Cold‐inducible RNA‐binding protein contributes to human antigen R and cyclin E1 deregulation in breast cancer

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress‐induced RNA‐binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF‐7 breast cancer cells by stabilizing its mRNA. We show here that cold‐inducible RNA‐binding protein (CIRP) co‐regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co‐regulate cyclin E1, HuR and CIRP levels were altered in MCF‐7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co‐precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co‐localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR‐containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets. © 2009 Wiley‐Liss, Inc.     

Trichostatin A and 5 Aza-2′ deoxycytidine decrease estrogen receptor mRNA stability in ER positive MCF7 cells through modulation of HuR

Trichostatin A (TSA) and 5-Aza 2'deoxycytidine (AZA), two well characterized pharmacologic inhibitors of histone deacetylation and DNA methylation, affect estrogen receptor alpha (ER) levels differently in ER-positive versus ER-negative breast cancer cell lines. Whereas pharmacologic inhibition of these epigenetic mechanisms results in re-expression and increased estrogen receptor alpha (ER) levels in ER-negative cells, treatment in ER-positive MCF7 cells results in decreased ER mRNA and protein levels. This decrease is dependent upon protein interaction with the ER 3'UTR. Actinomycin D studies showed a 37.5% reduction in ER mRNA stability from 4 to 1.5 h in AZA/TSA treated MCF7 cell lines; an effect not seen in 231ER + cells transfected with the ER coding region but lacking incorporation of the 3'UTR. AZA/TSA do not appear to directly interact with the 3'UTR but rather decrease stability through altered subcellular localization of the RNA binding protein, HuR. siRNA inhibition of HuR expression reduces both the steady-state and stability of ER mRNA, suggesting that HuR plays a critical role in the control of ER mRNA stability. Our data suggest that epigenetic modulators can alter stability through modulation of HuR subcellular distribution. Taken together, these data provide a novel anti-estrogenic mechanism for AZA and TSA in ER positive human breast cancer cells.     

Cytoplasmic HuR expression correlates with poor outcome and with cyclooxygenase 2 expression in serous ovarian carcinoma

Cyclooxygenase-2 (COX-2) expression has been shown to associate with poor prognosis in ovarian cancer, and an mRNA stability protein HuR has been shown to enhance expression of COX-2 in tissue culture conditions. We found cytoplasmic immunoreactivity for HuR protein in 52% (233 of 445) of serous-type ovarian carcinoma specimens, and it associated with high COX-2 expression (P = 0.0045) and with clinicopathological variables, including poor prognosis (P < 0.0001) and high grade (P < 0.0001). In ovarian cancer cells in vitro, a small interfering RNA against HuR and leptomycin B, an inhibitor of nucleocytoplasmic translocation of HuR, inhibited COX-2 expression. Our results show that cytoplasmic HuR expression associates with poor outcome in ovarian cancer, and one plausible explanation for this finding may be related to the ability of HuR to induce COX-2 expression.     

Cytoplasmic expression of the ELAV-like protein HuR as a potential prognostic marker in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most frequent cancers and a leading cause of death from cancer in China. The human ELAV-like protein HuR has been found to contribute to cancer development and progression through stabilizing a group of cellular mRNAs of cancer-related genes. In this study, we investigated the expression of HuR in a cohort of ESCC patients using immunohistochemical staining. HuR detected in the cytoplasm of cancer cells was positive in 46.6 % of 58 ESCC specimens; 75.9 % of these specimens had nuclear immunoreactivity for HuR. Cytoplasmic HuR expression was higher in cancer tissues compared to 20 matched adjacent noncancerous tissues. A clinicopathological study showed that cytoplasmic HuR expression was positively associated with lymph node metastasis, depth of tumor invasion, and advanced stage, whereas nuclear HuR expression was not correlated with any clinicopathological factors. Patients positive for cytoplasmic HuR expression had a cumulative 5-year survival rate of 25.3 %, whereas it was 43.8 % for patients negative for cytoplasmic HuR expression. In a multivariate analysis, cytoplasmic HuR expression was an independent prognostic factor, whereas nuclear positivity for HuR was not. Our results indicate that high cytoplasmic HuR expression is associated with positive lymph node metastasis, deep tumor invasion, high stage, and poor survival in ESCC. Thus, HuR is the first mRNA stability protein whose expression is associated with poor survival in esophageal cancer.     

Role of the RNA-binding protein HuR in colon carcinogenesis

Immunohistochemical analysis of paired tumor and normal tissue specimens revealed that the expression and cytoplasmic abundance of the RNA-binding protein HuR increased with malignancy, particularly in colon carcinomas. Interventions to modulate HuR expression in human RKO colon cancer cells altered gene expression profiles and identified beta-catenin mRNA as a novel HuR target. Subcutaneous injection of HuR-overexpressing RKO cells into nude mice produced significantly larger tumors than those arising from control populations; conversely, RKO cells expressing reduced HuR through small interference RNA- or antisense HuR-based approaches developed significantly more slowly. We propose that HuR-regulated target mRNA expression contributes to colon cancer growth. Our results suggest a pivotal function for HuR in colon carcinogenesis.     

HuR contributes to cyclin E1 deregulation in MCF-7 breast cancer cells

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.     

Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast carcinoma.

PURPOSE: The human ELAV (embryonic lethal abnormal vision)-like protein HuR stabilizes a certain group of cellular mRNAs that contain AU-rich elements in their 3'-untranslated region. Cell culture studies have shown that the mRNA of cyclooxygenase (COX)-2 can be stabilized by HuR. EXPERIMENTAL DESIGN: To investigate a possible contribution of dysregulation of mRNA stability to the progression of cancer and to overexpression of COX-2, we studied expression of HuR in 208 primary breast carcinomas by immunohistochemistry. RESULTS: There were two different staining patterns of HuR in tumor tissue of breast carcinomas: nuclear expression was seen in 61% of cases; and an additional cytoplasmic expression was seen in 30% of cases. Expression of HuR was significantly associated with increased COX-2 expression; this association was particularly significant for cytoplasmic HuR expression (P < 0.0005). We further observed a significant association of cytoplasmic (P = 0.002) or nuclear HuR (P = 0.027) expression with increased tumor grade. Only 13% of the grade 1 carcinomas showed cytoplasmic expression of HuR, compared with 46% of the grade 3 carcinomas. There was no significant correlation between HuR expression and other clinicopathological parameters such as histological type, tumor size, or nodal status as well as patient survival. CONCLUSIONS: Our results suggest that overexpression of HuR in tumor tissue may be part of a regulatory pathway that controls the mRNA stability of several important targets in tumor biology, such as COX-2. Based on our results, additional studies are necessary to investigate whether HuR might be a potential target for molecular tumor therapy.     

Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression

The human embryonic-lethal abnormal vision-like protein HuR is involved in the regulation of mRNA turnover and serves as a shuttling protein between the nucleus and the cytoplasm that stabilizes mRNAs containing adenine- and uridine-rich elements in their 3' untranslated region. We have shown recently that expression of cyclooxygenase (COX)-2 is related to poor prognosis in ovarian carcinoma. Other studies have shown that the COX-2 mRNA contains an adenine- and uridine-rich element and is stabilized by HuR. In this study, we investigated the expression and cellular distribution of HuR in 83 primary ovarian carcinomas, 16 borderline tumors of the ovary, 3 normal ovaries, and 9 ovarian carcinoma cell lines. Expression of HuR was detected in all cell lines on the mRNA and protein level and showed a predominantly nuclear staining in OVCAR-3 cells by confocal microscopy. In an immunohistochemical evaluation of human ovarian carcinomas, HuR showed a nuclear expression in 81% of tumors. In addition, a cytoplasmic expression of HuR was observed in a subgroup of 45% of ovarian carcinomas. Nuclear as well as cytoplasmic expression of HuR was significantly increased in ovarian carcinomas compared with borderline tumors or normal ovaries. In univariate analysis, a significant association between cytoplasmic HuR expression and increased COX-2 expression (P = 0.025) as well as between histological grade (P = 0.008) and mitotic activity (P = 0.002) was observed, although nuclear expression of HuR was not correlated with COX-2 expression or other clinicopathological parameters. In Kaplan-Meier survival analysis, increased cytoplasmic expression of HuR was a significant prognostic indicator for progression-free survival (P = 0.03) as well as overall survival (P = 0.007). In multivariate analysis using the Cox regression model, cytoplasmic expression of HuR was an independent prognostic parameter for reduced overall survival with a relative risk of 2.62 (95% confidence interval, 1.32-5.19). Our results suggest that there is a dysregulation of cellular distribution of the mRNA stability factor HuR in a subset of invasive ovarian carcinomas. This dysregulation appears to result in an increased expression of COX-2, an increased proliferative rate, and may lead to a reduced survival time. Additional studies are required to analyze the downstream effects of increased cytoplasmic expression of HuR. In addition, it would be interesting to investigate the prognostic role of increased cytoplasmic expression of HuR in prospective studies.     

Expression of the ELAV-like protein HuR in human prostate carcinoma is an indicator of disease relapse and linked to COX-2 expression

The human ELAV-like protein HuR is involved in the stabilization of the mRNAs of a group of genes implicated in the regulation of cellular growth, angiogenesis and rapid inflammatory response. HuR is a nuclear shuttling protein, translocating bound mRNAs from the nucleus to the cytoplasm. We have previously observed an increased expression of cyclooxygenase-2 (COX-2) in prostate cancer while cell culture studies have shown that HuR stabilizes the mRNA of COX-2. Based on these mechanistic data, we aimed to investigate the role of HuR in prostate cancer by a tissue-based analysis combined with functional evaluation using a cell culture approach. Investigating 104 primary prostate carcinomas by immunohistochemistry, we found HuR expression to be shifted from a nuclear staining in normal prostate glands to a cytoplasmic staining in carcinoma tissue (p<0.0001). Cytoplasmic HuR expression was significantly correlated with COX-2 expression (p=0.005). Loss of nuclear HuR expression was an indicator of earlier PSA-relapse both in univariate (p=0.04) and multivariate survival analysis (p=0.04). HuR inhibition by Leptomycin B reduced the inducibility of COX-2 in PC-3 prostate cancer cells. We found that the subcellular localization of HuR is deregulated in a subset of prostate carcinomas, and that this deregulation is linked to an altered expression of the tumorigenic COX-2 protein as well as to an adverse patient prognosis. Our results point to a potential prognostic role of HuR expression in prostate cancer diagnostics and propose HuR as a future therapeutic target in prostate cancer therapy.