MiR-122 Inhibits Cell Proliferation and Tumorigenesis of Breast Cancer by Targeting IGF1R

miRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, microRNA-122 (miR-122) has been proved to play an important role in hepatocellular carcinoma, but its functions in the context of breast cancer (BC) remain unknown. In this study, we report that miR-122 is commonly downregulated in BC specimens and BC cell lines with important functional consequences. Overexpression of miR-122 not only dramatically suppressed cell proliferation, colony formation by inducing G1-phase cell-cycle arrest in vitro, but also reduced tumorigenicity in vivo. We then screened and identified a novel miR-122 target, insulin-like growth factor 1 receptor (IGF1R), and it was further confirmed by luciferase assay. Overexpression of miR-122 would specifically and markedly reduce its expression. Similar to the restoring miR-122 expression, IGF1R downregulation suppressed cell growth and cell-cycle progression, whereas IGF1R overexpression rescued the suppressive effect of miR-122. To identify the mechanisms, we investigated the Akt/mTOR/p70S6K pathway and found that the expression of Akt, mTOR and p70S6K were suppressed, whereas re-expression of IGF1R which did not contain the 3′UTR totally reversed the inhibition of Akt/mTOR/p70S6K signal pathway profile. We also identified a novel, putative miR-122 target gene, PI3CG, a member of PI3K family, which further suggests miR-122 may be a key regulator of the PI3K/Akt pathway. In clinical specimens, IGF1R was widely overexpressed and its mRNA levels were inversely correlated with miR-122 expression. Taken together, our results demonstrate that miR-122 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting IGF1R and regulating PI3K/Akt/mTOR/p70S6K pathway. Given these, miR-122 may serve as a novel therapeutic or diagnostic/prognostic-target for treating BC.     

The PI3K/Akt pathway contributes to arenavirus budding

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a significant public health concern in regions where they are endemic. On the other hand, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway participates in many cellular processes, including cell survival and differentiation, and also has been shown to play important roles in different steps of the life cycles of a variety of viruses. Here we report that the inhibition of the PI3K/Akt pathway inhibited budding and to a lesser extent RNA synthesis, but not cell entry, of LCMV. Accordingly, BEZ-235, a PI3K inhibitor currently in cancer clinical trials, inhibited LCMV multiplication in cultured cells. These findings, together with those previously reported for Junin virus (JUNV), indicate that targeting the PI3K/Akt pathway could represent a novel antiviral strategy to combat human-pathogenic arenaviruses.     

Role for the phosphatidylinositol 3-kinase-Akt-TOR pathway during sindbis virus replication in arthropods

The efficient transmission of alphaviruses requires the establishment of a persistent infection in the arthropod vector; however, the nature of the virus-arthropod host interaction is not well understood. The phosphatidylinositol 3-kinase (PI3K)-Akt-TOR pathway is a signaling pathway with which viruses interact to manipulate cellular functions. The viral activation of this pathway can enhance translation and inhibit apoptosis, potentially promoting viral replication; conversely, repression can enhance cell death. Using a system to study Sindbis virus RNA replication in Drosophila melanogaster, we found that the overexpression of Akt enhanced Sindbis virus replication. In contrast, a decrease in viral replication was observed for flies hypomorphic for the Akt gene. Infection of cultured Drosophila cells led to the phosphorylation and activation of Akt. The chemical inhibition of PI3K, Akt, and TOR in mosquito cells reduced virus replication, suggesting that this pathway is proviral. Early after infection, there was an increase in the TOR-dependent phosphorylation of 4E-BP1 in mosquito cells and a consequent increase in the translation of a capped reporter mRNA. In contrast, no change in 4E-BP1 phosphorylation was seen in mammalian cells, and the level of translation of the reporter decreased following infection. Finally, we found that the increase in the phosphorylation of 4E-BP1 was stimulated by replicon RNA but not by UV-inactivated virus. Our data indicate that Sindbis virus replication complex formation in mosquito cells activates the PI3K-Akt-TOR pathway, causing the phosphorylation of 4E-BP1 and increasing the formation of eukaryotic initiation factor 4F (eIF4F), which promote cap-dependent translation. This virus-induced increase in cap-dependent translation allows the efficient translation of viral mRNA while minimizing the burden on the cell.     

Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway during Porcine Circovirus Type 2 Infection Facilitates Cell Survival and Viral Replication

Virus infection activates host cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates diverse cellular activities related to cell growth, survival, and apoptosis. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wasting syndrome, which is an emerging and important swine disease, can transiently induce the PI3K/Akt pathway in cultured cells at an early step during PCV2 infection. Activation of the PI3K/Akt signal was also induced by UV-irradiated PCV2, indicating that virus replication was not required for this induction. Inhibition of PI3K activation leads to reduced virus yield, which is associated with decreased viral DNA replication and lower virus protein expression. However, inhibition of PI3K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase and caspase-3 as well as DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining during the early stage of PCV2 infection. Furthermore, the pancaspase inhibitor zVAD.fmk alleviated the reduction in Akt phosphorylation levels by inhibiting PI3K activation, indicating that the signaling promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by PCV2 infection to suppress premature apoptosis for improved virus growth after infection, extending our understanding of the molecular mechanism of PCV2 infection.     

Autophagy–virus interplay in plants: from antiviral recognition to proviral manipulation

Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to ‘non-self’ such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy–virus interplay and its consequences for host immunity and viral pathogenesis in plants.     

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.     

A new player in a deadly game: influenza viruses and the PI3K/Akt signalling pathway

Upon influenza A virus infection of cells, a wide variety of antiviral and virus-supportive signalling pathways are induced. Phosphatidylinositol-3-kinase (PI3K) is a recent addition to the growing list of signalling mediators that are activated by these viruses. Several studies have addressed the role of PI3K and the downstream effector protein kinase Akt in influenza A virus-infected cells. PI3K/Akt signalling is activated by diverse mechanisms in a biphasic manner and is required for multiple functions during infection. While the kinase supports activation of the interferon regulatory factor-3 during antiviral interferon induction, it also exhibits virus supportive functions. In fact, PI3K not only regulates a very early step during viral entry but also results in suppression of premature apoptosis at later stages of infection. The latter function is dependent on the expression of the viral non-structural protein-1 (A/NS1). It has been shown that PI3K activation occurs by direct interaction of A/NS1 with the p85 regulatory subunit and interaction sites of A/NS1 and p85 have now been mapped in detail. Here, we summarize the current knowledge on influenza virus-induced PI3K signalling and how this pathway supports viral propagation.     

Varicella-zoster virus requires a functional PI3K/Akt/GSK-3alpha/beta signaling cascade for efficient replication

Successful replication of Varicella-zoster virus (VZV) relies upon strategies to counteract host defense mechanisms. This can be achieved by modulating host cell signaling pathways, which regulate apoptosis and cell survival. The Akt cascade is crucial for the regulation of cell survival since it controls factors such as Bad, FOXO1, mTor and GSK-3alpha/beta. These factors are involved in the regulation of cell death, cell cycle and translation. Here, we report i) that the VZV infection of MeWo cells caused a 9 to 18-fold increased phosphorylation of Akt. This phosphorylation was independent from PI3K inasmuch as the PI3K phosphorylation pattern differed strongly from the one of Akt. Bad, FOXO1 and mTor showed also variations in their phosphorylation patterns: phosphorylation of Bad (ser-136) decreased during the infection while phosphorylation of ser-2448 of mTor and of ser-256 of FOXO1 increased. The phosphorylation of GSK-3alpha/beta remained relatively stable during the infection. ii) Inhibition of PI3K, Akt or GSK-3alpha/beta prior to infection resulted in a severe decline of viral replication. The inhibition of Akt resulted also in an increased apoptotic response. iii) Transfection studies using plasmids coding for functional or inactive VZV protein kinases, pORFs 47 and 66, demonstrated an increase in Akt phosphorylation. Infection of MeWo cells with VZVDelta47 and VZVDelta66 resulted in a decline of Akt and GSK-3alpha/beta phosphorylation. These results suggest i) an essential role of PI3K/Akt/GSK-3alpha/beta signaling for a successful replication of VZV and ii) a key function of VZV kinases pORFs 47 and 66 to activate this pathway.     

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

MicroRNAs and human retroviruses

MicroRNAs (miRNAs) are small non-coding RNAs that control a multitude of critical processes in mammalian cells. Increasing evidence has emerged that host miRNAs serve in animal cells to restrict viral infections. In turn, many viruses encode RNA silencing suppressors (RSS) which are employed to moderate the potency of the cell's miRNA selection against viral replication. Some viruses also encode viral miRNAs. In this review, we summarize findings from human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) that illustrate examples of host cell miRNAs that target the viruses, of RSS encoded by viruses, and of host cell miRNA profile changes that are seen in infected cells. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Autophagy and RNA virus interactomes reveal IRGM as a common target

Several intracellular pathogens have the ability to avoid or exploit the otherwise destructive process of autophagy. RNA viruses are constantly confronted with cellular autophagy, and several of them hijack autophagy during the infectious cycle to improve their own replication. Nevertheless, our knowledge of viral molecular strategies used to manipulate autophagy remains limited. Our study allowed the identification of molecular interactions between 44 autophagy-associated proteins and 83 viral proteins belonging to five different RNA virus families. This interactome revealed that the autophagy network machinery is highly targeted by RNA viruses. Interestingly, whereas some autophagy-associated proteins are targeted by only one RNA virus family, others are recurrent targets of several families. Among them, we found IRGM as the most targeted autophagy-associated protein. Downregulation of IRGM expression prevents autophagy induction by measles virus, HCV and HIV-1, and compromises viral replication. Our work combined interactomic and analytical approaches to identify potential pathogen virulence factors targeting autophagy.     

PI3K/Akt/mTOR信号通路与自噬及肿瘤的关系

自噬是一种以胞质内出现双层膜结构包裹长寿命蛋白和细胞器的自噬体为特征的细胞“自我消化”过程,在维持细胞内稳态、发育、肿瘤发生和感染中发挥重要作用。近来,诸多研究表明,自噬作为一把“双刃剑”,对肿瘤的发生发展既有促进作用,也有抑制作用。PI3K/Akt/mTOR通路由PI3激酶(PI3K)、蛋白激酶B（PKB/Akt）和哺乳动物类雷帕霉素靶蛋白（mTOR）3个作用分子组成,是一个中心的调节机构,对肿瘤细胞的生长与增殖有促进作用,同时对自噬进行抑制。本文就PI3K/Akt/mTOR通路与自噬及肿瘤发生发展的关系作一综述。     

Autophagy and RNA virus interactomes reveal IRGM as a common target

Several intracellular pathogens have the ability to avoid or exploit the otherwise destructive process of autophagy. RNA viruses are constantly confronted with cellular autophagy, and several of them hijack autophagy during the infectious cycle to improve their own replication. Nevertheless, our knowledge of viral molecular strategies used to manipulate autophagy remains limited. Our study allowed the identification of molecular interactions between 44 autophagy-associated proteins and 83 viral proteins belonging to five different RNA virus families. This interactome revealed that the autophagy network machinery is highly targeted by RNA viruses. Interestingly, whereas some autophagy-associated proteins are targeted by only one RNA virus family, others are recurrent targets of several families. Among them, we found IRGM as the most targeted autophagy-associated protein. Downregulation of IRGM expression prevents autophagy induction by measles virus, HCV and HIV-1, and compromises viral replication. Our work combined interactomic and analytical approaches to identify potential pathogen virulence factors targeting autophagy.