Hypoxia-inducible Factor Subunits Research Articles

Hypoxia-Inducible Factor: Pattern of the Effects and Their Dualism

This review describes the peculiarities of the molecular structure of hypoxia-inducible factor (HIF) and the mechanisms of activation of a complex including HIF, a key factor of adaptation of the cell to ischemic and hypoxic injuries. The tissue specificity of expression of different subtypes of the α HIF subunit and most typical target genes activated by this complex are described. In addition, the specificities of HIF-mediated cell responses related to ischemic damage (the latter, at present, forms one of the main medical/clinical problems) are analyzed; the expedience and prospects of further studies of the signal pathways involving the mentioned transcription factor are emphasized.

The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.

Gain of HIF-1α under Normoxia in Cancer Mediates Immune Adaptation through the AKT/ERK and VEGFA Axes

Adaptation to host immune surveillance is now recognized as a hallmark of cancer onset and progression, and represents an early, indispensable event in cancer evolution. This process of evolution is first instigated by an immune selection pressure imposed by natural host surveillance mechanisms and may then be propagated by vaccination or other types of immunotherapy. We developed a system to simulate cancer evolution in a live host and to dissect the mechanisms responsible for adaptation to immune selection. Here, we show that the oxygen-sensitive α subunit of hypoxia-inducible factor 1 (HIF-1α) plays a central role in cancer immune adaptation under conditions of normal oxygen tension. We found that tumor cells gain HIF-1α in the course of immune selection under normoxia and that HIF-1α renders tumor cells resistant to lysis by tumor-specific cytotoxic T lymphocytes (CTL) in culture and in mice. The effects of HIF-1α on immune adaptation were mediated through VEGFA-dependent activation of the AKT and ERK signaling pathways, which induced an antiapoptotic gene expression network in tumor cells. Our study therefore establishes a link between immune selection, overexpression of HIF-1α, and cancer immune adaptation under normoxia, providing new opportunities for molecular intervention in patients with cancer.

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

Abstract 1014: Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies

Abstract Traditional anticancer therapies are limited in their efficacy because they often lead to drug-resistant tumors and display toxicity to normal cells. Control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.Chronic hypoxia, a hallmark of many solid tumors is responsible for the elevated levels of VEGF and other pro-proliferative factors. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, and pro-proliferative mechanisms that promote tumor growth. The specific hypothesis behind the proposed research is that angiogenesis could be effectively controlled by down-regulation of hypoxia-inducible gene expression via a disruption of key transcription factor-coactivator interactions with designed molecules. The team at BioCycive and its collaborators at USC are currently developing such compounds that block hypoxia-inducible transcription of key genes. We have designed and synthesized a novel dimeric epidithiodiketopiperazine, ETP2, which targets pleiotropic coactivator p300 disrupts HIF1α-p300 complex in vitro and effectively down-regulates hypoxia-inducible signaling and gene expression. ETP 2 effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of breast and lung cancer cell lines. We have shown excellent antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. A significant decrease in the vascularization of these tumors upon treatment with ETP 2 was also noticed, which is consistent with the proposed mechanism of inhibition of vascularization via down-regulation of hypoxia-inducible signaling.Our efficacy studies with renal carcinoma cells lines resulted in similar findings, where we successfully reached the primary endpoint of efficacy (reduction in tumor volume of >50%) and secondary endpoint of survival. Nude mice engrafted with 786-O (renal cell carcinoma) were treated with ETPs at 1 mg/kg given IV every three days for 45 days. All tumors were imaged at the end of the study and harvested for histopathology studies. MTD studies clearly indicated that ETP 2 was non-toxic within the range of injected doses, with an average MTD of 21.1 mg/kg. Our preliminary PK studies show that the ETP 2 is a lipophilic molecule, with a plasma half-life of 3.5-5 days. Our results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance. Citation Format: Ramin Dubey, Ivan Grishagin, Usha Nagavarapu, Chenera Balan, Shalabh Gupta, Bogdan Z. Olenyuk. Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies. [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 1014. doi:10.1158/1538-7445.AM2014-1014

Abstract 4388: Targeted VHL activation by RNAa inhibits renal cell carcinoma

Abstract Von Hippel Lindau (VHL) is a tumor suppressor protein classically known to target the α subunit of hypoxia inducible factor (HIF) for proteasomal degradation. Emerging evidence has underscored a novel role for VHL in both cell cycle regulation and extracelluar matrix assembly. Recent studies have reported that chemically synthesized small duplex RNAs complementary to target promoter sequence, also known as small activating RNA (saRNA), can specifically induce gene expression in cancer cells, which might be associated with epigenetic changes and exhibited long-lasting gene activation for nearly 2 weeks. In the present study, we designed 5 candidate dsRNAs (dsVHL-58, dsVHL-93, dsVHL-395, dsVHL-515 and dsVHL-675) targeted for the VHL promoter at sites ranging from -675 to -58 relative to the transcription start site. Compared with controls, dsVHL-675 induced VHL expression by approximately 10-fold in renal cell carcinoma 769-P (VHL methylated) cells. Transfection of a dsVHL-675 into 769-P cells significantly inhibited cell proliferation in dose dependent manner. In addition, single transfection of dsVHL-675 induced VHL gene expression for 14 days. Functional analysis of dsVHL-675 transfected cells revealed that dsVHL-675 induced apoptosis by activating caspase 3 in dose dependent manner. Knockdown experiments confirmed that induction of apoptosis by dsVHL-675 required Ago2 and TNRC6A. This study suggests that targeted activation of VHL by dsVHL-675 may be explored as a novel therapy for the treatment of renal cell carcinoma. Citation Format: Jong Soon Kang, Moo Rim Kang, Jieun Yun, Soo Jin Oh, Ki Hwan Park, Chang Woo Lee. Targeted VHL activation by RNAa inhibits renal cell carcinoma. [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 4388. doi:10.1158/1538-7445.AM2014-4388

Isoflurane reduces the ischemia reperfusion injury surge: A longitudinal study with MRI

BackgroundRecent studies show neuroprotective benefits of isoflurane (ISO) administered during cerebral ischemia. However, the available studies evaluated cerebral injury only at a single time point following the intervention and thus the longitudinal effect of ISO on ischemic tissues remains to be investigated. Objective: The objective of the present study was to investigate the longitudinal effect of ISO treatment in counteracting the deleterious effect of ischemia by evoking the transcription factor, hypoxia inducible factor-1 (HIF-1), and vascular endothelial growth factor (VEGF). Methods: Focal cerebral ischemia was induced in 70 rats by filament medial cerebral artery occlusion (MCAo) method. MCAo rats were randomly assigned to control (90min ischemia) and MCAo+ISO (90min ischemia+2% ISO) groups. Infarct volume, edema, intracerebral hemorrhage (ICH), and regional cerebral blood flow (rCBF) were measured in eight in vivo sequential MR imaging sessions for 3 weeks. Western blot analysis and immunofluorescence were used to determine the expression level of HIF-1α (the regulatable subunit of HIF-1) and VEGF proteins. Results: ISO inhalation during ischemia significantly decreased the surge of infarct volume, edema, ICH, and reduced the mortality rate (p<0.01). ISO transiently altered the rCBF, significantly enhanced the expression of HIF-1α and VEGF, and decreased the immune cell infiltration. Locomotor dysfunction was ameliorated at a significantly faster pace, and the benefit was seen to persist up to three weeks. Conclusion: Treatment with ISO during ischemia limits the deadly surge in the dynamics of ischemia reperfusion injury with no observed long-term inverse effect.

Prolyl-hydroxylase PHD3 interacts with pyruvate dehydrogenase (PDH)-E1β and regulates the cellular PDH activity

Cells are frequently exposed to hypoxia in physiological and pathophysiological conditions in organisms. Control of energy metabolism is one of the critical functions of the hypoxic response. Hypoxia-Inducible Factor (HIF) is a central transcription factor that regulates the hypoxic response. HIF prolyl-hydroxylase PHDs are the enzymes that hydroxylate the α subunit of HIF and negatively regulate its expression. To further understand the physiological role of PHD3, proteomics were used to identify PHD3-interacting proteins, and pyruvate dehydrogenase (PDH)-E1β was identified as such a protein. PDH catalyzes the conversion of pyruvate to acetyl-coA, thus playing a key role in cellular energy metabolism.PDH activity was significantly decreased in PHD3-depleted MCF7 breast cancer cells and PHD3−/− MEFs. PHD3 depletion did not affect the expression of the PDH-E1α, E1β, and E2 subunits, or the phosphorylation status of E1α, but destabilized the PDH complex (PDC), resulting in less functional PDC. Finally, PHD3−/− cells were resistant to cell death in prolonged hypoxia with decreased production of ROS.Taken together, the study reveals that PHD3 regulates PDH activity in cells by physically interacting with PDC.

Sumoylation of hypoxia inducible factor-1α and its significance in cancer.

Hypoxia-inducible factor-1 (HIF-1) is a key heterodimeric transcription factor for the cellular adaptive response to hypoxia, a common feature of the microenvironment in solid tumors. The transcriptional activity, protein stabilization, protein-protein interactions and cellular localization of HIF-1α, an oxygen-sensitive subunit of HIF-1, are mainly modulated by various post-translational modifications. Recently, we reported that polycomb chromobox 4 (Cbx4) governs the transcriptional activity of HIF-1α by enhancing its sumoylation at K391 and K477, through which Cbx4 potentiates angiogenesis of hepatocellular carcinoma. This review summarizes the current knowledge of HIF-1α sumoylation and its roles in the pathogenesis of cancer.

Ischemia induces different levels of hypoxia inducible factor-1α protein expression in interneurons and pyramidal neurons.

IntroductionPyramidal (glutamatergic) neurons and interneurons are morphologically and functionally well defined in the central nervous system. Although it is known that glutamatergic neurons undergo immediate cell death whereas interneurons are insensitive or survive longer during cerebral ischemia, the protection mechanisms responsible for this interneuronal survival are not well understood. Hypoxia inducible factor-1 (HIF-1) plays an important role in protecting neurons from hypoxic/ischemic insults. Here, we studied the expression of HIF-1α, the regulatable subunit of HIF-1, in the different neuronal phenotypes under in vitro and in vivo ischemia.ResultsIn a primary cortical culture, HIF-1α expression was observed in neuronal somata after hypoxia (1% oxygen) in the presence of 5 or 25 mM glucose but not under normoxia (21% oxygen). Interestingly, only certain MAP2-positive neurons containing round somata (interneuron-like morphology) co-localized with HIF-1α staining. Other neurons such as pyramidal-like neurons showed no expression of HIF-1α under either normoxia or hypoxia. The HIF-1α positive neurons were GAD65/67 positive, confirming that they were interneuron-type cells. The HIF-1α expressing GAD65/67-positive neurons also possessed high levels of glutathione. We further demonstrated that ischemia induced significant HIF-1α expression in interneurons but not in pyramidal neurons in a rat model of middle cerebral artery occlusion.ConclusionThese results suggest that HIF-1α protein expression induced by ischemia is neuron-type specific and that this specificity may be related to the intracellular level of glutathione (GSH).

Hsp27 and its expression pattern in diffusely infiltrating astrocytomas.

Heat shock protein 27 (Hsp27) is induced by cell stress conditions. In the presence of oxidative stress it functions as an antioxidant. To study the putative expression patterns and clinical significance of Hsp27, we assessed the associations between Hsp27, R132H mutation of Isocitrate dehydrogenase1 (IDH1-R132H), Hypoxia-inducible factor subunit alpha (HIF-1 alpha), Carbonic anhydrase IX (CA IX), and patient prognosis in astrocytic gliomas. Tissue micro-array samples of 295 grade II-IV astrocytomas were stained immunohistochemically for Hsp27, IDH1-R132H, HIF-1 alpha, and CA IX. We tested their relationship with clinicopathological features and patient survival. There was a significant correlation between Hsp27 expression and increasing WHO grade (p<0.001). Hsp27 expression correlated significantly with IDH1 mutation when studied within the entire cohort (p<0.001) as well as separately in WHO grade II and III tumors (p=0.006 and 0.002, respectively). IDH1 mutation and HIF-1 alpha positive staining were detected simultaneously (p<0.001). In IDH1 mutated tumors, positive HIF-1 alpha staining correlated with CA IX expression (p=0.027), whereas no such correlation was found in IDH1 non-mutated tumors. IDH1 mutation was associated with a low cell proliferation index (p=0.001) and HIF-1 alpha with increasing proliferation (p = 0.003). Hsp27 expression was associated with a shorter rate of patient survival in univariate survival analysis (p=0.001). In multivariate survival analysis, patient age, IDH1 mutation and HIF-1 alpha appeared as independent prognostic factors (p<0.000, <0.000 and 0.011 respectively) Hsp27 expression is associated with increasing WHO grade and patient prognosis in astrocytic gliomas. The results suggest that IDH1 mutation may have an effect on the expression pathways of Hsp27 and CA IX.

An Insertion/Deletion Polymorphism Within the Proximal Promoter of EGLN2 Is Associated With Susceptibility for Gastric Cancer in the Chinese Population

Gastric cancer (GC) is among the most common human malignancies and the second leading cause of cancer-related death worldwide. Accumulated evidence from molecular genetics indicates that an individual's genetic factors are involved in their susceptibility to GC. Hypoxia is a common feature of cancer and the hypoxia-inducible factor (HIF), a transcription factor that regulates oxygen homeostasis, plays key roles in the growth of solid tumors and regulating cellular responses to hypoxia. Prolyl hydroxylase (PHD1, also known as EGLN2) is one of the three enzymes capable of hydroxylating the alpha subunit of HIF and results in polyubiquitinylation and proteasomal degradation of HIF. A case-control study, including 415 GC patients and 830 healthy controls, was conducted to investigate the association between GC susceptibility with a 4-bp insertion/deletion polymorphism (rs10680577) in the proximal promoter of EGLN2. Logistic regression analysis showed that the heterozygote and the homozygote 4-bp del/del confer a significantly increased risk of GC after controlling for other covariates (adjusted odds ratio [OR]=1.35, 95% confidence interval [CI] 1.05-1.75, p=0.017; OR=2.19, 95% CI 1.15-4.18, p=0.009, respectively). Carriage of the 4-bp deletion allele was associated with a greatly increased risk of developing the disease (OR=1.38, 95% CI 1.12-1.70, p=0.002). Moreover, stratification analysis showed that the association was more prominent in smokers (adjusted OR=2.09, 95% CI=1.40-3.12, p for heterogeneity=0.01). Our data suggested that common genetic polymorphisms in EGLN2 may influence GC risk in the Chinese population. Considering the relative small sample size, replication in other populations with a larger sample size and further functional analysis are required for fully understanding the roles of EGLN2 polymorphisms in predisposition for GC.

Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer

IntroductionAlthough aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2.MethodsIn vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance.ResultsBasal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP.ConclusionsOne of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.

P-212 A Role for Hypoxia-Inducible Factor (HIF) in Intestinal Epithelial Junctional Assembly and Barrier Function

The gastrointestinal mucosa constitutes the largest mucosal surface found in multicellular organisms, and provides the primary physical barrier against gut luminal contents. Intestinal epithelial cells that line the GI tract selectively permit flux of nutrients and water, while protecting against omnipresent luminal antigens. In mucosal diseases such as inflammatory bowel disease (IBD), hypoxia and inflammation occur coincidentally at the level of the epithelium. Adaptive transcriptional responses to oxygen deprivation are mediated primarily through the hypoxia-inducible factor complex (HIF), comprising an oxygen-labile “alpha” subunit and constitutively expressed “beta” subunit. A role for HIF has been implicated in orchestrating a signaling program that promotes intestinal epithelial function and homeostasis. Disruption of the epithelial barrier by proinflammatory cytokines such as IFN-gamma defines a major pathophysiological consequence of intestinal inflammation in IBD. We previously demonstrated that HIF activity and expression in intestinal epithelial cells (IECs) is prominently repressed at the transcriptional level by IFN-gamma. Given the concurrent expression of both HIF-1alpha and HIF-2alpha isoforms of the HIF transcriptional complex in IECs, we sought to define the relative contribution of HIF-1 and HIF-2 to epithelial barrier function. Human intestinal epithelial cell lines T84 and Caco-2 were used throughout. Stable knockdown lines targeting individual HIF subunits were generated using shRNA lentiviral transduction particles (MISSION TRC). To monitor apical junction assembly after calcium switch, T84 cells plated on transwell inserts were incubated for 16h in low Ca2+ medium (LCM) or 5 mins in HBSS with 2mM EDTA before switching to normal Ca2+ conditions (1.8 mM). Transepithelial resistance (TER) was measured over time using a voltohmeter, and expressed in ohm.cm2. Stable shRNA-mediated knockdown of HIF-1alpha, HIF-2alpha and HIF-1beta in model T84 epithelial cells resulted in significantly increased transcellular and paracellular permeability, as measured by transepithelial resistance and FITC-dextran flux assays. Immunolocalization studies of tight junction and adherens junction markers ZO-1 and E-cadherin, respectively, revealed non-uniform, undulating junctions in HIF-1alpha depleted T84 monolayers, consistent with attenuated intercellular tension and apical junction formation under conditions of altered HIF signaling. IFNgamma treatment of shControl IECs resulted in decreased barrier function and a junctional morphology similar to that observed in HIF-1alpha knockdown epithelia. Moreover, western blot analyses of shControl and knockdown lysates revealed increased myosin light chain (MLC) phosphorylation in HIF-1alpha knockdown IECs, indicative of an activated myosin II motor and increased perijunctional actomyosin contractility. Finally, inhibition of myosin light chain kinase (MLCK) or the Rho-associated protein kinases (ROCK) in HIF knockdown epithelia attenuated barrier defects and improved junctional morphology. These results define a role for HIF signaling in maintenance of apical IEC junctions at the level of the perijunctional actomyosin ring, and may in part explain the pathomechanism of IFN-gamma mediated loss of junctional integrity.

Is NMR Fragment Screening Fine-Tuned to Assess Druggability of Protein–Protein Interactions?

Modulation of protein–protein interactions (PPIs) with small molecules has been hampered by a lack of lucid methods capable of reliably identifying high-quality hits. In fragment screening, the low ligand efficiencies associated with PPI target sites pose significant challenges to fragment binding detection. Here, we investigate the requirements for ligand-based NMR techniques to detect rule-of-three compliant fragments that form part of known high-affinity inhibitors of the PPI between the von Hippel–Lindau protein and the alpha subunit of hypoxia-inducible factor 1 (pVHL:HIF-1α). Careful triaging allowed rescuing weak but specific binding of fragments that would otherwise escape detection at this PPI. Further structural information provided by saturation transfer difference (STD) group epitope mapping, protein-based NMR, competitive isothermal titration calorimetry (ITC), and X-ray crystallography confirmed the binding mode of the rescued fragments. Our findings have important implications for PPI druggability assessment by fragment screening as they reveal an accessible threshold for fragment detection and validation.

Hypoxia and HIFs in regulating the development of the hematopoietic system

Many physiologic processes during the early stages of mammalian ontogeny, particularly placental and vascular development, take place in the low oxygen environment of the uterus. Organogenesis is affected by hypoxia inducible factor (HIF) transcription factors that are sensors of hypoxia. In response to hypoxia, HIFs activate downstream target genes - growth and metabolism factors. During hematopoietic system ontogeny, blood cells and hematopoietic progenitor/stem cells are respectively generated from mesodermal precursors, hemangioblasts, and from a specialized subset of endothelial cells that are hemogenic. Since HIFs are known to play a central role in vascular development, and hematopoietic system development occurs in parallel to that of the vascular system, several studies have examined the role of HIFs in hematopoietic development. The response to hypoxia has been examined in early and mid-gestation mouse embryos through genetic deletion of HIF subunits. We review here the data showing that hematopoietic tissues of the embryo are hypoxic and express HIFs and HIF downstream targets, and that HIFs regulate the development and function of hematopoietic progenitor/stem cells.

Senescence-associated protein p400 is a prognostic marker in renal cell carcinoma

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause hereditary and sporadic renal cell carcinomas (RCCs). The best characterized function of VHL protein is suppression of the α subunit of hypoxia inducible factor (HIF). Additional VHL functions have been reported, including induction of senescence upon loss of VHL mediated by downregulation of the chromatin remodeling factor p400. Induction of senescence either by oncogene activation or inactivation of tumor suppressors is considered a critical feature of mammalian cells by which to suppress tumorigenesis. In the present study, we investigated the relationship between the expression of p400 and patient survival following RCC diagnosis taking advantage of a large and well-documented series of RCC patients with long-term follow-up information. The expression of p400 was measured by immunohistochemistry using a tissue microarray containing tumor tissue samples from 868 RCC patients. Chi-squared tests, Kaplan-Meier curves, Cox regression models and Spearman's rank correlation estimates were used to investigate the possible relationship between p400 expression and Ki-67 proliferative index, clinical and pathological characteristics and patient survival. Complete loss of p400 expression was detected in 64% of all tumor specimens, and decreased p400 expression was associated with advanced tumor stage, higher grade of malignancy and regional lymph node metastasis. Among well-differentiated RCCs, high proliferation (Ki-67 index>10) was found in 12% of carcinomas with an increased p400 expression, compared to 5% of RCCs with decreased p400 expression. Multiple Cox regression indicated that patients with low proliferative tumors and increased p400 expression had a 60% lower cancer-specific mortality risk compared to those affected by low proliferative RCCs with decreased p400 expression. In summary, patients affected by highly proliferative tumors with decreased p400 expression exhibit a poor prognosis by multiple Cox regression. Our data suggest that the highly proliferative, decreased-p400 subgroup of RCCs represents tumors that are characterized by a loss of the tumor-suppressive mechanism of senescence.

CAMP-response Element-binding Protein (CREB) and NF-κB Transcription Factors Are Activated during Prolonged Hypoxia and Cooperatively Regulate the Induction of Matrix Metalloproteinase MMP1

Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, whereas CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix and pulmonary metastasis in nude mice were inhibited after depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells.

English

Introduction Hypoxia-inducible factor is a key transcriptional factor involved in the cellular response to low levels of oxygen, hypoxia. Moreover, hypoxiainducible factor has been recently associated with a role in inflammation and immunity. Importantly, hypoxiainducible factor is regulated by the major inflammatory responsive transcription factor, nuclear factor-κB. These two major pathways have been intimately linked. On one hand, they share a number of common target genes; on the other hand, physical interactions between hypoxiainducible factor subunits and nuclear factor-κB have been observed. Even though the role of nuclear factor-κB over hypoxia-inducible factor is fairly well-known, the involvement of hypoxia-inducible factor over the nuclear factor-κB pathway is not. Given the overlap between these pathways, it would not be surprising to find a functional involvement of hypoxiainducible factor in processes where nuclear factor-κB is involved. In this review, we will describe the communalities between hypoxia-inducible factor and nuclear factor-κB pathways, highlighting the crosstalk that occurs in a variety of conditions. Conclusion Taken together all the communalities between hypoxia-inducible factor and nuclear factor-κB pathways, there is no doubt that a crosstalk occurs, which can potentially bring new insights for therapeutic intervention in situations of disease such as cancer, stroke or rheumatoid arthritis. Introduction Oxygen is essential for multicellular organisms. As such, being able to respond to variations in oxygen availability is a requirement for the survival and homoeostasis of the organism. Sensing and responding appropriately to oxygen changes is important for a variety of important physiological processes, which include high altitude living, intense exercise and embryo development. However, lowering of the oxygen concentration or availability (hypoxia) is part and/or contributes to a number of human pathologies, such as cancer, stroke/infarction, diabetes and ageing1–2. Understanding the molecular mechanisms controlling the cellular response to hypoxia is, therefore, of great importance. One master regulator of the cellular response to oxygen changes is the family of transcription factors, hypoxia-inducible factor (HIF). However, HIF activity has been associated with additional stimuli that do not involve changes in oxygen, such as infection and inflammation3. These findings led to the discovery that HIF is controlled by a transcription factor, mostly involved in immune responses, nuclear factorκB (NF-κB). In this review, we will highlight the shared features of these transcription factors, from activating stimulus to common targets. Discussion The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. HIF pathway At the molecular level, the cellular response to hypoxia relies on HIF. HIF was first identified in 1995 together with hypoxia response element (HRE, 5ʹ-RCGTG-3ʹ) of the erythropoietin gene (EPO). Further studies revealed that HIF is actually a heterodimeric complex comprising an αand a β-subunit, which exist as a series of isoforms: -1α, -2α, and 3α. HIF-1α is constitutively expressed, while HIF-2α and HIF-3α expression is restricted to a subset of tissues. Even though HIF-1β expression and protein are not dependent on oxygen changes, HIF-α subunits are extremely labile at normal oxygen levels. This occurs mostly at the protein level, with HIF-α half-life being very short (~ 5 min), while transcription changes in response to oxygen have not been widely reported thus far. The activity of the complex HIF-1α– HIF-1β is determined by the stabilisation of the α subunit during hypoxia. In the presence of oxygen (normoxia), HIF-α is regulated by a class of dioxygenases called prolyl hydroxylases (PHDs), of which four isoforms have been identified so far (PHD1, PHD2, PHD3 and PHD4). These proteins use iron, 2-oxoglutarate, ascorbate and molecular oxygen as co-factors to catalyse the hydroxylation of HIF-α. The hydroxylation of specific prolyl residues promotes the interaction of HIF with von Hippel-Lindauprotein (VHL) containing E3 ligase complex, which mediates proteasomalmediated * Corresponding author Email: s.rocha@dundee.ac.uk Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow street, DD1 5EH, United Kingdom

Abstract 95: Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics.

Abstract Although aromatase inhibitors (AIs) have been shown to be highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Studies suggest that acquired resistance to AIs (i.e., letrozole) involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has also linked hypoxia inducible factor 1 (HIF-1) and its target gene breast cancer resistance protein (BCRP, a stem cell marker) to HER2 and AI resistance. Other studies have associated each of these factors with cancer stem cells (CSCs),, which have been implicated in drug resistance. Thus, the purpose of this study is to 1) determine whether HIF-1 expression is enriched in the CSC subpopulation of letrozole-resistant breast cancer cells; and 2) determine the effects of inhibiting HIF-1 on CSC characteristics. The in vitro model used for acquired letrozole resistant breast cancer is the LTLTCa cell line, which was obtained through long-term letrozole treatment of MCF-7Ca xenograft tumors. The CSC subpopulation in LTLTCa cells was isolated by flow cytometry based on their aldehyde dehydrogenase (ALDH) expression, and the CSC characteristics studied include mammosphere formation, side population percentage, and CD44+/CD24+ expression. Inhibition of HIF-1 expression was accomplished using either commercially available HIF-1α siRNA or EZN-2968, a specific RNA antagonist against HIF-1α currently in clinical trials. HIF-1α is the inducible subunit of HIF-1. RT-PCR indicates that mRNA expression of HIF-1α and stem cell markers, such as BCRP, BMI-1, and Nanog, were significantly higher (p&amp;lt;0.05) in ALDHhigh (CSC) vs. ALDHlow (non-CSC) cells. HIF-1 siRNA and EZN-2968 decreased HIF-1α mRNA and protein expression in LTLTCa cells within 48h (0.4 to 0.01-fold vs. negative control siRNA/RNA antagonist). This correlated with decreases in mRNA expression of stem cell markers BCRP and BMI-1, and TWIST, an EMT marker and regulator of BMI-1. HIF-1α inhibition also reduced mammosphere formation by 33% (p&amp;lt;0.05). Interestingly, preliminary results indicate that while EZN-2968 decreased the ratio of CD44+/24+ expression (28% vs. 52% in negative control RNA antagonist) and side population percentage (8% vs. 16% in negative control RNA antagonist) in LTLTCa cells, the commercially available HIF-1α siRNA had no effect. These results suggest that nonhypoxic HIF-1 is involved in regulating cancer stem cell characteristics in letrozole-resistant breast cancer cells, and that EZN-2968 should be further explored in the prevention and/or treatment of AI-resistant breast cancer. EZN-2968 kindly provided by Enzon Pharmaceuticals. This work is funded by DOD Breast Cancer Research Program Postdoctoral Award (BC1039031). Citation Format: Armina A. Kazi, Preeti Shah, Amanda Schech, Gauri Sabnis, Saranya Chumsri, Angela Brodie. Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2013-95