Nuclear Translocation Of HIF-1α Research Articles

Migration enhancement and HIF-1α nuclear translocation of vascular endothelial cells by flow and hypoxic exposure

Migration enhancement and HIF-1α nuclear translocation of vascular endothelial cells by flow and hypoxic exposure

Anti-Angiogenic Potential of Novel 31kDa Protein of Zanthoxylum Rhesta is Mediated by Inhibition of HIF-1α Nuclear Translocation in Vivo

Anti-Angiogenic Potential of Novel 31kDa Protein of Zanthoxylum Rhesta is Mediated by Inhibition of HIF-1α Nuclear Translocation in Vivo

Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions

We investigated the effects of hypoxia-inducible factor (HIF)-1α on articular cartilage under mechanical stimulation and the associated mechanisms. Chondrocytes, isolated from articular cartilage from the knee, hip, and shoulder joints of Wistar rats, were subjected to 20 % tensile stress under hypoxic (5% O2) conditions for 24 h. HIF-1α and aggrecan expression was significantly enhanced with mechanical stimulation under hypoxia but not significantly altered with mechanical stimulation under normoxia. The nuclear translocation of HIF-1α was enhanced by mechanical stress under hypoxia. Under both normoxia and hypoxia, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) 5 expression was significantly reduced with mechanical stimulation compared to that in the group without mechanical stimulation. However, HIF-1α knockdown mitigated changes in aggrecan and ADAM-TS5 expression mediated by mechanical stimulation under hypoxia. The effects of treadmill running on HIF-1α production in the articular cartilage of rat knee joints were also analyzed. HIF-1α production increased in the moderate running group and decreased to the same levels as those in the control group in the excessive running group. This suggests that HIF-1α regulates aggrecan and ADAM-TS5 expression in response to mechanical stimulation under hypoxia and general mechanical stimulation in articular cartilage under hypoxia, while controlling cartilage homeostasis.

Development of a constant pressure perfused ex vivo model of the equine larynx.

Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to check the vitality and functionality of the isolated larynx, the vessels´ reaction to norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents was tested. Additionally, the contractility of the CAD was checked via electrical stimulation. To determine the extent of hypoxic alterations, lactate dehydrogenase (LDH) and lactate were measured and an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α), a key transcription factor in hypoxia, was performed. For this, a hypoxia-induced cell culture for HIF-1α was developed. The application of NE led to an expected vasoconstriction while NP caused the expected vasodilation. During a perfusion period of 352 ±20.78 min, LDH values were in the reference range and lactate values slightly exceeded the reference range at the end of the perfusion. HIF-1α nuclear translocation could reliably be detected in the hypoxia-induced cell cultures, but not in sections of the perfused CAD. With the approach presented here, a solid basis for perfusing equine larynges was established and may serve as a tool for further investigations of equine larynx disorders as well as a transferrable model for other species.

Astaxanthin mediated regulation of VEGF through HIF1α and XBP1 signaling pathway: An insight from ARPE-19 cell and streptozotocin mediated diabetic rat model

Breakdown of outer blood-retina barrier (BRB) has been associated with the pathogenesis of diabetic retinopathy (DR) and diabetic macular edema (DME). Vascular endothelial growth factor (VEGF) might play a detrimental role in the pathogenesis of DME, a major clinical manifestation of DR. In the present study, we investigated the inhibitory mechanism of astaxanthin on VEGF and its upstream signaling pathways under in vitro and in vivo conditions. Astaxanthin has been observed to downregulate VEGF expression under hyperglycemic (HG) and CoCl2 induced hypoxic conditions in ARPE-19 cells. There were compelling pieces of evidence for the involvement of transcription factors like HIF1α and XBP1 in the upregulation of VEGF under HG and hypoxic conditions. Thus, we investigated the role of astaxanthin in the expression and nuclear translocation of HIF1α and XBP1. The activation and translocation of HIF1α and XBP1 induced by HG or CoCl2 conditions were hindered by astaxanthin. Additionally, treatment with HIF1α siRNA and IRE1 inhibitor STF-083010 also inhibited the expression of VEGF induced by HG and CoCl2 conditions. These results indicated that the anti-VEGF property of astaxanthin might be associated with the downregulation of HIF1α and XBP1. Furthermore, astaxanthin mitigated the enhanced migration of retinal pigment epithelial (RPE) cells under DR conditions. As well, astaxanthin protected disorganization of zona occludin-1 (ZO-1) tight junction protein in RPE and reduced HG or hypoxic induced permeability of RPE cells. In streptozotocin-induced diabetic rat model, astaxanthin reduced the expression of HIF1α, XBP1, and VEGF as well as protected the abnormalities in the retinal layers induced by diabetes condition. Thus, astaxanthin may be used as a potential nutraceutical to prevent or treat retinal dysfunction in diabetic patients.

PLCε regulates metabolism and metastasis signalingvia HIF-1α/MEK/ERK pathway in prostate cancer.

Phospholipase C-ε (PLCε) is frequently overexpressed in tumors and plays an important role in the regulation of tumorigenesis. Although great progress has been made in understanding biological roles of PLCε, the relevant molecular mechanisms underlying its pro-tumor activity remain largely unclear. Here, we demonstrated that PLCε knockdown reduced cell metastasis, glucose consumption and lactate production in a manner that depended on hypoxia inducible factor 1α (HIF-1α) expression in prostate cancer cells. Interestingly, our findings showed that the expression levels of PLCε were positively associated with those of HIF-1α in clinical prostate carcinomasamples. Knockdown of PLCε impaired HIF-1α levels and transcriptional activity by regulating the extracellular-signal-regulated kinase pathway, and blocking HIF-1α nuclear translocation. Furthermore, PLCε could interact with the von Hippel-LindauE3 ligase complex to modulate the stability of HIF-1α. Collectively, our findings demonstrate that PLCε could be a crucial positive regulator of HIF-1α, which would promote PLCε-enhanced tumorigenesis.

Hypercapnia Exacerbates the Blood-Brain Barrier Disruption Via Promoting HIF-1a Nuclear Translocation in the Astrocytes of the Hippocampus: Implication in Further Cognitive Impairment in Hypoxemic Adult Rats.

Hypercapnia in combination with hypoxemia is usually present in severe respiratory disease in the intensive care unit (ICU) and can lead to more severe cognitive dysfunction. Increasing evidence has indicated that the compromised blood–brain barrier (BBB) in the hippocampus in hypoxemia conditions can result in cognitive dysfunction. However, the role and underlying mechanism of hypercapnia in the BBB disruption remains poorly known. A rat model of hypercapnia was first established in this study by intubation and mechanical ventilation with a small-animal ventilator. After this, the cognitive function of the experimental rats was assessed by the Morris water maze test. The BBB permeability was evaluated by the Evans Blue (EB) test and brain water content (BWC). Western blot analysis was carried out to detect the protein expressions of total and nuclear hypoxia-inducible factor-1α (HIF-1α), matrixmetalloproteinase-9 (MMP-9) and Aquaporins-4 (AQP-4) in the hippocampus tissue. Double immunofluorescence further verified the protein expression of different biomarkers was localized in the astrocytes of the hippocampus. Hypercapnia alone did not disrupt the BBB, but it could further enhance the BBB permeability in hypoxemia. Concomitantly, up-regulation of nuclear HIF-1α, AQP-4, MMP-9 protein expression along with increased degradation of the occludin and claudin-5 proteins was found in the hypercapnia rat model, while the total HIF-1α remained unchanged. Interestingly, these changes were independent of the acidosis induced by hypercapnia. Of note, after premedication of 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α nuclear translocation), the disrupted BBB could be restored resulting in improvement of the cognitive impairment. Meanwhile, accumulation of nuclear HIF-1α, protein expression of AQP-4 and MMP-9 and protein degradation of the occludin and claudin-5 were decreased. Thus, our study demonstrated that hypercapnia can further disrupt the BBB through promoting HIF-1α nuclear translocation and up-regulation of AQP-4 and MMP-9 in hypoxemia. It is therefore suggested that the cascade of hypercapnia-induced nuclear HIF-1α protein translocation in hypoxia-activated astrocytes may be a potential target for ameliorating cognitive impairment.Electronic supplementary materialThe online version of this article (10.1007/s11064-020-03038-7) contains supplementary material, which is available to authorized users.

Enhanced glycolysis and HIF-1\u03b1 activation in adipose tissue macrophages sustains local and systemic interleukin-1\u03b2 production in obesity

During obesity, macrophages infiltrate the visceral adipose tissue and promote inflammation that contributes to type II diabetes. Evidence suggests that the rewiring of cellular metabolism can regulate macrophage function. However, the metabolic programs that characterize adipose tissue macrophages (ATM) in obesity are poorly defined. Here, we demonstrate that ATM from obese mice exhibit metabolic profiles characterized by elevated glycolysis and oxidative phosphorylation, distinct from ATM from lean mice. Increased activation of HIF-1α in ATM of obese visceral adipose tissue resulted in induction of IL-1β and genes in the glycolytic pathway. Using a hypoxia-tracer, we show that HIF-1α nuclear translocation occurred both in hypoxic and non-hypoxic ATM suggesting that both hypoxic and pseudohypoxic stimuli activate HIF-1α and its target genes in ATM during diet-induced obesity. Exposure of macrophages to the saturated fatty acid palmitate increased glycolysis and HIF-1α expression, which culminated in IL-1β induction thereby simulating pseudohypoxia. Using mice with macrophage-specific targeted deletion of HIF-1α, we demonstrate the critical role of HIF-1α-derived from macrophages in regulating ATM accumulation, and local and systemic IL-1β production, but not in modulating systemic metabolic responses. Collectively, our data identify enhanced glycolysis and HIF-1α activation as drivers of low-grade inflammation in obesity.

Mechanism of nuclear translocation of hypoxia-inducible factor-1α in influenza A (H1N1) virus infected-alveolar epithelial cells

Mechanism of nuclear translocation of hypoxia-inducible factor-1α in influenza A (H1N1) virus infected-alveolar epithelial cells

Role of Microtubule-Associated Factors in HIF1α Nuclear Translocation.

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.

Hypoxia-Induced ROS Contribute to Myoblast Pyroptosis during Obstructive Sleep Apnea via the NF-κB/HIF-1α Signaling Pathway.

Tissue hypoxia caused by upper airway collapse is a main cause of excessive oxidative stress and systemic inflammation in obstructive sleep apnea (OSA) patients. Increased reactive oxygen species (ROS) and inflammatory responses affect cell survival and ultimately contribute to tissue injury. In the present study, we proposed that the induction of ROS by hypoxia, as an intrinsic stress, activates myoblast pyroptosis in OSA. We found increased cell death and abnormal expression of pyroptosis markers in the skeletal muscle of OSA mice. In vitro studies showed hypoxia-induced pyroptotic death of C2C12 myoblasts, as evidenced by the activation of caspase-1 and gasdermin D (GSDMD). Hypoxia induced ROS overproduction and accumulation in myoblasts. More importantly, applying N-acetylcysteine (NAC), an ROS scavenger, rescued cell swelling, downregulated the inflammatory response, and prevented pyroptotic death in hypoxia-cultured myoblasts. Hypoxia stimulation promoted NF-κB P65 phosphorylation and HIF-1α nuclear translocation. Moreover, hypoxia increased the nuclear level of cleaved caspase-1 and GSDMD. NAC inhibited hypoxia-induced variations in the HIF-1α and NF-κB signaling pathway. Taken together, our results determined that hypoxia-induced ROS contribute to myoblast pyroptosis. Therefore, our findings suggest that ROS may be a potential therapeutic target for ameliorating hypoxia-induced cell death and tissue injury, especially in OSA and hypoxia-related diseases.

O-cyclic phytosphingosine-1-phosphate stimulates HIF1\u03b1-dependent glycolytic reprogramming to enhance the therapeutic potential of mesenchymal stem cells

O-cyclic phytosphingosine-1-phosphate (cP1P) is a novel chemically synthesized sphingosine metabolite derived from phytosphingosine-1-phosphate. Although structurally similar to sphingosine-1-phosphate (S1P), its biological properties in stem cells remain to be reported. We investigated the effect of cP1P on the therapeutic potential of mesenchymal stem cells (MSCs) and their regulatory mechanism. We found that, under hypoxia, cP1P suppressed MSC mitochondrial dysfunction and apoptosis. Metabolic data revealed that cP1P stimulated glycolysis via the upregulation of glycolysis-related genes. cP1P-induced hypoxia-inducible factor 1 alpha (HIF1α) plays a key role for MSC glycolytic reprogramming and transplantation efficacy. The intracellular calcium-dependent PKCα/mammalian target of the rapamycin (mTOR) signaling pathway triggered by cP1P regulated HIF1α translation via S6K1, which is critical for HIF1 activation. Furthermore, the cP1P-activated mTOR pathway induced bicaudal D homolog 1 expression, leading to HIF1α nuclear translocation. In conclusion, cP1P enhances the therapeutic potential of MSC through mTOR-dependent HIF1α translation and nuclear translocation.

Convulsive Stress Mimics Brain Hypoxia and Promotes the P-Glycoprotein (P-gp) and Erythropoietin Receptor Overexpression. Recombinant Human Erythropoietin Effect on P-gp Activity.

Erythropoietin (EPO) is not only a hormone that promotes erythropoiesis but also has a neuroprotective effect on neurons attributed to its known anti-apoptotic action. Previously, our group has demonstrated that recombinant-human EPO (rHu-EPO) can protect neurons and recovery motor activity in a chemical focal brain hypoxia model (Merelli et al., 2011). We and others also have reported that repetitive seizures can mimic a hypoxic- like condition by HIF-1α nuclear translocation and high neuronal expression P-gp. Here, we report that a single 20-min status epilepticus (SE) induces P-gp and EPO-R expression in cortical pyramidal neurons and only P-gp expression in astrocytes. In vitro, excitotoxic stress (300 μM glutamate, 5 min), can also induce the expression of EPO-R and P-gp simultaneously with both HIF-1α and NFkB nuclear translocation in primary cortical neurons. Primary astrocytes exposed to chemical hypoxia with CoCl2 (0.3 mM, 6 h) increased P-gp expression as well as an increased efflux of Rhodamine 123 (Rho123) that is a P-gp substrate. Tariquidar, a specific 3er generation P-gp-blocker was used as an efflux inhibitor control. Astrocytes treated with rHu-EPO showed a significant recovery of the Rho123 retention in a similar way as seen by Tariquidar, demonstrating for first time that rHu-EPO can inhibit the P-gp-dependent efflux activity. Taking together, these data suggest that stimulation of EPO depending signaling system could not only play a central role in brain cell protection, but this system could be a new tool for reverse the pharmacoresistant phenotype in refractory epilepsy as well as in other pharmacoresistant hypoxic brain diseases expressing P-gp.

Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination.

The long non-coding RNA H19 (lncH19) is broadly transcribed in the first stage of development and silenced in most cells of an adult organism; it appears again in several tumors where, through different molecular mediators, promotes cell proliferation, motility and metastases. LncH19 has been associated with hypoxia-inducible factor 1-alpha (HIF-1α) activation and, in some tumors, it has proved to be necessary and required to sustain hypoxic responses. Here we propose to investigate a putative role for the lncH19 in hypoxia induced multiple myeloma (MM) progression. Transcriptional analysis of MM cell lines (RPMI and MM1.S) exposed to normoxia or hypoxia (1% O2) was done in order to evaluate lncH19 levels under hypoxic stimulation. Then, to investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on hypoxia stimulated MM cell lines, silenced or not for lncH19. Our data demonstrated that hypoxic stimulation in MM cell lines induced the overexpression of lncH19, which, in turn, is required for the expression of the hypoxia induced genes involved in MM dissemination, such as C-X-C Motif Chemokine Receptor 4 (CXCR4) and Snail. Moreover, adhesion assays demonstrated that lncH19 silencing abrogates the increased adhesion on stromal cells induced by the hypoxic condition. Finally, Western blot analysis indicated that lncH19 silencing impaired HIF1α nuclear translocation. The LncH19, required for the induction of hypoxic responses in MM cells, could represent a new therapeutic target for MM.

Cryptotanshinone inhibits the growth and invasion of colon cancer by suppressing inflammation and tumor angiogenesis through modulating MMP/TIMP system, PI3K/Akt/mTOR signaling and HIF-1α nuclear translocation

The aim of this study was to evaluate the pharmacological effects of CPT on CT26 colon cancer cells in vivo and in vitro, and to reveal the potential mechanism. CPT suppressed the proliferation and growth of CT26 colon cancer in vitro and in vivo. CPT inhibited the invasion of CT26 cells in vitro, and decreased the protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 but increased those of tissue inhibitor of metallopeptidase-1 (TIMP-1) and TIMP-2 in vitro and in vivo. It also inhibited tumor cell-induced angiogenesis of endothelial cells in vitro and rat aortic ring angiogenesis ex vivo, and possibly by suppressing angiogenesis-associated factors. CPT suppressed the expressions of inflammatory factors in vivo and in vitro. Mechanism studies showed that CPT inhibited the PI3K/AKT/mTOR signaling pathway, as evidenced by decreased expressions of phospho-PI3K (p-PI3K), p-Akt and p-mTOR. Moreover, CPT significantly suppressed the nuclear expression but increased the cytosolic expression of hypoxia inducible factor-1α (HIF-1α). Collectively, CPT inhibited the growth, invasion, inflammation and angiogenesis in CT26 colon cancer, and at least partly, by regulating the PI3K/Akt/mTOR signaling and the nuclear translocation of HIF-1α.

Current insights and future perspectives of hypoxia-inducible factor-targeted therapy in cancer.

Hypoxia-inducible factors (HIFs) are transcription factors that are expressed in the hypoxic tumor microenvironment. They are involved in the cellular adaptations by improving the metabolism of glucose and enhance the expression of vascular endothelial growth factor, platelet-derived growth factor and angiopoietin, thereby they play a pivotal role in the angiogenesis. Hypoxia can increase the expression of nuclear factor-kappa B which promotes the pro-inflammatory status. Abnormally high angiogenesis, inflammation, antiapoptosis and anaerobic glycolysis can augment the progression and metastasis of tumor. Hence, HIFs remain one of the promising antiangiogenic agents as well as a direct target for interfering with the energetic of cancer cells in order to regulate the tumor growth. Previous studies found agents like topotecan, acriflavine and benzophenone-1B etc. to block the HIF-α mediated angiogenesis. The effect is mediated through interfering any one of the processes in the activation of HIF such as nuclear translocation of HIF-1α; dimerization of HIF-1α with β in the nucleus; HIF-1α/HIF-2α mediated induction of VEGF or translation of HIF-1α mRNA. Despite the experimental studies on the inhibitory molecules of HIFs, none of them are available for the clinical use. This review article discusses the recent update on the HIF-targeted therapy in cancer.

Abstract A083: Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3

Abstract Pten loss leading to constitutive PI3K/Akt/mTor activation is a well-characterized PCa survival pathway frequently activated in castration-resistant prostate cancer (CRPC). Unfortunately, targeted mono-therapies to PI3K/Akt/mTor with or without anti-androgen therapy failed to achieve significant response in clinical trials with CRPC patients. The mechanisms that drive PI3K resistance in CRPC are unknown. The androgen receptor (AR) is also a major driver of prostate cancer (PCa) survival, even in CRPC. However, surprisingly very little is known about how AR promotes PCa survival. We previously identified an AR-dependent survival pathway in androgen-dependent tumor cells whereby AR induction of integrin α6β1 and adhesion to laminin in the tumor microenvironment activates NF-κB to induce Bcl-xL. This pathway acts in parallel to the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin α6β1 is required to effectively kill tumor cells and suppress tumor growth. While this drug combination was effective in androgen-responsive tumors in vivo, it poorly suppressed CRPC. Thus, we hypothesized there are additional survival mechanisms operating in CRPC. To identify CRPC survival pathways, we mined the gene expression data comparing androgen-sensitive to CRPC tumors in a series of human PDX LuCaP models. We found the hypoxia-inducible protein, BNIP3, to be preferentially elevated in CRPC tumors, and this correlated with increased integrin α6β1 expression and enhanced AR signaling. Furthermore, elevated BNIP3 predicts for poor outcome in PCa patients. We discovered that androgen induces BNIP3 expression specifically in CRPC C4-2 cell lines, but not the androgen-dependent parental LNCaP cells. BNIP3 expression is dependent on AR induction of integrin α6β1, adhesion to laminin, and nuclear translocation of HIF-1α. The CRPC C4-2 cells are highly resistant to PI3K inhibition by PX866 compared to LNCaP when adherent to laminin, and siRNA-mediated loss of integrin α6 or BNIP3 sensitizes these cells to PI3K inhibition. Blocking Bnip3 expression in C4-2 cells injected orthotopically into mice reduced tumor growth and induced apoptosis. BNIP3 can promote survival by activating autophagy and/or mitophagy. Even though autophagic flux was specifically enhanced in laminin-adherent CRPC C4-2 cells, but not in LNCaP cells, this was not dependent on BNIP3. WT BNIP3 can restore PI3K drug resistance in shBNIP3-expressing cells, but the BNIP3 ΔLIR mutant, which is unable to target mitochondria to autophagosomes, cannot restore drug resistance. We have identified an AR-dependent survival pathway in CRPC involving BNIP3-induced mitophagy that is dependent on and mediated by integrin α6β1 adhesion to laminin that promotes resistance to PI3K inhibition. Laminin is abundant in prostate tissue, lymph nodes, and bone—the primary sites for PCa growth. Thus, laminin within the tumor microenvironment of both primary and metastatic PCa drives drug resistance in CRPC. Citation Format: Eric Nollet, Sourik Ganguly, Veronique Schulz, Eva Corey, Cindy K. Miranti. Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3 [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A083.

PO-162 Long non coding RNA H19 (IncH19) is required for the hypoxia mediated multiple myeloma progression

IntroductionMultiple myeloma (MM) is a malignant disorder of post-germinal centre B cells, characterised by the clonal proliferation of malignant plasma cells within the bone marrow (BM). The hypoxic condition that develops in the BM niche during progression of MM, has been shown to play a major role in i) the dissemination of MM, ii) the proliferation of MM cells and iii) the induction of drug resistance finally determining a poor prognosis for MM patients. The molecular mechanisms driving hypoxic responses is the activation and nuclear translocation of the Hypoxia-inducible factor 1-alpha, (HIF1α) that, in turns, induce the expression of genes controlling angiogenesis, hypermetabolism, stemnes maintenance, resistance to chemotherapy, and tumour metastasis. The lncRNA H19, an imprinted non-coding RNA which expression was found up-regulated in many tumours including MM, is among the targets of HIF1α.We previously attributed to the lncH19 a role into control of hypoxic response; in particular we demonstrated, in both glioblastoma and colon cancer, that the lncH19, is necessary and required to sustain HIF1α activity. Here we propose to investigate the role of lncH19 in hypoxia mediated MM progression.Material and methodsTranscriptional analysis (RT-PCR) of MM cell lines (RPMI and MM1S) exposed to normoxia or hypoxia (1% O2) was done in order to evaluate lncH19 levels under hypoxic stimulation. To investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on MM cell lines, silenced or not for lncH19, under normoxia or 24 hour hypoxic stimulation in low oxygen chamberResults and discussionsOur data indicate that MM cell lines respond to hypoxic stimulation by HIF1α nuclear translocation and activation of hypoxic responsive genes including the lncH19. Our data revealed that lncH19 silencing inhibits HIF1a nuclear translocation with a subsequent reduction in the expression of hypoxia induced genes, associated to MM progression, such as snail and VEGF. Moreover, adhesion assay of MM cells on Mesenchymal Stromal Cells revealed that lncH19 silencing abrogates the increased adhesion induced by hypoxic condition.ConclusionLncH19 is required for the induction of hypoxic responses in MM cells thus representing a new therapeutic target for MM. Further studies are required to better define the molecular mechanism through which H19 may control HIF1α activity.

PO-165 Estetrol reduce cell migration and proliferation in two/three-dimensional T47D breast cancer cell model

IntroductionMultiple myeloma (MM) is a malignant disorder of post-germinal centre B cells, characterised by the clonal proliferation of malignant plasma cells within the bone marrow (BM). The hypoxic condition that develops in the BM niche during progression of MM, has been shown to play a major role in i) the dissemination of MM, ii) the proliferation of MM cells and iii) the induction of drug resistance finally determining a poor prognosis for MM patients. The molecular mechanisms driving hypoxic responses is the activation and nuclear translocation of the Hypoxia-inducible factor 1-alpha, (HIF1α) that, in turns, induce the expression of genes controlling angiogenesis, hypermetabolism, stemnes maintenance, resistance to chemotherapy, and tumour metastasis. The lncRNA H19, an imprinted non-coding RNA which expression was found up-regulated in many tumours including MM, is among the targets of HIF1α.We previously attributed to the lncH19 a role into control of hypoxic response; in particular we demonstrated, in both glioblastoma and colon cancer, that the lncH19, is necessary and required to sustain HIF1α activity. Here we propose to investigate the role of lncH19 in hypoxia mediated MM progression.Material and methodsTranscriptional analysis (RT-PCR) of MM cell lines (RPMI and MM1S) exposed to normoxia or hypoxia (1% O2) was done in order to evaluate lncH19 levels under hypoxic stimulation. To investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on MM cell lines, silenced or not for lncH19, under normoxia or 24 hour hypoxic stimulation in low oxygen chamberResults and discussionsOur data indicate that MM cell lines respond to hypoxic stimulation by HIF1α nuclear translocation and activation of hypoxic responsive genes including the lncH19. Our data revealed that lncH19 silencing inhibits HIF1a nuclear translocation with a subsequent reduction in the expression of hypoxia induced genes, associated to MM progression, such as snail and VEGF. Moreover, adhesion assay of MM cells on Mesenchymal Stromal Cells revealed that lncH19 silencing abrogates the increased adhesion induced by hypoxic condition.ConclusionLncH19 is required for the induction of hypoxic responses in MM cells thus representing a new therapeutic target for MM. Further studies are required to better define the molecular mechanism through which H19 may control HIF1α activity.

Extracellular vesicles: emerging mediators of intercellular communication and tumor angiogenesis.

Rapidly expanding solid tumors demand large quantities of nutrients and oxygen. Growing tumors exploit pre-existing vessels and/or develop new vessels (vascularization) to obtain these nutrients. Vascularization is classified into two main processes, vasculogenesis and angiogenesis. Vasculogenesis is the formation of primitive blood vessels by endothelial progenitors, and angiogenesis encompasses the subsequent remodeling processes, including growth and migration of endothelial cells, sprouting, and stabilization of these sprouts by mural cells (1,2). Tumor cells utilize these processes to satisfy their needs and grow (tumor angiogenesis), especially under hypoxic conditions. One of the pro-angiogenic mechanisms exploited by hypoxic cancers is hypoxia-inducible transcriptional factor (HIF)-mediated signaling. Hypoxia leads to stabilization and nuclear translocation of HIF-1α, and subsequently increases transcription of pro-angiogenic genes, including vascular endothelial growth factor (VEGF) (3). Overexpression of HIF-1α has been reported in various solid tumors including brain, bladder, breast, colorectal, ovarian, pancreatic, and prostate cancers (4,5), and increased vascularity is a hallmark of poor prognosis.