The role of HuR in gemcitabine efficacy in pancreatic cancer: HuR Up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase.
Costantino Christina L,Witkiewicz Agnieszka K,Kuwano Yuki,Cozzitorto Joseph A,Kennedy Eugene P,Dasgupta Abhijit,Keen Judith C,Yeo Charles J,Gorospe Myriam,Brody Jonathan R
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.
Overexpression of ELAV-like protein HuR is associated with increased COX-2 expression in atrophy, high-grade prostatic intraepithelial neoplasia, and incidental prostate cancer in cystoprostatectomies.
Barbisan Francesca,Mazzucchelli Roberta,Santinelli Alfredo,Lopez-Beltran Antonio,Cheng Liang,Scarpelli Marina,Montorsi Francesco,Montironi Rodolfo
BACKGROUND:The human ELAV-like protein HuR regulates the stability of several mRNA targets, including that of cyclooygenase-2 (COX-2). Their expression in prostatic carcinogenesis is uncertain. OBJECTIVE:To analyze HuR and COX-2 expression in cystoprostatectomies (CyPs) with incidental prostate cancer and compare their expression with those in radical prostatectomies (RPs) with clinically detected cancer. DESIGN, SETTING, AND PARTICIPANTS:HuR and COX-2 were immunohistochemically evaluated in normal-looking epithelium (NEp), atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate carcinoma (PCa) in 20 CyPs and 20 RPs, both types of specimens with pT2a Gleason score 6 PCa. MEASUREMENTS:At least 1000 cells were counted in contiguous 400X microscopic fields in each case, separately for NEp, atrophy, HGPIN, and PCa. RESULTS AND LIMITATIONS:There was an increase in the percentage of secretory cells with cytoplasmic HuR staining from NEp to atrophy, HGPIN, and PCa. The mean percentages in NEp, atrophy, and HGPIN adjacent to PCa were greater than away from cancer, both in the CyP and RPs. There was a trend towards a reduced nuclear HuR expression in atrophy, HGPIN, and PCa, compared to NEp. COX-2 staining was seen in the cytoplasm of the basal and secretory cells. There was a reduction in the mean proportion of positive basal cells and progressive increase in the percentage of positive secretory cells from atrophy to HGPIN and PCa, compared to NEp. Cytoplasmic HuR overexpression was correlated with COX-2 expression. There was no difference in HuR and COX-2 expression between cancers with tumour volume <0.5 ccm or >0.5 ccm. The limitations of this study were the small number of cases investigated and lack of a control group without cancer. CONCLUSIONS:The secretory cells showed shift in HuR staining from nuclear in NEp to cytoplasmic in PCa. This is associated with a parallel shift in COX-2 expression from basal to secretory cells.
MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs.
Sun Qinyu,Tripathi Vidisha,Yoon Je-Hyun,Singh Deepak K,Hao Qinyu,Min Kyung-Won,Davila Sylvia,Zealy Richard W,Li Xiao Ling,Polycarpou-Schwarz Maria,Lehrmann Elin,Zhang Yongqing,Becker Kevin G,Freier Susan M,Zhu Yuelin,Diederichs Sven,Prasanth Supriya G,Lal Ashish,Gorospe Myriam,Prasanth Kannanganattu V
Nucleic acids research
Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.
LncRNA-HGBC stabilized by HuR promotes gallbladder cancer progression by regulating miR-502-3p/SET/AKT axis.
Hu Yun-Ping,Jin Yun-Peng,Wu Xiang-Song,Yang Yang,Li Yong-Sheng,Li Huai-Feng,Xiang Shan-Shan,Song Xiao-Ling,Jiang Lin,Zhang Yi-Jian,Huang Wen,Chen Shi-Li,Liu Fa-Tao,Chen Chen,Zhu Qin,Chen Hong-Zhuan,Shao Rong,Liu Ying-Bin
BACKGROUNDS:Long non-coding RNAs (lncRNAs) are essential factors that regulate tumor development and metastasis via diverse molecular mechanisms in a broad type of cancers. However, the pathological roles of lncRNAs in gallbladder carcinoma (GBC) remain largely unknown. Here we discovered a novel lncRNA termed lncRNA Highly expressed in GBC (lncRNA-HGBC) which was upregulated in GBC tissue and aimed to investigate its role and regulatory mechanism in the development and progression of GBC. METHODS:The expression level of lncRNA-HGBC in GBC tissue and different cell lines was determined by quantitative real-time PCR. The full length of lncRNA-HGBC was obtained by 5' and 3' rapid amplification of the cDNA ends (RACE). Cellular localization of lncRNA-HGBC was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. In vitro and in vivo assays were preformed to explore the biological effects of lncRNA-HGBC in GBC cells. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay were used to identify lncRNA-HGBC-interacting proteins. Dual luciferase reporter assays, AGO2-RIP, and MS2-RIP assays were performed to verify the interaction between lncRNA-HGBC and miR-502-3p. RESULTS:We found that lncRNA-HGBC was upregulated in GBC and its upregulation could predict poor survival. Overexpression or knockdown of lncRNA-HGBC in GBC cell lines resulted in increased or decreased, respectively, cell proliferation and invasion in vitro and in xenografted tumors. LncRNA-HGBC specifically bound to RNA binding protein Hu Antigen R (HuR) that in turn stabilized lncRNA-HGBC. LncRNA-HGBC functioned as a competitive endogenous RNA to bind to miR-502-3p that inhibits target gene SET. Overexpression, knockdown or mutation of lncRNA-HGBC altered the inhibitory effects of miR-502-3p on SET expression and downstream activation of AKT. Clinically, lncRNA-HGBC expression was negatively correlated with miR-502-3p, but positively correlated with SET and HuR in GBC tissue. CONCLUSIONS:Our study demonstrates that lncRNA-HGBC promotes GBC metastasis via activation of the miR-502-3p-SET-AKT cascade, pointing to lncRNA-HGBC as a new prognostic predictor and a therapeutic target.
HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients.
Richards Nathan G,Rittenhouse David W,Freydin Boris,Cozzitorto Joseph A,Grenda Dane,Rui Hallgeir,Gonye Greg,Kennedy Eugene P,Yeo Charles J,Brody Jonathan R,Witkiewicz Agnieszka K
Annals of surgery
BACKGROUND:Pancreatic ductal adenocarcinoma (PDA) is a devastating disease that killed nearly 38,000 people in the United States this past year. OBJECTIVE:Treatment of PDA typically includes surgery and/or chemotherapy with gemcitabine. No reliable biomarker exists for prognosis or response to chemotherapy. Two previously proposed prognostic markers, cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), are regulated by Hu protein antigen R (HuR), an mRNA binding protein that we have previously demonstrated to be a promising predictive marker of gemcitabine response. This study was designed to evaluate the clinical utility of HuR, COX-2, and VEGF as potential prognostic and predictive biomarkers for PDA. METHODS:A tissue microarray of 53 PDA specimens from patients who underwent potentially curative pancreatic resection was analyzed. HuR, COX-2, and VEGF status were correlated with clinicopathologic and survival data. We also performed ribonucleoprotein immunoprecipitation assays using an HuR antibody to assess VEGF and COX-2 mRNA binding to HuR in pancreatic cancer cells. RESULTS:Roughly 50% (27/53) of patients had high cytoplasmic HuR expression. These patients had worse pathologic features as assessed by T staging (P = 0.005). Only cytoplasmic HuR status correlated with tumor T staging, whereas VEGF (P = 1.0) and COX-2 (P = 0.39) expression did not correlate with T staging. Additionally, HuR status was an unprecedented positive predictive marker for overall survival in patients treated with gemcitabine, pushing median survival over 45 months in the high cytoplasmic HuR expressing patient population compared with less than 23 months in the low cytoplasmic HuR expressing patient group (P = 0.033 for log-rank test and P = 0.04 in a Cox regression model) for the low versus high cytoplasmic HuR expressing group. We also validated that mRNA transcripts for both VEGF and the gemcitabine metabolizing enzyme, deoxycytidine kinase, are specifically bound by HuR in pancreatic cancer cells. CONCLUSIONS:HuR is a useful prognostic biomarker for PDA patients as indicated by its association with higher tumor T stage. Additionally, HuR status is a robust predictor of outcome for patients with resected PDA in the setting of adjuvant gemcitabine therapy. Finally, HuR binds to VEGF mRNA implying that HuR, in part, regulates VEGF expression in PDA. This study supports the notion that HuR status should be used by clinicians for the individualized treatment of PDA in the future.
Complex HuR function in pancreatic cancer cells.
Brody Jonathan R,Dixon Dan A
Wiley interdisciplinary reviews. RNA
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with dismal patient outcomes. The underlying core genetic drivers of disease have been identified in human tumor specimens and described in genetically engineered mouse models. These genetic drivers of PDAC include KRAS signaling, TP53 mutations, and genetic loss of the SMAD4 tumor suppressor protein. Beyond the known mutational landscape of PDAC genomes, alternative disrupted targets that extend beyond conventional genetic mutations have been elusive and understudied in the context of PDAC cell therapeutic resistance and survival. This last point is important because PDAC tumors have a unique and complex tumor microenvironment that includes hypoxic and nutrient-deprived niches that could select for cell populations that garner therapeutic resistance, explaining tumor heterogeneity in regards to response to different therapies. We and others have embarked in a line of investigation focused on the key molecular mechanism of posttranscriptional gene regulation that is altered in PDAC cells and supports this pro-survival phenotype intrinsic to PDAC cells. Specifically, the key regulator of this mechanism is a RNA-binding protein, HuR (ELAVL1), first described in cancer nearly two decades ago. Herein, we will provide a brief overview of the work demonstrating the importance of this RNA-binding protein in PDAC biology and then provide insight into ongoing work developing therapeutic strategies aimed at targeting this molecule in PDAC cells. This article is categorized under: RNA in Disease and Development > RNA in Disease.
GPRC5A is a potential oncogene in pancreatic ductal adenocarcinoma cells that is upregulated by gemcitabine with help from HuR.
Zhou H,Telonis A G,Jing Y,Xia N L,Biederman L,Jimbo M,Blanco F,Londin E,Brody J R,Rigoutsos I
Cell death & disease
GPRC5A is an orphan G-protein coupled receptor with an intriguing dual behavior, acting as an oncogene in some cancers and as a tumor suppressor in other cancers. In the pancreatic cancer context, very little is known about GPRC5A. By analyzing messenger RNA (mRNA) expression data from 675 human cancer cell lines and 10 609 samples from The Cancer Genome Atlas (TCGA) we found that GPRC5A's abundance in pancreatic cancer is highest (cell lines) or second highest (TCGA) among all tissues and cancer types. Further analyses of an independent set of 252 pancreatic normal and cancer samples showed GPRC5A mRNA to be more than twofold upregulated in primary tumor samples compared with normal pancreas (P-value<10(-5)), and even further upregulated in pancreatic cancer metastases to various organs (P-value=0.0021). Immunostaining of 208 cores (103 samples) of a tissue microarray showed generally low expression of GPRC5A protein in normal pancreatic ductal cells; on the other hand, in primary and metastatic samples, GPRC5A protein levels were dramatically increased in pancreatic ductal cells. In vitro studies of multiple pancreatic cancer cell lines showed that an increase in GPRC5A protein levels promoted pancreatic cancer cell growth and migration. Unexpectedly, when we treated pancreatic cancer cell lines with gemcitabine (2',2'-difluorodeoxycytidine), we observed an increase in GPRC5A protein abundance. On the other hand, when we knocked down GPRC5A we sensitized pancreatic cancer cells to gemcitabine. Through further experimentation we showed that the monotonic increase in GPRC5A protein levels that we observe for the first 18 h following gemcitabine treatment results from interactions between GPRC5A's mRNA and the RNA-binding protein HuR, which is an established key mediator of gemcitabine's efficacy in cancer cells. As we discovered, the interaction between GPRC5A and HuR is mediated by at least one HuR-binding site in GPRC5A's mRNA. Our findings indicate that GPRC5A is part of a complex molecular axis that involves gemcitabine and HuR, and, possibly, other genes. Further work is warranted before it can be established unequivocally that GPRC5A is an oncogene in the pancreatic cancer context.
HuR posttranscriptionally regulates WEE1: implications for the DNA damage response in pancreatic cancer cells.
Lal Shruti,Burkhart Richard A,Beeharry Neil,Bhattacharjee Vikram,Londin Eric R,Cozzitorto Joseph A,Romeo Carmella,Jimbo Masaya,Norris Zoë A,Yeo Charles J,Sawicki Janet A,Winter Jordan M,Rigoutsos Isidore,Yen Timothy J,Brody Jonathan R
HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.
Reversible HuR-microRNA binding controls extracellular export of miR-122 and augments stress response.
Mukherjee Kamalika,Ghoshal Bartika,Ghosh Souvik,Chakrabarty Yogaditya,Shwetha Shivaprasad,Das Saumitra,Bhattacharyya Suvendra N
microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR-122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)-mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR-unbound miRNAs also reside. These MVB-associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR-122 level during starvation. Therefore, by modulating extracellular export of miR-122, HuR could control stress response in starved human hepatic cells.
The long intergenic non-protein coding RNA 707 promotes proliferation and metastasis of gastric cancer by interacting with mRNA stabilizing protein HuR.
Xie Min,Ma Tianshi,Xue Jiangyang,Ma Hongwei,Sun Ming,Zhang Zhihong,Liu Minjuan,Liu Yinghua,Ju Songwen,Wang Zhaoxia,De Wei
Multiple studies have revealed that long non-coding RNAs (lncRNAs) extensively participate in human cancer malignant progression. The long intergenic non-protein coding RNA 707 (LINC00707), 3087 bp in length, was recently reported to be an essential oncogene in promoting lung adenocarcinoma cell proliferation and metastasis. However, its role in gastric cancer (GC) remains unclear. In this study, we identified that LINC00707 was excessively expressed in GC tissues and correlated with advanced stage, larger tumor size, lymph node metastasis and poorer prognosis in GC patients. In vitro and in vivo assays showed that LINC00707 promote GC cell proliferation and metastasis. Mechanistically, LINC00707 could abundantly interact with mRNA stabilizing protein HuR; "LINC00707-HuR" coalition ulteriorly combined with VAV3/F11R mRNAs and increased their stability. Taken together, our findings prove that LINC00707 may act as an oncogene in GC by regulating mRNA stability and serve as a potential target for GC diagnosis and prognosis.
LncRNA B4GALT1-AS1 recruits HuR to promote osteosarcoma cells stemness and migration via enhancing YAP transcriptional activity.
Li Zhikun,Wang Yi,Hu Ruixi,Xu Ruijun,Xu Wei
OBJECTIVES:This study aims to reveal the roles and related mechanisms of LncRNA B4GALT1-AS1 in osteosarcoma (OS) cells stemness and migration. MATERIALS AND METHODS:Real-time quantitative PCR (RT-qPCR) was used to detect the expression of several LncRNAs in OS tissues and normal adjacent tissues and in OS mammospheres and cells. Cell viability, transwell migration, tumour spheres formation and in vivo tumour formation assays were used to examine the effects of LncRNA B4GALT1-AS1 on OS progression. In addition, RNA immunoprecipitation (RIP) and Luciferase reporter assays were performed to determine the binding site of RNA-binding protein HuR on B4GALT1-AS1 and transcriptional factor YAP. Immunofluorescence analysis was used to examine YAP nuclear-cytoplasm translocation. RESULTS:LncRNA B4GALT1-AS1 expression was significantly increased in OS tissues and cells spheres. Knockdown of B4GALT1-AS1 inhibited OS cells proliferation, migration, stemness and chemotherapeutic sensitivity. Mechanistically, B4GALT1-AS1 recruited HuR to enhance YAP mRNA stability and thus its transcriptional activity. CONCLUSIONS:We indicate that lncRNA B4GALT1-AS1 promotes OS cells stemness and migration via recruiting HuR to enhance YAP activity.
Cytoplasmic HuR Status Predicts Disease-free Survival in Resected Pancreatic Cancer: A Post-hoc Analysis From the International Phase III ESPAC-3 Clinical Trial.
Tatarian Talar,Jiang Wei,Leiby Benjamin E,Grigoli Amanda,Jimbo Masaya,Dabbish Nooreen,Neoptolemos John P,Greenhalf William,Costello Eithne,Ghaneh Paula,Halloran Christopher,Palmer Daniel,Buchler Markus,Yeo Charles J,Winter Jordan M,Brody Jonathan R
Annals of surgery
OBJECTIVES:We tested cytoplasmic HuR (cHuR) as a predictive marker for response to chemotherapy by examining tumor samples from the international European Study Group of Pancreatic Cancer-3 trial, in which patients with resected pancreatic ductal adenocarcinoma (PDA) received either gemcitabine (GEM) or 5-fluorouracil (5-FU) adjuvant monotherapy. BACKGROUND:Previous studies have implicated the mRNA-binding protein, HuR (ELAVL1), as a predictive marker for PDA treatment response in the adjuvant setting. These studies were, however, based on small cohorts of patients outside of a clinical trial, or a clinical trial in which patients received multimodality therapy with concomitant radiation. METHODS:Tissue samples from 379 patients with PDA enrolled in the European Study Group of Pancreatic Cancer-3 trial were immunolabeled with an anti-HuR antibody and scored for cHuR expression. Patients were dichotomized into groups of high versus low cHuR expression. RESULTS:There was no association between cHuR expression and prognosis in the overall cohort [disease-free survival (DFS), P = 0.44; overall survival, P = 0.41). Median DFS for patients with high cHuR was significantly greater for patients treated with 5-FU compared to GEM [20.1 months, confidence interval (CI): 8.3-36.4 vs 10.9 months, CI: 7.5-14.2; P = 0.04]. Median DFS was similar between the treatment arms in patients with low cHuR (5-FU, 12.8 months, CI: 10.6-14.6 vs GEM, 12.9 months, CI: 11.2-15.4). CONCLUSIONS:Patients with high cHuR-expressing tumors may benefit from 5-FU-based adjuvant therapy as compared to GEM, whereas those patients with low cHuR appear to have no survival advantage with GEM compared with 5-FU. Further studies are needed to validate HuR as a biomarker in both future monotherapy and multiagent regimens.
The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.
Blanco F F,Jimbo M,Wulfkuhle J,Gallagher I,Deng J,Enyenihi L,Meisner-Kober N,Londin E,Rigoutsos I,Sawicki J A,Risbud M V,Witkiewicz A K,McCue P A,Jiang W,Rui H,Yeo C J,Petricoin E,Winter J M,Brody J R
Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.
HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer.
Burkhart Richard A,Pineda Danielle M,Chand Saswati N,Romeo Carmella,Londin Eric R,Karoly Edward D,Cozzitorto Joseph A,Rigoutsos Isidore,Yeo Charles J,Brody Jonathan R,Winter Jordan M
Cancer cell metabolism differs from normal cells, yet the regulatory mechanisms responsible for these differences are incompletely understood, particularly in response to acute changes in the tumor microenvironment. HuR, an RNA-binding protein, acts under acute stress to regulate core signaling pathways in cancer through post-transcriptional regulation of mRNA targets. We demonstrate that HuR regulates the metabolic phenotype in pancreatic cancer cells and is critical for survival under acute glucose deprivation. Using three pancreatic cancer cell line models, HuR-proficient cells demonstrated superior survival under glucose deprivation when compared with isogenic cells with siRNA-silencing of HuR expression (HuR-deficient cells). We found that HuR-proficient cells utilized less glucose, but produced greater lactate, as compared with HuR-deficient cells. Acute glucose deprivation was found to act as a potent stimulus for HuR translocation from the nucleus to the cytoplasm, where HuR stabilizes its mRNA targets. We performed a gene expression array on ribonucleoprotein-immunoprecipitated mRNAs bound to HuR and identified 11 novel HuR target transcripts that encode enzymes central to glucose metabolism. Three (GPI, PRPS2 and IDH1) were selected for validation studies, and confirmed as bona fide HuR targets. These findings establish HuR as a critical regulator of pancreatic cancer cell metabolism and survival under acute glucose deprivation. Further explorations into HuR's role in cancer cell metabolism should uncover novel therapeutic targets that are critical for cancer cell survival in a metabolically compromised tumor microenvironment.
miR-519 suppresses tumor growth by reducing HuR levels.
Abdelmohsen Kotb,Kim Mihee M,Srikantan Subramanya,Mercken Evi M,Brennan Sarah E,Wilson Gerald M,Cabo Rafael de,Gorospe Myriam
Cell cycle (Georgetown, Tex.)
The RNA-binding protein HuR is highly abundant in many cancers. HuR expression was recently found to be repressed by microRNA miR-519, which potently lowered HuR translation without influencing HuR mRNA abundance. Here, we examined the levels of HuR and miR-519 in pairs of cancer and adjacent healthy tissues from ovary, lung, and kidney. In the three sample collections, the cancer specimens showed dramatically higher HuR levels, unchanged HuR mRNA concentrations, and markedly reduced miR-519 levels, when compared with healthy tissues. As tested using human cervical carcinoma cells, miR-519 reduced tumorigenesis in athymic mice. Compared with the tumors arising from control cells, cells overexpressing miR-519 formed significantly smaller tumors, while cells expressing reduced miR-519 levels gave rise to substantially larger tumors. Evidence that the miR-519-elicited reduction of HuR was critical for its tumor suppressor influence was obtained by reducing HuR, as HuR-silenced cells formed markedly smaller tumors and were unable to form large tumors even after lowering miR-519 abundance. Together, our data reveal that miR-519 inhibits tumorigenesis in large part by repressing HuR expression.
Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer.
Lan Yang,Xiao Xuewei,He Zhengchi,Luo Yu,Wu Chuanfang,Li Ling,Song Xu
Nucleic acids research
Overexpressed in colon carcinoma-1 (OCC-1) is one of the earliest annotated long noncoding RNAs (lncRNAs) in colorectal cancer (CRC); however, its function remains largely unknown. Here, we revealed that OCC-1 plays a tumor suppressive role in CRC. OCC-1 knockdown by RNA interference promotes cell growth both in vitro and in vivo, which is largely due to its ability to inhibit G0 to G1 and G1 to S phase cell cycle transitions. In addition, overexpression of OCC-1 can suppress cell growth in OCC-1 knockdown cells. OCC-1 exerts its function by binding to and destabilizing HuR (ELAVL1), a cancer-associated RNA binding protein (RBP) which can bind to and stabilize thousands of mRNAs. OCC-1 enhances the binding of ubiquitin E3 ligase β-TrCP1 to HuR and renders HuR susceptible to ubiquitination and degradation, thereby reducing the levels of HuR and its target mRNAs, including the mRNAs directly associated with cancer cell growth. These findings reveal that lncRNA OCC-1 can regulate the levels of a large number of mRNAs at post-transcriptional level through modulating RBP HuR stability.
HuR Suppresses Fas Expression and Correlates with Patient Outcome in Liver Cancer.
Zhu Haifeng,Berkova Zuzana,Mathur Rohit,Sehgal Lalit,Khashab Tamer,Tao Rong-Hua,Ao Xue,Feng Lei,Sabichi Anita L,Blechacz Boris,Rashid Asif,Samaniego Felipe
Molecular cancer research : MCR
UNLABELLED:Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3'-untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3'-UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties. IMPLICATIONS:Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism.
HuR Reduces Radiation-Induced DNA Damage by Enhancing Expression of ARID1A.
Andrade Daniel,Mehta Meghna,Griffith James,Oh Sangphil,Corbin Joshua,Babu Anish,De Supriyo,Chen Allshine,Zhao Yan D,Husain Sanam,Roy Sudeshna,Xu Liang,Aube Jeffrey,Janknecht Ralf,Gorospe Myriam,Herman Terence,Ramesh Rajagopal,Munshi Anupama
Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients.
Inhibition of Caspase-2 Translation by the mRNA Binding Protein HuR: A Novel Path of Therapy Resistance in Colon Carcinoma Cells?
Eberhardt Wolfgang,Nasrullah Usman,Haeussler Kristina
An increased expression and cytoplasmic abundance of the ubiquitous RNA binding protein human antigen R (HuR) is critically implicated in the dysregulated control of post- transcriptional gene expression during colorectal cancer development and is frequently associated with a high grade of malignancy and therapy resistance. Regardless of the fact that HuR elicits a broad cell survival program by increasing the stability of mRNAs coding for prominent anti-apoptotic factors, recent data suggest that HuR is critically involved in the regulation of translation, particularly, in the internal ribosome entry site (IRES) controlled translation of cell death regulatory proteins. Accordingly, data from human colon carcinoma cells revealed that HuR maintains constitutively reduced protein and activity levels of caspase-2 through negative interference with IRES-mediated translation. This review covers recent advances in the understanding of mechanisms underlying HuR's modulatory activity on IRES-triggered translation. With respect to the unique regulatory features of caspase-2 and its multiple roles (e.g., in DNA-damage-induced apoptosis, cell cycle regulation and maintenance of genomic stability), the pathophysiological consequences of negative caspase-2 regulation by HuR and its impact on therapy resistance of colorectal cancers will be discussed in detail. The negative HuR-caspase-2 axis may offer a novel target for tumor sensitizing therapies.
The RNA-Binding Protein HuR Confers Oxaliplatin Resistance of Colorectal Cancer By Upregulating CDC6.
Cai Jian,Wang Huaiming,Jiao Xiaodong,Huang Rongkang,Qin Qiyuan,Zhang Jianwei,Chen Honglei,Feng Dan,Tian Xin,Wang Hui
Molecular cancer therapeutics
Human antigen R (HuR) is an RNA-binding protein that posttranscriptionally regulates many cancer-trait genes. CDC6, a central regulator of DNA replication, is regulated by HuR. In this study, we investigated the role of HuR in colorectal cancer tumorigenesis and oxaliplatin (L-OHP) resistance, as well as the underlying mechanisms involving CDC6. We detected increased HuR and CDC6 expression, along with a positive correlation between the two in human colorectal cancer tissues. HuR overexpression increased colorectal cancer cell proliferation and xenograft tumor growth , and induced resistance to L-OHP. In contrast, HuR knockdown sensitized colorectal cancer cells to L-OHP. CDC6 overexpression increased while CDC6 knockdown decreased colorectal cancer cell malignant behaviors (growth, DNA synthesis, EMT, migration, and invasion) and L-OHP resistance Moreover, L-OHP resistance induced by HuR overexpression was reversed by CDC6 knockdown. Mechanistically, the results from our luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that HuR upregulates CDC6 by binding to CDC6 3'-UTR. Taken together, our findings identified HuR's regulation of CDC6 as an essential mechanism driving colorectal cancer tumorigenesis and L-OHP resistance, and this mechanism may represent a potential target for overcoming drug resistance in colorectal cancer.
LncRNA FAM83H-AS1 contributes to the radioresistance, proliferation, and metastasis in ovarian cancer through stabilizing HuR protein.
Dou Qianru,Xu Yang,Zhu Yuanyuan,Hu Yakun,Yan Yuchen,Yan Hongchao
European journal of pharmacology
Ovarian cancer (OC) is a major cause of cancer-related deaths in women all over the world. The easy metastasis of OC and the problem of radioresistance are serious issues remaining to be overcome. Thus, research on molecular mechanisms underlying is in urgent demand. Long non-coding RNAs (lncRNAs) are a class of RNAs without protein coding potential, which has been reported to participate in the regulation on multiple biological process in cancers, including cell radiosensitivity and metastasis. Present study aimed to explore the role of lncRNA FAM83-AS1 in radioresistance and metastasis of ovarian cancer. First of all, the obvious upregulation of FAM83H-AS1 was identified by qRT-PCR in OC tissues, especially in metastatic tissues, as well as in OC cell lines. Importantly, we confirmed the correlation of FAM83H-AS1 levels with both ovarian cancer cells and normal ovarian cells. And Kaplan-Meier analysis indicated FAM83H-AS1 as a potential target of poor prognosis of OC. Through loss-of-function assays, we validated the inductive effect of FAM83H-AS1 in OC cell metastasis and radioresistance. Through mechanism research on FAM83H-AS1, we confirmed its interaction with HuR using pull-down assay and RNA immunoprecipitation (RIP), and verified the stabilization of HuR protein by FAM83-AS1 through western blot with the addition of CHX. Finally, rescue assays showed that overexpression of HuR rescued the suppression on radioresistance and metastasis in OC cell caused by the silencing of FAM83H-AS1. In conclusion, present study proved that FAM83H-AS1 contributes to the radioresistance and cell metastasis in ovarian cancer through stabilizing HuR protein.
MiR-155-5p controls colon cancer cell migration via post-transcriptional regulation of Human Antigen R (HuR).
Al-Haidari Amr,Algaber Anwar,Madhi Raed,Syk Ingvar,Thorlacius Henrik
Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related deaths worldwide. Metastasis is the worst prognostic factor for patients with CRC. HuR (ELAVL1) is overexpressed in CRC and has been reported to promote colon cancer growth by targeting RNA in the cell cytoplasm. Herein, the role of miR-155-5p in regulating HuR expression and cell migration was examined in colon cancer cells. MiR-155-5p knockdown in serum-starved colon cancer cells decreased both colon cancer cell chemotaxis and cytoplasmic expression of HuR. Bioinformatics analysis predicted two putative binding sites in the AU-rich elements (AREs) at the 3'-UTR of HuR mRNA. MiR-155-5p binding to HuR was verified using specific target site blockers and functionally validated by use of RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of HuR expression is mediated by AREs. Targeting AREs with a specific blocker inhibited colon cancer cell migration. Taken together, these novel findings demonstrate that AREs mediate miR-155-5p positive regulation of HuR mRNA levels and translation as well as migration in colon cancer cells, suggesting that targeting miR-155-5p and/or Hur might be useful therapeutic strategies against colon cancer metastasis.
HuR controls apoptosis and activation response without effects on cytokine 3' UTRs.
Karginov Fedor V
RNA binding proteins regulate gene expression through several post-transcriptional mechanisms. The broadly expressed HuR/ELAVL1 is important for proper function of multiple immune cell types, and has been proposed to regulate cytokine and other mRNA 3' UTRs upon activation. However, this mechanism has not been previously dissected in stable cellular settings. In this study, HuR demonstrated strong anti-apoptotic and activation roles in Jurkat T cells. Detailed transcriptomic analysis of HuR knockout cells revealed a substantial negative impact on the activation program, coordinately preventing the expression of immune response gene categories, including all cytokines. Knockout cells showed a significant defect in IL-2 production, which was rescued upon reintroduction of HuR. Interestingly, the mechanism of HuR regulation did not involve control of the cytokine 3' UTRs: HuR knockout did not affect the activity of 3' UTR reporters in 293 cells, and had no effect on IL-2 and TNF 3' UTRs in resting or activated Jurkats. Instead, impaired cytokine production corresponded with defective induction of the IL-2 promoter upon activation. Accordingly, upregulation of NFATC1 was also impaired, without 3' UTR effects. Together, these results indicate that HuR controls cytokine production through coordinated upstream pathways, and that additional mechanisms must be considered in investigating its function.
RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.
Poria D K,Guha A,Nandi I,Ray P S
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.
Repression of caspase-3 and RNA-binding protein HuR cleavage by cyclooxygenase-2 promotes drug resistance in oral squamous cell carcinoma.
Janakiraman H,House R P,Talwar S,Courtney S M,Hazard E S,Hardiman G,Mehrotra S,Howe P H,Gangaraju V,Palanisamy V
A well-studied RNA-binding protein Hu Antigen-R (HuR), controls post-transcriptional gene regulation and undergoes stress-activated caspase-3 dependent cleavage in cancer cells. The cleavage products of HuR are known to promote cell death; however, the underlying molecular mechanisms facilitating caspase-3 activation and HuR cleavage remains unknown. Here, we show that HuR cleavage associated with active caspase-3 in oral cancer cells treated with ionizing radiation and chemotherapeutic drug, paclitaxel. We determined that oral cancer cells overexpressing cyclooxygenase-2 (COX-2) limited the cleavage of caspase-3 and HuR, which reduced the rate of cell death in paclitaxel resistant oral cancer cells. Specific inhibition of COX-2 by celecoxib, promoted apoptosis through activation of caspase-3 and cleavage of HuR in paclitaxel-resistant oral cancer cells, both in vitro and in vivo. In addition, oral cancer cells overexpressing cellular HuR increased the half-life of COX-2 mRNA, promoted COX-2 protein expression and exhibited enhanced tumor growth in vivo in comparison with cells expressing a cleavable form of HuR. Finally, our ribonucleoprotein immunoprecipitation and sequencing (RIP-seq) analyses of HuR in oral cancer cells treated with ionizing radiation (IR), determined that HuR cleavage product-1 (HuR-CP1) bound and promoted the expression of mRNAs encoding proteins involved in apoptosis. Our results indicated that, cellular non-cleavable HuR controls COX-2 mRNA expression and enzymatic activity. In addition, overexpressed COX-2 protein repressed the cleavage of caspase-3 and HuR to promote drug resistance and tumor growth. Altogether, our observations support the use of the COX-2 inhibitor celecoxib, in combination with paclitaxel, for the management of paclitaxel resistant oral cancer cells.
Understanding and targeting the disease-related RNA binding protein human antigen R (HuR).
Schultz Christopher W,Preet Ranjan,Dhir Teena,Dixon Dan A,Brody Jonathan R
Wiley interdisciplinary reviews. RNA
Altered gene expression is a characteristic feature of many disease states such as tumorigenesis, and in most cancers, it facilitates cancer cell survival and adaptation. Alterations in global gene expression are strongly impacted by post-transcriptional gene regulation. The RNA binding protein (RBP) HuR (ELAVL1) is an established regulator of post-transcriptional gene regulation and is overexpressed in most human cancers. In many cancerous settings, HuR is not only overexpressed, but it is "overactive" as denoted by increased subcellular localization within the cytoplasm. This dysregulation of HuR expression and cytoplasmic localization allows HuR to stabilize and increase the translation of various prosurvival messenger RNA (mRNAs) involved in the pathogenesis of numerous cancers and various diseases. Based on almost 20 years of work, HuR is now recognized as a therapeutic target. Herein, we will review the role HuR plays in the pathophysiology of different diseases and ongoing therapeutic strategies to target HuR. We will focus on three ongoing-targeted strategies: (1) inhibiting HuR's translocation from the nucleus to the cytoplasm; (2) inhibiting the ability of HuR to bind target RNA; and (3) silencing HuR expression levels. In an oncologic setting, HuR has been demonstrated to be critical for a cancer cell's ability to survive a variety of cancer relevant stressors (including drugs and elements of the tumor microenvironment) and targeting this protein has been shown to sensitize cancer cells further to insult. We strongly believe that targeting HuR could be a powerful therapeutic target to treat different diseases, particularly cancer, in the near future. This article is categorized under: RNA in Disease and Development > RNA in Disease NRA Turnover and Surveillance > Regulation of RNA Stability Translation > Translation Regulation.
HuR Small-Molecule Inhibitor Elicits Differential Effects in Adenomatosis Polyposis and Colorectal Carcinogenesis.
Lang Michaela,Berry David,Passecker Katharina,Mesteri Ildiko,Bhuju Sabin,Ebner Florian,Sedlyarov Vitaly,Evstatiev Rayko,Dammann Kyle,Loy Alexander,Kuzyk Orest,Kovarik Pavel,Khare Vineeta,Beibel Martin,Roma Guglielmo,Meisner-Kober Nicole,Gasche Christoph
HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APC mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of , and Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD. .
Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2.
Badawi Amel,Hehlgans Stephanie,Pfeilschifter Josef,Rödel Franz,Eberhardt Wolfgang
Increased abundance of the mRNA-binding protein human antigen R (HuR) is a characteristic feature of many cancers and frequently associated with a high grade malignancy and therapy resistance. HuR elicits a broad cell survival program mainly by stabilizing or increasing the translation of mRNAs coding for anti-apoptotic effector proteins. Conversally, we previously identified the pro-apoptotic caspase-2 as a novel HuR target which is mainly regulated at the level of translation. In this study, we investigated whether siRNA-mediated HuR knockdown interferes with cell survival and radiation sensitivity by monitoring apoptosis, DNA repair and three-dimensional (3D) clonogenic survival. We observed a significant elevation in caspase-2 upon HuR depletion and in turn, a sensitization of colorectal DLD-1 and HCT-15 cells to radiation-induced apoptosis as implicated by the dose-dependent elevation of sub-G phase cell entry and increased caspase-2, -3 and poly ADP-ribose polymerase (PARP)-cleavage, respectively. Coincidentally, HuR deficiency significantly elevated the number of radiation-induced γH2AX/53BP1-positive foci indicating an increase in DNA damage. Accordingly, the irradiation-dependent reduction in clonogenic cell survival was further impaired after knockdown of HuR. Importantly, HuR knockdown remained ineffective to radiation-induced cell responses after additional knockdown of caspase-2. Furthermore, by using RNA-pull down assay we demonstrate that irradiation (6 Gy) robustly increased HuR binding to caspase-2 mRNA. Collectively, sensitization of colon carcinoma cells to radiation-induced cell death and DNA-damage by HuR knockdown critically depends on caspase-2 and may represent a valuable approach to intervene with therapy resistance of colorectal cancer (CRC).
HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting.
Mubaid Souad,Ma Jennifer F,Omer Amr,Ashour Kholoud,Lian Xian J,Sanchez Brenda J,Robinson Samantha,Cammas Anne,Dormoy-Raclet Virginie,Di Marco Sergio,Chittur Sridar V,Tenenbaum Scott A,Gallouzi Imed-Eddine
Proceedings of the National Academy of Sciences of the United States of America
Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the () mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the () located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.
CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype.
Lal Shruti,Cheung Edwin C,Zarei Mahsa,Preet Ranjan,Chand Saswati N,Mambelli-Lisboa Nicole C,Romeo Carmella,Stout Matthew C,Londin Eric,Goetz Austin,Lowder Cinthya Y,Nevler Avinoam,Yeo Charles J,Campbell Paul M,Winter Jordan M,Dixon Dan A,Brody Jonathan R
Molecular cancer research : MCR
Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, , as HuR-deficient MIA PaCa-2 (MIA.HuR-KO) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO) showed significantly reduced tumor growth compared with controls (HCT.HuR-WT). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes. The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. .
Posttranscriptional Regulation of mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors.
Chand Saswati N,Zarei Mahsa,Schiewer Matthew J,Kamath Akshay R,Romeo Carmella,Lal Shruti,Cozzitorto Joseph A,Nevler Avinoam,Scolaro Laura,Londin Eric,Jiang Wei,Meisner-Kober Nicole,Pishvaian Michael J,Knudsen Karen E,Yeo Charles J,Pascal John M,Winter Jordan M,Brody Jonathan R
The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9-mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase () mRNA, by binding a unique sequence embedded in its 3' untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR-PARG axis as an opportunity to enhance PARPi-based therapies. .
Intestinal epithelial HuR modulates distinct pathways of proliferation and apoptosis and attenuates small intestinal and colonic tumor development.
Giammanco Antonina,Blanc Valerie,Montenegro Grace,Klos Coen,Xie Yan,Kennedy Susan,Luo Jianyang,Chang Sung-Hee,Hla Timothy,Nalbantoglu Ilke,Dharmarajan Sekhar,Davidson Nicholas O
HuR is a ubiquitous nucleocytoplasmic RNA-binding protein that exerts pleiotropic effects on cell growth and tumorigenesis. In this study, we explored the impact of conditional, tissue-specific genetic deletion of HuR on intestinal growth and tumorigenesis in mice. Mice lacking intestinal expression of HuR (Hur (IKO) mice) displayed reduced levels of cell proliferation in the small intestine and increased sensitivity to doxorubicin-induced acute intestinal injury, as evidenced by decreased villus height and a compensatory shift in proliferating cells. In the context of Apc(min/+) mice, a transgenic model of intestinal tumorigenesis, intestinal deletion of the HuR gene caused a three-fold decrease in tumor burden characterized by reduced proliferation, increased apoptosis, and decreased expression of transcripts encoding antiapoptotic HuR target RNAs. Similarly, Hur(IKO) mice subjected to an inflammatory colon carcinogenesis protocol [azoxymethane and dextran sodium sulfate (AOM-DSS) administration] exhibited a two-fold decrease in tumor burden. Hur(IKO) mice showed no change in ileal Asbt expression, fecal bile acid excretion, or enterohepatic pool size that might explain the phenotype. Moreover, none of the HuR targets identified in Apc(min/+)Hur(IKO) were altered in AOM-DSS-treated Hur(IKO) mice, the latter of which exhibited increased apoptosis of colonic epithelial cells, where elevation of a unique set of HuR-targeted proapoptotic factors was documented. Taken together, our results promote the concept of epithelial HuR as a contextual modifier of proapoptotic gene expression in intestinal cancers, acting independently of bile acid metabolism to promote cancer. In the small intestine, epithelial HuR promotes expression of prosurvival transcripts that support Wnt-dependent tumorigenesis, whereas in the large intestine epithelial HuR indirectly downregulates certain proapoptotic RNAs to attenuate colitis-associated cancer. Cancer Res; 74(18); 5322-35. ©2014 AACR.
Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.
Gauchotte Guillaume,Hergalant Sébastien,Vigouroux Charlène,Casse Jean-Matthieu,Houlgatte Rémi,Kaoma Tony,Helle Déborah,Brochin Lydia,Rech Fabien,Peyre Matthieu,Labrousse François,Vallar Laurent,Guéant Jean-Louis,Vignaud Jean-Michel,Battaglia-Hsu Shyue-Fang
The Journal of pathology
HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10 ) and IOMM-Lee (p = 4 × 10 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth.
Huang Yu-Hung,Peng Weidan,Furuuchi Narumi,Gerhart Jacquelyn,Rhodes Kelly,Mukherjee Neelanjan,Jimbo Masaya,Gonye Gregory E,Brody Jonathan R,Getts Robert C,Sawicki Janet A
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.
The Jun/miR-22/HuR regulatory axis contributes to tumourigenesis in colorectal cancer.
Liu Yanqing,Chen Xiaorui,Cheng Rongjie,Yang Fei,Yu Mengchao,Wang Chen,Cui Shufang,Hong Yeting,Liang Hongwei,Liu Minghui,Zhao Chihao,Ding Meng,Sun Wu,Liu Zhijian,Sun Feng,Zhang Chenyu,Zhou Zhen,Jiang Xiaohong,Chen Xi
BACKGROUND:Colorectal cancer (CRC) is a severe health problem worldwide. Clarifying the mechanisms for the deregulation of oncogenes and tumour suppressors in CRC is vital for its diagnosis, treatment, prognosis and prevention. Hu antigen R (HuR), which is highly upregulated in CRC, functions as a pivotal oncogene to promote CRC progression. However, the underlying cause of its dysregulation is poorly understood. METHODS:In CRC tissue sample pairs, HuR protein levels were measured by Western blot and immunohistochemical (IHC) staining, respectively. HuR mRNA levels were also monitored by qRT-PCR. Combining meta-analysis and microRNA (miRNA) target prediction software, we predicted miRNAs that targeted HuR. Pull-down assay, Western blot and luciferase assay were utilized to demonstrate the direct binding of miR-22 on HuR's 3'-UTR. The biological effects of HuR and miR-22 were investigated both in vitro by CCK-8, EdU and Transwell assays and in vivo by a xenograft mice model. JASPAR and SABiosciences were used to predict transcriptional factors that could affect miR-22. Luciferase assay was used to explore the validity of putative Jun binding sites for miR-22 regulation. ChIP assay was performed to test the Jun's occupancy on the C17orf91 promoter. RESULTS:We observed a significant upregulation of HuR in CRC tissue pairs and confirmed the oncogenic function of HuR both in vitro and in vivo. We found that an important tumour-suppressive miRNA, miR-22, was significantly downregulated in CRC tissues and inversely correlated with HuR in both CRC tissues and CRC cell lines. We demonstrated that miR-22 directly bound to the 3'-UTR of HuR and led to inhibition of HuR protein, which repressed CRC proliferation and migration in vitro and decelerated CRC xenografted tumour growth in vivo. Furthermore, we found that the onco-transcription factor Jun could inhibit the transcription of miR-22. CONCLUSIONS:Our findings highlight the critical roles of the Jun/miR-22/HuR regulatory axis in CRC progression and may provide attractive potential targets for CRC prevention and treatment.
Deletion of the RNA regulator HuR in tumor-associated microglia and macrophages stimulates anti-tumor immunity and attenuates glioma growth.
Wang Jiping,Leavenworth Jianmei W,Hjelmeland Anita B,Smith Reed,Patel Neha,Borg Ben,Si Ying,King Peter H
Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are prime targets, being drawn into the tumor and stimulated to produce factors that support tumor growth and evasion from the immune system. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout (KO) of HuR led to reduced tumor growth and proliferation associated with prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor-associated macrophages (TAMs) were decreased, more polarized toward an M1-like phenotype, and had reduced PD-L1 expression. There was an overall increase in infiltrating CD4 cells, including Th1 and cytotoxic effector cells, and a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells. Molecular and cellular analyses of HuR KO TAMs and cultured microglia showed changes in migration, chemoattraction, and chemokine/cytokine profiles that provide potential mechanisms for the altered tumor microenvironment and reduced tumor growth in HuR KO mice. In summary, HuR is a key modulator of pro-glioma responses by microglia/macrophages through the molecular regulation of chemokines, cytokines, and other factors. Our findings underscore the relevance of HuR as a therapeutic target in glioblastoma.