Extracellular Vesicles Inhibit the Response of Pancreatic Ductal Adenocarcinoma Cells to Gemcitabine and TRAIL Treatment.

Pancreatic ductal adenocarcinoma remains an aggressive cancer with a low 5-year survival rate. Although gemcitabine has been a standard treatment for advanced pancreatic cancer, patients often develop resistance to this therapeutic. We have previously shown that treating pancreatic cancer cells in vitro with a combination of gemcitabine and the cytokine TRAIL significantly reduced both cell viability and survival. The data presented here demonstrate that this response to treatment is inhibited when cells are incubated with a conditioned medium derived from untreated cells. We show that this inhibition is specifically mediated by extracellular vesicles present in the conditioned medium, as seen by a significant decrease in apoptosis. Additionally, we further demonstrate that this effect can be reversed in the presence of GW4869, an inhibitor of exosome biogenesis and release. These results show that pancreatic cancer cell-derived extracellular vesicles can confer resistance to treatment with gemcitabine and TRAIL. The implications of these findings suggest that removal of EVs during treatment can improve the response of cells to gemcitabine and TRAIL treatment in vitro.

Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL.

Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1.

DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1.

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.

Modulation of the sensitivity of Jurkat T-cells to inhibition of protein synthesis by tumor necrosis factor α-related apoptosis-inducing ligand.

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in Jurkat T lymphoma cells. One of the characteristics of the phase preceding overt apoptosis is the marked downregulation of protein synthesis. We have investigated factors that can influence this response and have explored some of the signaling pathways involved. We show that interferon-α (IFNα) pretreatment desensitizes Jurkat cells to TRAIL-induced inhibition of protein synthesis, such that the concentration of TRAIL required for 50% inhibition is increased by 10-fold. The inhibition of translation is characterized by dephosphorylation of the eIF4E-binding protein 4E-BP1 and IFNα desensitizes Jurkat cells to this effect. IFNα also inhibits TRAIL-mediated dephosphorylation of the growth-promoting protein kinase B (Akt). Since Jurkat cells are defective for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and therefore have constitutive phosphoinositide 3-kinase (PI3K) activity, we investigated the consequences for protein synthesis of inhibiting PI3K using LY294002. Inhibition of PI3K partially inhibits translation, but also enhances the effect of a suboptimal concentration of TRAIL. However, LY294002 does not block the ability of IFNα to protect protein synthesis from TRAIL-induced inhibition. Data are presented suggesting that IFNα impairs the process of activation of caspase-8 within the TRAIL death-inducing signaling complex.

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

Metformin inhibits follicle-stimulating hormone (FSH) action in human granulosa cells: relevance to polycystic ovary syndrome.

BACKGROUND: Women with anovulatory polycystic ovary syndrome (PCOS) are generally insulin-resistant and as a consequence are often treated with the biguanide metformin. Results with metformin have, however, been variable with some studies demonstrating induction of regular cycles and an increase in ovulation, whereas others do not. Hence more understanding is needed regarding the mechanism of metformin's actions in ovarian granulosa cells especially in light of previous demonstrations of direct actions. OBJECTIVE: The aim of this study was to investigate metformin's interaction with the FSH/cAMP/protein kinase A pathway, which is the primary signaling pathway controlling CYP19A1 (aromatase) expression in the ovary. METHODS: The effect of metformin on FSH and forskolin-stimulated aromatase expression in human granulosa cells was measured by quantitative real-time PCR. Activity was assessed after transfection with a promoter II-luciferase construct, and by an RIA measuring conversion of androgen to estrogens. The effect on FSH receptor (FSHR) mRNA was assessed by quantitative PCR. Levels of phosphorylated cAMP response element binding protein (CREB) and CREB-regulated transcription coactivator 2 (CRTC2) were measured by Western blotting and cAMP by a bioluminescent assay. RESULTS: Metformin markedly reduced FSH but not forskolin-stimulated aromatase expression and activity. This effect was exerted by inhibition of basal and ligand-induced up-regulation of FSHR expression. Metformin also reduced FSH-induced phosphorylation of CREB and hence CRE activity, which could potentially disrupt the CREB-CREB-binding protein-CRTC2 coactivator complex that binds to CRE in promoter II of the aromatase gene. This is mediated in an AMP-activated protein kinase-independent manner, and does not involve alteration of cAMP levels. CONCLUSION: These finding have implications for the use of metformin in the treatment of anovulation in women with PCOS.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis.

BACKGROUND INFORMATION: Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. RESULTS: We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. CONCLUSION: These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication.

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling.

Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1).

BACKGROUND INFORMATION: The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. RESULTS: We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. CONCLUSIONS: The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.

Interferon-alpha induces sensitization of cells to inhibition of protein synthesis by tumour necrosis factor-related apoptosis-inducing ligand.

Tumour cells are often sensitized by interferons to the effects of tumour necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). We have demonstrated previously that TRAIL has an inhibitory effect on protein synthesis [Jeffrey IW, Bushell M, Tilleray VJ, Morley S & Clemens MJ (2002) Cancer Res62, 2272-2280] and we have therefore examined the consequences of prior interferon-alpha treatment for the sensitivity of translation to inhibition by TRAIL. Interferon treatment alone has only a minor effect on protein synthesis but it sensitizes both MCF-7 cells and HeLa cells to the downregulation of translation by TRAIL. The inhibition of translation is characterized by increased phosphorylation of the alpha subunit of eukaryotic initiation factor eIF2 and dephosphorylation of the eIF4E-binding protein 4E-BP1. Both of these effects, as well as the decrease in overall protein synthesis, require caspase-8 activity, although they precede overt apoptosis by several hours. Interferon-alpha enhances the level and/or the extent of activation of caspase-8 by TRAIL, thus providing a likely explanation for the sensitization of cells to the inhibition of translation.

Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1.

Epstein-Barr virus (EBV) is a potent mitogenic and antiapoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phenotype and that this may be related to inhibition of the pro-apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 prevents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation.

Recent studies have suggested a role for the Epstein-Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein-Barr virus, its expression is not always sufficient for malignant transformation.