Global analysis of the abundance of AU-rich mRNAs in response to glucocorticoid treatment.

Glucocorticoids (GC) like dexamethasone (Dex) are potent anti-inflammatory agents with diverse cellular functions including the potentiation of the activity of AU-rich elements (AREs). AREs are cis-acting instability sequence elements located in the 3'UTRs of many inflammatory mediator mRNAs. Here, available RNA-seq data were used to investigate the effect of GCs on the ARE-mRNA-transcriptome. At a global scale, ARE-mRNAs had a tendency to be downregulated after GC-treatment of the A549 lung cancer cell-line, but with notable cases of upregulation. mRNA stability experiments indicated that not only the downregulated, but also the upregulated ARE-mRNAs are destabilized by Dex-treatment. Several of the most upregulated ARE-mRNAs code for anti-inflammatory mediators including the established GC targets DUSP1 and ZFP36; both code for proteins that target ARE-containing mRNAs for destruction. GCs are widely used in the treatment of COVID-19 patients; we show that ARE-mRNAs are more likely to regulate in opposite directions between Dex-treatment and SARS-CoV-2 infections compared to non-ARE mRNAs. The effect of GC treatment on ARE-mRNA abundance was also investigated in blood monocytes of COVID-19 patients. The results were heterogeneous; however, in agreement with in vitro observations, ZFP36 and DUSP1 were often amongst the most differentially expressed mRNAs. The results of this study propose a universal destabilization of ARE-mRNAs by GCs, but a diverse overall outcome in vitro likely due to induced transcription or due to the heterogeneity of COVID-19 patient's responses in vivo.

Comprehensive Transcriptome and Pathway Analyses Revealed Central Role for Fascin in Promoting Triple-Negative Breast Cancer Progression.

Recent years have witnessed major progress in development of novel therapeutic agents such as chemotherapy, targeted therapy and immune checkpoint inhibitors for breast cancer. However, cancer-related death remains high especially in triple-negative breast cancer (TNBC) due limited therapeutic options. Development of targeted therapies for TNBC requires better understanding of biology and signaling networks that promote disease progression. Fascin, an actin bundling protein, was identified as a key regulator of many signaling pathways that contribute to breast cancer progression. Herein, fascin ShRNA was used to generate stable fascin knockdown (FSCN1KD) in the MDA-MB-231 TNBC cell line and then were subjected to comprehensive mRNA and miRNA transcriptome analysis. We identified 129 upregulated and 114 downregulated mRNA transcripts, while 14 miRNAs were differentially expressed in FSCN1KD. Ingenuity pathway analysis (IPA) was used to predict the impact of differentially expressed transcripts on signaling pathways and functional categories and to construct miRNA-mRNA regulatory networks in the context of FSCN1 knockdown. Compared to FSCN1KD, fascin-positive (FSCN1CON) breast cancer cells showed enrichment in genes promoting cellular proliferation, migration, survival, DNA replication and repair. Expression of FSCN1high (identified in BRCA dataset from TCGA) in conjunction with elevated expression of the top 10 upregulated or decreased expression of the top 10 downregulated genes (identified in our FSCN1CON vs. FSCN1KD) correlates with worst survival outcome. Taken together, these data confirmed fascin's role in promoting TNBC progression, and identified a novel opportunity for therapeutic interventions via targeting those FSCN1-related transcripts.

Eugenol modulates genomic methylation and inactivates breast cancer-associated fibroblasts through E2F1-dependent downregulation of DNMT1/DNMT3A.

Active cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment, which promote carcinogenesis and modulate response to therapy. Therefore, targeting these cells or reducing their paracrine pro-carcinogenic effects could be of great therapeutic value. To this end, we sought to investigate the effect of eugenol, a natural phenolic molecule, on active breast CAFs. We have shown that decitabine (5-Aza-2'-deoxycytidine, DAC) and eugenol inhibit the expression of the DNA methyltransferase genes DNMT1 and DNMT3A at both the protein and mRNA levels in breast CAF cells. While the effect of eugenol was persistent, DAC had only a transient inhibitory effect on the mRNA level of both DNMT genes. Furthermore, eugenol and DAC suppressed the invasive/migratory and proliferative potential of CAF cells as well as their paracrine pro-carcinogenic effects both in vitro and in humanized orthotopic tumor xenografts. Interestingly, these inhibitory effects of decitabine and eugenol were mediated through E2F1 downregulation. Indeed, ectopic expression of E2F1 upregulated both genes and attenuated the effects of eugenol. Additionally, we provide clear evidence that eugenol, like DAC, strongly modulates the methylation pattern in active CAF cells, through methylating several oncogenes and demethylating various important tumor suppressor genes, which affected their mRNA expression levels. Importantly, the E2F1 promoter was also hypermethylated and the gene downregulated in response to eugenol. Together, these findings show that the active features of breast CAF cells can be normalized through eugenol-dependent targeting of DNMT1/DNMT3A and the consequent modulation in gene methylation.

Fascin Activates ß-Catenin Signaling and Promotes Breast Cancer Stem Cell Function Mainly Through Focal Adhesion Kinase (FAK): Relation With Disease Progression.

Cancer stem cells (CSCs), a rare population of tumor cells with high self-renewability potential, have gained increasing attention due to their contribution to chemoresistance and metastasis. We have previously demonstrated a critical role for the actin-bundling protein (fascin) in mediating breast cancer chemoresistance through activation of focal adhesion kinase (FAK). The latter is known to trigger the ß-catenin signaling pathway. Whether fascin activation of FAK would ultimately trigger ß-catenin signaling pathway has not been elucidated. Here, we assessed the effect of fascin manipulation in breast cancer cells on triggering ß-catenin downstream targets and its dependence on FAK. Gain and loss of fascin expression showed its direct effect on the constitutive expression of ß-catenin downstream targets and enhancement of self-renewability. In addition, fascin was essential for glycogen synthase kinase 3ß inhibitor-mediated inducible expression and function of the ß-catenin downstream targets. Importantly, fascin-mediated constitutive and inducible expression of ß-catenin downstream targets, as well as its subsequent effect on CSC function, was at least partially FAK dependent. To assess the clinical relevance of the in vitro findings, we evaluated the consequence of fascin, FAK, and ß-catenin downstream target coexpression on the outcome of breast cancer patient survival. Patients with coexpression of fascinhigh and FAKhigh or high ß-catenin downstream targets showed the worst survival outcome, whereas in fascinlow, patient coexpression of FAKhigh or high ß-catenin targets had less significant effect on the survival. Altogether, our data demonstrated the critical role of fascin-mediated ß-catenin activation and its dependence on intact FAK signaling to promote breast CSC function. These findings suggest that targeting of fascin-FAK-ß-catenin axis may provide a novel therapeutic approach for eradication of breast cancer from the root.

The AU-rich element landscape across human transcriptome reveals a large proportion in introns and regulation by ELAVL1/HuR.

Adenylate-uridylate (AU)-rich elements (AREs) are sequence instability elements that are known to be located in the 3' untranslated regions (UTR) in thousands of human transcripts. AREs regulate the expression of many genes at the post-transcriptional level, and they are essential for many normal cellular functions. We conducted a transcriptome-wide screen for AREs and found that they are most abundant in introns, with up to 25% of introns containing AREs corresponding to 58% of human genes. Clustering studies of ARE size, complexity, and distribution revealed that, in introns, longer AREs with two or more overlapping repeats are more abundant than in the 3'UTR, and only introns can contain very long AREs with 6-14 overlapping AUUUA pentamers. We found that intronic sites of the ARE binding proteins HuR/ELAVL1, ZFP36/TTP, AUF1, and BRF1/ZFP36L1 overlap with the intronic AREs with HuR being most abundant. Accordingly, RNA-IP experiments demonstrated a specific association of HuR with reporter and endogenous pre-mRNAs that contain intronic AREs. Moreover, HuR knockdown led to a significant general reduction in the mRNA levels of genes that contain intronic AREs and to a specific reduction in the expression of ARE-intronic reporters. The data represent bioinformatics analysis for key RNA-binding proteins interactions with intronic AREs and provide experimental evidence for HuR binding to AREs. The widespread distribution of intronic AREs and their particular association with HuR and HuR binding sites indicates that more than half of human genes can be regulated post-transcriptionally by AREs.

Systematic Analysis of AU-Rich Element Expression in Cancer Reveals Common Functional Clusters Regulated by Key RNA-Binding Proteins.

Defects in AU-rich elements (ARE)-mediated posttranscriptional control can lead to several abnormal processes that underlie carcinogenesis. Here, we performed a systematic analysis of ARE-mRNA expression across multiple cancer types. First, the ARE database (ARED) was intersected with The Cancer Genome Atlas databases and others. A large set of ARE-mRNAs was over-represented in cancer and, unlike non-ARE-mRNAs, correlated with the reversed balance in the expression of the RNA-binding proteins tristetraprolin (TTP, ZFP36) and HuR (ELAVL1). Serial statistical and functional enrichment clustering identified a cluster of 11 overexpressed ARE-mRNAs (CDC6, KIF11, PRC1, NEK2, NCAPG, CENPA, NUF2, KIF18A, CENPE, PBK, TOP2A) that negatively correlated with TTP/HuR mRNA ratios and was involved in the mitotic cell cycle. This cluster was upregulated in a number of solid cancers. Experimentally, we demonstrated that the ARE-mRNA cluster is upregulated in a number of tumor breast cell lines when compared with noninvasive and normal-like breast cancer cells. RNA-IP demonstrated the association of the ARE-mRNAs with TTP and HuR. Experimental modulation of TTP or HuR expression led to changes in the mitosis ARE-mRNAs. Posttranscriptional reporter assays confirmed the functionality of AREs. Moreover, TTP augmented mitotic cell-cycle arrest as demonstrated by flow cytometry and histone H3 phosphorylation. We found that poor breast cancer patient survival was significantly associated with low TTP/HuR mRNA ratios and correlated with high levels of the mitotic ARE-mRNA signature. These results significantly broaden the role of AREs and their binding proteins in cancer, and demonstrate that TTP induces an antimitotic pathway that is diminished in cancer. Cancer Res; 76(14); 4068-80. ©2016 AACR.

AU-rich transient response transcripts in the human genome: expressed sequence tag clustering and gene discovery approach.

Transient response genes regulate critical biological responses that include cell proliferation, signal transduction events, and responses to exogenous agents such as inflammatory stimuli, microbes, and radiation. An important feature that ensures a timely response is the short half-life of the messenger RNA (mRNA), which is thought to be predominantly mediated by adenylate uridylate-rich sequence elements (AREs) in the 3' untranslated region (3' UTR). The repertoire and extent of transient response genes in the human genome are not known. We used a computational approach to delineate those genes that code for transient ARE mRNAs. We utilized a 3' UTR-specific ARE motif to retrieve and cluster 3'-end ESTs using a refined extraction protocol. With the availability of the entire human genome, we were able to utilize ARE EST clusters for further mining and computational prediction of ARE genes. The described approaches led to the finding of more than 1500 ARE genes in the human genome. In particular, "hidden" ARE mRNAs and alternative forms due to 3'UTR completeness, variant polyadenylation, and splicing were uncovered.

Selection of AU-rich transiently expressed sequences: reversal of cDNA abundance.

Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3' untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, beta-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes.

Quick gene expression profiling of promyelocytic cell line HL-60.

OBJECTIVE: A number of techniques have been developed to perform gene expression profiling. We report preliminary results from our exploratory study, using sequential analysis of gene expression (SAGE) technique, to profile the undifferentiated and differentiated HL-60 cells in line with our interest to characterize the cancer phenotype. The aim of the study is to evaluate the technique and to understand the molecular bases of these 2 states of cells. METHODS: HL-60 cells were differentiated after treatment with dimethyl sulfoxide. Tag libraries were prepared from the messenger RNAs of the undifferentiated and differentiated cells according to the SAGE protocol. The search for genes corresponding to the tags was carried out using SAGE software. The tags and the genes from the 2 libraries were compared for their levels of expression. The study was carried out at the King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia during the year 2001. RESULTS: A comparison of tags from the 2 libraries revealed that 151 tags corresponding to 57 genes expressed differentially: 60 tags were elevated and 59 were repressed in the undifferentiated cells. Thirty-two tags were equally expressed in both types of cells. Of the corresponding genes, 25 were expressed at higher, 17 at lower, while 15 were expressed at comparable levels in both cell types. In the profile of undifferentiated cells, the genes involved in mitochondrial function and protein synthesis were prominent, while in the differentiated cells, the genes coding for proteins associated with cell membranes, signal transduction and for cell specific functions were prominent. The genes, expressed equally in both the cell types, were concerned with the maintenance of the living state. CONCLUSION: Sequential analysis of gene expression is a useful technique for gene expression profiling. As previously indicated by others, a dedicated team can generate useful data within reasonable time limits.

p38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts.

Adenylate/uridylate-rich element (ARE)-mediated mRNA turnover is an important regulatory component of gene expression for innate and specific immunity, in the hematopoietic system, in cellular growth regulation, and for many other cellular processes. This diversity is reflected in the distribution of AREs in the human genome, which we have established as a database of more than 900 ARE-containing genes that may utilize AREs as a means of controlling cellular mRNA levels. The p38 mitogen-activated protein kinase (MAP kinase) pathway has been implicated in regulating the stability of nine ARE-containing transcripts. Here we explored the entire spectrum of ARE-containing genes for p38-dependent regulation of ARE-mediated mRNA turnover with a custom cDNA array containing probes for 950 ARE mRNAs. The human monocytic cell line THP-1 treated with lipopolysaccharide (LPS) was used as a reproducible cellular model system that allowed us to precisely control the conditions of mRNA induction and decay in the absence and presence of the p38 inhibitor SB203580. This approach allowed us to establish an LPS-induced ARE mRNA expression profile in human monocytes and determine the half-lives of 470 AU-rich mRNAs. Most importantly, we identified 42 AU-rich genes, previously unrecognized, that show p38-dependent mRNA stabilization. In addition to a number of cytokines, several interesting novel AU-rich transcripts likely to play a role in macrophage activation by LPS exhibited p38-dependent transcript stabilization, including macrophage-specific colony-stimulating factor 1, carbonic anhydrase 2, Bcl2, Bcl2-like 2, and nuclear factor erythroid 2-like 2. Finally, the identification of the p38-dependent upstream activator MAP kinase kinase 6 as a member of this group identifies a positive feedback loop regulating macrophage signaling via p38 MAP kinase-dependent transcript stabilization.