Overexpression Of HuR Research Articles

The RNA-binding protein ELAVL1/HuR is essential for mouse spermatogenesis, acting both at meiotic and postmeiotic stages

Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accurate protein synthesis during germ cell development relies on RNA binding proteins that control the storage, stability, and translation of mRNAs in a tightly and temporally regulated manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regulation in somatic cells but the function of which during gametogenesis has never been investigated. In this study, we have used conditional loss- and gain-of-function approaches to address this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads to male but not female sterility. Mutant males are azoospermic because of the extensive death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter defect is also observed upon HuR overexpression. To elucidate further the molecular mechanisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2, a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifically binds hspa2 mRNA and controls its expression at the translational level in germ cells. Our study provides the first genetic evidence of HuR involvement during spermatogenesis and reveals Hspa2 as a target for HuR.

The RNA-Binding Protein HuR Promotes Glioma Growth and Treatment Resistance

Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. We previously found that the mRNA-binding protein, ELAV 1 (HuR), is upregulated in primary brain tumors and stabilizes growth factor mRNAs such as VEGF and IL-8. To better understand the role of HuR in brain tumor growth, we altered levels of HuR in glioma cells by short hairpin RNA or ectopic expression and measured tumor cell phenotype using in vitro and in vivo models. In HuR-silenced cells, we found a significant decrease in anchorage-independent growth and cell proliferation with a concomitant induction of apoptosis. Using an intracranial tumor model with primary glioblastoma cells, HuR silencing produced a significant decrease in tumor volume. In contrast, overexpression of HuR produced in vitro chemoresistance to standard glioma therapies. Because bcl-2 is abundantly expressed in glioma and associated with tumor growth and survival, we determined the impact of HuR on its regulation as a molecular validation to the cellular and animal studies. Using UV cross-linking and RNA immunoprecipitation, we show that HuR bound to the 3'-untranslated region of all bcl-2 family members. Silencing of HuR led to transcript destabilization and reduced protein expression. Polysome profiling indicated loss of HuR from the translational apparatus. In summary, these findings reveal a HuR-dependent mechanism for cancer cell survival and sensitivity to chemotherapeutic drugs suggesting that HuR should be considered as a new therapeutic target.

Mechanistic Roles of SRC Kinase, VE-Cadherin and Caveolin-1 in Early Increased Vascular Permeability in Experimental Ulcerative Colitis

flux assay. Subcytoplasmic CUGBP1mRNA distribution and its colocalizationwith processing bodies (PBs) were examined by MS2-system and immunoflurescence staining. Results: CUGBP1 bound GREs located in the occludin 3'UTR rather than coding region. CUGBP1 overexpression increased its association with occludin mRNA, repressed occludin translation, decreased its protein level (by ~60%), and disrupted the barrier function as indicated by an increase in paracellular permeability (by ~2-fold). In contrast, CUGBP silencing reduced its binding to occludin mRNA, promoted occludin translation, and induced its protein (by ~2.5-fold), thus enhancing the barrier function. Neither intervention altered occludin mRNA stability and its total levels. Interestingly, the inhibitory effect of CUGBP1 on occludin translation was abolished by HuR. HuR overexpression or increasing its phosphorylation by elevating Chk2 repressed occludin mRNA interaction with CUGBP1, enhanced its association with HuR, and promoted occludin translation in cells overexpressing CUGBP1, whereas HuR silencing increased CUGBP1 binding to the occludin mRNA, decreased the abundance of [HuR/ocludin mRNA] complex, and inhibited occludin translation. Furthermore, CUGBP1 increases the recruitment of occludin mRNA to PBs, which was prevented by HuR overexpression. Conclusions: These results indicate that induced CUGBP1 represses occludin mRNA translation by competitively inhibiting HuR binding to the occludinmRNA, thereby impairing the epithelial barrier function.

Knock-Down of Irbit (Inositol-1,4,5-Trisphosphate Receptor Binding Protein Released With IP3) Increases Epithelial Paracellular Permeability

flux assay. Subcytoplasmic CUGBP1mRNA distribution and its colocalizationwith processing bodies (PBs) were examined by MS2-system and immunoflurescence staining. Results: CUGBP1 bound GREs located in the occludin 3'UTR rather than coding region. CUGBP1 overexpression increased its association with occludin mRNA, repressed occludin translation, decreased its protein level (by ~60%), and disrupted the barrier function as indicated by an increase in paracellular permeability (by ~2-fold). In contrast, CUGBP silencing reduced its binding to occludin mRNA, promoted occludin translation, and induced its protein (by ~2.5-fold), thus enhancing the barrier function. Neither intervention altered occludin mRNA stability and its total levels. Interestingly, the inhibitory effect of CUGBP1 on occludin translation was abolished by HuR. HuR overexpression or increasing its phosphorylation by elevating Chk2 repressed occludin mRNA interaction with CUGBP1, enhanced its association with HuR, and promoted occludin translation in cells overexpressing CUGBP1, whereas HuR silencing increased CUGBP1 binding to the occludin mRNA, decreased the abundance of [HuR/ocludin mRNA] complex, and inhibited occludin translation. Furthermore, CUGBP1 increases the recruitment of occludin mRNA to PBs, which was prevented by HuR overexpression. Conclusions: These results indicate that induced CUGBP1 represses occludin mRNA translation by competitively inhibiting HuR binding to the occludinmRNA, thereby impairing the epithelial barrier function.

Abstract 3083: The RNA stability factor HuR promotes an angiogenic switch in colon cancer

Abstract The initiation and progression of colorectal tumorigenesis is characterized by alterations in gene expression that allow for the overexpression of oncogenic and tumor-promoting factors. In normal cells, the mRNA transcripts of these cancer-associated genes are targeted for rapid decay through AU-rich elements (ARE) present in the mRNA 3’ untranslated region (3′UTR). However, growing evidence reveals that the loss of ARE-mediated mRNA decay is a characteristic feature contributing to pathogenic gene expression in colorectal cancer. Our prior work identified enhanced expression of the RNA-binding protein HuR to occur during colon tumorigenesis and HuR overexpression is correlated with poor clinical prognosis. In this capacity, HuR promotes overexpression of many cancer-associated factors by binding to their ARE and interfering with subsequent rapid mRNA decay. To determine the functional significance of HuR overexpression in vivo, a tissue-specific HuR-transgenic mouse (HuR-Tg) was created that overexpresses HuR in the epithelium of the small intestine and colon. HuR-Tg mice displayed normal growth and did not reveal any morphological changes in the gastrointestinal tract. To test if HuR overexpression can impact tumorigenesis downstream a tumor initiating event, crosses between HuR-Tg mice and the APCMin/+ mouse, a genetic model of gastrointestinal tumorigenesis, were performed. As early as 12 weeks of age, HuR-Tg/APCMin/+ lines display a 2- to 4-fold increase in tumor burden and size in both the colon and small intestine compared to APCMin/+control mice. Based on HuR's ability to bind various ARE-containing mRNAs associated with angiogenesis, vascular density was assayed in tissue derived from HuR-Tg/APCMin/+ mice. Micro-vessel density was 2-fold greater in the tumors and surrounding tissue in APCMin/+ mice overexpressing HuR. Angiogenic mRNA array analysis of tumors derived from HuR-Tg/APCMin/+ mice showed enhanced expression of multiple ARE-containing angiogenic genes including the HuR target genes vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). Furthermore, dietary treatment of HuR-Tg/APCMin/+ mice with celecoxib attenuated HuR-dependent tumor formation and suppressed micro-vessel density and VEGF expression. These findings indicate that HuR functions during colon tumorigenesis downstream of a tumor-initiating event to stabilize ARE-containing mRNAs associated with tumor vascularization and identify this mRNA stability factor as a new molecular target for therapeutic intervention in controlling angiogenic gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2011-3083

Abstract 5416: The low-molecular-weight compound MS-444 selectively targets cancer cells via inhibition of the mRNA stability factor HuR

Abstract Colorectal cancer (CRC) is the third most common cancer among American adults and a leading cause of cancer-related mortality. The initiation and progression of colorectal tumorigenesis is characterized by genetic alterations that promote aberrant expression of oncogenic and tumor-promoting factors. In normal cells, the mRNAs of these inflammation- and cancer-associated genes are targeted for rapid decay through AU-rich elements (ARE) present in the mRNA 3’UTR. However, loss of ARE-mediated mRNA decay is a characteristic feature contributing to pathogenic gene overexpression in colorectal cancer. Our prior work identified enhanced expression of the RNA-binding protein HuR to occur during colon tumorigenesis and HuR overexpression is correlated with poor clinical prognosis. In this capacity, HuR acts as a mRNA stability factor by promoting ARE-containing mRNA stabilization. Therefore, HuR has emerged as a novel pharmacological target with potential selectivity for cancer cells. This was examined by treating HuR-overexpressing CRC cell lines HCT116, SW480, RKO, and HT-29 with the small molecule inhibitor of HuR MS-444. Growth inhibition was observed in all CRC lines with IC50 values of 7-12 µM and cancer cell selectivity was observed with 4- to 5-fold increased MS-444 IC50 in non-transformed intestinal and colonic epithelial cells. HuR is predominantly nuclear in non-transformed cells, whereas in CRC cells aberrant cytoplasmic HuR trafficking is observed indicating that cytoplasmic localization is a necessary component for HuR-mediated ARE-mRNA stabilization and cancer cell survival. In HCT116 cells treated with MS-444, complete inhibition of cytoplasmic HuR was observed along with induction of apoptosis. Similarly, long-term siRNA-mediated knockdown of HuR in HCT116 cells resulted in cellular apoptosis, and cells with attenuated levels of HuR exhibited a 5-fold higher sensitivity to MS-444 treatment. Expression profiling revealed increased expression of many pro-apoptotic proteins to be present in colon cancer cells resulting from MS-444 treatment. The expression of Fas ligand, a TNF superfamily member, is significantly increased, accompanied by elevated expression of caspase 8 and cell death effectors such as Bax and Bid. These results indicate that MS-444 treatment triggers a Fas-dependent signaling cascade mediated through cellular caspases, thereby resulting in cell death. Taken together, these findings indicate that small molecule-based inhibition of HuR results in selective cancer cell death while also indicating that overexpression of HuR is a necessary component for colorectal tumor cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2011-5416

Abstract 3084: The role of RNA-binding protein HuR in lung metastasis of triple-negative breast cancer

Abstract Breast cancer is the second most common cancer among women worldwide. It is estimated that approximately one million women are diagnosed with breast cancer annually. Furthermore, more than 410,000 women will die each year from the disease primarily due to tumor metastasis. Currently, even the most effective treatments often result in recurrence and metastasis, in part due to genetic factors. Therefore, understanding the mechanism of breast cancer related gene regulation is crucial for the development of novel treatments and preventive strategies to those who are predisposed to developing breast cancer. Previously, we have shown that the RNA-binding protein HuR posttranscriptionally regulates various proto-oncogenes by stabilizing their mRNAs and facilitating their translation into proteins. Additionally, HuR upregulation and cytoplasmic localization has been associated with invasive cancer progression and poor prognosis. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. HuR regulates genes involved in angiogenesis, cell growth and cell cycle regulation including VEGFα, TSP1, HIF1α, CDKN1A(p21) and β-catenin. In this study, we investigated the role of HuR in an aggressive triple-negative (estrogen receptor ER-, human epidermal growth factor receptor 2 (HER2)- and progesterone receptor (PR)-) breast cancer cell subline LM-2. The LM-2 cells were derived from triple-negative breast cancer cells MDA-MB-231 which were isolated from two rounds of in vivo selection of the cancer cells which metastasized to the lungs. The LM-2 cells were retrovirally transduced with triple-fusion protein reporter construct encoding thymidine kinase1, green fluorescent protein (GFP) and firefly luciferase in order to obtain nuclear imaging, fluorescent and bioluminescent properties for in vivo and in vitro tracking. LM2 cells were further transfected with a plasmid containing HA-HuR or empty vector control to investigate the function of HuR in LM2 cells. Two clones of HuR overexpressing LM2 cells were shown to grow faster in vitro compared to the empty vector control. Also, HuR overexpression significantly facilitates tumor invasion in vitro by matrigel invasion assay. Further analysis of HuR overexpressing LM2 cells using in vivo imaging system (IVIS) will reveal the role of HuR in breast cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2011-3084

Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements and related motifs present in the 3'untranslated region (UTR) of mRNAs. We postulate that HuR critically regulates the epithelial response by associating with multiple ARE-bearing, functionally related inflammatory transcripts. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNF-α plus IFN-γ, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein complexes from resting and cytokine-treated cells were immunoprecipitated using anti-HuR and isotype-control Ab, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1, and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control immunoprecipitation. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene ribonucleoprotein-immunoprecipitation and shown to be 3'UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic. Conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. HuR-mediated regulation in airway epithelium appears broader than previously appreciated, coordinating numerous inflammatory genes through multiple posttranscriptional mechanisms.

RNA-binding protein HuR mediates cytoprotection through stimulation of XIAP translation.

Expression of the intrinsic cellular caspase inhibitor XIAP is regulated primarily at the level of protein synthesis. The 5' untranslated region harbours an Internal Ribosome Entry Site (IRES) motif that supports cap-independent translation of XIAP mRNA during conditions of cellular stress. In this study, we show that the RNA-binding protein HuR, which is known to orchestrate an antiapoptotic cellular program, stimulates translation of XIAP mRNA through XIAP IRES. We further show that HuR binds to XIAP IRES in vitro and in vivo, and stimulates recruitment of the XIAP mRNA into polysomes. Importantly, protection from the apoptosis-inducing agent etoposide by overexpression of HuR requires the presence of XIAP, suggesting that HuR-mediated cytoprotection is partially executed through enhanced XIAP translation. Our data suggest that XIAP belongs to the HuR-regulated RNA operon of antiapoptotic genes, which, along with Bcl-2, Mcl-1 and ProTα, contributes to the regulation of cell survival.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFa, HIF1a, and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER-) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, down-regulation of VEGF, a target of HuR which it normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Loss of Repression of HuR Translation by miR‐16 May Be Responsible for the Elevation of HuR in Human Breast Carcinoma

Elevated levels of RNA binding protein HuR were found in various human cancers. However, the mechanisms underlying HuR over-expression in cancers have not been fully elucidated. Here, we show that miR-16 acts as a novel post-transcriptional regulator for HuR. Knockdown of miR-16 increased HuR protein levels in MDA-MB-231 cells, while over-expression of pre-miR16 reduced HuR expression. Neither knockdown nor over-expression of miR-16 could alter the mRNA levels of HuR. Instead, knockdown of miR-16 increased the level of de novo synthesized HuR protein. Importantly, mechanistic studies showed that miR-16 associated with the 3'UTR of HuR, and knockdown of miR-16 markedly increased the luciferase activity of a HuR 3'UTR-containing reporter. We further demonstrate that the level of miR-16 was inversely correlated with HuR protein level in human breast carcinoma. Together, our results suggest an important role of miR-16 in regulating HuR translation and link this regulatory pathway to human breast cancer.

Abstract 3643: Manipulation of HuR expression alters the efficacy of PARP-inhibitors in pancreatic and ovarian cancer cells

Abstract PARP-inhibitors (PARPi) are a novel class of agents that target cancer cells deficient in DNA repair. Early phase trials show promising success with PARP-inhibitors, yet drug resistance mechanisms and off-target pathways affected by drug exposure have not been explored. HuR (ELAV1) is a protein that binds and stabilizes mRNA transcripts upon certain stress stimuli. These HuR mRNA targets result in an increase in protein expression; and thus, protein function. Recently, we demonstrated that HuR binds and regulates deoxycytidine kinase, the gemcitabine metabolizing enzyme. Thus, HuR expression enhances gemcitabine efficacy against pancreatic cancer cells. We subsequently screened other chemotherapeutics that may stimulate the HuR stress response. In isogenic pancreatic cancer cell lines, stable HuR overexpression rendered cells up to 2-fold more sensitive to PARPi compared to control cells. Accordingly, siRNA knock down of HuR expression in both pancreatic and ovarian cancer cell lines caused PARPi resistance compared to a scramble-sequence control siRNA. In a pancreatic cancer cell line, MiaPaCa2, a 2.5-fold siRNA knock down of HuR mRNA expression detected by qPCR rendered cells approximately 4-fold more resistant to PARPi. Similarly, a modest siRNA knock down of HuR in an ovarian cancer cell line, A2780, rendered cells more resistant to PARPi. Immunofluorescence studies indicate that a 5 hour treatment with 75 µM PARPi directly caused HuR to transport from the nucleus to the cytoplasm, presumably moving ARE-rich mRNA cargoes after drug exposure. Focused gene expression analysis on HuR bound, immunoprecipitated RNA defined functional HuR downstream targets (including deoxycytidine kinase). Current studies will reveal and validate the importance of these downstream mRNA targets in PARP-inhibitor metabolism and efficacy. We hypothesize that HuR may be central to PARPi effectiveness and may be useful in understanding PARPi de novo drug resistance mechanisms. Future work will reveal whether HuR status can be utilized as a predictive marker for PARP-inhibitor-based therapy and if all clinically available PARP-inhibitors engage HuR's regulation of unique mRNA cargoes in cancer cells upon treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3643.

Post‐Transcriptional Regulation of CD38 expression in human airway smooth muscle (HASM) cells

CD38 is a membrane protein expressed in HASM cells and its induction by TNF‐α involves transcriptional and post‐transcriptional mechanisms. CD38‐null mice develop attenuated airway hyperresponsiveness compared to the wild‐type mice. TNF‐α causes greater elevation of CD38 expression in asthmatic HASM cells than in non‐asthmatic cells. The objectives of this study were to determine the role of 1) RNA binding protein HuR and 2) microRNAs in post‐transcriptional regulation of CD38 expression in HASM cells. Expression and sub‐cellular localization of HuR were determined by western blotting. CD38 mRNA stability was determined in HASM cells transiently transfected with HuR. Binding of HuR to an oligonucleotide representing the AU‐rich motif (ARE) of CD38 mRNA was determined by a pull‐down assay. Expression of the miRNA miR‐140‐3p was determined by qRT‐PCR. In HASM cells, HuR was localized to the nuclei and its expression was unaltered by TNF‐α, although TNF‐α exposure increased HuR binding to the oligo mimicking ARE of the CD38 3′UTR. CD38 mRNA decay and HuR levels were comparable in non‐asthmatic and asthmatic cells. HuR overexpression did not alter CD38 mRNA decay. MiR‐140‐3p expression was unaltered in non‐asthmatic cells by TNF‐α, whereas in asthmatic cells it was reduced. The differential induction of CD38 expression by TNF‐α in asthmatic ASM cells may arise from regulation by miR‐140‐3p, but not HuR, expression.

Reduced nuclear export of HuR mRNA by HuR is linked to the loss of HuR in replicative senescence

The RNA-binding protein, HuR, associates with the HuR mRNA, but the consequences of this interaction are unknown. Here, we use human diploid fibroblasts (HDFs) and cervical carcinoma cells to study this regulatory paradigm. Ectopic overexpression of HuR potently enhanced the translation and cytoplasmic levels of endogenous HuR, but did not affect HuR mRNA levels. Inhibition of CRM1 function by Lemptomycin B or by knockdown of CRM1 greatly diminished the cytoplasmic levels of endogenous HuR mRNA and hence blocked the induction of endogenous HuR by exogenous HuR. Further studies showed that HuR interacted with the 3′-untranslated region (UTR) of HuR and that overexpression of HuR increased the cytoplasmic levels of a chimeric luciferase-HuR 3′-UTR reporter transcript, as well as luciferase activity; conversely, HuR knockdown reduced both parameters. Moreover, the loss of HuR in senescent, late-passage HDFs was accompanied by a reduced cytoplasmic presence of endogenous HuR mRNA, ectopic Luc-HuR-3′UTR reporter transcript, and luciferase activity relative to what was observed in young, early-passage cells. Our results reveal a positive feedback mechanism for the regulation of HuR, which may play an important role in the regulation of HuR during replicative senescence.

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines.

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3′-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

The Role of HuR in Gemcitabine Efficacy in Pancreatic Cancer: HuR Up-regulates the Expression of the Gemcitabine Metabolizing Enzyme Deoxycytidine Kinase

RNA-binding protein HuR binds U- or AU-rich sequences in the 3'-untranslated regions of target mRNAs, stabilizing them and/or modulating their translation. Given the links of HuR with cancer, we studied the consequences of modulating HuR levels in pancreatic cancer cells. HuR-overexpressing cancer cells, in some instances, are roughly up to 30-fold more sensitive to treatment with gemcitabine, the main chemotherapeutic component of treatment regimens for pancreatic ductal adenocarcinoma (PDA), compared with control cells. In pancreatic cancer cells, HuR associates with deoxycytidine kinase (dCK) mRNA, which encodes the enzyme that metabolizes and thereby activates gemcitabine. Gemcitabine exposure to pancreatic cancer cells enriches the association between HuR and dCK mRNA and increases cytoplasmic HuR levels. Accordingly, HuR overexpression elevates, whereas HuR silencing reduces, dCK protein expression in pancreatic cancer cells. In a clinical correlate study of gemcitabine treatment, we found a 7-fold increase in risk of mortality in PDA patients with low cytoplasmic HuR levels compared with patients with high HuR levels, after adjusting for other treatments and demographic variables. These data support the notion that HuR is a key mediator of gemcitabine efficacy in cancer cells, at least in part through its ability to regulate dCK levels posttranscriptionally. We propose that HuR levels in PDA modulate the therapeutic efficacy of gemcitabine, thus serving as a marker of the clinical utility of this common chemotherapeutic agent and a potential target for intervention in pancreatic cancer.

Correlation of HuR cytoplasmic expression in pancreatic cancer and overall patient survival when treated with gemcitabine in the adjuvant setting

11097 Background:The Hu/ELAV mRNA binding protein HuR is normally localized to the nucleus and is activated upon stress. Previously, we observed that HuR expression is predominantly cytoplasmic in aggressive forms of pancreatic ductal adenocarcinomas (PDA). Additionally, we have shown that overexpression of HuR in pancreatic cancer cell lines renders them hypersensitive to gemcitabine (GEM). GEM is the standard first line therapy used against PDA. This study was designed to determine the relationship between endogenous HuR expression levels and patient outcome. Methods: We analyzed tissue from 29 resected PDA patients who did not receive neoadjuvant therapy. We determined the intensity of cytoplasmic HuR expression. All patients received adjuvant GEM, alone or in combination with radiation therapy or other chemotherapeutics. Correlation between HuR expression levels and overall survival was evaluated. Results: The median overall survival was 20.6 months, with 18 deaths observed. Median overall survival for low HuR expression was 15.3 months. Median overall survival for high HuR expression was not reached at a follow up of 40 months. A univariate Cox regression model gives a hazard ratio of low to high HuR of 3.36 (p=0.025) [95% CI 1.09–10.35]. Adjusting for age, sex, and radiation therapy in this patient group gives an adjusted hazard ratio of 5.04 (p = 0.03) [95% CI 1.15–22.02]. This indicates a 5-fold increase in risk of death in patients with low HuR levels compared to high HuR levels among patients receiving GEM. Conclusions: This is the first report of a correlation between levels of cytoplasmic HuR expression and overall survival in GEM treated patients. Together with our previously reported experimental data, HuR is regulating the key metabolic enzyme for GEM activation (deoxycytidine kinase). Therefore, HuR is a marker for therapeutic efficacy of GEM based regimens and is a candidate target for the optimization of GEM based treatment strategies. [Table: see text] No significant financial relationships to disclose.

S2037 Overexpression of the RNA-Binding Protein HuR in Esophageal Cancer Cells Contributes to Increased Levels of Survivin Protein Through Enhanced Survivin mRNA Stability

S2037 Overexpression of the RNA-Binding Protein HuR in Esophageal Cancer Cells Contributes to Increased Levels of Survivin Protein Through Enhanced Survivin mRNA Stability

The RNA stability regulator HuR regulates L1 protein expression in vivo in differentiating cervical epithelial cells

Human papillomavirus (HPV) L1 and L2 capsid protein expression is restricted to the granular layer of infected, stratified epithelia and is regulated at least partly at post-transcriptional levels. For HPV16, a 79 nt late regulatory element (LRE) is involved in this control. Using W12 cells as a model for HPV16-infected differentiating cervical epithelial cells we show that HuR, a key cellular protein that controls mRNA stability, binds the LRE most efficiently in nuclear and cytoplasmic extracts of differentiated cells. Further, HuR binds the 3′ U-rich portion of the LRE directly in vitro. Overexpression of HuR in undifferentiated W12 cells results in an increase in L1 mRNA and protein levels while siRNA knock-down of HuR in differentiated W12 cells depletes L1 expression. In differentiated cervical epithelial cells HuR may bind and stabilise L1 mRNAs aiding translation of L1 protein.