Hydroxychloroquine attenuates double-stranded RNA-stimulated hyper-phosphorylation of tristetraprolin/ZFP36 and AU-rich mRNA stabilization.

The human innate immune system recognizes dsRNA as a pathogen-associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK-MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay-promoting protein that controls the stability of AU-rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti-malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID-19 disease. We found that HCQ reduced the dsRNA-dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)-induced cytokines and inflammatory mRNAs like TNF, IL-6, COX-2, and IL-8 in THP-1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti-inflammatory role of HCQ at post-transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

Human Cytokinome Analysis for Interferon Response.

UNLABELLED: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-ß and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-ß response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities. IMPORTANCE: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control.

Assessment of post-transcriptional control relies on use of transcriptional inhibitors and is masked by copious and cryptic transcriptional induction. We screened several cellular promoters that are constitutively active yet noninducible to external stimuli. The ribosomal protein RPS30 promoter was chosen; its TATA signal and sp1 site location were optimized. The modified promoter (RPS30M) is selective to post-transcriptional effects of AU-rich elements (ARE) in the 3'UTR, while it is not transcriptionally responsive to a wide variety of agents including pro-inflammatory cytokines and RNA-binding proteins. Specific cis-acting elements can be appended to RPS30M by a cloning-free approach to allow coupled transcriptional/post-transcriptional assessment, as demonstrated with NF-kappaB and beta-catenin/wnt signaling experiments. Moreover, efficient tetracycline-regulated RPS30M was created for quantitative assessment of the half-lives of mRNAs containing AREs. The described approach provides enhanced versatility and suitability for selective post-transcriptional assessment with or without transcriptional induction.

Kinome inhibition reveals a role for polo-like kinase 1 in targeting post-transcriptional control in cancer.

Dysfunctions in post-transcriptional control are observed in cancer and chronic inflammatory diseases. Here, we employed a kinome inhibitor library (n = 378) in a reporter system selective for 3'-untranslated region-AU-rich elements (ARE). Fifteen inhibitors reduced the ARE-reporter activity; among the targets is the polo-like kinase 1 (PLK1). RNA-seq experiments demonstrated that the PLK1 inhibitor, volasertib, reduces the expression of cytokine and cell growth ARE mRNAs. PLK1 inhibition caused accelerated mRNA decay in cancer cells and was associated with reduced phosphorylation and stability of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). Ectopic expression of PLK1 increased abundance and stability of high molecular weight of ZFP36/TTP likely of the phosphorylated form. PLK1 effect was associated with the MAPK-MK2 pathway, a major regulator of ARE-mRNA stability, as evident from MK2 inhibition, in vitro phosphorylation, and knockout experiments. Mutational analysis demonstrates that TTP serine 186 is a target for PLK1 effect. Treatment of mice with the PLK1 inhibitor reduced both ZFP36/TTP phosphorylation in xenograft tumor tissues, and the tumor size. In cancer patients' tissues, PLK1/ARE-regulated gene cluster was overexpressed in solid tumors and associated with poor survival. The data showed that PLK1-mediated post-transcriptional aberration could be a therapeutic target.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Promyelocytic leukemia zinc finger triggers ATP-binding cassette subfamily E member 1-mediated growth inhibition in breast cancer cells.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor that is involved in a number of biological processes, including those regulating cellular growth; however, little is known regarding how it achieves its inhibitory effect in different cell and tissue types. It has previously been demonstrated that PLZF expression levels become diminished during the oncogenic transformation of certain tissue types and thus, may serve as a hallmark for tumor aggressiveness. To examine this in breast cancer, survival curves from available oncology databases were analyzed and demonstrated that PLZF expression was positively associated with increased survival in patients with breast cancer. The mRNA and protein levels of PLZF were also revealed to be associated with the tumorigenicity of four breast cancer cell lines. Since ATP-binding cassette subfamily E member 1 (ABCE1), also known as RNase L inhibitor, has been determined to be a target gene of PLZF, the present study also investigated whether the tumor suppressive effect of PLZF was associated with ABCE1 expression. PLZF was revealed to downregulate the expression of ABCE1 in vitro, which relieved the inhibitory effect of ABCE1 on the ribonuclease L enzyme. Finally, it was concluded that PLZF expression caused an ABCE1-mediated increase in cellular cytotoxicity, as demonstrated by a reduction in the proliferation rate of breast cancer cell lines. The results of the present study are important for understanding how PLZF exerts its final inhibitory actions in breast cancer cells, and potentially in other solid tumors, through the modulation of immunological pathways.

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.