HuR Expression Research Articles

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth.

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

Insights from HuR biology point to potential improvement for second-line ovarian cancer therapy.

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices.

Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1.

RNA-binding protein HuR mediates transforming growth factor (TGF)-β1-induced profibrogenic actions. Up-regulation of Sphingosine kinase 1 (SphK1) is involved in TGF-β1-induced activation of hepatic stellate cells (HSCs) in liver fibrogenesis. However, the molecular mechanism of TGF-β1 regulates SphK1 remains unclear. This study was designed to investigate the role of HuR in TGF-β1-induced SphK1 expression and identify a new molecular mechanism in liver fibrogenensis. In vivo, HuR expression was increased, translocated to cytoplasm, and bound to SphK1 mRNA in carbon tetrachloride- and bile duct ligation-induced mouse fibrotic liver. HuR mRNA expression had a positive correlation with mRNA expressions of SphK1 and fibrotic markers, α-smooth muscle actin (α-SMA) and Collagen α1(I), respectively. In vitro, up-regulation of SphK1 and activation of HSCs stimulated by TGF-β1 depended on HuR cytoplasmic accumulation. The effects of TGF-β1 were diminished when HuR was silenced or HuR cytoplasmic translocation was blocked. Meanwhile, overexpression of HuR mimicked the effects of TGF-β1. Furthermore, TGF-β1 prolonged half-life of SphK1 mRNA by promoting its binding to HuR. Pharmacological or siRNA-induced SphK1 inhibition abrogated HuR-mediated HSC activation. In conclusion, our data suggested that HuR bound to SphK1 mRNA and played a crucial role in TGF-β1-induced HSC activation.

Abstract B41: Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation

Abstract Introduction: Isocitrate dehydrogenase 1(IDH1) has been prioritized in the recent cancer biology literature because of mutations occurring in some tumor types. Additionally the wild type isoenzyme is important for metabolic reprogramming under hypoxic stress. Importantly, the role of IDH1 under other forms of metabolic stress, such as nutrient deprivation (hallmark of the PDA microenvironment) has not been explored, and the regulatory mechanism of IDH1 expression remains unknown. We recently showed that the regulatory RNA binding protein, HuR, binds and directly regulates IDH1 expression in multiple PDA cell lines. The regulatory protein becomes biologically engaged under nutrient deprivation by translocating with the IDH1 transcript form the nucleus to the cytoplasm under glucose deprivation, and HuR silencing sensitized cells under these conditions. Here, we hone in on the regulatory HuR binding site to the IDH1 transcript, explore the importance of HuR in the context of additional metabolic stressors and utilize stable isotope metabolomic profiling to gain mechanistic insight into how HuR is protective under nutrient deprivation. Methods: IDH1 and HuR expression were knocked down by siRNA, and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Immunofluorescence was used to image HuR subcellular localization under glutamine deprivation. Based on computational predictions of 5 HuR binding sites in the IDH1 mRNA 3'UTR, we subcloned this entire region into a luciferase reporter construct to further study this regulatory interaction. In order to determine the impact of HuR expression in cellular metabolism targeted tracer fate association studies were performed using GC-MS of pellets from cells cultured with 13C-labeled glucose and glutamine in BxPC3 pancreatic cancer cells that differed only in HuR expression. We calculated Pearson correlations of measured metabolites after HuR silencing. Results: Cell viability was impaired by depletion of both IDH1 and HuR (vs. controls) upon glutamine and glucose withdrawal. Moreover, HuR silencing resulted in potent suppression of IDH1 at the mRNA and protein levels. HuR silencing substantially decremented luciferase activity in the IDH1 3'UTR construct compared to the control (> 2-fold decrease). HuR translocated to the cytoplasm under glutamine deprivation, as previously published observed with glucose deprivation by our group. Metabolic tracer fate association studies revealed that HuR silencing impaired carbon flux from glutamine into fatty acid end products (myristate and palmitate, > 0.9-Pearson's Correlation) under low glucose conditions, implicating HuR's regulatory role in the IDH1-mediated reductive carboxylation step of this metabolic pathway. Additionally, HuR silencing impaired ribose and glycogen synthesis from glucose, and futile carbon exchange fluxes were prevalent. Conclusions: HuR is important for pancreatic cancer cell survival under glutamine deprivation, as previously observed for glucose deprivation. Carbon flux from glutamine to fatty acid end products suggests a role for HuR in reductive carboxylation of glutamine-derived α-ketoglutarate by IDH1, as a way to maintain adequate lipid synthesis under glucose deprivation. Our Studies provide a rationale to pursue pharmacologic strategies that target HuR or its regulation of IDH1 as a novel treatment of PDA. Citation Format: Mahsa Zarei, Fernando F. Blanco, Laszlo G. Boros, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B41.

Expression of ARE-binding proteins AUF1 and HuR in follicular adenoma and carcinoma of thyroid gland.

Both adenylate-uridylate rich elements binding proteins AUF1 and HuR may participate in thyroid carcinoma progression. In this study we investigated the expression of both factors on aprotein level with aspecial focus on follicular adenoma and follicular thyroid carcinoma. By employment of immunofluorescence and western blot on 68 thyroid tissues including 7 goiter, 16 follicular adenoma (4 adenomatous hyperplasia), 19 follicular thyroid carcinomas, 13 papillary thyroid carcinomas and 14 undifferentiated thyroid carcinomas we investigated protein expression of AUF1 and HuR. In addition to previous results we demonstrated that AUF1 and HuR are significantly up-regulated in carcinoma tissues as compared with follicular adenoma or goiter tissues. Furthermore, by evaluation of AUF1 or HuR expression, or combination of both proteins on total tissue lysates, we were able to demonstrate asignificant difference between follicular adenoma and follicular thyroid carcinoma. Overexpression of AUF1 and HuR is acommon finding observed in thyroid malignancy. Analysis of the tissues obtained by surgical resection as demonstrated in this study is comparable to afine needle aspiration and in combination with AUF1/HuR immuno-analysis may support the conventional immunohistological investigations. The promising results of this study were performed on relatively small collective, but justify future development of aquick thyroid diagnostic test on larger cohort of the patients, especially for thyroid samples which are inadequate for histological examinations.

A Feed-Forward Regulatory Loop between HuR and the Long Noncoding RNA HOTAIR Promotes Head and Neck Squamous Cell Carcinoma Progression and Metastasis

Background/Aims: The lncRNA Homeobox (HOX) transcript antisense RNA (HOTAIR) is overexpressed in numerous cancers. HuR is also overexpressed during tumourigenesis and is abnormally present within the cytoplasm, where it binds to AU-rich elements in the 3′UTRs of target mRNA and post-transcriptionally regulates the expression of its target genes. However, whether HOTAIR is regulated and the mechanisms by which it affects head and neck squamous cell carcinoma (HNSCC) are not well understood. Methods: MTT, cell cycle arrest and apoptotic assays were used to examine the effects of HOTAIR and HuR on cell viability in SCC25 and FaDu cells. Wound healing and transwell invasion analysis were performed to detect the effects of HOTAIR and HuR on cell migration and invasion. The interaction between HuR and HOTAIR was confirmed via qRT-PCR, western blots, luciferase reporter and RIP assays. Finally, qRT-PCR analysis was used to detect the levels of HuR and HOTAIR in HNSCC tumours and adjacent normal tissues. Results: Knockdown of HOTAIR and HuR decreased cell viability, cellular migration and invasion. Moreover, HuR interacted and stabilized HOTAIR stability and thus promoted HOTAIR expression. Notably, HOTAIR acted as a miRNA sponge for HuR. HuR also reinforced HOTAIR sponge activity through miRNA recruitment, thus enhancing HuR expression in turn. Finally, HuR and HOTAIR levels were positively correlated and significantly up-regulated in tumours samples. Conclusion: We demonstrated the existence of a regulatory loop in which the expression of HOTAIR and HuR is reciprocally and temporally regulated during the metastasis and progression of HNSCC.

MiR-519 inhibits gastric cancer cell activity through regulation of HuR expression

To investigate the role of miR-519 in regulating the cell activity of gastric cancer. The expression of miR-519 was measured by qPCR. The protein expression of HuR was detected by Western blot. MTT assay was used to detect the cell activity of gastric cancer cells. The protein level of HuR in the gastric cancer cells was higher than that in the control cells. Over-expression of HuR resulted in the increase in the cell activity of gastric cancer (P<0.001). Suppression of HuR decreased the cell activity of gastric cancer (P=0.001). The miR-519 level in the gastric cancer cells was lower than that in the control cells (P=0.001). Over-expression of miR-519 decreased the protein level of HuR and the activity of gastric cancer cells. MiR-may inhibit the gastric cancer cell activity through suppression of HuR expression.

Abstract B2-13: AU-rich expression in invasive ductal breast cancer: A systems biology approach in post-transcriptional regulation

Abstract AU-Rich element (ARE)-dependent post-transcriptional regulation mechanisms are increasingly emerging as key regulators that are influencing our perception of aberrant gene expression in cancer. AREs are instability determinant sequences located in the 3' UTR of mRNAs and are fundamental to the maintenance of transient expression of gene products that regulate various cellular functions such as cell proliferation, apoptosis, inflammation and immunity. Here we used a systems biology approach to examine the patterns of ARE-containing gene expression that is perturbed in invasive ductal breast cancer (IDBC). Based on IDBC mRNA expression profiles obtained from The Cancer Genome Atlas (TCGA) database, we intersected 2396 over-expressed genes with the AU-rich element database (ARED) and found that 414 comprise IDBC upregulated ARE-containing genes. Functional classification using the gene ontology tool, DAVID, showed the highest enrichment of ARE-genes in IDBC in chromosome segregation (5-fold, p&amp;lt;0.001). AREs are recognized by RNA binding proteins such as tristetraprolin (TTP, ZFP36), that targets mRNAs for decay, and HuR (ELAVL1), an mRNA stabilizing protein. Abnormal expression patterns of both these proteins, i.e. TTP deficiency coupled with HuR overexpression, are found in many cancers leading to prolonged expression of factors contributing to the maintenance and progression of tumors. We examined the relationship between the IDBC ARE-genes, TTP and HuR expression and the TTP/HuR ratio by looking at their correlations (R-values) with each other. Results indicated that the upregulated ARE-genes have a greater tendency to correlate with low TTP and elevated HuR expression than non-ARE genes. Using clustering assessment, we derived an 11-gene cluster from the 414 that function in chromosome positioning and segregation and mitotic kinase activity. The genes are CDC6, CENPA, CENPA, KIF11, KIF18A, NCAPG, NEK2, NUF2, PBK, PRC1, and TOP2A and their mRNA expression correlates negatively with the TTP/HuR ratio, i.e. their mRNA expression increases as TTP is deficient and HuR is simultaneously elevated (r = -0.53 to -0.66). Examination of the mRNA expression patterns of the 11 genes in the invasive breast cancer cell line MDA-MB-231 compared to MCF-7 cells and normal-like mammary cell lines, MCF10A and MCF12A showed that all of these genes were increased in MDA-MB-231 cell line compared to MCF10A by at least 2-fold (p&amp;lt;0.001). Immunoprecipitation of TTP protein in MDA-MB-231 cells followed by examination of associated mRNAs showed that 7 of the 11 gene-products were significantly associated with TTP (greater than 2-fold, p&amp;lt;0.01) compared to the control suggesting post-transcriptional regulation by TTP. Moreover, reporter experiments confirmed the functionally of the AREs. Upon examining the METBRIC breast cancer database, a larger database, we found that not only do the 11 chromosomal segregation genes overexpress, they also tend to be upregulated simultaneously in the same patients indicating that they participate in common regulatory mechanisms. Furthermore, the 11-gene cluster also correlates negatively with the TTP/HuR ratio further alluding to TTP regulation. Kaplan Meier survival analysis demonstrated a higher likelihood (1.6 times) of death due to co-overexpression of the 11 genes compared with a lower-expressing cohort. The data presented here suggest novel regulatory mechanisms related to mitosis and cellular proliferation that might be targeted by the mRNA decay-promoting protein TTP. Moreover, TTP deficiency in IDBC may, at least in part, be responsible for the aberrantly prolonged expression of factors that actively participate in chromosomal segregation and in turn, lead to enhanced maintenance and progression of tumors. Citation Format: Norah A. Alsouhibani, Edward Hitti, Tala Bakheet, Khalid SA Khabar. AU-rich expression in invasive ductal breast cancer: A systems biology approach in post-transcriptional regulation. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-13.

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

Abstract 14356: Blood Tests That Improve the Accuracy of a Brugada Syndrome Diagnosis

Objectives: To discover the role of altered gene expression regulation in Brugada Syndrome (BrS) and to find biomarkers for BrS diagnosis. Methods: Twenty-five control patients (Control), 25 BrS patients without SCN5A mutation (SCN5A(-)) and 20 BrS patients with SCN5A mutation (SCN5A(+)) were included in this study. Specified gene expression of white blood cells (WBC) were measured by RT-qPCR using TaqMan® Gene Expression assay. Results: MEF2C and MESP1 are the two major cardiac specific transcription factors expressed in WBC. The mRNA expression levels of SCN5A, MEF2C and HuR, one of mRNA stabilizers, were decreased in the SCN5A (+) group (P=0.047, 0.02, 0.000 vs. control group, respectively). The mRNA expression of MESP1 in WBCs was significantly lower in both SCN5A(-) (P=0.012 vs. control) and SCN5A(+) (P=0.000 vs. control) groups. There was no difference between the two BrS groups in MESP1 expression (P=0.215). The area under the Receiver Operating Characteristics (ROC) analysis curve for prediction of BrS using MESP1 levels was 0.775 (95% CI 0.668, 0.882, asymptotic Sig.=0.000). At the optimal cutoff, the corresponding maximum sensitivity and specificity were 0.62 (95% CI: 0.47, 0.76) and 0.88 (0.69, 0.97), respectively. The diagnostic odds ratio (DOR) of MESP1 for BrS diagnosis was 11.96 (95% CI: 5.79, 24.73). The assessment of the mRNA levels in blood SCN5A, MEF2C and HuR were useful for predicting BrS patients with an SCN5A mutation. The area under the ROC analysis curve for prediction of BrS with an SCN5A mutation using SCN5A, MEF2C and HuR mRNA levels in WBCs was 0.847 (95% CI 0.752, 0.942, asymptotic Sig.=0.000), 0.685 (95% CI 0.542, 0.828, asymptotic Sig.=0.016) and 0.777 (95% CI 0.652, 0.902, asymptotic Sig.=0.000), respectively. At the optimal cutoff, the DOR of SCN5A, MEF2C and HuR for SCN5A(+) BrS diagnosis was 17.5 (95% CI: 8.06, 37.86), 4.9 (95% CI: 2.61, 9.17) and 23.5 (95% CI: 9.39, 58.80), respectively. Conclusions: Our results suggest that assessment of circulating MESP1 may be used as a biomarker for BrS diagnosis while decreased SCN5A, MEF2C and HuR mRNA in WBCs is associated with BrS patients with an SCN5A mutation. Our results also suggest that decreased expression of SCN5A, MEF2C, MESP1, and HuR may be pathophysiologically related to BrS.

HUR mRNA expression in ovarian high-grade serous carcinoma effusions is associated with poor survival

The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

HuR Mediates Changes in the Stability of AChR β-Subunit mRNAs after Skeletal Muscle Denervation.

Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR β-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.

Abstract 5133: The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the Unites States, with an overall 5-year survival rate of &amp;lt;6%. The ubiquitously expressed RNA-binding protein HuR (ELAVL1) has been implicated in numerous cancer types as a post-transcriptional facilitator of tumorigenesis, through its stabilization of various proliferation-, survival-, and metastasis-associated mRNA transcripts. Our lab has previously demonstrated that elevated HuR expression in human resected PDA samples is associated with poor prognostic factors (e.g. increased T stage). Here, we explored the role of HuR expression in human PDA growth and metastasis by generating isogenic PDA cell lines with induced silencing or overexpression of HuR. Methods: Stable MiaPaCa2 cell lines were generated that produce two different shRNAs against the HuR coding sequence (labeled Mia.sh290 and Mia.sh700) in response to doxycycline (DOX) induction. HuR silencing was validated by RT-qPCR and Western blotting. Similarly, a DOX-inducible MiaPaCa2 cell line that overexpresses HuR cDNA (Mia.HuR) was generated and validated. Sustained ∼50% knockdown and 1.5-2-fold overexpression were achieved, respectively. Following characterization of HuR expression in these cell lines under ±DOX conditions, we performed a 7-day cell proliferation (PicoGreen) assay and assessed anchorage-independence over a 4-week period by soft agar colony formation assay. Metastatic potential was measured by Matrigel invasion and in vitro scratch test assays. Results: Upon DOX induction, Mia.sh290 and Mia.sh700 cell lines showed significantly reduced cell proliferation over 7 days (22% and 15% reduction compared to non-induced in sh290 and sh700 respectively, p&amp;lt;0.001). Accordingly, HuR overexpression in Mia.HuR showed slightly increased cell proliferation over 7 days (15% increase compared to non-induced, p&amp;lt;0.001). HuR silencing resulted in a dramatic reduction of colony formation in soft agar over 4 weeks (57% and 71% reduction compared to non-induced control cells, p&amp;lt;0.05). HuR silencing also reduced cells’ ability to invade through Matrigel (41% and 56% reduction compared to non-induced control cells, p&amp;lt;0.05), whereas HuR overexpression (Mia.HuR) resulted in a dramatic increase in invasion (1,250% increase compared to control, p&amp;lt;0.05). In a complementary in vitro scratch test, cell migration was reduced by HuR silencing and increased by HuR overexpression (p&amp;lt;0.01 for both). Conclusion: Taken together with our previous findings, our work demonstrates that manipulation of HuR expression affects key characteristics of tumorigenesis (i.e. proliferation, invasion, and migration). These findings, along with ongoing in vivo work, continue to support the notion that targeting HuR in PDA cells may be a promising therapeutic strategy. Citation Format: Masaya Jimbo, Fernando F. Blanco, Brad Screnci, Gabriela Cosma, Vitali Alexeev, Yu-Hung Huang, Jordan M. Winter, Charles J. Yeo, Janet A. Sawicki, Jonathan R. Brody. The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma. [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 5133. doi:10.1158/1538-7445.AM2015-5133

Abstract 1191: The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism

Abstract Introduction: Few proteins have been found to be master-regulators of pancreatic ductal adenocarcinoma (PDA) cell metabolism (e.g., LKB1, KRAS). We recently demonstrated that the regulatory RNA binding protein, HuR, binds to numerous metabolic mRNA transcripts and regulates their expression. Additionally, HuR silencing with small interfering RNAs(siRNAs) inhibited growth of PDA cells in vitro under glucose deprivation. Herein, we manipulated HuR expression levels in PDA cells and performed isotope tracer fate association studies to better understand HuR's role in metabolic reprogramming. Methods: We used 13C-labeled nutrients to map carbon flux in BxPC3 pancreatic cancer cells that were either transiently transfected with siRNA oligos against HuR (siHuR) or scrambled control (siCTRL). Cells were incubated with standard DMEM or mild glucose deprivation (5 mM glucose) for 48 hours, and media were supplemented with [1,2-13C2]-D-glucose or [U-13C5]-L-glutamine tracers for the final 24 hours. A total of 24 samples were snap frozen and prepared for GC-MS isotopomer analysis (3 replicates X 2 different siRNA oligos X 2 isotope tracers X 2 glucose concentrations). Transfection experiments were validated for reduced HuR expression (&amp;gt;70% reduction) and reduced protein expression of at least one metabolic and established HuR target (e.g., IDH1) by immunoblot. Results: HuR silencing directly impaired fatty acid (Table, line 1-3), ribose (line 5) and glycogen synthesis (line 6). Reductive carboxylation of glutamine-derived isocitrate was impaired and futile carbon exchange fluxes were prevalent (not shown). Conclusions: HuR enhances metabolic efficiency in PDA cells by directly regulating multiple metabolic pathways. Ongoing microarray studies will highlight which metabolic transcripts are post-transcriptionally regulated by HuR, resulting in the observed phenotype. Altered metabolites due to HuR silencing in PDA cells ([1,2-13C2]-D-glucose tracer)PathwayMetabolitesiHuR 25mM glucosesiHuR 5 mM glucoseCorrelation1Myristate (C:14) Intracell13C enrichment70.271.01.02Myristate (C:14) intracellFNS (direct)56.060.41.03Oleate (C:18-1) IntracellIndirect synthes-m187.689.51.04Glutam extracell [C2-C5]13C-m1 (m/z198)97.497.91.05RNA-ribose Intracell [C1-C4]13C-m3(m/z242)99.399.10.9846Glucose intracell [C3-C6]Peak area, Glycog-gluc74.180.10.983 Citation Format: Fernando F. Blanco, Mahsa Zarei, Jonathan R. Brody, Laszlo G. Boros, Jordan M. Winter. The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism. [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 1191. doi:10.1158/1538-7445.AM2015-1191

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&amp;lt;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

Abstract 3542: Inhibition of HuR effectively suppresses ovarian tumor growth in mice

Abstract Ovarian cancer is the most deadly gynecologic cancer. To address the clinical need for a more effective therapy than currently exists, this investigation focused on targeting a post-transcriptional (RNA to protein) regulatory RNA-binding protein, HuR (Human antigen R, aka ELAVL1). HuR regulates the expression of multiple genes known to function in tumor cell survival and in drug resistance. It is abundant and hyper-functional in ovarian cancer cells as compared to normal cells, thus providing a therapeutic window. We investigated the effect of inhibiting HuR expression in ovarian tumor cells in culture and in ovarian tumors in mice. We generated A2780 and OVCAR5 cells that constitutively express either shHuR or a control shRNA, and assayed cell proliferation and the ability of these cells to produce colonies in a soft agar. Cell proliferation in both shRNA-expressing cell lines was significantly reduced compared to control shRNA-expressing cells (19% and 11% in A2780 and OVCAR5, respectively; p&amp;lt;0.005). A2780-shRNA cells produced 65% fewer colonies in soft agar a compared to parental A2780 cells and control shRNA-expressing cells. To evaluate the effect of HuR suppression on tumor growth, C57BL/6 female mice were injected intraperitoneally with ID8-luc murine ovarian cancer cells; mice developed tumors throughout the peritoneal cavity. Approximately 4 weeks following cell injection, tumor-bearing mice were optically imaged to establish baseline biolumiscence, an indicator of ID8-luc-derived tumor load. A novel CpG-free DNA dendrimer vector, 3DNA, was derivatized with folate, aiming for tumor-targeted siHuR delivery upon systemic administration to mice. Mice were treated twice a week for four weeks (retro-orbital injection) with one of three 3DNA formulations or with 0.9% saline (n = 5-6/group). The 3DNA formulations were: 1) Folate-derivatized 3DNA/siHuR; 2) 3DNA/siHuR; and 3) Folate-derivatized 3DNA/control siRNA. Tumor response to treatment was assessed once a week by optical imaging. Using baseline tumor load as the comparator, tumor growth was suppressed ∼3-fold in mice treated for 4 weeks with folate-derivatized 3DNA/siHuR, the highest suppression of all treatment groups, including non-folate derivatized 3DNA/siHuR (p = 0.01), folate-derivatized 3DNA/control siRNA (p = 0.05), and 0.9% saline (p = 0.04). No overt toxicity was observed. A biodistribution study in which CY3-labeled folate-derivatized 3DNA was systemically administered resulted in very high fluorescence in ovarian tumors with little to no fluorescence in normal tissues, thus supporting the tumor-targeting potential of folate and the enhanced therapeutic effect observed in folate-derivatized 3DNA/siHuR treated mice. These results support further pursuit of HuR-targeted therapy as a complementary approach to current, active therapies for ovarian cancer for overcoming drug resistance and inhibiting tumor growth. Citation Format: Janet A. Sawicki, Yu-Hung Huang, Jonathan R. Brody, Robert C. Getts, Kelly Rhodes, Jackie Gerhart. Inhibition of HuR effectively suppresses ovarian tumor growth in mice. [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 3542. doi:10.1158/1538-7445.AM2015-3542

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.

Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3′-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3′-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a ‘miRNA sponge‘ to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.

Abstract 255: Hur/ppar-gamma Mediates Posttranscriptional Regulation of Hyperglycemia-induced Pro-inflammatory Response

Metabolic disorders including diabetes and obesity are associated with persistent activation of the innate immune response, which leads to low-grade inflammation and increased cardiovascular risk. The RNA-binding protein HuR binds to and post-transcriptionally regulates inflammatory cytokines, mRNA stability and gene expression, although it’s exact role in the diabetic condition remains unclear. Bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) displayed elevated expression of HuR and reduced expression of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) as compared to low glucose (LG, 5mM D-glucose)-treated cells. The increase in HuR expression was associated with an increase in the gene expression of various inflammatory cytokines including TNF-alpha, MCP-1, IL-1beta and IL-6. At the molecular level, macrophages activated with LPS showed enhancements in expression of inflammatory mRNAs and also promoted their stability. Conversely, knockdown of HuR induced post-transcriptional silencing, reduced the mRNA stability and expression of inflammatory cytokines. Macrophages treated with selective antagonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW9662 displayed increases in HuR expression and of various inflammatory cytokines. In an in vivo model of myocardial infarction, mice receiving intramyocardial injection of HuR-specific shRNA displayed a reduced inflammatory cytokine profile in the myocardium as compared to control shRNA treated mice. Taken together, these data highlight the role of HuR as a homeostatic coordinator of cytokine mRNA that regulates innate inflammatory effects and demonstrates the potential of modulating the effects of HuR for clinical benefit against pathologic inflammation in diabetes/obesity.

Methyl(R217)HuR and MCM6 are inversely correlated and are prognostic markers in non small cell lung carcinoma.

In non small cell lung carcinoma (NSCLC), earlier studies supported a prognostic value of intra-cytoplasmic HuR expression. HuR is a RNA binding protein previously shown to stimulate proliferation, but the link between HuR and proliferation in NSCLC has not yet been evaluated. The first objective of this study was to analyze the expression of HuR in a series of NSCLC and to correlate this to two proliferation markers, Ki-67 and MCM6. As potential post-transcriptional regulatory mechanisms for HuR expression, two miRNAs, miR16 and miR519, were also analyzed. Finally, because HuR methylation could be involved in its nucleocytoplasmic shuttling, the expression of methyl(R217)HuR and its relation to cancer survival were determined. Immunohistochemistry was used to evaluate the expression of HuR, methy(R217)HuR, Ki-67 and MCM6 in a series of 190 NSCLCs. The level of miR16 and miR519 was determined by qRT-PCR. Higher cytoplasmic HuR staining was found in tumor vs. control paired normal lung (p<0.0001), but without correlation with survival. The level of methyl(R217)HuR was correlated both significantly with intra-cytoplasmic HuR staining (p<0.001), and overall survival (p=0.01). MCM6 correlated to a poorer overall survival (p<0.01). Both MCM6 and Ki-67 were positively correlated with HuR nuclear staining (p<0.0001 and p<0.001, respectively). On the contrary, MCM6 and Ki-67 correlated inversely to methyl(R217)HuR (p<0.001 and p=0.01, respectively). The levels of miR16 and miR519 were significantly lower in tumor tissue vs. paired normal lung (p<0.0001), but only miR519 correlated inversely to HuR expression (p=0.01). While overall cytoplasmic HuR level was higher in tumor tissues, we found unexpectedly that methyl(R217)HuR was a marker of good prognosis. Furthermore, our data suggest that HuR level could be regulated by miR519. Finally, we demonstrated that Ki-67 and MCM6, both correlated with HuR, are valuable markers of poor prognosis in NSCLC.