HuR Localization Research Articles

T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.

Activation of the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.

Abstract 1961: Gaps in the armor: Targeting HuR to sensitize pancreatic cancer

Abstract HuR is an RNA binding protein involved in a coordinated cellular response to stressors. Upon insults such as chemotherapy or radiation treatment, HuR translocates from the nucleus to the cytoplasm where it binds the 3'UTR of target mRNAs. HuR's interaction with target mRNAs leads to the upregulation of target genes and ultimately treatment resistance. This is particularly relevant in the case of pancreatic ductal adenocarcinoma (PDA). PDA is highly resistant to standard chemotherapy such as FOLFIRINOX or gemcitabine/nab-paclitaxel. Using a tumor microarray (TMA), we found 79% of patient tumor samples (n=70) were positive for active cytoplasmic HuR, while little to no cytoplasmic localization was detected in normal tissue. In addition, HuR CRISPR knockout cell lines have a xenograft lethal phenotype. Previously published data also demonstrated that reduction of HuR in xenografts with a DOX-inducible shRNA system caused a 3.6 fold decrease in tumor size. HuR reduction was also shown to potentiate a PARP-inhibitor (olaparib) treatment from a 5.6-fold reduction alone to 9.3-fold reduction in tumor size when combined with shHuR, demonstrating the role of HuR in drug resistance. The aim of our current study was: A) to target HuR directly using nanoparticle delivery of siRNA against HuR; and B) to use the FDA approved small molecule pyrvinium pamoate (PP) to inhibit HuR's translocation and sensitize PDA cells to concurrent therapies. Using 3DNA, a 60nm nanoparticle composed of a sphere of crosslinked DNA, we have successfully delivered siRNA against HuR in vivo utilizing targeting moieties against receptors known to be overexpressed on the surface of PDA cells: EGFR, folic acid receptor, and transferrin receptor. Bi-weekly IP treatment of siHuR bound to 3DNA was safe and effective at extending life in a xenograft model as indicated by Kaplan Meier analysis (p=0.01). We are currently testing siHuR-3DNA dendrimer therapy's ability to sensitize PDA cells to oxaliplatin or olaparib in vivo. We are also investigating the use of PP to target HuR's localization. PP has previously been shown in bladder cancer to inhibit the translocation of HuR in vitro and in vivo. We have reproduced this finding in PDA cells, and have shown impressive drug potency with IC50s as low as 38nM in 2D cultures and 16nM in a 3D mouse PDA organoid model. Combination index (CI) values determine drugs interactions where 1 is additive, <1 is synergistic and >1 is antagonistic. We have determined that PP can enhance therapies such as gemcitabine (CI of 0.55), olaparib (CI of 0.40) and palbociclib (CI of 0.37) in vitro, and are currently validating these findings in vivo. Taken together, our data demonstrate that HuR inhibition via 3DNA delivery of siHuR and/or PP treatment can sensitize PDA cells to chemotherapy and targeted therapies. By inhibiting a resistance driver (HuR) in pancreatic cancer we aim to improve current therapies for this devastating disease. Citation Format: Christopher W. Schultz, Kevin O'Hayer, Teena Dhir, Oloruntoba Bolaji, Kathryn M. Bormes, Samantha Z. Brown, Henry Thomsett, Saswati Chand, Aditi Jain, Wei Jiang, Grace McCarthy, Charles J. Yeo, Austin Goetz, Avinoam Nevler, Jonathan R. Brody, Jordan M. Winter, Ranjan Preet, Dan Dixon, Jessica Bowers, Kelly Rhodes, Robert Getts. Gaps in the armor: Targeting HuR to sensitize pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1961.

PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells.

The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway.

Abstract 5476: A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the United States, and the 3rd most common cancer associated with BRCA mutations. Frontline therapies have significant toxicities and only minimally extend overall survival, highlighting the need to optimize targeted therapies. Poly-ADP ribose polymerase (PARP) inhibitors (PARPi), a ‘poster child’ for personalized medicine, depend on the concept of synthetic lethality where the combined perturbation of DNA repair genes, via genetic mutations within the tumor cells, and pharmacological PARP inhibition effectively targets BRCA-deficient tumors. Although PARPi have delivered promising preclinical and clinical results, initially- responsive patients ultimately develop resistance. A unique mechanism elucidated by our lab demonstrates that the mRNA-binding protein HuR mediates resistance to DNA damaging agents through post-transcriptional regulation of select mRNA cargo. Predominantly expressed in the nucleus, HuR translocates to the cytoplasm upon tumor-associated stress. Cytoplasmic HuR binds and stabilizes unique pro-survival transcripts, resulting in resistance to a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi response. Methods and Results: Through immunofluorescence and western blot of fractionated lysates, we demonstrate that the PARP inhibitors Veliparib, Olaparib, and Rucaparib induced cytoplasmic HuR localization. Conversely, pre-treatment with MS-444 (Novartis), an established small molecule inhibitor of HuR, abrogated its nuclear export induced by PARPi treatment. Consistent with these findings, the growth-inhibitory effects of PARPi treatment were significantly potentiated upon HuR silencing whereas ectopic HuR overexpression promoted resistance, as observed in short term cell survival and long-term anchorage-independent growth assays. Additionally, silencing of HuR enhanced PARPi-induced cytotoxicity, assessed by increased accumulation of DNA damage (γH2Ax) foci and Poly ADP-ribose (PAR) polymers. Ribonucleotide protein immunoprecipitation (RNP-IP) assays demonstrated that HuR binds and upregulates Poly-ADP Ribose Glycohydrolase (PARG) mRNA, the major enzyme responsible for catabolism of PAR. Taken together, when PDA cells are exposed to PARPi, HuR mediates upregulation of PARG, thereby decreasing PARylation and facilitating DNA repair. Conversely, HuR inhibition results in detrimental accumulation of PAR and enhanced DNA damage, which ultimately leads to increased PARPi-conferred cytotoxicity. Discussion: These results demonstrate that HuR imposes a significant barrier to PARPi therapy by orchestrating a strong chemoresistance mechanism. Thus, we provide evidence that HuR (and/or its target) inhibition via an HuR inhibitor (MS-444) can optimize PARPi-based therapies for better patient outcomes. Citation Format: Saswati N. Chand, Akshay R. Kamath, Nicole Meisner-Kober, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR. [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 5476. doi:10.1158/1538-7445.AM2015-5476

HuR mediated post-transcriptional regulation as a new potential adjuvant therapeutic target in chemotherapy for pancreatic cancer.

To investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA) and to assess the effects of HuR silencing on the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) and the in vitro response to gemcitabine (GEM) treatment in pancreatic cell lines. We compared the expression of HuR, COX-2, and HO-1 in PDA and normal pancreatic tissue using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. In addition, the HuR, COX-2 and HO-1 were analyzed in four types of cancer cell lines (MiaPaca2, Su.86.86, Capan-1, and Capan-2) with and without GEM treatment. Immunocytofluorescence analysis was used to investigate HuR localization in cells. Cell viability and response to GEM after HuR silencing were determined with the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide test and the crystal violet clonogenic assay, respectively. To measure apoptosis, activation of caspases 3/7 was evaluated using immunofluorescence. In PDA tissue obtained from patients not treated with GEM, HuR mRNA expression was 3.2 times lower (P < 0.05) and COX-2 and HO-1 mRNA expression was 2.3-fold and 7.2-fold higher (P < 0.05), respectively, than normal pancreatic tissue (from organ donor). qRT-PCR analysis showed that HuR, COX-2, and HO-1 mRNA were overexpressed in all cancer cell lines treated with the half maximal inhibitory concentration (IC50) dose of GEM compared with control cells (P < 0.05). Western blot analysis revealed that COX-2 and HO-1 levels were significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing increased the response to GEM treatment and decreased cell viability by 11.6%-53.7% compared to control cell lines. Caspases 3 and 7 were activated after HuR silencing and GEM treatment in all pancreatic cancer cell lines. In comparison, treatment with GEM alone did not activate caspases 3 and 7 in the same cell lines. HuR mediated post-transcriptional upregulation of COX-2 and HO-1 expression after GEM treatment in pancreatic cancer cells. HuR silencing significantly increased the effectiveness of GEM treatment in vitro.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

Abstract 1957: MiR-200c can mediate either poor or good outcome in ovarian cancer depending on HuR localization in the cell.

Abstract MicroRNAs (miRNAs) in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors in independent studies. Expression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Luciferase reporter assay and ribonucleic-immuno-precipitation assay was used to in vitro characterise the complexes between miR-200c, HuR and class III β-tubulin (TUBB3). Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients. Using such approach, we demonstrated that overexpression of miR-200c in ovarian cancer resulted in poor or good outcome depending on interaction with the RNA binding protein HuR. When the localization of HuR was confined in the nucleus, high expression of miR-200c suppressed class III β-tubulin (TUBB3) expression and resulted in a good prognosis, whereas when HuR occurred in cytoplasm, the same miRNA enhanced TUBB3 expression and produced a poor outcome. Indeed, in an ovarian cancer cell line model, the miR-200c increased HuR binding on TUBB3 mRNA in stressing microenvironmental conditions. This study demonstrated how the prognostic power of a single factor can depend on interactions with additional factors. Through a multidimensional analysis of all components, the actual mechanism(s) underlying the aggressive biological behavior of ovarian cancer can be unraveled, and the prognosis of ovarian cancer patients can be rendered based on rational explanations. Citation Format: Silvia Prislei, Enrica Martinelli, Marisa Mariani, Giuseppina Raspaglio, Steven Sieber, Gabriella Ferrandina, Shohreh Shahabi, Giovanni Scambia, Cristiano Ferlini. MiR-200c can mediate either poor or good outcome in ovarian cancer depending on HuR localization in the cell. [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 1957. doi:10.1158/1538-7445.AM2013-1957

MRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing adenylate/uridylate-rich element (ARE) located at the 3' untranslated region (3'UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using tissue microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer.

Abstract 3161: The RNA binding protein HuR stabilizes the ornithine decarboxylase (ODC) transcript in non-melanoma skin cancer

Abstract Non-melanoma skin cancer (NMSC), comprised of both basal cell carcinoma and squamous cell carcinoma, is the most common malignancy in the United States. The work described here focuses on understanding the underlying mechanisms of this disease using a mouse keratinocyte model comprised of two cells lines that represent distinct stages in skin carcinogenesis, initiation and progression, denoted as C5N and A5 respectively. Previous results have shown that induction of ornithine decarboxylase (ODC) enzyme activity is both necessary and sufficient for the promotion of skin tumors. As expected, we saw a dramatic increase in ODC enzyme activity in A5 spindle carcinoma cells when compared to C5N keratinocytes. Interestingly, this increase in ODC enzyme activity correlates with an increase in ODC stability in A5 cells compared to C5N cells. Thus, we sought to investigate whether ODC mRNA interacted with the RNA binding protein HuR, which is known to bind to and stabilize its target mRNA transcripts. HuR has been shown by others to localize to the cytoplasm where it stabilizes numerous transcripts corresponding to proteins that are important in the development of a variety of malignancies. Moreover, studies involving human tumor samples from breast cancer, ovarian cancer, and Merkel cell carcinoma patients have shown a correlation between high cytoplasmic HuR presence and increased tumor grade. By conducting biotin-pulldown assays, which use a synthetic ODC transcript to test for RNA-protein interactions, as well as mRNP assays, which identify endogenous RNA-protein interactions, we confirmed that HuR is able to bind to the ODC 3’UTR in A5 cells but not in C5N cells. Immunofluorescence results showed that HuR localization between the two cell lines was different, with A5 cells displaying a diffuse HuR presence in both the nucleus and cytoplasm while C5N cells exhibited nuclear localization of HuR. In order to definitively link HuR to ODC mRNA stability, knockdown experiments were conducted using a short interfering RNA (siRNA) that was specific for the coding region of HuR. These knockdown experiments confirmed our hypothesis that HuR is able to bind to and stabilize ODC mRNA in A5 cells, as a 60% knockdown of HuR protein correlated with a profound decrease in ODC mRNA half-life and reduced intracellular ODC protein. These results suggest that the stabilization of ODC mRNA in A5 spindle carcinoma cells is regulated directly by changes in localization of the HuR protein. These data are novel, as they show for the first time that an RNA binding protein is able to bind to and regulate ODC mRNA stability. Knowledge from these and subsequent studies could be used to design RNA binding protein-targeted therapies to combat not only NMSC but other epithelial cancers and/or non-malignant anomalies that display an increased presence of cytoplasmic HuR. 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 3161.

Regulation of HuR by DNA Damage Response Kinases

As many DNA-damaging conditions repress transcription, posttranscriptional processes critically influence gene expression during the genotoxic stress response. The RNA-binding protein HuR robustly influences gene expression following DNA damage. HuR function is controlled in two principal ways: (1) by mobilizing HuR from the nucleus to the cytoplasm, where it modulates the stability and translation of target mRNAs and (2) by altering its association with target mRNAs. Here, we review evidence that two main effectors of ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR), the checkpoint kinases Chk1 and Chk2, jointly influence HuR function. Chk1 affects HuR localization by phosphorylating (hence inactivating) Cdk1, a kinase that phosphorylates HuR and thereby blocks HuR's cytoplasmic export. Chk2 modulates HuR binding to target mRNAs by phosphorylating HuR's RNA-recognition motifs (RRM1 and RRM2). We discuss how HuR phosphorylation by kinases including Chk1/Cdk1 and Chk2 impacts upon gene expression patterns, cell proliferation, and survival following genotoxic injury.

Temporally Regulated Traffic of HuR and Its Associated ARE-Containing mRNAs from the Chromatoid Body to Polysomes during Mouse Spermatogenesis

BackgroundIn mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs).Methodology/Principal FindingsUsing a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation.Conclusions/SignificanceOur results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation.

Cytoplasmic Expression of HuR is Related to Cyclooxygenase-2 Expression in Colon Cancer

HuR, human family embryonic-lethal abnormal vision-like protein, can bind to mRNA and stabilizes the nucleic acid in the cytoplasm, resulting in more efficient translation. HuR is predominantly present in the nucleus and shuttles between the nucleus and cytoplasm. HuR stabilizes cyclooxygenase-2 (Cox-2) mRNA in several cancers, including breast, stomach, lung and brain cancer. We investigated the expression and cellular location of HuR, as well as evaluated Cox-2 expression in 79 colorectal cancer patients with the use of immunohistochemical methods. The biological implications of HuR localization and Cox-2 expression in colorectal carcinoma were evaluated. Nuclear HuR expression was observed in 59 (74.7%) tumors and cytoplasmic HuR expression was seen in 25 (31.6%) tumors. Cox-2 immunoreactivity was noted in 42 (53%) tumors. The expression of cytoplasmic HuR was significantly associated with Cox-2 expression (p=0.004). Cytoplasmic expression of HuR showed a correlation with lymphatic invasion (p=0.025) and the presence of a lymph node metastasis (p=0.027). The presence of nuclear HuR showed no correlation with Cox-2 expression or any other of the clinicopathological parameters that were examined. These results suggest that cytoplasmic translocation of HuR is associated with Cox-2 expression for some colorectal carcinomas.

Expression of the embryonic lethal abnormal vision‐like protein HuR in human mesothelioma

The human embryonic lethal abnormal vision (ELAV)-like protein HuR is a messenger RNA (mRNA)-binding protein that controls the stability of certain transcripts, including cyclooxygenase2 (COX-2). To investigate a possible contribution of dysregulation of mRNA stability to the progression of cancer and to COX-2 over expression in mesothelioma, the authors studied expression of COX-2 and HuR in 5 mesothelioma cell lines (MSTO, NCI, Ist-Mes1, Ist-Mes2, and MPP89) and in a group of 29 human mesothelioma specimens that were characterized previously for COX-2 expression. All 5 cell lines expressed HuR, whereas COX-2 was not detectable in MSTO or NCI cells. Treatment with cytokines induced a shift in systolic HuR protein levels in MPP89 and Ist-Mes2 cells that was accompanied by an increase in the expression of COX-2 mRNA and protein. In Ist-Mes1 cells, cytokine stimulation did not cause the passage of HuR from nucleus to cytoplasm, and the synthesis of COX-2 did not increase. In tumor tissues, immunohistochemistry revealed a positive, statistically significant correlation between high COX-2 expression and cytoplasmic localization of HuR (P = .016). Moreover, on univariate analysis, overall survival was found to be influenced strongly by cytoplasmic HuR localization (P = .004). The current results suggested that HuR plays a role in tumor progression in mesothelioma and that COX-2 may be a target of its activity in neoplastic cells. Together, these observations indicate that strategies aiming toward the modulation of HuR may have a potential clinical benefit in mesothelioma.

Modification at HuR(S242) alters HuR localization and proliferative influence

HuR is predominantly nuclear but following exposure to stress and mitogens, it can translocate to the cytoplasm where it stabilizes target mRNAs and/or modulates their translation. Several phosphorylation sites in a central 'hinge' region of HuR have been reported to affect its nucleocytoplasmic shuttle: phosphorylation by PKC at serine (S)221 and by Cdk1 at S202. Here, we investigated if there are additional putative phosphorylation sites within the HuR hinge region capable of influencing its cytoplasmic abundance. We systematically mutated all seven serine residues within the shuttling hinge domain to the nonphosphorylatable residue alanine (A), S197A, S202A, S221A, S229A, S232A, S241A, and S242A. Using HeLa cells as the study system, we found that the HuR(S242A) mutant was more abundant in the cytoplasm in both untreated cells and in cells treated with short-wavelength ultraviolet light or with an inhibitor of Cdk1. Conversely, mutation of S242 to aspartic acid (D), rendered the phosphomimetic HuR(S242D) nuclear under all treatment conditions. S242 mutations did not influence HuR stability, but HuR(S242A) showed increased association with target cyclin A2 and cyclin B1 mRNAs. Accordingly, expression of HuR(S242A) led to increased cyclin mRNA stability and heightened cell proliferation rates. Our findings suggest that HuR phosphorylation at S242 hinders its cytoplasmic localization, its function as a posttranscriptional regulator, and its proliferative influence.

Gastrin-Mediated Interleukin-8 and Cyclooxygenase-2 Gene Expression: Differential Transcriptional and Posttranscriptional Mechanisms

Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin-dependent gene expression. AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor kappaB (NF-kappaB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3' kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2-mediated prostaglandin E(2) synthesis. In the presence of an NF-kappaB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-kappaB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively.

S–Adenosylmethionine Regulates Cytoplasmic HuR Via AMP–Activated Kinase

After liver injury, hepatic S-adenosylmethionine (SAM) content decreases, and the blockage this molecule imposes on hepatocyte proliferation is released, facilitating liver regeneration. This activity of SAM is important for normal liver function because mice deficient in hepatic SAM display abnormal liver regeneration and develop hepatocellular carcinoma. How SAM regulates hepatocyte growth is unclear, but because SAM blocks hepatocyte growth factor (HGF)-induced cyclin D1 expression and DNA synthesis without affecting HGF-induced extracellular signal-regulated kinase phosphorylation, the mitogen-activated protein kinase (MAPK) pathway is probably not the target. The effects of SAM on AMPK, HuR localization were assessed in rat hepatocytes after HGF, AICAR, and SAM treatment. We show here that HGF and 5-aminoimidazole-4-carboxamide-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), induce the phosphorylation of AMPK in hepatocytes and that SAM blocks this process. We also show that HGF- and AICAR-induced AMPK activation stimulate the transport from nucleus to cytoplasm of HuR, an RNA-binding protein that increases the half-life of target mRNA such as cyclin A2, and that SAM blocks this process. We found that, in hepatocytes, AICAR increases HuR binding to cyclin A2 messenger RNA (mRNA) as well as the expression and stability of this mRNA and that SAM blocks these events. Consistently, we found that AICAR induces hepatocyte proliferation and that SAM blocks this effect. Finally, we found that liver AMPK phosphorylation, cytoplasmic HuR, and binding of HuR to HuR-target mRNA and the steady-state levels of these mRNA are increased in knockout mice deficient in hepatic SAM. Our results yield novel insights about the mechanism by which SAM inhibits cell-cycle progression in the liver.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.