Tommy Alain

mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs

Proliferation Control The protein complex mTORC1, which contains the protein kinase known as mammalian target of rapamycin, is an important regulator of cell proliferation and cell size. Among many targets, mTORC1 phosphorylates the eukaryotic translation initiation factor eIF4E–binding proteins (4E-BPs), thus controlling translation of proteins that regulate proliferation. Dowling et al. (p. 1172) used mice lacking expression of the 4E-BPs to show that these proteins contribute to mTORC1s activation of cell proliferation, but are dispensable for the effects of mTORC1 on cell growth. The latter required another mTORC1 target—the ribosomal protein S6 kinase. mTORC1 inhibitors are being explored as potential anticancer agents, and the presence of 4E-BPs was necessary for mTORC1 inhibitors to reduce the number and size of colonies formed by transformed mouse cells. Control of cell proliferation and cell size is separately regulated in mammals. The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogen and nutrient signals to control cell proliferation and cell size. Hence, mTORC1 is implicated in a large number of human diseases—including diabetes, obesity, heart disease, and cancer—that are characterized by aberrant cell growth and proliferation. Although eukaryotic translation initiation factor 4E–binding proteins (4E-BPs) are critical mediators of mTORC1 function, their precise contribution to mTORC1 signaling and the mechanisms by which they mediate mTORC1 function have remained unclear. We inhibited the mTORC1 pathway in cells lacking 4E-BPs and analyzed the effects on cell size, cell proliferation, and cell cycle progression. Although the 4E-BPs had no effect on cell size, they inhibited cell proliferation by selectively inhibiting the translation of messenger RNAs that encode proliferation-promoting proteins and proteins involved in cell cycle progression. Thus, control of cell size and cell cycle progression appear to be independent in mammalian cells, whereas in lower eukaryotes, 4E-BPs influence both cell growth and proliferation.

mTORC1 Controls Mitochondrial Activity and Biogenesis through 4E-BP-Dependent Translational Regulation

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

Distinct perturbation of the translatome by the antidiabetic drug metformin

Metformin has been reported to lower cancer incidence among type II diabetics. Metformin exhibits antiproliferative and antineoplastic effects associated with inhibition of mammalian target of rapamycin complex 1 (mTORC1), but the mechanisms are poorly understood. We provide a unique genome-wide analysis of translational targets of canonical mTOR inhibitors (rapamycin and PP242) compared with metformin, revealing that metformin controls gene expression at the level of mRNA translation to an extent comparable to that of canonical mTOR inhibitors. Importantly, metformins antiproliferative activity can be explained by selective translational suppression of mRNAs encoding cell-cycle regulators via the mTORC1/eukaryotic translation initiation factor 4E-binding protein pathway. Thus, metformin selectively inhibits translation of mRNAs encoding proteins that promote neoplastic proliferation, which should facilitate studies on metformin and related biguanides in cancer prevention and treatment.

eIF4E/4E-BP Ratio Predicts the Efficacy of mTOR Targeted Therapies

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.

Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSVΔM51), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSVΔM51 selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSVΔM51.

Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFβ) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFβ requires translational activation via the non-canonical TGFβ signaling branch acting through eIF4E phosphorylation.

The helicase protein DHX29 promotes translation initiation, cell proliferation, and tumorigenesis

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5′UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.

Structure-Activity Analysis of Niclosamide Reveals Potential Role for Cytoplasmic pH in Control of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling

Background: mTORC1 is dysregulated in human disease, and there is an interest in the development of mTORC1 inhibitors. Niclosamide inhibits mTORC1 signaling, but its mode of action remains unclear. Results: Niclosamide extrudes protons from lysosomes, thus lowering cytoplasmic pH and inhibiting mTORC1 signaling. Conclusion: Cytoplasmic acidification inhibits mTORC1 signaling. Significance: Our findings may aid the design of niclosamide-based anticancer therapeutic agents. Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.

Proteolytic disassembly is a critical determinant for reovirus oncolysis.

See page 1406 Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Myxoma Virus Virotherapy for Glioma in Immunocompetent Animal Models: Optimizing Administration Routes and Synergy with Rapamycin

Oncolytic myxoma virus (MYXV) is being developed as a novel virotherapeutic against human brain cancer and has promising activity against human brain tumor models in immunocompromised hosts. Because an intact immune system could reduce its efficacy, the purpose of this study was to evaluate the oncolytic potential of MYXV in immunocompetent racine glioma models. Here, we report that MYXV infects and kills all racine cell glioma lines and that its effects are enhanced by rapamycin. Intratumoral administration of MYXV with rapamycin improved viral replication in the tumor and significantly prolonged host survival. Similarly, coadministration via a method of convection-enhanced delivery (CED) enhanced viral replication and efficacy in vivo. Mechanisms by which rapamycin improved MYXV oncolysis included an inhibition of type I IFN production in vitro and a reduction of intratumoral infiltration of CD68(+) microglia/macrophages and CD163(+) macrophages in vivo. Our findings define a method to improve MYXV efficacy against gliomas by rapamycin coadministration, which acts to promote immune responses engaged by viral delivery.