Abstract C77: Tristetraprolin suppression is associated with advanced stage colorectal cancer
Esther A. SuswamBalanada Dhurjarti Kumar PutchaKiera WalkerLaJessica JohnsonJ. Harrison HowardEdward E. PartridgeMona N. FouadSejong BaeUpender Manne
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Abstract Background: Interleukin (IL)-8, vascular endothelial growth factor (VEGF), and IL-6 contribute to the colorectal cancer (CRC) progression by inhibiting apoptosis and by promoting angiogenesis and tumor proliferation. We have found that the tristetraprolin (TTP) gene attenuates these processes [J Neurooncol. 2013; 113(2):195-205]. TTP expression is lost or reduced in many cancers, including CRCs, and loss of TTP is thought to contribute to tumorigenesis. We hypothesized that low TTP levels favor expression of growth factors and correlate with CRC progression. In addition, we suggest that TTP modulates CRC growth through negative regulation on cell survival and/or anti-apoptotic factors in the NF-kB pathway. We tested this hypothesis by analyzing mRNA expression of TTP and its targets in primary CRCs of African American (AA) and Caucasian American (CA) patients. Methods: We analyzed frozen primary tissues from 45 CRC patients (AA=26 and CA=19), each with corresponding normal (benign/control) tissue. cDNAs were reverse-transcribed from total RNA; mRNA levels of TTP and its target genes (IL-8, VEGF, IL-6) were quantified by the qPCR sybr-green method. Expression levels were normalized to GAPDH. To assess TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116, SW480, and LoVo) were stimulated with TNF-α for 0-24 hr, and total RNA was analyzed for TTP, IL-8, IL-6, VEGF, and cIAP2 expression by qRT-PCR. Levels of HuR mRNA in cells were also assessed. Extracts from the cells were immunoblotted with anti-TTP and antiHuR antibodies. Results: We observed down-regulated expression of TTP mRNA in primary CRCs (31 of 45), and decreased TTP levels correlated with advanced tumor stage. Low levels of TTP were found in 21 of 26 AAs and 12 of 19 CAs. In both racial groups, there was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. Studies with cultured colon cancer cells demonstrated that TTP mRNA levels inversely correlated with levels of IL-8, IL-6, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with cell survival factors. Western blot analyses confirmed TTP expression levels in these cells. Conclusions: For both racial groups, TTP expression was lower in tumor tissues relative to normal tissues; the difference was more pronouced in CRCs of AAs. Further, lower TTP levels correlated with advanced tumor stage; and TTP negatively regulated the expression of IL-8, VEGF, and cIAP2 in cultured cells. These studies were supported by a pre-pilot project of the UAB/TU/MSM Partnership grant of NIH/NCI, U54-CA 118948. Citation Format: Esther A. Suswam, Balanada Dhurjarti Kumar Putcha, Kiera D. Walker, LaJessica Johnson, Jasmine Howard, Edward E. Partridge, Mona N. Fouad, Sejong Bae, Upender Manne. Tristetraprolin suppression is associated with advanced stage colorectal cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C77. doi:10.1158/1538-7755.DISP13-C77Keywords:
Tristetraprolin
To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy.Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes (including Beclin1, LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor-α (TNF-α) on the expressions of nuclear factor-κB (NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting.The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells (P < 0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ at the mRNA and protein levels (P < 0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel (P < 0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression (P > 0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-Ⅱ/LC3-Ⅰ ratio at both the mRNA and protein levels (P < 0.05).The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.
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Native nano ESI-mass spectrometry can be applied to track the formation of “Gold Fingers” formed when AuIII-terpy reacts with the non-classical zinc finger protein tristetraprolin. This powerful MS technique can also be utilized to probe reactivity of RNA bound zinc fingers, and it is reported that RNA has a protective effect towards Gold Finger reactivity with tristetraprolin. For more details, see the Full Paper by S. L. J. Michel and co-workers on page 1535 ff.
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Copyright information: Taken from Regulation and localization of endogenous human tristetraprolinArthritis Research & Therapy 2003;5(4):R214-R225.Published online 15 May 2003PMCID:PMC165067.Copyright © 2003 Fairhurst et al., licensee BioMed Central Ltd The cells were incubated with LPS or TNF in the absence or presence of blocking TNF antibody (infliximab [Remicade]), and TTP levels were measured at specific times. Results are expressed as percentage change from baseline expression (mean ± SE). Experiments were performed in duplicate.
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Tristetraprolin is a zinc‐finger‐containing RNA‐binding protein. Tristetraprolin binds to AU‐rich elements of target mRNAs such as proto‐oncogenes, cytokines and growth factors, and then induces mRNA rapid degradation. It was observed as an immediate‐early gene that was induced in response to several kinds of stimulus, such as insulin and other growth factors and stimulators of innate immunity such as lipopolysaccharides. We observed that tristetraprolin was briefly expressed during a 1–8 h period after induction of differentiation in 3T3‐L1 preadipocytes. Detailed analysis showed that tristetraprolin mRNA expression was stimulated by fetal bovine serum and differentiation inducers, and was followed by rapid degradation. The 3′UTR of tristetraprolin mRNAs contain adenine‐ and uridine‐rich elements. Biochemical analyses using RNA pull‐down, RNA immunoprecipitation and gel shift experiments demonstrated that adenine‐ and uridine‐rich element‐binding proteins, HuR and tristetraprolin itself, were associated with tristetraprolin adenine‐ and uridine‐rich elements. Functional characterization confirmed that tristetraprolin negatively regulated its own expression. Thus, our results indicated that the tight autoregulation of tristetraprolin expression correlated with its critical functional role in 3T3‐L1 differentiation.
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The expression of human inducible NO synthase (iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogen-activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominant-negative p38 MAPKα protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, quantitative real-time polymerase chain reaction, and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580 incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP), which is known to recruit mRNAs containing AU-rich elements to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580 treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Because tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.
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Inflammatory diseases place a heavy burden on the American health care system. Tristetraprolin, a zinc-dependent mRNA binding protein decreases the stability of mRNAS coding for some proinflammatory cytokines. Tristetraprolin-deficient mice develop a profound inflammatory syndrome. Tristetraprolin is a potential cancer therapy due to its control of vascular endothelial growth factor mRNA stability. Cinnamon extract stimulates the expression of antiinflammatory tristetraprolin. Bioactive compound(s) in cinnamon extract define its molecular mechanisms. Cinnamon is potentially important in tristetraprolin-mediated inflammatory diseases.
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An excess of interleukin 17 (IL‐17) may contribute to chronic inflammatory disorders, but mechanisms that regulate IL‐17 in immune cells are unclear. Here we report that tristetraprolin (TTP) inhibits IL‐17 production in human T cell lines. Overexpression of TTP decreased the expression of IL‐17. Conversely, TTP inhibition by siRNA increased IL‐17 production. IL‐17 mRNA contains eight AREs within its 3′UTR. TTP bound directly to the IL‐17 mRNA 3′UTR at a location between the fourth and seventh AREs and enhanced decay of IL‐17 transcripts. These results suggest that TTP could control IL‐17‐mediated inflammation.
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AU-rich element-binding proteins (ARE-BP) regulate the stability and/or translational efficiency of mRNAs containing cognate binding sites. Many targeted transcripts encode factors that control processes such as cell division, apoptosis, and angiogenesis, suggesting that dysregulated ARE-BP expression could dramatically influence oncogenic phenotypes. Using several approaches, we evaluated the expression of four well-characterized ARE-BPs across a variety of human neoplastic syndromes. AUF1, TIA-1, and HuR mRNAs were not systematically dysregulated in cancers; however, tristetraprolin mRNA levels were significantly decreased across many tumor types, including advanced cancers of the breast and prostate. Restoring tristetraprolin expression in an aggressive tumor cell line suppressed three key tumorgenic phenotypes: cell proliferation, resistance to proapoptotic stimuli, and expression of vascular endothelial growth factor mRNA. However, the cellular consequences of tristetraprolin expression varied across different cell models. Analyses of gene array data sets revealed that suppression of tristetraprolin expression is a negative prognostic indicator in breast cancer, because patients with low tumor tristetraprolin mRNA levels were more likely to present increased pathologic tumor grade, vascular endothelial growth factor expression, and mortality from recurrent disease. Collectively, these data establish that tristetraprolin expression is frequently suppressed in human cancers, which in turn can alter tumorigenic phenotypes that influence patient outcomes.
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Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.
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Genetic loss or mutations in tumor suppressor genes promote tumorigenesis. The prospective tumor suppressor tristetraprolin (TTP) has been shown to negatively regulate tumorigenesis through destabilizing the messenger RNAs of critical genes implicated in both tumor onset and tumor progression. Regulation of TTP has therefore emerged as an important issue in tumorigenesis. Similar to other tumor suppressors, TTP expression is frequently downregualted in various human cancers, and its low expression is correlated with poor prognosis. Additionally, disruption in the regulation of TTP by various mechanisms results in the inactivation of TTP protein or altered TTP expression. A recent study showing alleviation of Myc-driven lymphomagenesis by the forced expression of TTP has shed light on new therapeutic avenues for cancer prevention and treatment through the restoration of TTP expression. In this review, we summarize key oncogenes subjected to the TTP-mediated mRNA degradation, and discuss how dysregulation of TTP can contribute to tumorigenesis. In addition, the control mechanism underlying TTP expression at the posttranscriptional and posttranslational levels will be discussed.
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