Regulation Of HuR Research Articles

HuR Plays a Positive Role to Strengthen the Signaling Pathways of CD4+ T Cell Activation and Th17 Cell Differentiation.

After antigen and/or different cytokine stimulation, CD4+ T cells activated and differentiated into distinct T helper (Th) cells via differential T cell signaling pathways. Transcriptional regulation of the activation and differentiation of naïve CD4+ T cells into distinct lineage Th cells such as Th17 cells has been fully studied. However, the role of RNA-binding protein HuR in the signaling pathways of their activation and differentiation has not been well characterized. Here, we used HuR conditional knockout (HuR KO) CD4+ T cells to study mechanisms underlying HuR regulation of T cell activation and differentiation through distinct signaling pathways. Our work showed that, mechanistically, HuR positively promoted CD3g expression by binding its mRNA and enhanced the expression of downstream adaptor Zap70 and Malt1 in activated CD4+ T cells. Compared to WT Th0 cells, HuR KO Th0 cells with reduced Bcl-2 expression are much more susceptible to apoptosis than WT Th0 cells. We also found that HuR stabilized IL-6Rα mRNA and promoted IL-6Rα protein expression, thereby upregulating its downstream phosphorylation of Jak1 and Stat3 and increased level of phosphorylation of IκBα to facilitate Th17 cell differentiation. However, knockout of HuR increased IL-22 production in Th17 cells, which was due to HuR deficiency in reducing IL-22 transcription repressor c-Maf expression. These results highlight the importance of HuR in TCR signaling and IL-6/IL-6R axis driving naïve CD4+ T cell activation and differentiation into Th17 cells.

RNA-binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration.

ObjectivesDiabetes is a risk factor for intervertebral disc degeneration (IVDD). Studies have demonstrated that diabetes may affect IVDD through transcriptional regulation; however, whether post‐transcriptional regulation is involved in diabetic IVDD (DB‐IVDD) is still unknown. This study was performed to illustrate the role of HuR, an RNA‐binding protein, in DB‐IVDD development and its mechanism.Materials and MethodsThe expression of HuR was evaluated in nucleus pulposus (NP) tissues from diabetic IVDD patients and in high glucose‐treated NP cells. Senescence and autophagy were assessed in HuR over‐expressing and downregulation NP cells. The mRNAs that were regulated by HuR were screened, and immunoprecipitation was applied to confirm the regulation of HuR on targeted mRNAs.ResultsThe results showed that the expression of HuR was decreased in diabetic NP tissues and high glucose‐treated NP cells. Downregulation of HuR may lead to increased senescence in high glucose‐treated NP cells, while autophagy activation attenuates senescence in HuR deficient NP cells. Mechanistic study showed that HuR prompted Atg7 mRNA stability via binding to the AU‐rich elements. Furthermore, overexpression of Atg7, but not HuR, may ameliorate DB‐IVDD in rats in vivo.ConclusionsIn conclusion, HuR may suppress senescence through autophagy activation via stabilizing Atg7 in diabetic NP cells; while Atg7, but not HuR, may serve as a potential therapeutic target for DB‐IVDD.

SEMA4D under the posttranscriptional regulation of HuR and miR-4319 boosts cancer progression in esophageal squamous cell carcinoma

ABSTRACTEsophageal squamous cell carcinoma (ESCC) is the major type of esophageal carcinoma, one of the main reasons of cancer-caused death. While the therapeutic effect on ESCC patents is still unsatisfactory as a result of tumor aggression, recurrence and metastasis. RNA-binding proteins, microRNAs and specific genes get involved in tumorigenesis and development of tumors in a large proportion. In several reports, SEMA4D is an oncogene and miR-4319 is a tumor suppressor. We discovered the interaction of SEMA4D with HuR and miR-4319, whereas the detailed mechanism in ESCC was yet to be researched. At first, SEMA4D was significantly overexpressed in ESCC cells, and its knockdown repressed cell proliferation and migration as well as accelerated cell apoptosis. And then HuR was proved to stabilize SEMA4D mRNA by binding to its 3ʹUTR. In addition, miR-4319 targeted and degraded SEMA4D. Taken together, SEMA4D was regulated competitively by HuR and miR-4319. Collectively, HuR and miR-4319 co-regulating SEMA4D affected cell proliferation, apoptosis and migration in ESCC. This research explored the regulatory mechanism on SEMA4D in ESCC and provided optional therapeutic targets for ESCC patients.

The RNA-Binding Protein HuR Confers Oxaliplatin Resistance of Colorectal Cancer By Upregulating CDC6.

Human antigen R (HuR) is an RNA-binding protein that posttranscriptionally regulates many cancer-trait genes. CDC6, a central regulator of DNA replication, is regulated by HuR. In this study, we investigated the role of HuR in colorectal cancer tumorigenesis and oxaliplatin (L-OHP) resistance, as well as the underlying mechanisms involving CDC6. We detected increased HuR and CDC6 expression, along with a positive correlation between the two in human colorectal cancer tissues. HuR overexpression increased colorectal cancer cell proliferation in vitro and xenograft tumor growth in vivo, and induced resistance to L-OHP. In contrast, HuR knockdown sensitized colorectal cancer cells to L-OHP. CDC6 overexpression increased while CDC6 knockdown decreased colorectal cancer cell malignant behaviors (growth, DNA synthesis, EMT, migration, and invasion) and L-OHP resistance in vitro Moreover, L-OHP resistance induced by HuR overexpression was reversed by CDC6 knockdown. Mechanistically, the results from our luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that HuR upregulates CDC6 by binding to CDC6 3'-UTR. Taken together, our findings identified HuR's regulation of CDC6 as an essential mechanism driving colorectal cancer tumorigenesis and L-OHP resistance, and this mechanism may represent a potential target for overcoming drug resistance in colorectal cancer.

Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage

SummaryExpression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.

HuR controls apoptosis and activation response without effects on cytokine 3’ UTRs

ABSTRACT RNA binding proteins regulate gene expression through several post-transcriptional mechanisms. The broadly expressed HuR/ELAVL1 is important for proper function of multiple immune cell types, and has been proposed to regulate cytokine and other mRNA 3′ UTRs upon activation. However, this mechanism has not been previously dissected in stable cellular settings. In this study, HuR demonstrated strong anti-apoptotic and activation roles in Jurkat T cells. Detailed transcriptomic analysis of HuR knockout cells revealed a substantial negative impact on the activation program, coordinately preventing the expression of immune response gene categories, including all cytokines. Knockout cells showed a significant defect in IL-2 production, which was rescued upon reintroduction of HuR. Interestingly, the mechanism of HuR regulation did not involve control of the cytokine 3′ UTRs: HuR knockout did not affect the activity of 3′ UTR reporters in 293 cells, and had no effect on IL-2 and TNF 3′ UTRs in resting or activated Jurkats. Instead, impaired cytokine production corresponded with defective induction of the IL-2 promoter upon activation. Accordingly, upregulation of NFATC1 was also impaired, without 3′ UTR effects. Together, these results indicate that HuR controls cytokine production through coordinated upstream pathways, and that additional mechanisms must be considered in investigating its function.

RNA-Binding Protein HuR Regulates Both Mutant and Wild-Type IDH1 in IDH1-Mutated Cancer.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). Although pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently, it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient-deprived cancer cells without IDH1 mutations, by stabilizing WT.IDH1 transcripts. In the present study, a similar regulatory effect on both mutant (Mut.IDH1) and WT.IDH1 transcripts in heterozygous IDH1-mutant tumors is observed. In ribonucleoprotein immunoprecipitation assays of IDH1-mutant cell lines, wild-type and mutant IDH1 mRNAs each bound to HuR. Both isoforms were profoundly downregulated at the mRNA and protein levels after genetic suppression of HuR (siRNAs or CRISPR deletion) in HT1080 (R132C IDH1 mutation) and BT054 cells (R132H). Proliferation and invasion were adversely affected after HuR suppression and metabolomic studies revealed a reduction in Pentose Phosphate Pathway metabolites, nucleotide precursors, and 2-HG levels. HuR-deficient cells were especially sensitive to stress, including low glucose conditions or a mutant IDH1 inhibitor (AGI-5198). IDH1-mutant cancer cells were rescued by WT.IDH1 overexpression to a greater extent than Mut.IDH1 overexpression under these conditions. This study reveals the importance of HuR's regulation of both mutant and wild-type IDH1 in tumors harboring a heterozygous IDH1 mutation with implications for therapy. IMPLICATIONS: This study highlights the HuR-IDH1 (mutant and wild-type IDH1) regulatory axis as a critical, actionable therapeutic target in IDH1-mutated cancer, and incomplete blockade of the entire HuR-IDH1 survival axis would likely diminish the efficacy of drugs that selectively target only the mutant isoenzyme.

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells.

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

Abstract 2113: Targeting tumor-associated hypoxia to overcome chemoresistance in pancreatic ductal adenocarcinoma (PDA)

Abstract Background: PDA will become the 2nd leading cause of cancer-related mortality in the US by 2020. A recent Phase III randomized controlled trial revealed a 4 month overall survival benefit in metastatic PDA with FOLFIRINOX (Folinic acid, 5-Fluorouracil (5FU), Irinotecan, and Oxaliplatin) compared to gemcitabine, the standard of care. However, the long-term clinical efficacy of FOLFIRINOX and other chemotherapy regimens are limited by tumor-associated drug resistance driven by factors in the tumor microenvironment (e.g., hypoxia). We identified a novel drug resistance mechanism driven by the hypoxia-inducible pro-oncogenic kinase PIM1 and regulated by the RNA binding protein HuR. Herein, we launched into developing a strategy to target this tractable mechanism in an effort to optimize current therapeutic treatments for PDA. Methods: To model hypoxia, PDA cells were incubated in 1% O2 and responses to 5FU or oxaliplatin assessed to obtain IC50 doses. Stabilizing interactions between the RNA-binding protein HuR and PIM1 mRNA were quantified in vitro through binding assays, and confirmed in patients by immunohistochemistry in resected PDAs (n = 44). The contribution of HuR-mediated regulation of PIM1 to resistance to 5FU or oxaliplatin in hypoxia was examined using MS-444 (Novartis), a low-molecular-weight HuR inhibitor. Results: In response to hypoxia, HuR translocates from the nucleus to the cytoplasm where it binds and stabilizes the PIM1 mRNA transcript, thus amplifying PIM1 translation and protein expression. Clinically, we identified a positive correlation (p = 0.011) between cytoplasmic HuR and PIM1 protein expression in a cohort of PDA patients from our institution. In vitro mechanistic studies demonstrated that hypoxia-mediated induction of PIM1 overexpression enhanced DNA repair and evaded the apoptotic response elicited by hypoxic stress. Targeted inhibition of HuR by the HuR inhibitor MS-444 abrogated hypoxia-induced PIM1 overexpression, enhancing PDA cell sensitivity to oxaliplatin and 5FU (P<0.001). Conclusion: The mRNA-stability factor HuR post-transcriptionally induces PIM1 expression under hypoxic conditions, and thereby promotes hypoxia-induced chemoresistance. Ongoing pre-clinical studies will evaluate pharmacologic inhibition of HuR's regulation of PIM1 (e.g., MS-444) as a novel modality to enhance the therapeutic value of FOLFIRINOX for the treatment of metastatic PDA. Citation Format: Fernando F. Blanco, Masaya Jimbo, Liz Enyenihi, Nicole Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand Risbud, Peter McCue, Charles Yeo, Jordan Winter, Jonathan R. Brody. Targeting tumor-associated hypoxia to overcome chemoresistance in pancreatic ductal adenocarcinoma (PDA). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2113. doi:10.1158/1538-7445.AM2015-2113

MicroRNA-16 modulates HuR regulation of cyclin E1 in breast cancer cells.

RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

Growth factor dependent regulation of centrosome function and genomic instability by HuR.

The mRNA binding protein HuR is over expressed in cancer cells and contributes to disease progression through post-transcriptional regulation of mRNA. The regulation of HuR and how this relates to glioma is the focus of this report. SRC and c-Abl kinases regulate HuR sub-cellular trafficking and influence accumulation in the pericentriolar matrix (PCM) via a growth factor dependent signaling mechanism. Growth factor stimulation of glioma cell lines results in the associate of HuR with the PCM and amplification of centrosome number. This process is regulated by tyrosine phosphorylation of HuR and is abolished by mutating tyrosine residues. HuR is overexpressed in tumor samples from patients with glioblastoma and associated with a reduced survival. These findings suggest HuR plays a significant role in centrosome amplification and genomic instability, which contributes to a worse disease outcome.

Abstract A05: The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1

Abstract Pancreatic ductal adenocarcinoma (PDA) is expected to become the 2nd leading cause of cancer-related mortality in the United States by 2020. One of the reasons PDA remains one of the most lethal malignancies is due to drug resistance mechanisms to both conventional and targeted chemotherapeutic regimens. Previously, it has been shown that PDA tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Importantly, chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in a widely understudied phenomenon known as hypoxia-induced chemoresistance. Recently, the hypoxia-inducible, serine-threonine, pro-oncogenic kinase PIM1 has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. While its role in therapeutic resistance has been previously described, the molecular mechanism(s) behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present in the PIM1 mRNA 3′UTR mediate a regulatory interaction with the mRNA-stability factor HuR in the context of tumor hypoxia. We have previously demonstrated that HuR, a predominantly nuclear protein, translocates to the cytoplasm upon acute cancer-associated stress and selectively stabilizes key survival-associated transcripts. Here, we demonstrate by immunofluorescence and western blot analyses of fractionated lysates that PDA cells in hypoxic conditions mobilize HuR to the cytoplasm, where it binds and stabilizes the PIM1 transcript. The functional consequence of this interaction is observed with a 5-fold increase in PIM1 protein expression (p≤0.001). PIM1 imparts its oncogenic role by phosphorylating and inactivating key apoptotic proteins (e.g., BAD) and proteins involved in the DNA repair pathway. In this regard, siRNA-mediated knockdown of HuR abrogates hypoxia-induced PIM1 expression and its downstream phosphorylation of the pro-apoptotic protein BAD. As a result, cells undergo growth arrest and succumb to apoptosis. Most importantly, we demonstrate that HuR's regulation of PIM1 modulates a potent chemoresistance phenotype. HuR-mediated stabilization of PIM1 promoted dramatic resistance to 5-Fluorouracil (5-FU) and Oxaliplatin, two critical components of the promising combination therapy FOLFIRINOX. We employed confocal microscopy to analyze DNA damage foci formation by tracking and quantifying γH2AX foci in response to 5-FU and oxaliplatin. As a result of PIM1 up-regulation in hypoxia, or forced PIM1 overexpression in normoxia, PDA cells exhibit fewer DNA damage foci (p≤0.01) due to PIM1's ability to regulate DNA repair. From a translational standpoint, we demonstrate that disruption of the HuR:PIM1 axis by HuR knockdown results in PDA cell growth inhibition and enhances sensitivity to cytotoxic chemotherapeutic agents (5-FU and oxaliplatin). Similarly, pharmacological inhibition of HuR by MS-444 (Novartis), a small molecule that inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and dramatically enhances PDA cell sensitivity to oxaliplatin and 5-FU under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has pro-oncogenic properties in PDA cells by enabling them with selective growth advantages in stressful tumor microenvironment niches. Accordingly, our studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in a hypoxic tumor microenvironment. Citation Format: Fernando F. Blanco, Nicole C. Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand V. Risbud, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A05.

HUR DEPENDENT REGULATION OF CENTROSOMES

BACKGROUND: The RNA-binding protein, HuR is over-expressed in glioma and controls mRNA stabilization and translation in centrosomes. Alterations in HuR phosphorylation promote centrosomal abnormalities. METHODS: Primary and established glioma cell lines were utilized. Subcellular fractionation and immunofluorescence techniques were used to confirm localization of HuR in the PCM. Immunoprecipitation and kinase assays were used to document HuR interaction with and phosphorylation by SRC and ABL1. RESULTS: HuR association with the pericentriolar matrix (PCM) is growth factor dependent and facilitates centrosome amplification and genomic instability in glioma. The SRC and ABL1 kinases are possible regulators of the HuR association with pericentriolar matrix and phosphorylate HuR. CONCLUSIONS: HuR is involved in the regulation of genomic instability through growth factor and centrosome dependent pathways in glioma. We provide the evidence of HuR regulation by SRC and ABL1 kinases in U251 cells and discuss molecular and cellular consequences of this interaction. SECONDARY CATEGORY: n/a.

HSF1 regulation of β-catenin in mammary cancer cells through control of HuR/elavL1 expression.

There is now compelling evidence to indicate a place for heat shock factor 1 (HSF1) in mammary carcinogenesis, tumor progression and metastasis. Here we have investigated a role for HSF1 in regulating the expression of the stem cell renewal factor β-catenin in immortalized human mammary epithelial and carcinoma cells. We found HSF1 to be involved in regulating the translation of β–catenin, by investigating effects of gain and loss of HSF1 on this protein. Interestingly, although HSF1 is a potent transcription factor, it was not directly involved in regulating levels of β-catenin mRNA. Instead, our data suggest a complex role in translational regulation. HSF1 was shown to regulate levels of the RNA binding protein HuR that controlled β-catenin translation. An extra complexity was added to this scenario when it was shown that the long non-coding RNA molecule lincRNA-p21, known to be involved in β-catenin mRNA (CTNNB1) translational regulation, was controlled by HSF1 repression. We have shown previously that HSF1 was positively regulated through phosphorylation by mTOR kinase on a key residue, serine 326 essential for transcriptional activity. In this study we found that mTOR knockdown not only decreased HSF1-S326 phosphorylation in mammary cells, but also decreased β-catenin expression through a mechanism requiring HuR. Our data point to a complex role for HSF1 in the regulation of HuR and β-catenin expression that may be significant in mammary carcinogenesis.

Altered Regulation of ELAVL1/HuR in HLA-B27–Expressing U937 Monocytic Cells

ObjectiveTo investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response.MethodsU937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA–B27, or mutated HLA–B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA.ResultsFull length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27.ConclusionResults show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

Multiple Functions of the RNA-Binding Protein HuR in Cancer Progression, Treatment Responses and Prognosis

The human embryonic lethal abnormal vision-like protein, HuR, is a member of the Hu family of RNA-binding proteins. Over the past decade, this ubiquitously expressed protein has been extensively investigated in cancer research because it is involved in the regulation of mRNA stability and translation in many cell types. HuR activity and function is associated with its subcellular distribution, transcriptional regulation, translational and post-translational modifications. HuR regulation of target mRNAs is based on the interaction between the three specific domains of HuR protein and one or several U- or AU-rich elements (AREs) in the untranslated region of target mRNAs. A number of cancer-related transcripts containing AREs, including mRNAs for proto-oncogenes, cytokines, growth factors, and invasion factors, have been characterized as HuR targets. It has been proposed that HuR has a central tumorigenic activity by enabling multiple cancer phenotypes. In this review, we comprehensively survey the existing evidence with regard to the diverse functions of HuR in caner development and progression. The current data also suggest that HuR might be a novel and promising therapeutic target and a marker for treatment response and prognostic evaluation.

Abstract 628: A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells.

Abstract Introduction: We explored HuR's (an RNA binding protein) significance in the DNA damage response (DDR) in pancreatic ductal adenocarcinoma (PDA) cells. Experimental Procedures: We treated PDA cells with DNA damaging agent's mitomycin C (MMC), oxaliplatin, cisplatin, carboplatin, gemcitabine and a PARP inhibitor (ABT-888). HuR subcellular localization was assessed by immunoblotting. Additionally, we measured γH2AX foci, a marker of DNA damage, by immunofluorescence. Drug sensitivity assays were performed in isogenic PDA cells that differed in HuR expression (siRNA oligos against HuR, cDNAs encoding HuR for overexpression, and appropriate controls). A BrdU/PI pulse chase experiment monitored the cell cycle progression after treating cells with low (150nM) and high (1μM) doses of MMC. Ribonucleoprotein-immunoprecipitation (RIP) assays were used to isolate mRNAs bound to HuR to identify HuR targets in the DDR. Results: Enhanced-HuR cytoplasmic protein expression was observed in PDA cells exposed to all DNA damaging agents. Under high doses of certain DNA damaging agents (e.g., MMC) HuR was cleaved in a caspase-dependent manner. To assess HuR's role in the DDR, isogenic PDA cells lines manipulated with HuR levels were treated with MMC (150nM) for 2 hrs. γH2AX foci assay revealed a 3-fold increase in foci number with MMC treatment in the HuR depleted cells, as compared to the control cells, suggesting that DNA repair was delayed in the absence of HuR. Cell cycle analyses demonstrated that HuR-depleted PDA cells progressed through the G2/M checkpoint and into mitosis upon MMC treatment. A combinatorial approach with RIP-sequencing and an siRNA library screen for genes resistant to DNA damaging agents was performed, and WEE1, a mitotic inhibitor kinase, was identified as a novel HuR target. A separate RIP-qPCR experiment validated WEE1 as an HuR binder with 56-fold more expression upon MMC stress, compared to an IgG control-IP. WEE1 protein expression and Cdk1 (a target of the WEE1 kinase and G2/M phase regulator) phosphorylation was reduced in HuR depleted cells, validating HuR as an upstream post-transcriptional regulator of WEE1 and an indirect inhibitor of the cdk1-cyclin complex in the face of DNA damage. Conclusions: We demonstrate that HuR-deficiency allows PDA cells to enter the cell cycle more efficiently upon DNA damage. This event is dependent on HuR's regulation of WEE1 leading to activation of the CDK1-cyclin complex. These data support a model where HuR is integral to the G2/M checkpoint, forcing a cell cycle recovery response which is critical for PDA cells to avoid DNA damage. Therefore, HuR promotes survival of PDA cells through its acute activation of the DDR pathway. Disruption of HuR's molecular interaction with WEE1 represents a therapeutic opportunity to enhance clinically utilized chemotherapeutic DNA damaging agents for PDA. Citation Format: Shruti Lal, Richard A. Burkhart, Zoe Norris, Neil Beeharry, Vikram Bhattacharjee, Isidore Rigoutsos, Timothy Yen, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells. [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 628. doi:10.1158/1538-7445.AM2013-628

Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin

We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element-binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100μg/ml (0.61mM) for isoeugenol, 100μg/ml (0.58mM) for DEM, 3μg/ml (14.8μM) for DNCB, and 250μg/ml (1.81mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release.

High-constitutive HuR phosphorylation at Ser 318 by PKCδ propagates tumor relevant functions in colon carcinoma cells

Overexpression of the messenger RNA (mRNA)-binding protein HuR is an important feature of many tumors and in most cases correlates with a high-grade malignancy. Since phosphorylation of HuR by protein kinase C δ (PKCδ) at serine (Ser) 318 implies an important mode in HuR regulation, we studied its functional role in dysregulated HuR and related functions in colon carcinoma cells. Coimmunoprecipitation experiments revealed a high-constitutive association of nuclear PKCδ with HuR. Using a phospho-Ser 318-specific HuR antibody, we found a strong increase in nuclear HuR phosphorylation in DLD-1 cells when compared with nontransformed CCD 841 colon epithelial cells. Importantly, a strong increase in HuR phosphorylation at Ser 318 was also found in tissue specimen from human colon carcinomas. Employing ribonucleoprotein-immunoprecipitation, we show that DLD-1 cells displayed a strong and constitutive RNA binding of HuR to cyclooxygenase-2 (COX-2) and cyclin A encoding mRNAs that was strongly impaired by rottlerin, an inhibitor of novel PKCs. Accordingly, rottlerin accelerated the decay of COX-2 and cyclin A encoding mRNAs concomitant with a reduced expression of both genes. Functionally, migration and invasion is similarly impaired in PKCδ- or HuR-small interfering RNA-depleted cells and in tumor cells transfected with a nonphosphorylatable serine-to-alanine 318 HuR construct. Conversely, expression of a phosphomimetic Ser 318 aspartic acid (D) HuR caused a significant increase in migration and proliferation of CCD 841 cells. Our data suggest that the increased HuR phosphorylation at Ser 318 by PKCδ reflects an important regulatory paradigm for aberrant HuR functions and emphasize the antitumorigenic potential of PKCδ inhibitory strategies.

The mRNA-binding protein HuR is regulated in the menstrual cycle and repressed in ectopic endometrium.

Cytokines modulate turnover of the endometrium during the menstrual cycle and contribute to the pathogenesis of endometriosis. Gene expression for cytokines is often regulated by proteins that bind to adenosine- and uridine-rich elements (AREs) in their transcripts to stabilize or destabilize bound messenger RNAs (mRNAs). HuR/ELAVL1 is an RNA-binding protein that stabilizes ARE-containing mRNAs. We hypothesized that HuR might play a role in regulating cytokine expression during the menstrual cycle and in endometriosis and characterized the expression and regulation of HuR in eutopic and ectopic human endometrium. Tissue sections obtained from normal (n = 23) and ectopic (n = 16) endometrium were immunostained for HuR, and staining intensity was evaluated by HSCORE. Cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (E2), progesterone (P), E2 + P, tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) for 24 hours, and HuR expression was determined by Western blot. HuR immunoreactivity was significantly lower in the early proliferative and late secretory phases (157.5 ± 11.08 and 190.0 ± 15.2, respectively), compared to the mid-late proliferative (270.0 ± 8.0) and early-mid secretory phases (256.6 ± 20.2; P < .01, analysis of variance [ANOVA]). Furthermore, HuR expression was significantly lower in ectopic endometrial cells compared to normal endometrium in mid-late proliferative and early-mid-secretory phases (P < .01). Estrogen, P, or cytokines did not alter HuR expression in cultured endometrial stromal cells. Increased HuR levels in the mid-menstrual phases are likely to contribute to reduced mid-cycle cytokine expression and enhanced cellular survival in eutopic endometrium. In ectopic endometrium, elevated cytokine levels associated with endometriosis likely reduce HuR expression.