Human Antigen R Protein Research Articles

Phosphorylation at the disordered N-end makes HuR accumulate and dimerize in the cytoplasm.

Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuRtargeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24h, 48h, 72h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.

Eltrombopag Inhibits Metastasis in Breast Carcinoma by Targeting HuR Protein

Eltrombopag is a small molecule TPO-R agonist that has been shown in our previous studies to inhibit tumor growth by targeting Human antigen R (HuR) protein. HuR protein not only regulates the mRNA stability of tumor growth-related genes, but it also regulates the mRNA stability of a variety of cancer metastasis-related genes, such as Snail, Cox-2, and Vegf-c. However, the role and mechanisms of eltrombopag in breast cancer metastasis have not been fully investigated. The purpose of this study was to investigate whether eltrombopag can inhibit breast cancer metastasis by targeting HuR. Our study first found that eltrombopag can destroy HuR-AU-rich element (ARE) complexes at the molecular level. Secondly, eltrombopag was found to suppress 4T1 cell migration and invasion and inhibit macrophage-mediated lymphangiogenesis at the cellular level. In addition, eltrombopag exerted inhibitory effects on lung and lymph node metastasis in animal tumor metastasis models. Finally, it was verified that eltrombopag inhibited the expressions of Snail, Cox-2, and Vegf-c in 4T1 cells and Vegf-c in RAW264.7 cells by targeting HuR. In conclusion, eltrombopag displayed antimetastatic activity in breast cancer in an HuR dependent manner, which may provide a novel application for eltrombopag, hinting at the multiple effects of HuR inhibitors in cancer therapy.

The Prognostic Role of HuR Varies Between Different Subtypes of Breast Cancer Patients: Data Mining and Retrospective Analysis.

Human-antigen R (HuR) is an RNA-binding protein, which regulates the expression of several oncogenes and tumor suppressor genes through post-transcriptional mechanisms. But the role of HuR in breast cancer remains controversial. The aim of this study was to verify the association between cytoplasmic HuR level and prognosis of breast cancer patients. Data mining from the Human Protein Atlas (HPA) and Kaplan-Meier Plotter (KMP) databases was performed. Then, 394 patients with stage I-III primary breast cancer were enrolled between January 2005 and December 2016. We investigated the association between cytoplasmic HuR level and clinicopathological characteristics or survival of these patients. Immunohistochemical analysis was performed to determine HuR expression level. SPSS 21.0 statistical software was used for analysis. In the HPA and KMP datasets, HuR protein and mRNA expression level were not significantly associated with overall survival of all breast cancer patients enrolled. Results from our 394 patients indicated that higher expression level of cytoplasmic HuR was associated with larger tumor size, lymph node positive, ER negative and triple-negative subtype. For all patients enrolled, the results indicated that compared with HuR negative patients, the DFS (disease-free survival) of HuR 1+ was longer (60.5% vs 78.8, P=0.053, HR=0.616, 95% CI: 0.378-1.005), the P value was borderline. In the triple-negative breast cancer (TNBC) subgroup, HuR positive patients had significantly longer DFS than HuR negative patients (65.5% vs 30.8%, P=0.001, HR=0.345, 95% CI: 0.180-0.658). In the HR+HER2- subgroup, HuR low (0~1+) patients had significantly longer OS than HuR high (2+~3+) patients (97.0% vs 89.5%, P=0.033, HR=2.482, 95% CI: 1.074-5.736). In conclusion, our results revealed that higher expression level of HuR was related to aggressive biological characteristics which supported the findings from previous researches. In the HR+HER2- subgroup, lower HuR expression level patients had better survival time, while in the TNBC subgroup we got the opposite results. Our work indicated that HuR might play different roles in different breast cancer subtypes.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

CircREOS suppresses lipid synthesis and osteosarcoma progression through inhibiting HuR-mediated MYC activation.

MYC proto-oncogene (MYC) is a transcription factor among the most commonly activated oncoproteins, playing vital roles in lipid metabolism and tumor aggressiveness with broad effects. However, it is still largely unknown about the regulating mechanisms of MYC in osteosarcoma (OS). In this study, we identify a circRNA with Reduced Expression in OS (termed as circREOS) generated from MYC gene, as a novel regulator of MYC and OS progression. CircREOS is down-regulated in OS cells and localized in the nucleus. CircREOS suppresses MYC expression, lipid metabolism and growth, invasion in OS cells. Mechanically, circREOS physically interacts with HuR (human antigen R) protein, and subsequently restrains its binding and activation on the 3'-UTR (untranslated region) of MYC mRNA, resulting in down-regulation of MYC and inhibition of OS. Moreover, circREOS serves as a tumor suppressor via targeting lipid metabolism. CircREOS reduces FASN expression and lipid accumulation through inhibiting MYC-facilitated FASN regulation. Taken together, these results indicate that circREOS suppress lipid synthesis and OS progression through inhibiting HuR-mediated MYC activation, providing a potential therapeutic target for OS.

Effects of artesunate on the malignant biological behaviors of non-small cell lung cancer in human and its mechanism

ABSTRACT We aimed to assess the effects of artesunate (ART) on the proliferation, migration, invasion and apoptosis of the non-small cell lung cancer cells A549 and H1299. The effects of ART and carboplatin (CBP) alone or in combination on the viability of A549 and H1299 cells were evaluated by MTT assay. The effects of 30 μg/ml ART on cell invasion, migration and apoptosis were evaluated by Transwell assay, scratch assay and flow cytometry, respectively. The protein expressions of human antigen R (HuR) and MMP-9 after treatment with 30 μg/ml ART for 48 h were detected by Western blotting. After 48 h of treatment, 9 μg/ml ART in combination with 7 μg/ml CBP exerted a mild synergistic effect on cell viability. The migration rates of cells treated with 30 μg/ml ART and number of invasive cells were significantly lower, and the apoptosis rates were higher than those of the DMSO-treated group. HuR and MMP-9 expressions in cells treated with 30 μg/ml ART for 48 h were significantly lower than those of the DMSO-treated group. ART suppresses the proliferation, migration and invasion of A549 and H1299 cells and induces their apoptosis, probably being associated with decreased expressions of HuR and MMP-9 proteins.

HuR as Therapeutic Target in Cancer: What the Future Holds.

ELAV-like protein 1 or HuR (human antigen R) is an RNA-binding protein that in humans is encoded by the ELAVL1 gene, and one of its best functions is to stabilize mRNAs in order to regulate gene expression. HuR protein overexpression has undoubtedly been linked to an increased risk of tumor growth, progression and metastasis, rendering it a potential therapeutic target candidate in cancer. Novel agents, interfering with HuR expression, have been tested, both in vitro and in vivo, with promising results. The aim of this paper is to review the existing literature regarding the potential agents that could actively act on and inhibit HuR expression. HuR molecule controls the expression of various proto-oncogenes, cytokines and growth factors, representing a major player in tumor progression, invasion and metastasis and constituting an emerging target for cancer therapy. PubMed database was thoroughly searched, and all published articles providing scientific data on molecules that can exhibit antitumorigenic effects via HuR inhibition were included. According to these data, HuR inhibition should be a promising target in cancer therapeutics.

Inhibition of GATA3 and Th2 cytokines expression in type 2 high asthmatics by a small molecule targeting RNA-binding protein HuR

Abstract RNA-binding protein human antigen R (HuR) is a master regulator of gene expression in human pathophysiology. Our animal studies in the past showed multiple critical roles for HuR in regulating proinflammatory markers in CD4+ T cells. We hypothesized HuR plays an important role in expression of GATA3 and Th2 cytokines in type 2 high asthma and its inhibition impairs these Th2 inflammatory responses. Peripheral CD4+ T cells were isolated from 26 healthy controls and 32 type 2 high asthmatics (blood eosinophil ≥300 cells/μl or FeNO levels ≥25ppb) and stimulated with anti-CD3/CD28 for 4 days, followed by determination of GATA3 and cytokines expression by flow cytometry and LEGENDplex. HuR levels were quantitated by Western blot, flow cytometry, and qPCR. Inhibition of inflammatory responses was evaluated using a cumarin-derived small molecule named CMLD-2 (a HuR-specific inhibitor). Actinomycin D treatment was used to measure mRNA decay rates of targeted HuR mRNAs. HuR protein levels in CD4+ T cells was significantly higher in type 2 high asthmatics compared to healthy controls. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared to controls. Our further mRNA and protein studies in type 2 high asthmatics showed reduced expression of GATA3 and Th2 cytokines after CMLD-2 treatment. There was also a significant reduction in gata3 mRNA stability with CMLD-2 treatment, compared with vehicle in the asthmatics. Our study is the first to demonstrate the role of RNA-binding protein HuR in type 2 high asthmatics and that interfering with HuR action using CMLD-2 ameliorates GATA3 and Th2 cytokines production. This data suggests HuR may serve as a novel and powerful therapeutic target to treat type 2 high asthmatics.

Muscone derivative ZM-32 inhibits breast tumor angiogenesis by suppressing HuR-mediated VEGF and MMP9 expression

Inhibition of tumor angiogenesis is a highly effective strategy for cancer treatment. Human antigen R (HuR), an RNA-binding protein, is overexpressed in many cancers and regulates the mRNAs of multiple angiogenic factors by binding to the adenylate-uridylate-rich element in their 3′ untranslated region. HuR protein has been demonstrated to be an important regulatory factor in macrophage-mediated angiogenesis, a process in which macrophages are critical for tumor progression. Muscone is a synthetic equivalent of musk, and recent studies have shown that it has a regulatory effect on angiogenesis. In this study, we synthesized five series of muscone derivatives and discovered that compound ZM-32 was effective in preventing HuR RRM1/2-Vegf-a mRNA complex formation. ZM-32 bound to HuR RRM1/2 protein with a KD value of 521.7 nmol/L. Furthermore, ZM-32 inhibited endothelial cell proliferation, migration, and tubule formation, and suppressed the VEGF/VEGFR2/ERK1/2 signaling axis mediated by macrophages in vitro. We also demonstrated that ZM-32 effectively prevented the proliferation and migration of breast cancer cells and inhibited the growth and angiogenesis of MDA-MB-231 xenograft tumors without any obvious toxicity in vivo. Mechanistically, exposure to ZM-32 influenced the mRNA stability of Vegf-a and Mmp9 in a HuR-dependent manner in both macrophages and MDA-MB-231 cells. Thus, in this study we identified a new muscone derivative, ZM-32, with anti-angiogenesis effects mediated via targeting HuR in breast cancer, that may become a potentially valuable lead compound for anti-cancer angiogenesis.

AChR β-Subunit mRNAs Are Stabilized by HuR in a Mouse Model of Congenital Myasthenic Syndrome With Acetylcholinesterase Deficiency.

Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR β-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR β-subunit transcripts. This HuR/AChR transcript interaction induces AChR β-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR β-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.

CircDCUN1D4 suppresses tumor metastasis and glycolysis in lung adenocarcinoma by stabilizing TXNIP expression

Aberrant expression of circular RNAs (circRNAs) is involved in cancer progression through interaction with RNA-binding proteins (RBPs). Herein, we screened circRNA expression of A549 cells in circBase and the crosslinking immunoprecipitation (CLIP) data of human antigen R (HuR), an extensively studied RBP, and identified a circRNA, circ-defective in cullin neddylation 1 domain containing 4 (circDCUN1D4), originating from the DCUN1D4 gene transcript. circDCUN1D4 is downregulated in tumor samples under the mediation of DExH-box helicase 9 (DHX9), which inhibits the formation of circRNA by binding inverted repeat Alus (IRAlus) in flanking sequences. circDCUN1D4 depletion promoted invasion in vitro and metastasis in vivo. Importantly, the interaction between circDCUN1D4 and HuR increased the transportation of HuR to the cytoplasm. circDCUN1D4 acts as a scaffold to facilitate the interaction between the HuR protein and thioredoxin-interacting protein (TXNIP) mRNA, which enhances the stability of the TXNIP mRNA. Additionally, circDCUN1D4 directly interacts with TXNIP mRNA through base complementation, indicating the formation of the circDCUN1D4/HuR/TXNIP RNA-protein ternary complex. Furthermore, circDCUN1D4 suppressed metastasis and glycolysis of lung cancer cells in a TXNIP-dependent manner. Clinically, the downregulated expression of circDCUN1D4 was more prevalent in lymph node metastatic tissues and served as an independent risk factor for the overall survival of lung adenocarcinoma (LUAD) patients. These findings demonstrated that a novel circRNA, circDCUN1D4, is involved in the metastasis and glycolysis of LUAD.

Abstract B45: A feedback gene regulatory mechanism between YAP1 and the RNA-binding protein, Human Antigen R (HuR), in pancreatic cancer cells: Implications for a context-dependent pancreatic cancer cell survival network

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the U.S. In roughly 95% of cases, gain-of-function mutations in KRAS combine with loss of tumor suppressors to progress preinvasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important facilitators of KRAS function, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is an RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target prosurvival mRNAs upon stress. YAP1 is a transcriptional coactivator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular crosstalk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood. Our previous work has shown that overexpression of cytoplasmic HuR correlates strongly with tumor staging. Low levels of HuR correspond to early PanINs with staining steadily increasing in late-stage PanIN lesions and gross overexpression in invasive adenocarcinoma. Conversely, YAP1 overexpression seems to be most critical for initial development and expansion of the tumor cells, while it converts to a maintenance role once PDAC is fully developed. Ongoing studies will address whether the temporal regulation of these proteins could explain their overexpression patterns in pancreatic pathologic stages as they relate to cooperating with KRAS activity. YAP1 was first identified as a HuR target via ribonucleoprotein-immunoprecipitation assays in which HuR-bound mRNAs were run on a whole-transcriptome microarray. YAP1 mRNA was significantly bound to HuR as compared to the IgG isotype control (13.2-fold) and was in line with previously established mRNA targets (WEE1, 3.2-fold; PIM1, 13.9-fold). YAP1 mRNA bound to HuR is abolished when treated with a known HuR inhibitor, pyrvinium pamoate, even in the presence of an established HuR stressor (i.e., oxaliplatin). Actinomycin D chase assays demonstrated that YAP1 mRNA stability is significantly dependent on HuR proficiency. We validated that both YAP1 mRNA and protein expression levels are dependent on HuR via real-time quantitative PCR and Western blot analysis. Impact of YAP1 transcriptional activity was evaluated both by measuring total expression of canonical YAP targets (i.e., CTGF and CYR61) and by using a TEAD reporter construct (i.e., 8xGTIIC). Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as established HuR targets, WEE1 and PIM1. Treatment with small-molecule inhibitors verteporfin and CA3, which target the interface of YAP1’s transcription-factor binding domain, recapitulated these effects in a dose- and time-dependent manner. We are currently cloning HuR’s promoter region into a luciferase reporter in order to evaluate the impact of YAP1 on HuR transcriptional expression. Citation Format: Samantha Z. Brown, Christopher W. Schultz, Avinoam Nevler, Tianyun Li, Aditi Jain, Raymond O’Neil, Wei Jiang, Eric Londin, Dan A. Dixon, Liang Xu, Charles J. Yeo, Jonathan R. Brody. A feedback gene regulatory mechanism between YAP1 and the RNA-binding protein, Human Antigen R (HuR), in pancreatic cancer cells: Implications for a context-dependent pancreatic cancer cell survival network [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B45.

Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease

ObjectiveGlomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. MethodsWe studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. ResultsWe demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3′ untranslated region (3′-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3′-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. ConclusionsWe established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.

Abstract 4388: A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the U.S. In roughly 95% of cases, gain-of-function mutations in KRAS combine with loss of tumor suppressors to progress pre-invasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important mediators of KRASfunction, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is a RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target pro-survival mRNAs upon stress. YAP1 is a transcriptional co-activator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular cross-talk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood. YAP1 was first identified as a HuR target via ribonucleoprotein-immunoprecipitation assays in which HuR-bound mRNAs were run on a whole-transcriptome microarray. YAP1 mRNA was significantly bound to HuR as compared to the IgG isotype control (13.2-fold), and was in line with previously established targets (WEE1, 3.2-fold; PIM1, 13.9-fold). YAP1 mRNA bound to HuR is abolished when treated with a known HuR inhibitor, pyrvinium pamoate, even in the presence of an established HuR stressor (i.e., oxaliplatin). Actinomycin D chase assays demonstrated that YAP1 mRNA stability is significantly dependent on HuR proficiency. We validated that both YAP1 mRNA and protein expression levels are dependent on HuR via real-time quantitative PCR and Western blot analysis. Transcription of downstream YAP1 targets, CTGF and CYR61, was also significantly affected by knockdown of HuR. Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as HuR targets, WEE1 and PIM1. Treatment with small-molecule inhibitors, Verteporfin and CA3, which target the interface of YAP1’s transcription-factor binding domain, recapitulated these effects in a dose and time-dependent manner. We are currently using chromatin immunoprecipitation sequencing to identify HuR’s promoter/enhancer domains for YAP1-mediated transcription. Our previous work has shown that overexpression of cytoplasmic HuR correlates strongly with tumor staging. Low-levels of HuR correspond to early PanINs with staining steadily increasing in late-stage PanIN lesions and gross overexpression in invasive adenocarcinoma. Conversely, YAP1 overexpression seems to be most critical for initial development and expansion of the tumor cells, while it converts to a maintenance role once PDAC is fully developed. Ongoing studies will address whether the temporal regulation of these proteins could explain their overexpression patterns in pancreatic pathologic stages as they relate to cooperating with KRAS activity. Citation Format: Samantha Z. Brown, Christopher W. Schultz, Tianyun Li, Aditi Jain, Raymond O'Neill, Wei Jiang, Charles P. Yeo, Jonathan R. Brody. A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4388.

Abstract 3614: Targeted 3DNA-siHuR nanocarrier therapy for pancreatic ductal adenocarcinoma

Abstract The poor overall survival rate of pancreatic ductal adenocarcinoma (PDAC), less than 10% over 5 years, is largely due to the majority of patients presenting with overt metastatic burden or post-surgical recurrence of micrometastases. Due to these factors, new treatment methods are needed to effectively target both primary and metastatic tumors. We have previously established that the mRNA binding protein Human antigen R (HuR), is an important molecule for PDAC progression. In a tumor microarray of matched normal and malignant patient tissues, we found active HuR staining in 79% of tumor samples, which was not seen in normal pancreatic tissue (n=70). We have shown that homozygous CRISPR knock-out of HuR results in xenograft lethality, as well as sensitization of PDAC cell lines to standard-of-care chemotherapy. Therefore, in the current experiments we have utilized a targeted 3DNA nanocarrier for the delivery of an RNA silencer against HuR in order to enhance tumor susceptibility to chemotherapy. Treatment of PDAC cell lines with this 3DNA nanocarrier was highly effective in knocking down both HuR and established HuR targets (DCK, IDH1, PIM1). To increase the specificity of the 3DNA nanocarrier, we sought to discover targeting moieties specifically upregulated in PDAC. We found increased folic acid receptor (FAR) expression in HS 766T, MIA PaCa-2, and PANC-1 PDAC cell lines with an average fold change of 2.6, in comparison to normal pancreatic cells. Additionally, patient tissue samples showed elevated FAR levels in 100% of tumors (n=80). Thus, by attaching folic acid to the 3DNA nanocarrier (FA-3DNA), we were able to target the nanocarrier specifically to tumors, both primary and metastatic. This was detected by tagging FA-3DNA with Alexa750 fluorophore and monitoring its movement in mice bearing luciferase expressing-MIA PaCa-2 tumors. FA-3DNA had a longer half-life and increased delivery to tumors, as compared to non-targeted 3DNA. We also demonstrated that treatment of mice with FA-3DNA conjugated with an RNA silencer for HuR (FA-3DNA-siHuR) led to a significant decrease in HuR mRNA and protein, in comparison to mice treated with 3DNA containing a control RNA silencer (FA-3DNA-siControl). Subsequent in vivo experiments showed prolong survival of mice treated with FA-3DNA-siHuR, as compared to mice treated with FA-3DNA-siControl (p=0.04). Our previous work has established HuR as a promising therapeutic target in PDAC. In both in vitro and in vivo models, we have shown FA-3DNA-siHuR is capable of targeting tumors directly for the inhibition of HuR. These results suggest treatment with FA-3DNA-siHuR will sensitize tumors to standard of care chemotherapy, which can be introduced systemically or incorporated into the 3DNA nanocarrier for direct delivery. The latter could potentially increase the efficiency of the chemotherapy, and also of critical importance, reduce the toxic adverse effects of systemic chemotherapy. Citation Format: Grace McCarthy, Christopher Schultz, Aditi Jain, Teena Dhir, Charles Yeo, Jessica Bowers, Lou Casta, Kelly Rhodes, Lori Getts, Robert Getts, Trevor Baybutt, Adam Snook, Jonathan Brody. Targeted 3DNA-siHuR nanocarrier therapy for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3614.

Effect of Inhibiting p38 on HuR Involving in β-AChR Post-transcriptional Mechanisms in Denervated Skeletal Muscle.

Previous studies reported that RNA-binding protein human antigen R (HuR) mediates changes in the stability of AChR β-subunit mRNA after skeletal muscle denervation; also, p38 pathway regulated the stability of AChR β-subunit mRNA in C2C12 myotubes. However, the relationship between HuR and p38 in regulating the stability of AChR β-subunit mRNA have not been clarified. In this study, we wanted to examine the effect of inhibiting p38 on HuR in denervated skeletal muscle. Denervation model was built and 10% DMSO or SB203580 were administered respectively follow denervation. Tibialis muscles were collected in 10% DMSO-administered contralateral (undenervated) leg, 10% DMSO-administered denervated leg, SB203580-administered contralateral (undenervated) leg, and SB203580-administered denervated leg, respectively. P38 protein, β-AChR mRNA and protein, HuR protein, β-AChR mRNA stability, and HuR binding with AChR β-subunit mRNAs were measured. Results demonstrated that the administration of SB203580 can inhibit the increase of β-AChR protein expression and mRNA expression and stability, and RNA-binding protein human antigen R (HuR) expression, in cytoplasmic and nuclear fractions in skeletal muscle cells following denervation. Importantly, we observed that SB203580 also inhibited the increased level of binding activity between HuR and AChR β-subunit mRNAs following denervation. Collectively, these results suggested that inhibition of p38 can post-transcriptionally inhibit β-AChR upregulation via HuR in denervated skeletal muscle.

PF364 BORTEZOMIB LOWERS THE STAT3 PATHOGENETIC ROLE IN T‐LGLL NEUTROPENIA DEVELOPMENT BY RESTORING MIR146B EXPRESSION

Background:Symptomatic or severe neutropenia is the most common indication for treatment in T‐Large Granular Lymphocytes Leukemia (T‐LGLL) patients. To date, the therapeutic strategy mostly relies on immunosuppressive agents, however the ORR is quite low (<60%). A targeted therapy for neutropenic patients has not been developed yet, due to the lack of knowledge about the pathogenesis of this cytopenia. One of the most supported hypotheses relies on Fas Ligand (FasL) involvement, since high levels of this protein, accounting for regulation of neutrophils lifespan, were detected in sera of neutropenic patients. Of note, we recently found that the increased FasL transcription was induced by the high STAT3 activation that specifically characterizes T‐LGLs of neutropenic patients. We also showed that a microRNA(miR)146b down‐regulation, that represents a signature of these patients, led to the translation of Human antigen R (HuR), an essential FasL mRNA stabilizer.Several drugs are under pre‐clinical evaluation to improve the therapeutic possibilities for T‐LGLL. Among these, the proteasome inhibitor Bortezomib (Bz) has been demonstrated to affect in‐vitro leukemic LGL viability by increasing the levels of the pro‐apoptotic protein BID and by inhibiting the constitutive activation of NF‐kB pathway. Despite its promising effects, up to date Bz has never been tested in T‐LGLL patients and its effects on STAT3‐axis have never been investigated.Aims:Since in different neoplastic conditions miR146b expression is epigenetically silenced and in malignant T‐lymphocytes the DNA Methyl‐Transferase 1 (DNMT1) is reported to be STAT3‐induced, our work aims to investigate the potential linkage between STAT3 and miR146b and to evaluate whether Bz might prevent STAT3 pathogenetic role in neutropenia development.Methods:T‐LGL were immunomagnetically purified from peripheral blood of untreated T‐LGLL patients. The methylation status of miR146b promoter was assessed by Methylated DNA Immuno‐Precipitation (MeDIP). T‐LGL were treated for 72h‐2.5 μM 5‐aza‐2’‐deoxycytidine (DAC), an inhibitor of methyl‐transferase activity, or 24h‐15 μM STATTIC, a STAT3 inhibitor, or 48h‐5 μM Bz. Transcriptional and protein expression were evaluated by RTq‐PCR and western blot assays, respectively.Results:MeDIP analysis highlighted a significantly higher level of 5‐methyl‐cytosine in −687/−496 bp (+141%) and −149/+98 bp (+58%) regions upstream the miR‐146b transcription start site in T‐LGL of neutropenic as compared to non‐neutropenic patients. DAC treatment restored miR146b primary transcript expression in T‐LGL of neutropenic patients, suggesting that it was epigenetically silenced.By specifically inhibiting STAT3 activity, we showed a 58% DNMT1 transcriptional decrease in the STATTIC treated T‐LGL, as compared to controls, providing proof of a molecular linkage, likely mediated by DNMT1, between STAT3 activation and miR146b promoter methylation.Regarding Bz evaluation, a reduction of STAT3 activation (70%) and of DNMT1 transcription (67%) was observed in treated vs untreated conditions. Among miR146b downstream targets, a decrease of HuR protein (44%) and, thus, of FasL mRNA (43%) was observed in Bz‐treated T‐LGL, consistent with the restoration of miR‐146b expression.Summary/Conclusion:Our data demonstrate the presence of a STAT3‐miR46b‐FasL axis in neutropenic T‐LGLL patients. We also show for the first time that Bz down‐regulates STAT3 activation in in‐vitro treated leukemic T‐LGL, offering the rationale for testing Bz in clinical trials to manage this high‐risk subset of patients.

Gossypol but not cottonseed extracts or lipopolysaccharides stimulates HuR gene expression in mouse cells

Abstract Human antigen R (HuR) stabilizes cytokine mRNAs and plays an important role in tumorigenesis and inflammation. Plant extracts and compounds that can regulate HuR gene expression may have nutritional and therapeutic value, but little research has been done in this area. This study evaluated the effect of cottonseed extracts, anti-nutritional gossypol and endotoxin lipopolysaccharides (LPS) on HuR gene expression in mouse macrophages. Mouse RAW264.7 macrophages were treated with multiple concentrations of glanded and glandless cottonseed extracts, gossypol and LPS for 2–24 h. qPCR assay showed that cottonseed extracts and LPS had minor effect on HuR gene expression. However, gossypol increased HuR mRNA levels 16 fold in cells treated for 24 h. Immunoblotting confirmed that gossypol increased HuR protein in macrophages. These results showed that gossypol is a strong stimulator of HuR gene expression.