC. It conducted a meta-analysis of relevant publications searched in the Web of Science, PubMed, and ISI Web of Knowledge databases. Totally, this study identified 12 relevant articles reporting a total of 1112 patients. This analysis revealed a significant association between increased risk of mortality (RR = 1.20; 95% CI 0.74-1.95; I (2) 85.4%) and overexpression of HIFs. Furthermore, different HIF isoforms were associated with overall survival [HIF-1α (RR = 1.18; 95% CI 0.66-2.11; I (2) 87.2%) and HIF-2α (RR = 1.40; 95% CI 0.93-2.09; I(2) 0.0%)]. These results show that overexpression of HIFs, regardless of whether the HIF-1α or HIF-2α isoforms are overexpressed is significantly associated with increased risk of mortality in OSCC patients. In this study, the funnel is symmetric, suggesting existed no publication bias.

Selection, Analysis and Improvement of Anti-Angiogenesis Compounds Identified by an Anti-HIF-1α Screening and Validation System.

Cancer cells resort to activating hypoxia-inducible factor-1 (HIF-1) as one of several responses to hypoxic conditions. Overexpression of HIF-1, the transcriptional regulator for a group of malignant-pathway related genes including vascular endothelial growth factor (VEGF), is associated with increased tumor growth, vascularization, and metastasis. HIF-1 is composed of an inducible subunit, HIF-1α and a constitutively expressed subunit, HIF-1ß. HIF-1 activity is mainly dependent on the level of HIF-1α protein, the inducible and regulatory subunit of the HIF-1 heterodimer complex; thus, identification of novel anti-HIF-1α agents will lead to effective blockage of the HIF-1 (HIF-1α)-mediated “switch-on” function for those malignant-pathway related genes and suppression of the HIF-1α/VEGF-mediated signaling pathway that promotes cancer progression and metastasis.While there is an extremely large number of small molecule compounds in the database (compound libraries), the currently existing screening system is inefficient and time-consuming; or, at best, the application of the existing screening system is very limited as it is usually not coupled with biological validation processes. The further development of potential drugs is partly hindered due to the cumbersome steps in between the primary screen and consequent validation: the slow, exhausted and sometimes lack of a linked biological validation process contributes to the dismal fate of scant compounds uncovered in the primary screen.To improve upon the status quo, we developed a prototype screening system that is coupled anti-HIF-1α primary screen with secondary anti-VEGF/anti-angiogenesis validation screens. We used breast cancer cells as the model to select potent anti-HIF-1α small-molecule compounds by their abilities to inhibit transactivation of a VEGF promoter fused to a luciferase reporter gene under hypoxia. Positive compounds were then validated by a series of assays that confirm compounds' anti-HIF-1α activities including measurement of their effects on HIF-1α downstream VEGF gene expression and angiogenic ability of breast cancer cells. Moreover, we demonstrated that we could further improve the compound's potency of anti-HIF-1α and anti-angiogenesis by modifying the identified lead to synthesize a superior (novel) drug.

Patterning hypoxic multicellular spheroids in a 3D matrix - a promising method for anti-tumor drug screening.

3D multicellular spheroid models are of great value in the investigation of tumor biology and tumor responses to chemotherapy and radiation. To establish a mimicking tumor microenvironment in vitro, we developed a straightforward method by patterning hypoxic multicellular spheroids in a 3D matrix. The efficacy of this approach was evaluated by characterizing spheroid formation, invasive capability and phenotypic transition in aggressive human glioma cells. We observed enhanced cell proliferation, spheroid formation and invasive capability in U87 glioma cells transfected with hypoxia-inducible factors (HIFs) compared with non-treated cells. We also demonstrated that the overexpression of HIFs in hypoxic glioma cells may promote cell migration by epithelial-mesenchymal transition within the 3D matrix. Compared with conventional 3D culturing techniques, the simple operation, rapid prototyping, low cost and high throughput format of the micro-patterning method facilitates the characterization of cell proliferation, migration, phenotypic function and drug evaluation in physiologically relevant 3D microenvironments. This in vitro 3D system can recapitulate the physiologically relevant tumor microenvironment and is a promising method for 3D anti-tumor drug screening and the identification of novel targets for tumor invasion and angiogenesis.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth.

Hypoxia is a characteristic feature of solid tumors leading to the over expression of hypoxia-inducible factor (HIF)-1α protein and therefore to a specific cellular behavior. However, even though the oxygen tension in tumors is low (<5 %), most of the cell lines used in cancer studies are grown under 21 % oxygen tension. This work focuses on the impact of oxygen conditions during in vitro cell culture on glucose metabolism using 1-(13)C-glucose. Growing U87-MG glioma cells under hypoxic conditions leads to a two- to threefold reduction of labeled glutamine and an accumulation of fructose. However, under both hypoxic and normoxic conditions, glucose is used for de novo synthesis of pyrimidine since the (13)C label is found both in the uracil and ribose moieties. Labeling of the ribose ring demonstrates that U87-MG glioma cells use the reversible branch of the non-oxidative pentose phosphate pathway. Interestingly, stereotactic implantation of U87-MG cells grown under normoxia or mild hypoxia within the striatum of nude mice led to differential growth; the cells grown under hypoxia retaining an imprint of the oxygen adaptation as their development is then slowed down.

Hypoxia in breast cancer.

Breast cancer continues to be one of the most common malignancies and is a serious problem particularly in women, although men may also be affected. Such lesions are commonly accompanied by hypoxia, and therefore hypoxia-dependent mechanisms, such as overexpression of hypoxia-inducible factor (HIF), the mechanisms are studied as part of the search for a novel method of cancer treatment. Blocking the activity of HIF and HIF-dependent molecular changes raises hopes for identification of a molecular target to inhibit the tumor growth or even to completely prevent its progression. However, this is difficult due to the crucial role HIF plays in numerous processes occurring not only in cancer cells but mostly in healthy systemic cells in physiological conditions.

Oligomer procyanidins (F2) isolated from grape seeds inhibits tumor angiogenesis and cell invasion by targeting HIF-1α in vitro.

Overexpression of hypoxia-inducible factor-1 (HIF-1) α, a transcription factor which immortalizes tumors by inducing expression of the genes involved in cell survival, migration and angiogenesis, is closely associated with poor prognosis, increased risk of metastasis and increased mortality. Oligomer procyanidins (F2), a natural fraction from grape seeds, has been demonstrated to have antioxidant and antitumor activities, however the antitumor effect of F2 targeting HIF-1α remains unknown. The present study showed that F2 markedly decreased HIF-1α and the expression of its target genes in cancer cells through inactivating the EGFR-PI3K-AKT-mTOR and MAPK-ERK1/2 pathways. Moreover, F2 suppressed vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 expressions, followed by the inhibition of tumor angiogenesis and cell invasion in a HIF-1α-dependent manner. Collectively, these findings indicate that the antitumor effect of F2 is, at least in part, mediated by suppressing HIF-1α-dependent pathway, and suggest that F2 may be a potentially useful agent for treatment of human cancer.

Deletion of the von Hippel-Lindau gene causes sympathoadrenal cell death and impairs chemoreceptor-mediated adaptation to hypoxia.

Mutations of the von Hippel–Lindau (VHL) gene are associated with pheochromocytomas and paragangliomas, but the role of VHL in sympathoadrenal homeostasis is unknown. We generated mice lacking Vhl in catecholaminergic cells. They exhibited atrophy of the carotid body (CB), adrenal medulla, and sympathetic ganglia. Vhl-null animals had an increased number of adult CB stem cells, although the survival of newly generated neuron-like glomus cells was severely compromised. The effects of Vhl deficiency were neither prevented by pharmacological inhibition of prolyl hydroxylases or selective genetic down-regulation of prolyl hydroxylase-3, nor phenocopied by hypoxia inducible factor overexpression. Vhl-deficient animals appeared normal in normoxia but survived for only a few days in hypoxia, presenting with pronounced erythrocytosis, pulmonary edema, and right cardiac hypertrophy. Therefore, in the normal sympathoadrenal setting, Vhl deletion does not give rise to tumors but impairs development and plasticity of the peripheral O2-sensing system required for survival in hypoxic conditions.

Abstract 1824: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to chemotherapeutic agents and are associated with poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three dimensional (3D) cultures are decreased under hypoxic conditions (1%O2) Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. Moreover, hypoxia-mediated activation of ERK signaling, but not AKT, is required for lapatinib resistance as MEK inhibitors (including clinically approved tramatinib) reverses hypoxia-mediated lapatinib resistance in standard and 3D cultures. HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway via its regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression via RNAi in ErbB2-positive breast cancer cells is sufficient to reduce lapatanib-mediated effects under normal oxygen. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting MEK/ERK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. Citation Format: Sergey Karakashev. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2014-1824

Hypoxia inducible factor (HIF)-1α directly activates leptin receptor (Ob-R) in pancreatic cancer cells

The aim of this study is to investigate the regulatory mechanism of leptin receptors (Ob-R) in pancreatic cancer. We found that the over-expression of hypoxia inducible factor (HIF-1)α and hypoxia up-regulated the expression of Ob-R in pancreatic cancer cells. When HIF-1α gene was silenced in vitro, the expression of Ob-R was significantly decreased. Xenograft mouse models showed that the inhibition of HIF-1α resulted in the concomitant decrease of Ob-R in vivo. In addition, HIF-1α expression was correlated with Ob-R in pancreatic cancer tissues by immunohistochemical staining. Clinical data showed that over-expression of HIF-1 was associated with pathological tumor node metastasis stage, lymph node metastasis and overall survival. HIF-1α directly bound to the hypoxia-responsive element (HRE) located in Ob-R gene promoter (−828/−832) and activated the transcription. Finally, we demonstrated that the silence of HIF-1α gene reversed the inhibitory effect of leptin/Ob-R in pancreatic cancer cells. Taken together, our results indicate that HIF-1α directly regulated Ob-R expression in pancreatic cancer, which might be a valuable therapeutic target for pancreatic cancer.

Synthesis and structure–activity relationships of novel, potent, orally active hypoxia-inducible factor-1 inhibitors

Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC50=560nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with anti-HIF-1 activities in vitro (IC50=21 and 0.47nM, respectively), and in vivo. Additionally, 43a (12.5–100mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.

HIFs enhance the migratory and neoplastic capacities of hepatocellular carcinoma cells by promoting EMT.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers. Although the involvement of the epithelial-to-mesenchymal transition (EMT) in HCC progression is established, the mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, modulate the EMT through Twist1, a regulator of the EMT. The levels of HIF-1α and HIF-2α in HCC tissues were higher than those in matched, non-tumor surrounding tissues. In HCC samples, the levels of HIF-1α and HIF-2α negatively correlated with the levels of E-cadherin but positively correlated with the levels of vimentin. In highly metastatic MHCC97H cells, the levels of HIF-1α, HIF-2α, and Twist1 were higher than those in weakly metastatic HepG2 cells. In HepG2 cells, over-expression of HIFs enhanced levels of Twist1 and the EMT, which elevated the migratory and neoplastic capacities of cells. For MHCC97H cells, inhibition of HIFs reduced Twist1 levels and the EMT, which reduced their migratory and neoplastic capacity. Thus, the promotion of EMT by HIFs via Twist1 enhanced the migration and neoplastic capacities of HCC cells.

The Role of Hypoxia-Inducible Factor in Wound Healing

Significance: Poor wound healing remains a significant health issue for a large number of patients in the United States. The physiologic response to local wound hypoxia plays a critical role in determining the success of the normal healing process. Hypoxia-inducible factor-1 (HIF-1), as the master regulator of oxygen homeostasis, is an important determinant of healing outcomes. HIF-1 contributes to all stages of wound healing through its role in cell migration, cell survival under hypoxic conditions, cell division, growth factor release, and matrix synthesis throughout the healing process. Recent Advances: Positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic ischemic wound closure and are currently undergoing clinical trials for treatment of several human-ischemia-based conditions. Critical Issues: HIF-1 deficiency and subsequent failure to respond to hypoxic stimuli leads to chronic hypoxia, which has been shown to contribute to the formation of nonhealing ulcers. In contrast, overexpression of HIF-1 has been implicated in fibrotic disease through its role in increasing myofibroblast differentiation leading to excessive matrix production and deposition. Both positive and negative regulators of HIF-1 therefore provide important therapeutic targets that can be used to manipulate HIF-1 expression where an excess or deficiency in HIF-1 is known to correlate with pathogenesis. Future Directions: Targeting HIF-1 during wound healing has many important clinical implications for tissue repair. Counteracting the detrimental effects of excessive or deficient HIF-1 signaling by modulating HIF-1 expression may improve future management of poorly healing wounds.

Increased HIF-1α Expression Correlates With Cell Proliferation and Vascular Markers CD31 and VEGF-A in Uveal Melanoma

Overexpression of hypoxia inducible factor-1 α (HIF-1α) has been found in several cancers and is thought to correlate with aggressive disease. The purpose of our study was to investigate the influence of HIF-1α on clinical outcome in uveal melanoma (UM) along with proliferative (MIB-1) and vascular (CD31, VEGF-A) markers. A retrospective analysis was carried out on UM tumors from 88 patients. HIF-1α, MIB-1, CD31, and VEGF-A expression, as well as necrosis, were assessed by immunohistochemistry and hematoxylin/eosin on paraffin-embedded UM tumor sections by using a tissue microarray. The bivariate analysis involving HIF-1α expression and clinicopathologic covariates was performed by using the χ(2) test. The association of clinicopathologic covariates and HIF-1α expression with patient survival was evaluated by using the Kaplan-Meier approach and Cox proportional-hazards regression analysis. Among our study population, 56 patients (63.6%) had high levels of HIF-1α expression. High expression of HIF-1α was associated with high expression of MIB-1 (P = 0.04), CD31 (P = 0.03), and VEGF-A (P < 0.0001), as well as necrosis (P = 0.04). However, high HIF-1α expression was not correlated with cell type, largest macroscopic tumor dimension or thickness, anterior margin, pigmentation, or mitotic figures. Patients with high HIF-1α expression did not show a reduced survival when compared to patients with low HIF-1α expression (P = 0.92). Finally, HIF-1α expression was not increased after irradiation. An increase in HIF-1α expression was significantly associated with proliferative (MIB-1) and vascular (CD31 and VEGF-A) markers, as well as necrosis, in UM. However, there was no correlation between high HIF-1α expression and patient survival.

Abstract PR15: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, metabolism, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib (Tykerb), a small-molecule kinase inhibitor that specifically blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the exact mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to different chemotherapeutic agents and poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition is reduced under hypoxic conditions (1%O2). Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. One possible mechanism of how HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting ERK/MAPK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. This abstract is also presented as poster C63. Citation Format: Sergey V. Karakashev, Reginato J. Mauricio. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR15.

HIF-1-modified BMSCs improve migration and reduce neuronal apoptosis after stroke in rats

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated ameliorating neurologic deficits after stroke. Hypoxia-inducible factor-1 (HIF-1), the key regulator of cellular responses to low oxygen concentration, can activate multiple genes involving in crucial aspects for neurologic recovery. In this study, we present that rat BMSCs overexpression of HIF-1 α showed higher expression of HIF-1 target genes in HIF-1-BMSCs, including CXCR4, EPO, and VEGF. BMSCs-mHIF-1α also exhibited an enhanced mobility towards the ischemic area within rat brain. Neural cell apoptosis in ischemic brain shown less severe in rats transplanted with HIF-1-BMSCs. Furthermore, the number of cells expressing neural progenitor markers PAX6 and DCX were increased in BMSCs-mHIF-1α-transplanted rats. These results suggest that HIF-1α in BMSCs reduces neuronal apoptosis and promotes neurogenesis after stroke in rats.

Hypoxia-Inducible Factors Activate CD133 Promoter through ETS Family Transcription Factors

CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1–P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

Expression of aldehyde dehydrogenase after neoadjuvant chemotherapy is associated with expression of hypoxia-inducible factors 1 and 2 alpha and predicts prognosis in locally advanced breast cancer

OBJECTIVE:To analyze the expression of hypoxia-inducible factors (hypoxia-inducible factor 1A and hypoxia-inducible factor 2A) and aldehyde dehydrogenase proteins in patients with locally advanced breast carcinoma who were subjected to neoadjuvant chemotherapy.METHODS:We included 90 patients with histologically confirmed stage II and III breast carcinoma who were treated with neoadjuvant chemotherapy between 2000 and 2005. Immunohistochemistry for aldehyde dehydrogenase, hypoxia-inducible factor 1A, and hypoxia-inducible factor 2A was performed before and after neoadjuvant chemotherapy. We analyzed the influence of clinical and pathological features on clinical and pathological response, disease-free survival, and overall survival.RESULTS:An objective clinical response to neoadjuvant chemotherapy was observed in 80% of patients, with 12% showing a complete pathological response. Among all clinical and pathological parameters, only the expression of hypoxia-inducible factor 1A was associated with a pathological response. A positive association was found between expression of aldehyde dehydrogenase and that of hypoxia-inducible factor 1A before and after chemotherapy. Aldehyde dehydrogenase expression was associated with expression of hypoxia inducible-factor 2A in tumors after neoadjuvant treatment. In a univariate analysis, prognosis was influenced by age, pathological response, metastasis to axillary lymph nodes after neoadjuvant chemotherapy, overexpression of hypoxia-inducible factor 2, and the presence of aldehyde dehydrogenase-positive cells within the primary tumor after neoadjuvant chemotherapy. In a multivariate analysis, only age and the presence of aldehyde dehydrogenase-positive cells after chemotherapy were associated with reduced overall survival.CONCLUSION:The presence of aldehyde dehydrogenase-positive cells within the residual tumor after neoadjuvant chemotherapy is associated with an increase in the expression of hypoxia-inducible factor 2A and with poor prognosis in patients with locally advanced breast cancer.

The Repressor Element‐1 Silencing Transcription Factor (REST) regulates the Hypoxia Inducible Factor (HIF) network through a novel negative feedback loop

Hypoxia induces a number of metabolic changes with profound consequences for cell physiology and pathophysiology. Our understanding of the complex response to hypoxia has been focused on the Hypoxia‐Inducible Factor (HIF) and its regulation by prolyl‐hydroxylases (PHD). In solid tumours, both intra‐tumoral hypoxia and mutations in tumour suppressor genes can lead to the overexpression of HIF, a feature that has been associated with poor prognosis.The Repressor Element‐1 Silencing Transcription Factor (REST), is best characterized as a transcriptional repressor of neuronal genes in non‐neuronal tissues. More recently, REST was found to be affected by ischemia and to be down regulated in breast and colon cancer. This lead us to investigate if REST was an hypoxic sensitive transcription factor and if it interacts with the HIF pathway.Hypoxia induced an increase in REST translocation to the nucleus. siRNA knockdown of REST increased HIF, PHD2 and PHD3 protein expression. REST was found to be recruited to the HIF promoter, and HIF was recruited to the REST promoter in a hypoxia dependent way, indicating a novel negative feedback loop involving REST and HIF. In conclusion, REST was identified as a novel hypoxia‐sensitive transcriptional repressor that negatively regulates HIF, PHD2/3, acting as a novel negative feedback loop in the hypoxia response.

Artemisinin and Rapamycin Compromise Nitric Oxide-driven and Hypoxia-triggered Acute Articular Synovitis in Mice

To investigate the etiological cause of rheumatoid arthritis (RA) and reveal the mechanism underlying the artemisinin and rapamycin against RA, the present work has established a mouse model of the complete Freund’s adjuvant (CFA)-induced acute arthritis (CIAA). The inflammation morphological and histopathological comparisons of CIAA with the classic collagen-induced arthritis (CIA) indicated that CIAA perfectly simulates CIA in articular erythema and edema, synovial hyperplasia, and inflammatory infiltration in a substantially shortened modeling time (from 28 to 3 d). Intra-articular injection with CFA allows global up-regulation of pro-inflammatory cytokine genes, dramatic burst of nitric oxide (NO), overexpression of hypoxia inducible factor 1 alpha (HIF-1α) and vascular epithelial growth factor (VEGF), and synovial angiogenesis and hyperplasia. Articular injection of mice with the NO donor compound sodium nitroprusside (SNP) also causes articular erythema and edema, whereas co-administration of CFA with the NO synthetic inhibitor NG-monomethyl-L-arginine (L-NMMA) abrogates articular inflammation. SNP activates pro-inflammatory cytokines but suppresses inducible NO synthase within three days, and upregulates HIF-1α and VEGF within two weeks. Pre- and post-injection of modeling CIAA mice with artesunate and/or rapamycin reverses hypoxia-induced expression of HIF-1α and VEGF, synovial angiogenesis, tumor-like hyperplasia, and lymphocytic inflammatory infiltration. While artesunate fails to completely repress CFA-induced immune activation, rapamycin down-regulates CFA-triggered expression of pro-inflammatory cytokines. Nevertheless, both artesunate and rapamycin are able to inhibit NO production. Conclusively, NO serves as a pivotal mediator of RA pathogenesis, and artemisinin and rapamycin, by blocking NO generation, hold potential promise in prophylactic and therapeutic interventions of RA patients.

The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.