The NMD Pathway Regulates GABARAPL1 mRNA during the EMT.

EMT is a reversible cellular process that is linked to gene expression reprogramming, which allows for epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance and it is known that, during the EMT, many stress response pathways, such as autophagy and NMD, are dysregulated. Therefore, our goal was to study the regulation of ATG8 family members (GABARAP, GABARAPL1, LC3B) by the NMD and to identify molecular links between these two cellular processes that are involved in tumor development and metastasis formation. IHC experiments, which were conducted in a cohort of patients presenting lung adenocarcinomas, showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. We observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors in A549 cells in vitro. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3'UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3 that are potentially involved in the recognition of this transcript by the NMD pathway. Our study suggests that 3'UTR-dependent NMD might be an important mechanism that is involved in the induction of autophagy and could represent a promising target in the development of new anti-cancer therapies.

GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback.

The pathway of selective autophagy, leading to a targeted elimination of specific intracellular components, is mediated by the ATG8 proteins, and has been previously suggested to be involved in the regulation of the Epithelial-mesenchymal transition (EMT) during cancer's etiology. However, the molecular factors and steps of selective autophagy occurring during EMT remain unclear. We therefore analyzed a cohort of lung adenocarcinoma tumors using transcriptome analysis and immunohistochemistry, and found that the expression of ATG8 genes is correlated with that of EMT-related genes, and that GABARAPL1 protein levels are increased in EMT+ tumors compared to EMT- ones. Similarly, the induction of EMT in the A549 lung adenocarcinoma cell line using TGF-ß/TNF-α led to a high increase in GABARAPL1 expression mediated by the EMT-related transcription factors of the SMAD family, whereas the other ATG8 genes were less modified. To determine the role of GABARAPL1 during EMT, we used the CRISPR/Cas9 technology in A549 and ACHN kidney adenocarcinoma cell lines to deplete GABARAPL1. We then observed that GABARAPL1 knockout induced EMT linked to a defect of GABARAPL1-mediated degradation of the SMAD proteins. These findings suggest that, during EMT, GABARAPL1 might intervene in an EMT-regulatory loop. Indeed, induction of EMT led to an increase in GABARAPL1 levels through the activation of the SMAD signaling pathway, and then GABARAPL1 induced the autophagy-selective degradation of SMAD proteins, leading to EMT inhibition.

(±)-BIGI-3h: Pentatarget-Directed Ligand combining Cholinesterase, Monoamine Oxidase, and Glycogen Synthase Kinase 3ß Inhibition with Calcium Channel Antagonism and Antiaggregating Properties for Alzheimer's Disease.

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3ß and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and ß-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.

The exon junction complex core factor eIF4A3 is a key regulator of HPV16 gene expression.

High-risk human papillomavirus (hrHPVs), particularly HPV16 and HPV18, are the etiologic factors of ano-genital cancers and some head and neck squamous cell carcinomas (HNSCCs). Viral E6 and E7 oncoproteins, controlled at both transcriptional and post-transcriptional levels, drive hrHPVs-induced carcinogenesis. In the present study, we investigated the implication of the DEAD-box helicase eukaryotic translation initiation factor 4A3 (eIF4A3,) an Exon Junction Complex factor, in the regulation of HPV16 gene expression. Our data revealed that the depletion of the factor eIF4A3 up-regulated E7 oncoprotein levels. We also showed that the inhibition of the nonsense-mediated RNA decay (NMD) pathway, resulted in the up-regulation of E7 at both RNA and protein levels. We therefore proposed that HPV16 transcripts might present different susceptibilities to NMD and that this pathway could play a key role in the levels of expression of these viral oncoproteins during the development of HPV-related cancers.

DNA demethylation agent 5azadC downregulates HPV16 E6 expression in cervical cancer cell lines independently of TBX2 expression.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells. It has been previously reported that the treatment of HPV-positive cervical cancer cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5azadC) caused the downregulation of E6 expression due to mRNA destabilization that was mediated by miR-375. Recently, the T-box transcription factor 2 (TBX2) has been demonstrated to repress HPV LCR activity. In the current study, the role of TBX2 in E6 repression was investigated in HPV16 cervical cancer cell lines following 5azadC treatment. A decrease of E6 expression was accompanied by p53 and p21 restoration. While TBX2 mRNA was upregulated in 5azadC-treated SiHa and Ca Ski cells, TBX2 protein was not detectable. Furthermore, the overexpression of TBX2 protein in cervical cancer cells did not allow the repression of E6 expression. The TBX2 transcription factor is therefore unlikely to be associated with the repression of E6 following 5azadC treatment of SiHa and Ca Ski cells.

Comparative RNA sequencing reveals that HPV16 E6 abrogates the effect of E6*I on ROS metabolism.

High-risk Human Papillomavirus infections are responsible for anogenital and oropharyngeal cancers. Alternative splicing is an important mechanism controlling HPV16 gene expression. Modulation in the splice pattern leads to polycistronic HPV16 early transcripts encoding a full length E6 oncoprotein or truncated E6 proteins, commonly named E6*. Spliced E6*I transcripts are the most abundant RNAs produced in HPV-related cancers. To date, the biological function of the E6*I isoform remains controversial. In this study, we identified, by RNA sequencing, cellular targets deregulated by E6*I, among which genes related to ROS metabolism. Concomitantly, E6*I-overexpressing cells display high levels of ROS. However, co-overexpression of both E6 and E6*I has no effect on ROS production. In HPV16-infected cells expressing different E6/E6*I levels, we show that the newly identified targets CCL2 and RAC2 are increased by E6*I but decreased by E6 expression, suggesting that E6 abrogates the effect of E6*I. Taken together, these data support the idea that E6*I acts independently of E6 to increase ROS production and that E6 has the ability to counteract the effects of E6*I. This asks the question of how E6*I can be considered separately of E6 in the natural history of HPV16 infection.

Isolation and Characterization of an HLA-DRB1*04-Restricted HPV16-E7 T Cell Receptor for Cancer Immunotherapy.

High-risk human papillomavirus (HPV) infection is a causal factor in oropharyngeal and gynecological malignancies, and development of HPV-targeted immunotherapy could be used to treat patients with these cancers. T cell-mediated adoptive immunotherapy targeting E6 and E7, two HPV16 proteins consistently expressed in tumor cells, appears to be both attractive and safe. However, isolation of HPV-specific T cells is difficult owing to the low frequency of these cell precursors in the peripheral blood. In addition, HPV-positive cancer cells often down-regulate major histocompatibility complex (MHC) class I expression ex vivo, limiting the efficacy of MHC class I-restricted approaches. Of particular interest is that both CD4 and CD8 T cells can mediate the responses. Given that CD4 T cells play a critical role in coordinating effective antitumor responses, the generation of a T helper response in patients with HPV16-associated malignancies would unleash the ultimate potential of immunotherapy. In this view, T-cell receptor (TCR) gene transfer could be a relevant strategy to generate HPV16-E7-specific and MHC class II-restricted T cells in sufficient numbers. An HPV16-E7/HLA-DRB1*04 TCR has been isolated from a cancer patient with complete response, and retroviral particles encoding this TCR have been produced. The transgenic TCR is highly expressed in transduced T cells, with a functional inducible caspase-9 suicide gene safety cassette. TCR transgenic T cells are HPV16-E770-89 specific and HLA-DRB1*04 restricted, as determined by interferon (IFN)-γ secretion. CD8 and CD4 T cells are equivalently transduced and secrete interleukin-2 and IFN-γ when cultured with appropriate targets. We also demonstrate that TCR transgenic T cells recognize the endogenously processed and presented HPV16-E770-89 peptide. In conclusion, our data indicate that the production of MHC class II-restricted HPV16-E7-specific T cells is feasible through TCR gene transfer and could be used for immunotherapy.

5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression.

High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression.To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2'-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis.

Overexpression of MLN51 triggers P-body disassembly and formation of a new type of RNA granules.

Metastatic lymph node 51 (MLN51, also known as CASC3) is a core component of the exon junction complex (EJC), which is loaded onto spliced mRNAs and plays an essential role in determining their fate. Unlike the three other EJC core components [eIF4AIII, Magoh and Y14 (also known as RBM8A)], MLN51 is mainly located in the cytoplasm, where it plays a key role in the assembly of stress granules. In this study, we further investigated the cytoplasmic role of MLN51. We show that MLN51 is a new component of processing bodies (P-bodies). When overexpressed, MLN51 localizes in novel small cytoplasmic foci. These contain RNA, show directed movements and are distinct from stress granules and P-bodies. The appearance of these foci correlates with the process of P-body disassembly. A similar reduction in P-body count is also observed in human HER2-positive (HER2(+)) breast cancer cells overexpressing MLN51. This suggests that P-body disassembly and subsequent mRNA deregulation might correlate with cancer progression.

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition.

Recognition of the methyl-7-guanosine (m(7)G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m(7)G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m(7)G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

Substrate determinants in the C99 juxtamembrane domains differentially affect γ-secretase cleavage specificity and modulator pharmacology.

The molecular mechanisms governing γ-secretase cleavage specificity are not fully understood. Herein, we demonstrate that extending the transmembrane domain of the amyloid precursor protein-derived C99 substrate in proximity to the cytosolic face strongly influences γ-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Aß42, whilst lowering production of Aß40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the ε-site is a critical determinant of γ-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Aß37 and reduced Aß42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct γ-secretase modulators (GSMs), we elucidated the contribution of K28 to the modulatory mechanism. Surprisingly, whilst lowering the potency of the non-steroidal anti-inflammatory drug-type GSM, the K28E mutation converted a heteroaryl-type GSM to an inverse GSM. This result implies the proximal lysine is critical for the GSM mechanism and pharmacology. This region is likely a major determinant for substrate binding and we speculate that modulation of substrate binding is the fundamental mechanism by which GSMs exert their action.

The oncogene eIF4E reprograms the nuclear pore complex to promote mRNA export and oncogenic transformation.

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC), which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin, Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

Perispeckles are major assembly sites for the exon junction core complex.

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites.

Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: involvement of apoptosis inducing factor and caspase-12.

Flavaglines constitute a family of natural anticancer compounds. We present here 3 (FL3), the first synthetic flavagline that inhibits cell proliferation and viability (IC(50) approximately 1 nM) at lower doses than did the parent compound, racemic rocaglaol. Compound 3 enhanced doxorubicin cytotoxicity in HepG2 cells and retained its potency against adriamycin-resistant cell lines without inducing cardiomyocyte toxicity. Compound 3 induced apoptosis of HL60 and Hela cells by triggering the translocation of Apoptosis Inducing Factor (AIF) and caspase-12 to the nucleus. A fluorescent conjugate of 3 accumulated in endoplasmic reticulum (ER), suggesting that flavaglines bind to their target in the ER, where it triggers a cascade of events that leads to the translocation of AIF and caspase-12 to the nucleus and probably inhibition of eIF4A. Our studies highlight structural features critical to their antineoplastic potential and suggest that these compounds would retain their activity in cells refractory to caspase activation.

Tumor necrosis factor receptor-associated factor 4 is a dynamic tight junction-related shuttle protein involved in epithelium homeostasis.

BACKGROUND: Despite numerous in vivo evidences that Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) plays a key biological function, how it works at the cellular and molecular level remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we show using immunofluorescence and immuohistochemistry that TRAF4 is a novel player at the tight junctions (TJs). TRAF4 is connected to assembled TJs in confluent epithelial cells, but accumulates in the cytoplasm and/or nucleus when TJs are open in isolated cells or EGTA-treated confluent cells. In vivo, TRAF4 is consistently found at TJs in normal human mammary epithelia as well as in well-differentiated in situ carcinomas. In contrast, TRAF4 is never localized at the plasma membrane of poorly-differentiated invasive carcinomas devoid of correct TJs, but is observed in the cytoplasm and/or nucleus of the cancer cells. Moreover, TRAF4 TJ subcellular localization is remarkably dynamic. Fluorescence recovery after photobleaching (FRAP) experiments show that TRAF4 is highly mobile and shuttles between TJs and the cytoplasm. Finally, we show that intracellular TRAF4 potentiates ERK1/2 phosphorylation in proliferating HeLa cells, an epithelial cell line known to be devoid of TJs. CONCLUSIONS/SIGNIFICANCE: Collectively, our data strongly support the new concept of TJs as a dynamic structure. Moreover, our results implicate TRAF4 in one of the emerging TJ-dependent signaling pathways that responds to cell polarity by regulating the cell proliferation/differentiation balance, and subsequently epithelium homeostasis. Drastic phenotypes or lethality in TRAF4-deficient mice and drosophila strongly argue in favor of such a function.

The exon-junction-complex-component metastatic lymph node 51 functions in stress-granule assembly.

Metastatic lymph node 51 [MLN51 (also known as CASC3)] is a component of the exon junction complex (EJC), which is assembled on spliced mRNAs and plays important roles in post-splicing events. The four proteins of the EJC core, MLN51, MAGOH, Y14 and EIF4AIII shuttle between the cytoplasm and the nucleus. However, unlike the last three, MLN51 is mainly detected in the cytoplasm, suggesting that it plays an additional function in this compartment. In the present study, we show that MLN51 is recruited into cytoplasmic aggregates known as stress granules (SGs) together with the SG-resident proteins, fragile X mental retardation protein (FMRP), poly(A) binding protein (PABP) and poly(A)(+) RNA. MLN51 specifically associates with SGs via its C-terminal region, which is dispensable for its incorporation in the EJC. MLN51 does not promote SG formation but its silencing, or the overexpression of a mutant lacking its C-terminal region, alters SG assembly. Finally, in human breast carcinomas, MLN51 is sometimes present in cytoplasmic foci also positive for FMRP and PABP, suggesting that SGs formation occurs in malignant tumours.

Intergenic transcription is not required in Th2 cells to maintain histone acetylation and transcriptional permissiveness at the Il4-Il13 locus.

Noncoding RNA transcripts mapping to intergenic regions of the Il4-Il13 locus have been detected in Th2 cells harboring transcriptionally permissive Il4 and Il13 genes but not in Th1 cells where these genes are repressed. This correlation has given rise to the idea that intergenic transcription may be involved in maintaining the "open" chromatin structure of the Il4-Il13 locus in Th2 cells. We present evidence from real-time RT-PCR, nuclear run on, chromatin immunoprecipitation and 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside-mediated transcriptional inhibition analyses that argue against this hypothesis. Instead, our results are consistent with an alternative role for intergenic transcription in the maintenance of transcriptional silence in Th1-primed cells.

The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity.

The multiprotein exon junction complex (EJC) is assembled on mRNAs as a consequence of splicing. EJC core components maintain a stable grip on mRNAs even as the overall EJC protein composition evolves while mRNAs travel to the cytoplasm. Here we show that recombinant EJC subunits MLN51, MAGOH and Y14, together with the DEAD-box protein eIF4AIII bound to ATP, are necessary and sufficient to form a highly stable complex on single-stranded RNA. Cross-linking and RNase protection studies indicate that this recombinant complex recapitulates the EJC core. The stable association of the recombinant EJC core with RNA is maintained by inhibition of eIF4AIII ATPase activity by MAGOH-Y14. We elucidate the modalities of EJC binding to RNA and provide the first example of how cellular machineries may use RNA helicases to clamp several proteins onto RNA in stable and sequence-independent manners.

EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells.

Differentiation of naïve CD4 T cells toward the T helper 1 (T(H)1) and T helper 2 (T(H)2) fates involves the transcriptional repression and enhancement, respectively, of Il4 and Il13, adjacent chromosome 11 genes encoding the canonical T(H)2 cytokines interleukin-4 and interleukin-13. Proper execution of this developmental fate choice during immune responses is critical to host defense and, when misregulated, leads to susceptibility to infectious microbes and to allergic and autoimmune diseases. Here, using chromatin immunoprecipitation and real time reverse transcription PCR we identify the Polycomb family histone methyltransferase EZH2 as the enzyme responsible for methylating lysine 27 of histone H3 at the Il4-Il13 locus of T(H)1 but not T(H)2 cells, implicating EZH2 in the mechanism of Il4 and Il13 transcriptional silencing.

A Leishmania major response locus identified by interval-specific congenic mapping of a T helper type 2 cell bias-controlling quantitative trait locus.

The propensity of naive CD4 T cells to become T helper (Th) type 2 cells correlates with susceptibility to infection by the protozoal parasite Leishmania major. Using genetic linkage analysis, we earlier identified Dice1 as a Th2 cell bias-controlling quantitative trait locus on chromosome 16. Using interval-specific congenic mapping, we now resolve Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which control Il4 expression from naive Th cells and thereby indirectly control Th2 cell bias. Interestingly, only one of the two congenic intervals containing Dice1.1 and Dice1.2, respectively, also contained an L. major response locus, indicating that L. major responsiveness can be insensitive to determinants that influence Th2 cell bias by controlling naive T cell Il4 expression. These results lay the groundwork for identifying the Dice1.1 and Dice1.2 genes controlling naive T cell Il4 expression and L. major responses, and for testing whether these control other Th2 cell-dependent processes such as worm expulsion, allergic asthma, and dermatitis.