Christoph Moroni

Rapamycin passes the torch: a new generation of mTOR inhibitors

Mammalian target of rapamycin (mTOR) is an atypical protein kinase that controls growth and metabolism in response to nutrients, growth factors and cellular energy levels, and it is frequently dysregulated in cancer and metabolic disorders. Rapamycin is an allosteric inhibitor of mTOR, and was approved as an immuno-suppressant in 1999. In recent years, interest has focused on its potential as an anticancer drug. However, the performance of rapamycin and its analogues (rapalogues) has been undistinguished despite isolated successes in subsets of cancer, suggesting that the full therapeutic potential of targeting mTOR has yet to be exploited. A new generation of ATP-competitive inhibitors that directly target the mTOR catalytic site display potent and comprehensive mTOR inhibition and are in early clinical trials.

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

To identify regulators of AU‐rich element (ARE)‐dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin‐3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor‐1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame‐shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3‐kinase, while mutation of the zinc fingers abolished both function and ARE‐binding activity. This approach, which identified BRF1 as an essential regulator of ARE‐dependent mRNA decay, should also be applicable to other cis‐elements of mRNA turnover.

Parallel and independent regulation of interleukin-3 mRNA turnover by phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase.

ABSTRACT AU-rich elements (ARE) present in the 3′ untranslated regions of many cytokines and immediate-early genes are responsible for targeting the transcripts for rapid decay. We present evidence from cotransfection experiments in NIH 3T3 cells that two signaling pathways, one involving phosphatidylinositol 3-kinase (PI3-K), and one involving the p38 mitogen-activated protein kinase (MAPK), lead to stabilization of interleukin-3 mRNA in parallel. Stabilization mediated by either of the two pathways was antagonized by tristetraprolin (TTP), an AU-binding protein known to promote constitutive decay of ARE-containing transcripts. Remarkably, the stabilizing AU-binding protein HuR, in collaboration with p38 MAPK but not with PI3-K, could overcome the destabilizing effect of TTP. These data argue that the stabilizing kinases PI3-K and p38 MAPK do not act through direct inactivation of TTP but via activating pathway-specific stabilizing AU-binding proteins. Our data suggest an integrated model of mRNA turnover control, where stabilizing (HuR) and destabilizing (TTP) AU-binding proteins compete and where the former are under the positive control of independent phosphokinase signaling pathways.

c-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells.

Whereas signalling pathways involved in transcriptional control have been studied extensively, the pathways regulating mRNA turnover remain poorly understood. We are interested in the role of mRNA stability in cell activation and oncogenesis using PB‐3c mast cells as a model system. In these cells the short‐lived interleukin‐3 (IL‐3) mRNA is stabilized by ionomycin treatment and following oncogenesis. To identify the signalling pathways involved in these mechanisms, we analysed the effect of different kinase inhibitors. SB202190 and wortmannin were shown to antagonize ionomycin‐induced IL‐3 mRNA stabilization in PB‐3c cells in the presence of actinomycin D, and this effect coincided with their ability to inhibit c‐jun N‐terminal kinase (JNK) activation by ionomycin. Moreover, transfection of activated MEKK1 amplified ionomycin‐induced IL‐3 mRNA expression at the post‐transcriptional level, and a dominant‐negative mutant of JNK counteracted mRNA stabilization by ionomycin. Taken together, these data indicate that JNK is involved in the regulation of IL‐3 mRNA turnover in mast cells. In addition, transfection experiments revealed that the cis‐acting AU‐rich element in the 3′ untranslated region of IL‐3 mRNA is necessary and sufficient to confer JNK‐dependent mRNA stabilization in response to cell activation.

Somatic mRNA Turnover Mutants Implicate Tristetraprolin in the Interleukin-3 mRNA Degradation Pathway

ABSTRACT Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and proto-oncogenes. Regulation involves an AU-rich element (ARE) in the 3′ untranslated region (3′UTR) and cognatetrans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked to the IL-3 3′UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.

The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc‐finger proteins, which bind to mRNAs containing an AU‐rich element (ARE) in their 3′ untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE‐containing transcripts by a process thought to involve phosphorylation of ARE‐binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE‐containing mRNA (ARE‐mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14‐3‐3. In vivo and in vitro data support a model where PKB causes ARE‐mRNA stabilization by inactivating BRF1 through binding to 14‐3‐3.

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

The occurrence of pathologically stable mRNAs of proto-oncogenes, growth factors and cyclins has been proposed to contribute to experimental and human oncogenesis. In normal resting cells, mRNAs containing an AU-rich element (ARE) in their 3′ untranslated region are subjected to rapid degradation. Tristetraprolin (TTP) is an RNA-binding zinc-finger protein that promotes decay of ARE-containing mRNAs. Here we report that TTP acts as a potent tumor suppressor in a v-H-ras-dependent mast cell tumor model, where tumors express abnormally stable interleukin-3 (IL-3) mRNA as part of an oncogenic autocrine loop. Premalignant v-H-ras cells were transfected with TTP and injected into syngeneic mice. TTP expression delayed tumor progression by 4 weeks, and late appearing tumors escaped suppression by loss of TTP. When transfected into a fully established tumor line, TTP reduced cloning efficiency in vitro and growth of the inoculated cells in vivo. Transgenic TTP interfered with the autocrine loop by enhancing the degradation of IL-3 mRNA with concomitant reduction of IL-3 secretion. Our data establish the ARE as an antioncogenic target in a model situation, underline the importance of mRNA stabilization in oncogenesis and show for the first time that tumor suppression can be achieved by interfering with mRNA turnover.

A constitutive decay element promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway

ABSTRACT Tumor necrosis factor alpha (TNF-α) expression is regulated by transcriptional as well as posttranscriptional mechanisms, the latter including the control of mRNA decay through an AU-rich element (ARE) in the 3′ untranslated region (UTR). Using two mutant cell lines deficient for ARE-mediated mRNA decay, we provide evidence for a second element, the constitutive decay element (CDE), which is also located in the 3′ UTR of TNF-α. In stably transfected RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), the CDE continues to target a reporter transcript for rapid decay, whereas ARE-mediated decay is blocked. Similarly, the activation of p38 kinase and phosphatidylinositol 3-kinase in NIH 3T3 cells inhibits ARE-mediated but not CDE-mediated mRNA decay. The CDE was mapped to an 80-nucleotide (nt) segment downstream of the ARE, and point mutation analysis identified within the CDE a conserved sequence of 15 nt that is required for decay activity. We propose that the CDE represses TNF-α expression by maintaining the mRNA short-lived, thereby preventing excessive induction of TNF-α after LPS stimulation. Thus, CDE-mediated mRNA decay is likely to be an important mechanism limiting LPS-induced pathologic processes.

BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

mRNA stability and cancer: an emerging link?

Many oncogenes, growth factor, cytokine and cell-cycle genes are regulated post-transcriptionally. The major mechanism is by controlling the rate of mRNA turnover for transcripts bearing destabilising cis-elements. To date, only a handful of regulatory factors have been identified that appear to control a large pool of target mRNAs, suggesting that a slight perturbation in the control mechanism may generate wide-ranging effects that could contribute to the development of a complex disorder such as cancer. In support of this view, mRNA turnover responds to signalling pathways that are often overactive in cancer, suggesting a post-transcriptional component in addition to the well-recognised transcriptional aspect of oncogenesis. Here the authors review examples of deregulated post-transcriptional control in oncogenesis, discuss post-transcriptionally regulated transcripts of oncologic significance, and consider the key role of signalling pathways in linking both processes and as an enticing therapeutic prospect.