MicroRNAs (miRNAs) are small regulatory RNAs that can regulate gene expression by binding to mRNA sequences and repressing target-gene expression post-transcriptionally, either by inhibiting translation or promoting RNA degradation. We have analysed expression of 328 known and 152 novel human miRNAs in 10 benign peripheral zone tissues and 16 prostate cancer tissues using microarrays and found widespread, but not universal, downregulation of miRNAs in clinically localized prostate cancer relative to benign peripheral zone tissue. These findings have been verified by real-time RT-PCR assays on select miRNAs, including miR-125b, miR-145 and let-7c. The downregulated miRNAs include several with proven target mRNAs whose proteins have been previously shown to be increased in prostate cancer by immunohistochemistry, including RAS, E2F3, BCL-2 and MCL-1. Using a bioinformatics approach, we have identified additional potential mRNA targets of one of the miRNAs, (miR-125b) that are upregulated in prostate cancer and confirmed increased expression of one of these targets, EIF4EBP1, in prostate cancer tissues. Our findings indicate that changes in miRNA expression may have an important role in the biology of human prostate cancer.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-alpha and IFN-beta), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Endoplasmic reticulum (ER) stress-mediated apoptosis may play a crucial role in loss of pancreatic beta cell mass, contributing to the development of diabetes. Here we show that induction of 4E-BP1, the suppressor of the mRNA 5' cap-binding protein eukaryotic initiation factor 4E (eIF4E), is involved in beta cell survival under ER stress. 4E-BP1 expression was increased in islets under ER stress in several mouse models of diabetes. The Eif4ebp1 gene encoding 4E-BP1 was revealed to be a direct target of the transcription factor ATF4. Deletion of the Eif4ebp1 gene increased susceptibility to ER stress-mediated apoptosis in MIN6 beta cells and mouse islets, which was accompanied by deregulated translational control. Furthermore, Eif4ebp1 deletion accelerated beta cell loss and exacerbated hyperglycemia in mouse models of diabetes. Thus, 4E-BP1 induction contributes to the maintenance of beta cell homeostasis during ER stress and is a potential therapeutic target for diabetes.

The splicing factor SF2/ASF is an oncoprotein that is up-regulated in many cancers and can transform immortal rodent fibroblasts when slightly overexpressed. The mTOR signaling pathway is activated in many cancers, and pharmacological blockers of this pathway are in clinical trials as anticancer drugs. We examined the activity of the mTOR pathway in cells transformed by SF2/ASF and found that this splicing factor activates the mTORC1 branch of the pathway, as measured by S6K and eIF4EBP1 phosphorylation. This activation is specific to mTORC1 because no activation of Akt, an mTORC2 substrate, was detected. mTORC1 activation by SF2/ASF bypasses upstream PI3K/Akt signaling and is essential for SF2/ASF-mediated transformation, as inhibition of mTOR by rapamycin blocked transformation by SF2/ASF in vitro and in vivo. Moreover, shRNA-mediated knockdown of mTOR, or of the specific mTORC1 and mTORC2 components Raptor and Rictor, abolished the tumorigenic potential of cells overexpressing SF2/ASF. These results suggest that clinical tumors with SF2/ASF up-regulation could be especially sensitive to mTOR inhibitors.

Active-site mTOR inhibitors (asTORi) hold great promise for targeting dysregulated mTOR signaling in cancer. Because of the multifaceted nature of mTORC1 signaling, identification of reliable biomarkers for the sensitivity of tumors to asTORi is imperative for their clinical implementation. Here, we show that cancer cells acquire resistance to asTORi by downregulating eukaryotic translation initiation factor (eIF4E)-binding proteins (4E-BPs-EIF4EBP1, EIF4EBP2). Loss of 4E-BPs or overexpression of eIF4E renders neoplastic growth and translation of tumor-promoting mRNAs refractory to mTOR inhibition. Conversely, moderate depletion of eIF4E augments the anti-neoplastic effects of asTORi. The anti-proliferative effect of asTORi in vitro and in vivo is therefore significantly influenced by perturbations in eIF4E/4E-BP stoichiometry, whereby an increase in the eIF4E/4E-BP ratio dramatically limits the sensitivity of cancer cells to asTORi. We propose that the eIF4E/4E-BP ratio, rather than their individual protein levels or solely their phosphorylation status, should be considered as a paramount predictive marker for forecasting the clinical therapeutic response to mTOR inhibitors.

BACKGROUND

Despite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, TSC1 and TSC2, and other members of this pathway in tumorigenesis of OSCC.

METHODS

Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the TSC1, TSC2 and PTEN candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the TSC2 promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).

RESULTS

The semi-quantitative RT-PCR analysis showed downregulation of TSC1, TSC2, EIF4EBP1 and PTEN, and upregulation of PIK3C2A, AKT1, PDPK1, RHEB, FRAP1, RPS6KB1, EIF4E and RPS6 in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the TSC2 gene only. In order to confirm the results in patient tumor samples, the methylation status of the TSC2 gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.

CONCLUSIONS

Our study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.

Large tracts of extended homozygosity are more prevalent in outbred populations than previously thought. With the advent of high-density genotyping platforms, regions of extended homozygosity can be accurately located allowing for the identification of rare recessive risk variants contributing to disease. We compared measures of extended homozygosity (greater than 1 Mb in length) in a population of 837 late-onset Alzheimer's disease (LOAD) cases and 550 controls. In our analyses, we identify one homozygous region on chromosome 8 that is significantly associated with LOAD after adjusting for multiple testing. This region contains seven genes from which the most biologically plausible candidates are STAR, EIF4EBP1, and ADRB3. We also compared the total numbers of homozygous runs and the total length of these runs between cases and controls, showing a suggestive difference in these measures (p-values 0.052-0.062). This research suggests a recessive component to the etiology of LOAD.

Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy. Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy. We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype. Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice. There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1⁻/⁻ and Bcl-3⁻/⁻ mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3. All of the p50 gene targets were contained in the Bcl-3 gene target list. Most genes were involved with protein degradation, signaling, translation, transcription, and transport. To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy. Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading. Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3. This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse.

Protein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor(+/-) mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock.